Chemical and Enzymatic Properties of 60Co γ-Ray Irradiated Subtilisin BPN'

When subtilisin BPN' solutions were irradiated with 60Co γ-rays, three hydrolytic activities of the subtilisin toward N-acetyl-L-tyrosine ethyl ester (ATEE), N-benzoyl-L-arginine ethyl ester (BAEE), and casein were found to be lost at different rates with doses of 30 to 300 k rads. The esterase activity toward ATEE was most radio-sensitive and the protease activity toward casein was most radio-resistant. Behaviors of the irradiated subtilisins on electrophoresis showed that these enzymes had more electronegative charges than native ones. Two tyrosine, one tryptophan, one methionine, and one histidine residues were lost from the irradiated subtilisin which had 55% survival activity of the hydrolysis of ATEE after exposure to 110k rads of γ-rays, but still had 70% BAEE, 75% casein hydrolytic activities and 85% active site which was determined by using diisopropyl fluorophosphate. These results suggest that the radiolysis of these several amino acid residues in subtilisin carries about these new features of irradiated subtilisin against the three kinds of substrates.