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化学学报
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化学学报 ›› 2011, Vol. 69 ›› Issue (22): 2703-2709. 上一篇    下一篇

研究论文

血清蛋白对麦角甾-4,6,8,22-四烯-3-酮荧光增强作用的研究及应用

孙洋1，赵英永2，刘璐莎1，魏嵩1，梁旭华1，谭春雷1，胡晓云3，樊君*,1   

  1. (1 西北大学化工学院 西安 710069)
    (2 西北大学生命科学院 西安 710069)
    (3 西北大学物理系 西安 710069)
  • 投稿日期:2011-04-01 修回日期:2011-06-18 发布日期:2011-07-20
  • 通讯作者: 樊君 E-mail:fanjun@nwu.edu.cn
  • 基金资助:

    国家自然科学基金;科技部“重大新药创制”科技专项

Studies on the Fluorescence Enhancement Effect of Ergosta-4,6,8(14),22-tetraen-3-one with Serum Albumin and Its Application

Sun Yang1 Zhao Yingyong2 Liu Lusha1 Wei Song1 Liang Xuhua1 Tan Chunlei1 Hu Xiaoyun3 Fan Jun*,1   

  1. (1 School of Chemical Engineering, Northwest University, Xian 710069)
    (2 Biomedicine Key Laboratory of Shaanxi Province, Northwest University, Xian 710069)
    (3 Department of Physics, Northwest University, Xian 710069)
  • Received:2011-04-01 Revised:2011-06-18 Published:2011-07-20

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1373

被引次数

12

麦角甾-4,6,8,22-四烯-3-酮(ergone)为猪苓中主要甾体之一, 具有多种药理和生理活性. 研究了不同极性溶剂中ergone的光学性质, 考察了人血清蛋白(HSA), 牛血清蛋白(BSA)对ergone荧光和紫外光谱的影响, 结果表明血清蛋白加入后ergone的光谱信号强度显著增强, 并发生蓝移. 根据Benesi-Hildebrand方程求得了结合常数和自由能变. 基于血清蛋白对ergone具有良好的荧光增强作用, 在模拟生理条件下以ergone为荧光探针, 建立了一种灵敏的蛋白质定量分析方法, HSA和BSA线性响应浓度范围分别为(0.38～16.67)×10－7 mol•L－1和(0.42～15.25)×10－7 mol•L－1, 检测限(3σ)分别为1.01×10－10和1.22×10－10 mol•L－1. 考察共存物质对测定结果的影响中发现Fe3＋会显著淬灭ergone荧光. 该方法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: 麦角甾-4,6,8,22-四烯-3-酮, 血清蛋白, 吸收光谱, 荧光探针, 定量分析

Ergosta-4,6,8(14),22-tetraen-3-one (ergone) from many medicinal plants has been demonstrated to possess a variety of pharmacological activities in vivo and in vitro, including cytotoxic, diuretic and immunosuppressive activity. The effect of different solvents on spectral characteristic of ergone was investigated. The interactions between ergone and human serum albumin (HSA) and bovine serum albumin (BSA) had been studied by using absorption and fluorescence spectroscopy. Absorption and fluorescence spectral studies showed that binding to the serum albumins leaded to a blue shift of ergone together with a notable intensity change. Furthermore, the number of binding sites (n) was identified by the absorption spectra. The binding constant (Ka) and the free energy changes (ΔG) were obtained by analysis of fluorescence data of the ergone in the absence and presence of HSA and BSA according to Benesi-Hildebrand equation. Compared to BSA, HSA associated with ergone in a stronger way. A new fluorescence quantitative determination of proteins has been developed by using ergone as a fluorescence probe. Good calibration curves of the proteins were obtained in the range of (0.38～16.67)×10－7 mol•L－1 for HSA with detection limits (3σ) of 1.01× 10－10 mol•L－1, and (0.42～15.25)×10－7 mol•L－1 for BSA with detection limits of 1.22×10－10 mol•L－1. Most metal ions had no notable effect on the determination of proteins except Fe3＋ ions which could quench the fluorescence intensity of ergone. Determination of proteins in human serum by this method gived results which were very close to those obtained by Coomassie Brilliant Blue colorimetry.

Key words: ergosta-4,6,8(14),22-tetraen-3-one (ergone), serum albumin, absorption spectroscopy, fluorescence probe, quantitative assay