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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 quantity and existence of disulfide bonds in recombinant Candida urate oxidase by liquid chromatography-mass spectrometry. Methods The recombinant Candida albicans urate oxidase was alkylated and denatured. The average molecular weight of uricase oxidase treated by untreated and two different methods was determined by liquid chromatography-mass spectrometry (LC-MS / MS). According to the determination results Its disulfide bond number and existence mode. Results The average relative molecular mass of the untreated sample was 34 076.80, indicating that the protein existed as a monomer without interchain disulfide bonds. The average molecular weight of the directly alkylated sample was 34 133.40. Only one free sulfhydryl group was alkane The average relative molecular mass of the alkylated sample after denaturation was 34 304.80. Four free sulfhydryl groups were all alkylated, and there was no intrachain disulfide bond in the protein. Conclusion Four cysteines in the recombinant Candida urate oxidase do not form inter-chain and intra-chain disulfide bonds.