Rennin from Mucor pusillus has been irradiated in 0.1 M phosphate buffer in the presence of air at 20°C. The enzyme was inactivated with a yield corresponding to G = 0.36 at pH 6.4. The extent of inactivation was the same whether the activity was measured from the ability of the enzyme to clot milk or from the enzymatic aggregation of κ-casein. Evidence was obtained that the inactivation was due to reaction of OH radicals with the enzyme. The rate of interaction of the OH radical with the enzyme was calculated to be $2.1\times 10^{10}\ M^{-1}\ {\rm sec}^{-1}$ . Disc gel electrophoresis of the macropeptides released upon incubation of native and irradiated enzyme with acid casein and κ-casein revealed no effect of irradiation on the products. Thus, the specificity of the enzyme seems to be unaffected by irradiation. The radiation inactivation was associated with a significant after-effect as well as with a decrease in the normal heat stability of the enzyme. The enthalpy of activation and increase in entropy for the aftereffect was independent of the extent of radiation inactivation, while ΔH* and ΔS* for the heat denaturation of the enzyme decreased. Addition of cysteamine after irradiation decreased both the rate and the extent of the aftereffect, as well as the rate of heat-denaturation.

Glutamate dehydrogenase from beef liver was irradiated with X-rays in solution in the presence of air. The ability of the enzyme to catalyze the oxidative deamination of glutamate and the reductive amination of pyruvate was inactivated with the same yield (G = 0.3 per protomer). The radiation inactivation of the modifying effects of ADP, GTP, sodium chloride, sulfhydryl blocking and substrate inhibition was measured. When assayed with glutamate as substrate, the different allosteric functions had the same or higher radiosensitivity than the catalytic activity. The most radiosensitive function was the substrate inhibition which was destroyed at a yield 3-4 times that of the catalytic activity. When measured with pyruvate as substrate only the GTP inhibition was more sensitive than the catalytic activity. The ADP and the sodium chloride inhibition was less radiosensitive. Thus, the allosteric effect of each particular effector showed widely different radiosensitivity when the enzyme was assayed with the two different substrates. The allosteric effects of ADP and GTP were affected in a similar way by X-rays and sodium chloride, both with glutamate and pyruvate as substrate, while sulfhydryl blocking had a different effect. The results indicate that destruction of sulfhydryl groups by X-rays is of minor importance for the radiation induced inactivation of the catalytic and allosteric activity of the enzyme.

Pectin methyl esterase from tomatoes has been irradiated in dilute aqueous solution in the presence of air at 20°C. The enzyme was inactivated with a yield corresponding to G = 0.36. Evidence was obtained that the loss of activity was due to the interaction of OH radicals with the enzyme. Cations which activate the enzymatic hydrolysis of pectin did not affect the radiation sensitivity of the enzyme. The Michaelis Menten constant for the substrate pectin was unaffected by irradiation, while <tex-math>$K_{{\rm CaCl}_{2}}$</tex-math> for the activating effect of calcium ions decreased upon irradiation. The radiation inactivation was associated with a significant aftereffect. The relative extent of the aftereffect increased nearly proportionally with the inactivation. The aftereffect was completed after 5 minutes at 55°C. The extent and rate of the aftereffect was independent of the presence of cysteamine and catalase. The enzyme showed an increased sensitivity to heat-denaturation after irradiation. The increased heat lability was associated with a decrease in ΔH* and ΔS* for heat-denaturation. Addition of cysteamine immediately after irradiation reduced the heat sensitivity to that of the unirradiated control while catalase had no effect on the heat-sensitivity.

Electron spin resonance studies have been carried out on irradiated molecular mixtures containing a macromolecule (Sephadex, CM-Sephadex or DEAE-Sephadex) and a sulfur compound (reduced or oxidized penicillamine). The pH prior to freeze-drying was in all cases adjusted to predetermined values. The irradiation as well as the electron spin resonance measurements were carried out at 77°K. The transfer of radiation energy from uncharged Sephadex occurred more efficiently to oxidized penicillamine than to reduced penicillamine when the samples were freeze-dried from acid solutions, while the opposite was the case when the samples were freeze-dried from alkaline solutions. The pH prior to freeze-drying influenced greatly the temperature range where the major part of the transfer of unpaired spins occurred from the macromolecule to the disulfide. While no further energy transfer occurred to oxidized penicillamine when the samples were heated above room temperature, an extensive transfer of unpaired spins took place from the macromolecule to the thiol in the temperature range 293°K to 333°K. This transfer seemed to involve mainly the dissociated form of the thiol. The results suggest that the pH prior to lyophilization influences the course of the secondary radical reactions occurring upon heat treatment of the samples while it has only a minor influence on the stability of the radicals present immediately after irradiation at 77°K.

Electron spin resonance studies have been carried out on frozen aqueous suspensions of different derivatives of crosslinked dextran (Sephadex, Carboxymethyl-Sephadex, and Diethylaminoethyl-Sephadex). The samples were irradiated in vacuum at 77°K with x-rays to a dose of 0.5 Mrad and the measurements were carried out at 77°K immediately after irradiation and after subsequent heat treatments. The total number of radicals stabilized in the suspensions after irradiation at 77°K was significantly higher than expected on the basis of the yields for the components. The qualitative spectra indicated that the "excess radicals" stabilized in the suspensions were mainly solute radicals. The results obtained upon heat treatment of the samples indicated that the water radicals stabilized at 77°K reacted only to a very small extent with the solute molecules. The number of "excess radicals" formed was found to depend on the electric charge of the macromolecules. Thus, many more radicals were stabilized in suspensions of the positively charged DEAE-Sephadex than in those containing negatively charged CM-Sephadex, uncharged DEAE-Sephadex, or ordinary Sephadex. The data indicate that the high yield of solute radicals observed in frozen suspensions is caused primarily by interaction of water radicals formed in the bound water with the solute. The possibility is considered that "excited water" may also contribute to the formation of solute radicals.

Electron spin resonance studies have been carried out on dry molecular mixtures containing the disulfide, oxidized penicillamine, and a macromolecule (Sephadex, DEAE-Sephadex, CM-Sephadex, Thiogel, or DNA). The samples were irradiated in vacuum with x-rays at 77°K and measurements were carried out immediately after irradiation and after heating of the samples for 10 minutes at 333°K. All electron spin resonance spectra were recorded at 77°K. The radical yields after irradiation at 77°K were in all the mixtures higher than expected from the composition. The "excess radicals" were in most cases localized on the penicillamine molecules and the number of macromolecule radicals was close to that expected. In the mixtures of CM-Sephadex and oxidized penicillamine, however, the excess radicals were localized on the macromolecules. Heat-treatment of the samples resulted in a considerable increase in the number of penicillamine radicals while the majority of the unpaired spins on the macromolecules disappeared. On the basis of the results it is suggested that in certain cases up to 60% of the total transfer of radiation energy from the macromolecules to the disulfide observed after heating had actually occurred during irradiation at 77°K. To account for the transfer of unpaired spins taking place during the heating, 40-100% of the macromolecule radicals which disappeared had to interact with the disulfide.

The sensitivity of Escherichia coli B to x-rays, in the presence and in the absence of added compounds, was studied at different irradiation temperatures in the range 0° to - 196°C. All irradiations were carried out under anoxic conditions. The radiation sensitivity was reduced by approximately 50% when the irradiation temperature was reduced from 0° to - 15°C. Concomitantly, the protective action of ethanol disappeared almost completely. Glycyl-glycine, which offered no protection at 0°C, gave increasing protection at decreasing irradiation temperatures. The protection of E. coli B at 0°C increased with increasing ability of the compounds to react with OH radicals. No correlation was found between protective ability and rate of interaction with ${\rm e}_{{\rm aq}}^{-}$ The results indicate that, under anoxic conditions, the portion of the indirect effect in E. coli B that can be abolished by freezing or high concentrations of radical scavengers contributes approximately 50% to the lethal damage and that the indirect effect is due to the action of OH radicals. The protection to be expected at 0°C by different concentrations of ethanol was calculated with the aid of reaction kinetics. The calculated results were in good agreement with those observed. The diffusion length of the OH radicals involved in the destruction of the target molecules was calculated to be about 25 Å. None of the compounds tested protected to a greater extent than could be predicted from their ability to scavenge OH radicals.

Electron spin resonance studies have been carried out on frozen aqueous mixtures, containing a macromolecule (Sephadex or Thiogel) and a sulfur compound (reduced or oxidized penicillamine). Prior to freezing, the pH of the solutions was adjusted to 3.8, 7.4, or 9.3. Similar experiments were carried out on the individual components. The samples were irradiated in vacuum at 77°K with X-rays to a dose of 0.5 megarad, and the measurements were carried out immediately after irradiation and after subsequent heat-treatments to temperatures in the range 77° to 263°K. After heat-treatment the samples were brought back to 77°K and the spectra were recorded. The intermolecular transfer of unpaired spins from Sephadex or Thiogel to reduced penicillamine was found to be much greater in the acid than in neutral and alkaline solutions. In contrast, the transfer to oxidized pencillamine was independent of pH. The results indicate that the transfer of unpaired spins from the macromolecule is greater to the undissociated than to the dissociated thiol group, and that hydrogen transfer repair reactions may occur in frozen aqueous mixtures.

The mechanism of formation of secondary radicals in x-irradiated proteins has been studied by electron spin resonance spectroscopy. Ribonuclease, thiolated gelatin, and egg albumin were irradiated at 77°K, alone, as well as in the presence of oxygen or adenine. The spectra were recorded immediately after irradiation and after heat-treatment to predetermined temperatures. The presence of high concentrations of adenine during irradiation prevented the formation of sulfur radicals in ribonuclease and doublet-type radicals in thiolated gelatin. The presence of oxygen did not alter the radical yield or the qualitative spectra observed at 77°K. Oxygen, like adenine, prevented the formation of the typical secondary radicals on heat-treatment. Qualitative spectra were observed which were ascribed to transient HO 2 radicals, presumed to be formed by decomposition of organic peroxide radicals. The results are interpreted to mean that in proteins irradiated in vacuum the secondary radicals formed on heat-treatment arise largely by intermolecular reactions mediated by diffusible radicals, primarily H atoms, which can be scavenged by a second component like adenine.

Electron spin resonance studies have been carried out on frozen aqueous mixtures containing a macromolecule (Sephadex or Thiogel) and the thiol, penicillamine. The samples were irradiated in vacuum with X-rays at 77°K, and the measurements were carried out immediately after irradiation and after subsequent heat treatments. All electron spin resonance measurements were carried out at 77°K. Before heat treatment the radical yields were only slightly higher than expected on the basis of the composition of the mixtures. However, after heat treatment to 223°K the observed number of unpaired spins remaining in the mixture was much greater than expected. Decomposition of the spectra demonstrated that the excess radicals were localized on penicillamine. For low concentrations of penicillamine the number of penicillamine radicals in the mixture was twelve to seventeen times as great as that found when a pure penicillamine solution was studied. However, when the penicillamine concentration exceeded a certain value, the efficiency of the transfer of unpaired spins to penicillamine decreased. The results show that in frozen aqueous mixtures of macromolecules and a thiol an appreciable transfer of unpaired spins may occur from the macromolecule to the thiol. The data indicate that this transfer occurs mainly from a limited number of sites on the macromolecules.

Solid mixtures of Sephadex and penicillamine, irradiated at 77°K, were studied by ESR technique. Sephadex is a modified dextran containing a network of interstices and pores. The degree of mixing between Sephadex and penicillamine was altered by varying the amount of water in the suspension prior to lyophilization. It was found that the qualitative and quantitative ESR spectra depend strongly on the extent of contact between the two components of the mixture. Evidence was obtained that secondary reactions, resulting in transfer of radiation energy from Sephadex to penicillamine, take place at 77°K. On heat-treatment of the mixtures, irradiated at 77°K, the organo-sulfur radicals <tex-math>${\rm R}-{\rm C}({\rm CH}_{3})_{2}-{\rm S}\cdot $</tex-math> are formed to a much greater extent than if no intermolecular reactions had taken place. The results demonstrate that the formation of sulfur radicals increases with the degree of mixing between the two components.