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 CH2 > 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 for BP...