Characterization of Active Derivatives Produced by Acetamidination and Selective Autolysis of Bovine Trypsin

Abstract

A commercial preparation of bovine trypsin was treated with methyl acetimidate-HCl, and most of the lysine residues were converted to trypsin-resistant residues retaining their cationic charges. The modified preparation was then fractionated by ion-exchange chromatography on SE-Sephadex C-50 into two active components, amidinated α- and amidinated β-trypsins. The latter component (Am-β-trypsin), which consisted of a single polypeptide chain, was al-lowed to autolyze at pH 7.8, 25°C for 3.5h and a new active component named Am-δ-trypsin was isolated from the autolysate. Several lines of experimental evidence indicated that Am-δ-trypsin was derived by primary cleavage of the bond Arg_l05-Val₁₀₆. Cleavage at Arg₅₅ Leu₅₆, on the other hand, appeared to lead to inactivation of Am-β-trypsin.
The kinetic properties of the catalytic hydrolyses of synthetic substrates and the affinity to Gly-Gly-Arg immobilized on Sepharose were compared among Am-δ-, Am-β-, and Am-α-trypsins. Am-δ-trypsin resembled Am-β-trypsin in these properties, but did not resemble Am-α-trypsin which had a cleavage at Lys₁₃₁-Ser₁₃₂.