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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...

The adsorption of the enzymes, subtilisin BPN' and lysozyme, onto model surfaces was examined using attenuated total reflectance (ATR) infrared (IR) spectroscopy. Using a cylindrical internal reflection (CIRcle) cell with a Germanium (Ge) internal reflection element (IRE), model hydrophilic surfaces were made by plasma cleaning the IRE and model hydrophobic surfaces were made by precoating the IRE with a thin film of polystyrene. Gas chromatography (GC)-IR data collection software was used to monitor adsorption kinetics during the first five minutes after injection of the enzyme into the CIRcle cell. It was found that for both lysozyme and BPN', most of the enzyme that was going to adsorb onto the model surface did so within ten seconds after injection. Nearly an order-of-magnitude more BPN' adsorbed on the hydrophobic Ge surface than the hydrophilic one, while lysozyme adsorbed somewhat more strongly to the hydrophilic Ge surface. Overnight, the lysozyme layer continued to increase in thickness, while BPN' maintained its initial coverage. The appearance of carboxylate bands in some of the adsorbed BPN' spectra suggests the occurrence of peptide bond hydrolysis. A Au/Pd coating on the CIRcle cell o-rings had a significant effect on the adsorption of BPN'. (This coating was applied in an attempt to eliminate interfering Teflon absorption bands.) An apparent electrochemical reaction occurred, involving BPN', Ge, Au/Pd, and the salt solution used to stabilize BPN'. The result of this reaction was enhanced adsorption of the enzyme around the coated o-rings, etching of the Ge IRE at the o-ring site, and some autolysis of the enzyme. No such reaction was observed with lysozyme.