Enzymes heparinase

Extracts of Flavobacterium heparinum that had been induced to grow on heparin-like polysaccharides contain a number of enzymes that may ultimately degrade heparin and heparan sulfate to monosaccharides.137,145 240 The enzymes that cause the primary cleavage of heparinlike chains are heparinase (EC 4.2.2.7) and heparanase (EC 4.2.2.8, formerly called heparitinase241,242). 

Scheme 5.—Typical Cleavage of Heparin (Arbitrary Sequence) with Heparinase (HEPase) and Heparanase (HSase). [Fragments longer than a tetrasaccharide may be obtained by separate use of each enzyme. The combined use of the two enzymes produces only disaccharides.]...

Although the size distribution of fragments from heparinase (and hep-aranase) digests reflects the relative content of regular sequences 5 in different heparin preparations and fractions, these sequences may be quantitated only when the enzyme efficiency is high, and products are... 

The sensitivity of this method is directly related to the apparent molar enthalpy of reaction, so that very endo- or exothermic reactions will be most readily followed. Examples of the application of this method to the determination of enzyme kinetic parameters include dihydrofolate reductase, creatine phosphokinase, hexo-kinase, urease, trypsin, HIV-1 protease, heparinase, and pyruvate carboxylase. 

An abscess begins by the combined action of inflammatory cells (such as neutrophils), bacteria, fibrin, and other inflammatory mediators. Bacteria may release heparinases that cause local thrombosis and tissue necrosis or fibrinolysins, collagenases, or other enzymes that allow extension of the process into surrounding tissues. Neutrophils gathered in the abscess cavity die in 3 to 5 days, releasing lysosomal enzymes that liquefy the core of the abscess. A mature abscess may have a fibrinous capsule that isolates bacteria and the liquid core from antimicrobials and immunologic defenses. 

In developing a reactor such as the one just described, it is important to understand important design parameters, such as the radial distribution of the enzyme within the catalyst particles, the kinetics of heparin degradation catalyzed by immobilized heparinase, the flow properties in the reactor, and the effect of in vivo factors such as blood proteins which bind to the substrate. These parameters and how they can be evaluated are now discussed. 

The heparin degradation rate at any radial position inside the catalyst particle is proportional to the bound heparinase concentration at that position. If the immobilized enzyme concentration is not uniform, the conventional analysis of simultaneous diffusion and reaction within a porous catalytic particle must be modified. The reaction rate within the catalyst particle will have an explicit radial dependence introduced via the enzyme concentration, as well as a dependence on the substrate concentration. 

The first step in characterizing the heparinase binding rate to the catalyst particles is to establish experimental conditions where neither enzyme denaturation or external mass transfer are important. This can be accomplished by controlling the duration of immobilization, the mixing rate, and the catalyst particle size. In the absence of diffusional limitations and enzyme denaturation effects, the disappearance of enzymatic activity from the bulk phase equals the rate at which the enzyme binds to the catalyst particle. The molar conservation equation for heparinase in the bulk phase is given by... 

A model of the actual immobilization process with intact spherical catalyst particles was developed using the experimentally determined binding kinetics (48). The system was treated as a group of porous spheres suspended in a well-mixed solution of heparinase. The enzyme diffused through the porous network, where it reacted with the surface cyanate esters to produce the bound enzyme. 

Kp is a partition coefficient of the enzyme in agarose and kg is a mass transfer coefficient. Although predictions can be made about the bound-enzyme profile, it is not possible to directly verify them experimentally because of the protein impurities in heparinase. However, it is possible to measure the bulk heparinase concentration during the course of an immobilization and compare it to the model predictions. A series of immobilizations were conducted using intact agarose spheres with different cyanate ester concentrations and the bulk heparinase concentration monitored. 

Fig, 7. Data and model predictions of dimensionless bulk heparinase concentration during course of enzyme immobilization at 4°C, pH 7.0. Vf = 6.8 mL, VB = 3.6 mL, and Cc = = 9.8 iimol/g. Each point is mean of three independent experiments, and all samples assayed in duplicate. Error bars are size of points. Line is model prediction [from Bernstein el al. (48)]. 

The degradation of heparin by the reactor is a multistep process. Heparin and the heparin-antithrombin complex must first diffuse from the bulk phase to the surface of the immobilized enzyme particle. The two species diffuse into the agarose particles where they encounter immobilized heparinase. The heparin-anti thrombin complex is assumed to be sterically inhibited from binding to immobilized heparinase, and under these conditions only unbound heparin is enzymatically degraded. As unbound heparin is consumed, heparin dissociates from the heparin-antithrombin complex to generate more free heparin. The breakdown of heparin is given by the following chemical reaction ... 

Several other reactors for immobilized heparinase have been designed (53,54). The initial reactor (47) caused no more blood damage than conventionally used extracorporeal devices such as the artificial kidney machine (54a). By controlling the mode of immobilized enzyme bead suspension, all blood damage can be essentially eliminated (54). The FDA... 

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. 

IEF also was applied towards the HA-purified enzyme to obtain highly pure heparinase. The enzyme was loaded onto a prefocused acrylamide gel at pH 7.0. After IEF, the enzymatic activity was recovered at pH 8.5 0.5. The resulting enzyme had a specific activity of about 5000 units/mg protein, having undergone an enrichment of 50-fold (21). 

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 enzyme is very specific, acting only on heparin (Km = 4.2 X 10-5M) and heparin monosulfate. Heparinase acts endolytically as an a-1, 4-elimi-nase cleaving heparin (MW = 10,000) at 7 to 8 sites (42). 

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