Liu Jiaqin, Tian Jianniao, Li Ying, Yao Xiaojun, Hu Zhide, Chen Xingguo
Mianyang Teacher's College, Mianyang 621000, PR China.
Macromol Biosci. 2004 May 17;4(5):520-5. doi: 10.1002/mabi.200300109.
Daphnetin (7,8-dihydroxycoumarin), one of the major bioactive components isolated from Daphne koreane Nakai, has been used in traditional Chinese medicine for the treatment of coagulation disorders. It is also a chelator, an antioxidant and a protein kinase inhibitor. In this paper, a combination of intrinsic fluorescence, Fourier transform infrared (FT-IR) spectroscopy and circular dichroic (CD) spectroscopy has been used to characterize the binding between daphnetin and human serum albumin (HSA) under physiological conditions with drug concentrations of 6.7 x 10(-6) - 2.3 x 10(-5) mol x L(-1), and a HSA concentration of 1.5 x 10(-6) mol x L(-1). Changes in the CD spectra and FT-IR spectra were observed upon ligand binding, and the degree of tryptophan fluorescence quenching did change significantly in the complexes. These data have proved the change in protein secondary structure accompanying ligand binding. The change in tryptophan fluorescence intensity was used to determine the binding constants. The thermodynamic parameters, the enthalpy change (DeltaH) and the entropy change (DeltaS) were calculated to be -12.45 kJ x mol(-1)and 52.48 J x mol(-1) x K(-1) according to the van't Hoff equation, which indicated that hydrophobic and electrostatic interactions played the main role in the binding of daphnetin to HSA, in accordance with the results of calculations performed on a Silicon Graphics Ocatane2 workstation. In addition, the binding distance between daphnetin and HSA was obtained (4.02 nm) based on the Forster energy transfer theory.
瑞香素（7,8 - 二羟基香豆素）是从朝鲜瑞香中分离出的主要生物活性成分之一，在传统中药中用于治疗凝血紊乱。它也是一种螯合剂、抗氧化剂和蛋白激酶抑制剂。本文采用内源荧光、傅里叶变换红外（FT - IR）光谱和圆二色（CD）光谱相结合的方法，在生理条件下，药物浓度为6.7×10⁻⁶ - 2.3×10⁻⁵ mol·L⁻¹，人血清白蛋白（HSA）浓度为1.5×10⁻⁶ mol·L⁻¹时，对瑞香素与人血清白蛋白的结合进行了表征。配体结合后观察到CD光谱和FT - IR光谱的变化，并且复合物中色氨酸荧光猝灭程度确实发生了显著变化。这些数据证明了配体结合伴随蛋白质二级结构的改变。利用色氨酸荧光强度的变化来确定结合常数。根据范特霍夫方程计算出热力学参数，焓变（ΔH）和熵变（ΔS）分别为 - 12.45 kJ·mol⁻¹和52.48 J·mol⁻¹·K⁻¹，这表明疏水作用和静电作用在瑞香素与HSA的结合中起主要作用，这与在硅图Ocatane2工作站上进行的计算结果一致。此外，基于福斯特能量转移理论获得了瑞香素与HSA之间的结合距离（4.02 nm）。
