ABSTRACT INACTIVATION 0F ENZYMES BY IRRADIATION WITH LOW-ENERGY ELECTRONS BY Frank Melvin Whitaker The interaction of high energy ionizing radiation with matter produces a flux of low-energy electrons. In order to study the bio- logical damage produced by these electrons, thin films of enzymes were irradiated with electrons with an energy range of 20 to 2,000 eV. Two enzymes were used as indicators of biological damage: ribonuclease and trypsin. They were plated on either stainless steel disks, or the steel disks coated first with graphite. These samples were irradiated directly with electrons in a high vacuum. The stainless steel or graphite coated disks were coated with the enzymes by dipping them into bulk solutions of the enzymes, and then pulling them out slowly with an electric motor and pulley. This procedure deposited the surface layer of protein on the disk, and gave uniform films of molecular thickness. The film thickness could be varied by changing the bulk solution concentration of enzyme and the pulling speed from the solution. Data on this effect are presented. An o-ring sealed vacuum tank was built with a support table capable of holding 12 samples and rotating them one at a time beneath Frank Melvin Whitaker the electron gun. An electron gun was built which used a rare earth oxide—coated filament as an electron source. The gun provided current densities at the sample of up to lua/cm2 with uniform electron dis- tribution over its surface. The vacuum system was capable of pumping down to and holding a working pressure of 1 x 10.7 mm-Hg. A 2.25 inch diameter area was irradiated on the sample disk, and a 2.00 inch area was assayed for enzymatic activity. The 2.00 inch area was defined on the sample disk by an o-ring seal which also formed an assay cell, over the sample disk, into which the assay solutions were poured. The tryptic esterase activity was assayed using the BAEE assay, and the activity of the ribonuclease was determined by measuring its rate of depolymerization of yeast ribonucleic acid. The inactivation cross section, 0, was found to be energy de- pendent, and it increased rapidly with increasing energy from 20 eV up to a broad peak value in the electron energy range of 300-500 eV. Ribonuclease was most sensitive to 300 eV electrons, and trypsin was most sensitive to 500 eV electrons. With further increases in electron energy the inactivation rate decreased with increasing energy, but at a much slower rate than the initial rapid increase. The graphite coating was applied to the steel disks to test for a possible support material effect. A 40% reduction in the inactivation rate was observed for both enzymes on the graphite; however, a similar protective effect was observed for inactivation caused by irradiation with 254nm UV light. Therefore, the protective effect observed on graphite is not unique to the electron inactivation process, and a possible explana- tion is offered in the text. Frank Melvin Whitaker The two most important results of this study are the fact that the enzymes are most sensitive to electrons in the 300-500 eV energy range, and that the two enzymes had different peak energies for in- activation. A qualitative discussion is presented which postulates that this difference can be explained by the relative amounts of energy absorbed by electronic excitation, ionization, and excitation following ionization. The observed cross section can be expressed as 0 = pt(E)0 = pe(E)oe + pi(E)oi + pie(E)oie, where 0t is the total energy absorption cross section, and 0e, oi, and die are the reSpec— tive energy absorption cross sections for excitation, ionization, and ionization followed by excitation. The terms pt(E), pe(E), pi(E), and pie(E) are the respective probabilities of each energy absorption process causing an inactivation. A quantitative discussion is also presented which shows that the energy dependence of O can be expressed as<5= A/E 1n BE for E :_250 eV, and by o = kEa for E < 250 eV, where A, B, k, and a are all constants, and E is the electron energy. Using published data for electron energy absorption by solid materials, the energy absorbed per molecule inactivated was deter- mined. Using this data it was also shown that the energy absorbed per molecule inactivated approached a constant value just above the peak inactivation energy. For ribonuclease this energy was about 30-50 eV absorbed, and for trypsin it was about 50-100 eV absorbed per molecule inactivated. INACTIVATION OF ENZYMES BY IRRADIATION WITH LOW-ENERGY ELECTRONS BY Frank Melvin Whitaker A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biophysics 1974 This work is dedicated to the memory of Dr. Leroy G. Augenstein ii ACKNOWLEDGMENTS I would like to acknowledge the true friendship and generous help given to me by Dr. Richard M. Roppel, who acted as my advisor during the final stages of the writing of this thesis. Without Dr. Roppel's encouragement and reassurances I probably would not have finished this work. Next, I would like to thank the people of the Biophysics Department for their support throughout the years of my graduate program at Michigan State University, and for their under- standing and willingness to let me come back for another try after my leave of absence. A Special thanks is extended to Dr. Edward Eisenstein who stood by me through some especially hard times, to Dr. Barnett Rosenberg who has been a good friend throughout my years of graduate training, and to Dr. Eloise Kuntz who has also been a good friend and given me a great deal of help with various problems in my research work. Another thank you is due Dr. Edward Yeargers for his help during the early stages of writing this thesis. I would like to acknowledge the beautiful machine work done by the Physics Machine Shop in building my equipment. And finally a heartfelt thanks to all the beautiful pe0ple of the Biomechanics Department for their generous support and making my final days at MSU a joy. iii TABLE OF CONTENTS LIST OF TABLES . LIST OF FIGURES . . . . . . Chapter I. INTRODUCTION Background Categories of Radiation Subexcitation particles Electronic excitation 1. UV radiation . . . . . . 2. Subionizing particulate interactions Ionizing radiation 1. Electromagnetic photons . . . . 2. Charged particles 3. Neutrons . Ionizing Radiation—~The Four Temporal Stages of Radiobiological Reactions . Physical stage . . . Physicochemical stage . . Chemical stage Biochemical stage Interactions of Radiation with Biological Materials . . . . . . . . . . Ionizing radiation iv Page xi xii uw Uibub .ncfi ‘OCD‘JU'I 10 Chapter EM photons . Charged particles ”Track effects" . Linear energy transfer . ACNNb—o Subionizing interactions 1. Interactions of UV with proteins and nucleic acids 2. Energy transfer . . . . 3. Production of triplet states . . . Energy involved in ionizing radiation interactions . . . Biological Irradiation Studies Subionizing Studies UV studies on proteins UV studies on nucleic acids Low-Energy Electron Studies Prokaryotic cells Electrons as structural probes Protein irradiation . Ionizing Radiation Studies Mechanisms to Explain Bi010gical Radiation Damage General Requirements . Target Theory and the One- Ionization Model . Nonspecific Ionizing Radiation Damage Models Excited triplet state intermediate product Specific Damage Model . Protein Structure and Active Site Damage Experimental Study The Problem . . . . . . . . Irradiation System . . . . . . . . Page 10 11 12 13 14 14 IS 18 19 22 22 23 25 26 26 27 28 28 29 29 30 32 33 33 34 35 35 36 Chapter Radiation used . . . . . . . . . Samples . 1. Choice of enzymes as radiation victims . 2. Relation to normal biological processes Sample support materials II. MATERIALS AND METHODS Samples . . . . . . . . . . Sample Support Disks Sample Disk Cleaning . Sample Disk Graphite Coating . Enzyme Coating . . . . Dipping apparatus . . . . . . Enzyme dipping solutions . . . . . Dipping procedure . . . . . . Irradiation System and Procedure Vacuum Tank. . . . . . . . . . . Tank body . . . . . . . . . . . . Bottom tank cover . . . . . . . . . .. Top tank cover . . . . . . . . . . . Sample table . Vacuum pumping system Electron Gun and Tower Assembly . Electron gun Filament . . . . . . . . . . . Electron "emission coating” . . . . . . Gun mount and t0p plate . . . . . . Bellows housing . . . . . . . . . Top and bottom glass housings . . . . . . Control grids . . . . . . . . . . . Sample shields . . . . . . . . . . . Power Supplies and Instrumentation . . . Main high voltage power supply . . . . Filament current supply circuit . . . . Main control panel and auxillary power supplies . . . . vi Page 36 36 37 38 39 42 42 42 43 44 45 45 47 47 48 49 49 49 49 SO 52 52 52 52 54 55 55 57 57 S8 60 60 60 62 Chapter Galvanometer . Anode current microammeter Voltmeter . . .. . . . . . Irradiation Procedure Filament coating . . . . . Vacuum pump- -down . . . . . . Filament coating activation . Gun operational stabilization Primary beam current measurement Beam expansion to cover sample . Beam current monitor . . . . . System operating parameters . Irradiation procedure . . . Final steps . . . . . . . Assay System . . Assay Trays . . . . . . . . . . Trypsin Assay . . . . . . . . Principle . . . . . . . Reagents . . . . . . . . . Procedure . . Ribonuclease Assay . Principle . Reagents Procedure . Folin-Ciocalteu Protein Determination Principle . Reagents . . Procedure . . . . III. RESULTS . . . . Sample Preparation . . . Enzyme Solution Surface Layer of Protein Preparation Sensitivity of Graphite Coating Surface Properties of Stainless Steel and Polished Graphite Disks . . . Folin Protein Determination . . . . vii Page 63 64 64 65 65 65 66 67 67 69 70 71 73 73 74 74 75 75 75 75 76 76 76 77 78 78 78 78 80 80 80 81 83 84 Chapter Protein deposited as a function of dipping speed and concentration . . . . . . Protein deposited on irradiation samples . Specific Activity of Trypsin on Sample Disks Enzyme Activity Assays . . . . . . . . . Choice of Enzymes . . . . . . . . . . Statistical Analysis . , Factors Affecting Activity Values . Age of dipping solution and protein depletion Age of prepared samples . . . . . Vacuum effect . . . Complete removal of enzyme by the assay procedure . . . . . . . . . . . Expected Error in Sample Activity Values Operational PrOperties of the Irradiation System and Samples . . . . . . . . . Beam Energy Spread . . . . . . . . . Contact Potentials . . . . . . . . Beam Penetration and Charging of Sample . Secondary Emission and Backscattered Electrons from the Sample . . . . . . The Earth's Magnetic Field Effect . . . . Inactivation Results . Data 0 O O I O O O C O 0 O O 0 Data recorded . . . . . . . . . . . Nonlinear plots Control blank errors . . . . . Data selection and rejection . . . . Data Analysis . . . . . . . . . . Inactivation cross section model General linear statistical model . . . Linear regression analysis . . . . . viii Page 84 87 88 88 88 90 92 92 93 95 99 99 103 103 104 105 106 106 106 106 106 107 109 110 111 111 116 119 Chapter IV. Inactivation Cross Section Plots . . Inactivation Rate Modifying Factors . Exposure to hot filament . Contamination by pump oil and filament activation products . . . . . . Dose rate effects . . . . Nonuniform dose distribution over the sample surface . . . . Irradiation with secondaries returned to, or removed from the samples . . Positive ion current to the sample Age of dipping solution used to prepare samples . . . . . . . . . . Different Inactivation Rates on Stainless Steel and Graphite . . . . . . . Electron irradiation . . . . Ultraviolet irradiations . . . . Total reflectance Spectra . Film density and vacuum inactivation . DISCUSSION. . . . . . . . . Discussion of Present Results . . Possible Importance of Low-Energy Electrons in an Excited Triplet State Inactivation Mechanism . . . . . . . . Inactivation cross section plots Inactivation rate per unit electron energy . Energetic Considerations . . . Electron energy absorption by protein 1. Electron energy loss rate Energy absorbed by enzyme films 1. Shifts in absorption and inactivation peaks . . . . . . . . . Electrons incident per molecule inactivated Energy absorbed per molecule inactivated Inactivation rate per unit energy absorbed . ix Page 122 125 125 125 126 127 128 129 130 130 130 131 133 138 139 139 139 140 141 143 143 144 144 148 150 153 156 Chapter Page Enzyme Energy Absorption and Degradation Spectrum . . . . . . . . . . . . . . 158 Ionization and Secondary Electron Emission . . . . 160 Energy Absorption Mechanisms . . . . . . . . . 163 Excitation versus ionization . . . . . 165 Energy dependence of the inactivation process . . 167 Different Inactivation Rates for Different Samples . . . . . . . . . . . . . . . 171 Support material effects . . . . . . . . . 172 1. Enzyme plating observations . . . . . . 172 2. High vacuum exposure activity loss . . . . 172 3. UV reflectance spectra . . . . . . . . 173 Between different enzymes . . . . . . . . . 174 Future Work . . . . . . . . . . . . . . . 174 Apparatus . . . . . . . . . . . . 17S Electron Microscope Studies . . . . . . . . . 176 Irradiation Victims . . . . . . . . . . . . 177 Amino Acid Analysis . . . . . . . . . . . . 177 Conclusions . . . . . . . . . . . . . . . 178 APPENDIX . . . . . . . . . . . . . . . . . . . 180 REFERENCES . . . . . . . . . . . . . . . . . . 184 Table 1. LIST OF TABLES Protein Present on Assay Area for Five Sample Types . . . . . . . . . . . Analysis of Variance for Trypsin Vacuum Loss on Three Support Materials . . . . Vacuum Loss of Trypsin Films of Two Thicknesses on $8 and G Disks . . . . . . . . Comparisons Between All Possible Combinations of Trypsin Vacuum Loss Means Analysis of Variance for Average Standard Deviations of Trypsin Control Blanks . . . . Estimated Variation Expected in Each Trypsin Sample Type . . . . . . . . . Voltage Range Below Which Nonlinear Plots Were Observed . . . . . . . . . . . Molecular Thickness of Enzyme Films . . . . . Values of the Constants in Equations (38) and (39) xi Page 87 97 98 98 101 101 108 151 170 LIST OF FIGURES Figure 1. 2. 10. 11. 12. 13. 14. 15. 16. 17. 18. Dipping Apparatus . Vacuum Tank Body and Top Cover Sample Table. . . Sample Table Bearing and Contact Block . Electron Gun Electron Gun Mounting Gun Tower . . Assay Tray . . . Functional Pictoral of Gun and Tower Trypsin Plated on SS Function of Dipping Speed, Fixed Concentration . . . Specific Activity of Trypsin on Sample Disks . Trypsin Inactivation Cross Sections, 0 . Ribonuclease Inactivation Cross Sections, 0 Absorption and Reflectance Spectra Inactivation Rate Per Unit Energy--Trypsin, O/E . Inactivation Rate Per Unit Energy--Ribonuc1ease, O/E Electron Total Absorption Range . . Bulk Stopping Power For Electrons in Protein, dE/dX xii Page 46 46 51 51 53 53 56 56 59 85 89 123 124 135 142 142 145 145 Figure 19. 20. 21. 22. 23. 24. 25. 26. 27. Electron Energy Absorbed by Protein Films . Number of Electrons Incident on a Trypsin Molecule Per Inactivation, Y‘l Number of Electrons Incident on a Ribonuclease Molecule Per Inactivation, Y'1 Energy Absorbed Per Trypsin Molecule Inactivated, Eabs . . Energy Absorbed Per Ribonuclease Molecule Inactivated, Babs . . . Inactivation Rate Per Unit Energy Absorbed Per Trypsin Molecule Inactivated, O/Eabs . Inactivation Rate Per Unit Energy Absorbed Per Ribonuclease Molecule Inactivated, o/EabS Fit of Trypsin Data to the Born Approximation Fit of Trypsin Data to a Simple Power Law . xiii Page 147 152 152 154 155 157 157 169 169 CHAPTER I INTRODUCTION The central problem in understanding the effects of ionizing radiation on living systems is the elucidation of the events leading up to the observed biological damage or changes. This information coupled with a detailed knowledge of the nature of the actual biologi- cal damage produced by these radiations will lead to a complete under- standing of the radiation hazards to which all living things are exposed. Hopefully, this understanding will lead to the development of protective and curative measures against radiation damage, and also possibly tora better understanding of the life process itself. Experi- mental work directed at investigating the initial processes involved in producing the observed biological effects in radiation damage are at the frontier of our technical abilities in many areas because of the exceedingly brief duration of these events. The physical proper- ties of the various ionizing radiations are fairly well understood, as are the end results of their actions on living systems, in biologi- cal terms. But the nature and importance of the many steps between the initial interaction of the radiation and its final observable expression are unknown, or at best Speculative. Great strides forward in knowledge applicable to the radiation biological problem have been made in both theoretical and experimental radiation physics and radiation chemistry. These, coupled with the great advances made in biochemical techniques and molecular bi010gy in the past two decades, have made significant contributions to our understanding of the problem. The work to be presented here was directed at the basic ques- tion of what are some of the important factors which contribute to the observed biological damage produced by ionizing radiation. One of the end results of the action of this radiation on matter is a large flux of low-energy electrons. The problem attacked by this research was the possible importance of these low-energy electrons in producing the observed biological damage. The biologic effect of low-energy electrons was studied by irradiating enzymes directly with mono- energetic electrons in the energy range of 20 to 2,000 electron volts, and studying the loss of the enzyme's catalytic ability. Background Categories of Radiation Radiation of general interest to this work consists of particles of atomic and subatomic dimensions and electromagnetic (EM) photons. Only radiation able to deposit enough energy in a cell, by the action of a single unit of radiation, to disrupt its normal life processes will be discussed here. Therefore, radiation with an energy below that sufficient to produce electronic excitations in molecules will not be discussed, with one exception that follows next. Subexcitation particles.--Biological material is always subject to subexcitation EM radiation in the form of heat and radio waves, but these are either beneficial or have no known deleterious effects at normal background levels. However, it is also possible for a biologi- cal material to have a flux of energetic particles present in it, as the result of higher energy radiation's interactions, with an energy below the lowest electronic excitation potential of the biological molecules. The only ones of recognized importance at the present time are subexcitation electrons, whose existence was postulated by Platzman.1 The effects of subexcitation electrons will be discussed below. Electronic excitation.--Radiations which can cause electronic excitation in a molecule are EM photons and particulate radiation (electrons, ions, etc.). Although the energy imparted by either must be in the range of 3.5 to 10-15 electron volts (eV), the mode of interaction is fundamentally different, and each will be discussed separately. If Platzman's theory of superexcited states2 (to be discussed below) is taken into account, the above energy range would have to be extended upwards to 20 or 30 eV. 1. UV radiation. EM photon radiation in the energy range of 3.5 to 10-15 eV is in the ultraviolet (UV), and when absorbed by bi010gical material causes excitation of molecular electrons. Ab- sorption of an EM quantum of UV radiation is an all-or-nothing process in which the photon is completely absorbed, and this absorption is governed by quantum mechanical selection rules.3 2. Subionizingparticulate interactions. Molecular electrons can also be raised to excited energy levels by collisions with ener- getic particulate radiation. However, with particulate radiation, in a single collision, the only restriction on energy abSorption is that the particle have at least enough energy to excite an electron to its lowest excited energy level in a molecule. After a collision, a particle continues on as an energetic particle minus the energy im- parted to the molecule as excitation and/or vibrational energy. The factors governing these interactions will be discussed below. Ionizing radiation.~-Ionizing radiation consists of EM photons and charged particles. Although neutrons do not ionize directly, their interactions with matter usually result in multiple ionizations. l. ElectromagneticPhotons. EM photons are called either x-rays or y-rays depending on their origin. If they originate from atomic electronic transitions and are in the energy range of from about 101 to 106eV they are called x-rays. If they originate from atomic nuclear transitions and have an energy from about 104 to 107eV they are called y-rays. Gamma rays with energies much higher than these originate in outer Space and are called cosmic rays. Their origin is in doubt.4 2. Chargedparticles. The energetic charged particles which are considered to be ionizing radiation are electrons, ions, and nuclei; they have energies from thermal to a few hundred million eV (MeV). There are also particulate cosmic rays of much higher energies. 3. Neutrons. Neutrons are uncharged particles which interact almost exclusively with atomic nuclei and do not ionize directly. However, these interactions produce unstable nuclei (radioactive) which produce high energy charged particles and y-rays, and more neutrons by nuclear fission and relaxation processes (radioactive decay).4 Ionizin Radiation-~The Four Temporal Stages ofRadiobiOIOgical Reactions . The direct action of ionizing radiation on matter undoubtedly proceeds in a series of complicated steps. Those products which are usually called primary radiation products--ions, radicals, solvated electrons, etc.--are probably produced by interactions removed from the true primary interactions with the radiation by many complex steps. PlatzmanS has suggested that these reaction steps can be divided into four temporal stages of radiochemical reactionszg physical (primary), physicochemical, chemical, and followed by a fourth, the biochemical Stage. Physical stagg:--The physical stage includes all the primary interactions with the incident radiation as well as the production of secondary radiations. All of these reactions occur on a time scale on the order of the vibrational period of atomic electrons, i.e., from -15 to 10"14 seconds, and leave in their wake various highly excited 10 molecules and atoms (Platzman considers ionization as an excitation into the unbound state, or continuum). These reactions involve changes in atomic or molecular electron configurations only, because the time scale is too short for atomic nuclei to rearrange and produce molecular modifications--according to the Franck-Condon approximation. Platzman has been able to deduce that the products of the physical stage should be in the form of highly excited and unstable molecules.6 He demonstrates this in terms of his theory of super- excited states of molecules by Showing that the effect produced by a passing charged particle on a molecule is approximately equal to that produced by a beam of white light incident on the molecule. The num- ber of photons of a given energy in the beam is proportional to the reciprocal of the energy, and the maximum energy of the virtual photons in the beam is given by hum = hv/p, where h is Planck's constant, vm is the maximum frequency of the photons, v is the velocity of the charged particles, and p is the distance of approach (the "impact parameter") of the particle. The probability of absorption of a photon of energy E5 is proportional to the oscillator strength, £5, for an electronic transition of energy ES. Therefore, the number of primary interactions of energy ES is given by NS é constant x fs/Es' This equation is known as the optical approximation and is the basis for Platzman's predictions of excitations and energies of interaction of ionizing radiation. He notes that a large proportion of the total oscillator strength of molecules of biological interest lies at higher excitation levels. Because of the much smaller ES for valence elec- trons, most of the excitations lie in the 10 to 30 eV range, which frequently produce states of excitation which lie above the ionization energy level of these molecules. The existence of these superexcited states for periods of time long enough for the excitation energy to be degraded by means other than autoionization has been demonstrated by Jesse7 and discussed by Boag.8 Physicochemical stag_3--In this Stage the highly excited, ionized and unstable molecules formed in the primary interactions undergo various molecular rearrangements and adjustments to compensate for the altered electronic configurations. The time scale would be expected to start with that of molecular bond vibrations, which is on the order of 10"14 to 10'13 seconds, and progress through the various energy dissipation processes to about the lifetime of the lowest triplet excited states which can be on the order of seconds. If superexcited states are formed in abundance in the physical stage, as Platzman predicts, then they would be an important part of the physicochemical stage. One immediate possibility is autoionization which forms a positive ion, which would then have a high probability of being followed by a molecular rearrangement or possible dissociation.5’8 Another important possibility is that the superexcited state may be stable long enough to allow other energy degradation processes to take place. These processes could be molecular dissociation, rearrangement, or possible energy transfer to nearby molecules and other, as yet unknown, reactions driven by the large excitation energy of the super- excited states. A number of more conventional possible degradation pathways were suggested by Boag8 in an article on possible reactions in the physicochemical stage. They occur, in decreasing order of probability, 15 14 with the time scales indicated: 1) autoionization, 10' to 10' seconds; 2) molecular rearrangements and 3) molecular dissociations, 14 both of the order of 10- to 10.13 seconds; 4) radiationless energy transfer, probably very fast, but possibly up to 10.10 seconds; 13 11 5) conversion of excitation to vibrational energy, 10‘ to 10' seconds; 6) intersystem crossing to a low lying triplet excited state, 10'10 to 10.9 seconds, or less, to compete with; 7) radiative emission of singlet excitation energy, 10'9 to 10.8 seconds; and finally emission from triplet states which can have a lifetime of up to 10 seconds, or more. Probably another important damage process is the neutralization of positive ions by free electrons, which could be followed by molecular dissociation or rearrangement in response to the regained ionization energy. The time factor in the recombination process is difficult to assess because recombination depends on the availability and mobility of the electrons. Franck and Platzman9 and l . . . . . Dale 0 prov1de general reV1ews of the react1ons 1n these f1rst two stages. Chemical stage.--Following the physicochemical stage the rela- tively stable products can enter into the more conventional chemical reactions of the chemical stage. It is at this point that the most advanced detection techniques begin to measure the radiation products. The mass spectrograph used in conjunction with high speed sampling techniques can be used to isolate break-up products of excited ions free from reactions with other molecules.11 Low temperatures can be used to arrest various stages and their intermediate products. High speed flash techniques can be used in conjunction with optical and ESR Spectrographic techniques to detect reactive Species. The time scale of the chemical stage is that of normal chemical reactions--which can be as short as 10”6 seconds and as long as seconds or even hours. The results of the chemical stage can be detected as biological damage directly (e.g., by the loss of biOIOgical function), by changes in optical or ESR spectra, and by the increase in new chemical substances. Biochemical stage.--The fourth, or biochemical stage, is simply characterized by some observable change in normal biological function that can be correlated with the incidence of ionizing radiation. The expression of such changes may take from seconds to years. Okada12 presents an excellent up-to-date discussion of these topics. Interactions of Radiation with Biological Materials The interaction of radiation with biological material is almost always with an atomic or molecular electron. Depending on the energy of the radiation, the interaction either ionizes the molecule, or leaves it in an electronically excited state--or both--and when enough energy is available it also produces or leaves a radiation of lower energy. The main types of molecules found in living cells are carbo- hydrates, lipids, proteins, nucleic acids, and some prosthetic groups.13 They are usually found in an aqueous environment, but pro- teins may be found at a lipid interface in membranes. A great simplification on Studying the biological radiation problem can be made if isolated and purified biological materials are studied. An even greater simplification is made if radiation effects are studied on these compounds in the dry state, thereby eliminating 10 interactions with solute and solvent molecules activated by the radiation. Proteins and nucleic acids are the most commonly studied biological materials for radiation effects, and since dry enzymes were used in this work, the following discussion will focus mainly on radiation effects produced in proteins or those expected to be pro- duced directly in the material being studied. Ionizing radiation.--The action of ionizing EM photons and charged particles is basically different, and will be discussed separately. Two important properties of the interaction of ionizing radiation are: 1) the effects are not randomly scattered throughout the material, but are localized along the path of travel of the radiation; and 2) these localized interactions remove energy from the radiation, and depending on its properties have varying rates of energy loss in the material. 1. EM photons. An x-ray (or y—ray) is a localized EM dis- turbance with oscillating electric and magnetic fields, but has no mass associated with it. If a photon passes closely enough to a molecular electron its rapidly varying fields can impart energy to the electron in a process that is more of a resonant transfer than a collision process. The interaction of an x-ray and electron always results in the production of a fast electron, and, if the original photon's energy is high enough, another photon of lower energy. Depending on the incident photon energy, there are three basic processes involved in x-ray interactions with matter. They are, in order of decreasing x-ray energy: pair production; Compton scattering, 11 each of which produces a lower energy x-ray; and photoelectric ejec- tion, which produces only an energetic electron. The energy ranges for the three processes in carbon are: pair production for photons greater than about 1-2 MeV, Compton scattering from 10 MeV down to about 20 Kev, and photoelectric ejection at energies below about 20 KeV.14 Except for very high energy x-rays, energy degradation is by repeated Compton scatterings, each producing a high energy electron and a lower energy scattered x-ray. The process is terminated when the scattered x-ray is absorbed in a photoelectric reaction and only an electron is ejected. The result of x-ray interaction is, therefore, a number of high energy electrons (the number depending on the incident x—ray's energy) which then go on to interact as charged particles. 2. Charged particles. Charged particles carry a static electric field associated with their electric charge, and interact with atomic electrons directly through this field. The interaction is very much like a collision process, and the energy of the inter- action depends on three factors: the particle charge, speed (energy) and distance of approach ("radius of action" or "impact parameter"). Interactions in which a charged particle passes at a distance large enough so that energy in the range of a few eV up to 100 eV is trans- ferred are the most frequent, and are called glancing collisions by Fano.14 Included in this energy range are interactions which produce electronic excitation, including Platzman's superexcited states, and ionized atoms and molecules. "Knock-on" collisions are produced by a fast particle making a close approach, and the ejected electron 12 leaves at a much higher energy than in the "glancing" type collision. A charged particle making a near "head-on" collision can eject an inner shell electron, followed by an emission of an x-ray or another electron by the Auger process.14 As in the interaction of a photon with an electron, the inci- dent particle continues on, minus the energy given the electron, to make more collisions. The electrons ejected in x-ray interactions are usually of relatively high energy, which then proceed to act as charged particles as described immediately above. Since the majority of charged particle interactions are of a relatively low energy, the end result of either photon or charged particle interactions is the production of many low energy electrons and excited molecules lying along and around a well-defined track. 3. "Track Effects.” As was pointed out above, ionizing radiation leaves a track of ionized and excited molecules in its wake, as well as free energetic electrons. These electrons branch off the main particle track and are called delta rays or Spurs. The initial "track effect" is confined to a narrow path--with delta rays branching off-~but the effected area is broadened either by direct physical interactions with nearby molecules, or by other energy transfer processes. Work with the electron microscope and cell fractionation techniques have made it apparent that the living cell is a highly organized structure with its functional components organized in compartments and on surfaces, as well as being contained in smaller 13,15 bodies called organelles. The "track effects" are an important 13 factor to consider when trying to explain the biological effects of ionizing radiation because they represent an intense localized deposition of energy. Important characteristics of the particle track are ionization and excitation density, which are determined by the rate of energy transfer in the interactions with electrons of indi- vidual molecules in the physical stage of the interaction. 4. Linear energy transfer. An important prOperty of charged particle radiation interaction is the rate at which it loses energy to the irradiated medium. This is determined as a linear rate of energy transfer, or LET. It is independent of the mass of the charged particle, but increases as the square of the charge on the particle.16 However, the LET increases with decreasing speed of the particle, but in a way that must be determined experimentally. Experimental deter- minations of LET values can be used to determine both the energy deposited in a medium by an ionizing particle as well as the Spacing of the interactions. The Spacing of the ionizing radiation's inter- actions can vary, on the average, from under 10A per ionization event for a-particles, to a few hundred A for x-rays and thousands of A for 1 MeV electrons. These spacings were computed using the LET values given by Zirkle16 and assuming 100 eV/ionization cluster—-ionizations usually occur in tight clusters with an average of three ionizations per cluster.17 However, these spacings will grow less as the energy of the particle falls, growing less by a factor of 10 to 20 for the 0's and x-rays, and by a factor of over 100 for the electrons.14 These ranges place the total interaction ranges within the typical dimensions 14 of a living cell, and the potential targets in the water solvent and other solutes are many indeed. Subionizing interactions.--The studies of UV effects on pro- teins and nucleic acids have brought to light an important fact which has a bearing on ionizing radiation damage: energy absorption by molecules of quanta of energy less than that required to ionize can have lethal effects on them. The ubiquitous use of the 254nm mercury vapor lamp as a germicidal agent attests to this fact. This is impor- tant because it shows that subionizing excitations produced by ionizing radiation must also be considered as a potential biOIOgical damage mechanism. 1. Interactions of UV with proteins and nucleic acids. The action of UV with biological molecules is randomly distributed, as opposed to the highly localized "track effects" seen with ionizing radiation. UV absorption is, however, dependent on the Optical ab- sorption characteristics of the molecules present. Both proteins and nucleic acids absorb UV in the range of about 320nm down to 240nm, at which wave length both Species show a minimum. Below 240nm both show a rapidly increasing UV absorption. Absorption in the range of 320nm to 240nm is strongly dependent on pH, because each species contains ionizable chromophores. In addition, phase (crystallinity), tempera- ture, solvent, and helicity can have large effects on the absorption spectra of these molecules.18 All four of the bases of DNA and RNA are strong absorbers in the near-UV, whereas only 2 of the 20 amino acids—-tryptophan zuui tyrosine--are strong absorbers. Two other 15 amino acids--phenylalanine and cysteine--are weak absorbers at neutral pH. Therefore, nucleic acids show about 20-100 times as strong near-UV absorption as proteins.13 The near-UV absorption of proteins is almost the sum of the absorption characteristics of their amino acids. Nucleic acids, on the other hand, show a 20 to 40% increase in near-UV absorption when they are degraded to mononucleotides.18 This property is called hyperchromicity, and is significant to the radiation problem because it indicates that there is a large interaction between the bases in the polymerized nucleic acid molecules, and hence a good potential for energy transfer processes exists between them. 2. Energy transfer. Conceptually the simplest form of reso- nance energy transfer is the case in which a photon emitted by one molecule is absorbed by another molecule. A more important type is the nonradiative transfer of energy between two chromophores which have overlapping emission and absorption spectra. The model usually applicable to biological systems is the one proposed by FSrster,19 which assumes a relatively weak interaction between the chromophores. In his model the excitation energy is transferred by an electrostatic (dipole-dipole) interaction, and the transferred energy is called an exciton. The effect is well established in organic crystals, where light is absorbed and then transferred to an impurity (low concentra— tion) molecule in the crystal, which then emits most of the trans- ferred energy in the form of light of a longer wavelength. A classic example of energy transfer in a biOIOgical system is the photo- 0 decomposition of carboxyhemOglobin (CO-myoglobin complex).2 The 16 bound carbon monixide is released in high yield regardless of whether the light is absorbed in the heme (Amax = 418nm) or in the protein (Amax = 280nm).13 Augenstein §£_§l.,21 have shown that 15 to 20% of incident 254nm light absorbed in cystine disulfide residues of trypsin and ribonuclease (RNase) caused disruption of the -S-S- bond directly. The remainder of the photons are absorbed in the other chromOphores of the enzyme and appear to migrate preferentially to a tyrosine in RNase, and to a tryptophan in trypsin, which are adjacent to a critical cystine in each enzyme, and cause -S—S- bond rupture.88 Another energy transfer phenomenon observed is the apparent transfer of excitation energy absorbed in tyrosine to a tryptophan residue, when both resi- dues are present in a protein or amino acid mixtures. The enzymes a-chymotrypsin and trypsin contain both tyrosine and tryptophan and show almost complete quenching of tyrosine fluorescence20 and phos- phorescence,22 and the partial quenching of tyrosine phosphorescence in other enzymes has been demonstrated.22 The degree of quenching is probably a function of both the spatial orientation of the chromo- phores and the perturbations from being bound in the protein,22 if quenching is due to energy transfer, but the tyrosine quenching could 23,24 has also occur via other mechanisms. In addition Augenstein reported that thermoluminescence (TL) from trypsin irradiated with x-rays is characteristic of the TL from the tryptophan residues. The presence of TL and the predominant tryptOphan TL both implicate a possible energy transfer mechanism, but this time under the influence of ionizing radiation. l7 ESR studies have contributed more evidence of possible intra- and inter-molecular energy transfer in ionizing radiation effects. Irradiation of cystine-containing proteins at 77°K produced an un- identifiable singlet ESR pattern. Upon warming these samples to room temperature the ESR pattern changed to a fine structured composite pattern expected of sulfur and glycine radicals.25 Presumably the low temperature arrests a transfer process that leads to the produc- tion of the ESR pattern at room temperature, therefore indicating a transfer of electronic rearrangement energy. Intermolecular transfer of energy is evidenced by irradiation of glyceraldehyde dehydrogenase, which produces no detectable sulfur radicals, but the enzyme contains -SH groups. Addition of cysteamine produces a sulfur radical signal five times as intense as would be expected from the cysteamine alone.26 Since the enzyme contains -SH this indicates that cysteamine transfers energy to the enzyme causing sulfur radicals to appear in the enzyme too. Intra- or intermolecular charge transfer is another means of transferring energy in a biological system. It would seem an eSpe- cially probable event in the violent action of ionizing radiation, but it has also been shown to take place under milder conditions. Rosenberg27 demonstrated that charge transfer processes can take place in essentially dry proteins at subionization energies. He did so by demonstrating semiconduction in hemoglobin crystals, and further, that the conduction was via electrons. He also demonstrated that the activation energy for semiconduction was dependent on the water of hydration of the crystals, varying from 2.3 eV for dry 18 crystals to 1.45 eV for crystals containing about 7.5% water. He attributes the lowering of the activation energy to a decrease in the dielectric constant of the crystal by the water, which, in effect, decreases the electrostatic potential the conduction electron must overcome to be free. The high molecular weight, compact structure, and degree of organization of enzymes have prompted a great deal of speculation on the probability of their having crystal-like prOperties.28 In particu- lar, the a-helical sections look like regions in which electron or proton transfer can take place, and Mason29 has postulated a proton tunneling mechanism along the peptide chain (see also references 20 and 30). 3. Production of triplet states. Theoretical considerations predict, and experimental evidence confirms, that electrons of energy below about 100 eV can excite optically forbidden transitions in gases.31 Optically forbidden transitions in ethylene in the energy range of 4.4 to 10.5 eV were observed for incident electron energies of from 40 to 60 eV.32 The mechanism proposed for these excitations was direct spin interchange between the incident electron and an electron bound to the participating molecule. The particularly significant finding is that electrons with an energy about 50 eV above the excitation energy can still produce these forbidden transi- tions, and, as will be discussed below, energies in this range are expected to predominate in the electron spectrum produced by the cascade effect. 19 Experimental evidence linking the low energy electron spectrum to the production of triplet states was obtained by Augenstein et_al:33 They compared the ratio of phosphorescence/fluorescence (P/F which represents the ratio of emissive triplet/singlet states excited, if it is assumed that emitted light is a good index of the numbers of excited states formed) for near-UV and x-ray irradiation of the aromatic amino acids and trypsin. They found that the P/F increased with increasing photon energy and that it was at least ten times greater for x-rays in all compounds. A possible mechanism to explain the enhanced triplet state excitation is a direct excitation by the low energy electrons produced by the x-rays. However, other inter- pretations were also proposed. Energy involved in ionizingradiation interactions.--The earlier discussion of the events of the physical and physicochemical stages of the interaction of ionizing radiation points out that the transition energy transfer is accomplished in a violent and direct way. In the discussion of UV effects it was shown that other energy transfer processes can also take place in a more subtle way and at lower energies by resonance and charge transfer mechanisms. The relative importance of the initial high energy interactions of the ionizing radiation and the lower energy interactions, which might be considered as intermediate steps to the observed biological changes, is an Open question. The events of the first two temporal stages would be expected to take place along the path of these intense reactions-~the particle "track"--and the spacings of these reactions are related to the LET of the radiation, as opposed to the random 20 absorption of UV light, which is controlled by the light absorption properties of the molecular Species present. Experimental determinations of the energy expended per ion pair formed by electrons passing through gases support the low energy nature of most interactions, as mentioned above, and yield values of 30 to 35 eV per ionization event.34 Measurements of the inelastic collisional energy losses of 20 Kev electrons passing through very thin films of formvar (130A), polystyrene (480A), and carbon (310A) all showed that the greatest number of energy loss events was in the 20 to 30 eV range. Their mean energy losses per event were 62, 61, and 52 eV, reSpectively.35 In all three materials over 90% of the inelastically scattered electrons lost under 100 eV. The above numerical examples are indications of the general picture of the interaction of charged particles with matter (including the high energy electrons produced in the interaction of x- and y-rays). The charged particle passes through the material making a large proportion of low energy "glancing" collisions with atomic electrons.36 When an atom is approached more closely an energetic electron is ejected, and it in turn starts making low energy inter- actions. The end result of this process, sometimes called a cascade effect, is a very large number of low energy electrons and also a large number of ionized and probably excited atoms and molecules. A knowledge of the relative numbers and energies of these products would constitute an energy degradation Spectrum which is, unfor- tunately, unknown at this time.8 21 An important consideration in Platzman‘s concept of the physi- cal stage of radiation action is the distribution of the numbers and energy of the secondary electrons produced in the cascading effect. Fano36 estimates that the majority of the interactions involving the production of secondary electrons are of the low energy "glancing" type. Energies transferred in these types of collisions are expected to be on the order of 100 eV or less. He estimates that about 2/3 of the primary incident ionizing radiation energy is dissipated by secondary electrons. Once the secondary electrons have drOpped in energy by multiple inelastic collisions to below the lowest excitation potential in the material, they show a drop in the rate of energy loss by about 10'3 and are called subexcitation electrons by Platzman.37 Fano estimates that electrons with an energy below 100 eV will have a maximum range of about 10A in condensed matter and that the subexcita- tion electrons could have a range of up to 100A because of the sharp decrease in LET for an electron with an energy below the lowest ex- citation energy of the material.14 Platzman37 estimates that 15 to 20% of the incident radiation energy might be dissipated by the sub- excitation electrons by heating, since their primary mode of energy loss is to be vibrational and rotational modes of molecular energy. However, they may be important in the radiation damage process by ferming excited states by recombining with positive ions and thereby reclaiming most of the ionization energy. This excited molecule could also be in a triplet configuration if the electron were gained with its Spin unpaired. Subexcitation electrons are probably more important in in-solution processes because they are likely to form hydrated electrons.38 22 Biological Irradiation Studies Bi010gical irradiation studies can be broken down into two categories by the radiation used: ionizing and subionizing, and further into two subcategories by the irradiatiOn victim used: living organisms and subcellular components. There is a vast literature on biological irradiation studies, and a large proportion of it is de- voted to high energy ionizing radiation effects on a living system-- from single celled animals to man. The present studies evolved from those of gross physioIOgical effects in whole animals to the search for a correlation of damaged cellular components to observed physio- logical effects. Analysis of the effects of high energy radiations for basic mechanisms of action are at best exceedingly difficult because of the inherent complexity of the interactions involved and their very Short duration, as outlined in the last section. Subionizing Studies Subionizing radiation Studies on living systems range all the way from effects produced by electrostatic fields to those produced by EM photons in the near-UV. The studies in this area that are the most relevant to the ionizing radiation problem are those which involve radiations of sufficient energy to affect molecular binding forces, from about 0.1 to 1 eV (for apolar, ionic, and hydrOgen bonds) up to about 10 eV (the ionization energy) and are performed on biological molecules or model systems. Spectrographic techniques are especially valuable in these Studies, as well as to those utilizing higher energy radiations, because they can give valuable information on the energies of excited states produced as well as possible absorbed 23 energy degradation and utilization pathways. Optical Spectrographic techniques can yield information on the absorbed energy available to cause damage by observing both the absorption and reemission of energy--which is then unavailable to do damage.' ESR techniques can yield information about electronic configuration changes induced by the radiation directly. NMR techniques can yield information about nuclear environments and changes in nuclear interactions brought about by molecular rearrangements caused by the radiation. UV studies on proteins.--The most studied class of molecules for UV radiation effects is proteins and their constituent amino acids. They are studied both in the dry state and in solutions, and the major changes observed in them are: Changes in amino acid content.--Amino acids are changed or destroyed selectively because of their absorption characteristics. However, they are not always affected in proportion to their absorp- tion characteristics; in fact, even nonabsorbing amino acids in pro- teins are frequently destroyed, e.g., methionine and histidine do not absorb UV, but are frequently destroyed.30 Also the cystine disulfide bridge is ruptured to a far greater extent than would be predicted on the basis of its UV absorption alone.‘ Observations of these kinds are one of the main arguments supporting energy transfer in UV irradiated proteins. Increase in total absorbance and Shifts in absorption spectra.--Proteins usually show an increase in UV absorption after long exposures to UV. Part of this increase is due to light scattering 24 which is increased by aggregation of the protein molecules induced by the UV. However, increases are also observed which are correlated with shifts in the absorption spectrum, i.e., absorption at a shorter or longer wavelengths increase. These changes can be caused by the creation of new chromophores, or by changes in the molecular environ- ment of a chromophore, which can have drastic effects on its absorption spectrum. Spectral Shifts can also be caused by the loss of a par- ticular chromophore, decreasing absorption at a particular wavelength. The effects of being bound in a molecule are amply demonstrated by these studies, because much larger changes in absorption spectra and absorbance are observed for amino acids in proteins than an equivalent amino acid mixture irradiated with UV.18 Decreases in solubility.--Some proteins show marked decreases in solubility in response to UV irradiation. This is observed as a precipitation of protein from solution, or failure to dissolve again when irradiated in the dry state. These changes are attributed to aggregation of protein molecules and changes in amino acid side chains. After a very long exposure to UV some proteins again become soluble, an effect probably due to degradation of the aggregates and molecules. Increase in viscosity and decrease in sedimentation rates.-- Both these effects are caused by UV-induced aggregation of molecules. The opposite effect is also observed when UV causes loss of protein structure by breaking weak intramolecular bonds which allows unfolding of the molecules. Breakage of the peptide backbone of the molecule . . 18 by direct action of near-UV photons has not been demonstrated, but 25 molecular fragmentation can take place by energy transfer and secondary processes. Loss of enzyme activity.--Perhaps one of the most studied UV effect on proteins is the loss of enzymatic catalytic activity. Enzymes provide an ideal radiation probe because they have a unique biological function, and are very sensitive to disruptive forces, losing their activity in reSponse to them. The usual procedure is to try to correlate the loss of activity with some observed physical expression of the UV's action on the protein. For example, rupture of -S-S- bridges can be correlated with loss of molecular configura- tion by changes in viscosity, solubility, and sedimentation rates. However, some of the physical attributes of UV action on proteins are not correlated with the loss of activity. For instance changes in viscosity are not observed until 95% of the activity has been lost. The UV inactivation process is fairly inefficient, with inactivation yields on the order of 10'2 molecules inactivated per photon absorbed.30 UV studies on nucleic acids.--The action of UV on nucleic acid is generally similar to that on protein. However, the bases of nucleic acids do not seem to be as susceptible to destruction as amino acids in proteins. This is probably a reflection of the strong inter- action between bases evidenced by the hypochromicity of nucleic acids. UV does cause the rupture of the hydrogen bonding holding the two strands of DNA tOgether, thereby causing an increase in absorption, shifts in the Spectrum, and a decrease in viscosity of a DNA solution. Very long exposures of nucleic acids will cause an apparent drOp in 26 the molecular weight of nucleic acids, probably reflecting depoly- merization of the molecules. Also long exposures of nucleic acids to UV in the dry State will cause a loss of solubility, presumably due to cross-linking reactions intra- or intermolecularly. However, as with proteins,at doseswhich are able to cause observable biological effects none of the above physical effects are observed. Low-Energy Electron Studies An area of irradiation work that remains almost unexplored is that from an energy of about 25-50 eV up into the low kilovolt (KeV) range. Light sources down to about 50nm (25 eV) are fairly reliable, and with great difficulty 20 to 10nm (about 100 eV) can be achieved.40 A unique radiation source that can give any energy desired above a few eV up to the low KeV region--and well above--with ample intensity is an electron gun with a thermionic electron source. The low elec- tron energy and thermionic source limit work to high vacuum condi- tions, which severely restricts the potential samples that can be irradiated. These restrictions coupled with the difficult technical problems encountered in building a working system are probably the reason for the paucity of experimental work using low-energy electrons. The use of low-energy electrons as radiation offers the possi- bility of studying one component of many present in a medium exposed to high energy ionizing radiation, and thereby Simplifying the problem of interpretation of results. The following is a brief summary of some of the work that has been done in this area. Prokaryotic cells.--Bacterial Spores are excellent radiation victims because they are tolerant of extremes in the physical 27 environment, but are susceptible to radiation effects; and unaffected Spores regain activity if placed in a suitable culture medium. They have been used extensively in high energy radiation work, and occasion- ally in the low energy range of interest here. 'In an early work in 1930 using low-energy electrons, Wells41 was able to Show that Staphylococcus albus cells exhibited a threshold killing energy of 25 to 30 eV with electrons. Later Fitzpatrick et a1.42 confirmed this work, and demonstrated that the yeast Saccharomyces cerevisiae exhibited threshold killing by 300 eV electrons. They also demon- strated that killing of these cells at a fixed electron energy, above threshold killing, was dependent on the total energy incident on the cells. However, these results were only for total killing (i.e., failure to grow on a culture medium), therefore, a mechanistic interpretation of the results is difficult. Electrons as structural probes.--One particularly useful ap- plication of electron irradiation has been the application of the "target" theory to determinations of molecular weights of various large, bi010gically active molecules.43’44 Various workers in the biophysics group at Yale University have also reported work using low- energy electrons as probes to determine the location of radiation sensitive structures in bacterial Spores and virus particles, as well as to obtain information on their Structure. Using the enzyme inver- tase in layers of varying thickness, Davis was able to determine the range of low-energy electrons in proteins. Her values were within an additive constant (-100A) of Lea's computed values using the Bethe stopping equation.45 Low-energy electrons were used as probes to 28 inactivate sensitive units in bacterial Spores and viral particles, and these range/energy determinations were used to determine how far they were from the surface“.50 These investigations used low-energy electrons as a tool to determine the relative pOSitions of functional components in biological systems, rather than to study damage mechanisms.51 Protein irradiation.--Hutchinson undertook the only low-energy electron study of damage mechanisms to a biological molecule known to this author. He irradiated bovine serum albumin (BSA) with electrons with energies from a few eV up to about 250 eV, and then studied the loss of antigenic specificity to homologous rabbit antibody. He found a small, but real, inactivation cross section below 10 eV, with a sharp rise above 10 eV to about 100 to 150 eV. The inactivation cross section approached a constant value above 150 eV. Hutchinson attributed the rapid increase in cross section above 10 eV to the onset of ionization effects, and suggested that the constant cross section observed above 150 eV corresponded to the physical size of the BSA molecule.$2 IonizingRadiation Studies Although the mechanisms and primary interactions are undoubtedly very different for ionizing radiation than for UV on biological mater— ial, the end results observed are very similar. The main differences observed are in efficiency, because quantum yields increase from about .01 for UV to about one molecule inactivated per incident radiation particle absorbed for ionizing radiation. The yields approach unity 29 as soon as the radiation's energy is high enough to cause ionization, i.e., around 10 eV and above. Also ionizing radiation damage to amino acids and nucleic acid bases appears to be more random in nature, and more intense; and peptide chain scissions and depolymerization of nucleic acids are commonly observed. Generally, interpretation of the effects of ionizing and sub- ionizing radiation on the simplest of molecules has been aided by a continuously developing theoretical framework, and the relative simplicity of the model atoms and small molecules Studied. However, as the systems studied become more complex and eventually reach Sizes of biological interest (e.g., proteins and nucleic acids) interpreta- tion of radiation effects becomes increasingly difficult, if not impossible. When high energy radiations are used the added complexity of the many possible interactions and secondary interactions compound the difficulties. Mechanisms to Explain Biological Radiation Damage General Requirements A radiation inactivation mechanism model has to explain how the energy deposited in a biological functional unit is able to cause the observed damage. A successful model also has to explain the effects of at least two well established modifying factors of radiation damage: temperature and added molecules. The inactivation rate of an enzyme has been shown to increase five-fold as the temperature in- creased from 77°K to 375°K.53 In general the changes in rate are not this large, but the effect is generally well established. Irradiations 30 performed in the presence of gasses like 02 and NO frequently show an increased damage rate, and the presence of solids like -SH compounds and metal ions like iron and copper frequently have a protective effect.54 The effects of additives can change the rate either way, but usually by a factor less than two.24 Target Theory and the One-Ionization Model The hit theory developed by DessauerSS in 1922 was notable in that it recognized that radiation interacted with matter in discrete localized steps, and directed the attention of researchers toward smaller affected biological units. The hit theory led to the develop- ment of the target theory by Crowther,S6 and others, in 1924, which prompted the search for target structures within the living cell. Simply phrased, the target theory states that a hit-~or ionization-- anywhere within a sensitive target is enough to destroy its activity. The attendant search for these sensitive targets as well as develop- ments in biochemical techniques, enabled researchers to isolate and identify the damaged cellular components, and shifted the search for target structures to the molecular level. Lea,17 in his classic book of 1946, developed the target theory further, and described the division of radiation effects into direct and indirect components. Direct effects are those produced in molecules by the radiation without the intervention of outside activated molecules, and can therefore be studied by irradiating in the dry state. Indirect effects refer to those effects produced by the activated radiation products of water and other solute molecules. 31 In his book Lea set forth the one-ionization model which has been notably successful in explaining many of the characteristics of the radiation effect. It simply assumes that an ionization produced anywhere in the sensitive target volume is effective in the destruction of the biological activity. Further, it assumes that ionizations are distributed energetically (with a density correction) and Spatially as they are in gaseous systems. Therefore, it would be expected that about 100 to 110 eV are expended per ionization event, and it is known from cloud chamber studies and film work that there are about three ionizations produced per ionization event, on the average.44 There- fore, it is assumed that about 35 eV are expended per ion produced in biolOgical material. The action in the one-ionization model is non- specific by assumption. The main failings of the one-ionization model are its failure to predict temperature and added molecule effects. Since the number of ionizations produced in a molecule by ionizing radiation should be temperature independent the model does not provide an explanation of temperature effects. In the case of added metal ions, the electron density of added metal ions could have a local effect on the atomic level, but it seems unlikely that they could affect the ionization rate over a large molecule. This is especially so because they are added in small numbers per molecule-~usually one or two metal ions per molecule,54 and therefore would only affect localized areas of a large protein molecule. 32 NonSpecific Ionizing Radiation DamageModels Platzman and FranckS7 pr0posed a model to explain the effects of ionizing radiation based on the introduction of positive charge at an unspecified location in the molecule as a result of ionization. They proposed that inactivation caused by the act of ionizing a mole- cule had a low probability, but that the crucial event was the wave of polarization produced by the sudden introduction of a positive charge in a molecule by the ionization process. They postulated that this wave caused a profound disturbance in the molecular organization, and caused the rupture of many weak secondary and tertiary bonds. This mechanism could easily include a temperature effect, because the weak bonding should be expected to be made weaker by increasing the thermal energy of the molecule. Pollard e£_21358 later introduced an ionizing radiation model which attempted to include more factors of the inactivation process, notably charge transfer and the triplet state as an intermediate in- activation step. They proposed that at least two ionizations at unspecified locations were necessary for inactivation. The positive charge introduced is considered as excess charge that could move about the molecule by an electronic hole conduction process. This charge transfer could result in the breakage of a covalent bond in the poly- peptide chain. The triplet state was postulated to be a long-lived intermediate in the energy migration chain. These two factors allowed both a temperature and additive effect prediction. 33 Excited triplet state intermediate product.--There has been a considerable amount of speculation that low-lying excited triplet states might be important intermediates in the radiation damage pro- cess. This Speculation is based on the fact that excited triplet States can be quite long—lived relative to excited Singlet states because radiative transitions to the ground state are optically for- bidden. Triplet states have lifetimes of from 10-3 to 10 seconds, even at room temperatures. Although excited singlet States have lifetimes 9 to 10'8 seconds, and the fastest chemical reac- on the order of 10' tions take place on the order of about 10"6 seconds,3 the importance of excited states in chemical reactions is amply demonstrated by the many photochemical reactions that require activation by absorption of a photon. Therefore, excited triplet states provide an attractive long-lived "activated" Species to take part in chemical reactions. However, clear experimental evidence on this possible mechanism is lacking at the present time. Specific Damage Model In contrast to the models above, Augenstein59 proposed a "weak link" model in which radiation damage in a protein molecule was caused by the rupture of a Specific critical "weak link." The "weak link" was postulated to be essential in maintaining the configuration of the active site, and was composed of a disulfide bridge (-S-S-) of cystine and a few essential hydrogen bonds. The model postulated that energy absorbed elsewhere in the molecule could be funneled to the "weak link" and cause its rupture. This model predicts a temperature effect by weakening the hydrOgen bonds, and additives could modify the 34 radiation action either by acting as energy sinks or as energy ab- sorbers, or by interfering with the energy transfer required. Although Augenstein proposed the "weak link" model to explain UV radiation damage to proteins, the mechanism of the model Can also be used to attempt to explain ionizing radiation damage. The models above, and others which attempt to bring other factors of radiation action into them are discussed critically in reviews by Augenstein.23’30 Protein Structure and Active Site Damage. Enzymes are usually large globular proteins with molecular weights from tens of thousands to hundreds of thousands. The basic structure of proteins is a linear chain of the 20 naturally occurring amino acids, joined by amide bonds between the carbonyl group on one amino acid and the a-amino group of the next. These bonds, called peptide bonds, form a repeating structure of [-NH—CHR-CO—]n, where R represents one of the 20 various side chains of the amino acids. This linear sequence of amino acids is called the primary structure of the protein. Coiling and folding of this linear chain forms the secondary structure and is maintained by the formation of hydrogen bonds between the carbonyl oxygen and the amide nitrogen atoms of the chain. The coiling usually takes the form of an u-helix, and the folding gives rise to the pleated sheet structure.60 Folding of these secondary structures and the primary chain give rise to the tertiary structure, which gives the protein molecule its overall three-dimensional Shape, called its conformation. The main forces responsible for maintaining the tertiary structure in all proteins are imparted by hydrOgen bonds, 35 ionic bonds, and apolar or hydrophobic bonds. Also, in cystine- containing enzymes the disulfide bridge is an important factor in maintaining the molecular conformation.13 The generally accepted model for enzyme action is the active site model. The model maintains that, due to the Specific folding of the tertiary structure, there are groups of amino acid side chains located so that they can preferentially bind Specific substrates.61 The complete Structure and mechanism of enzyme action have been worked out for lysozyme, and the details fit the active Site model with con- formation changes and electronic interactions. Based on the active site model of enzyme action there are only two basic mechanisms of enzyme inactivation. External stimuli cause: 1) the enzyme to lose its active conformation (usually called denatura- tion), which disrupts the active site, or 2) the destruction of the amino acids in the active Site or of those amino acids that are essen- tial to the enzyme in some functional way. Various chemical tests can be applied to demonstrate the various possible routes of inactivation, and provide known mechanisms for comparison with radiation induced damage--e.g., hydrogen bond rupture with reagents like 8M urea pro- duces a known denaturation effect on an enzyme to compare with an un- known radiation effect.l3’18 Experimental Study The Problem In the sections above it was pointed out that the net result of the interaction of ionizing radiation in a dry material is a large flux of low-energy electrons. In addition the possibility of exciting 36 an electron into a low-lying excited triplet state directly was shown to exist. Because excited triplet States provide a long-lived excited molecule it has been postulated that they are important intermediates in the radiation damage process.62’63 The research to be reported here was undertaken to investigate the role played by low-energy electrons in radiation damage to pro- teins. Electrons in the energy range of 20 to 2,000 eV were used as the radiation, and provide energy well above the energy range of interest in the production of triplet states.31 The energy range was extended to 2 KeV to provide a wider spectrum so that the postulated triplet state inactivation involvement could be tested by comparison with inactivations at higher energies in which ionization and more energetic processes would be expected to dominate.31 Irradiation System Radiation used.--The unique electron energy range of the irradiation system involves energies from about 50 eV up into the low kilovolt range, as discussed earlier. The range provided is unique in the sense that any energy can be selected from a continuous spec- trum, with ample intensity (particle flux). The irradiation model is based on the idea that using electrons directly provides for isolation of one active component in a very complex sequence of reaction steps. The isolated component is the low-energy electron flux whose inter- action can be studied down to 20 eV, the low energy limit of the system. Samples.--The use of electrons for irradiation requires that the samples be in a vacuum, and hence in a dry state, because of the 37 limited range of low-energy electrons in a gas. This limited range also requires that the samples be present in a film about one molecule thick to insure that one molecule won't shield another, or at least will limit this effect. Therefore, enzyme samples were prepared by dipping stainless Steel disks into bulk enzyme solutions, and then drying them, which provided thin films of molecular dimensions. 1. Choice of enzymes as radiation victims. Practically speaking, there is a wide variety of enzymes commercially available fer which there exists well developed assay techniques.64 Although some enzymes will catalyze more than one Specific reaction, the reactions catalyzed are generally quite Similar, and it is generally believed that each catalytic function is localized in one area called the active site,60 discussed above. Although the ability to catalyze more than one reaction violates the Single unique function criterion, each function can usually be tested separately, if desired, and if only part of a number of functions is lost then an even finer indica- tion of radiation damage is available. In addition to strictly bio- logical assay for damage, other physical properties of proteins can be used to give an indication of radiation damage. For example the optical absorption Spectrum of a protein has been shown to change in 18 and the response to exposure to both ionizing radiation and UV, denaturation/precipitation temperature can Show a decrease upon exposure to radiation,13 however, both these effects require longer exposures than required for loss of biological activity. The molecular Species considered to be the most important in the expression of cellular radiation damage is DNA.12 However, DNA 38 is the genetic code carrier which controls the production of many proteins, and therefore has many specific functions that could be affected by radiation. Radiation damage to DNA can be determined by measuring its various physical properties (e.g., viscosity or mole- cular weight), but these are not expressions of specific biological functions and not very sensitive to small changes. Biological assays are also available, but are complicated and involve living systems. Hutchinson52 used an antigen/antibody system as an indicator of bi010gical radiation damage, but he irradiated the antigen (BSA) and exposed it to the antibody (the functional biological species) to determine biological damage. From the antigen/hapten studies on anti- body specificity, in which a small molecule, the hapten, confers the specificity of the antigen/hapten to the antibody, it might be con- cluded that antigen binding to an antibody is governed by only one small part of the antigen.65 Therefore, the antibody-antigen binding may not be very sensitive to small changes in the antigen. But, in any event, the loss of antibody specificity by changes induced in the antigen does not indicate a loss of potential biological function. If the loss of binding Specificity were caused by irradiation of the antibody this would demonstrate biological function loss. 2. Relation to normal biological_processes. The most obvious method of studying the biological significance of a particular radia- tion is to study the loss of some biological function in reSponse to the radiation. Therefore, the best subject to study would be a bi010gical entity that has only one bi010gical function, which would make possible a direct cause and effect correlation between the 39 incidence of the radiation and the functional loss that it causes. This criterion limits such a study to subcellular components, and of these enzymes are the only ones that have a unique and easily measur- able single function. This function is the catalysis of the chemical reactions in a cell, and each enzyme is usually quite Specific in the type of reaction that it will catalyze. Enzymes are proteins and can be isolated and purified, and since they are catalysts in biological reactions which are amenable to assay techniques, their functional integrity can be determined. Sample support materials.--The question of the relevancy of the environment of the sample molecules in this work to one in a living cell is difficult to answer. A very large proportion of enzyme func- tion and radiation damage studies has been carried out in dilute aqueous solutions, and this too probably represents a biologically questionable situation. Studies, in the past decade, of cell struc- ture using the electron microscOpe and differential centrifugation techniques have made it clear that a living cell is a highly structured and compartmentalized system with many coordinated enzyme systems bound to internal membrane surfaces, and yet the parts interact intimately.15 It was pointed out earlier that the effect of the action of ionizing radiation can be modified by the close association with other molecules, either those in the solvent phase or those supporting a molecule if it is attached to something. The diffusion of activated radiation products in liquid systems adds another radiation effect 40 component to be considered. Energy or charge transfer processes are possible in either liquid or solid systems, but would be expected to be greater in the bound state because of the stronger interaction due to being in closer association. Physical binding of a large protein molecule to a surface could also possibly modify its reSponse to radiation by constraining unfolding of the molecule in response to radiation induced disruptive effects--this effect is often called a "cage effect." Holladay e£_el:66 demonstrated a solvent "cage effect" with trypsin/agar complexes in solution in response to 250kvp x-rays. They feund a three—component effect: I) molecules that were slightly less sensitive to the x-rays, 2) molecules that required 3 to 4 times the uncomplexed enzyme dose for inactivation, and 3) molecules that were apparently inactivated by complexing. L8froth and Augenstein67 demon- strated that trypsin was inactivated at a slightly Slower rate on quartz or glass Slides than it was in solution by UV light. In general, however, radiation sensitivity can be affected either way by inter- actions with a binding material, but data are not plentiful and those that are available are often contradictory, Showing either increased or decreased sensitivity.67"69 The irradiation sample model used in this work, a thin layer of enzyme on a solid surface in a vacuum, completely eliminates inter- actions with any material above the sample molecules; therefore, in effect, interactions with a solvent phase are partially eliminated. There are two possible situations in regard to radiation interaction with the sample/support combination: 1) the possibility of 41 modification of the direct effects of the electrons on the sample molecules, and 2) possible effects on the sample by interactions of the electrons and the support material. The first possibility will be studied by supporting the enzyme samples on two Support materials: the stainless steel disks mentioned above, and a graphite coating applied to the disks, which is more like a biological environment because it is both carbonaceous and composed of low-atomic weight material. The interaction of the incoming electrons with the sample support material of concern would be either heating or backscattering, which would introduce the possibility of a double-dose effect. At the incident flux and energies used enzyme and support material heating by electron bombardment are completely negligible.52 The backscattered effect is well studied and represented in the literature, and, therefore, is a component of known properties which can be corrected for as necessary. CHAPTER II MATERIALS AND METHODS Samples The biological molecules used as radiation targets were the enzymes trypsin and ribonuclease. They were plated in films of mole- cular thickness onto sample support disks 2.50 inches in diameter. The area assayed for biological activity was the central area 2.00 inches in diameter, the 1/4 inch margin being left to compensate for possible edge effects. Two materials were used for the support surface: polished stainless steel and polished graphite. Each disk was coated by dipping it into a bulk solution of the enzyme and then pulling it out slowly with a motor and pulley. It is known that protein molecules in solution form a concentrated layer at an air/water interface,68 and it is this layer that was transferred to the disks by the dipping technique. Sample Support Disks The sample disks were cut in 2.50" diameter disks from 16 ga. (1/16" thick) #316 Stainless Steel plate. This material was chosen because it can withstand cleaning with concentrated nitric acid and because it has a hard surface that will take and hold a high polish 42 43 and is inert to the enzymes used. Two pairs of 1/16'| holes, l/4" apart, and 1/16" in from the edge were drilled diametrically opposite. Needle-nosed pliers, pointed tweezers, or a double hook could be in- serted into either of these pairs of holes to pick the disk up or suspend it. After cutting and drilling, the disks were buffed with a buffing wheel and white jewelers rouge. The buffing marks were then removed by hand polishing with a fine metal polish (Semichrome Polish; Competition Chemicals; Iowa Falls, Iowa). Sample Disk Cleaning. A sample disk must be clean enough to hold a film of water after wetting in order to plate with protein properly. The disks were made from stainless steel so that they could be cleaned by dipping them in boiling nitric acid. This method was used, but only with moderate success, and was finally abandoned because it was inconvenient and dangerous. Next a 200 watt ultrasonic probe with a flat 1/2" tip was used. The probe's tip was placed about 1/2" below the surface of a strong laboratory detergent solution and the disk moved about under the tip for 60 seconds. This technique was also only moderately successful in producing wettable disks, but was much easier to use and completely safe. The technique finally adopted was both the most successful in producing clean surfaces, and the one that would seem most unlikely to succeed. The disk was simply placed on a paper towel, a wet thumb was thrust into the dry laboratory detergent powder (Haemo-Sol; Scientific Products; Evanston, Illinois), and the surface of the disk rubbed vigorously with the thumb and detergent paste. After this the 44 disk was again "thumbed" under warm running tap water, and the edge holes cleaned with a fresh pipe cleaner. The disk was then held by the edge holes with pointed tweezers and rinsed under running dis- tilled water for 30 seconds. The disk was then held vertically and the water allowed to drain off. (Touching the rounded tip of a glass rod to the edge of the disk Speeds draining.) The disk was then placed on edge on 4 layers of paper towels, leaning against a glass support beaker to dry. During the draining and drying steps the disk was watched constantly. The criteria for cleanliness were 1) that it dried evenly with no pulling-in of the water film at the edges, and 2) that as drying proceeded down the disk, interference fringes appeared in the film. The drying film with interference fringes made an exquisitely sensitive detector of any surface-active contaminants. Drying irregu- larities usually showed up in the glass rod draining stage, but the drying had to be watched at least until the interference fringes started moving down the disk. If any irregularities were spotted the disk was recycled through the "thumbing" process. When not in use the disks were stored in Haemo-Sol solution containing 2 oz. detergent per gallon. Sample Disk Graphite Coating_ The graphite coating was applied in two coats by spraying it from an aerosol can of Aerodag G (Acheson Colloids, Port Huron, Michigan). The disks were Sprayed while lying flat on clean paper towels. The first coat was a light priming coat; the second was heavy enough to cover any signs of the polished steel surface. The second 45 coat had to be allowed to dry undisturbed and as Slowly as possible. After drying about 15 minutes the disks were placed on a metal cookie Sheet covered with paper towels and baked in a laboratory oven at 110°C for 30 minutes to remove all the remaining solvent. After baking, the graphite coating was polished with the disk lying flat on a paper towel. (A couple of drops of water on the towel held the disk in place.) The graphite was polished by placing a doubly folded Kimwipe (type 900-8; Kimberly-Clark Corporation; Neenah, Wisconsin) over the index finger and rubbing vigorously with as much pressure as possible. The Kimwipes were changed frequently, and polishing continued until the surface was smooth and had a deep, black Shine. After polishing, the disks were rinsed in distilled water for 30 seconds. The round-tipped glass rod was again a help in draining the dr0p of liquid from the bottom of the disk, but the drying criteria did not apply to the graphite because the polished surface was com- pletely hydrophobic. The disks were again dried on edge on paper towels leaning against a glass support beaker. Enzyme Coating_ DippingapparatuS.--The electric motor and pulley shown in Figure l were used to pull the sample disks slowly from the dipping solution. Two motors (model DA 1 r.p.m. and CA l/3 r.p.m.; Hurst; Princeton, Indiana) in conjunction with pulley radii of 1/4", 3/8", and l/2" to 4" in 1/2” Steps, provide a wide range of dipping speeds. The coupling between the motor and pulley Shaft Slipped under moderate torque so that the pulleys could be turned by hand. The double pulleys and pull cords provided torsional stability to the hanging disk. 46 um>ou new use Atom xcme Easom> N anamsm maumuwmm< wcwmmwo H wHDMHn— 47 Enzyme dippinggsolutions.--All enzymes were dissolved in pure water to keep the protein films as free of contaminants as possible. Solutions were made up in 220 ml lots using glass distilled water, and were stored in Nalgene centrifuge bottles at 2-3°C. Mixing was accomplished by placing the enzyme on top of the water in the bottle, and allowing it to dissolve undisturbed. Both enzymes were obtained from the Worthington Biochemical Corporation; Freehold, New Jersey, and used as obtained. The trypsin, from bovine pancreas, code TRL, is a twice crystallized, dialyzed salt-free, lyOphilized powder. The ribonuclease, also from bovine pancreas, Code R, is twice crystallized from ammonium sulfate and once from dilute ethyl alcohol, and comes in powdered form. The trypsin was used in two concentrations: 60 and 120 uM; the ribonuclease only 60 uM. Dipping procedure.--The amount of protein plated on the disk was dependent on both the solution's protein concentration and the pulling Speed. Stainless steel (SS) and graphite (G) disks were plated by being pulled at 10.6 cm/min. (1/3 r.p.m. motor, 2" radius pulley) from 60 uM trypsin. These will be denoted as T-S-52 and T-G-40, respectively. To obtain a coating with twice as much protein as the T-S-SZ disks, SS disks were pulled at 16 cm/min. (l r.p.m. motor, 1" radius pulley) from 120 uM trypsin. These disks will be denoted as T-S-llZ. SS and G disks were pulled from 60 uM ribo- nuclease (RNase) at 16 cm/min., and these will be denoted as R-S-31 and R-G-38, reSpectively. The number in the sample designation denotes the film thickness, which will be shown below. 48 The dipping solutions were taken from the refrigerator at least 3 hours prior to dipping and were warmed by a small fan. When using RNase the dipping beaker was coated with Silicone using a 1% solution of Siliclad in water (Clay Adams; Parsippany, New Jersey) before filling with RNase solution because RNase is known to have a high affinity for glass.70 The dipping beaker was kept covered when not in use. When a disk was to be dipped it was hooked by its edge holes, as Shown in Figure l, and lowered into the solution by hand. The disk was then allowed to stand in the solution, undisturbed, for 20 seconds before the pull was started by motor. After the pull, the disk was placed on edge on paper towels leaning against a glass sup- port beaker to dry. The uniform drying of the protein film was used as the criterion for a successful dip. Drying was a little more erratic on the coated disks and interference fringes could not be seen on G disks, but improperly plated areas were very easy to spot during the drying process. The disks were stored tightly covered under room conditions between all preparation steps, and after dipping in the enzyme solutions. Irradiation System and Procedure The irradiation system can be broken down into three main component groupings for description: 1) the vacuum tank, sample support table, and pumping system; 2) the electron gun and its housing, the gun tower, with its electrostatic control elements; and 3) the electrical power supplies and instrumentation. The sample table accommodated 10 irradiation samples and 4 vacuum blanks. The pumping system routinely pumped down to below 2 x 10.7 torr (mmHg) in 4 to 8 49 hours, and maintained this pressure during irradiations. The thermionic electron gun irradiated a 2.25” diameter sample area uniformly with a total beam current of from 0.50 to 10.0 x 10'6 amps. The control ele- ments located in the gun tower provided for control of the primary beam and the backscattered (secondary) electrons. The beam current and net current to the sample could be monitored at all times. Vacuum Tank Tank body.--The tank body is Shown in Figure 2 and was a 2.75" length cut from a 16" I.D. brass pipe. The flanges were cut from a 1/2" thick brass plate, and were Silver soldered to the tank body. The flanges increased the internal height to 3.25". A Simple o-ring sealed valve was fitted in the side of the tank for an air inlet. Bottom tank cover.-—The bottom cover, shown in place in Figure 2, was cut from 1/2" brass plate. An o—ring groove was cut in it to provide for a 1/4" o-ring to seal against the tank body's flange. A 5.75" opening was provided for a stand-off pipe and flange to mount the optical baffle for the diffusion pump. A 1/2" centered dowel was provided to locate the sample table bearing, and an electrical feed- through was fitted in the cover for the sample and sample table elec- trical connections. Top tank cover.--The top cover is Shown to the rear in Figure 2. It was cut from l/2" brass plate and had a o-ring groove to provide for a 1/4" o-ring seal to the top tank body flange. A 5.00" access port was fitted with an o-ring sealed cover plate. Next 50 to the access port was a 3.30" Opening for the gun tower which was placed diametrically Opposite the baffle opening in the bottom cover. An o-ring sealed fitting for the ionization gauge was installed, and a threaded port was provided for the thermocouple gauge tube. At the center of the table was the o-ring sealed motion feed-through to rotate the sample table. An indexed handwheel was provided which indicates which sample position was under the gun. Sample table.--The sample table is shown in place in Figure 3 with 4 control vacuum blanks placed in its center and 2 radiation sample disks in place. The samples rested in teflon cups which pro- vided for electrical isolation, and made contact to the outside via the cup's metal bottom disk. The screw attaching the metal disk to the teflon cup passed through the cup's bottom and made electrical contact to the contact block located directly below the gun. Shown in place over the sample disk to the right is a guard ring which masked the 2.25" diameter area on the sample disk that was exposed to the beam. The guard rings were insulated from the samples and made con- tact through the table circuit. The functional relationship of these elements is Shown in Figure 9. There was an indexing "V" groove cut vertically in the edge Of the sample table for each of the 12 sample positions. A phosphor bronze Spring strip with a ball bearing soldered at its tip was attached to the contact block below the gun to engage the "V" grooves and located the samples below the gun accurately. This indexing ball also made the external electrical contact to the sample table and guard rings. The table was supported by the center bearing and insulated from it by a teflon Spacer and 51 xoofim Dowacoo mam mewumom OHQOH mfimEmm q unawam tsnme mimetm m atswsm 52 nylon screws. The bearing, spacer, contact block, and underside of the table Showing the teflon cups and contact screws are Shown in Figure 4. The table itself was machined from 1/4" aluminum plate and the holes cut in it were to reduce the pumping impedance to the gun and samples. Vacuumgpumping system.--The pumping system can be seen in Figure 7 and consisted of an NRC VHS-4 1200 l/sec. diffusion pump backed by a Welch Duo-Seal 1402 mechanical vacuum pump. The diffusion pump was fitted with a conduction cooled "cold cap" and was baffled by a water cooled NRC HW4 optical baffle. The system pressure was moni- tored by an NRC type 518 ionization gauge and 2 NRC type 501 thermo- couple gauges. These were controlled and read by an NRC type 710 gauge control. The NRC components were manufactured by Norton Equipment Company; Newton, Massachusetts, and the mechanical pump by Welch Company; Chicago, Illinois. Electron Gun and Tower Assembly Electron gun.--The gun was taken from an RCA 2APlA cathode ray tube, and the deflector plates, filament assembly and all leads were removed. Figure 5 shows a prepared gun with the gun mounting clamps attached at the center. The clamps were machined from l/2" brass rod. Filament.--The filament used in the original CRT was an in- directly heated oxide coated unit; and the oxide coating is destroyed upon exposure to the atmosphere. It was replaced by a filament fashioned from .002" pure tungsten Stock. The tungsten was Obtained in a 1" wide Strip from the NRC Metals Division, Norton Company; 53 saucsoz :50 aouuoofim o Ouswfim doc cobwooflm n musmsc 54 Newton, Massachusetts. It was malleable before heating and could be cut with scissors. A 0.1" x 1" strip of this stock was bent into a "V" shape with a 0.1" flat area at the apex. This area was narrowed to about .030" wide with a high Speed hand grinder. The narrow bridge concentrated the IR heating in this area. This narrow bridge was the source of thermally ejected electrons and was centered over the hole in the gun's grid cup (Figure 9), which was the exit hole for electrons accelerated to the anode. The filament was clamped in stainless steel clamps attached to the mica disk shown in Figure 5. This disk was in turn attached to two stainless Steel blocks by two screws which allowed the disk considerable freedom of movement to align the filament in the grid cup. These two blocks seated on two .060" steel drill rods against set screws in the blocks which set the filament to grid-cup— hole distance. The steel rods were clamped to the gun by two stainless steel clamps which can be seen in Figure 5. Electron "emission coating:"--The filament could be made to emit electrons by heating it to a "white heat," and although an ade- quate supply of electrons could be obtained this way, the high tempera- tures generated caused problems. The major concern was direct heating damage to the sample, but the heating also caused long-term beam cur- rent drifting problems--presumably due to thermal expansion and shifting in the gun elements. A rare earth oxide "emission coating," type 56, was obtained in a liquid form from Kulite Tungsten Company; Ridgefield, New Jersey. A small amount of the liquid was applied to the filament bridge with a pointed stick, and when pr0perly activated provided a copious supply of electrons with the filament at a just barely visible 55 red glow. Long term beam current stability was excellent using the coating and the thermal problems were completely eliminated. The coating was destroyed by exposure to the atmOSphere and had to be re- newed each time the system was Opened. The filament mounting described above was designed for easy filament removal and accurate positioning upon reinstallation. The effort of recoating was more than compensated for by the operational stability gained. Gun mount and top plate.--The gun mounting carriage was de- signed so that the gun could be tilted to avoid exposing the samples directly to the filament. The carriage provided for movement in two dimensions in the horizontal plane and the gun bracket provided for vertical adjustments. The carriage slid in a rectangular hole in the pyrex plate which provided electrical isolation of the gun. This entire assembly is shown mounted on the top plate in Figure 6. The top plate was fitted with electrical feedthroughs for the gun leads and an o—ring groove for the vacuum seal. Bellows housing,~-The bellows housing provided for mechanical aiming of the beam by three adjustment screws 120° apart. The screws were threaded into the tOp flange and were pointed at the lower ends to seat in radial "v" grooves on the top edge of the lower flange. The lower flange had an O-ring groove for the vacuum seal to the next flange. The bellows was a one-ply brass unit and provided il/Z" ad- justment range about its neutral length. The bellows housing can be seen in Figure 7, which shows the entire gun tower. S6 >mue zmmm< w UHDMHE uo3c9 :30 A mtsmsa 57 Top and bottom glass housing§,--The glass housings were used to allow for visual alignment of the beam on the sample using a ZnS fluorescent screen. The housing was made in two pieces to mount a lens assembly at the center of the gun tower to Spread the beam when the gun was operated in a tilted position. The potential on the center flange could be used to control beam spread and to a small extent, beam shape. The housings, actually two Kovar metal-tO-glass seals, were made by H. S. Martin and Son, Evanston, Illinois, and the flanges, provided by me, were silver soldered to the seals by them. The lower housing had 11 electrical leads through the glass for the lens (not used) at the tOp, and 2 electrical leads at the bottom for the two control grids. The o-ring seals at the center and lower tower flanges were formed by concentric rings placed between the flanges, which tOgether with the flanges provided for proper o-ring compression for a vacuum seal. The housings are shown in Figure 7. ControlgridS.--Two grids were included in the beam's path, just above the sample, to control backscattered electrons from the sample. The top grid, G1, acted as a potential shield for the gun tower so that changing the potential on the lower grid, G would not 2. affect the beam above 61' The lower grid was larger in diameter than the top one to allow for beam Spreading in a retarding field. The top grid was 2-1/4" I.D., and the lower 2-3/4" I.D. The grid holder, which was machined from plexiglas, was cone Shaped internally to match these Sizes and was coated with graphite and electrically connected to the bottom grid, G2. 58 The grids were formed by Stringing .001" uninsulated tantalum wire on brass rings. The wires were spaced at .040" intervals by two perpendicular sets of grooves cut with a 0.1 mm circular saw. This provided a fine square mesh grid that was 95% transparent. The lower grid in conjunction with the sample Shield (described below) formed, in effect, a hemiSpherical retarding potential spectrometer for electrons backscattered from the sample. However, since the incident beam also contributed current to the grid and caused secondary electron emission from it, precise analysis of the currents to it was not possible. Therefore, determination of quantitative backscattered Spectra was not possible. However, qualitative results were obtained and they greatly helped in understanding the operational characteristics of the system. It was found that the grids caused uneven electron dose dis- tribution by casting shadows and focusing effects. Therefore, the grids were removed for all electron irradiation work. Secondary emissions were controlled by the potential on the tank and sample shield which were Operated at the same potential. The functional and spatial relationship of all elements of the gun and gun tower are illustrated in Figure 9. Sample shield.--During early attempts to determine primary and backscattered current components to the sample, large discrepancies were found between them. It was finally realized that there was a large leakage of backscattered electrons to the tank which had to be Operated at 2.0KV relative to the cathode because of the main power supply's grounded chassis. This problem was cured in two ways. First, the sample shield, illustrated in Figure 9, was installed which Center Tap g) ér 0V Reference Filament 0 If Grid Cup } ’ V i I g 5 A Anode ———+ ‘ VA Focus Ring— *-" —'————’ V O ‘ O. Center—.009,» - : i m Vcenter Flange G & G Lead Shield 1 2 (Screen) 3.. Grid Support (Plexiglass) >-—- --..J Tank Cover 1 EW Graphite Coating-— Sample Shield”aru (Screen) \5 Sample Table} i\\\\\\\\\\\\\\\\f\_\ Teflon Cup Guard Ring Sample Contac t——‘>' . Contact Block Figure 9 Funct'on' icLOr 1 f u nd Tower 60 enclosed the sample entirely with an electrostatic shield. It was attached to the grid support housing and connected electrically to the lower grid. When the grids were removed for irradiations another means of suppressing secondary emission was required to get an accurate measurement Of the primary beam current to the sample. This was accom- plished by the modification to the main power supply described next. It made the power supply's output completely floating, hence the tank could be set at any potential desired and the loss problem was controlled. Power Supplies and Instrumentation Main high voltage power supply.--The supply is a large vacuum tube unit which will deliver 2KV at 200ma, with its output voltage adjustable about 2KV by thOV. Its voltage output was very stable and showed less than 100mV drift in 3 hours. The ripple in the output was less than lmV when the supply was delivering lOma at 2KV. The supply was modified for the reason given above, to have a completely floating output. This was accomplished by mounting all the circuit chassis on phenolic spacers that gave at least 1/2" clearance between any chassis element and the grounded cabinet. The output of the supply was applied across a 200K ohm dropping network of 20 10K ohm resistors in series. These provided for taps with 100V steps, and with the high-voltage supply modification any point on the bridge could be grounded safely. Filament current supply circuit.--This circuit turned out to be the most troublesome link in the system as far as beam current 61 stability was concerned. A typical filament drew 8 amps with a voltage drop of only about 0.1 volt, which meant that every connection in the high current circuit had to be absolutely clean and very tight. The connections in the gun chamber were the mOSt critical because of thermal expansion and the requirement of removing the gun for each run. The filament was powered by a Chicago Stancor P4086 filament transformer, insulated for lOKV isolation, capable of 14 amp output at 5 volts, and center tapped. The center tap was used as the 0V reference point for the electrical system Since it was at the potential of the center of the filament, assuming that most of the voltage drop in the filament circuit was across the narrow filament bridge. How- ever, since the voltage drop across the filament was only 0.1—0.2 volts this was not a critical point. The input to the filament transformer had to be absolutely stable, but also had to be variable in small increments that were stable once set. Variations in the line voltage were controlled by a Sola CVS constant voltage transformer with an output capacity’of 250va (Sola Electric Company; Elk Grove, Illinois). The output from the Sola was applied to the primary of a transformer that had an output of 48V in four 12V steps. The output was selected by moving a lead on a heavy duty terminal strip. This reduced voltage was supplied to the input of a Powerstat type 116 autotransformer (Superior Electric Company; Bristol, Connecticut). The Powerstat was modified by filing down the moving contact brush until it contacted one wire at a time on the transformer coil sector. The contact was set on a single wire visually (cover removed), and this procedure eliminated the occasional 62 uncertainty in a Powerstat's output which is inherent in its design, because the movable contact overlapped 3 or 4 contacts on the sector. Finally, ultrafine current adjustments were provided by a 1 Ohm, 50 watt rheostat in series with the primary of the filament trans- former. The rheostat was conveniently located and provided for the only necessary adjustment once the gun had warmed up and stabilized. Current through the filament was measured on a 0-10 amp ammeter, but its value was only a reference guide since the critical variable was the beam current. Main control panel and auxillary power supplieS.--This panel was essentially a junction box which had taps for each of the gun and control tower elements, and taps from the voltage divider for the main power supply which were in 100V steps, from 0 to 2KV. The OV tap was connected to the center tap of the filament transformer, and an addi— tional resistor prior to this point in the voltage divider provided -50V for beam turn Off at the gun grid cup (Figure 9). The focus element of the gun (Figure 9) required down to -210V to Spread the beam, so a set of 10 batteries, a lO-position switch, and a potentiometer provided down to -236V for this purpose. Two Heathkit IP-32 regulated 0-400V power supplies (Heath Company; Benton Harbor, Michigan) which were isolated from ground potential by line isolation transformers, had their inputs, outputs, and voltage controls terminated on the control panel. They were used to provide fine control for the sample and ground potentials and provide more flexibility in operation Of the system. Also provided on the control panel is a switch which applies a fixed sample and tank potential for 63 beam current measurements. (Its use will be described below.) All connections were made via external patch cords between the jacks for various elements and the voltage divider taps to retain maximum flexi- bility with easy system set-up. Galvanometer.--The sample current was a critical factor, and this was measured on a Rubicon D'Arsonval galvanometer (Catalogue No. 3414; Rubicon Company; Philadelphia, Pennsylvania) with a rated sensitivity of .0015 ua/mm (1mm = smallest linear scale division). A simple electromechanical meter was chosen over a more sophisticated electronic unit because its sensitivity was more than adequate, and Since the input could be iZKV d.c. with reference to ground, difficult isolation and drift problems were avoided. The galvanometer's sensitivity was attenuated with a D'Aryton Shunt which provided a constant resistive load to the galvanometer equal to its critical damping resistance. The resistors used in the Shunt were chosen to give an adequate attenuation range-~full sensitivity = 1.42 x 10.5 amp/div. down to 8.03 x 10'7 amp/div. in many small range steps. This had the disadvantage of not giving a direct reading scale, but the advantage of keeping deflections always nearly full scale. The galvanometer/shunt combination was calibrated using 3 General Radio precision decade resistance boxes (0.1% toler- ance) and a dry cell whose voltage was monitored to lmv on a Leeds and Northrup potentiometer. The decade resistance boxes and battery were set up as a precision vOltage source and the galvanometer current/ deflection ratios calculated by Ohm's law. The galvanometer's re- sistance was checked by Obtaining a full scale deflection and then 64 shunting it with one of the decade boxes and reducing the deflection to one—half the full scale value. When the resistance Of the Shunt was equal to the resistance of the galvanometer's coil, one-half the current flowed through each, and the deflection was reduced to one- half. This determination was reproducible to about $2 Ohms in 400 Ohms, or il/2%. I would, therefore, estimate the overall calibration of the galvanometer/shunt to be good to a tolerance of il%. To compensate for small leakage currents a bucking voltage was applied to the input of the shunt from a pair of dry cells and two potentiometers. Anode current microammeter.—-The beam current was monitored by measuring the current to the anode. Once the gun was warmed up and Operation was stable, any changes in the beam current were reflected by changes in the anode current, Since they had to originate from changes in the emission current from the filament. The anode current was read on a 0-50ua Weston model 301 microammeter (Weston Instrument Company; Newark, New Jersey). Since the readings were reference values only, they were kept on scale by placing a variable resistance Shunt directly across the meter. Voltmeter.--TO avoid high voltage isolation problems either a simple electromechanical meter or a battery Operated electronic meter was required. Except for battery operated meters costing many times more than a simple VOM, there was not much to be gained in accuracy by going to a battery meter, and sensitivity was no problem. Therefore, a simple VOM (a Triplett model 630 PLK, Triplett Electric Instrument 65 Company, Bluffton, Ohio) was used in the early stages of the irradia- tion work to measure the sample potential. The results of this work made it apparent that a more expensive 1% tolerance electronic volt- meter was not required because the inactivation cross section did not change rapidly with electron energy, and the Triplett VOM was used throughout this work. Irradiation Procedure Filament coating.--The filament had to be recoated with "emission coating” before each run was started. The gun was usually removed at the end of each run to reduce the time the system was Open to the atmOSphere before starting a new run. Once the coating was applied and dry, it was sensitive to atmospheric water. Therefore, it was usually coated while the dipped enzyme samples were drying. When the filament was coated and the gun reassembled, the gun and samples were loaded into the vacuum tank as rapidly as possible. Vacuumpump-down.-7Freshly dipped samples were always placed in the vacuum tank and pumping started within 1 to 2 hours after dipping was completed. The system was rough pumped 10 minutes prior to turning the cooling water and diffusion pump on. The diffusion pump required 10 minutes to heat up and was usually down to 10.5 torr in 20 to 30 minutes. After this the rate of pressure decrease was dependent on how long the system had been Open to the atmosphere and how humid it had been. The vacuum criterion was 2 x 10.7 torr, but the system usually reached 1.2 x 10.7 torr in 8 hours, the usual pump—down time. In very humid weather pump-down could take as long as 12-16 hours. 66 Filament coatingfiactivation.--In the freshly applied form the "emission coating" was in an acetate binder and the oxide was present as a carbonate. To activate the filament the acetate binder had to be driven off first by heating at a low temperature. If the coating was heated at too high a temperature the coating would flake Off at this stage. In the second stage the carbonates were broken down to the oxides and emission started. The binder usually Started breaking down at about 50% operating current, and the coating activation began at about 90-95% Operating current. The activation process was monitored by the increase in system pressure on the ionization gauge. The limit I found that worked best was never to let the pressure go above 3-4 x 10.7 torr during the process. The high voltage supplies were turned on at the start of the activation process and the filament Powerstat turned up slowly until a reSponse was seen on the ionization gauge. After this the Powerstat was advanced one or two segments at a time (see Filament Current Supply Circuit, above). Again the increase in filament current was limited by the increases in the system pressure. It usually took about an hour to reach Operating current. Once the coating Started emitting, the filament current was increased to about 105% its Operating value and held there until the coating was emitting strongly--usually in 2-5 minutes. Occasionally a coating would start emitting, then fall to practically zero emission and then take about 30 minutes at the 105% current to start emitting strongly. Occasionally a coating would fail altogether. Gun Operational stabilization.—-There was a period of an hour or two after the filament Started emitting in which the beam current 67 drifted around quite badly. I found that the ultimate stability was improved if the emission current was kept reasonably constant during this time. To avoid long exposure effects on either the screen or tune-up disk (to be explained below), the gun was left on but the beam was Stopped at the center flange by applying -300V to it from an auxillary battery (U200, "B” battery) referenced to 0V. During irradiation, however, the beam was turned Off by applying -50v to the gun's grid cup (Figure 9). After this initial period of an hour or two, the beam current was checked every hour or two until the gun had been on 6 to 8 hours. After this period, if the anode current held the desired value for at least 15 minutes without drifting, the gun was considered ready to calibrate for the run. Primary beam current measurement.-—During irradiations G1 and 62 were removed and VGl’ vtank’ and VGz were all set equal to vtank (Figure 9). This placed everything immediately above the sample at the same potential and the grid support graphite coating and sample shield were able to control backscattered electrons. Sample positions #1 and #2 were specially prepared for setting the system up for an irradiation run. Position #1 contained a SS disk coated with a thin layer of finely powdered activated ZnS to act as a fluroescent screen to visualize the beam pattern on the sample. The guard rings for positions #1 and #2 were also coated with ZnS to visualize beam overlap onto the rings. Position #2 contained a clean SS disk which was prepared with the sample set but not dipped. A SS disk was always used in #2 position, and was called the tune-up disk. It served as a collector for the primary beam current, and a clean 68 SS disk was found to be resistant to changes in secondary emission properties under electron bombardment, whereas a protein coated SS disk or clean G disk was subject to considerable change in emission properties under long exposure to lkv electrons.' The primary beam current was always measured with Vs = lKV and vtank = 700V. These potentials were applied by switching the ”set/run" switch to "set" (see Main Control Panel and Auxillary Power Supplies, above). With all the elements above the collector disk at -300V with respect to the disk, backscattered electrons were forced back to the collector (see Figure 9). The exceptions to this would be 1) elas- tically reflected electrons, and 2) inelastically scattered electrons with energy greater than 300 eV. Fortunately the number of elastically reflected electrons is known to be a minimum in this energy range, and 31’71 The second possibility must is probably only a few per cent. have been completely negligible because the same beam current was measured with a fixed VS of lKV as Vtank was varied from l-2V up to about 900V. If there had been elastically scattered electrons with an energy of (300+x)V then they would have been repelled by the tank potential below (300+x)V and attracted away by the tank potential above (300+x)V, thereby causing variations in the measured beam current. Also the same beam current was measured for any Vs over about 300V as long as V was 100V lower than VS. Here the -100V tank above the sample disk repelled the low energy backscattered component, but would have allowed any higher energy electrons to escape and de- crease the beam current read as IS. The above Observations were also independent of the beam's expansion on the sample, from a small Spot in the center to being Spread out to the guard ring. 69 Beam expansion to cover sample.--The size and position of the beam Spot on the screen were dependent on V5 and Vtank' Therefore, in the step above, the beam alignment was only checked to be sure that it was roughly centered on the screen. Except for a VS of less than 100V in the run mode, vtank = VS + 200V. The magnitude of V had little effect on the net IS when it was over VS + 50V for a V5 of tank 400V and over. However, when VS = 50V the beam would not Spread to cover the disk for a V1: of less than 500V. This was due to the ank focusing effect of vtank' For uniformity over most of the irradia- tion range Vtank = VS + 200V was chosen as the Operating tank voltage. For VS over 500V the tank potential could be operated below V5, and secondaries would be forced back to the sample. However, again for uniformity of irradiation conditions, the lower Vs problem restricted irradiations over the entire Vs Spectrum to conditions where secondar- ies were removed. Comparisons were made at 500 eV under conditions which caused electrons to be removed and returned (V and > tank vs vtank “mauom mum mumm AN .o.m in .quman mumnamoam 2 «o. a“ cfimnmuw AH cowumuucoocoo voxwm «OOOQWImemmwQ mo Gowuocsm mm :o poumHm awmmhue s OH ousmfim Ou2c«8\eo u voomm mawamfia 00H ea“ mug Nag co ow «0 ad mm 0H m .01» .om ofi 5N on 00 oh eexv Kessv plepuens uo utsdfiil Ed on GO 00H 86 comes out completely wet, and therefore, the amount deposited is a function of the dipping solution concentration only. The drying of these completely wet disks show some running effects, and hence the variation in values is larger. A few assays were tried at very slow speeds, down to 0.8 cm/min. It appeared that the amount of protein deposited leveled off at about one-half the amounts shown in Figure 10 for the 8 cm/min. dips (lowest Speed shown on plot), at a Speed of . about 2 cm/min. for the two concentrations used: 60 and lZOuM. 3 The error involved in these determinations has two main i i a components: 1) the total of all the errors in the Steps involved in determining the Folin assay product's 0.0.; and 2) the errors involved in the determination of the standard curve which is used to convert the product 0.D.'s to ug of protein. Both these component errors will be estimated from consideration of the actual deviations observed in these measurements. To determine 1) a subjective estimate had to be made since there were not enough values for any sample available from which to make a meaningful Statistical calculation. Considering the deviations indicated in Figure 10, and deviations observed in later work on samples dipped from water solutions, I estimated that these values are reproducible to i 3%. During the tests represented in Figure 10, six standard curves were run. The average slope for these curves was determined and the standard error of the mean, expressed as a coefficient of variation, was found to be i 1.5%. Therefore, the mean value for the standard curve slope was estimated to be within 1: 3% (i 2 x Standard deviation of the mean). 87 Since the mass of protein was determined by multiplying the Folin product 0.0. by the SIOpe of the standard curve, the two frac- tional errors determined above were added. The overall estimate of error involved in the Folin protein determinations was therefore i 6%. Protein deposited on irradiation samples.--The protein de- posited on the assay area was determined for the 5 sample types used. These are symbolized as T-S—SZ and T-G-40 for SS and G disks pulled from 60uM trypsin at 10.6 cm/min.; T-S-llZ for SS disks pulled from lZOuM trypsin at 16 cm/min.; and R-S-3l and R-G-38 for SS and G disks pulled from 60uM ribonuclease at 16 cm/min. Using molecular weights of 23,700 for trypsin and 13,700 for RNase, and a value of 1.27 for the average density Of protein, the values in Table l were calculated from the mass of protein on the 20.48cm2 assay area. TABLE 1 Protein Present on Assay Area for Five Sample Types Y Sample pg Protein molec./cm2 Film Thickness x 101:5 A T-S-52 13.5 1.67 52 T-S-112 29 3.58 112 T-G-40 10.4 1.28 40 R-S-31 8.2 1.76 31 R-G-38 9.8 2.10 38 . . .‘_—_—-—.-—_ “A: _—x -A“MA-; 0‘ 0') 88 Specific Activipy of Trypsin on Sample DiSks 1.1: '1: ar' used in the irradiations and on Gu was determined by running parallel The specific activity of the trypsin on the three sample types activity assays with the Folin assays reported in Table l. The SS samples were carried out over a range of 6 dipping speeds to see what E Happens at higher dipping speeds. G and Gu were only determined at the 1 dipping speed used and one speed on either Side. These tests were 1' limited in scope to save time for more directly related problems. 1 E Figure 11 presents the results of these tests. As an estimate of the errors involved, the i 6% error, due to the Folin assay, was added to the actual deviations of the sample activity assay. (Again two samples per datum point were run.) Enzyme Activity Assays Choice of Enzymes The choice of enzymes that were used in this work was in- fluenced by many considerations. The main considerations were the sensitivity of the enzyme to the sample preparation, and the adapt- ability of the activity assay to the assay tray technique. An enzyme's sensitivity to preparation conditions could be estimated from data on its sensitivity to metal ions and inhibitors, and by its long term Storage stability. The availability of an enzyme in a dry form indicated that it might be able to withstand drying on the sample disks. If lyOphilization was used in its preparation this indicated that the enzyme might withstand vacuum exposure. The assay criteria were less demanding, but the time factor was important because the 9 8 Full-1'. 11 All! 111.! .1le .mcowum:HEuouov cwououm one huw>wuom ego “Om vo>uomno muouuo Hmaowuomum emu mo 83m Oau >2 o=~m> muw>wuom oawwoomm ego wcwmfiaaufiaa >9 pocfiEuouom mmz non uouum on» mo guwcofi Hmuou may .mos~m> mow>Huom Oawwooam ego cw nouuo man no oumaflumo am one moon Ono ha poumowvcw mosam> mo Owcmu one mxmwa OHQEMm co cummmue uo muH>fiuo< Owwaooam u fia ousmwm ouacwaxao t vmoam wafimmwo oo mm «m ca c.0H m.n d u q 4 AN 1 \D a .. N234 M/M ——1 +— —l FIZJ-—+ F—El\-——‘l El ——1 w L snrun Kiezntqiv - Antntqov orgtoeds l-—-E—--l 5: 3-0-9 ousedfio Bamzom 8 ._. 3 285 3ng some: a F—O—H F—O—4 8L1 is 90 assays had to be run right after an already long, drawn-out irradia- tion procedure. Trypsin was the better choice of the two enzymes used because it was more stable on the sample disks, and because the assay was simpler to perform, and the course of the reaction was fOllowed spectrometrically. However, under the conditions of the experiment, the ribonuclease also proved to be a suitable enzyme for an inactiva- tion study. Other enzymes that looked promising for this type of work were lysozyme, from chicken egg white; polyphenol oxidase, from mushroom; lipoxidase, from soybean; and peroxidase, from horseradish. The peroxidase would be an especially interesting subject for a comparative Study because it is a heme containing enzyme, and it would be inter- esting to see if the porphyrin ring gave the enzyme any radiation protection. StatiStical Analysis The statistical analysis used in this work utilized standard statistical tests and techniques and, therefore, they will not be reproduced here. The formulas used in the computations and a brief outline of the techniques used are given in the Appendix, and the formulas will be referred to by their formula numbers A.xx. All statistical tests were conducted at a 5% significance level (the probability of rejecting the null hypothesis when it is true). All samples (measured experimental values) are assumed to be randomly selected variables from normal populations with mean u and variance 02. Since the concept of variance and standard deviation will be used frequently below, they will be introduced here. 91 The concept of variance is used in Statistical discussions to describe the variation in the values of random variables. The mean value of n variables xi is n §'= l/n Z x. (1) A frequently used statistic is the sum of the Squared deviations (SS) of the xi from their mean value, 55 = (x1 - 362 (2) IIMD i and from this the sample estimate, 52, of the population variance, 02, is given by $2 = SS/(n-l) = SS/degrees of freedom (3) The degrees of freedom available in a computation can usually be determined by counting the number of independent variables (samples here) in the computation, and subtracting one for each parameter computed from them (-1 for x'in the SS). The standard deviation 5 of a sample value from the sample mean is given by, s = /fS§. (4) and gives a measure Of the variation of the sample values about the sample mean in the dimensional units of the sample. Since the samples are assumed to be selected from a normal population, 95% of the sample values would be expected to lie in a range of x'i 1.965. The variance of the mean value, E; is given by75 s 2 = s /n (S) I.--“ . manna-v M; .6.“- ‘4—' 92 and the standard error of the mean, SEM, is given by SEM = V s:_ = s//7F (6) and gives an estimate of the error expected in a mean value. These concepts are presented here in the briefest of detail, and the reader 75 76 5 is referred to the text by Ostle and a review article by Stearman for details on these concepts and those to be used below, and on their applications. Factors AffectingiActivity Values The sample preparation, storage, irradiation, and assay pro- cedures were designed to eliminate environmental and procedural variations from run to run, as much as possible. The affects of aging on both enzyme dipping solutions and prepared samples were Observed and their possible effects evaluated. Another factor that would be expected to cause some loss of enzyme activity-~probab1y by denatura- tion caused by dehydration--would be the exposure to a high vacuum. This effect was also evaluated, and will be discussed below. Age of dipping solution and protein depletion.--Both trypsin and ribonuclease appear to be stable in unbuffered water solutions at 2-3°C for at least two months. It appeared that the unbuffered enzyme solutions were able to combat microorganisms, because no growths were ever observed in the water solutions. Phosphate buffered trypsin solutions at pH 3.0 were quite prone to mold formation because growths usually formed within 2-3 weeks. However, one two-year old lZOuM trypsin water solution was found still clear and apparently free of any growths. 93 Solution aging was checked over a 21-day period in which 6 trypsin irradiation runs were completed. A total of about 150 disks were dipped, and the activity of the control blank fell at a uniform rate. The last-dipped control disks gave 93% of the activity of the first dipped disks. If it is assumed that the solution's protein concentration is lowered by the amount of protein plated on the total area of the disks dipped (drOps clinging to the disk deplete only the solution's bulk), only about one-third of the 7% loss in activity can be explained by this mechanism. However, disks dipped in a solu- _‘L I- .' .' ' tion of trypsin Stored for two months, unused, showed the same activity as disks dipped prior to the storage period, so simple aging effects are not critical. Although my experience with ribonuclease did not extend over the period of years of my trypsin experience, I did find the RNase solutions were similarly stable. As a matter of fact, I found that a fresh RNase solution had to be aged in the refrigerator for a day or two to gain full activity. Age of prepared samples.--Aging effect on trypsin samples were never specifically checked because once samples were dipped they were placed in the vacuum system, the irradiation sequence completed, and the assay begun immediately. However, from a great deal of ex- perience and surveying all the trypsin data, I would estimate that the trypsin loses activity at a maximum of l%/day. The RNase disks, on the other hand, lose activity at a rapid rate at first, and then more Slowly. During the development stage of using RNase samples, activity data were obtained for both SS and G disks, dipped at 10.6 and 16 cm/min. These disks were 2 hours, 94 2 days, and 57 days old. The two older sample sets also had a vacuum disk of each type. Empirically, a plot of ln Age vs. activity for these disks gave a reasonably linear plot for almost all the data (one datum point out of 18 fell off the straight line), with a negative slope of k. Therefore, the disks age according to In Age = -k activity, which can be written as ln(l/T) = kA, where T corresponds to age and A to activity. By differentials: d[1n(l/T)] = -(l/T)dT = de dA/dT = -l/k x l/T. Therefore the loss in activity over a small time interval (dA/dT) for a given age (T) film can be compared to the loss of an identical film of a different age by forming a ratio of the two loss rates. During a typical run the RNase assay is performed 48 to 72 hours after the disks are dipped. Relative to a 2 hour old disk‘s rate of activity loss (dA/dT), the rate of 48 hours would be (1/48)/ (1/2) x 100 = 4% of the 2 hour old's rate; and at 72 hours it would be (1/72)/(1/2) = 1.4% of the 2 hour old disk's rate. From the above mentioned plots, the loss between the second and third hours would be 7%. Therefore, the rate of activity loss at 48 and 72 hours would be approximately .04 x 7%/hr. = 0.28%/hr. and .014 x 7%/hr. = 0.l%/hr. Therefore, the change in activity of the RNase samples during the assay period Of several hours is entirely negligible for 48 to 72 hour Old samples. Since the estimated loss/hr. for trypsin would be l%/24/hr., activity loss of these samples would also be entirely negligible. The question of time changes in the damage produced by the electron irradiation, between the time of exposure and assay, was not investigated. However, the assay for both enzymes was always started 95 within one hour of the exposure, so the time factor should have been unimportant. Vacuum effect.--The effect of exposing prepared samples to a high vacuum was evaluated for the three types of trypsin samples only, because extra samples could be carried in the trypsin BAEE assay with- {1 out major procedural changes. During 19 T-S-52, 19 T-G-40, and 1 6 T-S-llZ runs an extra set of air control blanks—~usually 4 disks-- 3, were carried along for each set of 4 vacuum blanks assayed (see page 87 a for sample notation). These gave three sets of paired air and vacuum i_ samples to compare for vacuum loss on the three sample support mater- ials. The comparison made was the subtraction of the average vacuum blank values, for one run, from the corresponding air blank average value, and then division by the air blank value and multiplication by 100 to give the per cent activity loss due to vacuum affects. These losses will usually be denoted as d, but with subscripts, dxx’ when needed for clarity. These three sets of d values can be tested statistically to see if there is any significant difference between them, if some basic assumptions are made about the d values. Each of the three sets con- sist of two subsets of values of the trypsin's activity on a common support material, one subset the air values and the other those exposed to a high vacuum. It will be assumed that the values measured are random samples from a normal distribution with mean ui, i=l,2,3, and variance 02, as is usually done for data of this type. Thus, since each of the three sample sets use the same enzyme from the same source, but placed on different support material, it will also be assumed that 96 the d's for the three sample sets have a common variance. This amounts to the assumption that the variance in each subset is due to the variance in the trypsin's activity determination only, and not due to support material interactions with the trypsin. Since the individual activity values are from (assumed) normal populations, the d values, which are linear combinations of values from normal distributions, are also normally distributed. Converting the d values to per cent loss relative to the air values weights each d value relative to 100 for complete loss, for comparison's sake, and does not change the normality of d. The information desired from the d values is an estimate of d for each support material, for trypsin, and its standard error. A comparison between the d's to see if the trypsin loses activity at differing rates on the different support materials is also of interest. The three sets of d values could be tested to see if they were significantly different from each other, under the assumptions dis- cussed above (normal distribution and common variance), by the tech- nique of the analysis of variance. The null hypothesis tested was the sample means were equal, d1 = d2 = d3, and if the hypothesis was re- jected it was assumed that at least one of the d's was Significantly different from the others. The analysis was conducted at a 5% signi- ficance level. If the null hypothesis was rejected the various d values could be compared to see if they were significantly different from each other using Scheffé confidence intervals. Confidence intervals for the differences between any combination of d values were determined. If a confidence interval did not contain zero 97 75’77 Confidence then the difference was considered to be significant. levels of 95% were used. The analysis of variance was carried out on the three sets of d values discussed above. Formulas A.2 through A.8 were used, and the results of the computations are summarized in Table 2. TABLE 2 u ”A.“ A‘“, Analysis of Variance for Trypsin Vacuum Loss on Three Support Materials Fug—v.21— menu“; .- c l . Source of Degrees of Sum of Mean F Variation Freedom Squares Square Between sample sets 2 1176 588 Within sample 21°6 sets 4l_ 1114 27.2 Total 43 2290 3.22 / F.05(2,41) = The ”Total" values were computed separately and serve as computational 2 checks on the "between" and "within" values. Since F = sg/s W = 21.6 > 3.22, the hypothesis that the d's are equal was rejected. The mean values for the vacuum loss of trypsin for each sample type and its expected error, determined from the analysis of variance computations and A.ll are presented in Table 3. The analysis of variance indicated that there was a difference between the d's, therefore, comparisons between all possible combina- tions of the d's were made using A.9. The Significance of these com- parisons, at a 95% confidence level, were made using the Scheffé 98 confidence interval given by A.10. The results of these tests are presented in Table 4. TABLE 3 Vacuum Loss of Trypsin Films of Two Thicknesses* on SS and G Disks Sample** Mean Activity Loss Standard Error of Mean dSSZ (thin) 14.0% 1.2% dSllZ (thick) 0.3% 2.1% dG40 (thin) 5.5% 1.2% *See Table l for thicknesses. **See page 87 for sample notation. TABLE 4 Comparisons Between All Possible Combinations of Trypsin Vacuum Loss Means Comparison Scheffé Comparison* % Interval Conclusion % dSSZ - dG40 8.5 4.3 Different dSSZ - dSllZ 13.7 6.2 Different dG40 - dSllZ 5.2 6.2 No Difference dSSZ + dG40 - 2dSllZ 18.8 8.8 Different dSSZ + dSllZ - 2dG40 3.3 7.5 No Difference dSllZ + dG40 - 2d852 22.2 7.5 Different *The sample and comparison designations use the sample notation (page 87 above) for sample types. The d's are denoted as: dSSZ = [(T-S-SZ-air) - (T-S-52-vac)]/(T-S-SZ-air) x 100; dSllZ = [(T-S-llZ-air) - (T-S-llZ-vac)]/(T—S-112-air) x 100; dG40 = [(T-G-40-air) - (T-G-40-vac)]/(T-G-40-air) x 100. 99 The comparisons indicated in Table 4 and the values for the d's given in Table 3 indicate that the thin SS films lose activity faster than thin films on G or thick films on SS, but that the thin films on G do not lose activity faster than thick films on SS. The multiple comparisons in Table 4 compare one d value to the average of the other two d values. These comparisons are of questionable Signi- ficance to these tests, but are included to illustrate the method, and to clarify the results in the next Scheffé test used. Complete removal of enzyme by the assay procedure.--The possibility that some protein might not be removed during the activity or protein content assays was carefully checked, and eliminated as a possibility. This was done by Ixipeting known amounts of enzyme on the two types of sample disks (SS and G), allowing it to dry, and then performing the assays. In all cases the activity or protein con- tent was as expected from the known amount of enzyme pipeted on the disks. Also a refilling of the assay cell with BAEE immediately after a trypsin assay produced no Significant activity in the second assay on the same disk. Therefore, it was assumed that all the active protein was removed by the assay procedure. Expected Error in Sample Activity Values The error expected in an assayed activity value has two components: 1) the deviations produced by nonuniformity in the enzyme film on the sample disk, and 2) the variations introduced by the assay procedure. Although these components could be estimated separately, what actually is wanted is an estimate of the total error that might 100 be expected in the activity values. This total error can be estimated from the actual experimental variations observed. These variations were determined for each sample set, for each run, by calculating the standard deviation by equation (4). These calculations provide six sets of treatment statistics for the trypsin samples and two sets for the RNase samples (only vacuum blanks were assayed for the RNase samples because of experimental difficulties). The s values for each set of control blanks for each run was expressed as a per cent of the average value of the activity for that run. The average of the 5's for each sample type blanks will then give an estimate of the per cent error that might be expected for any sample of that type for all the factors that normally affect its activity value. If it is assumed that the s values determined are, in effect, randomly selected from the population of all s values for either trypsin or RNase, that these populations are normally distributed, and that the 5's for each enzyme have a common variance, then the data for each enzyme can be analyzed statistically to see if their mean standard deviations differ or not. Since there are only two sets of RNase data, the RNase mean values were tested for difference by the t-test for significance of the difference between two means using A.12. The trypsin data was amenable to analysis using the technique of analysis of variance by the above assumptions. The null hypothesis tested was that all the S35 were equal, and it was tested at a 5% Significance level. Rejection of the null hypothesis would be taken as evidence that at least one of the 3's differs Significantly from the others. In this eventuality, the various differences between combinations of 101 the S35 could be tested by the Scheffé test as was done in the last section. The results of the analysis of variance computations are presented in Table 5. TABLE 5 Analysis of Variance for Average Standard Deviations of Trypsin Control Blanks Source of Degrees of Sum of Mean F Variation Freedom Squares Square Between sample sets 5 90 18.0 2.68 Within sample sets 81 545 6.73 Totals 86 635 / F.05(5,81) = 2.33 The mean of each standard deviation for each sample type con- trol blank was determined in the above computation, and its SEM was computed using A.11. These results are presented in Table 6. TABLE 6 Estimated Variation Expected in Each Trypsin Sample Type Sample Type* Average Standard Deviation SEM (96) (%) SS-SZ-air 2.5 0.6 SS—SZ-vac 4.8 0.6 SS-llZ-air 1.3 1.1 SS-llZ-vac 2.1 1.1 G-40-air 2.7 0.6 G—40-vac 3.3 0.6 *See page 87 for sample notation. 102 The F value computed in the analysis of variance was greater than the F table value for F therefore it was assumed that .05(5,81)’ at least one of the 5‘s was Significantly different from the others. Therefore, the Scheffé test was applied to all possible comparisons between the various S‘s, as was done in the last section for the re- sults presented in Table 4 using equation A.9. 0f the many possible comparisons between the S‘s (all of them were checked), the only ones that were potentially significant to the data being compared are the following: comparisons between each S'and every other one singly; between each S'and the sum of all the others, which compares each to the average of the other five 5‘5; and the comparison of each air-vac pair with each other air-vac pair to see if any sample support material has a lower S'in comparison to the other materials. Of all the possible comparisons only one showed a significant difference, and it was one of the above: the T-S-SZ-vac disks Showed a higher devia— tion when compared to the average of the remaining five disks. How- ever, the comparison between it and each other disk singly revealed no Significant differences, therefore, these results seem to be incon- clusive. A possible interpretation might be that an average of the five remaining disks might be used as an average value for all of their expected errors, which, from Table 6 is 2.4%. The value of'S for T-S-SZ-vac is 4.8%, which is twice the average for the others, and by the Scheffé test is a Significant difference. The standard deviations for 11 R-S-3l and 4 R-G-38 vacuum blanks were compared to see if their mean values were Significantly different using the t-teSt given in equation A.12. A t value of 0.57 103 was computed with 13 degrees of freedom. The t table value for 13 degrees of freedom at the 5% level is 2.16, therefore it is assumed that RNase has the same expected error on either SS or G in a vacuum. An estimate of this variation is the average value of the two sample types which is 7.0% t 0.8%--the SEM is the denominator of equation A.12, which gives an estimate by "pooling" the two variances of the 3‘s. It should be noted that the RNase expected error is three times as large as the average of the five trypsin samples, and appears to be quite significant by considering the sum of the expected errors for each enzyme ' s S" s . Operational Properties of the Irradiation System and Samples Beam Energy Spread The energy homOgeneity of the electron beam is not a problem in this irradiation system because the sample molecules are physically attached to the equipotential surface that determines the energy of the incident electrons. The energy of the electrons striking the sample surface is a function of the potential difference between the filament bridge (zero reference point by center tapped transformer) and sample disk only, and is unaffected by any other potentials in the electron's path of travel. This is a consequence of a basic law of physics which States that the work in moving a body between two points in a conservative force field is independent of the path taken. Since the only forces acting on an electron traveling in the evacuated gun tower are electrostatic and magnetic, which are conservative, the net work done on an electron accelerating it from the filament to 104 the sample is eVS. The one exception to this argument would be elec- trons losing energy in collisions with gas molecules, but these colli- sions are very infrequent, because the collision mean free path at the system's operating pressure is many times greater than the path of travel of the electrons in the gun tower, and only result in an energy loss and probably scatter the electron out of the beam.. Another source of lower energy electrons would be backscattered electrons returning to the sample, but these were attracted away by a positive potential above the sample in all irradiations. The only source of energy Spread in the electrons arriving at the sample is the thermal Spread in energy of electrons ejected from the hot filament. The oxide-coated filament operates at a dull red glow which correSponds to a temperature of about 1,100°K,78 which gives a kT = 0.1eV. Since the thermionic emission utilizes the high energy tail of the Boltzmann energy distribution, the energy spread is on the order of 0.3eV.79 A tungsten filament emitting directly operates at about 2,700°K and has an energy spread of about 0.8eV.79 Therefore, an oxide-coated filament has an Operational advantage of reducing the beam's energy spread. Contact Potentials The presence of contact potentials in this type of system could be demonstrated by Observing the collected electron current with the sample at near zero potential. In my system the current to the assayed sample area, (15), Started falling off rapidly at 0V, and was near zero at -l.0V. There was a just-detectable small current tail out to 105 —1.2V, which probably corresponded to the thermal energy spread discussed above. Beam Penetration and Charging of Sample The argument above (that energy is established by the sample potential) would be invalidated if the protein charged up with elec- trons from the beam. If electrons with energy below the minimum energy necessary to penetrate a protein film were incident on it, and if the protein was a perfect insulator, then the surface would charge up to a potential that would just stop the beam and no more electrons could reach the sample. Since protein is generally considered to be a nonconductor, it would be expected that the protein films would charge up under electron bombardment below a certain incident energy. Above this threshold energy the electrons would be able to penetrate the protein film and pass on to the conductor below, and would not be expected to cause charging up. If a clean SS disk and One coated with a thick film of trypsin (T-SS-112, 112 A thick) was alternately placed under the beam, no difference in IS could be detected, even down to 2-3eV. As an extreme test, a very thick trypsin coating was prepared by pipeting 360uM trypsin directly on the disk and allowing it to dry. The thickness of this film was calculated to be about 30,000 A, and by comparison with a clean SS disk, no difference in Is could be detected down to energies of 25eV. Below this energy a Small attenuation in 15 could just be seen, and at lOeV, IS was down by about 25%. At 2-3eV Is was only down about 50%. From these observations it seems very unlikely that any of the protein films used in the inactivation studies were charging up, even 106 . 5 . . . at the lowest energy used of 20eV. Hutchlnson 2 arr1ved at a S1m1lar conclusion in his work with low energy electrons on bovine serum albumin. Secondary Emission and Backscattered Electrons from the Sample The only problem caused by secondary emission from the sample disk was its interference with the primary beam current measurement. Any electrons leaving the sample as a backscattered component subtract from the measurement of the primary beam current. These losses were controlled by forcing the backscattered electrons back to the sample by a negative potential above the sample (see below). During a radi- ation run secondary electrons were prevented from returning to the sample by a positive potential above the sample to avoid a double dose effect due to the electrons returning to the sample. The Earth's Magnetic Field Effect Deflections of the beam by the earth's magnetic field were compensated for in the beam aiming procedure. The beam was aimed by aligning it on a sample disk coated with a thin layer of powdered ZnS to act as a fluorescent screen, and therefore any deflections of the beam caused by external influences were corrected for. Inactivation Results Data Data recorded.--The primary inactivation data produced in this research are the per cent activity remaining on a fixed area on a sample disk for a given dose delivered to the disk in electrons/cmz. 107 Each run produced two sets of data at different energies. Each set consists of 5 irradiated values and a control blank value-~each set usually on the same support material. In the earlier work a run was made at one energy for two support materials (SS 8 G) for a particular enzyme, but this was changed later to two energies for one support material to give more control blank values. There were from one to five runs per energy; multiple runs were made at one energy when ex- perimental difficulties were encountered or at energies at which a more accurate inactivation rate was required, e.g., to determine the energy or energies which produced a maximum inactivation rate. The data for each run were plotted on semi-log paper, and the data generally gave very good linear plots with a slope of -o. There- fore the data fit an empirical relationship of the form In N/No = ~00, or N/No = e-OD. The inactivation cross-section for each energy, the Slope of the semi-log plot, -0, was determined by the method of a least squares fit using an electronic computer. This method will be discussed below. Nonlinear plots.--In early exploratory work with the irradia- tion system it was found that all data curves were nonlinear on semi- lOg paper (a few data sets gave linear log-log plots). This non- linearity was traced to a nonuniform dose distribution effect, and will be discussed below. When the dose distribution was made uniform over the sample surface all RNase runs gave linear semi-10g plots at all energies. The trypsin runs gave linear 1n per cent activity versus dose plots down to some minimum electron energy, and below this energy the plots were linear for the first part, and then Started 108 curving upwards at longer exposures. The curvature usually started between 40% and 70% activity remaining, and as the incident energy decreased the onset of curvature occurred at a higher percentage activity. The RNase plots probably did not shOw nonlinearity at the lowest energies because the exposures were not made long enough to drop the activity into the range of the onset of curvature-~at energies below 100eV the inactivation rate was so low that even a 1 hour exposure was not long enough to reduce activity to below 70%. Although data were not good enough to give exact values for the energy at which the curvature just appears, they were good enough to give a general picture of the effect. Inspection of all the trypsin inactivation plots revealed the ranges of incident energy for the onset of nonlinearity, and they are given in Table 7. TABLE 7 Voltage Range Below Which Nonlinear Plots Were Observed Sample Voltage Range *Film Thickness (A) Thin trypsin film on C 200-300 40 Thin trypsin film on SS 150-200** 52 Thick trypsin film on 55 400-500 112 *From Table l. **The onset of curvature was not clearly enough defined to say that there is a significant difference between the thin films on G and SS; they probably both start at about 200 volts. The overall picture presented by the nonlinear plots is one of a two component process, the initial fast decrease in activity with increasing dose representing the inactivation rate at the given 109 energy, and the curvature being produced by a more Slowly inactivated, or perhaps an unexposed component. Working on this assumption, only the first linear portion of the data plots were used to determine the inactivation SlOpe for the lower energy data with plots that show non- linearity. NO attempt was made to correct for the slower component by subtraction because the curvature was severe only at the lowest ener- gies, and the inactivation rates were so low at these energies that the correction would have had little consequence in the over-all picture. Hutchinson came to a similar conclusion in his low-energy . . . . . 52 electron 1nact1vatlon work on bov1ne serum album1n. Control blank errors.--A frequent observation in the earlier work where only two vacuum blanks for each sample type were used as a non 100% activity intercept at zero dose. Although the cause was never learned, the average activity value for the sample disks placed at the center of the sample table (see Figure 3) was clearly not one that would be expected from the intercept of the semi-log plot on the activity axis. However, any error in the vacuum control blank activity (No) would cause a multiplication error in each activity value, and hence would only shift a semi-log plot up or down by an additive con- stant. Actually the runs could have just as well been made without the control blanks, but they were included as a check on the samples and because they yielded information on the behavior of the sample/ support system. The intercepts were far off only in the earliest 1/3 of the runs, and the 100% points were not used in determining the cross section for these runs. 0n the later runs the problem was much less, 110 :and only about 10% of the runs showed a large deviation in the activity :intercept. In these cases the 100% point was also rejected. Data selection and rejection.--My experience with the sample :system used in this work has been that occasionally a sample disk will :show a large deviation in its value from that expected from the semi- 110g plot of the data obtained from other samples prepared at the same ‘time. Looking over all the data for the inactivation runs 1 would destimate that this happened to about 5% of the sample activities ob- :served. As discussed above, the standard deviation for the activity nualues for each vacuum blank set was computed, and gives an estimate <3f the variation expected for that set of sample disks. A good working :rule-of—thumb is that 95% of the values Observed should fall within 2: 2 standard deviations of the mean value for the set. If the standard «deviation is expressed as a percentage of the mean value for the blanks, ‘then any other sample's deviation would also be expected to fall within ‘this percentage range. If a sample's activity value deviates from that ]predicted from the semi-log plot by more than i 2 standard deviations, ‘then its variation is probably due to causes other than just random ‘variations. This criterion was used to reject a datum point if it lappeared to be far off from the values predicted by the remaining data ]points on the semi-log plot. The points rejected in this way were tusually well outside the i 2 Standard deviation range. The data for all the runs were surveyed at the same time for 'Jejection of points to keep the treatment as uniform as possible. All ‘the activity versus dose values were listed in tabular form, and then ‘the plots for each data set were consulted for possible point rejection. 111 A minimum of four points was used to establish the slope for a par- ticular run, and for most runs 5 or 6 points were used. As mentioned in the section immediately above, all of the 100% points were rejected for the first 1/3 of the runs, and of the remaining 60 runs only 7 of the 100%-points were rejected. These selected data were subjected to the least squares analysis which will be described below. Data Analysis Inactivation cross section model.--In the section on data re- corded, above, the percent activity remaining on the sample area was denoted as N/No(x 100 per cent), where N is the number of active mole- cules present after exposure to a radiation dose D (e'/cm2), which is proportional to the activity present on the fixed assay area, and No is the number of active molecules present before exposure to the radia- tion, which is proportional to the activity present on the fixed assay area of a control blank. The data were observed to conform to a relationship of the form N/No = e-OD over most of the energies inves- tigated. It will now be shown that o in the empirical relationship is a measure of the radiation-sensitive area on a molecule affected by the incident electrons. To start off, assume that the molecules used in the model are radiation sensitive (i.e., irradiation with electrons can cause loss of biological activity), and that an electron incident in some area of the molecule will cause inactivation. The possibility that a molecule is hit by more than one electron is left open, the only assumption made is that at least one electron is incident in some area, say A, on the molecule will cause inactivation. The case of multiple sensitive 112 areas, or one area that requires more than one hit will be considered later. Assume that these molecules all have the same sensitive area A (i.e., have uniform radiation sensitivity), and that they are uni- formly distributed on a conducting surface. ASSume that this surface is bombarded by a uniform flux of electrons, f (e-/cm2‘sec), of energy eVS, where V5 is the potential on the conducting surface rela- tive to the point of zero energy of the electrons. Since the molecules and electrons are present and incident at random on the surface (uni- formity assumption), the probability of a molecular area A being hit by an electron is the same for all A's. The average dose delivered to each area A in a time t is f x t x A = Ne' Since we are dealing with a random process involving average numbers of discrete events in discrete areas, the Poisson probability distribution can be used to give the probability of an electron landing in an area A. If Ne is the average number of elec— trons incident in an area A, then the probability of p electrons landing in area A is given by P(p) = Nge'Ne/p! (7) The probability of A receiving no hits is, therefore, P(O) = e-Ne. The experimental relationship found is also a probability relationship, in which the probability of survival after receiving a dose D is given by N/No = e-CD. But the probability of survival after receiving a total dose D is the same as the probability of receiving no hits, P(0), therefore e.Ne = e-OD, if it is assumed that the model system proposed above represents the true physical situation. Since in the actual physical Situation the sample preparation procedure was 113 developed to give a uniform coating of enzyme, and the beam spreading technique gave a uniform beam distribution visible on the ZnS screen, the physical Situation is a good approximation to the model system. Therefore, Ne = OD, and since Né = f x t x A = A x (f x t) = A x D it follows that o = A, which by definition in the model is the radiation sensitive area of the molecule. Therefore the slope of the inactiva- tion plot on semi-log paper gives a measure of the radiation sensitive area, as defined in the model, above. In radiation biology o is usual usually called an inactivation cross section. In the model the assumption was made that one electron inci- dent anywhere in the area A caused an inactivation. If, instead, in general, it takes n electrons incident in area A to inactivate the molecule, then A can be subdivided into n subareas A' such that when one electron is incident in each subarea the molecule will be in- activated. Since the electrons are incident randomly and independent of each other, the probability of an electron landing in any of the A' equal areas is the same. However, once an area A' has been hit by an electron, its probability of being hit again is the same as the probability that any other of the areas will be hit. Therefore, from a probabilistic point of view, the probability of n separate Sites A' being hit once is the same as one A' being hit n times. Therefore, by an argument exactly parallel to the one given above, the probability of survival for one of the sites would be P(O) = e'OD, where o' = A'. The probability of an A' being hit is equal to the probability of all events (which includes being hit or not being hit), one, minus the - t probability of not being hit: 1 - e O D. Since the probability of 114 multiple independent events occurring is the product of the separate probabilities, the probability of the n sites being hit is -o'D n . . . . (l - e ) . The surv1val curve for a molecule with n sens1t1ve sites, each with an inactivation cross section 0', and each requiring one hit for the molecule to be inactivated; or of a single active Site with a cross section 0' requiring n hits, is given by p(0) = N/No = l - (1 - e'O'D)n (8) If there are n different sites with (possibly) different inactivation cross sections, then each will have, in general, a cross section of Oi' The probability of each not beinglfi1:is P(O) = e'oiD, and the probability of each being hit is l - e-oiD. If inactivation requires that each be hit once by an electron, then, as above, the probability n a of each being hit is n (l - e‘OID), i.e., the product of the indi- i=1 vidual probabilities. Therefore, the probability of survival for a molecule with n sensitive areas of inactivation cross section oi, each requiring one hit for inactivation is n P(0)=N/N =1- II (l-e' 0 . 1=1 OiD ) (9) By the assumptions of the model, if the inactivation process requires one hit on the sensitive area A, and if the probability of survival is given by P(0) = e.Ne = e.AXD = e'OD, as shown, then a plot of the log survival versus dose would give a linear plot with a Slope of -o. If either (8) or (9) apply to an inactivation model, then the inactivation is called a multiple hit process, and clearly a plot of survival on semi-log paper will not be linear over the entire dose range. Zimmer8O discusses analysis of multiple hit curves to 115 determine the multiplicity of the inactivation process, but since the data for this work gave linear semi-log plots, it will be assumed that the process observed in this work is one-hit. A concept that is useful in computing some quantitative radiation parameters of the process can be deduced from the develop- ment above. In the model the average number of electrons incident on the sensitive area of a molecule, Ne’ was shown to be equal to 0D. Therefore, for a one hit process (one hit on the average per molecule to cause an inactivation) CD = l and N/No = l/e = .37 or 37%. Call the dose that reduces the activity to 37% D37. Then, since OD37 = l, o = l/D37. Since 00 = the average number of hits per molecule, and since 0037 = l, 037 is the dose that, on the average, will cause one inactivating event, or hit, per sensitive site per molecule. Two useful parameters can be computed from D In Chapter II 37' the number of molecules per cm2 was determined as v molecules/cmz. The value of 037 gives the dose in e-/cm2 incident over the entire sample surface that, on the average, produces one hit per sensitive site per molecule. Since l/Y gives a measure of the area/molecule, 037/y gives a measure of the number of electrons incident on a mole- cule which cause, on the average, an inactivation-~i.e., the number of e- per unit area x area of a molecule. In studying radiation effects a radiation yield, Y, is usually stated, and gives the number of affected units per incident radiation particle. Since D37/Y gives the number of electrons per molecule inactivated, Y = v/D37 = 70 (molecules inactivated/incident e-) (10) 116 It Should be pointed out that the calculation of Y is based on the calculated value of v. Since Y was calculated on the assumption that the protein was present as a homogeneous film on the sample, it fails to take into account the fact that the enzyme molecules are probably globular in shape. Therefore the true physical area of a molecule is probably less than the area given by 1/7, and the estimate of the number of e- incident per molecule inactivated, 037/7, is probably high. Therefore the parameter Y is probably a low estimate of the true value. General linear statistical model.--The data prepared by the selection process, above, was analyzed to give the slope of a plot of the logarithm per cent activity remaining versus dose for each energy/sample type, and an error was estimated for each slope. In general the data can be represented by a series of linear plots (one for each run) with different SlOpes and intercepts. A linear re- gression analysis (least squares fit) of the data in the form In (N/No x 100) versus dose, applied to all the data points for each energy/sample-type set can give both the average slope and standard deviation for the slope, which is based on the scatter of the points about the linear regression line. However, the data as presented here will Show a larger scatter than is representative of the true situation because of the additional scatter introduced by the Shifting of the curves up and down by the different intercepts (the In of a multiplicative constant is added to each term). This scatter could be reduced by shifting each curve so that it has a 100% intercept at zero dose, and minimized by Shifting all the curves vertically and 117 horizontally so that they all intercept at their midpoints. A more straight-forward technique is provided by fitting the data to a general linear statistical model, writing the data matrices deduced from the model, and solving the resulting matrix equations for the regression coefficients which will give all the intercepts, a common slope, and an error estimate for all the coefficients. The general linear statistical model for m experimental random variables x1 is y = 80 + lel + . . . . . + Bmxm + e (12) The B's are parameters whose values are to be estimated from the experimental y and m xi values; and the c is an error term which accounts for the variation observed in the y value. The error term 6 is a random variable with mean u = 0 and variance oz. The expected value of y, E(y), is the equation of an m-dimensional hyperplane in (m +1) Space E(y) = 80 + 81x1 + . . . . + Bmxm (13) since E(c) = u = 0. If there are n points in the (m + 1) space (x11, x12, . . . . xim’ yi, w1th 1 = l to n) then the pred1ct1on equation obtained by fitting a straight line in the (m + 1) space to the n points is denoted as yi = 80 + 61x11 + 82xi2 + . . . . + Bmxim (14) The quantities BO, 81, . . . . , 8m are estimates of the true values of the 8's, and 91 is both an estimate of the expected value of y as well as a particular value of y Obtained by substitution of a particu- lar set of m x ij in (14). 118 The representation of one inactivation cross section from one run based on the general linear model (12) would be yik = Bk—l * kaik * 5k , (15) where k = the run number = l to m, and 1 indicates the particular datum point (inactivation cross section) (xik, yik) with i = l to n and n = total number of data points for a given energy/sample-type. Each of the k runs will have nk data points, and an = n. From this notation the prediction equation (14) for a single run, m = 1, re- duces to that for a Straight line in 2 dimensional Space: Y = 80 + 81x1 (16) with the k subscripts dropped. If it is wanted to combine the m runs at a given energy/sample-type in the general linear model (12), allowing each Bk-l in (15) to remain distinct but forcing a common SlOpe Bk on all the m run's x ik’ this can be done by reCOgnizing that each xi in (12) can represent a quantitative variable (e.g., the xik of (15), which represent dose values here) or a qualitative variable. The intercepts can be retained and kept distinct by defin- ing the qualitative variables Tk-l such that = . O I .- Tk-l 1 1f yik 15 one of the nk y S of the k th run = 0 if yik is not one of the nk y's of the k-th run (17) In (16) m = 1, therefore there is only one Tk-l’ T0 = 1. There is only one quantitative variable in the data, and we want to force it to have the same coefficient Bk in the general linear model (12) for all determinations of its value. For the m runs at a particular energy/sample-type the data can be expressed as 119 yik = B0T0 * B1T1 * ° ° ' ' * Bm-le-li'Bmximk * e1T0 * . + eka_1 (18) with i = 1 to n, n = total number of data points (xik, yik) and the T's the Tk-l defined in (17). The e's are not observable variables, but are included as error terms to explain the variations observed in the yik’ Linear regression analysis.--If a straight line is fitted to the n data points (xik’ yik) represented in equation (18) with m dummy variables Tk-l defined in (17) and xik always assigned to the xm qualitative variable with coefficient Bm’ then the line fitted to these points will be the prediction equation we are looking for yik = BOTO + BlTl . . . . . + B T + B m-l m-l (19) mximk In fitting a line to a set of data points the criterion usually used is to make the sum of the squares of the deviations of each yik from 9ik as small as possible. The deviation of each point yik from the prediction line is mA A yik ‘ yik = Yik " [kilsk-lTk-l * Bmximk] (3°) and the sum of squares of error, SSE, from the prediction line is m . A 2 SSE = [yik - (kilsk‘lTk'l + Bmximk)] (21) The "least squares" criterion can be found by solving 3555 = o i = o to m (22) 381 120 which gives m+1 equations in the m+l B's. At this point matrix notation will be introduced to simplify the notation, and put the data in a form amenable to computer appli- cation. It is assumed that the reader is familiar with matrix nota- tion and matrix prOperties, and only the notation used here will be stated. (Halmos81 presents an excellent introduction to matrix algebra, and Mendenhall82 presents an excellent introduction to the general linear statistical model.) A matrix will be denoted by an underlined symbol, and its elements by symbols with subscripts denoting the row (i), and the column (j) numbers The transpose of a matrix is Obtained by interchanging its rows and columns 0 = c = . . E_ (cij) cjl (24) 'Fhe inverse of a matrix Y_is denoted Yil and has the property c‘lc = c ‘1 = I. — T — (25) where I = I = E WFhe data for the n points (xik’ yik) represented by (19) can be put in matrix notation for convenience. The yik can be summarized in the (:olumn matrix :3, written here as its transpose to give a row matrix 1'=y.'=[y .y ..-.y 1k 11 21 n11,y(n1+1)2,. . "y(n1+n2)2, - Y (n1+n2+ . . .+nm_1+l)m , . . .,y ] (26) nm 121 and the matrix for the variable associated with the B coefficients 0 . . . . . . . . . 1 0 xlml 1 0 . . . . . . . . . 0 mel O . . O xnlml 9 1 ' 0 ¥(n1+l)m2 X=X : ° ° _' 13k 9 1 ° 9 ¥(n1+n2)m2 0 0 . . . . . . . . . l 5(n +n ...n . . . 1 2 m-l 0 0 . . . l x nmm i = row number j = column number k = run number i = l to n j = 0 to m x ll 1 to m The matrix Of the coefficients is A A g' = [811' = [80, 61, 82, . . . . Bm_1, 8m] Equation (19) can now be written in matrix notation as Y = Z(_ 8:. +l)mm ) (27) (28) The solution to the m+l equations in the m+l B's of equation (:22) is given in matrix notation by Mendenhall82 as = (g'pflé'l, if (11319-1 exists. lo» (29) 122 'Ihis gives an estimate of each of the 8's in (19), including the 8m \NhiCh gives the common slope desired. The variation in the yik values «expressed in (18) by the Ek’ are now contained in the SSE (21), which can be evaluated from 82 SSE = 1': - @353: (30) 'The E's were assumed to have a variance of 02, and an estimate of . 2 82 ‘this, s , can be computed from s2 = SSE/[n - (m+l)] (31) 'The B's are themselves not variables, but the Bi are estimates of ‘the "true" values, and the variation in the 8's is a result of the ‘variations in the yik values and hence, related to the ck. The “variance in any 81 can be shown to be given by82 vcsi) = ciisz (32) \where cij are the elements of the matrix (§j§)_1. Therefore an esti- lnate of the error expected in Bm’ the desired common SlOpe, is gaiven by s = / V(B ) = / c s2 (33) 8m m mm The data were subjected to the analysis outlined above using the Michigan State University CDC 3600 computer using the library Program AES Stat Package LS # 7. Inactivation Cross Section Plots ‘ The linear regression coefficients 8m from the computer Etnalysis for each energy/sample-type are plotted in Figures 12 and 13. 123 .uooHo«wwooo ooammouwou mom dome Odo mo uouuo uncommon N.H.uoOmoudou mums uouum Am .hwuoco ao>ww e on menu afio u0u muaowoammooo :Ofimmouwou umoewfi ago one muuwoa mama AH Nndmcowuoom mmouo :ofium>wuoman cwmmwue u NH moonwm OH x >0 l mmuoam coquOHM on O: 2 MN: 2 n; e m e m N s e - J a 4 4 q . q d 71 .Nx‘ . H m A \Cm W ._ .P a: j X L. .1 m a. . l—e-l F—;[4 /// KM l—«ei m i—x-i r—-a-—lr»Z{ //K0 d‘ I c: I E-‘ f—x-i N 6’ 01 x 28 - notices SSOJQ uotnehtnoeuI \0 oo Z- .uaowoawuooo dowmmouwou yew name one mo uouuo mandamum N M.ucomoudou moon uouum AN .mmuoco co>ww e um menu Ham wow unawowwwooo :ofimmouwou umocwfi use mum monOQ mama AH b dmeoHuoowmwouo cowwm>wuwme mammaoaconem n ma Ousmwm Nlofi x >0 1 Awuocm :ouuoon 8 O: 2 «.2 2 n; e n 1 q 1 q - fi 1 d ‘x‘f +03 124 N a) Z_OI X 28 - uoiuoas 55013 uoinehtnoeul O H a 125 Error bars are plotted with each 8m = o = the inactivation cross section, and represents 0 i 2 x standard error of the mean. The value of o = 8m is Obtained from equation (33), and the standard error Of the mean from equations (6) and (31), where n is the number of runs made at a particular energy. The plot of the 0's for T-S-52 is indi- cated in Figure 13 as a dashed line for comparison purposes. Inactivation Rate Modifying Factors Exposure to hot filament.--The apertures of the gun (Figure 9) limit the direct exposure of the sample to the hot filament to the central 2/3 area of the disk. Samples were exposed to the gun for 60, 90 and 120 minutes with the filament operating at 105% of the normal heating current, and with the beam off at the gun grid cup. No inactivation of the samples attributable to the exposure to the hot filament were observed. A small inactivation by the positive ion current was observed (see below). Contamination by pump oil and filament activation products.-- Clean undipped SS disks were placed in the vacuum system in sample positions and at the center of the table in control blank positions to test for pump oil contamination. The disks were left in the system for three days with the diffusion pump operating. After this period the disks were checked for contamination by rinsing the disks in dis- tilled water and watching for hydrophobic areas. Another set of clean SS disks stored covered at room conditions was also rinsed in distilled water at the same time to check for contamination. Both sets of disks showed very Slight contamination by having small areas 126 that did not wet well. Since this wetting criterion is known to give an exquisitely sensitive test for surface contamination of a hydro- phobic nature, it was assumed that contamination by pump oil was minimal because the vacuum disks appeared to be as clean as the non- vacuum set rinsed at the same time. During one run a sample disk was accidentally left below the gun during the filament activation process. It was discovered there just before the filament started to emit electrons and was rotated away. However, the disk was exposed directly to all the acetate binder break-down products and most of the oxide conversion products. The disk was used as a Short irradiation exposure sample, and upon assaying and plotting its N/Nb value vs. dose the activity appeared to be un- affected by the activation product exposure. Since activation was usually carried out over the ZnS screen it seems very unlikely that filament activation products had any effect on sample activity. Dose rate effects.--Samples of T-G-40 were exposed at 100eV to total beam currents of 1.0 and 10.0ua, and T-S-52 samples were exposed at 150eV to total beam currents of 0.5 and 5.0ua. The exposure times were adjusted so that each sample set, at a given Vs, received the same total dose. Parallel inactivation plots at each energy showed clearly that the inactivation rate was dose rate independent for these samples at these dose rates and energies. In another test T-S-52 disks were exposed to 2KeV electrons at 0.6ua. A second set was exposed to the filament directly, without the gun, with the sample acting as the anode at 2KV. The total cur- rent delivered to the samples in this way was 20ua. Irradiation in 127 the gun-less mode could only be accomplished with the sample near 2KV, because below this VS beam spreading and current measurement could not be accomplished. A comparison of the inactivation rates of these 2KeV runs at a 30:1 ratio in incident doSe rates revealed that the inactivation rates were within 5% of each other, the higher current producing lower inactivation rate (smaller cross section). Since heating due to energetic electron bombardment would be greater at the higher current, these results indicate that inactivation by direct electron heating probably is not an important inactivation mechanism. Hutchinson52 has shown by calculations that the tempera- ture rise of the sample due to the electron bombardment is entirely negligible. NOnuniform dose distribution over the sample surface.--During the early development stages of the irradiation work severe non- linearity of the inactivation plots was found at all energies. Part of the problem was found to be due to beam deflection by the G1 and G2 leads which caused an area at the edge of the sample to be left unexposed. Installation of the lead shield, shown in Figures 2, 3, and 9, reduced the problem, but nonlinearity was still observed at all energies. The problem was finally traced to the casting of shadows on the sample and focusing effects by the grids. Although the grids are 95% transparent each, they are rotated 45° with respect to each other, and, therefore, tOgether they can cast a real Shadow on the sample that shields 10% of the area from electron exposure. Shadows are cast only under the most favorable potential configuration of the 128 grids and sample. A more severe problem is caused when G2 has a retarding potential applied relative to the G1 potential. This type of configuration caused the beam pattern on the ZnS screen to appear as a square array of dots. Irradiation results produced under these focusing conditions showed large upward deviations from linearity at long exposures. Removal of the grids during irradiation eliminated the problem, except at low energies, as was discussed above. These observations are presented as experimental evidence that shielding of molecules on the sample surface during irradiation can cause nonlinearity in the inactivation plot. The nonlinearity produced this way appears to be identical to that observed at lower irradiation energies. Irradiation with secondaries returned to, or removed from the samples.--Any material bombarded by energetic electrons with an energy above about 20eV will start to emit secondary electrons. The secondary emission properties of materials under electron bombardment is a well studied phenomenon, and there is a large literature on it covering a wide range of materials, with metals and conductors especially well considered. At the energies used in this work the energy distribution of the secondary electrons is independent of the incident electron energy, and is peaked at only a few eV, with over 31’71 The effect of this 90% of them with an energy below 20eV. secondary electron component can be studied by forcing the secondar- ies back to the sample, or attracting them away from it. If a secondary electron leaves the sample with an energy ES and is forced back to the sample by a negative potential above, then 129 it will return to the sample with an energy 55' From the cross sec- tion vs. energy plots, above, it can be seen that electrons with an energy below 50eV have very little effect on the proteins relative to the effects above 200eV. If the energy distribution cited above is correct then irradiations at over 200eV under conditions in which secondaries are returned or removed should produce almost the same inactivation rate. Irradiation of T-S-SZ disks at 500eV with the potentials above at 300V and 700V (i.e., with secondaries returned and removed, reSpectively) gave inactivation rates with 2% of each other. Positive ion current to the sample.--In the normal irradia- tion configuration the potential above the sample is 200V positive with respect to the sample potential, to remove secondaries from the sample. This produces a 200 volt gradient in the sample area that could accelerate positive ions produced above the sample to the sample surface. To check for a possible positive ion effect, one of the "beam off" tests described above, was conducted with the beam on from the gun and the tank at +100V, but electrons were stOpped by applying a -400V to the sample. This provided a 500 volt potential gradient to accelerate positive ions produced by the beam in the area above the sample. Exposures of 30 and 60 minutes of T-S-52 disks produced an inactivation cross section of .033A2, which corresponds to an inactivation rate 145 times lower than that produced by 20eV electrons and 5,000 times lower than 100eV electrons. Since these positive ion accelerating conditions were more severe than ever 130 encountered in an actual irradiation run, the positive ion effect is considered to be entirely negligible. Age of dipping solution used to prepare samples.--To check for a possible age effect, two sets of T-S-SZ disks were prepared: one set from a freshly mixed 60uM trypsin solution, the other from a 30 day old solution. The average vacuum blank activities for these samples were 1268 i 2.3% and 1175 t 2.0%, respectively. These samples were exposed to 500eV electrons at a total beam current of l.Oua with secondaries removed. The inactivation rates were within 1.5% of each other for these two sets of samples. Different Inactivation Rates on Staifiless Steel and Graphite Electron irradiation.--In Figures 12 and 13 it can be seen that the inactivation rate on G is about 40% less than it is on $5 for both enzymes. Further, the SS and G curves are similar in shape, indicat- ing qualitatively that each enzyme seems to be inactivated by the same process, but the rate is attenuated on the G disks. The fact that both enzymes Show the same peak inactivation energy on both support materials, have a 40% slower inactivation rate on G relative to SS, and then approach the same inactivation rate at 2KeV seems to suggest that the slower inactivation rate on G is due to an interaction between G and the protein rather than between G and the electrons. The idea behind this speculation is that the protein film might be rela- tively opaque to the lower energy electrons, but the higher energy electrons can pass through the film with more energy left and perhaps disrupt an interaction between the graphite and protein. The work 131 described in the following two sections was undertaken in the hope of learning something about a possible difference in the interaction between trypsin and SS or G. Ultraviolet irradiations.--Ultraviolet (UV) irradiation has been used extensively to Study radiation damage in trypsin. It has been suggested that inactivation can be caused by denaturation of the molecule by radiation induced rupturing of weak bonds, such as hydro— gen and ionic bonds,18 or by rupture of the stronger disulfide bridge of cystine.21 Rupturing of these bonds can lead to loss of biological activity by allowing the molecule to physically unfOld and lose its active configuration. If the binding mechanism proposed above for the G surface could also act to constrain unfolding, to a greater extent than the interaction with the SS surface, then it might be predicted that inactivation by UV would also take place at a lower rate on the G surface under UV irradiation. There are too many im- ponderables in this speculation to reach any conclusions on the possi- bility of a G binding mechanism, but information as to whether there is a similar attenuation of inactivation on G relative to SS, or not, under the action of UV Should provide some valuable insight into the process. Therefore, some UV inactivation runs were made using trypsin on SS and G disks to compare the inactivation rates on each. Early UV tests using a bank of eight 8-watt germicidal lamps placed 25 cm from SS and Gu disks (dose not determined) indicated that UV inactivation at 254nm was 3-6 times slower on Gu than on SS disks. After the develOpment of the graphite polishing technique, the UV studies were repeated using SS, Gu’ and G samples dipped in 60uM 132 trypsin at 10.6 cm/min. The inactivation rates were different on the three materials, and in this order of magnitude: RSS>RG>RG , with ratios of l:0.77:0.25. Further, the semi-10g plots of the SSuUV data were linear down to the lowest activity of 5%, but the Gu and G plots Showed upward curvature starting at about 45% and 30% activity, re- spectively. The Cu plots showed a large curvature and the G plots showed a small one. The graphite spray is in an acetate binder, and therefore a G surface might be expected to have some cellulose acetate in it. The Gu surface is primarily of cellulose acetate, with graphite particles settled to the bottom, but it probably also has some exposed graphite on its surface. Therefore, if the inactivation rate on pure graphite is fast, and the rate on pure binder much Slower, then the curvature observations above could be explained as a two component process. The upward curvature would be caused by the domination of the Slower pro- cess at the longer exposure, i.e., the molecules on the graphite are essentially all inactivated at the faster rate and the residual activity is due to the more Slowly inactivated molecules on the ace- tate binder. To test this idea sets of Cu and G disks were exposed to 254nm UV under identical conditions, for times up to 180 minutes. The idea was to expose the disks long enough to eliminate the fast component and leave only the slow component, and then see if the slow component was the same for each disk. However, much to my surprise, the inactivation plots for both disks developed so much curvature that they hit a low value and started to increase in activity again at longer exposures. The G disks fell to 37% activity in 10 minutes exposure, Started to curve at 5% activity at 30 minutes, hit a 133 minimum of 1.1% at 90 minutes, and rose to 1.75% at 150 minutes. The Gu disks fell to 37% activity in 30 minutes and also started curving upward, fell to a minimum of 13% at 150 minutes, and rose to 18% activity at 180 minutes. Activity values below 5% are not reliable on an absolute scale, but on a relative basis the points below 5% on the G curve are very Smooth, and the reactivation process is unmis- takable. A component analysis was not possible because a slow com- ponent did not express itself, but the two curves did appear to have the same, slow, reactivation slope. The ratio of RG/RGu = 2.2. Total reflectance spectra.--The UV inactivation rate for T-S-SZ is only 1.3 times greater than that for T-G-40. Since the SS surface is polished and mirror-like it seems quite likely that the greater inactivation rate on 88 might be due to reflection and a double exposure effect. To check this idea, reflectance Spectra for the Cu and G disks were recorded on a Cary model 15 using a Cary total reflectance integrating sphere attachment. In addition, the absorption of trypsin films plated on each disk by dipping in 60uM trypsin at 10.6 cm/min. was checked by comparing the reflectance of unplated disks to the reflectance of plated disks. Since all reflectance readings (in 00) were within the 00 scale on a single plot, all Spectra were recorded relative to the same SS disk in the reference position, and all the spectra were traced on one plot. The appropriate reflectance values were read from the multiple plot by reading the difference in 00 between the appropriate uncoated disk and the plot of interest. The reflectance spectra for two dif- ferent SS disks were used as a baseline, and two tracings for two disks 134 of Gu and of G were used to determine the reflectivity of these disks. The two Gu and two G tracings were identical, but the two 38 tracings were Slightly different, and a line drawn midway between them was used as their average. TWO disks of the SS sample were run and only one disk of each graphite sample was used. The film's absorbance could be read quite easily because they were in the range of 0.1 to 0.2 00 dif- ference between the plated and unplated tracings. The OD difference readings were converted to percentage values, which gave per cent light reflected, and these values were converted to the per cent absorbed by subtracting them from 100%. The absorbance spectra for Gu and 6 relative to SS, and for the films of trypsin, T-S-52, T-Gu-48, and T-G-40, are presented in Figure 14. Values read from this plot for light absorption at 254nm (the wave length used in the UV work described above) are: reflectance of Gu relative to $8 = 61%, reflectance of G relative to 88 = 67%; and the absorptions of T-Gu-48 = 7%, T-S-52 = 21%, and TbG-40 a 28%. The absorption reading of any support disk relative to itself would be zero, and since the light travels through the film twice the absorption measurement is the difference between the light reflected from itself and when it is coated with protein. The light absorbed by the film in one pass can be determined by the fOllowing argument. Assume that the fraction of light absorbed in the film is the same at each pass through the film. (The light in both the irradiations and reflectance work was normally incident.) Then let y be the fraction of light transmitted by the film in one direction, and let x be the fraction of light absorbed in passing through the film twice (i.e., 13S .muaas Bouumufie a“ uuucxounu aaaw oumowvcw oahu nausea umumm muonadz AN .Hmfiumume uuomm=m mo oocmuoodwou ou o>wuu~ou pounmmoa ceaumuomnm EHHH :Hmmmue Ad muuoo w ooamuoofiwom can scan ponn< . ea unawam e: . sumac“ o>o3 oow onm oom omm ooq one cow onm coo ‘ a 0\0\¢ - 1 4\a1|||lnu1 C . - O \ \D b b 97:99....“ 2-....-elalulo1u >\b\l\b S \0 \U‘D‘U “\41QI‘I‘Q (1.1! fl. 0 \uxu (e 1.35an .73-“: \ \D\D Q\Q\Q D\D {ON 0 n— \0\ b\ \ K . x... 1.. Ole o\ \v v/«Séé . onm U\ KN, ”\D Mr 0 e%.\.vrex.. .9...“ \ \ > . / 1 o . \ x o \ D e /X OMM . .4 \. \ /. /x/ u x\\ D //. x/x NV \ / IX 10014 e O I .\ > /. x/x/ u e 03 am A O 3 am e £33m w I: o a u u o u /a t‘ ‘ .\ oo mmo : m o o m o G \D /./. C on s s um u >3 s 5 m. b IOL e O oocmuoofiwom A om ouwammuo vuamaaomnb .oo .oo LooH 136 the absorption measurements above). If 100 is the incident light in- tensity, then on passing through the film twice (100-y)-y is the light intensity that is transmitted, assuming no loss in reflection (disk compared to itself, therefore, no absorption). A reflectance measurement of the film's absorption would tell us that (100-100x) = 100(1-x) = the intensity of light that would be transmitted after re- flection through the film. These two quantities are the same, there- fore 100y2 = 100(l-x), or y = JTI:;) x 100. Substituting x = (% relative absorption of film)/100 from the values given above into the equation immediately above, gives the per cent of 254nm light that is absorbed in one passage through the film: T-Gu-48 = 3.5%, T-S-SZ = 11%, and T-G-40 = 15%. This result shows that there is an odd interaction between the trypsin and the support materials, because from Table l the T-S-52 film is 52A thick, the T-Gu-48 film is 48A thick, and the T-G-40 film is 40A thick. TherefOre, the thinnest film absorbs the most light. To see if the reflectance, or reflectance and absorption dif- ferences can be used to explain the different inactivation rates, the fOllowing calculations were made. The absolute reflectance of SS was 78 indicate that the reflectance for not measured, but published tables bright polished metals is about 35% at 251nm. If the trypsin is con- sidered to be irradiated by both the incoming and reflected light, then on SS the film will be exposed to (100 + 35) = 135% of the in- tensity of light that it would be exposed to in the absence of re- flection from the support material. Similarly, since Gu and G re- flect 61% and 67% as much 254nm light as $8, a trypsin film on them 137 will see (100 + 0.61 x 35) s 121% and (100 + 0.67 x 35) a 123% of the incident light intensity. If the assumption is made that the in— activation is prOportional to the amount of UV light that the film is exposed to, then the ratio of the light expoSure the film sees should be the same as the inactivation ratios Observed. The ratios of light intensities of Gu/SS = 121/135 = 0.90 and G/SS = 0.91 do not come close to explaining the difference between Gu/SS and G/SS rates of 0.25 and 0.77 actually seen. The calculated G/SS = 0.91 does pre- dict a lower inactivation rate for T-G-40, however. A more logical assumption would be to assume the UV inactiva- tion is prOportional to the UV light absorbed in the film. The film's absorption can be calculated by using the one-pass absorption figures fOund above and the SS reflectivity estimation of 35%. For SS it would be 0.11 x 100% + 0.11 x 35% = 14.9% of the incident light ab- sorbed by T-S-52. For T-Gu-48 it would be .035 x 100% + .035 x 0.61 x 35% a 4.3% absorbed, and for G it would be 0.15 x 100% + 0.15 x 0.67 x 35% a 18.5% absorbed. The inactivation ratios should be approximated by the absorbed light ratios of Gu/SS = 4.3/14.9 = 0.29 and G/SS = l8.5/l4.9 = 1.24. The absorption ratio for Gu of 0.29 comes reasonably close to the 0.25 absorption ratio Observed for RG but the u/RSS’ absorption ratio for G of 1.24 is off from the observed absorption ratio of 0.77 for RG/RSS. These results confirm the observation made above that there is indeed an odd interaction taking place between the trypsin film and the two graphite surfaces. In the case of the Gu' which is mainly a cellulose acetate surface, the low light absorption accounts for the 138 low inactivation rate. In the case of the G disk, which is primarily graphite, the film of trypsin on it iS 22% thinner than that on the SS disk, but it absorbs 20% more light than the trypsin on SS. These anomalies will be discussed in the next chapter. Film density and vacuum inactivation.--The results presented in Vacuum Effect above, further indicate that an interaction between the support material and enzyme can decrease inactivation. In that section it was shown that a T-S-52 film loses 14% activity upon being exposed to a high vacuum for 2 days, but that a T-S-112 film loses no activity upon a similar vacuum exposure. Table 1 gives the 13 T—S-52 film molecular density as 1.67 x 10 molec./cm2 and the 13 molec./cm2. This could T-S-llZ film molecular density as 3.58 x 10 indicate that packing the molecules closer together produces a "cage effect" and prevents vacuum stress inactivation. (This could be caused by loss of configuration due to dehydration.) A T-G-40 film 13 molec./cm2 which is 22% lower than that has a density of 1.28 x 10 fer T-S-52 film, but a T-G-40 film loses only 5.5% activity upon exposure to a vacuum, as compared to the 14% loss fer the more densely packed T—S-52 film. Therefore the G surface could bind the trypsin molecules in a way that prevents loss of configuration, or at least inactivation by the vacuum stress. The observations in the first section of this chapter on the hydrophobic nature of the G surface are also relevant here, because they suggest that the enzyme binds to the graphite surface. CHAPTER IV DISCUSSION Discussion of Present Results In this section I will first discuss only results obtained in this work and how they apply to the question asked at the outset of this research project. Then I will bring in some published data on energy absorption by proteins from energetic electrons and try to ask pertinent questions and draw some conclusions about the inactivation process observed in trypsin and ribonuclease. Possible Importance of Low-Ener Electrons in an Excited Triplet State Inactivation Mechanism The original purpose fOr undertaking this research was to see how important low-energy electrons might be in causing the observed radiation damage produced in biOIOgical systems by ionizing radiation. As was pointed out in the Introduction, there are both theoretical and experimental reasons to believe that electrons with energies below 100eV are able to produce excited triplet states in biological mole- cules directly. Since excited triplet states are very long-lived relative to excited singlet states, and Since it is generally believed that molecules in an electronically excited state are a more reactive Species, it seems reasonable to postulate that if excited triplet 139 140 states are efficiently produced by low-energy electrons, then they might be an important primary radiation damage product. Inactivation cross section plotS.--Figures 12-13 present the inactivation cross section (0) vs. electron energy (E) for all the enzyme samples irradiated. The inactivation cross section can be interpreted as a relative measure of how sensitive a molecule is to inactivation by exposure to an electron of a given energy: the larger the inactivation cross section, the more sensitive the molecule is. With this in mind, the 0 plots Show that trypsin is most sensitive to 500-600eV electrons, and RNase is most sensitive to 300-400eV elec- trons. In addition both enzymes are about six times as sensitive to electrons with energies in this peak energy range as they are to 100eV electrons, and the ratio increases rapidly to over 30 at the lowest energies used. These results do not rule out a possible triplet state mecha- nism, but they do Show clearly that there is a more important process taking place at energies above those at which the triplet state pro- duction would be expected to be important. The crucial question in assaying the importance of these two processes is the relative numbers of electrons in the 300-500eV range and those below lOOeV in the cas- cading effect in the degradation of energetic electron energy. How- ever, befOre a high-energy electron can be reduced in energy to below lOOeV-it must pass through the critical 300-500eV range, and it might be argued that the 300-500eV electrons will be effective befOre the electrons below 100eV have a chance. No data is available on the actual electron energy degradation spectrum in a material, but it is 141 generally believed that high-energy electrons passing through matter suffer a large prOportion of low-energy interactions (see Introduc- tion), probably most of these with an interaction energy of below 100eV. However, from the data of Cole to be discussed below, it will be apparent that because of the dimensions of a molecule, and the limited range of low-energy secondary electrons, in order for a low- energy electron to be present in a molecule it will have to be pro- duced in or very near to a molecule to affect it. Another important consideration at this point is how important the various energetic electrons are on a per unit energy basis. Inactivation rate per unit electron energy:--An immediate question that could arise from looking at the 0 vs. E plots is: wouldn't five 100eV electrons be as effective as, or more effective than, one 500eV electron in producing an inactivation? In terms of incident energy available to do damage, these would be equivalent situations. But which would be more damaging? The simplest way to answer this question is to divide each cross section by its corres- ponding electron energy, and thereby give an inactivation rate per unit energy for comparison. The results of such calculations are presented in Figures 15 and 16. These plots show an even more sharply peaked response, but with the peak at lower energies: at 300-400eV for trypsin and at 200-300eV fOr RNase. They again Show a very rapid rise from lower energies, and a rapid fall-off with increasing energy past the a peak. These plots indicate even more dramatically how im- portant electrons in the 200-500eV range can be in the inactivation process. 142 24’ . //9 20' o '- WT'S 51 ,4 16. T-5 "2‘ 13 b D m/ \ x n E 12. x X % X/ /\0 x \ t W a. / \a 8’0 111 T'G‘ X \ \ p x a \3\° x~——__.§-———_.$ I I J I I I L L I I I I U 2 a 6 7.5 10 12.5 15 17.5 20 Electron Energy - eV x 10.2 Figure 15 - Inactivation Rate at Unit Ener - Tr sin 0 E “’1 30- a4—R‘S 31 Rzlev x 10.1 ;7\ 0 R-G 3:::\ 10" RA 4 l L 1 3 5 7.5 10 15 Electron Energy - eV x 10 Figure 16 - Inactivation Rate Per Unit E er l -2 20 - Ribonuclease 143 When energy degradation Spectra are available fOr energetic electrons in solids, data like those presented above will be a valuable asset in determining biological radiation damage mechanisms. The question on the importance of the low-energy electrons and possible triplet state mechanism will be discussed in more detail after data on protein energy absorption from electrons is introduced in the next section. Energetic Considerations A recent publication by Cole83 has made it possible to take a detailed look at some of the energetic aSpects of the electron in- activation process observed. His paper contains data on the total absorption range fOr electrons in organic films (collodion) for ener- gies from 20eV to 50KeV. Fortunately the range of interest here, 20-2,000eV, is well covered by his data, and will be used in the dis- cussion under this heading. He also presented data on energy absorp- tion per ionization event, but this data was taken from ionizations produced in air, and therefOre applicability to ionizations produced in condensed matter is open to question. Electron energy absorption by protein.--Cole's data on total absorption range is presented as the thickness of unit density material that will attenuate the flux of incoming electrons by 99%. The only problem with applying his data to this work is in converting his unit density data to ranges in protein. Cole points out that the ratio of the stopping powers (dE/dX) of water to protein should be used, but these ratios are only known fOr electron energies above lOKeV. 144 TherefOre, the density correction used by Lea was used, which con- sisted of dividing the unit density ranges by the density of proteinsll7 An average protein density of 1.27 g/cm3 was used throughout this work. Figure 17 is a plot of Cole's data, corrected for protein density, over the electron energy range of 20-2,000eV. 1. Electron energy_loss rate. The rate at which an electron of energy E loses energy in protein can be determined by taking the reciprocal of the derivative of the absorption range curve in Figure 17. It is evaluated as AE/AX between the data points shown, and plotted against the midpoint energy of the energy interval of evaluation. These determinations are plotted in Figure 18, and are interesting because they Show a peak dE/dX at 100eV, with a decrease in value with a further decrease in incident electron energy. It should be pointed out that these determinations are for a bulk material and for linear absorber thickness. Therefore they are not the LET (linear energy transfer) measurements discussed in Chapter I, because those measure- ments were for electron path of travel, which can be far greater than the thickness of an absorber because of many deflections suffered by an electron. Epsrgy absorbed by enzyme films.--The energy absorbed by each enzyme film was calculated from Cole's data shown in Figure 17. A greatly expanded plot was used, and the energy absorbed by the films fer a given electron energy was determined by reading the difference in abscissa corresponding to a difference in ordinate of the film thickness. This can be denoted as: Y2'+ X2 = incident electron 145 H L r W V TT 9.. Q V U! Electron Range In Protein - R x it)"2 a a a I L l I 1 6 7.5 lio 12.5 7 15 17.3 20 I J I 0 EN 3‘ Electron Energy - eV x 10"2 Figgre 17‘- Electron Eggal'Absorptiqngggggg. Derived from data of Cole,83 corrected for protein density of 1.27g/cm3. 4)- )- 3p 0‘ \ > I 0 121' E N ‘ 1)- 1 l a 141 a a a! a_LLI I_LL_I 1 l 0 2 4 6 a lo 12 14 16 18 20 Electron Energy - eV x 10' Figure 18 - Bulk Stoppipg Power For Electrons In Protein, dEjdx Derived from data of Cole,83 corrected for protein density of 1.27g/cm3. 146 energy; Y2 - Y1 = enzyme film thickness; Y1 +-X1; x2 - X1 2 energy absorbed by the film from an electron of initial energy X2 and final energy X1. That this process does give the energy absorbed by the film can be seen by noting that an electron with an energy X2 will have just enough energy to penetrate a protein film of thickness Y2, and an electron of energy x1 will have just enough energy to penetrate a film of thickness Y1. Therefore the difference in energy X2 - X1 = Babs has to be equal to the energy lost in passing through a film of thickness = Y2 - Y1. A plot of the results of these determinations for each sample film thickness is shown in Figure 19. These curves give clues to two aSpects of the inactivation process. First it is logical to assume that inactivation is caused by energy absorbed from the electrons. The curves in Figure 19 are similar in shape to the 0 curves in Figures 12-13, with one important exception: they peak at lower energies and the curves for thinner films peak at the same energy. If inactivation were directly proportional to energy absorption then the thinner film curves should mimic the 0 curves. The thinner curve qualification is used because the thinner curves are close to being one molecule thick, and therefore reflect molecular absorption charac- teristics rather than characteristics of a film of some arbitrary thickness. As will be discussed below, a is a molecular parameter, and properties attributable to film properties need not be the same as molecular properties, i.e., energy absorbed by each will be dif- ferent.because of the different thicknesses. The 0 plots are sigmoid in shape up to their peaks, as Opposed to the linear absorption 147 .mao\mn~.H muwmcov van memoocxownu efimlam we meflau nweuoum new mwofiou we rump Eouu vo>wumo madam :Hououm he venuomn< mmwocu couuuofim a 0H enough «cod x >0 n mwuocm :ouuuofim ow n.nn nu n.~fi ea n.» w a N q q «T 8] uozaoalg JUOPIOUI/Z_OI x paqzoaqv he on if 148 curves, and, more importantly, do not fall off nearly as sharply with increasing energy after the peak as the absorption curves do. Another discrepancy between the two sets of curves is the inability of the absorption curves to explain the difference between the two enzyme's inactivation rates. Since the absorption curve is based on a general- ized protein absorption, it would not be expected to be possible to differentiate between them, but this difference is one that has to be incorporated in an explanation. This discrepancy points to a more fundamental question to be discussed below. 1. Shifts in absorption and inactivation peaks. At first glance it might appear that the 1121 film's absorption peak is shifted to higher energies. It is, relative to the thinner films, but closer inspection will reveal that maximum energy absorption for a film is at an electron energy which is about lOOeV (or less) greater than the energy that will just penetrate it. The linear portion of the curve at the low-energy end represents total energy absorption by the film, and the break point is at the point at which an electron just has enough energy to penetrate the film. The peaking at an energy beyond this point is due to two factors: 1) the increasing energy absorption rate with decreasing electron energy down to a point, and then the fall-off in this effect below lOOeV with a rapid decrease in the energy loss rate. This effect is shown in the dE/dx plot in Figure 18. Therefore, as electrons have enough energy to pass through the film they start losing more energy at the bottom of the film because there is a rise in the energy loss rate between OeV and lOOeV~-evidenced by the fall-off in the dE/dx curve below 100eV. When electrons leave the 149 film with about lOOeV they have passed through the maximum energy loss energy and the energy absorption rate starts decreasing again. The fact that the thick trypsin film on 88 (112A) has its peak shifted to higher energies and that the magnitude of the peak is below that of the thinner film on 88 is also related to the energy loss rate in the film, but not due to the mechanism described above. This film is, on the average, 3.54 molecules thick (as will be shown below), and a 500eV electron will lose 270eV in passing through the film (determined from Figure 17). Therefore the top layer of molecules will be exposed to 500eV electrons and show a corresponding 0, but those below will be exposed to lower energy electrons and have inactivation rates which will still be increasing with increasing incident electron energies. As the energy of the incident electrons increases from 500eV the inactivation rate of those in the top layer will start decreasing, but the rate of those below will still be increasing until they are finally exposed to 500eV electrons, at which point their rate will also start decreasing. By this argument it can be seen that the inactiva- tion rate peak will be shifted to higher energies and be of a lower magnitude. The fact that the 0 curves approach the same cross section at higher energies supports this argument because the energy loss in the film, as well as the increase in rate with decreasing energy, is less, and therefore the difference between the two values would be expected to be less. The smaller values for the inactivation rate in the thick film could also be partly due to the thick film protection effect to be discussed below. 150 Electrons incident per molecule inactivated.--The inactivation cross section, 0, is a molecular prOperty, and its determination is independent of the particular arrangement of molecules in the film. This is true because N/Nb = e’UD, or 0 = l/D lniNO/N, which is independent of film thicknesan/l‘lo = fraction of activity remaining, and is determined from the enzyme activity values. The inactivation yield, Y, given by equation (10) as Y = YO (molecules inactivated/ electron) is dependent upon the molecular arrangement in the film because of y, the molecular surface density. If Y (molecules/cmz) was determined for a film one molecule thick, then l/y would give the sur- face area occupied by one molecule. If 7 was determined for a film n molecules thick, then l/y would be small by a factor of n, and n/Y would give the surface area for one molecule. The reciprocal of Y, Y.1 = l/yo = 037/7 (1/0 = 037 = dose that produces one inactivation event per molecule) would give the electrons incident per molecule in- activated if the film was one molecule thick. If the film was n molecules thick, then Y"1 = 037 x n/y = dose that delivers one hit/ molecule (electrons/cmz) x area of one molecule in the surface (cmZ/ molecule) = electrons per molecule inactivated. Therefore, Y.1 = n/yo (electrons/molecule inactivated) (34) The thickness of the various films in units of molecular thickness was determined by dividing the film thickness determined for each enzyme (Table 1) by the thickness of a film calculated to be one molecule thick. This thickness was determined by assuming a molecule was a cube with a density of 1.27 g/cm3 and using a molecular weight 151 of 23,700 for trypsin and 13,700 for RNase.64 The area a molecule occupies in the surface is the film thickness squared. The results of these calculations are presented in Table 8. TABLE 8 Molecular Thickness of Enzyme Films 5...... “ii?“ ”°t§§‘fi§&§2i§§2§i§ “ T-G-4O 40 1.30 T-S-SZ 52 1.65 T-S-112 112 3.54 R-S-31 31.4 1.20 R-S-38 37.7 1.44 Notes: Unit Trypsin Film 31.5A Thick; Molecular Area = 990A2 Unit RNase Film 26.2A Thick; Molecular Area s 685A2 The values for the number of electrons per molecule inactivated were calculated using equation (34) and the values of n from Table 8. The results of these calculations are plotted in Figures 20 and 21. These curves indicate a surprising leveling off in the numbers of electrons required to produce an inactivation at energies above the peak in the 0 curves. They also indicate more dramatically how much more sensitive RNase is to low-energy electrons than trypsin (note expanded RNase ordinate, 2 x that of trypsin). The trypsin molecules took ten times as many 20eV electrons as the RNase molecules did for inactivation, and at lOOeV trypsin took about twice as many electrons as RNase. Number of Electrons/Molecule Inactivated Number of Electrons/Molecule Inactivated H H H O 0‘ 152 _ T-S-52 14_ (to 210 G 20eV) ' T-G-40 2' (to 258 O 20eV) 0h 8' X E .\&N——T-S-112 : \"\ a 0 a x _.____—x 2- _______ _.————- I I I I I I I I I I I I 0 2 4 6 7.5 10 12.5 15 17.5 20 Electron Energy - eV x 10'2 Figure 20 u r f 1 r0 _1 Trypsin Molecule Fer Inactivation, Y 8- 7. A‘—-R-S-31 6’ (to 21 a 20eV) 5. 4. 3» A 2- .,R-c-aa a/A 1. A .r'\./‘ _______——-—-A/ \a—a—a—A/A I I I I I I I I I I I I U '2 a 6 7.5 10 12.5 15 17.5 20 Electron Energy - eV x 10' Figure 21 r f den 0n -1 Ribonuclgase Molgculg ger Inactivation, Y 153 A possible discrepancy in the RNase data is apparent in Figure 21, and it also appears in the comparison of the molecular area for RNase in Table 8 and the 0's in Figure 13. The cross section for R-S-31 has a maximum value that is about twice the molecular area, and, therefore, it appears that only 1/2 electron is required to cause an inactivation in Figure 21 for R-S-3l. A possible explanation of this is the formation of RNase dimers. The enzyme is usually found in a dimeric state, but this dimerization does not seem to affect the enzyme's activity.64 If the binding of the molecules in the dimer were quite strong, then it seems feasible that inactivation of one molecule could also cause inactivation of the second, and thereby double the size of the affected units. If inactivation was caused by physical unfolding or loss of conformation, then this could easily affect a second molecule bound to the affected one. However, I have no direct evidence on this point, other than that of dimer formation. Engrgyabsorbedper molecule inactivated.--The quantity that would be the most interesting in these considerations would be an estimate of the actual energy absorbed per molecule inactivated. An average energy absorbed per molecule from one electron can be obtained from the data in Figure 17 by dividing the values for the film absorp- tion by the molecular depth of the film. Then if these values are multiplied by the respective number of electrons required to inactivate a molecule, a value of the energy absorbed per molecule inactivated is obtained. The results of these calculations are presented in Figures 22 and 23. Again note that the RNase ordinate is expanded 2 x the trypsin ordinate's scale. 154 one a oeum>wuomcu canoeaoz can uh nmm oenuomn< menu 1 «N unswwm «nod x >0 1 hwuocu couuuofim om n.5H ma n. NH o« m. n o e N d d a u u u u a d u q u NanmuH E X A>o ON @ oonu one ~m-m-a A>u am e oofis one os- -o-e\\1x 01 x panosqv Aa Z- 155 one n.su m queum>auuedH «fireman: ommufiuaconwm mom venuomn< Nunecm 1 mm ousmfim NnoH x >0 1 hmuocm couuuodm d d d 1 d d 1 # d 4 4 D II! lid! . . .9. . . M Innail wry; . .lnarlnie . . w / ./4/ As . .q ac.” mmdéllv In.” a... La. Hm-m-m N io.n 4n.n OI x paqaosqv as Z. 156 Again, as would be expected from Figures 20 and 21, these plots show clearly how much more sensitive RNase is to the low-energy electrons, and also that it takes about half the absorbed energy to produce an inactivation in RNase. The curves fer both enzymes show the leveling off above the peak 0 energies, as would be expected from Figures 20 and 21, but here there is a true leveling off, as opposed to a slight rise at the highest energies for the number of electrons required to cause an inactivation. The slight rise in numbers is offset by the decreasing energy absorption with increasing electron energy. In order to get a better look at the energy absorption re- quirements at lower energies, a O/Eabs plot will be used. Inactivation rate per unit energy absorbed.--If one process or energy absorption mechanism was responsible fer the inactivation process, then it would be reasonable to expect that there was some proportionality between the energy absorbed and the inactivation rate. This can be simply diSplayed, as in the O/E curves, by dividing the 0's by Babs at each energy. This was done, and the results are dis- played in Figures 24 and 25. If the inactivation rate was proportional to Babs’ then o/Eabs would have a constant value. A decreasing value of o/Eabs would indicate an increasing energy absorption requirement to do the job, or a decreasing inactivation efficiency; and an increasing value fer the ratio would indicate an increasing inactivation efficiency, or equivalently, a decreased energy absorption per inactivation event. The plots in Figures 24 and 25 indicate a rapid increase in efficiency from the lowest electron energies up to the 0 peak energy, N k H Rzlev Absorbed Per Molecule 2 H N O Rzlev Absorbed Per Molecule 0" N o p Q N Rb 4 O N O 157 ’ ' /.,4‘3' x\ " T" 5" “52\0// T- s- /11/::1 / x L/a/\\‘1Q——"’:/ ;//}7/ -G-40 x—r’x/ 4 I I I j I I $ L I J_ I 4 6 7. 5 10 12.5 $5 17.5 20 Electron Energy - eV x 10’ Figure 24 - Inactivation Rate Per Unit Energy Absorbed Per Trypsin Molecule Inactivated, OVE abs r >A r /A\ /A i A‘~~/R-s- 31 . A/A/ // r- .\, _ ' / \R-G-as ._-. I L I I 1 1 J 4 6 7.5 10 215 20 Electron Energy - eV x 10' W Figure 25 - Inactivation Rate Per Unit Energy. Absorbed Per RibonucleasegMolecule Inactivated, d[EabS 158 and then a slower but significant increase in inactivation efficiency with increasing energy above the peak.o energy. TherefOre, even though the 0 curves show a decrease in sensitivity to electrons at energies greater than those that cause a maximum inactivation rate, and the other energy plots show a leveling off in energy requirements, these plots indicate that the inactivation process becomes more efficient as the electron energy increases. Enzyme Energy Absorption and Degradation Spectrum With the aid of Cole's data, as presented in Figures 17 and 18, more can now be said about the significance of the O and all? curves discusses in the first section. The question being dealt with is the relative significance of electrons with an energy Em which yields the maximumio, and electrons with an energy below lOOeV, which would be expected to excite triplet states directly. The crucial question is still the relative abundance of electrons in the various energy ranges, which would be specified in the details of an energy degradation spectrum. However, with a knowledge of the absorption ranges of electrons, some deductions about their probability of affecting a molecule can be made, as well as their probable abundance in an energy degradation spectrum. In order for a electron to affect a molecule it has to be able to interact with it. Therefore, a crude approximation of the relative probability that electrons in two different energy range: have of affecting a molecule can be made by comparing their absorption ranges. In the case of trypsin, with an Em of 500eV, the range of a 500eV 159 electron is about 2003, from Figure 17, and the range of an electron below lOOeV is 403 or less. Therefore, a 500eV electron has 200/40 = 5 times the probability of reaching a molecule that a 100eV electron does. The ratio of the cross sections at these energies is about 6, which is also a relative probability measurement. Therefore, since the probability of occurrence of two independent events is the product of their separate probabilities (here relative probabilities), it can be estimated that a 500eV electron has about 5 x 6 a 30 times the probability that a lOOeV electron has of causing an inactivation while traveling through the material. Another important consideration in the relative effectiveness of electrons with less than lOOeV energy is their very limited absorp- tion range. Since their range is about the dimension of a molecule, a very low energy electron would have to be produced in or very close to a molecule to have any effect on it. Therefore, the concept of a medium filled with a swarm of low-energy electrons as a result of the cascading effect produced by high-energy radiation is not really meaningful with regard to the present work. For a low-energy electron to affect a molecule it would have to be produced in the molecule, which would mean that the molecule had already interacted with a higher energy electron, i.e., the act of producing the secondary is an ioniza- tion. From a strategic point of view, there may be lots of them around, but the fact that they are there prObably means that the damage has already been done, and whether they can cause damage or not is irrelevant. To answer the original question about the importance of very lowsenergy electrons to the overall picture of radiation damage 160 produced in biological molecules by ionizing radiation, it would seem that they are relatively unimportant in producing the primary radia- tion damage products. I feel that this is a consequence of their small inactivation cross sections, limited range, and dependence on being produced in or very close to a molecule to affect it. In terms of an isolated irradiation process, involving electrons incident on the molecules directly from a beam, the o and o/E plots also indicate that a higher energy process is of greater relative importance, i.e., the 300-SOOeV electrons which cause a maximum inactivation rate. Ionization and Secondary Electron Emission In his paper on energy absorption, Cole also presents data on the average energy expended per ionization produced in air. His measurements were made of the total ionization current, and he com- putes his values using the assumption that the total energy absorbed is expended in producing ionizations. At the present no way exists to measure ionization produced in a solid, so in computations requir- ing a value for the number of ionizations produced in a solid it is assumed that the energy per ionization is the same as that observed in the gaseous phase, and a value of 30-35eV is usually used. Cole gives values of this magnitude for electron energies above lKeV, but his values rise slowly to 43eV/ionization at lOOeV, and then they rise rapidly to 70eV/ion at 20eV. I have reason to believe that there is much more ionization produced in a solid surface than would be predicted on the basis of Cole's air measurements. With a protein plated sample disk in place under the electron beam, a negative potential (relative to the sample) 161 must be placed on the shield above the sample (Figure 9) to measure the beam current, because if a positive potential exists above the sample surface secondary electrons can escape and reduce the net current read. Secondaries start leaving the surface at an incident electron energy of 20eV, and by about 50eV as many electrons can leave the sample as arrive, as evidenced by a zero current in the anode (sample) circuit. At energies as low as lOO-lSOeV there are twice as many electrons leaving the surface as arrive, and the maximum secondary electron current saturates at this value. During actual irradiation runs the secondaries were removed by a positive potential on the shield above to eliminate any possible double dose effects. However, tests under conditions in which secondaries were both re- moved and returned to the sample produced no difference in the in- activation rate observed. The phenomenon of secondary electron emission under electron bombardment is well studied, and the energy distribution of secondar— ies that leave a bombarded surface is peaked at a few eV with over 31’71 Since electrons with 90% of them with an energy under 20eV. this energy have a very limited range, under 103 by Cole's data, the electrons that leave the surface must be produced very near the sur- face. As a rough estimate assume that secondaries are produced at the same rate throughout an enzyme molecule of 30A thickness, then for each incident electron with an energy above about 150eV there would be 2 (top 1/3 of molecule) + 4 (bottom 2/3 of molecule) = 6 secondary electrons produced per incident electron. If more elec- trons leave the surface than arrive, then there is only one place that they can come from: the molecules under bombardment themselves. If 162 an electron leaves a molecule, then by definition, the molecule is ionized. Because of the very low energy of the electrons involved, it seems quite unlikely that all of the secondary electrons released in the protein are able to reach the surface and escape. Therefore, the rough estimate made above is probably very conservative. From the literature, the secondary electron energy distribu- tion is almost independent of the material being bombarded, conduc- tors and nonconductors included, and also independent of the incident electron energy, at least up to the energies used in this work. The fact that the magnitude of the secondary electron current from the sample quickly reaches a maximum value with increasing incident electron energy, and then saturates at that value with increasing energy, seems to be at variance with the energy absorption picture plotted in Figures l7-23. This is so because as energy absorption varies, it would be expected that ionization should change also, not reach a constant value. However, this observation is in complete agreement with the literature on secondary electron emission. These observations and arguments seem to raise more questions than answers, but if one is willing to accept that the production of a secondary electron = an ionization in a surface molecule of protein, and I see no alternative to this, then ionization produced by ener- getic electrons in solid materials must be much greater than is usually assumed. Using Cole's data, the maximum energy absorption by a 31K film (Figure 17) is lOOeV from an incident 200eV electron. From his ionization data, a 200eV electron loses 43eV/ionization, and on this basis 100/43 = 2.3 ions are produced. From my observations 163 and by my estimate above, at least 6 ions should be produced by a 200eV electron, and therefore utilization of gaseous ionization data to determine ionization in solid material is quite possibly in error. For this reason, and because gaseous values are based on the idea of total energy absorption going to production of ions, I have not made an attempt to estimate ionization in my films. For reasons to be dis- cussed below, I believe a significant amount of energy absorption is by electronic excitation, as well as by ionization. Energy Absorption Mechanisms A logical point to start from is to make the assumption that the inactivation process observed is a consequence of energy absorbed in the enzyme molecules from the incident electrons. If the process was directly proportional to the energy absorbed in a molecule, Babs’ then the plots of'O/Eabs in figures 24 and 25 would be horizontal lines. They are not, but instead show two characteristics depending on whether the incident electron energy, E, is above or below the peak inactivation energy, Em. If E is below Em’ the process shows a rapidly increasing energy requirement with decreasing E. If E is above Em, then the process shows a decreasing energy requirement with increasing E, but at a much lower rate of change. Figures 22 and 23, which plot Babs vs. E, show a nearly constant energy absorption per inactivation above Em’ but a rapidly increasing energy requirement below Em' These Observations show clearly that the inactivation process is not pro- portional to the total electron energy absorbed by the enzyme, but must depend on how it is absorbed, which in turn is a function of E. 164 Regardless of how much energy it takes to inactivate a mole- cule, Babs’ a study of the kinetics of the inactivation process shows that it is a one-hit process, i.e., inactivation is caused by a single interaction with an electron and the radiation sensitive area of the molecule. This conclusion is based on the independence of the in- activation rate on the dose rate (over the rates checked) and the fact that the plots of the ln fractional activity remaining vs. dose were linear. Also at the beam currents used, there were fewer than 10 electrons incident on a molecule in a time period of from 1 to 5 minutes to produce one inactivation event per molecule-~based on D37 and the exposure times needed to deliver it. Therefore, at this low a dose rate, it seems safe to assume that energy absorption events were independent and well separated in time. Above Em the inactivation process can be visualized as one that becomes gradually more efficient in terms of energy utilization, but one that becomes less likely because of the decrease in energy absorp- tion which is governed by the decreasing dE/dX which is plotted in Figure 18. However, at Em, and below, something quite radical takes place, and the energy requirement starts increasing rapidly. Since Em is in the range of 300-500eV, this change cannot be related to changes in the rate of energy loss by the electrons, because the plot of dE/dx in Figure 18 shows a continuous increase in dE/dx for decreasing E down to lOOeV, where it peaks, and then declines rapidly for lower E. The peaks in the enzyme energy absorption vs. E plots, Figure 19, are related to the behavior of dE/dx with energy, and hence cannot explain the inactivation peaks, or decreases in 0 below Em. 16S Excitation versus ionization.--At the electron energies used here the only probable energy absorption mechanisms are electronic excitation and ionization. Thermal energies can also be transferred to molecular vibrational and rotational modes, but heating by the electron beam was completely negligible factor in this work as discussed in the fourth section of Chapter 111. If excitation is to one of Platzman's superexcited states, then ionization could follow excitation by an autoionization process, but the observed effect would be an ionization. However, an ionized molecule could also be left in an electronically excited state. If it is assumed that an energy absorption cross sec- tion, or relative probability of absorption, can be determined for excitation, ionization, and excitation fOllowing ionization, then the total energy absorption cross section, 0 , can be written as t 0t=0 +O.+O. e 1 ie The three component cross sections add because it is assumed that they are separate and independent events. New, working on the assumption that inactivation is a result of energy absorbed in a molecule, it follows that the inactivation cross section observed should be propor- tional to the total energy absorbed, at’ by 0 = ptot = pt(oe + a1 + die) Since it is assumed that each absorption process is independent, then each energy absorption event should have a probability of producing an inactivation, which can be denoted as pe(E), Pi(E)’ and pie(E). These probabilities are assumed to be energy dependent, and the 0's are 166 energy dependent by their nature. The dependence of the inactivation cross section on energy absorption can now be written as o = 13,0330, = pecswe + picfimi + pieumie (35) This equation says, in effect, that the inactivation process, as measured by an inactivation cross section 0, is proportional to the total energy absorbed in the molecule, but that energy absorption is partitioned between excitation, ionization, and excitation fOllowing ionization by 0 Ui’ and Gie' It further says that each of these 89 energy absorption processes has a probability of causing an inactiva- tion at a given energy E, which fer each process is pe(E), pi(E), and P1603)- The fact that the two enzymes have different 0 peaks, or Em's, indicates that how the energy absorption is divided between excitation and ionization is a molecular property, which is not an unreasonable prOperty to attribute to a system. Since it might be possible that excitation is a property that is more governed by certain molecular properties, i.e., by ffi, the optical oscillator strength, than ioniza- tion; then it might be possible that as E decreases excitation becomes the dominant energy absorption mode. Since quantum yields fer enzyme inactivation by 254nm UV light are on the order of .01, and the quantum yields observed in this work are on the order of 1 (see Figures 20 and 21); and since 254nm UV can only cause molecular excitation in a mole- cule, it might be reasonable to assume that inactivation initiated by energy absorption by excitation has a much smaller inactivation cross section. Then, if excitation started dominating as the energy 167 absorption mechanism at lower E's, this might be able to explain the decreasing 0's and increasing E observed with decreasing E. abs The above speculations are very iffy; however, some observa— tions on the energy dependence of O with E can be made, based on an experimentally verified theoretical relationship which predicts cross sections above a minimum energy range. Energy dependence of the inactivation process.--As a first estimate, the optical approximation used by Platzman in estimating radiation products in his paper on superexcited states,2 which was discussed in the Introduction, could be used: at = c fS/Es, where fs is the optical oscillator strength fer an interaction of energy Es. A more sophisticated approach is to use the Born approximation, whose application to cross section estimation was developed by Miller and Platzman,84 and whose application to measurement of ionization cross sections has been verified by many researchers. ‘In a paper developing a semiempirical equation to predict ionization cross sec- tions at low electron energies, Vriens85 gives a statement of the Born approximation applicable here: 2 2 e = 4H3oR fa. 1n ceE = fi§_ 1n ceE (36) E E E s and 2 o. = 4113eR M? 1n c.E = A1 1n c.E (37) 1 1 1 -—- 1 E E where 2 _ a5R df “i' E aEdE 168 and a0 = the radius of the first Bohr orbit of hydrogen (0.529A), R is the Rydberg energy (13.60eV), Es and E1 the excitation and ioniza- tion energies, reSpectively, and df/dE the differential oscillator strength. Equations (36) and (37) can be substituted in (35) to yield: 0 = k(Ae 1n c E + Ai 1n c.E) = A ln CE (38) E— e E— 1 E If this energy dependence has any relation to my data, then a plot of CE vs. 1n B should give a linear plot. Plots of my data in this form gave reasonably linear results for energies down to about 250eV for all the enzyme samples, and these plots for trypsin are shown in Figure 26. Therefore, it might be concluded that my excitation scheme has some relevance to my inactivation results, at least above electron energies of 250eV. A plot of my 0 vs. E data on log-log paper gave linear plots up to about 300eV and these are shown for trypsin in Figure 27. Therefore, for energies below 300eV the energy dependence of the in- activation process can be described by: a = kEa (39) The slopes of the OE vs.lnE plots were determined to obtain the value of A in (38). This value was substituted in (38) which was then solved fer C. The slopes of the log-log plots of 0 vs. E were deter- mined to find a in (39) and the value of k was adjusted to give the same values for equations (38) and (39) at E = 300eV. Table 9 gives the results of these calculations. 169 120 ' Y 105 \l \O UN 0 V 1 6E - 82 x eV x 10-4 as c> 45r 301- 15- 0 1 In E Figure 26 - Fit of Trypsin Data to theLBorn Approximation [Over the range of applicability: E = ZOO-2,000 eV, eq. (40)] 7r In E Figure 27 - Fit of Trypsin Data to A Simple Power Law [Over the range of applicability: E = 20-300 eV, eq. (40)] 170 TABLE 9 Values of the Constants in Equations (38) and (39) A c ‘ sample (x1o3eV) (eV'l) 3‘ k T-S-SZ 44s 1/134 1.6 .079 T-S-llZ 491 1/232 1.6 .0454 'r-c-4o 342 1/218 1.6 .040 11-3-31 426 1/139 1.4 .44 R-G-38 451 1/237 1.4 .175 In experimental work to determine ionization cross sections for noble and diatomic gases, Schram 3331.86 found that their data for lighter gases conformed to the Born approximation for electrons with an energy down to about 600eV. However, they found that for the heavier gases Kr and Xe the approximation held all the way down to 100eV. This indicates that the approximation might also apply to other large molecules at low electron energies. In a paper discussing target theory analysis, Marshal g£_21387 observed that Mi was pro- portional to the molecular weight for a large number of molecules, and that M = Mi/MW i 0.3. Solving equation (37) for A1 yields: 2 A. = (4Ha2R) x M.. 1 o 1 Substituting the values of the constants in this, and using my values of A, I found a value of ME for trypsin and RNase, and using a MW of 23,700 for trypsin and 13,700 for RNase, I found the values of M for these enzymes. These turned out to be 0.33 for trypsin and 0.65 for RNase. If it is assumed that the RNase is pre- sent in the dimeric form, then its MW would be 27,400, and M would then be 0.33. The agreement with the observed values of M for other 171 molecules is intriguing, but there is a large discrepancy between the values of C that I found and those observed for smaller molecules. Empirical values of C1 usually lie in the range of 1/20 to 1/5 eV-1, which is about an order of magnitude larger than those I observed. The relevance of equation (38), and through it equations (36) and (37), and the observations above to my data is questionable, but there is at least room for speculation in the matter. However, a combination of equations (38) and (39), over specific energy ranges, does give an expression for the energy dependence of the inactivation process observed in this work: Q ll %- 1n CE E 2 3008V (40) kEa E < 300eV Substitution of the values of the constants for the various enzyme samples from Table 9 into equation (40) gives a good fit to the 0 curves for them. Unfortunately the forms of equations (36) and (37) are the same, therefore, they are unable to shed any light on the sizes of the cross sections in equation (35), or to give an idea of the energy distribution to excitation and ionization. But they are only valid down to energies of about 300eV, and it is the relative distri- bution of absorbed energies below this energy that is of the most interest. Different Inactivation Rates for Different Samples Different inactivation rates were observed for the two enzymes used, and between sample support materials for the same enzyme. The 172 differing rates on the two support materials, SS and G, are not a unique property of the electron irradiation process because the same differences were observed for inactivation on these samples caused by UV irradiation with 254nm light. Support material effects.--There were three different types of observations that bear on the different inactivation rates observed between SS and G disks. They point to protection effects by binding, or "cage" type effects, and a possible energy transfer mechanism. The following is a summation of the observations discussed in the first two sections of Chapter III. 1. Enzyme platingobservations. Both sample support materials wet and plate well with both enzymes in solution, but the G disks are completely hydrophobic when dipped in pure water. This suggests that there is a binding mechanism between the G surface and the protein, and that there is probably a different molecular orientation on the two surfaces. 2. High vacuum exposure activity loss. A 52A thick film on SS loses about 14% activity upon being exposed to a high vacuum. A 1123 thick trypsin film on SS shows a negligible high vacuum loss. A 40A trypsin film on C shows a 5% activity loss in a high vacuum. I interpret these facts to indicate that the molecules in the thicker film on SS are more densely packed, and hence are able to restrain molecular unfolding or deformation due to dehydration in the high vacuum. The trypsin film on a G disk is 20% thinner than the trypsin film on a SS disk that lost 14% activity, but upon exposure to the high 173 vacuum the thinner film on G lost activity at 1/3 the rate trypsin did on SS. Therefore I postulate that the binding between the G surface and trypsin molecules must be able to restrain unfOIding induced by the vacuum dehydration. I further propose that this binding, or "cage effect," is also able to act as a protective mechanism to lower the radiation inactivation due to unfolding, or denaturation of the molecules. 3. UV reflectance spectra. The UV total reflectance work done on a Cary model 15, with a total reflectance attachment, was able to measure the UV absorption of the films plated on the two sample support materials. It was determined that a 52A trypsin film on SS absorbed 219s of the 254nm light incident on it, and that a 401 thick trypsin film on G absorbed 28% of the 254nm light incident on it. I suggest that this increased absorption on the G surface by the protein could be due to an energy transfer mechanism between the enzyme and the highly conjugated ring structures of the graphite. This type of structure would make an ideal energy sink if the energy can be trans- ferred to it from the protein. I propose that this is possible, and in fact is reSponsible for the unusually high UV absorption of the protein plated on it. The energy transfer is facilitated by the bind- ing process proposed above, and is partially responsible for the lower inactivation rate observed on the G disks. The Babs plots in Figures 22 and 23 indicate a larger energy absorption required for inactiva- tion of both enzymes on the G surfaces. From these observations I propose that the lower inactivation rate observed for both enzymes on the G disks is due to both a 174 protective binding to the graphite, and an energy transfer from the protein to the graphite that provides an alternate energy degradation pathway for the otherwise damaging excess energy. This energy transfer process could be facilitated by the protein binding process. Between different enzymes.--The most important result observed from using two different enzymes is that they have different inactiva- tion rates, at least as far as energy dependence goes. Ribonuclease appears to be uniformly more sensitive than trypsin to the electron irradiation, requiring about half the absorbed energy per inactivation required by trypsin over the entire energy range of electrons used. RNase also shows a larger inactivation cross section--an observation probably related to molecular size, i.e., RNase dimers-~and shows an inactivation rate peak at a lower energy than trypsin. This last ob- servation is the most important because it implies different energy absorption processes in each enzyme. Different energy absorption mechanisms imply that molecular physical properties enter into the process, rather than havingiflmamolecules acting like a homogeneous mass of protein. Studies involving other enzymes with known, and relevant, physical characteristic differences could provide more information of help in defining the inactivation mechanism. For example, horseradish peroxidase is a heme containing enzyme, and it would be interesting to see if the heme could act as an energy sink and confer a degree of radiation protection on the enzyme. Future Work There are two directions I feel future work in this area should take. First istfiuainclusion of more enzymes as radiation 175 victims, for reasons given above, and the amassing of more data to give a better picture of what is going on. The major part of the work done to date has been in perfecting the equipment and techniques, and I feel that they are good enough for a team effort to acquire enough data to get a more complete picture of the inactivation process. The second area of work that needs to be done is an attempt to see if any luminescence can be detected from the proteins under electron bombard- ment. If it can be observed, it would give valuable information about possible energy absorption pathways, and it should be analyzed both Spectrally and by lifetime studies to see if phosphorescence is pre- sent, which would indicate the involvement of excited triplet states. Another project that would be of great value in evaluating what is going on at the sample surfaces would be an electron microscope study of the surface both with and without enzyme present. Also an amino acid analysis of the enzymes after electron irradiation would be valuable in determining damage pathways. The following are a few Specific suggestions for future research efforts using this equipment and these research techniques. Apparatus The apparatus and techniques seem adequately developed to sup- port a small team approach to data collection. However, one improve— ment would provide a major improvement in the electron gun stability and freedom from gun teardown before each data run: the installation of a sliding-gate vacuum valve between the gun and sample chamber. This would also necessitate a separate vacuum pumping system for the gun tower, but it could be of small capacity because once the gun has 176 been Operated there is very little outgassing from it. One of the most frustrating aspects of running these experiments was the frequent failure of a newly coated filament to work properly, thereby causing an aborted run. Then, once a good filament coating was prepared and operated, it had to be destroyed by exposing it to atmOSpheric moisture when the sample chamber was opened. The gun and gun tower were designed as a separate unit, and could be installed on a McPherson vacuum grating Spectrophosphorimeter, or similar system. The gate valve and pumping system would be all that was required for such an installation, and this would make possible a study of luminescence under electron bombardment. The energy range available could also be extended up to about lOKeV by providing a power supply with this capacity. The only re- quirement on such a supply which could be a problem would be that it have a completely floating output with respect to ground, because of the gun set-up potential requirements. Electron Microscope Studies Any information that could be obtained about the physical environment of the enzyme molecules during irradiation, or regarding their physical placement on the sample support surface would be most valuable in further interpreting the results of these experiments. It would be especially desirable to know what the enzyme films actually looked like regarding uniformness, the presence of holes, etc. The ideal electron microscope to use for these samples would be a scanning type, because the sample can be used as it is, and the results would have a 3-dimensional perSpective. However the resolution of the 177 presently available scanning microscopes is not great enough to show molecular detail, and to do this would even be pushing the present transmission microscopy techniques. Irradiation Victims In view of the differing inactivation sensitivity and energy dependence of the two enzymes studied, it would be important to study the inactivation process in as many more enzymes as possible. Various alternative enzymes that seem amenable to the irradiation and activity assay systems were suggested in the last section of Chapter I, and would provide ample breadth to a future study. Amino Acid Analysis The amount of protein on one sample disk is not sufficient for an amino acid analysis using a Beckman Amino Acid Analyzer. However, the protein from about five sample disks would provide an adequate amount for such an analysis. Analysis of enzymes irradiated at (or below) lOOeV, at the peak inactivation energy, and at the highest energy used, 2KeV, might provide valuable clues to answering the ques- tion of the relevance of excitation and ionization in the inactivation process. For example, if a predominant loss of a particular amino acid at the low-energy were observed, Opposed to no Specific losses at the higher energies, it might support the dominance of excitation processes at the lower energies proposed above--provided such an effect was observed. 178 Conclusions The original goal of this research project has been achieved in that the relative importance of electrons with an energy below lOOeV in an inactivation process has been determined. It was found that in an electron bombardment of a protein molecule, trypsin and ribonuclease were the most sensitive to electrons in the energy range of 300 to 500eV. By considering the total absorption range of elec- trons with an energy below lOOeV, their relatively small cross sec- tions for inactivation, and deducing that in order for a low-energy electron to be present in a molecule it would just about have to be produced in the molecule--a process which would probably cause far more damage than the low-energy electron would be capable of--it was decided that these low-energy electrons were probably only of rela- tively minor importance as precursors of radiation damage produced in biological systems. Considering the isolated electron interaction with an enzyme again, it was observed that the maximum rates and energies at which they occurred were different for the two enzymes, therefore it was assumed that the inactivation process was governed to some extent by the molecular properties of the irradiation target. Because of this observation and the one showing a rapid increase in the energy ab- sorbed per molecule inactivated for energies below the energy produc- ing the maximum inactivation rate, an energy dependent inactivation mechanism was proposed. It is very general in nature, and states that inactivation is dependent on the total energy absorbed by the molecule, but the energy absorption is via electronic excitation, ionization, and ionization followed by excitation, each of which can 179 lead to inactivation, but probably at different rates. The overall energy dependence of the inactivation process is expressed through the sum of the independent energy absorption processes: Ge, oi, and Oie’ and their independent probabilities of causing an inactivation: pe(E), pi(E), and pie(E)’ which can be written as o = pe(E)oe + pi(E)oi + pie(E)oie. Unfortunately there are no reliable theoretical quantitative formulations which are able to predict any of these processes at the lowest energies, the ones of most interest here. Therefore, further interpretation of these results will have to await the availability of either theoretical or experimental information on these processes in the low-energy range. Based on an experimentally verified theoretical equation that predicts excitation and ionization at higher energies, equation (40) was arrived at to give a quantitative description of the energy de- pendence of the inactivation process. The low-energy limit was found to be 300eV, and for energies below this a simple power dependence on energy was found to give a very good approximation to the inactivation cross section curve. Again, unfortunately, these equations do not yield Specific information on either process, and the mechanism will have to remain descriptive at the present time. The inactivation cross section versus electron energy plots, and the plots of energy absorbed per molecule inactivated against electron energy will probably prove to be the most useful results from this research for immediate application, because they provide pre- viously unavailable details of electron interactions with biological molecules. APPENDIX APPENDIX The following is a brief outline of the statistical tech- niques and formulas used in Chapter III. The computational form for finding SS.(2) is 2 n xi - ( X xi)2/n A.1 1 i=1 as. (xi - {)2 = 1 i n u P1: n P13 1 The technique of the analysis of variance consists of breaking the total SS,for the deviation between each of the n observations xij and the grand mean X'into two components: 1) that between each sample in the j-th treatment and the treatment mean X}, the Within varia- tions; and 2) that between each treatment mean and the grand mean. There are a total of n observations, m different treatments each with nm observations. The overall method is formulated as: n m _‘ 2 n m __ 2 m __ __2 z 2 (xi. - X) = z 2 (xi. - x.) + z n.(X. - X) A.2 i=1 j=l 3 i=1 j=l 3 3 j=1 J 3 Total 8.8. Within S's. Between 8.8. The formulas used to compute each component are: 180 11 m -2 n m 2 n m 2 Total SS = Z Z (x - x) = Z Z x.. + ( X X x..) /n i=1 j=l 1=1 j=l 13 i=1 j=l 13 11 m 2 m 11 2 T1 2 Within as = z z (x - XI) = z [ X x.. - 2 x. /n ] i=1 j=1 13 3 3:1 i=1 13 1:1 13 3 m _ _2 m 11 2 n 2 Between SS = 2 n (X - X) = Z [ 2 x../n - Z x../n] j=l 3 J 1:1 1:1 13 3 i=1 13 The degrees of freedom for each component are: d.f. Total SS = n - 1 d.f. Within SS = n - m d.f. Between SS = m - 1 A.3 A.4 A.5 A.6 The basic assumption made in the technique of analysis of variance is that the samples are chosen from a pOpulation with a common variance. If this is so, then the variance estimated from the Within SS, 5 the variance estimated from the Between SS, 5:, W’ and should both be esti- . 2 . mates of the common variance 0 . The two estimates are computed from: EN Within, 5 Within SS/d.f. Within = WSS/(n - m) Between, 52 B Between SS/d.f. Between = WS§{(m - 1) The null hypothesis tested is that the means for each treatment, 15, A.7 are equal. If they are equal then the estimate of the common variance made from Between the treatments will not differ from that estimated from Within the samples by more than an amount that would be expected 182 from random variations alone. The significance of the difference between the two estimated variances can be tested by the F-test: 2 2 F - sB/s W’ with d.f.‘s of d.f. Between m - 1, A.8 and d.f. Within n - m If the F from A.8 is greater than the table value of F.05(m-l,n-m) then the difference between the two variance estimates is greater than that expected by chance alone, to a 5% significance level. Therefore variations observed must be due to differences between the means for the treatments. If the null hypothesis is rejected in the analysis of variance, then the Scheffé test can be used to test all possible differences between combinations of the various means. The test is based on an F-type confidence interval computed for each difference tested. Comparisons between all possible combinations of means can be formu- lated by: 1 A.9 Ho where ll M5 ll ME PI The Scheffé test for the significance of the C3. is, in terms of the above notation for the analysis of variance, to a 5% significance level75’76: 183 15- lCjl > (F.05) Scj where /r A.10 (F.05) = (”‘1)F.05(n-1,n-m) and //2 m = sw .2 new 3 i=1 Then C3. is considered to be significantly different from zero. The analysis of variance also yield estimates of the various Y}, the means for each treatment, and an estimate of the error for each is is obtained from the estimate of the common variance, where the 2 sw, number of samples included in the estimation of each mean is nj, is: s- = SEM for XI = / sz/n. A.ll X J J When only two treatments are to be tested for a difference in their means a simple t-test can be used. 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