The Pulse Radiolysis and Inactivation of Trypsin

Abstract

Pulse radiolysis of aqueous trypsin with 33 MeV electrons has identified the initial products with the OH radical and H atom adducts to tryptophan residues, and the electron adduct to cystine residues. Essentially, all OH reacts with tryptophan in the initial stage, with k(OH + trypsin) = 8·2 ± 1·2 × 10¹⁰M⁻¹ sec⁻¹. The disulphide bond electron adduct accounts for about 60 per cent of the primary electron yield, where k(e_aq⁻ + trypsin) = 3·5 ± 0·8 × 10¹⁰ M⁻¹ sec⁻¹. Measurements of tryptophan residue loss and sulphhydryl formation under different irradiation conditions show that the attack of OH radicals is partially reversed, except in the presence of oxygen, and that the permanent reduction of cystine residues is small compared to the initial yield and negligible when oxygen is present. There is no significant difference between the initial radiolysis reactions and permanent residue changes in trypsinogen and the active enzyme. The inactivation G value can be expressed as Σf_ig_i where g_i is the primary yield of species i under the irradiation conditions, and f_i is the probability that the attack of species i inactivates a trypsin molecule. A good fit obtains at pH 2·1 and pH 7·4 under all irradiation conditions investigated for: f_e = 0·09, f_OH = 0·05, f_H = 0·10, f_{H2O2 = 0·04, fHO2 = 0·04, and fO2 − = 0·03. The inactivation of trypsin by many radicals with low independent probabilities is explained by postulating that only a small fraction fi of the primary reactions influence the integrity of the active centre.}