血清蛋白对麦角甾-4,6,8,22-四烯-3-酮荧光光增强作用的研究及应用

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

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1.22×10-10mol·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.

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 im- munosuppressive activity. The effect of different solvents on spectral characteristic of ergone was investi- gated. 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 （AG） 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 （0.38～6.67）×10^-7 mol,L-1 for HSA with detection limits （3tr） of 1.01 × 10-10 mol·L-1, and （0.42～15.25）×10^-7 moloL-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 gave results which were very close to those obtained by Coomassie Brilliant Blue colorimetry.