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目的从中华眼镜蛇毒中分离纯化核糖核酸酶,并研究其生物学活性。方法以中华眼镜蛇毒为原料,采用SP-Trisacryl阳离子交换色谱、Sephadex G-75凝胶色谱、C8反相色谱等纯化方法,分离纯化具有核糖核酸酶活性的蛋白质,表征其酶学性质,检测其抑菌性、抗肿瘤细胞作用及抗氧化作用。结果 SDS-PAGE电泳显示纯化的中华眼镜蛇毒核糖核酸酶(Na-RNase)为相对分子质量为13 000的单一成分。该酶最适反应温度为40℃,最适pH值为6.5,米氏常数为3.67μmol/L,最大反应速率为3.52 pmol/s。在体外抑菌实验中,在8μmol/L的浓度下对大肠艾希菌和金黄葡萄球菌均未显示出显著抑制作用;在体外细胞毒性实验中,对于Hela肿瘤细胞和正常的人成纤维细胞在8μmol/L的浓度下无明显抑制作用;在抗氧化实验中,浓度达到71.5μmol/mL时,对小鼠肝微粒体脂质过氧化反应的抑制率为41.30%。结论中华眼镜蛇毒中的核糖核酸酶具有抗氧化作用,可能作为氧化还原的调节剂,在有关的疾病治疗方面具有应用的潜能。
Objective To isolate and purify ribonuclease from Chinese cobra venom and study its biological activity. METHODS: The Chinese cobra venom was used as raw material. The protein with ribonuclease activity was isolated and purified by SP-Trisacryl cation exchange chromatography, Sephadex G-75 gel chromatography and C8 reverse phase chromatography. Antibacterial activity, anti-tumor cell function and anti-oxidation. Results SDS-PAGE electrophoresis showed that the purified Na-RNase was a single component with relative molecular mass of 13 000. The optimal reaction temperature was 40 ℃, the optimum pH was 6.5, the Mk constant was 3.67μmol / L and the maximum reaction rate was 3.52 pmol / s. In in vitro antibacterial experiments, Escherichia coli and Staphylococcus aureus did not show significant inhibitory effect at the concentration of 8μmol / L; in vitro cytotoxicity experiments, Hela tumor cells and normal human fibroblasts in 8μmol / L had no obvious inhibitory effect. In the anti-oxidation experiment, the inhibitory rate of lipid peroxidation in mouse liver microsomes was 41.30% at the concentration of 71.5μmol / mL. Conclusion RNase in Chinese cobra venom has anti-oxidant activity and may serve as a redox regulator, which has potential application in the treatment of related diseases.