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

Abstract

Previous studies in this laboratory (1) on the inactivation of dilute solutions of crystalline trypsin in 0.005 N hydrochloric acid by x-radiation at room temperature showed that the reaction yield, that is, the number of micromoles of trypsin per liter inactivated per 1000 roentgens (r), increases markedly as the initial concentration of trypsin is increased from 0.1 to 200 micromolar (~) . On the other hand, Dale and his coworkers (2) demonstrated that the reaction yield for the inactivation of aqueous solutions of crystalline carboxypeptidase by x-rays is constant for concentrations of enzyme between 6 and 600 /~M. Only a slight rise in the reaction field for the inactivation of aqueous solutions of crystalline pepsin by high voltage electrons was observed by Bellamy and Lawton (3) when the concentration of pepsin was increased from 2.8 to 140 ~s. These are the only pure enzymes that have been studied extensively over a wide concentration range.Fricke's observations (4) on the reactivity of hydrochloric acid in radiation studies raised the question whether the continuous rise in reaction field with increasing concentration of trypsin obtained in the previous studies (1) was due to the presence of hydrochloric acid in the solutions. A detailed study was therefore made of the inactivation by x-radiation of dilute solutions of trypsin in diverse media under various experimental conditions. This showed that the chemical nature of the medium in which the trypsin is irradiated markedly affects the reaction field. When solutions of trypsin of various concentrations in 0.005 N hydrochloric, sulfuric, or nitric acid were irradiated, the reaction yields were found to increase as the concentration of enzyme was increased. The rise was less pronounced in the presence of sulfuric or nitric acid, however, than in the presence of hydrochloric acid. With concentrations of trypsin between 1 and 10/~M, the reaction yields obtained in 0.005 N hydrochloric acid

Inactivation of ribonuclease and other enzymes by peroxidizing lipids and by malonaldehyde

“…A linear relationship between log of enzyme activity remaining and oxygen uptake is observed. As this relationship is similar to that for enzymes inactivated by radiation, the ratios of mole of enzyme inactivated per mole of damaging species formed were calculated on similar principles (McDonald, 1955). Quantitative determinations of enzyme inactivation were calculated for the first 10% enzyme damage and are given in Table I.…”

“…The linear relationship between log of enzyme activity remaining and lipid peroxidation, indicates the similarity between inactivation of enzyme by lipid peroxidation and ionizing radiation damage to enzymes, proteins, and amino acids. For comparison with the values of enzyme inactivated in Table I are yield values of moles of enzymes inactivated per ion pair from radiation as follows: 0.05, 0.03, 0.10, and 0.48 for invertase, catalase, cytochrome c, and ribonuclease, respectively (Setlow and Pollard, 1962); 0.20 for trypsin (McDonald, 1955); 0.01,0.02, and 0.10 for aldolase, alcohol dehydrogenase, and ribonuclease, respectively (Romani and Tappel, 1959).…”