低分子肝素修饰超氧化物歧化酶及修饰酶的理化性质研究

Chemical Modification of Superoxide Dismutase by Low Molecular Weight Heparin and Their Physical and Chemical Properties

目的用低分子肝素(LMWH)化学修饰超氧化物歧化酶(SOD),并研究修饰酶(LMWH-SOD)的理化性质,以期获得高稳定性的酶。方法采用高碘酸钠法活化LMWH,与Cu,Zn-SOD共价结合;凝胶过滤层析法和SDS-PAGE法测定其相对分子质量 (Mr);分别用Folin-酚法、Elson-Morgan法、Bitter-Muir咔唑法、Teho法测定其酶蛋白、氨基葡糖、葡糖醛酸及硫酸基含量;用水浴、酸碱缓冲液及蛋白酶分别考察其耐热、耐酸碱及抗蛋白酶酶解能力。结果用LMWH成功修饰Cu,Zn-SOD;修饰酶Mr增大且不均一,耐热、耐酸碱及抗蛋白酶酶解能力均提高。结论用LMWH可成功修饰SOD,并改进SOD性能。

OBJECTIVE To chemically modify superoxide dismutase （SOD） with low molecular weight heparin（LMWH） and study the physical and chemical properties of the modified enzyme（LMWH-SOD）, in order to make the enzyme with higher stability.METHODS LMWH was activated by sodium periodate and then combined with Cu,Zn-SOD covalently. The relative molecular weight （Mr） of the modified enzyme was measured by gel filtration chromatography and SDS-PAGE, the contents of enzyme protein, aminoglucose, glucuronic acid and sulfate group were determined by Lowry＇s method, Elson-Morgan＇s method, Bitter-Muir＇s method and Teho＇s method, respectively, and the stabilities of Cu,Zn-SOD to heat, pH and protease digestion were studied under high temperature, acidic and basic buffer, pepsin and trypsin treating condition.RESULTS Cu,Zn-SOD was successfully modified by LMWH. The obtained LMWH-SOD had bigger Mr than Cu,Zn-SOD did and the Mr was heterogeneous. LMHW-SOD was evidently superior to unmodified SOD in terms of their tolerances to high temperature, acid, base, pepsin and trypsin. CONCLUSION LMWH can be used to modify SOD and improve the properties of the enzyme.