Changes in optical density, amino acid composition, and fluorescence of papain inactivated by hydroxyl radicals and hydrogen peroxide

Chromatography of irradiated papain on an affinity column with the Gly-Gly-Tyr(Bzl)-Arg inhibitor peptide gave rise to three clearly resolved peaks. The first one was relatively small and contained completely inactive nonreparable enzyme, which appeared to have suffered a massive conformational change or loss of several binding sites. The second contained the inactive sulfenic acid derivative, which can be reactivated with cysteine. The third peak was composed of nonrepairable enzyme as well as some repairable enzyme and some fully active papain. Changes in absorbance and amino acid analysis established a significant loss of tyrosine residues, while tryptophan destruction appeared to be insignificant up to 10 krad. Fluorescence measurements indicated changes in the active-site region, which are probably largely due to the inactivating modification of the Cys-25 sulfhydryl group, for which evidence has already been reported