ENZYMES ON IMMOBILIZED SUBSTRATE SURFACES - DIFFUSION

Our goal is to measure the influence of reaction on the lateral mobili ty of an enzyme on the surface of an immobilized substrate. We examine the mobility of collagenase on surfaces comprising immobilized peptid es susceptible to cleavage by collagenase. To probe the effect of reac tion on enzyme mobility, we study adsorption and subsequent movement o f both active and inactive collagenase on substrate surfaces. Using th e technique of total internal reflection fluorescence, we find that co llagenase adsorption onto the surface is transport limited under the f low 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 reflect ion and fluorescence recovery after pattern photobleaching. Active col lagenase moves laterally on the substrate surface more slowly than ina ctive collagenase at both low and high surface coverages indicating th e interplay between the processes of reaction and surface diffusion.