Protease Adsorption and Reaction on an Immobilized Substrate Surface

Abstract

Enzymatic reactions with surface-bound substrates present an interesting problem in biomolecular surface science, as they require us to consider traditional enzyme kinetics in the context of protein adsorption. These reactions are important in such applications as detergent enzyme additives, food processing, and contact lens cleaning. We study the interaction of a serine protease (subtilisin) with an immobilized substrate (bovine serum albumin) surface through the simultaneous use of surface plasmon resonance and surface plasmon enhanced fluorescence techniques. We measure adsorbed enzyme concentrations and substrate cleavage rates in situ and compare the reactivities with those in solution. By varying the ionic strength of the reaction environment and studying several single point mutations of subtilisin, we find the adsorption behavior of the enzyme is strongly influenced by its electrostatic interactions with the charged bovine serum albumin surface. The surface reactivity of each of the mutants is cou...