用同步辐射X-荧光定量测定电泳分离后蛋白条带内的微量元素

Analysis of Trace Elements in Protein Bands with Synchrotron Radiation X-ray Fluorescence after Electrophoresis Separation

建立了同步辐射X-荧光（SRXRF）定量测定生物样品等电聚焦（IEF）分离后蛋白条带内的微量元素Fe、Cu和Zn的方法.用薄层聚丙烯酰胺凝胶分离人血红蛋白后,用SRXRF测定了各亚型条带内的金属含量,用加一定量金属的含蛋白聚丙烯酰胺凝胶做SRXRF定量测定蛋白条带内微量元素Fe、Cu和Zn的定量标准,校准曲线线性回归系数r在0～8 μg/g范围内均大于0.99;检出限分别为2.43、1.12和0.96 μg/g;测定蛋白条带内Fe和Zn的回收率分别为90.4%和115.7 %.该联用技术可用于生物样品中微量元素的化学形态分析,同时给出蛋白质的微量元素组成和等电点等信息.

A procedure has been proposed for the determination of Fe, Cu and Zn in protein bands with synchrotron radiation X-ray fluorescence （SRXRF） analysis after isoelectric focusing （IEF） separation. Calibration standards were prepared by adding certain amounts of metal ions and free-metal proteins to electrophoresis gel. Human hemoglobin was separated with IEF, then Fe, Cu and Zn in protein bands were analyzed by SRXRF. The calibration curves can be obtained in a range of 0 -8 μg/g metals and linear relationship between dosage of metals and fluorescent intensity can be observed （ r 〉 0.99 ）. The method provides detection limits of 2.43, 1.12 and 0.96 μg/g for Fe, Cu and Zn, and recoveries of 90.4% and 115.7% for Fe and Zn, respectively. The combined technique of SRXRF and IEF have advantages of quantifying trace elements in proteins and identifying the proteins simultaneously. It can be employed for speciation analysis of trace elements in biological samples.