高速逆流色谱双水相体系分离蛋白质

利用多分离柱高速逆流色谱仪,研究了聚乙二醇1000(PEG1000)-磷酸盐双水相体系的固定相保留率及该体系对蛋白质混合物和鸡蛋清样品的分离。以14.0%PEG1000-16.0%磷酸盐体系的上相为固定相,在流速0.6 mL/min和转速900 r/min的条件下,固定相的保留率达到33.3%。在pH 9.2的PEG1000-磷酸盐双水相体系中，细胞色素C、溶菌酶和血红蛋白的分配系数差异最大,采用该pH值的14.0%PEG1000-16.0%磷酸钾盐双水相体系,在流速1.0 mL/min和转速850 r/min的条件下,成功地分离了这3种蛋白质的混合物。鸡蛋清中的主要蛋白质成分卵转铁蛋白、卵白蛋白和溶菌酶在pH 9.2的15.0%PEG1000-17.0%磷酸钾盐体系中也具有最大的分配系数差异。采用该体系,在流速1.0 mL/min和转速850 r/min的条件下,成功地分离了鸡蛋清样品,得到的卵白蛋白、溶菌酶和卵转铁蛋白的电泳纯度分别为100％,100％和60％,收率均大于90％。

Separation of Proteins in Aqueous Two-Phase Systems with High-Speed Counter-Current Chromatography

High-speed counter-current chromatography (HSCCC) is a continuous liquid-liquid partition chromatography, with remarkable advantages of high separation efficiency and no (adsorption) or denaturation by solid phase. The retention of stationary phase and the separation of proteins in polyethylene glycol 1000 (PEG1000)-phosphate aqueous two-phase system (ATPs) were studied with a multi-column high speed-counter-current chromatograph. The flow (direction) and speed of the mobile phase, and the rotation direction and speed of the apparatus showed different effects on the retention of the stationary phase, which reached the maximum at 33.3% with a flow rate of 0.6(mL/min) and a rotation speed of 900 (r/min) in 14.0%PEG1000-16.0%phosphate ATPs. Distinct differences in partition coefficients among cytochrome C, (lysozyme) and hemoglobin were found at pH 9.2 and these three proteins were successfully (separated) in 14.0% PEG1000-16.0% phosphate ATPs at pH 9.2 by HSCCC with the apparatus rotating at 850 (r/min) and the mobile phase flow rate of 1.0 (mL/min). The major protein (components) in hen egg white, including ovaltransferrin, ovalbumin and lysozyme also show (distinct) differences of partition coefficients in PEG1000-phosphate ATPs at pH 9.2. Ovalbumin and lysozyme were successfully purified to homogeneity and ovaltransferrin to ca 60% purity from the hen egg white sample with yields (over) 90% in 15.0% PEG1000-17.0% phosphate ATPs at pH 9.2 with the apparatus rotating at 850 (r/min) and mobile phase flow rate of 1.0(mL/min).