Purification and characterization of heparinase from Flavobacterium heparinum.
Purification and characterization of heparinase from Flavobacterium heparinum.
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发表时间：
1985-02
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通讯作者：
V. Yang;R. Linhardt;H. Bernstein;C. Cooney;R. Langer
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作者：
V. Yang;R. Linhardt;H. Bernstein;C. Cooney;R. Langer
从肝素黄杆菌分离的肝素酶（EC 4.2.2.7）通过羟基磷灰石层析、重复凝胶过滤层析和层析聚焦的组合纯化至均一。通过在十二烷基硫酸钠和酸-尿素凝胶电泳系统上存在单一条带来确定均一性。氨基酸分析表明，该酶含有相对较高量的赖氨酸残基（9%），与其阳离子性质（pI 8.5）一致，但仅含有4个半胱氨酸残基/多肽。肝素酶的分子量估计为42，900 +/-1，000道尔顿（凝胶过滤法）和42，700 +/-1，200道尔顿（十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法）。该酶是非常具体的，仅作用于肝素和硫酸乙酰肝素的12个类似的多糖底物测试。它在pH 6.5和0.1 M NaCl下具有最大活性，在pH 7.0和0.15 M NaCl下具有最大稳定性。Arrhenius活化能为6.3 kcal/mol。然而，该酶对热变性非常敏感，在超过40摄氏度的温度下非常迅速地失去活性。在37 ℃下肝素酶反应的动力学研究得到在0.5微克/毫升蛋白浓度下的Km为8.04 × 10（-6）M和Vm为9.85 × 10（-5）M/min。通过采用羟基磷灰石和QAE（季氨乙基）-Sephadex层析的分批程序，克量的肝素酶可以在4-5小时内纯化，几乎不含催化酶污染物。
Heparinase (EC 4.2.2.7) isolated from Flavobacterium heparinum was purified to homogeneity by a combination of hydroxylapatite chromatography, repeated gel filtration chromatography, and chromatofocusing. Homogeneity was established by the presence of a single band on both sodium dodecyl sulfate and acid-urea gel electrophoretic systems. Amino acid analysis shows that the enzyme contains relatively high amounts of lysine residues (9%) consistent with its cationic nature (pI 8.5) but contains only 4 cysteine residues/polypeptide. The molecular weight of heparinase was estimated to be 42,900 +/- 1,000 daltons by gel filtration and 42,700 +/- 1,200 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is very specific, acting only on heparin and heparan monosulfate out of 12 similar polysaccharide substrates tested. It has an activity maximum at pH 6.5 and 0.1 M NaCl and a stability maximum at pH 7.0 and 0.15 M NaCl. The Arrhenius activation energy was found to be 6.3 kcal/mol. However, the enzyme is very sensitive to thermal denaturation and loses activity very rapidly at temperatures over 40 degrees C. Kinetic studies of the heparinase reaction at 37 degrees C gave a Km of 8.04 X 10(-6) M and a Vm of 9.85 X 10(-5) M/min at a protein concentration of 0.5 microgram/ml. By adapting batch procedures of hydroxylapatite and QAE (quaternary aminoethyl)-Sephadex chromatography, gram quantities of heparinase that is nearly free of catalytic enzyme contaminants can be purified in 4-5 h.