Protease Activity on an Immobilized Substrate Modified by Polymers:  Subtilisin BPN‘

Abstract

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 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 (CH2 > 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. Materials and MethodsEnzyme Solution. The steps used to isolate, purify, and prepare a stock solution of the serine protease used here, subtilisin BPN′ (MW ) 27 534 g/mol), from Bacillus amyloliquefaciens are outlined elsewhere. 14 Prior to use, the enzyme stock solution is

Fluorescent-conjugated polymer superquenching facilitates highly sensitive detection of proteases

“…However, as was observed for EK and EK-1, addition of enzyme even at large excess failed to hydrolyze the peptide substrate when bound to either microsphere or solution sensor (monitored by HPLC and fluorescence recovery experiments). It is possible that the enzyme is hindered from accessing the binding site on the peptide either through steric interactions with the polymer or by means of the irreversible adsorption of the enzyme to the hydrophobic polymer (21,22). The introduction of a poly(ethylene glycol) functional group between the biotin and the peptide substrate was expected to improve the situation in one of the following ways: increase the distance between the cleavage site and the polymer to prevent steric hindrance to approach by the enzyme or reduce the hydrophobicity and, thus, reduce adsorption of enzyme to the polymer (23).…”

Immobilization of Lipid Substrates: Application on Phospholipase A2 Determination

“…On the contrary, the immobilization of the substrates for the determination of the enzymic activity has not met a wide implementation yet. With the advent of micro-array technology, a large variety of new applications of enzyme action on solid-phase supported substrates have been rapidly developed [16][17][18][19][20]. Substrate immobilization on solid surfaces is becoming increasingly important, especially in the case of photosynthetic materials which often present a relatively short active lifetime limiting their effective use [21,22].…”

Enhanced functionalization of Mn2O3@SiO2 core-shell nanostructures

“…Yoshitake et al [ 6 ] in their studies have shown that the captured transition metal ions on amino-functionalized silica act as adsorption centers for arsenate ions. Surface-functionalized silica particles have found applications in catalysis [ 7 - 9 ], sensors [ 7 , 10 ], and protein immobilization [ 11 , 12 ]. Also, functional groups have been incorporated into silicate surfaces to facilitate molecular imprinting of those surfaces to form highly specific biomimetic catalytic or adsorbent materials [ 13 - 15 ].…”