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化学学报
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化学学报 ›› 2011, Vol. 69 ›› Issue (08): 937-944. 上一篇    下一篇

研究论文

荧光素钠与牛血清蛋白相互作用的荧光光谱研究及其分析应用

孙洋1，樊君*,1，胡晓云2，刘璐莎1，胡春梅1，尹辰1，魏嵩1   

  1. (1 西北大学化工学院 西安 710069)
    (2 西北大学物理系 西安 710069)
  • 投稿日期:2010-10-08 修回日期:2010-11-10 发布日期:2010-12-05
  • 通讯作者: 樊君 E-mail:fanjun@nwu.edu.cn
  • 基金资助:

    西安市工业应用技术研发资助项目

Studies on the Interaction between Sodium Fluorescein and Bovine Serum Albumin by Fluorescence Spectroscopy and Its Analytical Application

Sun Yang1 Fan Jun*,1 Hu Xiaoyun2 Liu Lusha1 Hu Chunmei1 Yin Chen1 Wei Song1   

  1. (1 School of Chemical Engineering, Northwest University, Xi an 710069)
    (2 Department of Physics, Northwest University, Xi an 710069)
  • Received:2010-10-08 Revised:2010-11-10 Published:2010-12-05

PDF

2512

被引次数

32

采用荧光光谱研究了荧光素钠与牛血清蛋白(BSA)间的相互作用, 根据荧光淬灭相关方程分别计算了淬灭速率常数、结合常数、结合位点数及热力学参数, 确定了BSA对荧光素钠的淬灭机理及作用方式, 根据能量转移理论求得荧光素钠与BSA的结合距离及能量转移率, 结合三维、同步荧光光谱研究了荧光素钠对BSA构象的影响|在一定范围内荧光素钠的淬灭程度与BSA浓度成正比, 据此建立一种荧光素钠测定蛋白的方法, 线性范围为0.15×10－7～15× 10－7 mol•L–1, 方法具有高灵敏度, 检测极限为0.146×10－8 mol•L–1, 文中还考察了不同pH值和干扰物质对于测定结果的影响, 用于人血清中总蛋白含量测定结果与考马斯亮蓝法基本一致.

关键词: 荧光素钠, 牛血清蛋白, 荧光光谱, 定量分析

The interactions between sodium fluorescein (SoF) and bovine serum albumin (BSA) were investigated by fluorescence spectroscopy. According to the Stern-Volmer equation, the association constants, quenching constants and the numbers of binding sites were obtained. It is proved that the fluorescence quenching of SoF by BSA was a result of the formation of SoF-BSA complex. The thermodynamic parameters for the reaction were calculated according to van t Hoff equation. The distance and energy transfer efficiency between SoF and BSA were obtained according to the theory of non-radioactive energy transfer. The effect of SoF on the conformation of BSA was also analyzed using 3D fluorescence spectroscopy and synchronous fluorescence spectroscopy. The fluorescence intensity of SoF-BSA complex was proportional to the concentration of BSA, based on which, a new quantitative assay of protein was presented. The linear range was 0.15×10－7～15×10－7 mol•L–1, and the sensitivity of the method was high with detection limit of 0.146×10－8 mol•L－1. The effects of pH and interfering substance on the detection were also investigated. The results indicated that the most of the water-soluble amino acids, metal ion and antioxidant do not interfer or only interfer slightly under the permission of ±5.0% relative error, whereas SDS, Lecithin, APG, Cu2＋, Fe3＋, Sucrose and Tryptophane produced obvious interference. Determination of proteins in human serum by this method gave results which were very close to those obtained by Coomassie Brilliant Blue colorimetry.

Key words: sodium fluorescein, bovine serum albumin, fluorescence spectroscopy, quantitative assay