丹酚酸B与牛血清白蛋白相互作用的光谱学研究

丹酚酸B（SAB）是丹参中主要的水溶性成分之一，具有广泛的生物活性。血清白蛋白是哺乳动物体内血浆中含量最为丰富的蛋白质，约占血浆总蛋白的60%，能与许多内源及外源性物质相结合，发挥存储和转运的作用。丹酚酸B进入人体后，必然先与血液中的蛋白质相结合，然后才被转运到其受体结合部位，进而发挥其药理作用。为更好地了解丹酚酸B在体内的分布、转运及代谢，在模拟生理条件下，采用荧光光谱法、圆二色光谱法和核磁共振波谱法等方法研究丹酚酸B与牛血清白蛋白（BSA）的相互作用机制。结果表明：丹酚酸B与牛血清白蛋白的结合能有效地导致牛血清白蛋白的内源荧光猝灭，猝灭机制为以静态猝灭为主的联合猝灭方式。荧光光谱分析表明两者的结合常数分别为7.51 ×105（288 K），7.40 ×105（298 K）和5.57 ×105（308 K） L·mol -1，达到105 L·mol -1数量级，且随着温度的升高逐渐降低。Scatchard方法确定牛血清白蛋白与丹酚酸B相互作用时结合位点数约为1，说明两者之间可形成1∶1型非共价复合物。位点标记竞争实验表明丹酚酸B在牛血清白蛋白亚结构域IIA（Site I）的疏水腔内相结合。三维荧光光谱法和圆二色谱法实验结果显示，结合丹酚酸B后牛血清白蛋白中色氨酸和酪氨酸所处的微环境发生了一定的变化（即峰位置发生红移，色氨酸和酪氨酸残基周围微环境疏水性减小，极性增强），而二级结构和和三级结构的变化较小。此外，利用核磁共振波谱技术比较一定浓度的丹酚酸B在不同浓度的牛血清白蛋白溶液中的化学位移变化情况，研究表明H5”和H6”所在的苯环在牛血清白蛋白与丹酚酸B相互作用过程中发挥着重要的作用。该研究有助于了解丹酚酸B在机体内的作用机制以及对血清白蛋白结构和功能的影响，为丹酚酸B类新药的研发提供一定的理论参考。

Spectroscopic Studies on the Interaction Between Salvianolic Acid B and Bovine Serum Albumin

As the main water-soluble active ingredient from Salvia miltiorrhiza, salvianolic acid B (SAB) has a wide range of biological activities. Serum albumin is the most abundant protein in plasma (about 60%) and can bind with various endogenous and exogenous compounds, which can play a role in the storage and transport of compounds. When SAB enters into the human body, it must be combined with the protein in the blood system and then transported to its receptor site and played its pharmacological effects. Thus, in order to better understand the distribution, transport and metabolism of SAB in vivo, the interaction of SAB with bovine serum albumin (BSA) has been investigated using fluorescence spectra, circular dichroism (CD) and nuclear magnetic resonance spectra (NMR) under simulated physiological conditions. The results showed that the binding of SAB to BSA could quench the intrinsic fluorescence of BSA through the combined quenching mechanism (static and dynamic), but the static quenching was the primary one. The binding constant was 7.51×105 L·mol-1 (288 K), 7.40×105 L·mol-1 (298 K) and 5.57×105 L·mol-1 (308 K), respectively. It was found to be in the order of 105 L·mol-1 and decreased with the increasing temperature. The results of site marker competitive experiments indicated that SAB specifically bound to Site I of BSA in the hydrophobic pocket of sub-domain IIA. The stoichiometric ratio between BSA and SAB was calculated by using the Scatchard equation, and the result suggested that the SAB can form a 1∶1 type non-covalent complex with BSA. The three-dimensional fluorescence and CD studies indicated that SAB induced some microenvironmental changes of tryptophan and tyrosine in BSA. That was, the binding of SAB to BSA brought the tryptophan and tyrosine residues to a more hydrophilic environment, while the changes of secondary and tertiary structures of BSA were relatively small. Furthermore, the chemical shift of SAB at various concentrations of BSA was studied using NMR spectra, and the results showed that the benzene ring of H5” and H6” in SAB played a vital role in the binding process during the BSA-SAB complexation. This research will be helpful for understanding the mechanism of SAB in vivo and the influence of the binding of SAB to the conformation and function of serum albumin in biological processes, and can provide some theoretical basis for the development of SAB related new drugs.