ENZYMES ON IMMOBILIZED SUBSTRATE SURFACES - DIFFUSION

Our goal is to measure the influence of reaction on the lateral mobility of an enzyme on the surface of an immobilized substrate. We examine the mobility of collagenase on surfaces comprising immobilized peptides susceptible to cleavage by collagenase. To probe the effect of reaction on enzyme mobility, we study adsorption and subsequent movement of both active and inactive collagenase on substrate surfaces. Using the technique of total internal reflection fluorescence, we find that collagenase adsorption onto the surface is transport limited under the flow conditions used herein. After assessing the dependence of surface coverage on bulk concentration, we examine enzyme mobility at low and high surface coverages via a combined method of total internal reflection and fluorescence recovery after pattern photobleaching. Active collagenase moves laterally on the substrate surface more slowly than inactive collagenase at both low and high surface coverages indicating the interplay between the processes of reaction and surface diffusion.