摘要
目的采用液质联用法分析重组假丝酵母尿酸氧化酶的二硫键数量及存在方式。方法将重组假丝酵母尿酸氧化酶分别进行烷基化及变性后烷基化处理,液质联用法测定未处理及两种不同方法处理后的尿酸氧化酶的平均相对分子质量,根据测定结果判断其二硫键数量及存在方式。结果未处理样品的平均相对分子质量为34 076.80,表明该蛋白以单体形式存在,不存在链间二硫键;直接烷基化样品平均相对分子质量为34 133.40,仅有一个游离巯基被烷基化;变性后烷基化样品的平均相对分子质量为34 304.80,4个游离巯基全被烷基化,蛋白不存在链内二硫键。结论重组假丝酵母尿酸氧化酶中的4个半胱氨酸未形成链间及链内二硫键。
Objective To analyze the number and pattern of disulfide bonds in recombinant Candida uricase by ultraperformance liquid chromatography-mass spectrometry（UPLC-MS）. Methods Recombinant Candida uricase was alkylated directly and after denaturation respectively, and determined for mean relative molecular mass by UPLC-MS, using that untreated as control. The number and pattern were judged according to the determination result. Results The mean relative molecular mass of untreated uricase was 34 076. 80, indicating the protein as a monomer without inter-chain disulfide bond. However, only one free thiol group was alkylated in the directly alkylated sample with a mean relative molecular mass of 34 133. 40, while all the four free thiol groups were alkylated in the samples alkylated after dena-turation, with a mean relative molecular mass of 34 304. 80 and without intra-chain disulfide bond. Conclusion Neither inter- nor intra-chain disulfide bond was formed in recombinant Candida uricase.
出处
《中国生物制品学杂志》
CAS
CSCD
2015年第7期746-748,共3页
Chinese Journal of Biologicals
基金
十二五科技重大专项"生物技术药物质量标准和质量控制技术平台"(2012ZX09304010)
