咖啡因与牛血清和人血清白蛋白相互作用:荧光光谱“内滤光效应”的校正

用荧光光谱和紫外吸收光谱法研究咖啡因与牛血清白蛋白和人血清白蛋白的相互作用.实验结果表明,咖啡因与牛血清白蛋白的结合主要是静态猝灭过程,而与人血清白蛋白的结合则是动态猝灭过程.获得了不同温度下,咖啡因与血清白蛋白作用的结合常数以及ΔH、ΔG和ΔS等热力学参数.由于咖啡因在荧光激发波长处有吸收,其猝灭行为中包含有"内滤光效应",因此我们对荧光实验数据进行了校正,结果显示"内滤光效应"使咖啡因与血清白蛋白的结合常数升高.另外,用紫外光谱和圆二色谱考察了咖啡因对血清白蛋白二级结构的影响,位点竞争实验表明咖啡因与牛血清白蛋白的结合主要发生在siteⅠ(sub-domainⅡA)位.

Interaction of caffeine with human and bovine serum albumins： correction for inner filter effect in fluorescence spectroscopy

The interaction of caffeine with human serum albumin （HSA~ and bovine serum albumin （BSA） under physiological condition, was investigated by fluorescence, UV-vis absorbance and circular dichroism （CD） spectroscopy. It was proved that the fluorescence quenching of BSA by caffeine was mainly a static quenching process, whereas the mechanism of quenching of HSA fluorescence by caffeine was shown to involve a dynamic quenching procedure. The binding constant and corresponding thermodynamic parameters△H, △G, △S at different temperatures were calculated.But the results obtained by fluorescence spectroscopy were influenced by a non-negligible artifact, known as the inner filter effect due to the absorption of caffeine at the excitation wavelength. Therefore, a suitable correction of the obtained data was performed. Our findings showed that the binding constant at different temperatures derived from the corrected fluorescence intensities were decreased with respect to the ones of non-corrected. The results reflected that the inner filter effect should be taken into account before that the conclusion about fluorescence quenching of serum albumins by caffeine could be made. Furthermore, UV-vis and CD spectroscopy revealed that the secondary structure of serum albumins had been changed, site marker competitive experiment suggested that caffeine bound to BSA within site Ⅰ （sub-domain Ⅱ A）.