Protease activity on an immobilized substrate modified by polymers:: Subtilisin BPN′

We describe the adsorption and catalytic behavior of the serine protease subtilisin BPN' on controlled pore glass (CPG) beads with a short (aminopropyl) or a long (aminoalkyl CH(2) > 12) chain covalent link separating the reporter peptide succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide (sAAPFpNA) from the surface. The propyl-linked sAAPFpNA modified glass surface (aminopropyl CPG:sAAPFpNA) showed a 2-fold increase in protease adsorption over an aminopropyl-glass surface. In contrast, the sAAPFpNA surface with the long chain connector showed a 2-fold drop in adsorption relative to an aminoalkyl surface. BPN'-catalyzed hydrolysis rates showed an inverse relationship to adsorption. Water-soluble polymers [poly(vinylpyrrolidone) (PVP), poly(ethylene oxide) (PEO), poly(4-vinylpyridine-N-oxide) (PVPO) and a copolymer of 1-vinyl-2-pyrrolidone and 1-vinylimidazole (PVPVI)] neutralize the 2-fold increase in BPN' adsorption and provide more than a 3-fold increase in the initial rate of hydrolysis far BPN'-catalyzed cleavage of pNA. Another water-soluble polymer, poly(vinyl alcohol) (PVA), causes only a slight adsorption decrease and hydrolysis increase for the BPN', aminopropyl CPG:sAAPFpNA system. None of the polymers causes a significant change in BPN'-catalyzed hydrolysis of, or adsorption on, aminoalkyl (CH(2) > 12) CPG:sAAPFpNA. The apparent mechanism behind these effects is one in which the long alkyl chains and adsorbed polymers decrease the amount of adsorbed enzyme and increase the amount available for reaction in solution. A model is presented which describes the relationship between adsorption and surface hydrolysis.