Abstract: Bovine hemoglobin (BHb) is highly homologous to human hemoglobin and obtained easily, so it is often used in the study of binding of hemoglobin to small molecule compounds. Gallic acid (GA) is a polyphenolic small molecule compound, which can be used as drug model. The study on interaction between GA and BHb can provide a theoretical basis for the application of GA in the field of medicine. The interaction between GA and BHb was investigated at pH 7.0 by ultraviolet-visible (UV-vis) spectroscopy, fluorescence spectroscopy, Fourier transform infrared (FTIR) spectroscopy and circular dichroism (CD). The results of UV-vis spectroscopy showed that GA had no effect on oxygenation state of BHb, and did not deoxidize the oxygenated hemoglobin. Combined fluorescence spectra with calculation results of related formulas, it could be known that the interaction between GA and BHb resulted in fluorescence quenching, and the quenching constant increased with the increase of temperature, indicating that the quenching type of the interaction between GA and BHb was dynamic quenching. Then the binding constants of GA and BHb at different temperatures were calculated, which were K298a=7.941×103 L/mol, K308a=10.478×103 L/mol, respectively. And the binding process was a spontaneous molecular interaction procedure, in which hydrophobic interactions played major roles. The results also demonstrated that the microenvironment of tryptophan and tyrosine residues had been disturbed, under the same condition, variations of fluorescence absorption peak intensity of the tryptophan residue were greater than that of the tyrosine residue, indicating that the binding site of GA and BHb was closer to  the tryptophan residue. The results of FTIR spectroscopy and CD showed that the secondary structures before and after the interaction between BHb and GA were both dominated by α-helices, it meant that GA had little influence on the secondary structure of BHb molecules.

Key words: gallic acid, bovine hemoglobin, interaction, spectrometry

摘要： 牛血红蛋白（bovine hemoglobin，BHb）与人血红蛋白高度同源，且易获得，常用于血红蛋白与小分子化合物结合的研究。没食子酸（gallic acid，GA）作为一种多酚类小分子化合物，可被用作小分子药物模型，研究其与BHb的相互作用，可为其在医药领域的应用提供理论依据。运用紫外-可见光谱（UV-vis spectroscopy）、荧光光谱（fluorescence spectroscopy）、傅里叶变换红外（Fourier transform infrared，FTIR）光谱、圆二色光谱（circular dichroism，CD）等手段，在pH 7.0条件下，研究GA与BHb的相互作用。紫外-可见光谱的结果表明，GA对BHb的氧合状态没有影响，不会使氧合血红蛋白脱氧。结合荧光光谱和相关公式计算结果可知，GA与BHb相互作用发生了荧光猝灭，且猝灭常数随着温度的升高而增大，说明GA与BHb相互作用的猝灭类型为动态猝灭；通过计算得到了不同温度下GA与BHb相互作用的结合常数，分别为K298a=7.941×103 L/mol，K308a= 10.478×103 L/mol；GA与BHb之间主要靠疏水作用力结合，可自发发生反应；色氨酸和酪氨酸残基所处微环境受到扰动，色氨酸残基的荧光吸收峰强度比酪氨酸残基变化大，表明GA与BHb分子的结合位点更接近于色氨酸残基。FTIR光谱和CD的检测结果显示，与GA作用前后，BHb均以α-螺旋结构为
主，即GA对BHb分子的二级结构影响较小。

关键词: 没食子酸, 牛血红蛋白, 相互作用, 光谱测定