Stability heparinase

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. 

Detailed studies have been performed on the activity and stability of heparinase. Hydroxylapatite-purified heparinase (HA) is stable to freezethawing and freeze-drying, with 90 and 87% recovered activity, respectively. Highly purified heparinase requires the addition of BSA or polylysine (0.05%) and glycerol (7.5%) to permit 100% activity recovery on freezethawing. 

Studies of the effect of pH on HA heparinase activity and stability determined that the activity maximum occurs at pH 5.8, while the stability maximum occurs at pH 7.0. 

Figure 5. Stability of heparinase. Key A, half life of denaturation of native enzyme, and Q> half life of denaturation of Sepnarose-immobilized enzyme.

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. 

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