Further observations on the action of chymotrypsin on insulin

Abstract

Throughout the digestion of insulin by chymotrypsin, the enzyme remained almost fully active. The digestion rate was unaffected by the presence of urea or the products of the digestion. The pH-digestion rate curve for this proteolysis had 2 optima, at pH 8.6 and 9.5. The double optimum was not due to inactivation of part of the insulin or the enzyme, and the bonds split at pH 7.6 and 9.9 appeared to be the same. Insulin was inactivated at the same rate at pH 7.6 and 9.9 by chymotrypsin. The amino groups found by the proteolysis were mostly amino acids other than tyrosine and phenylalanine. The products gave indirect support to the specificity rule suggested for this enzyme by Bergmann and Fruton (1937). The degradation had the character of an "all-or-none" reaction as postulated by Tiselius and Eriksson-Quensel (1939). Every molecule degraded underwent the fission of 13 or 14 bonds irrespective of the number of intact insulin molecules present.