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 coupled to its adsorption properties. On the basis of these findings, we propose a modified Michaelis-Menten enzyme surface adsorption and reaction model.

Fluorescent-conjugated polymer superquenching facilitates highly sensitive detection of proteases

“…However, as was observed for EK and EK-1, addition of enzyme even at large excess failed to hydrolyze the peptide substrate when bound to either microsphere or solution sensor (monitored by HPLC and fluorescence recovery experiments). It is possible that the enzyme is hindered from accessing the binding site on the peptide either through steric interactions with the polymer or by means of the irreversible adsorption of the enzyme to the hydrophobic polymer (21,22). The introduction of a poly(ethylene glycol) functional group between the biotin and the peptide substrate was expected to improve the situation in one of the following ways: increase the distance between the cleavage site and the polymer to prevent steric hindrance to approach by the enzyme or reduce the hydrophobicity and, thus, reduce adsorption of enzyme to the polymer (23).…”

Label-Free High-Throughput Functional Lytic Assays

“…The digestion signature for biopolymers on a biosensor is a complex signal that is derived from a number of factors, such as enzyme surface activity, molecular stoichiometry of the enzyme to substrate reaction on a surface, substrate orientation on the surface, enzyme mobility on a surface, enzyme adsorptivity on a surface, number of cleavage sites in the substrate, size of fragments that leave the sensor surface, and the ability of fragments to leave the sensor. 17,18 The idiosyncrasies of each protein-specific digestion profile were found to be relatable to expected digestion-specific properties. The albumins and their modified forms offered a reasonable means of assessing the digestion signal on a biosensor.…”

Proteolytic cleaning of a surface-bound rubisco protein stain

“…Enzyme activities (proteolysis rates) in bulk and on surfaces are usually poorly correlated (Kim et al, 2002), and accordingly, bulk reaction studies cannot be used as indicators for reactions occurring on surfaces (Brode and Rauch, 1992;Gaspers et al, 1995). This is because in bulk, the enzyme molecules freely diffuse (in the case of stagnant solutions) until they find the protein molecules, leading to the formation of reactive complexes that dissociate into products and release the enzyme.…”

“…For example, Foose et al (2007) expressed the proteolysis (cleavage) rate of immobilised ovalbumin as the slope of the film thickness gradient with time. Kim et al (2002) limited their treatment of the proteolysis data of immobilised BSA to the low substrate concentration region, and therefore proposed a simple proteolysis rate equation similar to that obtained from the treatment of the proteolysis rate based on Lineweaver-Burk linearisation. Brode and Rauch (1992) studied the adsorption of subtilisin BPN onto an immobilised peptide substrate, however, they treated the reaction data in terms of Lineweaver-Burk linearisation method, without considering enzyme adsorption.…”

“…Therefore, these processes must be decoupled to obtain unique values of the adsorption and proteolysis rate constants. This decoupling might be achieved by using a fluorescence technique (Kim et al, 2002) in which either the enzyme or the protein is labelled irreversibly. However, labelling the enzyme can undesirably alter its activity, while protein labelling might modify protein-surface and protein-enzyme interactions.…”