聚唾液酸修饰CuZn-SOD及其形态和稳定性研究

Modification of CuZn-SOD by Polysialic Acids: Synthesis,Characterization and Enzyme Stability

摘要:

聚唾液酸(Polysialic acid,PSA)因其更为有效的安全性,被公认为传统药用缓释材料——聚乙二醇(PEG)的理想替代材料。作者对蛋白质药物——铜锌超氧化物歧化酶(CuZn-SOD)的聚唾液酸修饰进行了初步研究。研究了修饰反应的主要条件:聚唾液酸经高碘酸钾氧化后生成的活化体,与超氧化物歧化酶以40∶1的摩尔比反应24h,可以达到理想修饰效果,生成的聚唾液酸修饰的超氧化物歧化酶衍生物(PSA-SOD)平均交联比达到了3.9±0.3。该衍生物经过SDS-PAGE电泳和原子力显微镜(AFM)检测测得其平均相对分子质量为90 000～100 000,分子粒径为10～15nm,约为天然超氧化物歧化酶的4倍。除此,PSA-SOD呈单颗粒包覆和多颗粒包覆两种显微形态。PSA-SOD进行稳定性试验表明,经聚唾液酸修饰的超氧化物歧化酶的酸碱稳定性、热稳定性和抗酶解稳定性都有了较大程度的提高。

Abstract:

Polysialic acid(PSA) is considered an ideal alternative material of the polyethylene glycol(PEG) for protein or peptide modification due to its more effective safety in therapeutics.This research was a preliminary study of modification of CuZn superoxide dismutase(CuZn-SOD) with polysialic acid.The optimal conditions for the modifying reaction were as follows: PSA was activated by periodate oxidation,then coupled to CuZn-SOD in the PSA: SOD mole ratios of 40: 1 for 24h.It was shown that polysialylated macromolecule contained 3.9±0.3 molecules of PSA per molecule of SOD.SDS-PAGE electrophoresis and Atomic Force Microscopy(AFM) revealed that the molecular weight of polysialylated SOD was about 90~100kDa and the average size was 10~15nm,about four-fold of native enzyme size.Besides,it showed two kinds of microscopic spherical shape: coated single particle or coated multiple particles.As compared with native enzyme,the stabilities of modified enzyme against heat,acid,alkali and proteinases such as pepsin and trypsin were improved significantly.