Heparinase purification

We propose a new approach that would allow the full heparinization of the extracorporeal device, yet could enable, on-demand, elimination of heparin in the patient s bloodstream. This approach consists of a blood filter containing immobilized heparinase, which could be placed at the effluent of any extracorporeal device (Figure 1). Such a filter could theoretically be used to eliminate heparin after it had served its purpose in the extracorporeal device and before it returned to the patient. In this chapter we discuss our efforts to develop such a filter. Our work has focused on several areas (1) enzyme production (2) enzyme purification (3) characterization of heparinase (4) immobilization of heparinase and (5) in vitro testing of immobilized heparinase. 

As a further purification technique, affinity chromatography was explored. In preliminary experiments, a heparin-Sepharose column failed to bind heparinase. We therefore searched for a competitive and reversible heparinase inhibitor to act as a ligand. Three synthetic heparin substitutes— poly(vinyl sulfate), polyanethole sulfonate, and polystyrene sulfonate—met these requirements. The inhibitory effect of poly(vinyl sulfate) (K = 3.0 X 10 8M MW 10,000) appeared to be linked to the presence of sulfate groups because inhibition was lost when poly(vinyl sulfate) was hydrolyzed. 

The purification of heparinase has been followed by SDS gel electrophoresis. The crude sonicate gave more than 20 major bands the HA purified enzyme, 3 major bands and the IEF purified enzyme, 2 major bands. A summary of the specific activities, protein recoveries, and enzyme purity obtained using our purification procedures is listed in Table I. 

The development of the heparin removal system is still at an early stage. Work currently is being directed toward (1) completing the purification of heparinase, (2) immobilizing heparinase to additional supports, and (3) testing the blood compatibility and effectiveness of heparinase reactors in vitro and in vivo. 

One of the critical factors in our research has been the adaptation and use of multiple assays to follow heparinase activity. Particularly important were assays (e.g., Azure A) used in monitoring the fermentation and early stages of purification. By utilizing three different approaches for assaying heparin (disappearance of heparin, appearance of reaction products, and disappearance of heparin s biological activity), the occurrence of any arti-... 

While our studies on heparinase production and purification have been encouraging, less success has been achieved in the immobilization procedures (Table II). Studies are in progress to understand better the important parameters in immobilization procedures and in establishing new supports. Initial results indicate that a noncharged support with a high surface area is best (Table II). Additionally, our preliminary evidence is that high levels of heparinase (> 1 mg/mL) and the presence of substrate in the immobilization reaction enhance the recovery of immobilized enzyme activity. 

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