Distribution of heparinase covalently immobilized to agarose: Experimental and theoretical studies

Abstract

An immobilized enzyme reactor has been developed to remove heparin, the anticoagulant that is required in all extracorporeal devices for patients undergoing open‐heart surgery or kidney dialysis. The device uses the enzyme heparinase (EC 4.2.2.7), which is covalently linked to agarose with cyanogen bromide. A critical parameter in the development of a model for the degradation of heparin catalyzed by immobilized heparinase is the radial concentration profile of the enzyme within the agarose matrix. Experimental determinations of bound enzyme con centrations have been conducted previously for several enzyme systems using radioactive or fluorescent labels. For the development of the heparinase reactor it is necessary to use catalytically but not electrophoretically pure enzyme, and thus it is not possible to use the labeling techniques. To obtain information about the bound enzyme distribution, an experimental study of the intrinsic binding kinetics of heparinase to cyanogen bromide‐activated agarose was conducted. The binding reaction was studied as a function of both the concentration of heparinase and the gel‐reactive group. At conditions of functional group excess, the binding kinetics were pseudo first order in heparinase concentration with a rate constant equal to 0.12 C_cn (h⁻¹), where C_cn is the gel‐reactive group concentration. The reactive group concentration remained constant within the 2–4‐h experiments. Competitive binding between heparinase and the protein contaminants was unimportant. A model was formulated for the immobilization procedure based on the diffusion of heparinase within the porous network and the binding kinetics as determined above. The model predicted the immobilization of heparinase to be kinetically controlled and the enzyme to distribute uniformly within the agarose matrix. These experimental techniques could be applied to predict the immobilized enzyme distribution for different enzyme systems that are not electrophoretically pure.