The effects of surface and macromolecular interactions on the kinetics of inactivation of trypsin and α-chymotrypsin

Abstract

The autolysis of [bovine] trypsin and .alpha.-chymotrypsin is accelerated in the presence of colloidal silica and glass surfaces. Adsorption of the enzymes (favored by electrostatic factors) may result in a conformational change that renders the adsorbed enzyme more susceptible to proteolytic attack. Although the adsorbed enzymes are more susceptible to proteolysis, their activity towards low MW substrates is not affected, indicating a relatively minor conformational change on adsorption. The rates of autolysis in solution (i.e., in inert vessels) are 2nd-order for trypsin and .alpha.-chymotrypsin, with rate constants of 13.0 mol-1 .cntdot. dm3 .cntdot. s-1 for trypsin (in 50 mM-NaCl at pH 8.0 at 25.degree. C) and 10.2 mol-1 .cntdot. dm3 .cntdot. s-1 for .alpha.-chymotrypsin (in 0.1 M-glycine at pH 9.2 at 30.degree. C). In glass vessels or in the presence of small areas of silica surface (as colloidal silica particles), the autolysis of trypsin and .alpha.-chymotrypsin can show 1st-order kinetics. Under these conditions, saturation of the surface occurs and the fast surface proteolytic reaction controls the overall kinetic order. When greater areas of silica surface are present, saturation of the surface does not occur and, since for a considerable portion of the adsorption isotherm the amount adsorbed is approximately proportional to the concentration in solution, 2nd-order kinetics are again observed. A number of negatively charged macromolecules have been shown similarly to increase the rate of autolysis of trypsin: thus this effect, observed initially with glass and silica surfaces, is of more general occurrence when these enzymes adsorb on or interact with negatively charged surfaces and macromolecules. These observations explain the confusion in the literature with regard to the kinetics of autolysis of .alpha.-chymotrypsin, where 1st-order, 2nd-order and intermediate kinetics have been reported. A further effect of glass-surfaces and negatively charged macromolecules is to shift the pH-activity curve of trypsin to higher pH values, as a consequence of the effective decrease in pH in the microenvironment of the enzyme associated with the negatively charged surface or macromolecule.