Ivv 7 F7 G:=v4 F03?=-{ERABIE5SQN 555§CT5MT 655 05? K§ESERADISK 5’EEGXIBRSE A 55535:: 5265‘ 535m @595“ <25 55?. 53 5555655555555 STATE 85555153553555 535555 J. 555. =<::55.. 575» J MW! 51115115555551 WWW L L 3 1293 01085 0745 LI B RA R Y Michigan State ’ University ABSTRACT POST-IRRADIATION INACTIVATION 0F HORSERADISH PEROXIDASE BY Basil J. Macris Aqueous solutions of purified horseradish peroxidase (HRPO), com- posed of at least 11 different molecular forms, were found to lose activity not only during irradiation with gamma rays, but also during post-irradiation storage. A number of factors were found to affect the post-irradiation rate of inactivation. The radiation dose at which the enzyme solution was exposed, affected greatly the rates of post-irradiation loss of activi- ty. The higher the dose the faster the rate of post-irradiation inactivation. The rate of enzyme activity loss after irradiation was increased with the temperature of post-irradiation storage. There was no apparent difference in the rates of post-irradiation inactivation between pH 5 and 7; at pH 9 this rate was greater. Irradiation of different enzyme concentrations resulted in differ- ent rates of post-irradiation inactivation. The higher the enzyme con- centration the slower the rate of post-irradiation inactivation. Water or glycerol, added to the already irradiated enzyme solution resulted in decreased rates of post-irradiation loss of activity. Basil J3 Macris No appreciable activity was lost after irradiation when the enzyme solution was irradiated in the frozen state. Addition of intact enzyme to irradiated water or to irradiated enzyme solution did not result in inactivation of the intact enzyme, indicating that there were no radiolysis products originating from the water or the enzyme and capable of destroying the added intact enzyme. The following mechanism for the post-irradiation inactivation of HRPO is proposed: A portion of the enzyme which is not inactivated during irradiation is modified in such a way as to result in subsequent inactivation by reacting with itself. The free radicals formed during irradiation appear to be involved in the production of the metastable enzyme molecules. POST-IRRADIATION INACTIVATION 0F HORSERADISH PEROXIDASE BY _ , , \ \ Basil Ji 'Macris A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science 1970 ACKNOWLEDGEMENTS The author is greatly indebted and appreciative of his major professor, Professor P. Markakis, for guidance, encouragement and constructive criticism during his graduate work. He also wishes to express his appreciation to Professors Walter M. Urbain and John G. Scandalios for their help, advice and critical reading of this manuscript. ii TABLE OF CONTENTS Page INTRODUCTION. . . . . . . . . . . . . . . . . . . . . . . 1 LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . 3 METHODS AND MATERIALS . . . . . . . . . . . . . . . . . . 8 1. Materials. . . . . . . . . . . . . . . . . . 8 2. Assay Procedure of Horseradish Peroxidase. . . . 9 3. Preparation of Samples . . . . . . . . . . . . . 11 Effect of dose . . . . . . . . . . . ... . 11 Effect of temperature. . . . . . . . . . . 11 Effect of pH . . . . . . . . . . . . . . . 11 Effect of enzyme concentration . . . . . . 11 Effect of glycerol . . . . . . . . . . . . 12 Effect of freezing . . . . . . . . . . . . 12 Effect of dilution . . . . . . . . . . . 12 . Effect of irradiated water . . . . . . . 12 Effect of addition of intact enzyme to irradiated enzyme. . . . . . . . . . . . . 13 L—I-D‘OQH'AIDQOU'N RESULTS AND DISCUSSION. . . . . . . . . . . . . . . . . . 14 Effect of Radiation Dose . . . . . . . . . . . . 14 Effect of Temperature. . . . . . . . . . . . . . 16 Effect of pH . . . . . . . . . . . . . . . . . . 16 Effect of Enzyme Concentration . . . . . . . . . 18 Effect of Glycerol . . . . . . . . . . . . . . . 21 Effect of Freezing . . . . . . . . . . . . . . . 21 Effect of Dilution . . . . . . . . . . . . . . . 23 Effect of Irradiated Water . . . . . . . 23 Effect of Addition of Irradiated Enzyme on the Enzymatic Activity of Intact Enzyme Solution . . . . . . . . . . . . . . . . . . . . 27 \oooxsoxmwar-a 0 0 SUMMARY AND CONCLUSIONS . . . . . .‘. . . . . . . . . . . 31 LIEMTUM CITED 0 O O 0 C O O C O O O O O O O O O O O O O 33 APPENDIX. . . . . . . . . . . . . . . . . . . . . . . . . 36 iii LIST OF FIGURES FIGURE Page a. Automatic tracing and calculation of enzyme reaction rates. . . . . . . . . . . . . . . . . . 10 1. Effect of radiation dose on the post- irradiation inactivation of HRPO. . . . . . . . . 15 2. Effect of the post-irradiation storage temperature on the HRPO activity. . . . . . ... . 17 3. Effect of the post-irradiation pH on the HRPO actiVity O O O O O O O O O O O O O O O 0 O O 19 4. Effect of enzyme concentration on the post- irradiation inactivation of HRPO. . . . . . . . . 20 5. Effect of addition of glycerol to irradiated HRPO on the post-irradiation inactivation of the enzyme . . . . . . . . . . . . . . . . . . 22 6. Effect of freezing during irradiation on the post-irradiation inactivation of HRPO . . . . . . 24 7. Effect of dilution of the irradiated HRPO on its post-irradiation inactivation . . . . . . . . 25 8. Effect of addition of irradiated water on the actiVity 0f IRPO. O O O O O O O O O I O O O O 26 9. Effect of addition of irradiated HRPO on the enzymatic activity of non-irradiated HRPO . . . . 28 iv INTRODUCTION A characteristic property of ionizing radiation (X-rays,ar,§ , and.X’rays, high energy electrons) is to cause ionization of a receptor atom or molecule by ejecting one or more orbital electrons from the latter. The ionized species is reactive and leads to further chemical change. Since the discovery of X-rays by Roentgen and radioactivity by Bequerel (35) in 1895 an immense amount of research endeavor has been applied to the physics, chemistry and biology of ionizing radiations. Radiochemical reactions and the mechanism of energy transfer are now fairly well understood. Precise methods of irradiating biological units have afforded more definite notions of the effects on various cell constituents, such as the enzymes. The resistance of enzymes to radiation is much stronger in_§i£g than in cell free extracts or pure solutions. The mechanism of enzyme inactivation in heterogeneous systems is complicated by the effect of moisture content, the presence of other solutes and the possibility of the enzyme being absorbed at an inter- face. Of the three types of radiation used in radiation preservation of food, namely, high energy electrons, X-rays and gamma rays, the last one, emanating from Cobalt-60, was used in this research. Horseradish peroxidase is an enzyme which is highly resistant to heat inactivation and can be reactivated under certain conditions (40). The original objective of this research was to investigate the possibilities of reactivation of this enzyme after it had been inacti- vated by gamma radiation. In a number of exploratory experiments not only reactivation was not observed but the enzyme continued losing activity after irradiation at a rather rapid rate. It was then deemed desirable to study this post-irradiation inactivation in some depth and the results of this work are reported in the present thesis. LITERATURE REVIEW The earliest systematic studies regarding the effect of ionizing radiation on enzymes are attributed to Dale and his collaborators (18,19,20,21,22,23). They studied the effect of irradiation l2.!l££2 on certain purified enzymes, e.g., polyphenol oxidase, D-amino acid oxidase and carboxy peptidase and they found that enzymes in solution are inactivated by irradiation and the greater the dilution of the protein the greater was the percentage of inactivation. Irradiation of enzymes in the presence of substrate or coenzyme prevented inacti- vation partially or completely. Enzymes in solution lose activity upon exposure to ionizing radiation by two different mechanisms (10,21): (a) by direct action, in which the energy of the radiation is deposited directly on the enzyme molecule, (b) by indirect action, in which the radiation energy is deposited on water molecules and the enzyme changes are caused indirectly via the radiolysis products of the water. In the case of the enzyme inactivation by irradiation the rate of inactivation is commonly described by an exponential decrease in the activity with increasing dose. This follows from the direct action mechanism (32,34,38). It also applies to cases of indirect action by free radicals (30,33). One of the most sensitive sites of proteins is the sulfhydryl group. The oxidation of two sulfhydryl groups by hydroxyl free radi- cals results in a disulfide bridge. The results of this oxidation is the inactivation of certain enzymes (39). Augenstein (7) found that the destruction of trypsin and ribonuclease is directly correlated with the number of titratable SH groups. The inactivation of enzymes by ionizing radiation may be due to a sequence of specific modifications in molecular conformation as opposed to indiscriminate disruption of secondary bonds. Among the most prob- able sites for these structural modifications to occur are the clusters of weak bonds responsible for maintaining the proper conformation of the area of the active site. Absorption of enough energy in the cluster to disrupt all crucial bonds leads to inactivation (8). Butler (16) re- ported that the average energy of each ionization, taken as 32 eV or 1.25x10‘18 cal, is sufficient to raise the temperature of the whole protein molecule (MW up to 60,000) by 60 C. This rise can bring an appreciable part, though not the whole, of the molecule up to denatura- tion temperatures, as the absorption of 10"20 cal can break one hydrogen bond. Admittedly, the exact mechanism of enzyme inactivation by ionizing radiation is not completely understood. Some factors (9), affecting the enzyme inactivation by radiation, are the following: The free radicals produced during irradiation of aqueous solutions are very reactive and can act as reducing and oxidizing agents or cleave carbon-to-carbon bonds. In solutions, a fixed number of free radicals is produced by a given dose. If the action is indirect, the number of enzyme molecules inactivated will depend on the enzyme concentration in the irradiated volume and will be proportional to this concentration. In a number of enzyme solutions it was found that at low concentrations the enzyme sensitivity decreased as the concentration of the enzyme in- creased (28). Addition of other substances in the enzyme solution before irradi- ation will result in competition for the free radicals between the enzyme and the added solutes. This may lead to a "protection" of the enzyme from the irradiation effect. Two mechanisms of such chemical protec- tion have so far been discovered. a. The energy, which is deposited in a molecule by ionization, is transferred to the added new molecule. This new molecule plays the role of a second receptor and thereby protects the first receptor molecule (1). b. Repair of damaged molecule (2). If the chemical change pro- duced by radiation is reversible, there may be a short time during which the enzyme molecule can react with the added new molecule (protector) in such a way that the damaged enzyme molecule can be restored to its orig- inal state. In the absence of the protector it decomposes further and it is inactivated. Many chemical compounds have been shown to exert a protective effect against destruction of enzymes during irradiation (15). Sissakian (36) found that the anhydrous glycerine protects peroxidase and polyphenolo- oxidase against ionizing radiation. Cysteine, cystine and glutathione were reported to exert a protective effect against the destruction of catalase, irradiated with gamma rays, electrons and X-rays (22). Okada (29) found that the addition of glutathione, cysteine and homocysteine to irradiated Dnase solution failed to reactivate the enzyme. Almost all biological systems are more radiosensitive in the pres- ence of oxygen. The enhancement of the damage produced in the presence of oxygen during irradiation is believed to be accomplished: a. By converting the free radicals in the water into more reactive entities such as HOé and hydrogen peroxide (25). b. By enhancing the direct action of radiation on enzymes through formation of either an organic radical which reacts with oxygen or 02- radicals which combine readily with enzyme molecules (1,3,4,). Enhanced inactivation of trypsin by gamma radiation in the presence of oxygen was reported by Alexander (4). The temperature during irradiation plays an important role in the enzyme inactivation. If the temperature of the irradiated enzyme solu- tion is below the freezing point the diffusion of free radicals is hindered and the indirect process is sharply reduced. Therefore, if the diffusible free radicals formed in water play a part in the enzyme inactivation then the radiation damage is sharply decreased when the irradiated enzyme solution is frozen. In the case in which there is no such decrease, the indirect action is not occurring. The fact that pro- tein is less sensitive to irradiation damage at liquid air temperature than at room temperature was shown by Setlow (34). The radiation dose required to inactivate enzymes decreases with increasing temperature during irradiation as exemplified by studies with electron radiation of aqueous tyrosinase, polyphenoloxidase (13), milk peroxidase (15) and with deuteron irradiation of dry catalase (36,29). The radiation inactivation of enzyme in aqueous solutions is pH dependent. For trypsin the inactivation dose is decreased at pH values above and below 6 to 7 (12). Baron gt a1 (11) found that not only pH but also the nature of the buffer influenced the effect of X-rays on phosphoglyceraldehyde dehydrogenase and ribonuclease. Enzymes in crystalline form are generally more resistant to irradi- ation than they are in pure solutions (26,27,37). Brier and Nord (14) found that the radiation dose necessary to cause certain amount of inactivation in crystalline trypsin was 170 times higher than for a dilute solution. Phillips and Griffiths (31) found that the exposure of potato phosphorylase in the crystalline form to gamma rays did not modify the pattern of the enzyme action, but the efficiency of the enzyme in carrying out its normal function was reduced. The aqueous solution of this enzyme was more radiation sensitive. Anderson (5,6) found that the inactivation of pepsin by X-rays consisted of two separate parts, an immediate inactivation and a slow post-irradiation inactivation. He found that the inactivation, caused by the slow reaction, occurred at a rate which was dependent upon temper-. ature. The possible effect of H202, produced during irradiation, was examined. Incubation with concentrations of H202 similar to those found in irradiated solutions did not eliminate the post-irradiation loss of enzyme activity. The author suggested that the major factor in this post- irradiation inactivation of pepsin is a modification of the enzyme mole- cule which occurs during irradiation and which permits it to continue to act as an enzyme molecule yet makes it unstable. A continuous post-irradiation loss of the enzymatic activity and denaturation of alkaline phosphatase and -lactoglobu1in in cow's milk was observed, respectively (24). These post-irradiation changes were found to be dependent on storage temperature, presence of oxygen during irradiation and irradiation dose-rate. METHODS AND MATERIALS 1. Materials Peroxidase: Electrophoretically purified horseradish peroxidase from Sigma Biochemical Company. Its activity was 250 purpurogallin units per mg. This preparation was subjected to starch gel electrophoresis and was found to be composed of at least 11 different molecular forms. However, the composite enzyme was used in this research. Guaiacol: Reagent grade; Eastman Organic Chemical. Hydrogen Peroxide: 30% analytical reagent solution from J. T. Backer Chemical Company. Glycerol: Analytical reagent from Mallinckrodt Chemical Works. Phosphate buffer: 0.01M potassium dehydrogen phOSphate adjusted to pH 7 with NaOH. Water: Distilled water, redistilled in glass was used to prepare the enzyme and reagent solutions. Cobalt-60 source: A 50,000 curie (June, 1967) pool type source located in the Department of Food Science, Michigan State University. 2. Assay Procedure of Peroxidase Activity The guaiacol test of Delvin was used as reported by Chance and Maehly (17). The activity of peroxidase was measured in terms of increasing absorbance resulting from the oxidation of guaiacol (hydrogen donor) by hydrogen peroxide (hydrogen acceptor) in the presence of peroxidase. The absorbance measurements were made with a Beckman DU Spectrophotometer connected to a Ledland-log convertor and a Sargent recorder. The reaction mixture consisted of the following: 2.95 ml of 10 mM phosphate buffer, pH 7 0.05 ml of 20 mM guaiacol 0.01 ml of 40 mM H202 0.02-0.06 m1 of the horseradish peroxidase solution The H202 solution was the last component of the reacting mixture to be transferred into the 1 cm light path Beckman cuvette. For the transfer and mixing of peroxide solution, a square Teflon plunger, provided with a groove and three orifices and connected to a stainless steel handle, was used. The reaction was followed by automatically recording the change in absorbance at 470mm. The continuing change in absorbance was recorded as a straight line in all measurements. The slope of the straight line was used to measure the rate of the enzymatic reaction (Figure a). Absorbance at 470 nm (arbitrary units) 10 ’ q Figure a. 60 50 4O 30 20 10 Automatic tracing and calculation of enzyme reaction rates (numbers are arbitrary absorbance units at 470 nm per 0.5 min.) Reaction mixture: 2.95 ml 10 mM phosphate buffer, pH 7, 0.05 ml 20 M protein mM guaiacol, 0.01 ml 40 mM H202, 0.04 ml enzyme (3.0x10- in A and 1.0x10'7M protein in B triplicates). 7 11 Preparation of Samples Triplicate sets of 3 m1 aliquots of all samples were transferred to Pyrex glass test tubes, 7.5x0.8cm, and closed with rubber stoppers. Each set was irradiated in the center of the radiation facility well. After irradiation, all samples were kept at room temperature (20°C) except in the experiment in which the effect of post-irradiation temperature was studied. a. Effect of dose Fresh enzyme solution containing 7.5x10- M protein was irradiated with 0, 8, l6 and 24 Krad. Aliquots of all enzyme samples were measured for their activity immediately after irradiation and at 3, 6, 9, 12, and 24 hours. b. Effect of temperature Enzyme solutions containing 4.02x10-7M protein were prepared. The dose of radiation used was 24 Krad. All samples were measured for their enzymatic activity immediately after irradiation, at room temperature (20°C), and then were stored at 0, 10, 20 and 30°C. The enzyme activity during post-irradiation storage was measured at 3, 6, 9, 12 and 24 hours. c. Effect of pH Enzyme solution containing 3.75x10'7M protein was irradiated with 24 Krad. Immediately after irradiation equal volumes of irradiated enzyme solution and 20mm phosphate buffer, pH 5, 7 and 9 were mixed and the enzyme activity was measured. The post-irradiation enzyme activity was also measured at 3, 6, 9, 12 and 24 hours. d. Effect of enzyme concentration Enzyme solutions containing 7 - - -7 1.25x10 7, 2.5x10 and 7.5x10 M protein were irradiated with 16 Krad. 12 The enzyme activity was measured immediately after irradiation and at 3, 6, 9, 12 and 24 hours. e. Effect of glycerol. An enzyme solution containing 7.5x10-7M protein was irradiated with 16 Krad. The enzyme activity was measured immediately after irradiation. Then glycerol was added to the irradi- ated enzyme solution. Two glycerol concentrations, namely 30 and 60% by volume were prepared by mixing 0.8m1 of the irradiated enzyme solu- tion with 1.2m1 of 50 and 100% glycerol solutions. The enzyme concen- tration in each glycerol solution was 3.0x10-7M protein. Alliquots of all samples, taken at the end of 3, 6, 9, 12 and 24 hours of post- irradiation storage, were measured for their enzymatic activity. f. Effect of freezing. An enzyme solution containing 4.0x10'7M portion was frozen at -38°C and introduced to Co60 well at that temper- ature. There was no provision for maintaining that temperature during irradiation which lasted only 60 seconds. At the end of this time the sample was still frozen, although the temperature might have increased somewhat. The dose of radiation applied was 24 Krad. After irradi- ation the samples were thawed and the enzyme activity was measured. The post-irradiation enzyme activity was also measured at 3, 6, 9, 12 and 24 hours. g. Effect of dilution. A solution containing 7.5x10-7M protein was irradiated with 24 Krad. Immediately after irradiation the enzyme solution was diluted with redistilled water to contain 3.0x10-7M protein and the enzyme activity in both diluted and nondiluted samples was measured. The enzymatic activity was also measured at 3, 6, 9, 12 and 24 hours after irradiation. h. Effect of irradiated water. Redistilled water was irradiated 13 with 24 Krad and mixed with non-irradiated enzyme solution containing 10'6M protein. After mixing the final enzyme concentrations of 1.25x 10-7M and 2.50x10-7M protein were obtained. The preparation of these solutions was done by mixing one volume of the enzyme solution contain- 6M protein with 7 and 3 volumes of irradiated water. The enzyme ing 10- activity was measured at 0, 3, 6 and 9 hours after mixing. j- Effect of addition of intact enzyme to irradiated enzyme solution. An irradiated enzyme solution was prepared by dissolving 3.0x10'7M protein in redistilled water and irradiating with 24 Krad. Immediately after irradiation, a portion of irradiated enzyme was mixed with intact enzyme solution and the enzymatic activity of both irradi- ated enzyme and mixture of irradiated and intact enzyme solutions was measured. The addition of intact enzyme to irradiated enzyme solution was done by mixing 0.9 m1 of the irradiated enzyme and 0.1 m1 of intact enzyme solution containing 3.0x10-6M protein. The enzymatic activity of intact enzyme in the mixture was calculated from the following equation: Atotal - 0~9Airr. Aint. where: Aint. Activity of the intact enzyme in the mixture. Atotal = Enzyme activity of the mixture. irr. = Enzyme activity of the irradiated enzyme. The 10-fold enzyme concentration in the intact enzyme solution cancels the 1:10 dilution in the mixture of intact and irradiated enzymes. All samples were measured for their activity at 0, 3, 6 and 9 hours after mixing of irradiated enzyme with intact enzyme solution. RESULTS AND DISCUSSION The results of these experiments are expressed as percentage enzyme activity of that remaining after irradiation, i.e. the activity of enzyme solution immediately after irradiation was taken to be 100% and all subsequent measurements were calculated as percentage fractions of that activity. The loss of enzyme activity during irradiation is also given for all the experiments described in the following. 1. Effect of radiation dose. Triplicates of solutions containing HRPO were irradiated with 0, 8, 16 and 24 Krad of gamma rays. The loss of enzyme activity during irradiation was 51.6, 65.9 and 84.3% for the radiation dose of 8, l6 and 24 Krad, respectively. The results of the effect of dose on post-irradiation enzyme activity are shown in Figure 1. Detailed values are presented in Appendix, Table 1. The post-irradiation enzyme activity as a function of dose was found to decrease with increasing dose. The rate of decrease depended upon the dose and was faster at the early stages of post-irradiation storage. For example, the percentage loss in enzyme activity 3 hours after irradiation was 4.9, 7.9, 27.2 and 46.0% for radiation doses 0, 8, 16 and 24 Krad, respectively; whereas the loss of activity 12 hours 14 15 Enzyme activity (% of the remaining after irrn) 100 - 0 Krad o 80 ‘- o C) I 70 F 8 Krad o 1 6O -' (D 50 '- (D 40 ~ 16 Krad 1 30 '- 20 L. 24 Krad 10 r O 1 4 l I J 3 6 9 12 24 Time after irrn (hours) Figure 1. Effect of radiation dose on the post-irradiation inacti- vation of HRPO. 16 after irradiation was 6.6, 20.9, 44.9 and 75.0% for the same radiation doses, respectively. The percentage loss of enzyme activity during post-irradiation storage did not appear to be logarithmic. 2. Effect of temperature. A solution of HRPO was prepared and irradiated with 24 Krad. The percentage enzyme inactivation during irradiation was 80%. The effect of post-irradiation storage temperature on the enzyme activity remaining after irradiation is shown in Figure 2. Detailed results are presented in Appendix, Table 2. The post-irradiation rate of inactivation is greatly affected by temperature. The higher the post-irradiation temperature the greater the enzyme activity loss. As an example, the post-irradiation enzyme activity found 24 hours after irradiation was 53.3, 34.5, 19.0 and 3.2% for samples irradiated with 24 Krad and kept at 0, 10, 20 and 30C, respectively, while the control lost in the same time 10.4, 6.9, 7.3 and 13.8% at the same storage temperatures. On the other hand, the rate of loss in the enzyme activity during post-irradiation stor- age was greater at the earlier rather than the later stages following irradiation for all storage temperatures tested and depended upon the temperature. Plotting of these data on semi-log graph paper did not give a linear relationship between the percentage loss of enzyme activity and post-irradiation time. 3. Effect of pH. The effect of pH on post-irradiation drop of the enzyme activity is shown in Figure 3. Detailed values are presented in Appendix, Table 3. Enzyme activity (% of that remaining after irrn) l7 100 \V—\~ ====== EEEEEB. 90 0 Krad o 80 O 0°C . (5 10° C _ o 20°C 70 t. ’ Q 30°C 0 24 Krad 60 p 0 (3 o 50 C 24 Krad 40 1 30 1 ' 24 Krad 20 (D 10 24 Krad ‘0 0 1 jg; 1 1 .13 3 6 9 12 24 Time after irrn (hours) Figure 2. Effect of the post-irradiation storage temperature on the HRPO activity. 18 During irradiation the enzyme lost activity. The percentage of the activity lost during irradiation with 16 Krad was found to be 68.6%. The post-irradiation rate of inactivation varied when the irradir ated solution was adjusted to different pH values immediately after irradiation. The results indicate that at pH 5 and 7 the rate of de- crease in the post-irradiation enzyme activity appears to be the same. A greater rate of decrease was obtained at pH 9. For example, 3 hours after irradiation the percentage enzyme activity was 74.2, 75.7 and 54.6% for pH 5, 7 and 9, respectively, whereas the corresponding acti- vity values for the control, in the same time, were 94.4, 98.0 and 95.2% for the same pH values, respectively. 4. Effect of enzyme concentration. Three different concentrations of HRPO, namely 1.25x10-7, 2.5x10"7 and 7.5x10'7M protein were irradiated with 16 Krad. The enzyme activity loss during irradiation was 74.8, 64.4 and 48.7%, respectively. Dale (23) found also that water solutions of polyphenol oxidase, D-amino acid oxidase and carboxypeptidase are inactivated by irradiation and the greater the dilution of the protein the greater the percentage inacti- vation found immediately after irradiation. The resultant post-irradiation loss in enzyme activity is shown in Figure 4. In the Appendix, Table 4, detailed values are presented. There is a definite relationship between the rates of decrease in the enzyme activity and the concentration. The lower the concentration the faster the rate of post-irradiation loss in enzyme activity. Greater differences in the rate were obtained at the earlier rather than the later stages following irradiation. 100 90 ’3 H 3:. so H 0.) U '44 m 70 CO I: -.-1 G '3 60 E 0 H JJ ‘0 5 so 14.4 O N V 40 >5 U ~.-1 > -.-1 u g 30 Q) E. E‘ m 20 10 0 Figure 3. l9 I. O . ¥ 0 Krad l ' O 0 Krad O 0 Krad O ‘. (D 0 pH 5 8 0 pH 7 (D pH 9 \8 (I ‘9 l6 Krad 16 Kr d ' <’ a ‘_iD 1 1 1 l l 6 9 12 24 Time after irrn (hours) Effect of the post-irradiation pH on the HRPO activity. 20 Enzyme activity (% of that remaining after irrn) 100 0 I o ‘ 90 fl 80 - 0 0 Krad a o 16 Krad (1.25x10'7M protein) o 16 Krad (2.50x10'7M protein) 70 p (3 16 Krad (7.50x10'7M protein) ' o 60 - _ . . o 50 - Q o 40 - 30 - ' 20 _ 10 r 0 l J 1 1 3 6 9 12 24 Time after irrn (hours) Figure 4. Effect of enzyme concentration on the post-irradiation inactivation of HRPO. 21 The fact that dilution of the enzyme prior to irradiation resulted in a greater rate during irradiation shows that the prevalent mechanism of the enzyme inactivation upon irradiation is the indirect action. On the other hand, the differences in the post-irradiation rate of inacti- vation between the different concentrations support the following obser- vation: If there is a modification of the enzyme molecules during irradi- ation, resulting in continuous inactivation during post-irradiation storage, free radicals should contribute predominantly to this kind of modification. 5. Effect ofgglycerol. In this experiment glycerol was added to the enzyme solution after. irradiation. The radiation dose at which the enzyme solution was exposed was 16 Krad and the percentage inactivation during irradiation was 48.7%. The results of the effect of glycerol on the post-irradiation enzyme activity are shown in Figure 5. Detailed values are presented in Appendix, Table 5. Glycerol obviously exerts a great protection in the drop of post- irradiation enzyme activity. This protection was greater as the glycerol concentration was increased. For example, 24 hours after irradiation, 60% glycerol afforded almost complete protection to the enzyme from post- irradiation inactivation, whereas 30% protected the enzyme activity to the extent of 70 to 75%. 6. Effect of freezing. The effect of freezing during irradiation on the rate of post-irradi- ation inactivation of HRPO was studied in this experiment. Frozen (-38°C) enz e solutions were irradiated with 24 Krad. The loss of ym Enzyme activity (% of that remaining after irrn) 22 100 90 80 0 Krad (H20) 165Krad (H20) 16 Krad (30% glycerol) 16 Krad (60% glycerol) 70 - 00.0 60 b 50 b 40 =— 30 r 20 - 10 - Time after irrn (hours) Figure 5. Effect of addition of glycerol to irradiated HRPO on the post-irradiation inactivation of the enzyme. 23 enzyme activity during irradiation was found to be 19.4%, whereas that of the sample irradiated at room temperature (20°C) was 81-1%. The results of the effect of freezing on.post-irradiation inacti- vation are shown in Figure 6. In Appendix, Table 6, detailed values are presented. From these results it is clear that the post-irradia- tion enzyme activity was maintained in the samples irradiated in the frozen state, while that of samples irradiated at room temperature de- creased quickly with time. 7. Effect of dilution. An enzyme solution was irradiated with 16 Krad and the enzyme activity loss during irradiation was found to be 47.3%. Immediately after irradiation, the enzyme was diluted 2.5 times its volume with redistilled water. The results of the effect of dilu- tion on post-irradiation enzyme activity are shown in Figure 7. Detailed values are presented in Appendix, Table 7. The results of this experiment indicate that dilution of the irradiated enzyme solution reduced considerably the loss of post-irradi- ation enzyme activity. This is in contrast to the observation that dilution before irradiation resulted in greater inactivation during irradiation. The differences in the rate of post-irradiation inactivation in diluted and non-diluted samples are more obvious at the earlier rather than the later stages following irradiation. In both diluted and non- diluted samples, the rate of post-irradiation inactivation is not loga- rithmic. 8. Effect of irradiated water. The possible effect of radiolysis products on the enzyme activity 24 100 90 L :2 5 80 L H ~.-1 H 3 u.) 70 1" (0 00 .E O o Krad (-38°C) .9. 50 .. a o Krad ( 20°C) 2 a 24 Krad (-38°C) 8 e 24 Krad ( 20°C) .3 5o _ U ‘H O 5 40 _ >~. .LJ -.-1 > -.-4 t: 30 +- (U (D E a 20 - [:1 10 .. 0 1 I 1 1 1 Time after irrn (hours) Figure 6. Effect of freezing during irradiation on the post-irradiation inactivation of HRPO. 25 100 G - G - 0 Krad 90 =- a A O ‘7' O 5: so — -.-1 H 3 o ‘3 7o 1 o g) 16 Krad. Diluted "-4 .5. o g 60 — (D H 4..) (U '5 50 - 16 Krad. Not diluted 14-! O as: 40 1— >5 4.! ”-1 > ~.-I ‘5 30 .. co 5 (D E 2 20 L. Lt-‘l 10 _ O 1 1 l l l 3 6 9 12 24 Time after irrn (hours) Figure 7. Effect of dilution of the irradiated HRPO on its post- irradiation inactivation. 26 100 90 ’8 t: 80 h- -.-I H G) 4.) 3; 70 — O 0 Krad r020 0 Add. of irr. H20 '2 (1.25x10-7M) § (2.50x1o-7 M) 4.) E 50 - u 144 0 2‘3 40 _ >5 4.! -.-I .3 u 30 r- U (U 0) E r: 20 -— m 10 - 0 J 3 6 Time after irrn (hours) Figure 8. Effect of addition of irradiated water on the activity of HRPO. 27 was studied by irradiating redistilled water and mixing it with an enzyme solution. The results obtained appear in Figure 8 and details in Appendix, Table 8. In this experiment two enzyme solutions were prepared by mixing one part of intact enzyme solution with seven and three parts of irradiated water. From all results obtained it is clear that there are no radio- lysis products remaining in the irradiated water which are able to des- troy intact enzyme added to it. 9. Effect of addition of irradiated enzyme on the enzymatic activity of intact enzyme solution. Irradiated enzyme solution was mixed with intact enzyme solution. The radiation dose used was 24 Krad. During irradiation the enzyme lost 93% of its activity. The results of addition of irradiated enzyme to intact enzyme solution appear in Figure 9. In Appendix, Table 9 detailed values are presented. The purpose of this experiment was to determine whether radiolysis products originating from the irradiated enzyme solution could destroy the activity of the added intact enzyme. The results obtained indicated that this was not the case. There were no radiolysis products origina- ting from the irradiated enzyme solution capable of reacting and causing inactivation of intact enzyme. On the basis of these results the following mechanisms of inacti- vation of the enzyme horseradish peroxidase during and after irradiation are proposed. Indirect action must be the prevalent mechanism during irradiation, since (a) dilution of the enzyme prior to irradiation resulted in greater 100 90 g 80 H -.-4 H m U 'H 70 (I! no a ’E ‘H 60 CU B O) u J.) g 50 U ‘H 0 ES 40 >. U ‘H .3 U 30 0 CO “5’. N 20 c a: 10 0 Figure 9. 28 O ‘ 0 Krad O 24 Krad O Non-irradiated enzyme in the mixture I ‘ G I I _J 3 6 9 Time after irrn (hours) Effect of addition of irradiated HRPO on the enzymatic of intact HRPO. 29 rate of inactivation, and (b) irradiation of the enzyme solution in the frozen state resulted in a very substantial decrease of inactivation in comparison to irradiation at room temperature. During irradiation some enzyme molecules are subjected to suffi- cient change to become inactive. Other molecules remain intact and there must be a third category of molecules which although they have lost their activity, have become sensitized (excited, incompletely damaged, metastable) to further changes leading to inactivation during storage. The following two observations suggest the presence of this third category of molecules. Irradiated water added to non-irradiated enzyme did not cause inact- ivation. The existence, therefore, of stable radiolysis products ori- ginating from the water and capable of damaging the enzyme after irradi- ation is excluded. Non-irradiated enzyme added to irradiated enzyme did not result in inactivation of the non-irradiated enzyme; this indicated that no radiolysis products originating from the enzyme or the water are capable of reacting with intact enzyme molecules present in the irradiated enzyme solution. One is led to the thought that whatever partially modified enzyme molecules are present in the irradiated solution must react among them- selves in order for the enzyme inactivation to be continued after irradiation. Such a possibility is supported by the fact that dilution after irradiation resulted in lower rate of post-irradiation inactiva- tion. Incorporation of other solutes, such as glycerol, which might act as dampers in the collision between "activated" enzyme molecules, would be expected to slow down the post-irradiation inactivation; this also was observed. 30 The increase in the rate of post-irradiation inactivation as a result of increased temperature of post-irradiation storage is also compatible with this hypothesis, since higher temperatures would in- crease the effective collisions among the metastable enzyme species. These metastable enzyme Species must have been formed by indirect action (free radicals) during irradiation, since irradiation in the frozen state did not result in any appreciable post-irradiation inacti- vation and the more diluted the enzyme solution before irradiation the greater the post-irradiation enzyme activity loss. SUMMARY AND CONCLUSIONS The post-irradiation inactivation of aqueous solutions of purified horseradish peroxidase, composed of at least 11 different molecular forms, was studied. Gamma rays from Co60 were used. The following factors that might affect the post-irradiation inactivation were studied: dose of radiation; temperature during post-irradiation stor- age; pH of the enzyme solution after irradiation; concentration of the enzyme solution during irradiation; addition of glycerol to the irradi- ated enzyme solution; dilution of the enzyme solution after irradiation; freezing of the enzyme during irradiation; addition of irradiated water to intact enzyme solution; addition of intact enzyme to irradiated enzyme solution. The following results were obtained. 1. The rate of post-irradiation inactivation was increased by in- creasing the radiation dose. 2. When the post-irradiation storage temperature was increased, the rate of the enzyme inactivation was increased. 3. There was no significant difference in the rate of post-irradi- ation inactivation between pH 5 and 7. Greater rate of decrease in the enzyme activity was obtained at pH 9. 4. The higher the enzyme concentration the slower the rate of post- irradiation inactivation. 31 32 5. Glycerol added to the enzyme solution after irradiation resulted in lower rates of post-irradiation inactivation. This protection was greater as the glycerol concentration was increased. 6. The post-irradiation enzyme activity was maintained unchanged when the enzyme solution was irradiated in the frozen state (-380 C). 7. When the irradiated enzyme solution was diluted the rate of post-irradiation inactivation was considerably reduced. 8. Irradiated water added to intact enzyme solution did not cause any loss in enzyme activity. 9. Addition of irradiated enzyme to intact enzyme solution did not result in inactivation of the intact enzyme. The results suggest the following mechanism for the post-irradi- ation inactivation of HRPO: During irradiation, a portion of the enzyme which is not inactivated is modified in such a way as to result in subsequent inactivation by reacting with itself. Free radicals formed during irradiation, appear to be involved in the production of the metastable enzyme molecules. LITERATURE CITED 10. 11. 12. 13. LITERATURE CITED Alexander, P. and A. Charlesby. 1954. Nature 173, 578. Alexander, P. and A. Charlesby. 1954. Radiology Symposium, Liege, Butterworth, London 42.. Alexander, P. and D. Toms. 1956. J. Polymer Sci. 22, 343. Alexander, P. 1957. Radiation Research 6, 653. Anderson, R. S. 1947. A delayed effect of X-rays on pepsin. Biol. Bull. 93, 189. Anderson, R. S. 1954. A delayed effect of X-rays on pepsin. Brit. J. Radiol. 21, 56. Augenstein, L. and K. L. Grist. 1962. On the nature of enzyme inactivation by radiation. From "Biological Effect of Ionizing Radiation at the Molecular Level," M. Zeleny, ed. Intern. Atomic Energy Agency, Vienna. Augenstein, L. 1962. The effect of ionizing radiation on enzymes. Advan. Enzymol. 24, 359. Bacq, Z. M. and P. Alexander. 1966. From "Fundamentals of Radio- biology," revised second edition. The English Language Book Society and Pergamon Press, London. Bacq, Z. M. and P. Alexander. 1961. From "Fundamentals of Radio- biology," second edition. Pergamon Press, New York. Barron, E. S. G., S. R. Dickman, T. P. Singer and J. A. Munta. 1954. Effect of X-rays on the activity of enzymes. From "Biolog- ical Effect of External X- and gamma Radiation," R. E. Zirkle, ed. McGraw-Hill, New York. Bellamy, W. D. and E. J. Lawton. 1954. Problems in using high voltage electrons for sterilization. Nucleonics 12, (4) 54. Bellamy, W. D. and E. J. Lawton. 1955. Studies on factors affecting the sensitivity of bacteria to high velocity electrons. Ann. N. Y. Acad. Sci. 22, 595. 33 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 34 Bier, M. and F. E. Nord. 1952. On the mechanism of enzyme action. XLVII. Effect of high intensity electron bombardment on crys- talline trypsin. Arch. Biochem. Biophys. 35, 204.. Bier, M. and F. E. Nord. 1951. The effect of certain ions and of radiation on crystalline trypsin. Arch. Biochem. Biophys. 33, 355. Butler, J. V. A. 1956. The action of ionizing radiation on bio- logical material. Facts and Theories. Radiation Research 3, 20. Chance, B. and C. A. Maehly. Assay of catalases and peroxidases. ”Methods in Enzymology,” Academic Press, New York 3. Dale, W. M. 1942. The effect of X-rays on the conjugated protein D-amino-acid oxidase. Biochem. J. 33, 80. Dale, W. M., J. W. Davis and W. J. Meredith. 1949. Further observations on the protective effect in radiation chemistry. Brit. J. Cancer 3, 33. Dale, W. M. 1952. The indirect action of ionizing radiation of aqueous solution and its dependence on chemical structure of the substrate. J. Cellular Comp. Physiol. 33, Suppl. 1, 39. Dale, W. M. 1954. Basic radiation biochemistry. From "Radiation Biology," A. Hollander, ed. McGraw-Hill, New York. Dale, W. M. and C. A. Russell. 1956. A study of irradiation of catalase by ionizing radiation in the presence of cysteine, cystine and glutathionine. Biochem. J. 33, 50. Dale, W. M. 1940. The effect of X-rays on enzymes. Biochem. J. 34, 1367. Glew, G. April 8-12 1968. Changes in enzyme activity and protein solubility during storage after radiation treatment. Proceedings of a Panel, Vienna, Organized by the joint FAO/IAEA, Division of Atomic Energy in Food and Agriculture. Gray, L. H. 1954. Radiation Research 3, 189. Hannan, R. S. 1955. Scientific and technological problems involved in using radiation for preservation of food. Department of Sci- entific and Industrial Research Food Investigation, Special Report. Her Majesty Station Office, London 33. Jefferson, S. 1964. Food irradiation. From "Massive Radiation Techniques," George Newness Ltd., London. Lea, D. E. 1956. From "Action of Radiation on Living Cells," second edition. Cambridge University Press England. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 35 Okada, S. 1957. Inactivation of deoxyribonuclease by X-rays. III. Modification of the effect of radiation on the DNA-DNase system. Arch. Biochem. Biophys. 31, 113. Okada, S. 1955. Inactivation of deoxyribonuclease by ionizing radiation. 1. The kinetics of inactivation by aqueous solution. Cited by A. 0. Allen. Proc. Inter. Conf. Peaceful Uses of Atomic Energy 1, 520. Phillips, G. O. and W. Griffiths. 1965. Radiation inactivation of potato phosphorylase. Radiation Research 33, 363. Pollard, E. 1951. Ionizing radiation as a test of molecular organization. Am. Scientist 33, 99. Proctor, B. E. and S. A. Goldblith. 1952. Preservation of side effects in sterilization of food and drug by ionizing radiation. Nucleonics 39, (4) 64. Setlow, R. B. 1955. Radiation Studies of protein and enzymes. Ann. N. Y. Acad. Sci. 33, 471. Sigleton, W. R. 1958. Nuclear Radiation in Food and Agriculture. D. Van Norstand Co., Inc., Princeton, N. J. Sissakian, N. M. 1955. On the nature of change in metabolism under irradiation effect. Proc. Inter. Conf. Peaceful Uses of Atomic Energy 33, 248. Siu, R. H. 1957. Action of ionizing radiation on enzymes. "Radi- ation Preservation on Food," U. S. Govt. Printing Office, Wash- ington, D. C. Smith, C. L. 1953. The inactivation of deoxyribonuclease by electron bombardment, deuteron and head. Arch. Biochem. Biophys. £3, 83. Tievsky, G. 1962. Radiation enters the cell. "Ionizing Radia- tion," Charles C. Thomas Publ., Springfield, Illinois. Wilder, C. J. 1962. Factors affecting heat irradiation and partial reactivation of peroxidase purified by ion-exchange chromatography. J. Food Sci 31, 567-573. APPENDIX 36 Table 1. Effect of radiation dose on post-irradiation inactivation of HRPO. Time after Enzyme activity as Z of that measured irradiation Expt. immediately after irradiation (hours) 0 Krad 8 Krad 16 Krad 24 Krad O 1 99.3 96.7 95.2 2 99.2 104.6 102.4 3 101.5 98.7 102.4 Aver. 100.0 100.0 100.0 3 1 94.6 88.8 75 4 51.2 2. 95.7 92.7 71.6 58.5 3 98.0 94.7 71.6 58.5 Aver. 96.1 92.1 72.8 56.0 6 1 95.7 81.9 60.3 29.2 2 93.4 82.9 56.6 36 6 3 94.6 87.8 60.3 29.2 Aver. 94.5 84.2 59.0 31.6 9 1 94.6 79.0 55.7 23.0 2 93.4 78.9 54.0 25.8 3 94.6 80.0 55.8 24.3 Aver. 94.2 79.1 55.1 24 3 12 1 93.4 69.0 45 2 19.6 2 95.7 75.0 41 5 23 3 3 93.4 76.9 46 5 20.3 Aver. 94 1 73.6 44 4 21.0 24 1 92.3 64.8 28.9 8.5 2 92.3 64.4 28.9 13.3 3 92.3 68.3 37 7 12.1 > < (D H o \O N b) 0‘ 0‘ H w ...: oo ...: I—‘ 0 L0 1vity. Effect of post-irradiation storage temperature on the HRPO act Table 2. iation. ty as Z of that measured immediately after irrad 1vi Enzyme act 0°C 30°C 20”c 10°C Time after Expt. irradiation 24 Krad 0 Krad 24 Krad 0 Krad 24 Krad 0 Krad 24 Krad 0 Krad (hours) 103.9 100.6 103.9 100.6 103.9 100.6 103.9 100.6 98.8 98.7 100.7 98.8 98.7 100.7 98.8 98.7 100.7 98.8 98.7 100.7 97.3 100.0 97.3 100.0 97.3 100.0 ' Aver. 100.0 100.0 100.0 100.0 100.0 32.3 94.5 47.7 97.6 74.9 95.5 90.3 94.5 27.2 96.5 49.4 96.5 71.6 97.6 85.2 96.5 28.9 29.5 \0 In H Ln co 0‘ o l\ 0‘ 87 .4 Aver. 37 16.6 93.4 35.2 96.5 66.4 94.5 80.8 96.5 14.9 96.5 35.2 96.5 56.2 96.5 76.6 96.5 \0 0‘ Ln 0‘ [x on F} -¢ \0 ON Ln 0‘ Aver. 11.5 92.4 28.6 96.5 54.5 96.5 71.6 94.5 9.2 94.5 28.3 97.6 51.1 96.5 71.6 96.5 v—i r—l Aver. I-‘x'f (DE 90.4 90.4 23.5 24.5 92.4 92.4 49.6 43.4 94.5 96.5 68.1 71.5 94.5 92.4 12 4.5 91.7 26.5 24.8 96.5 93.7 45.0 46.0 94.5 95.1 64.7 68.1 94.5 96.5 Aver. 3.7 84.2 16.8 93.4 30.6 95.5 56.2 88.3 24 3.0 87.3 19.9 90.4 35.8 90.4 52.1 92.4 88.3 89.6 Aver. Table 3. Time after Enzyme activity as % of that measured immediately after irradiation 38 Effect of post-irradiation pH on the HRPO activity. irradiation Expt pH 5 pH'7 pH 9 (hours) 0 Krad 16 Krad 0 Krad '16 Krad 0 Krad 16 Krad o 1 101.4 101.7 100.5 102.0 97.6 103.0 2 98.2 101.7 103.4 100.0 103.3 97.0 3 100.4 96.6 96.1 98.0 99.1 100.0 Aver 100.0 100.0 100.0 100.0 100.0 100.0 3 1 94.3 77.5 97.5 76.7 96.2 57.7 2 95.0 77.5 102.0 79.4 93.4 53.3 3 93.9 67. 94.6 71.2 96.2 53.3 Aver. 94.4 74.2 98.0 75.7 95.2 54.6 6 1 88.2 65.4 97.5 65.7 93.4 46.6 2. 91.1 65.4 100.5 63.0 96.2 42.2 3 88.2 63.0 97.5 63.0 93.4 44.4 Aver. 89.1 64.6 98.5 63.9 94.3 44.4 9 1 85.7 48.4 96.1 52.0 92.0 35.5 2 91.1 50.9 96.1 49.3 96.2 35.5 3 87.9 54.5 96.1 52.0 92.0 31.1 Aver. 88.2 51.2 96.1 51.1 93.4 34.0 12 1 85.4 36.3 92.4 35.6 90.6 26.6 2 87.1 36.3 96.1 38.3 95.5 26.6 3 87.9 36.3 93.5 38.3 93.8 22.2 Aver. 86.8 36.3 94.6 37.4 93.3 25.1 24 1 82.5 21.8 93.1 24.6 92.0 22.2 2 82.5 24.2 91.6 27.3 90.6 22.2 3 79.7 24.2 91.6 27.3 89.2 22.2 Aver. 81.5 23.4 92.1 26.4 90.6 22.2 39 Table 4. Effect of enzyme concentration on the post-irradiation inactivation of HRPO. Enzyme activity as Z of that measured Time after immediately after irradiation irradiation Expt. 1.25x10'7M 2.5x10'7M 7.5x10'7M (hours) 0 Krad l6 Krad 16 Krad 16 Krad 0 1 98.4 100.0 93.2 104.4 2 100.0 103.5 103.4 97.0 3 101.6 96.5 103.4 98.6 Aver. 100.0 100.0 100.0 100.0 3 1 95.3 64.2 62.0 75.0 2 96 9 60.7 67.2 75.0 3 95.3 60.7 4.6 79.4 Aver. 95.8 61.8 64.6 76.4 6 1 92 3 57.1 58 6 66.1 2 96 9 54.7 59 4 64.7 3 96 9 55.9 60.3 58.8 Aver. 95 8 55.9 59.4 63 1 9 1 95.3 46 4 44.3 58.8 2 96.9 44 0 45.2 58 8 3 95.3 38.0 48.7 -- Aver. 95.8 42.8 46.1 58 8 12 1 92.3 41.6 42 6 54.4 2 93 8 36.9 41.8 57 3 3 -- 35.7 44.8 55.8 Aver 93 0 38.0 43.0 55 8 24 1 83.0 23.8 27 5 48.5 2 89.2 21.4 34 0 42.9 3 90.7 -- 33 8 44.1 Aver. 87.5 22.6 31.7 45.1 40 Table 5. Effect of addition of glycerol to irradiated HRPO on the post- irradiation inactivation of the enzyme. Enzyme activity as % of that measured Time after immediatelyyafter irradiation irradiation Expt. H20 30% glyc. 60% glyc. (hours) 0 Krad 16 Krad 16 Krad 16 Krad 0 1 98.5 104.8 95.8 101.0 2 100.0 96.2 105.2 101.0 3 101.5 99.0 99.0 98.0 Aver. 100.0 100.0 100.0 100.0 3 1 95.3 69.9 91.0 95.0 2 96.9 72.8 97.4 98.1 3 95.3 69.9 91.0 98.1 Aver 95.8 70.8 93.1 97.0 6 1 92.3 58 3 89.4 93.5 2 96.9 62 3 91.0 92.0 3 96.9 58.3 89.4 98.1 Aver 95.3 59.6 89.9 94.5 9 1 95.3 55.3 87.8 92.0 2 96.9 55.3 91.0 88.9 3 96.9 54.4 87.8 92.0 Aver. 96.3 55.0 88.8 90.9 12 1 92.3 48 5 84.6 90.4 2 93 8 51.4 87.8 88.9 3 -- 53.4 83.0 90.4 Aver 93 0 51.1 85 l 89 9 24 1 83.0 42.0 73.4 87.4 2 89.2 43.7 78.2 85.8 3 90.7 42.2 75 0 84.3 41 Table 6. Effect of freezing during irradiation on the post-irradiation inactivation of HRPO. Enzyme activity as % of that measured Time after immediately after irradiation irradiation Expt. 200/ c -38° 0 (hours) 0 Krad 24 Krad 0 Krad 24 Krad 0 1 98.7 98.0 97.5 98.4 2 101.3 98.0 99.0 100.8 3 100.0 104.0 103.5 100.8 Aver. 100.0 100.0 100.0 100.0 3 1 90.5 47.4 95.9 95.7 2 93.2 47.4 96.0 92.4 3 95.9 47.4 96.0 92.4 Aver. 93.2 47.4 95.9 93.5 6 1 91.8 36.4 91.4 90.7 2 93.2 33.0 92 9 94.1 3 94.2 30.5 94.5 90.7 Aver. 93.0 33.3 92.9 91.8 9 1 87.8 29.6 88.4 89.0 2 89.1 27.9 91.4 89.0 3 86.4 28.8 91.4 -- Aver. 87.7 28 7 90.4 89.0 12 1 87.8 18.6 88.4 87.3 2 86.4 16.9 89.9 89.0 3 -- 16.9 91.4 -- Aver. 87.1 17.4 90.0 88.1 24 1 87.3 9.3 86.8 84.0 2 85.1 12.7 89.9 87.3 3 85.1 11.8 91.4 -- Aver. 85.8 11.2 89 4 85.6 42 Table 7. Effect of dilution of the irradiated HRPO on its post- irradiation inactivation. Enzyme activity as % of that measured Time after immediately after irradiation irradiation Expt. 7.5x10’7M Diluted (2.5x10'7M) (hours) 0 Krad 16 Krad 16 Krad 0 1 100.6 102.0 103.2 2 99.4 97.5 101.6 3 100.0 100.5 95.2 Aver. 100.0 100.0 100.0 3 1 93.8 68.5 85.2 2 96.9 72.8 88.5 3 96.9 71.4 83.6 Aver. 95.9 70.9 85.7 6 1 95.3 55.3 81.9 2 95.3 61.2 85.2 3 95.3 61.2 81.9 Aver. 95.3 59.2 83.0 9 1 93.8 52.3 75.4 2 95.3 57.3 70.4 3 96.9 55.3 73.7 Aver. 95.3 54.9 73.1 12 1 89.2 49.5 69.8 2 95.3 52.4 68.1 3 93.0 51.4 69.5 Aver. 92.5 51.1 69.0 24 1 86.1 44.9 62.2 2 87 6 41.5 65.5 3 89 2 41.5 60.0 43 Table 8. Effect of addition of irradiated water on the activity of HRPO. Enzyme activity as Z of that measured immediately after irradiation Addition of irradiated H20 (24 Krad) 1 part enz.soln(10‘5M) 1 part enz.soln(10'9M) Time after plus 7 parts irr. H20 plus 3 parts irr. H20 irradiation Expt. Contro Final enzyme concentr. Final enzyme concentr. 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LIBRRRIES 31293010850745