Heparinase, immobilized

Heparinase Immobilization. Heparinase has been immobilized on a variety of supports, with a widely differing degree of success. The best results have been obtained on Sepharose and polyacrylamide. Low levels of activity recovery occurred on PHEMA. The other supports tested gave either no activity recovery or only barely detectable levels of activity (Table II). 

Heparinase, immobilized on Sepharose, has enhanced thermal stability. This effect is especially noticeable in the low-temperature storage of this enzyme. At 4°C the immobilized enzyme has a half life of denaturation of > 3600 h, compared with a 125-h half life of the native enzyme at the same temperature (Figure 5). The greater stability of the immobilized enzyme is also seen at higher temperatures 25°C, t /2 = 1,000 h 37°C, tl/2 = 15 h and 60°C, t1/2 = 0.2 h. 

Heparinase immobilized on a negatively charged support probably will result in substrate repulsion, and thus reduced activity due to the strong negative charge of heparin. This result may, in fact, explain the apparent poor activities of some of the immobilized heparinase preparations listed in Table II. 

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. 

Normally, the immobilization of heparinase to agarose catalyst particles is terminated after 4-5 h because greater than 85% of the initial heparinase is bound (49). Based on a cyanate ester stability study, the cyanate ester concentration drops to only 88% of its initial value. For modeling purposes, the cyanate ester concentration was assumed constant. In addition, because of its small size relative to the large molecular weight cutoff (1.5 x 106 daltons) of the catalyst particle, cyanogen bromide (MW 106) should diffuse rapidly into the particle and uniformly activate the matrix. 

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)]. 

Fio. 9. Effect of agitation speed on rate of heparin degradation catalyzed by immobilized heparinase at T = 37°C, pH 7.4, E = 230 units/mL, and Cb = 0.1 mg/mL. Ratio of volume of fluid phase to volume of beads, 200 1. Each point is mean of 10 independent experiments [from Bernstein et al. (50)]. 

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. 

An affinity column was prepared by immobilizing partially hydrolyzed poly(vinyl sulfate) on epoxy-activated Sepharose (25). Heparinase (HA purified) was bound to this column, and was released at either high or low pH (11 or 4) with 5-10% total activity recovery and up to 500% enrichment (21). 

To check several of the immobilization methods, chondroitinase ABC (from Proteus vulgaris) was used as a control. A summary of the activity recoveries of immobilized heparinase and chondroitinase is listed in Table II. 

Figure 4. Activity profile of heparinase. Key A, specific activity of native enzyme, and Q> specific activity of the Sepharose-immobilized enzyme.

The CNBr-activated Sepharose 4B support (1 g dry weight) was swelled in 25 mL of hydrochloric acid (0.001 Af), and then washed with 100 mL of 0.5M NaCl, 0.1 M NaHC03 buffer at pH 8.3. To this support 5.5 mL of hydroxylapatite-purified heparinase (0.2 mg/mL protein with an activity of 88 units/mg protein in 0.2M phosphate buffer at pH 7.0) and 60 mg of heparin were added. The mixture was shaken overnight at 4°C, after which the beads were washed and blocked overnight at 4°C with a solution of lysine at pH 8.2 in 0.5M NaCl, 0.1M NaHC03 buffer solution. This support showed an uptake of 87% of the protein and an immobilization of 91% of the heparinase activity. 

At present, we are continuing our efforts to immobilize heparinase to support materials in order to achieve higher yields. While this work is in progress, we have begun to explore the properties of immobilized heparinase using heparinase-Sepharose as a model. 

In Vitro Studies on Immobilized Heparinase. Initial experiments have been conducted to test the effectiveness of immobilized heparinase in removing heparin in vitro. Controls consisted of Sepharose-heparinase that was denatured by heating at 100°C for 30 min. In one set of experiments, both active and denatured immobilized heparinase were loaded into two columns, both with a 1.5-mL bed volume. Solutions of heparin, BSA (60... 

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. 

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. 

The initial tests of immobilized heparinase on heparinized blood were limited to short time periods (< 5 min). At later times, apparent decreases in heparin levels were observed in the control columns, although at a slower rate than with the active column. This effect may be due to blood damage occurring on the column. Such damage by Sepharose is not unexpected (37), and research is in progress to use either a different support with better blood compatibility or a Sepharose column with a lower bed-to-blood volume ratio. 

At present, synthetic blood filters are routinely placed at the effluent of extracorporeal devices such as the pump-oxygenator or artificial kidney to remove clots or aggregates formed during the perfusion. The filters used in oxygenators can be as large as 2 L, whereas those used in renal dialysis are only several milliliters. With further development, heparinase could be immobilized to polymers in these filters. In this case, the filter could remove both clots and heparin. 

Yan AX, Li XW, Ye YH (2002) Recent progress on immobilization of enzymes in molecular sieves for reactions in organic solvents. Appl Biochem Biotechnol 101 113—129 Yang VC, Bernstein H, Cooney CL et al. (1986) Removal of the emticoagulemt activities of the low molecular weight heparin fractions and fragments with flavobacterial heparinase. Thromb Res 44(5) 599-610... 

Heparinase, an enzyme that degrades heparin into small polysaccharides, has also been immobilized into an extracorporeal device (artificial kidney bioreactor) to eliminate the anticoagulant properties of heparin (used to prevent clotting in the device) before the blood returns to the patient. ... 

Shpigel, E. et al.. Immobilization of recombinant heparinase I fused to cellulose-binding domain, Biotechnol. Bioeng., 65, 17, 1999. 

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