Institute of Food Engineering, College of Life & Environment Science, Shanghai Normal University, 100 Guilin Rd, Shanghai 200234, PR China.
Spectrochim Acta A Mol Biomol Spectrosc. 2009 Nov;74(4):977-82. doi: 10.1016/j.saa.2009.09.003. Epub 2009 Sep 8.
The interaction between esculin and serum albumins was investigated to illustrate that the fluorescence resonance energy-transfer (FRET) affects the determination of the binding constants obtained by fluorescence quenching method. The binding constants (K(a)) obtained by the double-logarithm curve for esculin-BSA and esculin-HSA were 1.02x10(7) and 2.07x10(4)L/mol, respectively. These results from synchronous fluorescence showed that the Tyr and Trp residues of HSA were affected more deeply than those in BSA. The excitation profile of esculin showed that in the presence of BSA and HSA, the S(0)-->S(1) transition of esculin (lambda(ex)(max) approximately 340nm) appears, which is similar to the lambda(em)(max) of BSA and HSA. The critical distance (R(0)) between BSA and esculin is higher than that of HSA, which showed that the affinity of esculin and HSA should be higher than that of BSA. After centrifugation, the concentrations of esculin bound to albumins were determined by means of the fluorescence of esculin. It was found that much more esculin was bound to HSA than to BSA. However, the bound models for BSA and HSA are almost the same. The concentration of esculin bound to serum albumin at first decreased with the addition of esculin and then increased. From above results, it can be concluded that the affinity of esculin and HSA should be higher than that of esculin and BSA. This example showed that in the presence of FRET, the binding constants between ligands and proteins based on fluorescence quenching might be deviated.
研究了秦皮乙素与血清白蛋白的相互作用，说明了荧光共振能量转移（FRET）会影响荧光猝灭法测定结合常数的结果。秦皮乙素-BSA 和秦皮乙素-HSA 的双对数曲线得到的结合常数（K(a)）分别为 1.02x10(7) 和 2.07x10(4)L/mol。这些来自同步荧光的结果表明，HSA 中的 Tyr 和 Trp 残基比 BSA 中的更受影响。秦皮乙素的激发谱表明，在 BSA 和 HSA 的存在下，秦皮乙素的 S(0)-->S(1)跃迁（lambda(ex)(max)约为 340nm）出现，这与 BSA 和 HSA 的 lambda(em)(max)相似。BSA 和秦皮乙素之间的临界距离（R(0)）高于 HSA，这表明秦皮乙素与 HSA 的亲和力应该高于 BSA。离心后，通过秦皮乙素的荧光测定结合到白蛋白上的秦皮乙素浓度。结果发现，与 BSA 相比，更多的秦皮乙素与 HSA 结合。然而，BSA 和 HSA 的结合模型几乎相同。结合到血清白蛋白上的秦皮乙素的浓度最初随着秦皮乙素的加入而降低，然后增加。从以上结果可以得出结论，秦皮乙素与 HSA 的亲和力应该高于秦皮乙素与 BSA。这个例子表明，在存在 FRET 的情况下，基于荧光猝灭的配体与蛋白质之间的结合常数可能会出现偏差。
