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 alpha- and amidinated beta-trypsins. The latter component (Am-beta-trypsin), which consisted of a single polypeptide chain, was allowed to autolyze at pH 7.8, 25 degrees C for 3.5 h and a new active component named Am-delta-trypsin was isolated from the autolysate. Several lines of experimental evidence indicated that Am-delta-trypsin was derived by primary cleavage of the bond Arg105-Val106. Cleavage at Arg55-Leu56, on the other hand, appeared to lead to inactivation of Am-beta-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-delta-, Am-beta-, and Am-alpha-trypsins. Am-delta-trypsin resembled Am-beta-trypsin in these properties, but did not resemble Am-alpha-trypsin which had a cleavage at Lys131-Ser132.