PROTGBIOWEMISIRY - WEORETICAL AND EXPERIMENTAL CONSIDERATIONS GOENfiRNENG FRIMORDIAL BIQCHEMECAL DEVELOPMENT mesh {or the. Dam of M. 8'. MICHIGAN STA}? UNIVERSITY Gan: Sfeinmaa i963 LIBRARY Michigan State University ABSTRACT PDTOBIOCBEMISTRY - EIGHT!“ MD mum CONSIDERATIONS CONCERNING . 2mm. BlomBMICAL DEVEOPMENT Gary Stelnm AI exteeelve theery wee tormleted to deflne the reletlenehlp of lnfereetlee wen-- u entropy. From thle, the evolution e1 Ieee .4 energy wee prepeeed end the cainectlan of the electron to thle develop-at wee eleo eeggeeted. leewlee. the reletlmehlp a the cum te protehleeheelcel evolution wee indlceted. o- the bee“ et theee fend-um theoretlcel conelderetlene. expert-emu were prepeeed ad cerrled out to teet the flnel concept dleueeed in the theory perteleln; to the role of the electron in evelutlen. Bret of ell, lt wee ebeerved that equeeue eolutlcne of eleehele, flee eperhefidleyleyed the cherecterletlce of eerbehydretee. Glycerol me eelected ee the reectnt for the greeteet portlee of the etedy ad upee eperklu. lt reepaded positively to teete ueuelly qleyed to Identify due ensure each ee ninhydrln, Elem-Morgen, ad Pehlln; procedures. SeeeedIy, erg.“ eelde were eperked. {the eperked product of acetic eeld ledleeted e unaware nut-u. Ileelly. e fixture e! xlyeerel. ecetlc ecld, ethanol, end nun-uni- sun-m. en. eperhed to: e lees period of :1... yielded e huh eeleeeler weight predect. m- polyeerbllke euhetnee hed Icy of the eherecterletlce of memlyeeccherldee. '1 .fl . v ‘4.'_ -. --- 9‘ -.‘e :7 _ .. . . . _ _ ,. ‘_ ,_-. - 3.51.; I; 5 - n~i v ‘g‘..§"e .0 e '5‘.) a, ‘\’_‘ 1-2.." v'.'\ ‘ ‘ '* ;‘_(" J‘ ""-, 3- "I‘.' ""_ ‘...""_. .r. -'.I_ ',' 'I '1’" , n :33: .x that 3:31. 3 05’55393': .-s-r=..’.3 10 irol'xarg 33:30:: :5 2103 bQLJRQG Isa“; .1"m.r..':‘zl '4': manning ' " - ' ' . ' 1-‘ ’0' 9- a ..-‘ -‘ (e e :kx-Je'uajd': field “441‘. " v§'¢u\- C‘JE .jOU; M( ‘ 1:!) La £93 '11 $3119 105:. rigid s .31; quoaon it: Bf)ifii.i‘$€>33£-ffl3rf3 3:63 3-3 15:19.3: PIDIDBIOCHIHISIRX’- Immune». AND maximum dousmamnons coucnnuntc rnxubanrsL nzocanfixcan bészoéuznr by Gary 8mm A 1113318 mmtud to Michigan State Dakar“ ty in partial fulfill-n: of eh. ugh-cunts for the degru of man 01‘ SCIENCE We of Bioduuury ' 1963 ., _y. I r. .'..,-*1. A . p ; l.‘l l'tuilh .K. \ l . :v‘ x . .,'.‘. T '2 ‘n . 1" ""f ,(i f‘. I _- '1". Au ’! ks‘; .6 wall .-¢ .u V4 '4 L61... '5',(‘.~1\ nAts l I‘€¢°Pv\o. ‘ _."' '1'. .' If"\ ."-'Y‘.’ - .LIJ..1~.LI.'...... J '1‘]; £24: . .OOI" v a .,.. t. 4". -"\ 3:1)nJlA9-Ju ‘rthv .-,‘I -'* 0 'fl\ ‘ “*‘I‘i‘vz ‘ID 0:: 2193-1 hurt»? \jfilrxnvl'fii 3:113; [mfgidoh-i s-3:19:r:;.u'xlupo'x 9d: 320 tut-minim} mum; m“; 30 £35.13!ng ed: '10} ('1' '. :'-:_a t',’ (1" '3"! nuns-"uh.- 'n) In.» Lulu‘a 31': ;; a intofiz‘c 3: 3.0 3113.13 u: get} {9391 .-r-1 ' .0“, '.'-"‘I' “ran a"' r.- ..v.»'¢ -‘| 'rr - ' ' . ., . ,1 - . ; ,. ~ ‘ - .3. ~.\.|.a.\. .H- uni-Lllbula/‘v- 1.3.0.. \-.’JA .8. n.3- a“‘ ‘JD J..- r ‘ l w. .11. Acmgm EDGEMEN'I’S It is the authorfs belief that the type of counsel and direction provided by Dr. H.A. Lillevik of the Department of Biochemistry and Dr. J.R. Brunner of the Department of Food Science represents the essence of educational enlightenment. The expanse of the topics considered in this dissertation suggests the freedom of thought and experimental adventure encouraged by these professors. Acknowledgement for helpful evaluations of the ideas presented in the theoretical section of this work is due to Drs. Lillevik, Brunner, Dye, Kinsinger, Montgomery, Schlegel, Speck, and Scheraga. The author is grateful for the constant encouragement provided by his parents, brother, and fiancee. Portions of this work were supported by the National Science Foundation Undergraduate Research Program.administered by the Department of Chemistry, Tel Hashomer Hospital (Tel Aviv, Israel), and the Department of Biochemistry at Michigan State University for which gratitude is sincerely extended. I. II. 111. V. VI. “BL! OF CONTENTS “Ct‘ufllooooooooooooo Guardintrodnotim ......o.... Manna! nautical. Considerations . . Intonation. Entropy. and Organization fivointion of Entropy and Information . Absolute Diopooition . o Originofflnorgyondnou oooo ooo ‘ Tho Eloctron in Protobiochonicol Evolution Biotoricoiloviov..'............. lxporinonui Expotinonui Objoctivu . mod-onto! Moth“. o . Apparatus o o o o o alonicolo all lug-nu Pronouns-duct... hurl ad rhino “no N“ o Poly-or- o o o Discussion 000...... m an Conclusions . . o millmlphy Appondix I - musical Toohniquu Sonora O Appondiin-Vito oooooooo 11 17 27 38 38 39 39 43 47 65 78 88 9O 94 112 V) r, v- ’1 to ~- r'.‘ 5'1’.‘ rlu‘. V.) P“? 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Mitotivo inflation for tho prooonoo of o pontooo in Wdflmfll oooooooooooooooooooooooo Ioonlto of pa and lloon-Horunlotoroiootiono . oporkod oqluotglyoorolndoo-roo'onuinoxporiuntb. . o o o . . trod-nu- o! porotolnonoonlfonyl ohlorldo dorivotivoo tron tbooiotiontrootionootoporkodoooticocid .......... my o! pbyoioo-oboniool .olyooo of tbo oyntbooiood polyur o Coqorion of tho otfocto of tho photon no oloctrn W -mcn5RMoooooooooooooooooooooo A7 51 52 53 53 54 65 75 77 t4 (‘o \J A: (i. r . ' v ' o; N‘- I . - . I . ,. ‘w _ l o ' o L . L .H ....v s .--._‘-o-olnc". .v. In 0—- ’UI‘“ l l _ I _ _ I \ ’I .’. P ‘ g 1-. .. - a . —~ g - -§.' o ...u .1 . .' . v -. 4 .. I: LI' ‘1 433D} ‘ if”: :ida-To-N-n. ." (.j."J.s.L9 —:.'3 ac £( Loaf) ‘5'“).9 .'\ I , , ~~~ .' ‘ -. a ' 3-. . o” '\ o‘ 3 - V ... . , ‘ I J , . ‘4‘" .' .o-J {9"5 'f'd ”to—‘5: J'si' 1‘3 ’1.) D :jrlo,’; *J~‘) 0"-) “)5 fJ‘ 1 .Ja 4‘“: ’..51¢.‘"¢ ‘f{ 2.1:? f.) ?e- ‘.I.~'- f. A.‘.‘o."-.-_‘ v-n ,- ‘ .' 4 \ o .... .4.' D. ,' f-f ' ... “ "q I. 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'-. . - , ' s o. . -- o . o ‘ ‘ was? f=.rw.-:*&*<=;t.=;t=.;-3 an: :0 firm}; 31?. 1.o~....:.-..:.-r:.. lfig’ut. lo (Inna: Oi ..‘I'wod {Knit-sun ivxm :‘.x...".:.-I: .‘srx: go ;-..:'1-"".".'.'-3 “:33 30 m:ai‘1:..'.';;:03 II a o o o o o o o o o o o o o o o o o o Lfitriv233luia’x Skiing-[IO W3 bUNH OCNOU 10 ll 12 13 16 15 16 17 ll 1! 21 LIST OF FIGURES bletienship between entropy end intonation . . . . bastion of sbsolute disposition . e e . e . e . . Proposed orgsnissti‘on distribution of s finite system Proposed shift of the distributionsl center of gravity in s finite systen with tine (fro- Fig. 3) e e e e . Organisation of the primordisl energy systeo . . . e Heterislisetionefenergy ............. Proposed overell “ration of sbsolute disposition . Schmtic droning of electricsl spsrking system . . lsectionchanber..... e ........... Scholastic drains of reaction chamber . . . . . e . Experinentelsetupoooo....o....ooo. 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' . - I . a \ - ... n“. I - , - .4\ D- nu‘tjnJaxmubna “3.: ._,.;.r.i...‘1s..rm mi away mi‘xsafijm.’ 1.x: .s.:,._;_:..».-.33 "E‘ ... .. .'. 3—. 1..-. {b e e e e e e e e e e e e e e o e e e Eva-plug UMJ ..k- :u;A‘).e 13193511.)?! ml.) f-“inigmazm'a 111 but: winning 41.112331: ”3.5131? ‘F V .x g A 4“ e e e e e e e o e e e e e e e e o e e e IATfiitfl ‘1‘; .35} 3.1::51 . r..... t. . .... - ' .'. .- r ., . - “1.1,. ' 2'... M II'( nhigun iIL‘H‘J [18 Link ’29 X30 3'3 “45h! B “3531'. 31.30 3",}. .‘nT‘h-tfl.‘ 1.1:.ét’iGQU: 1 53 e e e e e e e e e e e e e e e e en 0 e e e e e e 0 9513833338 3'11!“ i "I “unto—h. ES 6 a _ n t.- La (1! m OJ 5 ’3 "Physical and chemical approaches to problems in biology have become increasingly productive in recent years. Major advances in the understanding of life processes have been made through research in such Specialties as biOphysical chemistry, molecular biology, biOphysics, and electr0physiology. Continuing progress will require an even more perceptive study of the interactions of matter, energy, and information in biological systems." - J.L.Oncley, editor, Biophysical Science “.é Studprrogram, John Wiley and Sons, Inc., New York, 1959, p.1. I. GEI‘TEPJJ. II‘JTRODU TION This thesis represents a systematic line of scientific reasoning and experimentation. It is designed to present plausible explanations for certain problems which science has tried to resolve for many years. The objectives of this work are threefold. First of all, from the standpoint of organic chemistry, the synthesis and analysiscf basic reactions will be investigated. A study of the effect of high voltage electrical Sparking on compounds of organic interest will augment present knowledge of the characteristics of these molecules. ‘ Secondly, laboratory experimentation will be used to demonstrate certain desired syntheses as a possible indication to the origin of biochemically significant structures and compounds. This would, of course, supply essential links in the explanation of the origin of biological life itself. These first two goals will be discussed more thoroughly in the section on eXperimental objectives. Finally, this thesis will be used to introduce a theory which is intended to provide a rational definition of some of the dynamics of the physical world. The significance of the photon as an energy supplier for promoting atomic and molecular evolution with the ultimate appearance of the electron and its assumption of the role of the photon as a reaction stimulator will be prOposed. The importance of the electron in relation to physically significant functions such as entrapy, information, and organization, as well as its possible connection to biOphysical evolution will also be discussed. II. FUNDAMENTAL THEORETICAL CONSIDERATIONS The real world, as is evident to any astute observer, is a con- glomerate of various physical and thermodynamic interrelationships. These would include material transfer, interconversion of the various forms of energy, constructive and destructive co-activities, and the like. A reflection upon these functions entices one to analyze the causes behind the observed effects. For example, the conception of the forces in the physical world rests entirely upon sense perception. Therefore, no matter what may be the actual conditions of a given event, absolutes, even into the realm of abstractions, must be proposed within the framework of sensual limitations, whether this be by first hand observation or with the assistance of analytical instruments and aids. Mental codification is based on interpretations of external impressions by which perception determines resultant values. Thus, for example, the degree to which an observer realizes the organization or disorganization of the units of a given system is subject to the manner in which the stimuli are received, the relative magnitudes of each class of data reaching the observer, and the value system he employs in drawing conclusions, whether this be arbitrary or prejudiced. Since man must draw conclusions from perceptions received, the exPanse of these con- ceptions is subject to the limitations and the inaccuracies of the receptor or the means of transmission so that what is concluded to occur in a certain physical event may either not represent the actual phenomenon or may not give a large enough overview of all the factors significant to defining the meaning and relevance of the given event. Based upon such functions, many aSpects of the physical environment have been perceived and concluded to be in continuous flux. It is possible, however, that the primary absolute which sense perceptions have failed to register because of a limited scope of observation is the tendency of progressive development to replicate primordial constancy, for if such change were unlimited and unidirectional, then primordial constancy would be dissipated forever. (This term will be defined later in this section). From this, it is possible to make the distinction between that which is absolute and that which is relative. Let it be assumed that the tendency toward primoridal constancy, the destined realm of potential development, is a reality which is so, with or without an observer to perceive it. This makes it an absolute for no matter how the observer goes about defining such a reality, the intrinsic properties of it remain unchanged. Should the observer, however, misinterpret the data which he receives concerning the characteristics of primordial constancy, the conclusions he draws will define it in a manner which does not represent a real picture of this phenomenon so that the observer's definition is relative to him and those he affects by his conclusions. Even in such a circumstance, the real character of the phenomenon remains an absolute and its definition is still a relative concept, dependent on the observer. Information, Entropyg and Organization A consideration of the essential features of change would include evalua- tion of the relationship between organization and disorganization. For example, metabolism is the result of the opposing forces of anabolism, or biological construction, and catabolism, the destruction of molecular complexes and food stores for the purpose of conversion to required energy supplies (1). Thus, in a developing bacterial cell, growth is readily apparent so that anabolism predominates whereas in a performing muscle, catabolism overrides its counter- part. The relative complexity of the physical world can be viewed as a reflection of a process similar to metabolism but entailing a much broader sc0pe. First of all, the buildup process may be interpreted as an accumulation of information. Information may be defined as that which sets or reflects the pattern of organization of a system. DNA (2:) carries the information which will ultimately set the pattern for the sequence of amino acids in cellular proteins as well as a template for its own duplication. Likewise, as a hereditary determinant, DNA establishes the manner in which the given cell can react to a variety of environmental possibilities. It is observed that the greater the com. plexity and abundance of proteins in a given cell, the more extensive and SOphisticated must be the inherent information of the nuclear DNA. Similarly the more functional the DNA molecule itself is, the greater must be the systemization of the units of its somposition. To clarify this point, one can consider a series of interlocking pieces of a jigsaw puzzle whose units form the number sequence 1-2-3-4-5. This series is composed of five members, each of which when standing alone bears little significance relative to the final pattern and when sacttered randomly, are found in almost complete disorder and disorgani- zation. The potential for creating many different sequences, each with 'its own particular, restricted significance, is initially large. From the disorganized conglomerate of the numbers of which the series 1-2-3-4-5 is composed could results such patterns as 5-1-2-3-4, 4-5-1-2-3, 3-4-5-1-2 and so forth. Each possibility is more than just a regular series of units since such a system can also eXpress a concept of something very real beyond the immediate association of parts. Then, in only one sequence of interactions by which the 2 follows the l and the 3 follows 2, is the series 1-2-3-4-5 formed. The first member bears the information that if the desired pattern is to be formed, a 2 must follow it. This, if all possible units were available to be in juxtaposition to the l, the inherent quality of this informational entity by which the desired sequence would be formed sets the pattern whereby only a 2 can and will follow it. Likewise, the 2 possesses such information by which only a 1 can come before it, and it, in turn, must follow the 1. Therefore, the information of the 1-2 system is threefold: l) The l, in order to lead to the formation of the series 1-2-3-4-5, must be the first of the sequence and by so doing, carry with it all its geometrical significance. 2) The 2, by this scheme, must appear in the system because of the architectural importance it entails as well as the restrictions it inherently possesses with regard to the types of combinations that may precede or follow it. 3) The sequence 1-2 is not 2-1 nor 1-3 nor 1-4 so that the creation of this sequence (1-2) deve10ps the information both as an extrinsic property of its immediate appearance and significance, and as an intrinsic prOperty by which only a 3 can follow it so that ultimately the desired series will be formed, similar to the theory of epigenesis (13). Now, let it be assumed that for every two members of the series which are placed in prOper sequence, one falls out of line. As more units become organized into the sequence, there are more places in which a disarrangement could result and thus a greater number of permutations of possible resultant distributions and errors in the system. Ultimately the desired series will be formed but initially it would appear to the neutral observer that all is chaos and confusion. It would seem to him that the process was more inefficient than the value any possible result could merit since he could not, at the point of one-third or even one-half completion, comprehend the enhanced information resulting from the process or the effect which such accumulation of information could have on overcoming the disorder noted at first. One case of this is found in proteins. This example is given merely as an illustration of the concept of information and its relationship to organization, not as a pr0posal to explain protein evolution. It is noted that nearly all natural proteins are composed of L-amino acids exclusively (6 ). It can be speculated that primordially there was an equal possibility of amino acid polymers being composed of D or L stereo- isomeric members. As it may have turned out, the first polypeptides were formed from L-amino acids. To maintain the stability of the a-helix, all other members of tha t polymer would also have to be L substances. From there, these initial proteins catalytically determined the L character of all other proteins to be synthesized. Thus, biological evolution utilized the proteins composed of L-amino acids because of their abundance. Initially the high randomness and low organization of the racemic modification indicated a small degree of information in the system. As polypeptides appeared with Specific characteristics, the information increased. Each L-amino acid had the inherent information that it could polymerize only with another L-molecule, but this system did not formerly have the information that nearly all such polymers would be of the L variety. As L polymers increased in number and size, this information became an inherent aspect of the system. Each polymer not only had the information that it was itself composed of L members but also that it could serve as a template for the systemization of the units of another polymer from unorganized masses of potential members. The information of the first members set restrictions and specifications on the future systems to be evolved. This anaIOgy can be used to explain certain preperties in the dynamics of original thinking. The nervous system is primarily a network of interconnected patterns by which apprOpriate stimuli are channeled to bring about the corresponding reSponse (5 ). Except for the autonomic system which is genetically determined, the patterning of reSponses is formed by trial and error. A baby f10ps its arms about until apprOpriate nerve patterns are established to distinguish the action of arms, fingers, and so forth. Thus, human experience patterns the nerve channels by which thinking itself originates. Concepts without suitable past experience and conditioning are almost untenable. For example, when the sound of the word "tuberculosis” reaches the brain by way of the auditory system, a prearranged pattern stimulates the response,"0h, that's a disease.” If additional patterning has set up the system of thought that diseases are evil, the stimulus of the original word will continue to meander through the brain's complex network and eventually come upon this pattern as well, yielding the correSponding verbal response. To an unenlightened aborigine, this word will most likely elicit no reSponse since no patterns for reaction have been deve10ped from his past experience. Deep original "thinking," the synthesis of new information, is not quite this Spontaneous since more prearranged patterns must be traversed before the "idea" emerges. An idea is original with a given person only by the fact that his unique experience and ability to establish correSponding patterns has set up the apprOpriate circuit to organize elements of information into a new system bringing about the emergence of the new concept to the corresponding stimulus. Therefore, inessence, thinking does not originate inside the brain but is merely a response to apprOpriate stimuli traveling through prearranged individu- alized nerve patterns. This concept can be extended to analyze certain prOperties in 9 physico—chemical processes. The first point of consideration is entropy (6). This is defined as the function by which the overall effect of physical inter- actions is to lead to greater disorganization, or increased randomness, so that entropy indicates a system's randomness or tendency toward disorganization. Thus, the eternal job of living cells, where energy flow is a homeostatic pro- cess, is to resist entropy which would, if unchecked, lead to the total disor- ganization of the cellular system. The second law of thermodynamics (6) may be viewed as that whenever energy is transferred from a state of higher temperature to one of lower temper— ature, it is impossible to regain all of the energy of the first state in the second since some of it will be lost in the process. AS=q/T Entropy may also be observed in the case of a freely expanding gas. Here the gas molecules are going from a state of less randomness to one of greater disorganization which implies increased entropy. ids = R ln Vz/V1 In this irreversible case, the probability of finding a given gas molecule within a certain unit volume is decreased by the process. Evolution of Entropy and Information Entropy, however, when considered in a much wider scope, is merely a relative concept. In other words, it is the observation of the results of a given number of phenomena during a certain time span that is relative to the observer. This becomes more readily apparent within the consideration of the concept of information. As noted, this value may be defined as that which sets or reflects the pattern of organization. For example, all of the units of information necessary to construct an atomic bomb were available to a prehistoric man. He did not, however, have the organizational information with which to pattern the units of information into a utilizable assembly. The units were already in existence, such as the physical 10 phenomenon of critical mass, but his level of eXperience restricted his capabilities to even recognize this information, an organizational process itself, so that it might be used effectively. Thus, because of a lack of organization of information, the units with which to assemble a greater system of information remained unorganized for a time. If information is that which sets the pattern for all organization or is a function of the organization itself, inherent information is necessary before energy acquired by agiven material system can be used to the advantage of the scheme. By this, information represents a degree of organiztion of units of information into a coordinated system. By a decreaseof randomness of a finite number of units through the evolution of coordinating information, there is a corresponding decrease in the choice of alternatives. This information serves to promote further quantitative and qualitative changes by systematizing a reSponse, inducing the formation of additional unique informational assembfies, duplicating information, coordinating and maintaining a system, and so forth. Therefore, information is seen to be an intrinsic characteristic of a system. It is that which not only indicates its own significance when standing alone but also its relationship to the total systemization of the parts, which is a unit of information itself, as well as determining and restricting possibflities of organization with other available units. As evolutionary time advanced, more units of information were recognized and organized but at the same time the possibility for this organization to be disrupted likewise increased as aresult of the expanded complexity of the system. Therefore, the permutations of potential dis- organizatibn increased as the number of organized units in the system increased. At this point, the much larger view of the total universe must be ll considered. With time, it may be noted that the increased organization of information comes into conflict with the increase of disorganization, or entropy. This is indicated in figure 1. Here the value, AN, is a function of organization whereas AS is a function of negative organization (AN = f(O); AS = -f(0')). Since the necessary condition for any equilibrium is f(0) = f(0') and by the graphic representation such a balance is observed to approach absolute existence as time becomes infinitely large, then it may be concluded that at such points in time the real world approaches homeostatic constancy because organizational variation is no longer an overall variable, thus replicating and recreating primoridal constancy. The progression of such sophistication is readily observed in biological nature and evolution. The amoeba carries on the same basic general functions as man in order to survive as a living organism such as respiration, excretion, reproduction and the like. However, the essential difference between man and the amoeba is that the former, through evolution, has become more complex overall and more specialized in each specific function. These functions individually operate at less relative efficiency and independence than the corresponding simplified functions in the amoeba but give man greater versatility as a result. The entropy, as a function of specific inefficiency, is greater in man than the amoeba but the organizational information wrapped up in man is evidently greater. Thus, maximized simplicity approaches the state of complete non-entropy. Absolute Disposition The entropy indicated here is actually relative entropy since it is that which appears to the neutral observer and is relative only to his point of reference and realm of observation. However, from the con- sideration of the counterforce of information, it may be concluded that 12 zofiumsmomnH Una amonpnm consume QHSwQOHpmHom I H .wfim coameLOQCH mo moapmmaemmmonno: compmmfinmeOIco: mo mpHns moposmnmm opmmwonooo mafia mmonpco HmcoaummanmmmOmHm mEHCDZ B / \ \\ D 13 the actual resultant of these two functions is a third value, absolute disposition (A) which expresses the change in the relationship of infor- mation and relative entrOpy to the total state of the physical world. Thus, it gives a means by which the actual order-disorder dichotomy can be elucidated in its true perspective. For example, as the imbalance between entrapy and information increases, absolute disposition increases. This relationship is indicated in figure 2. Whereas information or entrOpy may be thought of as a measure of the randomness of an isolated event or system of events, absolute disposition is taken as an overall indication of the relationship between information and entrepy of all isolated physical phenomena combined at any given instant. The absolute diSposition, with respect to the X and Y axis coordinates, approximately follows a normal Gaussian probability distribution (7'). In a continuum, the area under such a curve is taken as one square unit. Another way of viewing this concept would be through distribution functions. For example, if 0 is the coordinate of the organization para- meter and N(0) is the number of units in each organization segment, where it is assumed that the total number of units in the entire system is constant at all times, then figure 3 indicates such an arrangement. This would be taken as a representation of the units at a given time classified according to the degree of organization (or disorganization) relative to primordial (t=0) conditions. The point A on the x-axis represents the locus of all units of the system on the organization scale at time to. As time advances and thermodynamic processes take place, some units become relatively more organized because of enhanced information while the remaining units either do not vary or else decrease in organization taking on greater entrepy, such as at t=3. Because of the initial 14 coaufimommfia opsaomnm mo cofimenom I m .wam mmpoEmmmm opmnamnooo camp noapamommfim opsaompm E 15 overpowering effect of entrepy, the center of gravity shifts toward C. However, as higher positive organizational segments become more heavily pepulated with the complexation of information, the trend of the center of gravity of the units reverses back toward A. If this ever reached A, primordial constancy, the exact balance between information and entrepy, would be recreated, although it is here postulated that this could happen only when time would reach infinity. This indicates that enhanced complexation compensates fof earlier trends toward the overall increase of entrepy and Shifts the previous tendency of maximum disorganization into the Opposite direction. Since it is assumed that the primordial constancy is never completely recreated within finite time, the reversed trend is the tendency toward a homeostatic steady state by which greater numbers of units become more complex and more highly organized while other units possessincreased entrepy. Therefore, units are always available for greater or less organization. The expanded information of the system allows it to compensate for earlier accurmlations of disorganization and thus reverse the overall entrepy trend. Of course, if such a shift did not take place, a new constancy function would appear at C when all units would reach that level of organization at one time implying a breakdown of information complexes in the process. Then C could be taken as the new arbitrary zero and the process would start again. On. the other hand, if the shift did take place but did not stOp at A, a new arbitrary zero would be created at B as complexation reached an upper limit for all the units of the system and likewise the process could start again in either direction from B. If such shifts alternated, the center of gravity would vary about A as waves where A would serve as the mean center of gravity of these waves. The maximum of each successive wave would be slightly farther from A than 16 m sosmmm opaque a mo soapsnfinpmam nofiumwaemwno momoaonm I n .mam peosoaoea coupmnaewmno u o peosomoea soapmwdemmmo mom mpfie: uflovz Reap u p + ufl/ I/, 1, 8; 17 the previous corresponding counterpart so that greater degrees of complexation would be attained with each shift into the positive direction. If it is assumed that only one overall shift takes place and it is a composite of several oscillating waves, then figure 3 is upheld and the original hypothesis corroborated. It is important to note that during such a shift, the individual changes of entropy and information are, by themselves, unaltered but their overall effect, as reflected by the absolute disposition curve, is affected. Since at the present time it is found that entropy appears to over- shadow information accumulation in the physical world, it may concluded from this hypothesis that the world, within the realm of empirical observation, is either before or almost in the midst of this shift. Absolute disposition is a measure of the degree to which the center of gravity of distribution deviates from A whereby entropy and information are not in homeostatic balance. The return to A indicates a decrease in absolute disposition and the re-establish- ment of such a balance. If time is to be defined in terms of the trend of entropy, then after such a shift, time would be reversed due to the increasing prominence of information accumulation. Finally, since finite time, by defini- tion, never reaches infinity, the return to the homeostatic steady state of primordial constancy between entropy and information is never quite achieved. The curve note in figure 3 would approach the x—axis and continue to grow broader at its extremities but would never be exactly superimposed upon the axis. Origin of Energy and Mass The law of conservation of matter and energy is a law only as long as no observation is made of some phenomenon which disobeys it. Thus, even if such phenomena do occur but have not been observed either because cf limita- tions of observation or lack of sufficient sampling experience, the law would still be quoted even though it was not actually true. Also l8 an.mfim Sommv page Spas Sopmmm opaeam m ea hpfi>mm0 mo nopeoo anneapsefinpmfin one no pmagm momomone I m .mfim m 0 + 4 I O 0 II III!‘ . I‘ll 11 4‘! ill 1‘ 4‘ l i Ill, . ...?!503 ‘3‘: I}. 4‘. \Tr‘lt w v l9 it is possible that the law is true today but at some previous time it was not, yielding the plausibility of Spontaneous generation of mass and/or energy. In this case, a previously undefined concept of absolute nothingness would have to be postulated. This principle is more easily understood if instead_ of customarily accepting time as being infinitely divisible, it shodd rather be considered quantized. In other words, a point of division of time increments could be reached by which no further segmentation would be possible. The unit at that point would be the fundamental quantum of time. Combinations of these quanta constitute various segments of time passage such as the second. Undoubtedly, the fundamental quantum of time is much smaller than the second. It is here assumed that primordially the fundamental quantum was not the same as it is now and was in fact infinitely largd. Different velocities may be compared in two ways: 1) constant distance with variable time; 2) variable distance with constant time. The first case can be applied to the point at hand. Since c =Ax/At andAx, the distance traversed by a light photon during a quantum of time, is held constant, the only way c can vary would be if and when the quantum of time (At) is not constant because c is a function ofAt. Since it has been postualated already that primordially this at was infinite, by the Einstein expression, energy was then nonexistent: E me2 - m(Ax/At)2 - mxz/oo = 0. For time to pass, this quantum would have had to become finite which would likewise cause energy, and from this, mass, to appear. The primordial state of absolute nothingness, where the time quantum was infinitely large, was not a realm of an absence of something since time patterns were lacking and the realm itself did not even exist in ..v n. 3 which something could function in the first place. by this deficiency, physical laws were also nonexistent. This motionless primordial state gradually took on the dimension of moving time (AtW) which led to the appearance of energy. From the concept of absolute nothingness, it may be noted that energy is as interrelated with time as it is with mass. The apparent stationary character of the physical world today with regard to the constancy of total mass and energy would indicate that the quantum of time is, for all practical purposes, now constant on the Earth. The concept of quantized time could also be used to explain other physical phenomena. For example, it has been observed that light emitted from distant galaxies has a tendency to appear shifted toward the red end of the Spectrum when suitably analyzed (8 ). One explanation of this may be found in the following derivation where a hypothetical finite system is considered: h a Planck's constant Ax1= distance traversed by a light V = light frequency photon during a quantum of time L = light wavelength ,At 3 fundamental quantum of time 1) E = me2 2) E = hV 3) V = c/L 2 4) me 3 hV = hc/L 5) L - h/mc = h/mAxAAt h, an Ax.held constant 6) h/mAx = k 7) L = kat Thus, as At goes from large to small, L goes from I.R. to visible. It has been observed that the red shift increases the greater the distance of the light source is from our Earth (8 ). Therefore, 21 it may be hypothesized that somewhere in the vicinity of our solar system was the location of the first point in evolution where the quantum of time went from infinity to some finite value. By drawing concentric spheres about that point, the spread of this phenomenon may be visualized as outflowing waves. This would account for the observation of the increasing magnitude of the red shift with distance away from our Earth. Consequently, the universe is exapnding in relation to the rate at which the realm of absolute nothingness is oversome by the Spherically Spreading deviation of the quanta of time from infinity. If it is tentatively accepted that initially the real physical world was a giant blob of energy lacking mass, an additional factor must be included at this juncture. Such a possibility has empirical and theoretical foundations. The materialization of energy (9) has been observed in the case of high energy photons bombarded upon a heavy nucleus from which results a positron-negatron pair. In 1900, Planck preposed the concept of quantized energy which Einstein took up in 1905 to eXplain the photo- electric effect (6). This is quantitatively expressed by E = hV where V is the frequency, h is Planck's constant, and E is the energy of the photon. Thus, the photon is considered a quantum of energy which, when bombarded upon a suitable target, induces an expulsion of electrons (9). If the energy of the photon is above 1.02 Mev, the photon disappears within the field of the nucleus and a positron-negatron pair results, conforming to the equation hV = 2mc2 + £1 + E2 where E is the kinetic energy of the particle at the time of production, me2 equals 0.51 Nev or the rest-mass energy of each of the resultant particles, and hV is the energy of the photon. Annihilation of matter is observed when a positron collides with a negatron, yielding two gamma rays each of energy 0.51 Mev. Similarly, it is possible for a positron-negatron collision to result in the production of a neutrino-antineutrino pair which also has no mass (10). 22 The positron may be viewed as an electron of higher positive energy, greater than 2mc2, while bearing positive charge and the same EEEE EEEE as the electron (11). On the other hand, a photon has no charge, Eg,£g§£|gg§§, and possesses energy by virtue of its motion at the Speed of light. The photon is a quantum of energy at any one of a number of frequencies so that to define a gamma ray or an X-ray is merely to designate the frequency of the given group of photons. The production of a positron-negatron pair by photon-photon collision has been preposed (12). Such a process requires sufficient energy. It is also of significance that when the energy of the photons is of the order of billions of elecron volts (Bev), the products of their collision can be a proton and an antiproton, each possessing identifiable mass and charge. As a result of the collision process, both photons disappear. Pair production by photon-photon collision is very difficult to observe and perform eXperimentally because of the high energy (hv1+hv2)2mc2) and the very sensitive equipment required (13). However, positron-negatron pair production becomes appreciable in the interior of stars where radiation density and temperature are both very high. The value kT is 2 at T = 5 x 109C0 and above this the density of approximately equal to me beta particle pairs becomes equal to the density of the light quanta themselves. In photon-photon collision, the production of identifiable mass from energy without the need for an original mass has fundamental theoretical significance especially in the hypothesis now under conSideration. hot only does such a process give a plausible explanation for the materialization of non-matter from primordial energy sources but it also suggests strongly the real significance of the electrical charge dichotomy observable in nature. One of the most outstanding examples is the electron Eopmmm zweoem Hmaomoefinm one we coapmmfiemwmo I m .me Eopmmm mmnoeo ammomofimnm one no somemnaemMLOIeoe u z coapmmaemwAOIcoe mo means u .b B . n"..i| III, l.‘ .D / and proton. The graphic representation of the change of organization of the primordial energy system by photon conversion (figure 5) is given in organizational units at any given time. This indicates that the change of absolute disposition is affected by the change in the number of material units available to be ordered or disordered. As already noted, the number of material units is affected both by the appearance of energy with the change in the time quantum and the conversion of that energy to mass by photon-photon collision. If it is postulated that in the materialization of energy by photon- photon collision an instantaneous unstable intermediate particle N is formed, the relationship of absolute diSposition to information and relative entrepy is clarified. a“ w<:. @* @' Figure 6 - Materialization of Energy By this scheme, primordial energy E, representing two interacting photon quanta, is formed into a Single unit, the intermediate N. This is the ultimate of organization for this particular event. Disorganization ensues when the single particle divides into the beta pair. Information is greatest in the intermediate, but Since mass remains at the end of the event, absolute diSposition is altered by it even though some relative entr0py is noted. The more often such transformations take place in a given unit of time, the greater is the alteration of absolute disposition When considering only this type of event. Thus materialization of energy is an example of the increase of organization or information, whereas the annihilation increases disorganization, or entrepy. Of course, on a much broader, the systemization of the resultant 25 beta particles also affects the total absolute disposition, whether that be annihilation or further material construction. Haterialization of energy puts a greater number of units into the system to be affected by the interactions taking place. As long as materialization overrides annihilation, the flux of this factor is significant to the alteration of absolute disposition. It could be concluded from this theory that primordial energy lacked any semblance of order and organization. However, even in the greatest possible degree of disorder there is some measure of organization. This is partly based on the inherent information of each of the components of the system as well as the relationship of one seemingly unordered unit to another with regard to such interacting factors as energy, position, relative velocity, apparent momentum, and so forth. From this consideration, it may be noted that the primordial energy system possessed a measure of organization with regard to the state of its constituents and that as materialization progressed, with its resultant charge dichotomy and attraction-repulsion phenomena from particle interactions, this degree of organizatinn changed and, in turn, information fluxuated. When this aSpect is included on the original order-disorder graph (figure 1), a third dimension on the z-axis is introduced which directly affects the total change of absolute disposition with time by defining it over a surface of possible variations of organization at any instant. Ultimately, once the balance between materialization-dichotomdzation, annihilation, entrepy and information complexation is achieved, an overall steady state is established (figure 7). It is noted that as time approaches infinity, the absolute diSposition, or total interplay of order-disorder, reaches a homeostatic balance and all subsequent finite changes would alter the entire system in only infinitesimally small degrees. Thus, 27 absolute disposition is the true measure of the flux of the order-disorder dichotomy when considered on an overall universal scale and not relative to an observer's limited realm of perception. Absolute disposition, within the limits of finite time, never reaches 0 since the function approaches zero asymptotically. The instant at which absolute disposition reaches a maximum is likewise theoretically determinable and is the point in the time of the entropy shift noted earlier. The Electron in Protobiochemical Evolution Electricity is recognized as the flow ofelectrons in which negative and positive regions are defined by an abundance of or deficiency in electrons respectively (14). Here the electron serves not only as a unit of energy-mass but also as a transport medium for energy from one site to another. As noted earlier, the electron may be postulated to be the primary and most primitive mass particle in the physical world resulting from positron—negatron pair pro- duction by photon-photon collision. Thus, any subsequent transformations and fabrications which occurred depended on the electron both as a site of available mass and as an energy source. As the abundance of negative and positive particles increased in physical evolution, the importance of photons as reaction stimulators decreased and the subsequent importance of the electron as a reaction inducer became more significant and apparent. One of the most outstanding remnants by which the photon acts as a reaction inducer is in photosynthesis. The basis for chemical reactions is the activity of planetary electrons and spectroscopy depends on the phenomenon of electrons absorbing and emitting quanta of energy (6). Thus, the electron appears to serve as an energetic stimulator to induce and promote reactions whose course is predetermined by inherent patterns for organization, much the same as in the number example given earlier where the individual jigsaw pieces carried the inherent information by which the ultimate sequence of their interaction would be ordered. Assuming that the course of biochemical evolution reflects the inherent patterns within the constituents, it may be hypothesized that if basic structural units could be placed under the influence of electrical stimulus, the course of biochemical develOpment could be, at least in part, reproduced. Thus, primitive atoms and molecules seem to have had a wide range of possible forms to assumeupon complexing and evolving, but, much the same as in Darwinian-type survival of the fittest, only those which found use in the further develoPment of biochemically significant structures continued to appear repeatedly in the course of chemical sophistication and evolution. The degree to which a given atom or molecule served as a useful site for further evolution was determined by its natural tendencies as controlled by deve10ped intrinsic informational units as well. Thas, the biologically significant compounds and structures which appear today are those which evolution has found by experience to be the most efficient and most useful from the entire repertoire of possible combinations. The abundance of energy in an electrical environment permits reactions which would otherwise be prohibited without such conditions, and thus allows the reacting units to exhibit their inherent informational tendencies. Likewise, such an experiment would give some indication as to the significance of fundamental molecular units and structures to larger systems such as macromolecules, as well as shedding light on the plausibility of certain energy sources having been primordial reaction inducers. In any consideration of the origin of biological life, the means for the synthesis of biochemically significant structures must first be established. Therefore, if the postulation is tentatively accepted that electrons are among the principal stimulators for the fabrication of 29 fundamental structures from basic atomic and molecular units, then the projection of this concept into the realm of molecular biology provides a substantial foundation upon which to explain biochemical evolution. Thus, it is the essential purpose of this series of experiments to provide support to this specific hypothesis as well as to the general theory already prOposed. III. HISTORICAL REVIEW Even though the intended purpose of the experimental portion of this study is to deal with specific reactions within a highly Specialized realm of the physical sciences, the more important goal here proposed is to introduce some coordinating concepts from a broad point of view and thence provide Specific laboratory procedures which tend to substantiate the theory advanced. Paul Wiss, of the Rockefeller Institute, in his article, "Cellular Dynamics," has pointed out the need to attack big problems (15): "... we frequently try to fit our questions to the very limited answers which our fragmentary knowledge has been able to provide, instead of boldly facing the much broader questions posed by living systems and phrasing them in such a way that still more penetrating answers may be found in the future." Therefore, sinceathe theory put forth in this thesis concerns itself with a comprehensive tOpic of this nature, it is necessary to seek support and impetus not only from current efforts in the laboratory but also from the work of previous researchers whose findings have significant bearing upon the subject now under consideration. In this sense, the job of the researcher is to observe the trends in the present and from these, extrapolate backward and forward in order to legically theorize on various asPects of the physical world, its origin, dynamics, and destiny. The enigma of the origin of life and the materials necessary to sustain life has been pondered for many years. Some rational suggestions were advanced as early as the days of the ancient Greeks. For example, Democritus thought that life began in the water as the result of an inherent movement of the atoms (16). The question of Spontaneous generation engendered eSpecially high fervor during the debates between 31 Spallanzani and Needham in the 1760's (17). In 1931, Holiday and Nuttingham demonstrated that methane (believed to be one of the components in the primordial atmosphere), when heated to lOOOOC, is converted to acetylene (17). 2014 - G-iECH + 31-12 Tchitchibabin described how acetlene, in the presence of water, produces oxidized hydroxy derivatives, specifically acetaldehyde (18). CZECH + H20 = CH3CHO Oparin suggested that such reactions could also head to other oxidation products of hydrocarbons, such as alcohols, aldehydes, ketones and organic acids (17). He also noted that such oxidized derivatives can enter into a variety of chemical reactions with reduced forms of nitrogen and give rise to amines and other nitrogen-containing compounds. The Cannizaro type of reaction gives a Specific means by which aldehydes can react to form alcohols and organic acids (19). An oxidation- reduction reaction between two acetaldehyde molecules plus water can yield ethyl alcohol and acetic acid, according to Oparin (17). Acetaldehyde and formaldehyde can also react under the Cannizaro type of reaction to produce the polyhydroxy compound, pentaerythritol (20). Sabatier reported that aldehydes have a great tendency towards polymerization (17). Thus, it is reasonable to postulate that primordial conditions could have provided the necessary conditions and materials for the production of various types of alcohols and organic acids specifically. A number of chemically induced techniques have been found for the artificial 13,2lE52 synthesis of sugars. Kiliani discovered that it is possible to synthesize a sugar-life material by his cynamohydrin method, in which cyanide intermediates provide additional carbons to build up the size of the molecule (21). Similarly, under the influence 9—, 32 of a weak base, formaldehyde produces formose, a mixture of hexoses (22). Fischer found that a several step oxidation-reduction manipulation of glycerol resulted in the synthesis of hexose sugars which involves aldol condensations between carbonyl groups and the hydrogen atoms adjacent to the carbonyl groups(23). Acrolein, resulting from the oxidation of glycerol, also produces hexoses in alkaline solution due to the formation of glycerose, a mixture of glyceraldehyde and dihydroxyacetone, with subsequent condensation of these products to acrose. In 1902, Fischer and Leuche achieved the synthesis of glucosamine from arabinose (24). Peat found that by rupturing the Vic-oxide group of an anhydro sugar in the presence of ammonia, both a 2-amino sugar derivative and a 3-amino sugar derivative result (20). It has been determined that as part of the ig‘yixg photosynthetic carbon cycle, glyceraldehyde and dihydroxyacetone serve as significant intermediates within an enzyme regulated system. The production of hexoses is here induced and controlled by the input of energy, in this case, ligthS) . The original form of nitrOgen needed to produce amino groups, such as in amino acids, as well as the means by which such substances were synthesized has been a topic of investigation. Urey noted that molecular nitrogen could very well have been present in the primordial atmosphere (26). He also suggested that absorptionrf ultraviolet light in the upper atmosphere provided the free energy by which critical syntheses were induced through free radical reactions. S.L. Miller, a student of Urey, published; the results of an experiment in which he synthesized amino acids by passing a gaseous mixture of hydrOgen, methane, ammonia, and water vapor through an electric Spark (27). At the end of a week, glycine and alanine were identified in his solution 33 by chromatography. From these data he preposed that ammonia was the fundamental primordial source of nitrogen. Similar results have been observed when such a gaseous mixture was exposed to ultraviolet light QB) . Otozai and co-workers were able to polymerize glycine under the influence of a Tesla coil discharge (29). Hughes and Ingold synthesized alanine from lactic acid by means of bromination and azide formation (20). The product was unnatural since it turned out to be of the D-form. Natural L-alanine was prepared chemically and resolved by Wolfram using suitable cleavages of glucosamine with lead tetraacetateCZO). Fieser and Fieser have reviewed other chemical methods for the lgflylggg synthesis of sugars and amino acids (20). The Gabriel synthesis, utilizing potassium phthalimide and suitable halo esters, has been employed in the production of amino acids. Amination of aldehydes by means of cyanide addition to yield amino acids has been shown in the Strecker synthesis. For example, acetaldehyde would give alanine by this process. Suitable treatment of malonic ester, such as with phthalimide and nitrous acid, was shown to result in amino acids. Aldehyde condensations of apprOpriate aromatic reagents was used to synthesize amino acids with aromatic groups by making use of the activated methylene groups. The possible primordial sources of energy to bring about essential reactions has also been investigated quite extensively. While a number of experiments have been carried out on the effect of both ultraviolet light and electrical dishcarge, few have had significant bearing on the problem of the origin of life and the substances necessary for the generation of life. However, some workers such as Miller have shown that in the gaseous phase, cyanides and aldehydes play an important role as intermediate 3‘ products of these reactions. There appears to be little difference in the products of chemical transformation by various forms of high-energy bombardment (30). For example, it was observed that high-energy alpha particles, gamma rays, and ultraviolet light can all produce a variety of forms, such as formaldehyde and formic acid, from the same initial materials. In 1962, Calvin reported that he had submitted a gaseous mixture of methane, ammonia, hydrogen and water to alinear beta accelerator where C14 methane was employed. He found that the products of this reaction,when analyzed as an autoradiographic paper chromatogram, matched up favorably in certain cases with the product from the reaction of formaldehyde in base (31). This latter material is known to be a source of formose. In 1958, Hasselstron and co-workers irradiated aqueous solutions of ammonium acetate with the beta accelerator whereby glycine and some other amino acids were produced (32). Under the influence of ultraviolet rays of less than 2000 X, methanol and ethanol were found to yield formaldehyde and acetaldehyde reSpectively with a quantum yield between one and two(33), while iSOprOpyl alcohol gave acetone (34). In the presence of ultraviolet light and oxygen, Cartieni found that ethylene glycol was oxidized to a peroxide(35). Henri and Kane, in 1912, observed that irradiated glycerol gave a positive phloroglucinol reaction for glycerol aldehyde (36). Reactions induced by X-ray bombardment bear much resemblance to those produced by a stream of high velocity electrons (37). Fricke noted that a peculiarity of X-ray action is that it frequently involves the production of molecules with unusually high energy states, such as water molecules with at least 91 kilocalories per gram molecule (38). 35 Ellis and Wells noted that electromagnetic radiation of apprOpriate wavelength can be used to bring about chemical reactions involving free radicals (37). Such mechanisms are often initiated by the homolytic Splitting of a bond to yield free radicals which then proceed to various other reactions utilizing these reactive units (39). An example of this would be the photolysis of acetic acid where hydrogen free radicals play an important part in the essential production of ethane, water and carbon dioxide finally. Chain prOpagation is also possible in this general type of reaction (37). Glen and Hansen, in 1962, found that ionizing gamma radiation causes b-lactoglobulin to unfold. Since a free radical reaction ensued, the unfolded species tended to dimerize. Similar effects were observed under the influence of electrons from a linear accelerator (40). Dhar and Mukherjee, in 1934, claimed that exposure to sunlight or a mercury-vapor lamp of 0.5N solutions of ammonium, potassium or sodium nitrate containing glucose, tartaric acid, glycerol, arabinose, fructose, mannose or galactose gave positive colorimetric tests for amino acids where the best yields were with ammonium nitrateCél). This is similar in nature to the work carried out by Hasselstrom and co-workers already noted. Dhar and Mukherjee also found that the use of ammonium hydroxide as the nitrogen source proved unsuccessful. In 1866, Berthelot examined the behavior of alcohols, aldehydes, and organic acids in the presence of nitrogen under the influence of silent electrical discharge. 1e found that small amounts of nitrOgen became fixed to these substances in the processs(42). Also, nitrogen- containing molecules were found to fix more nitrogen in the effluve. In 1875, Cavendish discovered that an electric spark passed through a mixture of oxygen and nitrogen induced the formation of 36 nitric acid(53). Birkeland and Eyde deve10ped a high-tension discharge are furnace which formed nitrogen oxide from air containing nitrOgen and oxygen at 3000°C with yields of about 1%. When cooled, the nitric oxide combined with oxygen giving nitrogen dioxide and tetroxide. The dioxide, when dissolVed in water, formed nitric acid and, when brought into contact with slaked lime, yielded calcium nitrate. Lewis and Randall noted that thermodynamics has been reSponsible for the introduction of many significant concepts among which entropy is of noteworthy importance. The primciple resulted from the pioneering work of Carnot, Kelvin, and Clasius and ultimately found its place in physical science as the second law of thermodynamics (54). As already noted, this concept relates to the randomness of a system, although a precise interpretation of entrepy cannot be given easily. Gibbs, as well as Helmholtz and Maxwell freely employed this concept in their contributory works. Lewis and Randall commented on the deve10pment and significance of the first law of thermodynamics: "The first law of thermodynamics, or the law of conservation of energy, was universally accepted almost as soon as it was stated, not because the experimental evidence in its favor was at that time overwhelming, but rather because it appeared reasonable and in accord with human intuition. The concept of the permanence of things is one which is possessed by all." (The underlining in the previous quotation is that of the author of this thesis. The reader is here referred back to the section on theoretical considerations dealing with human observation in relation to absolute and relative concepts.) From these considerations, a very decisive point can be drawn: The origin of life and the primordial synthesis of biochemically significant compounds have been tepics of considerable scientific concern. 37 However, no set of eXperiments has thusfar decisively established the answer to this enigma, or has strictly limited the realm of speculation in this problem. Therefore, as a greater number of possible solutions are presented, the true eXplanation will eventually become clearer. The function of critical Speculation, based on sound scientific reasoning and empirical justification, is to present plausible perSpectives by which progress may be stimulated and the knowledge of man will ultimately elucidate the solutions to his deepest problems. IV . MERIMENTAL HI‘“ . 'I ' :T‘, ': 9. H" " :" ‘.~...; 011.1. ...—3...... .- ' - . ...”-..— 38 EXPERIMENTAL OBJECTIVES This series of eXperiments has been undertaken to investigate many aSpects of the topics under consideration: 1) To determine the feasibility of using high voltage electrical sparking to bring about the synthesis of biochemically significant compounds and functional groups in the aqueous phase such as: a) Primary amino groups from atmospheric nitrogen (and not ammonia) b) Amino acid-like molecules from organic acids c) Reducing aldehydic groups from hydroxyl groups d) Sugar-like molecules from mono- and poly-hydroxyl alcohols e) Amino sugarblike molecules f) High molecular weight polymers. 2) To investigate the effect of electrical Sparking on known molecules and polymers. 3) To present a possible means by which the compounds essential to the initiation of biological life under primordial conditions may have originated. 4) To lend initial support to an encompassing theory concerning basic concepts of the dynamics of the physical world as a plausible explanation for its origin, course, and destiny (see Theoretical Considerations). 5) To suggest a course of attack in solving associated problems in biOphysical and molecular research. 6) To gain experience and insight with analytical procedures which are important in organic, biochemical, and biOphysical research. :39 WW For expediency and clarity in explanation, the experimental procedures and data are presented tOgether in this section. Jhen analytical tests were run with a Specific purpose (e.g., to identify carbohydrates) and the test was positive, it was intended to show only that carbohydrate- like substances were present in the tested material. However, no single test can be taken as conclusive or absolute. References to "T-number” (e.g., T-l) indicate the analytical techniques and reagents described in detail in the Appendix. A. Apparatus The primary investigation problem, as noted in the objectives, was to examine the various aSpects of the use of electrical Sparking to initiate the synthesis of biochemically significant compounds. It is important here to distinguish between the linear beta accelerator, silent electrical discharge, which is found in vacuum tubes, and electrical Sparking, where a visible Spark jumps a gap between two electrodes as employed in this series of experiments. For this, closed setups were designed to approach this problem. The schematic drawing of the electrical system is shown in Fig. 8 . electrodes PP 7 Figp 8 - Schematic drawing of electrical sparking system. In this way, a common electrical source of 110 volts was utilized and suitably converted to supply a voltage high enough to are a gap of about 1 centimeter. Since all syntheses were carried out in the aqueous (7, f. . . I .I ‘- w Figure # 9 - Reaction Chamber 1: 2: 3: 4: 5: Connection for upper electrode Ventilation connections Upper electrode . Experimental solution level Connection for lower electrode 41 , Upper Electrode PDQ/Ventilation Connection +1 1 44.4.1qu Surface Lower Electrode / .L A , P1910 . Schematic drain; of reaction she-her Figure 1; 11- Experimental Setup 1: 2: 3: 4: 5: Reaction chamber Magnetic stirrer Power supply Induction coil Ventilation 43 phase, one of the electrodes was submerged within the solution and the other was suspended above. Thus, the entire solution served as one of the poles and a continuous electrical sparking current passed from the upper electrode directly to the surface of the test solution. Consequently, the molecules in the path of the current were directly bombarded with electrons. The test solution and electrodes were contained within the closed glass vessel and Open only to ventilation ports at the tOp of the vessel bringing in and releasing acid- and thymol-filtered air under pressure. This arrangement removed undesired gases resulting from the Sparking process. Filtration of the air before entry insured its microbiological purity. To accommodate large amounts of test reagents, vessels of l-liter and 2-liter capacity were used, whereas for smaller samples, a vessel of 10- to 12-ml. capacity was employed (see Fig.9,10 and Fig.11 ). B. Chemicals and Reagents The following list includes the experimental reagents of at least C.P. grade employed in this work. The chemicals starred were tested for purity before use in experiment no. 5, to be described. For a listing of analytical reagents and equipment, refer to the Appendix. Experimental equipment not mentioned in the Appendix will be cited in the appr0priate experiments. Methanol, absolute * (Central Scientific Company) Ethanol, 95%* (Commercial Solvents Corporation; C.P.) Ethylene glycol* (Fisher Scientific Company; Fisher Certified Reagent) n-PrOpanol* (Baker and Addison; C.P.) iso-Pr0pan01* (Baker and Addison) 1,2-Pr0panediol* (Eastman Organic Chemicals) 1,3-Pr0panediol* (Eastman Organic Chemicals) Glycerol* (Central Scientific Company; USP) 44 Acetic acid, glacial (Mallinchrodt Chemical Works; Analytical Reagent) Bovine blood serum, freshly drawn and citrated n-Butanol (Fisher Scientific Company; Fisher Certified Reagent) n-Amyl alcohol (S.T. Baker Chemical Company; Baker Analyzed Reagent) Ribose (Nutritional Biochemicals Corporation) Glucosamine-HCl (General Biochemicals) Ammonium sulfamate (Lafiotte Chemical Products Company) Glycine (Nutritional Biochemicals Corporation) Valine (Nutritional Biochemicals Corporation) Alanine (Nutritional Biochemicals Corporation) Histidine (Nutritional Biochemicals Corporation) Cysteine (Nutritional Biochemicals Corporation) Phenylalanine (Nutritional Biochemicals Corporation) Tyrosine (Nutritional Biochemicals Corporation) Lysine (Nutritional Biochemicals Corporation) Arginine (Nutritional Biochemicals Corporation) Nitric acid (Allied Chemical Company; C.P.) Phenylacetic acid (Eastman Organic Chemicals) C. Procedures and Data The experiments and data to be presented after experiment no.1 are arranged into three major groups of compounds produced by sparking techniques. 1) Sugars and amino sugars (experiments 2 to 10) ii) Amino acids (experiments 11 to 12) iii) Polymers (experiments 13a to 13c) Each experiment is introduced with a statement of its purpose, followed by the procedure, and concluded with the resulting data usually in tabulated fo n. The analysis and significance of these data is presented under the Discussion section. Hherever apprOpriate, certain experiments (such as 5 and 14) were included to rule out nonSpecific effects as well as to provide evidence for Specific points put forth in the theory and the discussion (such as in eXperiment no.15). Several measures were employed to minimize microbial contamination including use of high purity reagents as well as chemical and/or autoclave 45 sterilization of glassware. Standard bacteriological assays (see experiment no. 14) verified the effectiveness of these precautionary me th OdS o EXperiment 1 To carry out studies on the effect of electrical Sparking on solutions of known b1010gica1 substances or compounds, the following procedures were performed: a) Several known amino acids including glycine, alanine, histidine, cysteine, phenylalanine, tyrosine, lysine, and arginine were mixed together into aqueous solution so that the final concentration of each was 1% (w/v) and with no Sparking were tested by ultraviolet Spectro- phtometry in the Beckman Model DK-Z instrument by scanning between 220 mu and 340 mu (T-32). b) Amino acids such as in a) were mixed together into a solvent composed of glycerol:95% thalOI (50:50) instead of water and tested without sparking by ultraviolet SpectrOphotometry. c) A mixture like that in b) was prepared and heated at 1100C for 24 hours, not Sparked, and then submitted to ultraviolet SpectrOphotometry. d) A mixture of amino acids as in b) was Sparked for 18 hours and subsequently tested by ultraviolet spectrOphotometry. e) To 2 ml. of ethanol (95%) and 1 ml. of glycerol was added 7 m1. of a 1% (w/v) aqueous solution of alanine which in turn was examined as follows: (1) About 1/2 of this solution as a control was measured directly by ultraviolet spectrOphotometry between 220 mu and 340 mu. (2) The remaining portion of the solution was sparked for 2 hours and scanned similarily by ultraviolet SpectrOphotometry. 46 f) To 2 ml. of ethanol (95%) and 1 m1. of glycerol was added 7 ml. of a 1% (w/v) aqueous solution of phenylalanine and divided in the following manner: (1) About 1/3 of this solution as a control was directly scanned by ultraviolet SpectrOphotometry between 220 mu and 340 mu. (2) About 1/3 of this solution was Sparked for 2 hours and subjected to ultraviolet SpectrOphotometry. (3) The remaining portion of the solution was added to 2 ml. of hydrOgen peroxide (5%) and tested by ultraviolet Spectr0photometry. o) A sample of freshly drawn citrated bovine blood serum was divided into two portions: (1) The first portion served as a control and was analyzed for its protein fractions in a Perkin-Elmer Hodel 38 Tiselius electrOphoresis apparatus (T-26) at pH 8.6 and ionic strength of 0.01 in veronal buffer. (2) The second portion was sparked for 24 hours and submitted to Tiselius electr0phoresis under the same conditions. A summary of the results of the various parts of experiment 1 is;presented in table 1 0 meEl A COHPARISCN OF THE ETFECTS UV SPARK hG AN' OTHER PHYSICAL TRfiATkwN 5 ON SELECTED BIO-ORGANIC CCHPUUUDS Ah" SUBSTANCE‘ ‘ (table on next page) 47 A CCHPARISUK OF THE EFFECTS OF SPARKIEG AKD OIHER.PHYSICAL TflfliTMEKTS CR SELECTED BIO-ORGAEIC C‘MPOUHDS AHD SUBSTzYCES T —J Test Substance Treatment Analysis Change from Control a a.a.* none UV scan control b a.a. g.-e.** UV scan none c a.a. 1100C - 24 hr. UV scan none d a.a. Spark - 18 hr. UV scan yes e-l alanine none UV scan control e-2 alanine Spark - 2 hr. UV scan none f-l phenylalanine none UV scan control f-2 phenylalanine spark - 2 hr. UV scan yes f-3 phenylalanine H202 - 5% UV scan yes*** g-l serum none Tiselius several peaks 3-2 serum spark - 24 hr. Eiselius one peak * The solution was a mixture of a number of selected amino acids including glycine, alanine, histidine, cysteine, phenylalanine, tyrosine, lysine, and arginine each in 1% (w/v) concentration. The solvent was a 1:1 solution of glycerol and 95% ethanol instead of water. ***This differed from f-2 as well as from the control. I I ‘o.“o€ SUCARS AKD AHINO SUGARS Experiment 2 This experiment was undertaken to test for the production of reducing groups from various monhydroxy alcohols as well as olycerol by Sparking as well as changes in solubility as a result of this process. Aqueous solutions of each selected alcohol were arced for 30 minutes and then SpectrOphotometrically analyzed for reducing groups by the ferricyanide test (T-l). The results to this test are found in table 2 . r19, 1" 2‘1 .1.L)l..15 19"” ‘-r\ \? fly? ”1.7-. ~1.!v-mr ‘1 ~-‘/‘ I M" v vfi: 5 :\ ‘~_ 73 ‘1“";' r 7 (P "V i TTTTT in.“ .il'lurLui. v1.3 Lia: LJJU'LJCIIAIJ ‘J‘bVLJ‘. J a I.) L) )Ja-J-b)!“‘) BY -1. :3 LL‘JL :~I:-.LL IL ~71 "57‘ d‘~-1‘ m \w-r—w‘v 1 -v T'T sa “.T-r v-qty C v «~1- half ....)1. ALAH'J 1.4.111 Jul) In .‘..LJ..ILJ.LLI.L.L 110.414.3312 "¢'\ " A/ 71%- '§ .\'\"' Vf‘q‘f)'-.~.“-« .- \) 4‘fi'u‘\I§\U VilssLb K); 4L‘JV-~\a J Sample ‘Concentration Ferricyanide hiscibility (v/v) Absorbancy before _a££g£ blank - 0.03 nethanol 02 0.17 total total ethanol 1 0.24 total total n-prOpanol 102 0.32 total total n-butanol 103 0.20 partial total n-amyl alcohol 102 0.30 partial total glycerol* 10; inf. total total 1 * Glycerol was Sparkei for 60 minutes. .‘ Experiment 3 The purpose of this e1periment was to observe the rate of production of reducing groups from glycerol by Sparking and to note the effects of ammonia on this synthesis. A 10% (v/v) aqueous solution of glycerol was Sparked for 2 hours. Also, an aqueous solution of glycerol plus 2.5% aurionia was Sparlced for 2 hours. Samples were removed from both solutions after 0, 1, 5, 15, 30, 60, 90, and 120 minutes of Sparking. All samples were analyzed by paper chromat0graphy using ethyl acetate:pyridine:wa er (60:25:20) as solvent and benzidine to deve10p the reducing sugars (T-10). The ciromatogram from this experiment where only the 101 aqueous glycerol was Sparked .f' . 12. A.sample of the product from the Sparking or glycerol O is seen in Fig with ammonia gave the same kind of Cl. romatoSram as with glycerol alone. The samples from the Sp Mr :ing of 10% aqueous glycerol were further checked with Fehling's solution (T-B). These were made 49 $01.va BOUNDARY e I U . : "O5e e e e m e L 0 e o 1 1 60 90 120 MINUTES F 5 5 50 (SPARRING) Fig.12 - Sketch of a chromatogram on whatnot: #1 paper using ethyl acetatezpyridinesuater (60:25:20) solvent showing the increase of benzidine-positive material produced from glycerol with time of sparking. semi-quantitative by reading their absorbancy at 570 mu in the Spectro- photometer before the iron oxide precipitated completely out of the solution. The results are summarized in table 3 . PRDDUCTIUK or REDUCING SUBSTAKCES “Y AS NOTED dY SPECTROPHOTOMETRIC FEHLINGJ $83? PRODUCT TEE SPARRING 0F GLYCEROL READINGS OF THE fi_ ———_ WW fiamplg Sparking time (min.) Absorbancy» Approximate percent* 1 water blank 0.000 O-OOO 2 o 0.036 0.054 3 1 0.063 0.095 a 5 0.129 0.194 5 15 0.208 0.312 6 30 0.347 0.522 7 60 0.553 0.833 8 90 0.600 0.902 9 120 0.985 1.480 10 0.2% glucose 0.133 0.200 * The percentages are approximate since the readings are only semi- quantitative. The percentages are calculated in comparison to 0.2% (w/v) glucose. Sample calculation: 0.200/x = 0.133/0.600 x = 0.902 Experiment 4 The purpose of this experiment was to indicate the production of carbohydrate-like compounds from the sparking of glycerol. A 10% (v/v) aqueous solution of glycerol was arced for 2 hours and checked for sugars by the o-aminodiphenyl test (T-4), change in Optical rotation from the initial solution (T-6), and solubility of the product in ether (3-12). Also, solutions of known glucose concentration were included for comparison with the products from sparked glycerol. The possibility that the product might be ascorbic acid was 51 ruled out by a negative response to the dichlorOphenol-indOphenol test (T-8). TAJLE 4 TESTS ”OR THE PRODUCTION OF SUGARS FR“H THE SPARKIHG 0F GLYCERO '1 AS C01-lPA.lED l'JI'l‘ri 010C0513 Sample Concentration Sparking Dilution Absorbancy Ether Opt. time (after) (o-Aminodiphenyl) sol. rot. glucose 25 mg% 0 0 0.655* - + glucose 50 mg% 0 O 1.360 - + glucose 100 mg% 0 0 inf. - + glycerol 10% (v/v) 0 1:100 0.000 - none glycerol 10% (v/v) 120 min. 1:100 0.623* - none * Calculation (dilution factor = 100): 655/623 3 25/X x a 23.78 ng 23.78 X 100 = 2.38 ng Experiment 5 From the preliminary results on the alcohols in experiment 2, it became desirable to carry out a more systematic study of the effect of the spark on various mono-, di-, and tri-hydroxy alcohols. Selected 10% (v/v) aqueous alcohol solutions were each Sparked for 2 hours and checked by various tests for organic functional groups. Also, the purity of the reagents before use was ascertained. The results are found summarized from these tests in table 5 . Experiment 6 To test for the production of amino sugars from glycerol by Sparking with selected co-reagents and the effects of the arcing process on hydrogen ion concentration (pH), the following aqueous solutions in 10 m1. total volume were each sparked for 2 hours: a) glycerol (1 ml.) plus ammonium sulfamate (500 mgm.) b) glycerol (1 m1.) plus 95% ethanol (1 m1.) TABLE 5 Tests for the Appearance of Functional Groups in Sparked Alcohol Solutions from Experiment 5 -__ ~__-; -=_'__-__-—_‘-‘ Before Spark r4 Tests Alcohols .3 .4 Q? '3 .3 0 -r". H m 'H 'U g3 * '(3 (D 0.) a? x ’4 9 r+ ri : :1 gt a 8 '3 (L5 9 O 84 “3 8 OJ :0 c .2 d 5 <3 8‘ a. E; ii 8 ES 8* 8‘ d: c: ' £13 15 I 3-. x. I I (Y: 56 r7 83 a: 0; n; cu «\ a, .3 I O O O r4 r4 r1 r4 a: .4 r4 r4 3 Fehling (T—B) - - - - - - - - Elson-Morgan (T-15) - - - - + - - - 2,4-DNPH*(T—9b) — - _ - - - - _ Ninhydrin (T-13a) - - - - _ - _ _ After Spark helisch (T-Sa) + + + + + + + + Ninhydrin T-13a) + + + + + + + + Benzidine (T-lOa) + + + + + + + + Fehling (T-3) + + +++. +f +f +++. +++. +++. Osazone (T-9a) +0 +0 +++o. +0 +0 +++Y.+++0.-H+D. Elson-Morgan (T-15) - - HI - - +++l + +++I 2,4-DNPH*(T-9b) I . reaction +.Y +.Y .R +.R +.0 +.R +.Y +.D melting point 160 151 T 141 110 P245 7245 7245 Benzidine-IOu(T-10d) - - +0 - - +0 +d +d *KEY yellow precipitate double Spot immediate reaction positive reaction negative reaction gze:iter than green f = faint reaction . = precipitation without heating 0 = yellow-orange precipitate R = orange precipitate D = deep orange precipitate G a greenish yellow precipitate 2,4—0 EIPH = 2,4-dinitr0phenylhydrazone T = 155-v245 s3\ll 4-F40JH4 (D 2 53 Before and after sparking, pH readings were taken and the solutions were .2 analyzed by the Elson-Horgan test for amino sugars (T-lS). The results 0 were compared with data obtained from known samples, as noted in table 8 . EXDeriment 7 A-——A To test for the production of primary amines, a 10% (v/v) aqueous solution of glycerol was sparked for 2 hours and tested by the nitrous acid procedure (T-14). TABL E 6 RESULTS OF THE NITROUS ACID TEST FOR PRIMARY AMINES Sample Observation glyC1ne + Sparked glycerol + unsparked glycerol water - ) Experiment 8 In order to support the assumption that a portion of the Sparked glycerol products was a pentose, a 10% (v/v) aqueous solution arced for 2 hours was analyzed by the Molisch (T—Sa), Bial (T-Sb), and Tauber (T-Sc) methods. The findines are reported in table 7 . U F‘- -n'- .5? 7 1:3...)14 1.. ‘(Vi 7 r1 urn“ 17‘ ~yy-1‘R-lfl [I‘m :x--‘« fir“"‘ ,n-jv? ~2 .~:|‘--v.~ ‘ ”1*: ,- 11 _e ~‘7'I’.rfi‘f~ -~-‘\ - v: 2 1..“ \5‘1f‘rTV‘ I ‘-"r‘v:-3!a_ *{L.XLIJ.£\L.L)ILL I..UL'u4‘¢J.IL/.\:J L'Lt‘. L...“ l-sJ.JJu'.‘.bl.a UL‘ A Eu.‘.LK./Jz.a I.“ vI13-LL'..aJ GLLVuALa Test Sample Result —-——‘.——_ , -..—......- l) Holisch Sparked glycerol + ribose (1% solution) + 2) Bial Sparked glycerol + (for pentose) ribose (1% solution) + (for pentose) 3) Tauber Sparked glycerol + (for pentose) ribose (1% solution) + (for pentose) 54 Wms mm H .oamo Wms an 0 .ocoo "myopoam soapsanp spas Ms\ws mm H x HE\ms an n x ooa\x u mud.0\mmm.o om\x u NQN.o\mmm.o "mmnanmmm osflammoonam Hs\md OOH new Hs\mw on so momma mmeNMHdm Esfleosse spas Honmomaw Umxmmmm pom cowpwHSOHmo one ma msazoaaom one ** xemfip nape: new meapmmm I + amN.o ems OHHH .xnmam .h: N A>\>vmm\ Asxsv moa + mam.o .emp OHHH spasm.sn N n>\>vmm. AI\>V eon ++ .eaa amt came m.N anaam.na N A>\>vmm\ As\>v aoa I sodas» N.m xnmnm on A>\>me. Hosanna A>\>V nod Honmohao + .ammm.o amp OHHH .xhaam.nn N A>\svmm A>\>v floa + mad.o . new oaua xmmmm.hs N A>\3VWm A>\>V we ++ .mmfi pom moon m.N xmemunx N A>\3vmn _ N A>\>v on I soaflmm N.m xpmom on K>\3vmm Nmz omamz A>\>v wed Howmomaw + oqm.o was xnwnm on n\ms omH + nu:.o may xnmnm on Hs\md 00H + mwN.o pom thmm on asxmd on + mmH.o i may xnmmm on Hs\ms mN meusmmoodao I MNo.o nmmfio . xnmnm on xsmap poems cowmsaocoo ”madnhoen4 noaoo “magma“ mm whoapwmmoo .oaoo osmz .ocouc mama paws ammuochomHm soapsafio pcoManIoo mammmm JI- IIHII‘ “Ill 1; w psmeaummxw CH mucomwonIoo new Honoohao mo mmamswm Umxnmmm so meowpmcwsnmpom :wmpochomHm one mm mo mpasmmm mnmqmdfi 55 Lrpcriment 9 This experiment was performed for the purpose of testing the effect of Sparking on a known pentose sugar solution without glycerol in relation to the sugars synthesized from glycerol. A 10% (w/v) aqueous solution of ribose was sparked for 2 hours after which paper chromatOgraphy of the ribose product and that also synthesized from glycerol by sparking was carried out using ethyl acetate:pyridine:water (60:25:20) as solvent and ninhydrin as the spot(s) indicator (T-lOc). Several ninhydrin-positive Spots appeared from the Sparked ribose, two of which matched the sugars synthesized from sparked glycerol. The results are depicted in Fig.13 . Experiment 10 The purpose of this experiment was to effect separation of the sugar products of sparked alcohols by means of column chromatography. Separate 10 ml. 20% (v/v) aqueous solutions of glycerol and ethylene glycol were each Sparked for 4 hours and then applied to columns containing Dowex 50-X4 resin (T-17). The acid diSplacement eluates were tested by the ninhydrin (T-l3a), ferricyanide (T-l), and Elson-horgan (T-15) procedures. Also, a sample of Sparked glycerol was adsorbed on Amberlite IRA-400 and separated by gradient elution of the column. The results of the various tests on the eluates are presented in Figs.l4, 15, 16. A more Specific identification of the main sugar fraction eluted from the Dowex columns was carried out by paper chromatography using ethyl acetate:pyridine:water (60:25:20) as solvent and ninhydrin indicator (T-lOn). The fraction from Sparked glycerol showed an Rf value of 0.500 and that from ethylene glycol exhibited an Rf of 0.529. 56 Fig.13 - Approximate sketch of s pspcr chronotegrsn on which sparked ribose and spsrked glycerol hsvs been resolved ex. O‘Coorigln B - solvent houndsry l - spsrksd glycerol 2 - sparked ribose Solvent: ethyl seststs:pyridins: ester (60:25:20) Developer: Nishydrin sraphy rol C) yce SG—KD hromato ed 01 Column C of Spark Dowex on iglé - [14 Test 'drin ;.fi, . lson—norgan 4. . L150 ...Fer we, '.- ..I.. I? r...) 8. ac; O y‘ 58 PO 141:4)!“ dram»? \mxyfllfl.“ OHMWQ HE mv VHF...» Upwww'f (fr I14. I mN , 0 on ma 0» OK: I“. . dc P K1 ‘ .r \ w. ~— " "n ....J /\ ~f . omN _ , H _ a s . 8 _ . . , a _ a H a I _ z M 8m ._ l H “"1 O (D £+01 x 100v soot°’°.... .—_-—— .’ C) In ("\ \Y LU..-“ T———-—- O C) r4 meno...ueom+rm .. . .. . . . ._ . a. ,1, . u moacmmOmnnom II.I.II p . a at r o u L. at“ .I. ewnmmecfiz IIIIII r . uNHao I no (NH f... . F. _ OJ... \PUCE / . mu W... . U. I L. n. L. a i. 03H 03H .1 \ 4 \u s dqlom N®3OQ GO HOUWFUQCQHLALJHC MHsMflW .’ N . _ . . ES 0 I . fimthmm MO hLQNhMOfiMFGLSU L H U u’ Oud H, owe oma 59 mm mm mm :m coflyomwm Lomwdoz wzu mafia: Aoo:-amH moantmpe£mwpwoumE0pzu CESHoo Inumt weswwm 0: cm omH 00H com ij E+0I x Aonvauosav 65 TABLE 9 Pa Ducriuw 0F PAnAICLUEuasULFoxYL cuLonibr DEAIVATIVES FROM the ELUTIUN Faacriors or spannsa ACETIC ACID Sample State of derivative Melting_Point oC glycine (control) needles 145 tubes 5-9 oil (not determined) tubes 10-14 needles (not determined) tubes 15-20 needles 148 POLnggg Experiment 13 The purpose of this experiment was to search for a biOpolymer being formed by Sparking a solution of 5% (w/v) ammonium sulfamate, 5% (v/v) acetic acid, 2.5% (v/v) glycerol, and 52 (v/v) ethanol for 250 hours using inert platinum electrodes. dith dialysis followed by lyophilization, a large amount of white material assumed to be polymeric was isolated and analyzed by several tests to be described. Ihen the same eXperiment was repeated but with COpper electrodes, the white material was no longer found in the dried isolated product. Instead, a small quantity of a reddish-brown substance was observed and no further tests were performed on this product. a) General tests and characterizations A soluble concentrate of the pervaporated product solution Oave U a positive biuret reaction (3-28). Jhen the white polymer was checked by the tryptOphan-anthrone test (T234), an absorbency of 0.380 for he test product was observed at 520 mu against a water blank. 66 A 20 ml. portion of the isolated polymer dissolved in a 0.67% (w/v) aqueous solution was titrated with 0.1 g hCl. The titrant was added in 0.01 ml. aliquots with a microsyringc and the pH change was followed with a deckman heter equipped with a calomel electrode. Another 20 ml. portion of the polymer solution was titrated with 0.1 E KCH. To a third 20 ml. portion was added 2 m1. of 30% formaldehyde and then titrated with 0.1 E (CE. The data were plotted as in Fig. 21. ihe results indicated the typical graph for a dipolar ion as with amino acids or proteins. A standard Kjeldahl (T—31) determination indicated the polymer to contain 0.5% nitrogen. This value was confirmed by a Conway micro- diffusion adaptation of the sane method. b) Polysacgharide and AminOpolysaccharide Characteristics A sample of the white polymer was partially hydrolyzed by refluxing in 6 5 hCl for 30 minutes. Two aliquots of neutral hydrolysate were paper chromatographed side by side using n-butanol:acetone:water (4:1:5) as solvent after which one side was developed with silver nitrate and the other with ninhydrin (T-lO). Jach showed a common spot at an hf value of 0.10. Using n-butanolzacetic acid:water (4:1:5) as solvent for two similar parallel chromatograms of the same neutralized polymer hydrolysate, three spots were observed on one chromatogram after develOpment with the blson~fiorgan Spray with the strongest spot at Rf 0.64. fihen the other chromatogram was stained for acidic grOUps with toluidine blue, a single spot was nOted at hf 0.62. In order to further test for possible carbohydrate-like prOperties of the white polymer produced by sparking, a 0.1% (w/v) aqueous solution was divided into three eoual portions. Cne portion was oxidized by sodium periodate solution {2-30). The second was hydrolyzed in 6 3 H01 for 67 a.aa oo.oa omwm oo.w oo.n me O C J UN C) .C: :r O C) O M 00.0 Umnmmsmcm empmmnuncflfimELCL I-‘\/\ r‘i N vv neemfiod one we cowumnpfie mmmmuwflod u.mm.mwm “NV AHV (Isa) 010V 3 .0 r3 INH l r~. T~_v D "‘1' 14qu ..Ll -\ Tr} ' I a“ A VAL—L‘AV ff“ 'TX' cv 'JLL '\ 5.5 hours at 100°C. The third portion remained untreated and was used as a control. Both the periodate¢treated and control samples gave a positive Nolisch reaction (T-Sa) whereas the hydrolyzed sample did not. Also, the hydrolysate did not produce a positive benzidine reaction (T-lOa). To test for the possibility of linear polysaccharide-lihe structure within the polymer, a 50 mgm. portion of the product was submitted to periodate oz-u dation (1‘-30). The oxidized product became neutralized with 0.34 ml. of 0.01 5 sodium hydroxide solution. This result would suggest a molecular weight of 50 /0.0034 = 14, 706 if only one formic acid molecule were released per polymer chain. It was thU ;ht desirable to compare the hydrolysate of the white polymer to tne amino sugar-like products synthes'zed earlier as in experiment 3. A 10 ml. aqueous solution of 105 (V/v) glycerol was sparked for 60 minutes and analyzed for amino sugars by paper chi OJ””O“IJ)1V 1x1 I‘- ( \_/ P.) 0') O ...—a ’1 (I :5 CT I'\ i .1 l ....- C, \I O 0 v t. rr 0 '1 using ethyl acetate:pyridine::.'ater (30;' develOpment with ninhydrin on one st; ip and eiio‘at e benzidine on another strip, spots with identical hf values were obtained. A 11 (w/v) aqueous solution of the white p01): or was partially hydrolyze1 by refluxing for 30 minutes in 1 h hydrochloric acid and then submitted to paper chromatograpi y identical with that of the sparked glycerol sample. The outcome was the formation of a spot with the same Rf as from Sparked glycerol. C) 1hV31co-cnew1ca1 2;-alysis M‘ Tiselius electrOphoresis (T-Qé) of the wnlrmer product in 13 (w/v) . ‘- ' ._, '..‘..-- .g .* , 1‘. .17: . 'M‘f _ ‘ ‘1: . __ \ ,- ‘. a1ueous solution aftei d1 hal.s 3 against phosphate nutter \gh — u.za,l'/2 — 0.ul, _ - - _ v a .‘1 ...v v I o ' ‘-5 2 --l -1. gave a Single peas w1-n a mOJlllt} equal to 1.4c x 1t cw volt sec . She original product solution wasnnde 6 molar w'th respect to urea 6“ Q q ’ a \ ' . n "H ,‘v-‘ . - «.1- w .4 \ A ‘. u .. «1 . .- J-‘ . - . _ '1 -. ,-‘ I- 101 12 nears and then dialyzed ageinst LaLcl. finally, in the same prosphatc Figure #22 - Tiselius electr0phoresis pattern of the polymer in phosphate buffer at pH 6.75. 69 70 meAOHEHHHHE CH npmcmfio>gs _ 1 h Wan can can oon 0mm 9% Ohm com 03 h p P noEmHOQ 03p mo cnopumm oammeOuozaompoon upHOapmnuHD I mm .wfim '1“! CU fi' T 7 (eteos uorssrmsueaq quooaed e no) Koueqaosqv T 71 smsmaom exp mo shopped owmumEOPQSQOLpoomm eopmmm:H I mm.wfi muonOH: CH Sumneam>91 m L a fiH om m r1» f00.w l ”3.1 .usu I O '1‘ any ‘5 VI 0 5 notes 72 ’ .. . ’-. .‘o- 1 .- ,- w . 1 w- l _ I *_ ,. -. , 1‘ ‘ .1 - 1 - .. - " “ 1' "- ' ,- N "|"\ t " "1 v-r m . m r . m . ~1 f» ' {er ‘ , «5'1 ' ~ ; '1 r‘ -~‘4 , 1 , ,,. UJn-Ux, t.l\.‘ Leanne KILLal -Lgcb .lL1L3 l'J.’ LjLL.L\ heal; OI)U\ULV(JL" LA.I VJVLQA'-‘ ktJ—L—us K treatment. Age stability was tested after storinfi the sol"me“ solution at 4 c for one full year and then subjecting it to riselius electrOphoresis again. Cnce more the same unaltered single peak was observed as with In an effort to determine the essential reactants for the production of the polymer, a 1 liter aqueous solution of 150 ml. of glacial acetic acid and 150 m1. of glycerol was Sparked for 300 hours, dia ysed against water, concentrated by pcrvaporation, and tested by Tiselius electrOphoresis in phOSphate buffer buffer (pl 6.0;‘r72 = 0.01). 30 peak was observed as it had been in earlier syntheses. Ultraviolet and infrared double beam spectrOphotometric sea; patterns were determined for the dried pol;mer (T-32; see Figs. To further characterize the mass prOperties of the polymer, ultracentrifugation (T-24) was carried out on a 12 (w/v) solution of the isolated product in 0.1 d sodium chloride. Partial Specific volume was measured with a pycnometer and classical calculations were then made for sedimentation coefficient with data collected by sedimentation centrifugation. Molecular weight was determined by Archibald equilibrium centrifugation. The results obtained were as follows: 1) Sedimentation Coefficient (59,730 REL; see Fig.25 ) d log x/dt = 1.319 x 10- at 23 C , ..l. ' ”'- x/dt w2 = 11.319 x 10 ‘)(9.794 x 101”) -13 L W ll 0.. 5‘ ..J Ja = 1.29 x 10 sec = 1.29 520 = 335n25/n20 = (1.29)(o.3937)/1.005 = 1.15 3) Initial concentration ( low speed; see 913.25 ) “inner: 119.37zmn 1%1= 131.242m1 co =1Ax/Uofnf = (o.1)(73.105)/2.1o3 = 3.476 Fig. 25 - Uitracentrifugal patterns used in determining the sedimentation coefficient of the polymer. 73 Fig. 26 - Ultrocontrifugal patterns used in determining the molecular weight of the polymer. 74 75 a; ConCentration at the Jeniscus Cm = C0 - (1123“)(d::/1-£0)21{2A1' c1 = 0.03476 - 0.01(55.32953/41.033M2.103)= 2333 4) Kolecular Height 3 = RTCdc/dx)m/{l-v H = 8.314: {107(3 ”5) (lQC)(l.20$)/{1 - (9.343)(2.014))O.C347o(131. 24) U = 31,027 To test the polymer for immunogenicity, antibodies for the substance were prepared by the standard procedure (T- 27) with rabbits and run on Cuchterlony plates against the antigen. Initial experiments indicated no immunological specificity. ‘ LQSLJLS 10 SJLLAJZ CF IKYQICt-Cnaule. ALALYSJS CF TX; JYLIXEJILLD PCLTXZR Ana1"sis lgonccntration iglvcnt Treatment .LEQEE .EEEEQEiJflEEiEE Electroghoresis l”{v/v) IhOSphate buffer 1 u = 1.48 (pJ 6.75; u = 0.01) 12(w/V) Phosphate buffer; 1 u = 1.43 6 X urea-treated IZCw/v) Phospl Late buffer; 1 u = 1.4 1 year storage Ultracer trifugation 13 Ew/vg 0.1 M sodium chloride 1 39,— 1.14 1; w/v 0.1 U socium chloride 1 33 :031’327 nxnerinie1v;14 —.—J—.~—- The pu1 rpos e of this experiment was to test for microbial contamination in the reactants and the biological significance 0f the synthesized products in supportin life. Separate 10; (v/v) aqueous solutions of glycerol and acetic acid were each Sparked. During the Sparking period, samples were taken from the "lvcerol to check for the k) ‘ presence of ye s, molds, and bacteria (3-33 ' Lollowing the sparking period, both test solutions were exposed to the Open air for 43 hours and 76 then observed for colony growth. ho contamination was found in the glycerol sample by standard bacteriological techniques during the Sparking period. However, the xposed solutions were both observed to support microbial growth. In the case of the glycerol, the colonies were transrerred aseptically to a fresh solution of ooa (v/v) unSparked glycerol and 11 (v/v) nitric acid but growth was observed to cease entirely. experiment 15 A .' '11 ° nis eXperiment was carried out to compare the effects of electrons and photons on the Specific reagents used in this study. Samples of an aqueous 202 (v/v) solution of glycerol were each submitted to a separate type of treatment as described below. Also, portions of a 203 (v/v) aqueous solution of acetic acid were treated in a similar fashion. Jammie Breatment -..-“.-.... - . ...---...._... .. ‘. r~ ”a A ,- 6 -fi —‘ . . . -. . .. . 1. 1; glycerol 3.1tnlo neps.(seneral electric Linear “eta accelerator) 2) glycerol 4 hours of spark {electrical Sparking apparatus) 3) glycerol 21,030 roentgens (seniral electric K-ray Treatment apparatus) 4) glycerol 44 hours of UV light (ultra-Violet Products Inc. hineralight UV lamp - model RS - 0.6 amps) 5) acetic acid 3.72 x 10° Reps. (Linear Beta Accelerator) 6) acetic acid 2 hours of Spark (electrical Sparkin apparatus) (7 U I 7) acetic acid 45 hours of t? light (UV lamp) ‘ e The products were tested by the Lolisch (i—Sa), Tauber (l-Sc), L f i (.3 U} o “m. 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Bench ad . . 609a,, Data, 1957. . . 21) nun-1, n.‘ n c Chain . rum. 1... ad Paint, a. . Doc. Ruth c~." ”In, 1951. . 22) hadron, A. In Mn of mu. Opnfln, Ad. Dan: Publicnuan, . mo, In. 1.“. 1’53. . . . 23) night. 3.. and ram, J. :- Ema. Mar. 1... a . . rum, ll. me. Hench ad «may, Benton, 57. 24) “a... 3. 1w. hlnnr, 1... ad Mann. )1. - Doc. Ruth nay, Ionian, 1951. 25) Cnifln, I. lnntgy mention ad transfer In Photonynthnnu. In ”apical Sciacn - A Stud; groga. moldy, JJ... nd. Jenn ny a San. Inc., In Yo: , . 26) Urny. u.c. an en. uni added mum at m m ad eh. 021813 0‘ L110. Proc. "In. Mo 30‘. 0.8. 22. 351 (1952). 27) Minor. 3.1; Prodnctia of San Org-in CW and: Poona“. . mat!” Intel: Canaan. J. h. Gun. 80c. 11. 2351 (1955). 28) Grant, 1.1.3.. ad manna-nu. 'n. amoeba-nun rot-and out ' Organic Coqcnndn Iran lunar“ of 81.10 Conn. Plantar, Space “‘03. 79 (1,“).- 29) own. x.. .11. n1. Poly-annual by name uncut-p. Chan. Soc. 0‘ J” M10 279 ‘7‘ (19“). 30) Calvin, I. Wanda: tron Holconlu to Man. A138 Duncan. 3.5.” (1962). . 31) 2.1-, 6.. ad Calvin, I. run-nude: Orgaiznanantry. I. W balung tron uncut-a manna-£1. An. Gun. Soc. 8‘, 2115 (1962). . . 32) annulus-a; Inlnary. n.c.. ad an, I. Synthun of him Acidn by b.“ “Ct!“n “in“ 1.3;. 35° (1’57). 33) ma», 3.. ad am. 3.3. Stndinn o: inaction. Indnend by m hamttnua of tho fined: Molncnln. J. Chan. Physics 5. 418 (1936). , 34) rnrknn. 6., and auditors, Y. m hunch-tut nnccqnuua a! - Altpnguc Alcoholn 1n Aqnnonn Solnuan. J. h.,Chnn. Soc. 92. 2‘50 (1937 . - _ . . _ . II fl , '- - . - u . ' - ”' ‘ I .15"'|-)“‘ . .1 .33LIB(«i o. ..‘- § 1" ' ~ . n 1 11.. . .0‘- 'x L‘ t ‘JK:-;’3‘ “' o ‘J’ (; ‘.'.~ I ' ‘ . ' ' '1': I I .'.‘ - J. '4- b - w ' Q. ‘:t\ .‘Iily‘ ‘uladLA“H-fi . . ‘ " o. ‘7; f . '. . V ‘ 0 a- o . 3.1- ‘. '. :. . ...-‘- ,' ' ..:n':i‘.f.3;.-£.‘.rus. ~. a~ot1 .121’1”'~ . LL. a.) !;...;) .4... u. ¢".¥.’oa..ln'n-._. OJ. I-Q'wL-O -1-.. cu. a.‘..,.._— . ., .1 ‘. . _‘_ r... '_ -__ .... .' _ _ .3- '._ ..., . 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'0’" ’ .,-, ,_. 9 _ v y . u- -\ .' . , . , or ._. . . _ h“ :1 t HQ.)- f'agt «3.10:1..JLL—L ..U (395-433;. O\.-— ...! L11. . ~§”: .... .—)nv £15. xvi. ' f '. ’. ... ‘ , Pa". ..‘ ‘ , f. H ' f .v, _' ; r." 1.. .~ .2 3 -3... '.'. q. . .0.‘ .Q \,‘ L-.l~,' .f’ \‘..j ' V‘l‘ 0 4C4..-' .. .‘o O JJUDJL():; :{JJtava -34).] \J Am. ‘.:.-V.L....l‘- “L; f . .",’ O \'/‘-\' a" 3 ... ‘..' :v. _- , .- ‘.- ' y. .i.‘r.4r . -': '2 "' ,. ' 2 . .. "' t... ‘ . , '. ‘ :0 (2'13- r *.-".- .))w 4;... “-..-.1. 5L.- os {4'1 )’-ub...".s aw.“ c- .r ¢'-‘-"‘-~ \x‘ ~- '.'~' ~ . ~. :1 r' I. 'y ." I r ’..‘ ' r u ' :r. " ' ~.'. : --\.§‘- t . ...). '1. . ~-J-|J .l'll . ...: ..~'o.~. (4'1“.‘n 811‘!)U"-'. 0'.'. ‘;-.:~l~‘ ...}... .‘s. .3 uni‘l.‘ “‘ Q 92 35) Canaan, R. Oxydauoo daa Mothylalkohola decala nalakalaran Smaratofta dutch ultraviolattaa Licht. Bar. 292. 1101 (1936). 36) nun. v.. and me. A. In maxed nun-{ct nus-«1.1a a. . 3111a, c., and Walla, A. RaIEoH RENE!“ manual, ECU York, 1941. 37) 8111a, 0., and Hana, A. manual Action at Ultraviolat Ba 3. Rainhold Publiahing Corporation, Nov York, 14. 33) men... n. n annual mum at mtraviolat El” nus. c.. and , .. Hallo, A. natnhol Publi n; Corporation, Nov York, 1961. 39) loyala, 3.8. Advuoad manic Chasing. Pranuoa-nall Inc., , mlavood 611 a, Nan Jaraay. . 1'0) Glow. 6., at a1. Sana Effacl: So1ut1ona of b-Laoto 1511 Brain, 31131833, 1965. 41) that. 11.3., no mkharjaa,‘ 3.x. Photoa'ynthaaia ail-loo Aotda 3.5 vitro. Hacura L353. 499 (1934). 42) Clocklar, 6.. and Lind, s.c. ‘Blaoorocha-lat of Gaaaa and 0thar . Dialaetrtca. John Hilay and son, Ino., Na. YorE, I959. 43) Baoar, J.A. Ganaral Eng. rho-u Y. Crova11 Conny, lav York. 1929., ' ' U.) Lada. 6.11.. and Randall, M. mama“. McCain-11111 Book . _ Conan}. Ino., lav York. 1961. 45) Saffron, a. Iha Origin of mu. m Graduata «and 5. a: (1961). as) man. a. Cour-attic Analyala of Susan. 1. Mathoda an ' 232.133. Colo‘uck, 3.2., no Kaplan, 11.0., «I. made Praao “co, P“ 1“.”. NC. York. 1,570 47) Gaidall, S. Dataudnation at llama-in“. In Hathoda of Blochadoal Aoalzaia. click, 1).. ad. Intaraoianca Poul-£5311. Inc. Motor: _, 68) “teach, G. %%rinanta1 Btochadatg. John “Hay and Sons. Inc" . fl" rat, 1 a #9) Hanan, 1.3., at a1. Spactraphotouatrio Mathod for hater-tattoo of Supra. Anal. Chan. 33. 1916 (1956). . 50) non-u. a.a. Mocha-lea Laborato ’ Monica. John any and Son, , Inc., London. 19”. 51) up... 1!. ma Carbohzdratoa. Acadamic Press, Inc., Naw York, 1957. . I . . . . - . - - r . . .~ 4 .‘ . . n g A . n ‘ I u a b n I ‘ . . ‘ g . . .. g Q - ’ D .. ; . J - . . . .' .- ‘A '.. . . J < a - I . I .- . . ' ‘ . ' ' . ’ A . ,' ‘ . I I . v _Q u t . 0 it ' 4 a - .. ' . . u . . u . _ I . , . . c F . I I 7 II I ‘ ‘ . . - . , 1! 1 _ . \ ‘ ‘ ‘ A r v u‘ l? . l . _ . .I . . . . a II I ' . I g . . . o . . < . O o. . -. ‘r_.. Q Q a. v... ‘ u . . a , ' ' . - ... i. . . ‘ . c . . ¢ \ :m ' ..,‘ ’o . ~- . a.‘ ; ‘.‘ . s . . - - 1 ‘ . - .- o n v - ' 1 I . - . . «' * . j ‘ f. p. ' . . u . - . - ‘I u' .- «J “ v - ' a VI ~- ‘ .c' ..\\.‘I ‘ ‘. ‘.¢ - g - . > , O , I , - . C . ‘ ‘I ‘ ‘ . . H‘ r! ‘ I ' “ .. ; I " a ' s r’ ‘ .- ,3 . . - A c A o C o. . l‘.~4. a fl 1‘ .3. “ ..- _ fl. . . . v .- . VJ. -a-n.-.o,a~'o“ Q"-. - .-.-..4 .‘ - , . . . . .' t 'v ‘ . a ’ . -‘ v I I . n4 . n f . . .. , .. A. ... .? g- .. J ' "R"... 01"! > I _ ' '. - . . . . - V . . . ‘v ’ .. ‘ - ‘ . I . a ' v ' ' . ‘ ’r ‘ ' ~ 5. I A . ' . . ' 1 . ': ‘3" s.." I .75 .' . .v . . * "'.' ‘; . ' ‘ ‘ ~ an... . .,~,u . - '6 ‘ ~ - . ' V ' ' ‘- -} \ ‘ L ' Q ~ - .‘~" - . ' '_ x a .»A ~ 1 . O - ' l ‘ o .a-- ... \.-...-1.-. u I I ‘ - ~ _ I I' . _ ~ ‘3 — . r o . I ' QI 1 E" - '. . .- . A. ‘ ° .w-"' 1 ‘< ‘ I . . t —‘ -- .I . v t ‘ t3 ‘ I- a r 79:. z *7. . s", x. . . §"I~ ‘ I . n - - . I A I 5;“ U" 6" a.: t- . a ' . "' ‘ . 4' -' O . -.. ., " QI.‘.§ ,' (;e '-. .‘.’ k '- . r. . o . .. . ’ . . . .. . ‘4 L _.. ':.‘ .0 . ‘ “1‘ ' . .1 I.. , u 1' A A . . 6 - " '. ' ' ‘ . . . I 1 I I ' ‘ , I -. . V. - - , - ‘ t. t . v ,. ‘ - 7 . ...-.....- A1} .....- . ..x. a~-. ‘.-.5...- \ a ', g I . I If I. f 4 Q .. I- a ‘ ' I‘ I I ‘ II II I I. I II .I II'I I I I 'II “’ - 'I‘ ‘ « - ‘ 'II “I 7- ' ‘ 'I t l a 31:.“ I'. ‘ Ir’ ._ .. . . . . . . , V ~' . 5- ¥' v! ‘ \ ‘ , ‘. I. A t 1. __ 1 . . ‘. .. . . . . '3 3-. . - ,‘ £5, 4 ' a 9 la '. , V a ' . ' " q. .' ’. -.. ' ' .. '. ‘ C ii. .. .'r .‘ I J - . . ' ‘0 A 1.. .0 - - A <. a . o ‘ p . . , p o n - ‘1 I ' ‘ _ . _'. .‘ . l‘ 1. ., .J‘ ‘ . 7‘ ' ( } . . . . . , . -, J . . ‘ t. 3 . ' Q a ' ' . .» . '. I ‘ I I _ I ' o - - a 1 I ‘ O .. '1‘ . «I . P. . 'ua, ‘ ‘ ' ~ ‘. . J :‘ .I‘ ' D,‘ .‘ .~ . . - ”... I ) 5 0 .‘ ‘ ‘o . f v _ . - V '. . . H- n u f '. rv . . . I . _ . “I 0 ’A, - .‘ I ...i c ' . ’.,I ..y (no - , I ' t a . 4 3 :vv , a . . ‘ ’6. O ' .!. ‘ 1.. ' . ‘-.-J a I D I ‘ I I - - ova ‘ k h , A. t. . l , 3 .. , , \h ' a . _ .11... o - . " .1 r' .. a.» .v‘ " O C I“ a. . O . ' -«L—J‘ -.. - . g _ ‘ u I D a. I ‘ “I I ’I ’ .'. 5 e.. .. ~ . a - ‘ .._ J (- ...¢ . a J. 0‘. ' .‘ . .1.‘ I ‘ a ' . I C I- "‘ . . ~ ' ‘ 9 fl .a .‘g , . I ‘ I , - ” I R‘ ' ‘ q. .n ..4 ' -1 ‘ II x‘ ‘ . - . - p. . «- . a , w .. O . ~ I .i I ‘ . A '~ I I I .' x I I ' -.I . »», ‘ l —'. h. '- a » '- o 93 52) Mayor. 14.14., and Ksbst, LA. anti-anal I-nnochenista. A. Giarlas C. Tho-as, Pnblishar, Spring a , I ino s, 1. 53) Shrinar, 3.1... and nasal, LC. astountic Idantificstion of Organic . Cam“. John Uilay and Sons, Inc., New or , . 56) hith. I. Chronatogrggic and Blactroggorotic Iachnifls. Intarsciuca . ”bl1mr.9 mo. NC. Y0 , o 55) Mora. 8.. andIStain, ".11. Photonatric Ninhydrin liathod for Usa in tho alronotography of Amino Acids. J. Biol. Chan. 116, 367 (1966). 56) lobartsan, a.a. Laboratog Practice of Organic Chanistg. lha . MIG-111“ Cn‘y. N“ Y0 ’ O 57) Ladorar, 3.. and Lodarar, M. Giro-st r . Elsaviar Publishing Coqany, Nov York, 1957. I 58) Parkin-Blnar Corporation. Instruction Manual. Nor-alt, Con-action, 1951. 59) Biochanistry Division Staff, lbivarsity of Illinois (Drbsna). ¥Frinants1 Biochanistg. Stipos Publishing Coqsny. Main, I inois. 1 . w 60) Duiials, r., and Albany, 3... thsical a.a.-1.2g. John mm and . 3... m0. N” York. 19610 61) Danials,IB., at a1. garland hzsical Chanistg. McCray-Bill . _ Book Coqsny, Inc” Nov Yo , . 62) Graanbarg, 9.11.. and lothstain. M. In Hathods in 35313“. . Colo-dos. 3.1%, -d Kayla. II.0.. ad. c Pross Inc., Pnbliohars. lav York. 1957. 63) Moora. 8.. ad Stain, H.1i. Gruntography of Amino Acids on hltmtod Polystyrona mm. J. Biol. Chan. _1_2_2_, 663 (1951). 66) Difco Laboratorias, Inc. Difco Manual. Datroit. lichign, 1953. .9 \ I a. ‘. . II I. . “ . . . _.. . J o o . ‘ ; .' . Q f .... 'o ’s an- ‘ A up - d E . - H ' . . . .' ..- ‘ . I II ' n I o . I \ I n ’. . j ' _K. ‘ . ’ . . ‘ ’- .. O o -I. ., . . V . - ‘ a _ ( I ' Q a .1 o‘- a ‘. .,- o t» .- I a ‘ \‘ - .. A . i. Q I v N \..¢. .- . ~ A O O a "'s . ... . '0 C . . g'. 'o a ... . ‘v. .s‘.. ’ y -‘-"9 ,. 'i l I 1 . a u. . O. 4. s t . ‘~. . 'a n o‘- ....4. ' ‘ 4" O . g . . . \ f'. I ‘c. . § _ . , .- ‘ ‘ ‘ - a A . f I f w‘ J r k 74. I ' : - 9 v .a .. ’._ f , a v . ‘ - ‘ ‘ V J ‘I z .I 4 ‘ > 7.x ' .o' .. . ; I. .. . ‘ . . I. .i‘,‘ . . . . ‘ v a v . a -. . o. J ' . 1 APPENDIX I Analytical Techniques A. B. C. Index to Sugars and Amino Sugars 1. 2. 3. 4. S. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. Ferricyanide o o o o 0 Alkaline decomposition Fehling O o o o o o o Orthoaminodiphenyl . . a. Molisch . . . . be 3181 0 O O 0 o C. Tauber o o o 0 Optical rotation . Anthrone . . . . . Ascorbic acid . . a. Osazone . . . . b. 2,4-dinitr0phenylh Paper chromatography . pH measurement . . . . Solubility o o o o o o a. Ninhydrin - method b. Ninhydrin - method Nitrous 301d 0 o o o o Elson-Morgan . . . . . Sulfate O... .00.... Analytical Technigues Y o 0 CL. 0 o o o 1 2 o o r 2 o o O O o o o o O a o O o o o O o C). o o o o o o o b .0000... Ion exchange chromatography Amino Acids 18. Paper chromatography. . . . 19. Ninhydrin o o o o o o o o o 20. Nitrous 8C1do o o o o o o o 21. Ion exchange chromatography 22. p-Toluene sulfonyl chloride 23. (all previous apprOpriate tests) Polymer 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. ‘30 o o o o o o o 000...... ......OOOOOOOOOOOOOOO ......OOOOOOOO. ......OCOOOOOOOOOOOCO Ultracentrlfugation Q g o o o 0 o 0 0 Dialysis and isolation... . . . . . . Tiselius free-boundary electrOphoresis Immunological analysis. Biuret. o o o o o o Iurbidimetry . . . Periodate oxidation. . Kje1dah1 nitrogen. . . SpectrOphotometry . . Trichloroacetic acid . TryptOphan-anthrone . Microbial contamination 0.. 0.. 0.... ......OOOOOOOOCOOOOOO 0.... 0...... 0.00.00.00.00.. 0.... 0...... 00.000.00.00... 00.... OOO‘OOOOOOOOQ 0.... 94 808 103 103 103 103 103 104 106 107 107 108 109 110 111 111 111 9!. 95 A. Sugar and Amino Sugars 1. 2. 3. Ferricyanide test, based on the reduction of the ferricyanide ion (46) a. Reagents 1) 2) 3) A solution of 0.5 grams of potassium ferricynaide in 1 liter of water. A solution of 5.3 grams of sodium carbonate plus 0.65 grams of potassium cyanide in 1 liter of water. A solution of ferric ammonium sulfate in 1 liter of 0.05 normal sulfuric acid. b. Procedure 1) 2) 3) a) Add 1 milliliter of reagent 2 to l milliliter of sample and l milliliter of reagent 1. Heat in boiling water bath for 15 minutes. Cool and add 5 ml. of reagent 3. Read in spectrOphotometer at 690 mu after 15 minutes of development. Alkline decomposition, for total amino grps.(47) a. Reagents 1) 2) 3) Saturated solution of sodium phosphate and sodium borate. Nessler's reagent: 4.55 gm. of mercuric iodide plus 3.49 gm. of potassium iodide dissolved in as little water as possible, followed by 8 gm. of sodium hydroxide and made up to 100 ml. of final solution with water. A 2% boric acid solution. b. Prodedure 1) 2) 3) a) Add 2 ml. of sample to 5 ml. of reagent 1 and 2 ml. of water and 1 ml. of reagent 3. Heat in boiling water bath for 2 minutes. Cool in ice water bath and add 1 ml. of reagent 2. Stand for 30 minutes and then read in spectr0photometer at 490 mu. Fehling test, for aldehyde reducing groups (48) a. Reagents l) A solution of 34.6 gm. of capper sulfate in 500 ml. 2) of water. A solution of 173.0 gm. of sodium potassium tartrate plus 60 gm. of sodium hydroxide in 500 ml. of water. ‘ ?a\ b. 96 Procedure 1) Add 0.5 m1. of reagent 1 to 0.5 m1. of sample, 0.5 m1. of reagent 2, and 4 m1. of water. 2) Heat in boiling water bath for 15 minutes. 3) Observe for red precipitate of cuprous oxide. 4. Orthoaminodiphenyl test, for sugars (49) a. b. Reagent l) A 100 ml. solution of 0.4% o-aminodiphenyl in glacial acetic acid. Procedure 1) Add 1 ml. of sample to 5 ml. of reagnt 1. 2) Heat in boiling water bath for 45 minutes. 3) Read in spectr0photometer at 380 mu and observe the color of the solution. a) Arabinose, ribose, and xylose give a brownish yellow solution. b) Galactose and glucose give a green solution. c) Rhamnose gives a brown solution. 5a. Molisch reaction, for pentoses and hexoses (50) a. b. cflum A 3. U > colored products 0U J , yo (similar for 'a pentose) hexose hydroxymethyl furfural Reagent 1) A 5% solution of a-naphthol or thymol in 95% ethanol. Procedure 1) Add four draps of reagent to 2 ml. of sample. 2) Stratify solution over concentrated sulfuric acid. 3) Observe for violet ring at the plane of contact, an indication of the production and liberation of furfural. 5b. Bial reaction, for pentoses (48) 3 t 3' t .( HM U {,1 a blue- green as b. condensation pentose furfUral . product Specificity - for pentoses, 2-deoxypentoses, 6.deoxypentoses, hexuronic acids, trioses, and certain heptoses. Reagent 1) A solution of 1.5 gm. of orcinol and 30 drOps of a 0.1% aqueous solution of ferric chloride in 500 ml. of concentrated hydrochloric acid. 5c. 6. 7. 97 procedure 1) Add 1 ml. of sample solution to 3 m1. of reagent. 2) Heat in a boiling water bath until color change occurs. 3) Observe color produced. a) Yellow or brown - hexose, aldehyde, hydroxy aldehyde. b) Emerald green, blue-green, or blue - pentose. Taber test, for pentoses and hexoses (51) a. b. C. Mechanism - similar to the Molisch and Bial reactions. Reagent 1) A solution of 1.5 gm. of benzidine in 500 m1. of concentrated sulfuric acid. Procedure 1) Add 1 ml. of sample solution to 3ml. of reagent 1. 2) Heat in a boiling water bath until color change occurs. 3) Observe color produced. a) Cherry red - pentoses and uronic acids. b) Yellow or brown - hexoses. Optical rotation (48) incident _ Optically active beam of pl 4% ‘(,a»substance polarized light ' A‘EEE, ‘(///,deflected plane of light a. b. Theory " Optical rotation is a measure of a given molecule's asymmetry as defined by its ability to bend a plane of polarized light. For example, a carbon atom is asymmetrical if the four substituents bonded to it are all different in polarizability, as is the case with carbon 2 of glucose. A substance is known as dextrorotatory if it bends the polarized light to the right and levorotatory if to the left. The degree of rotation is a function of temperature, concen- tration, and solution density. An equal mixture of a dextrorotatory and a levorotatory Species of a given molecule will result in no Optical rotation in the polarimeter. Procedure 1) Fill cell and place in polarimeter. 2) Take reading and compare unknown to knowns. Anthrone reaction (52) 7. 8. 9a. 9b. a. b. C. 98 Reagent l) A 0.2% solution anthrone in 95% sulfuric acid. Mechanism - similar to Molisch and Bial reactions. Procedure 1) Add 2.5 m1. of sample to 5 m1. of reagent. 2) Heat in boiling water bath for 10 minutes. 3) Cool in cold water bath. 4) Read in spectrOphotometer at 620 mu and observe for color reaction Test for ascorbic acid (46) a. b. Reagent 1) A 5% metaphOSphoric acid solution. 2) A solution of 2.5 mg. of 2,6-dichlorphenol-indOphenol in 100 ml. of water. 3) A solution of 4.53 gm. of sodium acetate in 100 ml. of water plus 0.26 ml. of 0.5 normal acetic acid. Procedure 1) Mix equal volumes of all three reagents and sample. 2) Read in spectrOphotometer at 520 mu. Osazones, for a-hydroxyketone and b-hydroxyaldehydes (53) a. lfifo ya:y—Ny-¢ “of“ +2.~~-~~z ——-- “(PM ,1 a. I ’5 fl. Reagents 1) A solution of 0.4 gm. of phenylhydrazine plus 0.6 gm. of sodium acetate in 4 m1. of concentrated sulfuric acid. Procedure 1) Mix equal volumes of reagent 1 and sample. 2) Heat in boiling water bath and note time for yellow precipitate formation. 2,4-dinitrOphenylhydrazones (53) a. Mechnaism - same as with osazones except that 2,4-dinitro- phenylhydrazine is used and substitution occurs only once per molecule, at the carbon 1 position. b. Procedure 1) Mix equal volumes of reagent 1 and sample. 2) Heat in boiling water bath and note time for precipitate formation. 3) Collect precipitate, recrystallize from 95% ethanol, and take melting point. 10. Paper chromatography (54) a. b. Solvent systems 1) Ethyl acetate:pyridine:water (60:25:20) 2) ISOprOpanol:water (80320) 3) Butanol:pyridine:water (40:40:20) 4) Butanol:acetone:water (40:10:50) 5) Butanol:acetic acid:water (40:10:50) 6) Phenol:water (80:20) 7) Phenol:water (88:12) 8) Butanol:pyridine:water (60:60:60) Procedure 1) Spot sample on whatman #1 paper and set up for run. 2) Pass solvent either ascending or descending. 3) Dry and deve10p with apprOpriate indicator. a) Benzidine, for reducing groups (1) A solution of 0.1 gm. of benzidine in 4 m1. of glacial acetic acid plus 3 gm. of trichloro- acetic acid in 4 m1. of water. (2) Use as Spray and heat to develop. (3) Observe for brown Spots. b) Elson-Morgan, for amino sugars (1) Spray #1 - 0.5 m1. of a solution of 5ml. of 50% aqueous potassium hydroxide and 20 m1. of ethanol added to 10 m1. of a solution of 0.5 ml. of acetylacetone and 50 m1. of butanol. (2) Spray #2 - 1 gm. of dimethylaminobenzaldehyde in 30 m1. of ethanol added to 30 m1. of 99 hydrochloric acid and then to 180 m1. of n-butanol. (3) Spray with #1 and heat for 5 mdnutes. (4) Spray with #2 and heat for 5 Minutes. (5) Observe for red spots c) Ninhydrin, for amino groups. (1) A 0.2% solution of 1,2,3-triketohydrindene in n-butanol (2) Use as a Spray and heat to deve10p as purple spots. d) Periodate-benzidine, for viscinal glycols ( ) (1) Dip #1 - 0.5 gm.of sodium periodate in 100 ml. of water. (2) Spray #1 - 0.5 gm. of benzidine in in 100 m1. of ethanol-acetic acid (80:20). (3) Submerge in dip #1 and dry. (4) Coat with Spray #1 and observe for white Spots on a blue background. 100 e) Silver nitrate, for sugars (1) Spray #1 - 1 m1. of a saturated silver nitrate solution is added drOpwise to 20 ml. of acetone with stirring. (2) Spray with #1 and keep in a light-free ammonia atmosphere for one hour. (3) Heat to develop. (4) wash in 10% sodium thiosulfate solution (5) Wash in running water and dry. c. Calculation of Rf - Divide distance of migration of fraction spot by distance of migration of solvent front. 11. pH measurement (48) 12. a. Adjust meter with standard buffer. b. Submerge electrodes in test solution and take reading 0. pH - olog (H+) Solubility of a sugar 3. Dissolve sample in water. b. Bring solution into contact with another immiscible solvent such as ether. c. Test second solvent for presence of sugars by Molisch test (technique 5). 13a.Ninhydrin test, for amino groups - method #1 (55) 0 ML Q .7 I .01.} /( 4» k(”0 \ .7. W - :: Pam-c...» 21: 15} 14 (:6) A: C\ 1‘ C02, 6 g g Ninhydrin a. Reagents 1) A solution of 0.8 gm. of stannous chloride in 500 m1. of 4 molar sodium acetate buffer added to 20 gm. of 1,2,3-triketohydrindene in 500 m1. of methyl cellosolve. 2) A 50% solution of ethanol in water. b. Procedure 1) Add 1 m1. of reagent 1 to 1 m1. of sample sohtion. 2) Heat in boiling water bath for 20 minutes. 3) Add a 5 m1. portion of reagent 2 and stand for 15 minutes. 4) Read in spectrOphotometer at 570 mu. 13b.Ninhydrin test - method #2 (52) a. Reagents 1) Add 2 m1. of 0.01 molar potassium cyanide solution to 98 m1. 0f pyridine. 2) An 80% solution of phenol. 3) A 5% solution of 1,2,3,-triketohydrindene in absolute ethanol. 4) A solution of 60% ethanol. b. 101 Procedure 1) Add 0.5 m1. of sample solution to 0.8 m1. of reagent 1 and 0.8 ml. of reagent 2. 2) Heat in boiling water bath for 3 minutes. 3) Add 0.4 ml. of reagent 3. 4) Heat in boiling water bath for 5 minutes. 5) Add 7.5 m1. of reagent 4. 6) Read in Spectr0photometer at 570 mu. l4. Nitrous acid test, for primary amines (56) 1°: 2°: 3°: a. b. C. NaNOz + uc1 - NaCl + HNOZ R-NHZ + HONO - ROH + N2 Rz-NH + HONO - RzfiN-NO (yellow) R3-N + HONO a (no reaction) Reagents l) A 2 normal hydrochloric acid solution 2) Solid sodium nitrite. Procedure 1) Add 1 m1. of sample solution to 1 ml. of reagent 1. 2) Add 250 mg. of reagent 2. 3) Observe for release of nitrogen gas bubbles. Alternative application 1) Treat sample with nitrous acid to remove amino group and replace it with a hydroxyl group. 2) Use product for paper chromatography of non-aminated species. 15. Elson-Morgan reaction, for amino sugars (48) C1490” Cybofl v red ..C ...;- , . acoadensati on H56 My‘cocfl’ . \\A “#3 product t M <-c H 5 a. Reagents l) A solution of 0.75 ml. of acetylacetone in 25 m1. of 1.25 normal sodium carbonate. 2) A solution of 1.6 gm. of p-dimethylaminobenzaldehyde in 30 m1. of hydrochloric acid added to 30 ml. of 96% ethanol. b. Procedure 1) Add 0.5 m1. of sample to 0.5 ml. of reagent 1, heat in boiling water bath for 20 minutes, and add 4 m1. of 96% ethanol and 0.5 m1. of reagent 2. 2) Stand for 1 hour. 3) Read in Spectr0photometer at 525 mu and observe for red color. 102 16. Sulfate test (47) a. b. 170 1011 A. C. Reagent 1) A 10% aqueous solution of barium chloride Procedure 1) Add 1 m1. of sample and 1 m1. of water to 1 m1. of reagent 1. 2) Observe for turbidity and read in the spectr0photometer at 550 m 0 exchange chromatography for amino sugars and amino acids (48.57,) (63) Theory _ This procedure depends heavily upon the principle that Oppositely charged ions attract each other. The supporting material for the ion exchange is usually a macromolecular polyelectrolyte whose free groups are ionizable. Synthetic polyelectrolytes are generally employed with smaller molecules, such as amino acids and amino sugars. Examples of supporting resins are Dowex-50, a nuclear sulfonic acid cation exchanger, and Amberlite IRAP4OO, a strong base anion exchanger. Dowex-50 acts as a sulfonated cross-linked polystyrene resin, making it insoluble but permeable to other ions and capable of changing size by swelling. An ion is bound to the resin until a suitable solvent breaks the electrostatic bond by replacing the held ion with an exchanging ion of the solvent. The rate of elution is governed by temperature, swelling, pH, and ionic strength of the solvent, as well as the flow rate of the system. Preparation of Dowex 50-X4 (200-400 mesh) for amino sugar analysis. 1) Wash with a 2 normal sodium hydroxide solution. 2) Wash with a 2 normal hydrochloric acid solution. 3) Wash with water. 4) Repeat steps 1, 2, and 3 several times. 5) Wash with a 0.3 normal hydrochloric acid solution after finishing the last wash cycle with stepl, followed by Step 3. 6) Apply 28 cubic centimeters of resin as a 1:1 (v/v) suSpension with the 0.3 normal hydrochloric acid solution to the column. Preparation of Dowex 50-X4 (200-400 mesh) for amino acid analysis. 1) Prepare buffer #1 - 21.008 gm. of citric acid in 200 m1. of one normal sodium hydroxide solution is diluted to 500 m1. total volume and added to 110 m1. of one normal hydrochloric acid solution and 390 m1. of water (pH - 3.52). 0‘7 Co d. e. 103 2) Wash resin as for amino sugar analysis except that buffer 1 replaces the 0.3 normal hydrochloric acid solution. 3) Apply 90 cubic centimeters of resin to the column. Preparation of Amberlite IRA9400 (HSO3'form) for sugar analysis. 1) Wash resin as for Dowex 50-X4. 2) Apply 13.5 cubic centimeters of resin to column. Elution and collecition of fractions 1) Apply sample solution to column. 2) Apply suitable solvent. a) A 0.3 normal solution of hydrochloric acid for amino sugars on Dowex 50-X4. b) Buffer 1 (see 17.c.1) for amino acids on Dowex 50-X4. c) Gradient elution between 99.5% ethanol and 95.0% ethanol for sugars on Amberlite IRAP4OO. 3) Collect samples with an automatic fraction collector table based on siphon volume control (3 ml. or 10 m1. fractions). 4) Analyze fractions. B. Amino Acids 18. 19. 20. 21. 22. Paper chromatOgraphy (see technique 10). Ninhydrin (see technique 13). Nitrous acid (see technique 14). Ion exchange chromatography (see technique 17). p-Toluene sulfonyl chloride derivatives (53) a. b. 1°: p-CH3C6H4SOZC1 + R-NH2 - p-CH3C6H4SOZNH-R 2°: p-CH3C6H4SOZC1 + RZ-NH - p-CH3C6H4SOZNR2 3°: p-CH3C6H4SOZC1‘ + R3-N = (no reaction) Reagents 1) A 1 normal sodium hydroxide solution. 2) An 8% solution of p-toluenesulfonyl chloride in ether. Procedure 1) Add smaple to 20 m1. of reagent 1 and 25 m1. of reagent 2. 2) Stir for 4 hours and then acidify with dilute hydrochloric acid. 3) Filter, recrystallize from 60% ethanol, and take melting point. k d O ,A l 104 C. Polymer 23. (All appr0priate tests for amino acids, sugars, and amino sugars.) 24. Ultracentrifugation (52) a. Theory Ultracentrifugation can generally be divided into two sections: 1. Equilibrium centrifugation 2. Sedimentation centrifugation In the first type of study, a sufficient Speed is attained so that the diffusion of the substancd is exactly counterbalanced by centrifugal force. In the latter type of study, the speed is increased from that in the former study so that the molecules being investigated actually sediment out of solution. The cell of the ultracentrifuge is in the shape of a sector so that migration of the molecules away from the center of the rotor is not impeded by collision with the walls of the cell which would set up disturbing countercurrents within the solution. In sedimentation studies, the migration of particles under the influence of the high centrifugal field created by the speed of the moving rotor may be followed as the movement of the boundary between the plateau, the region of uniform distribution of molecules, and the solvent, in particular that portion which has been cleansed of the test molecules by the sedimentation toward the bottom of the cell. The Schlieren lens system of the ultracentrifuge is such that an image of the differential of concentration with distance is transmitted. Theoretically, this division would be an infinitely sharp line at a given distance from the center of the rotor. However, diffusion causes the peak to broaden out, giving significance to the area under the curve in various analytical considerations. The primary formula in ultracentrifugation is the Svedburg equation. With the contributions of Einstein and Sutherland, the Svedburg equation indicates the relationship between Species molecular weight and the sedimentation coefficient, which in turn is defined by the radial acceleration of the centrifugal field in a sedimentaition study. 3 a d long X(2.303) w‘ dt 105 S I V I: X -X1)2A ‘52; (x2+x1)w‘(t2-t1) RTS M I: Archibald has taken the Svedburg equation and applied it to equilibrium ultracentrifugation ' RT(dc/dx)m M (T-VSPP)W‘xmcm Several factors are necessary in the Archibald equation for which Shachmann and others have determined the means of calculation. 1. Partial specific volume _* v2 - vp(1/mo - 100(l/mo - 1/m)/p) v: - apparent partial specific volume vp - volume of the pycnometer mo - weight of contents with solvent m - weight of contents with solution p ' percent of macromolecules in solution Values are graphically extrapolated to 0 concentration to get partial Specific volume. 2. 3. 4. 5. Initial concentration (synthetic boundary cell, see figure 26) Readings of AY taken at regular intervals of X co - AX/Moflr Mo - 2.103 (magnification factor) Concentration at the meniscus (low speed equilibrium study, see figure 26) cm - co - (1/x3.)(dx/Mo)sx2u xm a distance of meniscus from center of rotor. The value of (dc/dx)m (equilibrium study) (dc/dx)tn - AYm - change of concentration at the meniscus Additional necessary values - gas constant - 8.314 x 107 ergs/CO/mole - absolute temperature - density of solution a radial velocity 8'08" 106 25. Dialysis and polymer isolation (52) a. b. Purpose - to separate small ions from solutions of macro- molecules and to determine the minimum value for molecular weight of the polymer. Procedure The solution containing the polymer and the undersired smaller ions and the initial reagents is placed in a dialyzing membrane which is suspended in water. The pores of the membrane chosen are of such size that molecules of less than 6,000 molecular weight diffuse out into the water and the macromolecules remain within the bag. The surrounding water is changed often. The resultant polymer solution is condensed by air pervaporation and the product is isolated as a dry solid by freeze-drying 1y0philization. 26. Tiselius free-boundary electrOphoresis (58) a. Theory Electr0phoresis may be defined as the migration of charged particles within the influence of an electrical field gradient. Tidelius, in 1937, developed a practical means for using electrOphoresis in the study of high molecular weight substances, which is now available as the Perkin-Elmer Model 38 Apparatus, among others. The cell is in the shape of a "U". When suitably prepared with samples and buffers, the migration of particles within the electrical field can be viewed through the Schlieren lens system in the apparatus. Since, at a given buffered pH, different proteins of a mixture, for example, exhibit correSpondingly different degrees of net charge, the rate of migration of each protein Species varies. Since the lens system depends on areas of more or less dense concentration, the movement of these proteins, seen as boundaries, can be quantitatively analyzed. The Schlieren lens system projects the change of concentration from point to point as a series of differential curves. Thus, as with the sedimentaflon coefficient in ultracentrifugation, the rate of migration of each Species is measurable and calculable. Procedure 1) Dialyze the sample solution in buffer. 2) Prepare and fill the cell. 3) Place the cell in the ice bath chamber filled with water within the apparatus and connect the electrodes. 4) Align the channels of the cell. 5) Compensate the initial boundary and photograph it. 6) Apply measured voltage and amperage, noting time as well. 7) Step theelectric current after suitable migration and photograph the peaks. 8) Measure the resistance of buffer in conductivity cell. 9) Measure distance of migration from the photOgraphS. 107 c. Calculations - dAK/tIRm C - mobility (cmzlvolt/sec) distance Of migration (cm) cross-sectional area of cell (0.30 cmz) conductivity cell constant (0.850) time (sec) current (amp) resistance of buffer (ohms) magnification factor of Optical system (1) aanK>ac 27. Immunological analysis (52) a. Theory A macromolecule often has the prOperty of inducing the production of specific antibodies against itself which may be analysed by the Ouchterlony plate technique. By this method, antigen diffuses from a source well through the agar of the plate toward the antibody diffusing from the other direction. At the points where specific antibodies meet correSponding antigens, precipitation occurs. Since different types of antigens diffuse at different rates, precipitation lines arise at different locations so that a count of these lines indicates the number of Specific components in the antigen mixture for which antibodies have been produced. b. Procedure 1) Inject a 5% solution of polymer in complete adjuvant:water (50:50) suspension into the rabbit. 2) Two weeks later inject a 5% solution of polymer in an incomplete adjuvant:water (50:50) suspension into the same rabbit. 3) One week later, bleed the rabbit, allow the blood to clot and settle overnight. 4) Collect serum and use against a 1% solution of polymer antigen in the Ouchterlony agar plates. 5) Observe the plates for the appearance of precipitation lines. 28. Biuret reaction 69) 1 0i ..." LL: \ ~ colored “fill!” \yfl‘ complex a. Theory Compounds containing two or more peptide bonds give a characteristic purple color when treated with dilute capper sulfate in an alkaline solution. The name of the reaction comes 29. 30. b. Co 108 from the compound biuret which gives a typically positive test. The color is apparently due to the coordination comples of the c0pper and four nitrogen atoms, two from each of the two peptide chains. Steric factors are also significant. Reagent 1) A 500 ml. solution is made up of 0.75 gm. of cOpper Sulfate, 3.00 gm. Of sodium potassium tartrate, 150 ml. of a 10% sodium hydroxide solution, and enough water tomake up the remaining volume of solution. Procedure 1) Add 1 ml. of sample solution to 5 ml. of reagent 1. 2) Stand for 30 minutes. 3) Read in photometer at 570 mu. Turbidimetry (59) a. Reagent 1) Low concentration solution of acetic acid and iron potassium cyanide. Procedure 1) Add 1 m1. of sample to 5 m1. of reagent 1. 2) Measure photometrically at 600 mu. Periodate oxidation (48,52) a. Theory Periodate ion oxidizes the carbon to carbon bond between combinations involving hydroxyls, aldehydes, ketones or primary amines. For example, the oxidation of a mole of ethylene glycol would yield two moles of formic acid. An a-acetal of a pyranose aldohexose is not affected by such action nor is the bond between C1 and C2 altered. However, cleaving the C2-C3 bond and C3- C4 bond produces a molecule of formic acid. If the hydroxyl group of C3 were an amino group, ammonia would be produced instead. . +%an¥ (14"1 1" “* In a polysaccharide such as amylose with its 1 ,4-g1ycosidic bonds, formic acid is liberated from both ends of the chain but from no internal members. Thus, the number of moles of formic acid produced can be used to determine the average chain length of a given sugar polymer. b. C. d. 109 Reagents 1) A 0.37 molar aqueous solution of sodium metaperiodate 2) Ethylene glycol 3) A solution of carbon dioxide-free 0.01 normal sodium hydroxide. Procedure 1) Dissolve 50 milligrams of sample in 5 milliliters of water. 2) Cool to 3°C and add 5 milliliters of reagent 1. 3) Keep in the dark for 30 hours under refrigeration. 4) Add 1 ml. of reagent 2. 5) Titrate potentiometrically to pH 6.0 with reagent 3. Calculations U - w/vNP UP - M . wlvN U - average number of units per chain w - weight of sample used v - volume of base used to titrate N - normality of base P - weight of each unit M - average molecular weight of chain 31. Kjeldahl nitrogen determination (52) a. b. C. Reagents l) Powdered sodium sulfate with some available mercuric ion contributor such as mercuric oxide. 2) Concentrated sulfuric acid. 3) A solution of 12.5 normal sodium hydroxide. 4) Boric acid, water saturated. 5) A mixture of 5 parts 0.2% brOmocresol green and 1 part 0.02% methyl red. Procedure 1) Add sample to 1 teaSpoon of reagent 1 and 12 m1. of reagent 2. 2) Digest for 1.5 hours. 3) Dilute with 200 m1. of water. 4) Add 30 m1. of reagent 3. 5) Distill into a flask containing 25 ml. of reagent 4 and 5 draps of reagent 5. 6) Titrate against standard acid. Chlculations % Nitrogen - ANM/W - volume of standard acid used to titrate - normality of standard acid milliequivalent weight of nitrogen (0.014) - dry weight of sample (gm) ZZZ> I 110 32. SpectrOphotometry (60.61) a. Theory The SpectrOphotometer, as the name implies, is an instrument for making quantitative measurements of the transmission Of light at various wavelengths by a given medium. The principal parts are the source of electromagnetic radiation, the mono- chromator (usually a prism or a grating), the cell compartment, the photoelectric detector, a variable slit to control light intensity and limit the Spread of wavelengths, and a device for indicating the signal from the detector such as an electrical meter, potentiometer, or recording potentiometer. Various chemical structures are known to absorb light at certain wavelengths. Therefore, when a solution containing a structure which absorbs light at a given wavelength, is placed in a beam of that wavelength, the dector notes that less light is transmitted than when the solution is not there. A double beam SpectrOphotometer is so constructed that absorbance due to the solvent is ruled out and only that due to the substance being tested is recorded. The Beer-Lambert law gives quantitative significance to SpectrOphotometry: log (Io/I) = A = abc where A is absorbancy, Io is the intensity of light of a particular wavelength with no sample in its path, I is the light intensity with the sample, a is the absorbancy index (a solute prOportionality constant which varies with light wavelength, temperature, and solvent), b is the thickness of the solution, and c is the concentration. Automatic Spectro- photometers are so constructed that they directly measure absorbance or percent transmission. It is possible to test a given substance in the ultraviolet, infrared, or visible range with a suitable SpectrOphotometer. In the ultraviolet and visible range, quartz cells are used; in the infrared range, sodium chloride cells are used so that all test materials must be emultions or solutions in nonpolar solvents. Rotation and vibration-rotation Spectra are observed in the infrared range whereas the ultraviolet and visible ranges exhibit the electronic spectrum of a given substance. In the Beckman DK-a (ultraviolet and visible) and Beckman IR-5 (infrared) SpectrOphotometers, it is possible to either take a reading at one wavelength or set the apparatus to automatically take readings within a range of wavelengths. b. Procedure 1) Adjust 0% and 100% transmission and fill cells. 2) Take necessary readings as absorbancy or percent transmission. 111 33. Trichloracetic acid precipitation, for proteins (62) 34. 35. a. Reagent l) A 5% aqueous solution of trichloroacetic acid. Procedure 1) Add 5 ml. of sample solution to 10 ml. of reagent 1. 2) Observe for precipitate. TryptOphan-anthrone test, for mucOpolysaccharides (52) 80 b. Reagents 1) Asolution of 95% ethanol. 2) A 0.1% aqueous solution of tryptOphan. 3) A 0.15% solution of anthrone in 95% sulfuric acid. Procedure 1) Add 2 ml. of reagent 1 and 1 ml. of reagent 2 to sample. 2) Chill for 15 minutes. 3) Add 6 ml. of reagent 3. 4) Heat in boiling water bath for 20 minutes. 5) Cool for 10 minutes and then read in SpectrOphotometer at 520 mu. Tests for solution contamination (64) a. b. Test for bacterial contamination 1) Add 23.5 gm. Of Plate Count Agar (tryptone glucose yeast agar - Difco Laboratories) to 1 liter of water. 2) Heat to dissolve and sterilize for 15 minutes at 15 pounds pressure and 121°C. 3) Aseptically mix the warm fluid agar in a Petri dish with 1 m1. of sample solution and allow to harden; store at constant 32°C. 4) After 3 days, inspect the dish for colonies. Test for yeast and mold contamination 1) Add 39 gm. of Potato Dextrose Agar (Difco Laboratories) and 1 ml. of a 10% solution of tartaric acid (to adjust to pH 3.5) to 1 liter Of water. 2) Heat to dissolve and sterilize for 15 minutes at 15 pounds pressure and 121°C. 3) Aseptically mix warm fluid agar in Petri dish with 1 ml. of sample solution and allow to harden. 4) Store at constant 23°C. 5) After 7 days, inspect the dish for colonies. 112 APPENDIX 11'. VITA he atber wee born in Detroit, maiga a June l,194l. He gredneted ee velediceeria fue‘niet Northern High School in June, l9” ad entered mm;- suc. new” the following 2.11 en es. Dietingniehed nun: Sebelerebip. ' in 1960, the ad»: entered en. um. cm... prom- ad leter bee“ e Univereity Scbeler. Hie endergrednete reeeerob wee eerried at under the enepieee e! the letiael Science randetia progr- et liicbiga Stete. lie elee wee eqleyed an e reeeerch eeeietat in tbe Depertant of Food Science. - hiring theieprieg ad a-er of 1961, be continued hie reeeereb .: m 3...... Seepitel a. M May. Tereel. 1. m of 1962, be entered the graduate progra- of the Depart-at .1 Biochemistry .1: mm... sac. Ihivereity. ' m. atbor plae te eerry a He: 12, 1963 ad eepeete to dealete both hie beebelere ad neetere degree in June, 1963. nee there. be will begin e dectorel progren in biephyeice. 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