Application of pulse radiolysis to the study of proteins: chymotrypsin and trypsin.

The one-electron reduction of chymotrypsin, trypsin, and their zymogens have been studied by pulse radiolysis. The optical spectra of the transient products from the two active enzymes display a pH-dependent band at 360 nm, associated with the histidine-electron adduct. The yield of the histidyl radical as a function of pH is consistent with a pK(a) less than 4.5, which suggests that the radical is located at the enzyme active site. The histidines of the proenzymes chymotrypsinogen and trypsinogen are unreactive towards the hydrated electron. We conclude that formation of the histidine-electron adduct at the serine protease active site is sensitive to the physical alterations which accompany protease activation.