The Inactivation of Dilute Solutions of Crystalline Trypsin by X-Radiation

Abstract

Numerous investigators (1) have shown that exposure of solutions of enzymes to ionizing radiation results in loss of enzymatic activity. Dale and his coworkers (2, 3) demonstrated that the enzyme molecules are not inactivated by x-rays directly, but indirectly, through reaction with intermediate products formed by the action of the ionizing radiations on the solvent. The hypothesis of the indirect action of radiation was originally proposed and developed in great detail by Risse (4) and Fricke et al. (5)(6)(7), who used principally inorganic and simple organic compounds as their test materials. Such indirect reactions are usually characterized (6, 8) by a reaction yield which is independent of the concentration of the solute when this is above a certain value characteristic of the system. For example, the ionic yield for the inactivation of solutions of carboxypeptidase by x-radiation was found (9) to be constant for all concentrations greater than 6 X 10 -6 M. This, however, is the only pure enzyme for which published data covering a wide range of concentrations are available. When preliminary experiments in this laboratory (10) on the inactivation of solutions of crystalline trypsin ranging from 1 X 10-7 to 2 X 10 -4 ~ demonstrated that the reaction yield increased continuously with increasing concentrations of trypsin, a detailed study of the phenomenon was undertaken. Some of the results of these investigations, using 0.005 ~r hydrochloric acid as the solvent, are presented in this paper. The results of experiments in which other solvents were used will be published subsequently. Materials and MethodsPreparation of Trypsin.--The trypsin used throughout this work was crystallized by the procedure of McDonald and Kunitz (11) from crystalline trypsinogen that