Kinetics of the Tryptic Hydrolysis of Zinc-free Bovine Insulin

Abstract

The kinetics of tryptic hydrolysis of the arginyl and lysyl bonds at positions B-22 and B-29 on zinc-free bovine insulin, studied as a function of insulin concentration and pH, was compared with the results of previous studies on the oxidized B chain of insulin. When the insulin concentration was low (0.1 mg per ml or less) at pH 9.0 and 30°, the time course of the reaction was found to be satisfactorily described by first order kinetics. Under these conditions the rate constants for the hydrolysis of the arginyl and lysyl bonds of insulin were 1/45 and 1/9, respectively, of those for the hydrolysis of the corresponding bonds in the oxidized B chain of insulin. These results indicate that when the B chain is folded into the secondary and tertiary structure characteristic of the insulin molecule, both of the basic residues become more inaccessible to the enzyme than the corresponding residues in the oxidized B chain, but the effect is much greater for the arginine than for the lysine residue. The initial velocity for the hydrolysis of these two bonds was measured as a function of insulin concentration at pH 9.0 and 10.5 over the substrate concentration range of 0.01 to 10.0 mg per ml. At pH 9.0 and substrate concentrations below 0.1 mg per ml, the initial velocity was directly proportional to the substrate concentration, indicating first order kinetics. Above this concentration of insulin, the increment in the initial velocity decreased with increasing substrate concentration up to a concentration of 2 mg per ml in a fashion similar to but not adequately described by Michaelis-Menten kinetics. At concentrations above 2 mg per ml, the initial velocity again became directly proportional to the substrate concentration, which was indicative of a second region of first order kinetics. Similar results were obtained at pH 10.5, with the exception that the two ranges of first order behavior were shifted to slightly higher substrate concentrations. It is proposed that the effects of increasing substrate concentration on the initial velocity of the reaction may be attributed to aggregation of insulin molecules. At an insulin concentration of 1 mg per ml, the optimum pH for the hydrolysis of insulin occurred at pH 8.5, a value somewhat higher than that (pH 7.8) observed for model peptides. A comparison of the relative rate of hydrolysis of the arginyl to lysyl bond as a function of pH showed this ratio to change from 3.5:1 at pH 8.0 to a value of 9:1 at pH 10.5. This change in the relative rates with pH is ascribed to partial deprotonation of the e-amino group of the lysine residue at high pH.