Enzymatic Attack on Immobilized Substrates

Abstract

1. The chyrnotrypsin-catalyzed hydrolysis of polyacrylamide-bound L-phenylalanine 4-nitroanilide was studied. As a spacer, one or two 6-aminohexanoyl residues were inserted between the matrix and ligand.2. In the course of the enzymatic hydrolysis of polyacrylamide-bound substrates, enzyme adsorption by the gel substrates was observed. A quasi equilibrium of enzyme partitioning was reached after approximately 20-min incubation time. The enzyme adsorption could be described by the Langmuir adsorption isotherm.3. The substrates attached via spacers to the matrix were completely hydrolyzed. 4. The initial course of the product vs time curves, as well as the dependence of the initial hydrolysis rates on enzyme concentration or substrate concentration, have been interpreted by the Nernst reaction theory. From the results obtained it has been concluded that the initial rate of the hydrolysis of polyacrylamide-bound L-phenylalanine 4-nitroanilide depends on the velocity of enzyme diffusion into the matrix.Results obtained with agarose-bound L-phenylalanine 4-nitroanilide show that the diffusion of the enzyme into the substrate gel does not influence the chymotrypsin-catalyzed hydrolysis to a significant degree. On the other hand, only a definite part of the bound ligand, dependent on the spacer length, is accessible to enzymatic attack [l]. This behaviour is presumably due to the structure of agarose [ 2 , 3 ] . The gross conformation of most synthetic polymers may be described by the model of random coil [4]. In comparison with agarose gels, synthetic crosslinked gels contain, on an average, smaller pores and a more flexible matrix. These parameters of synthetic gels depend on the degree of cross-linking.In the present paper we have tried to study the influence of a matrix different from agarose on the enzymatic hydrolysis of immobilized low-molecularweight substrates. For this purpose polyacrylamide (Biogel P100) was chosen. In polyacrylamide the interactions of the polymeric chains should be restricted to hydrogen bonds between the carboxyl amide groups Abbreviations. EAhx-Phe-Nan, 6-aminohexanoyl-~-phenylalanine 4-nitroanilide ; (EAhx)2-Phe-Nan, 6-aminohexanoyl-6-aminohexanoyl-L-phenylalanine 4-nitroanilide; P-, polyacrylamidebound; A-, agarose-bound; PhMeS03-, phenylmethanesulfonyl-.Enzyme. Chymotrypsin (EC 3.4.21.1).if cross-linking is neglected. As in our studies with agarose-bound substrates 111, L-phenylalanine 4-nitroanilide interposed by either one to two 6-aminohexanoyl residues was bound to polyacrylamide and used for the investigation of both the behaviour of protein adsorption and enzymatic hydrolysis. MATERIALS AND METHODS MaterialsBiogel PI00 was purchased from Bio-Rad Laboratories and crystalline chymotrypsin was purchased from Spofa (Prague). All other reagents were the purest commercially available. Synthesis of Polyacrylamide-Bound SubstratesBiogel P100, containing azide groups, was prepared according to Inman and Dintzis [5]. The ligands were coupled to the matrix in 0.2 M carbonate buffer/dimethy...