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[triple chemical bond]n) (h(-1)), where C(c[triple chemical bond]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.

Poly-L-lysine amplification of protamine immobilization and heparin adsorption

“…The feasibility and utility of such approaches in eliminating diffusion limits were demonstrated in our previous work. 18 During each step of the solid phase syntheses, the reaction products were characterized by IR spectrometric analysis (Fig. 2).…”

Immobilization alters heparin cleaving properties of heparinase I

“…Heparinase immobilization was developed long ago (Bhushan et al 2017) to effect de-heparinization of human blood (Langer et al 1982;Linhardt et al 1984). Systematic investigations of parameters related to immobilization homogeneity, catalytic activity, enzyme stability, types of supports and chemistries were performed (Bernstein et al 1987a(Bernstein et al , 1987bLeckband and Langer 1991), which led to the conclusion that CNBr-activated agarose was the most optimal support to ensure isotropicity, high efficiency and stability. In this work we show that heparinase I immobilization is intrinsically capable of producing longer oligosaccharides even under a batch process.…”