利用紫外-可见光谱、傅里叶变红外光谱、圆二色谱和分子建模技术研究了考马斯亮蓝G-250与牛血清白蛋白(BSA)的相互作用。紫外-可见光谱测定结果表明,随BSA的浓度的增加,以1:1形成复合物,在不同温度条件下,结合形成常数值分别为5.03×104、3.04×104、2.84×104和1.99×104 L/mol,随温度升高而减小,整个反应过程是焓、熵联合驱动自发进行,热力学焓变(ΔH)和熵变(ΔS)分别为-45.3229 k J/mol和-139.1847 J/(K·mol),说明分子间作用力以氢键和范德华力为主;采用傅里叶红外技术研究了作用前后BSA中α-螺旋特征酰胺带(I和II)的变化,结果表明酰胺带I由1650 cm-1移动到1710 cm-1,酰胺带II从1544 cm-1移动到1573 cm-1,α-螺旋结构降低;圆二色谱测定结果和分子对接技术进一步验证了BSA结构的变化,α-螺旋含量由42.15%下降至1.27%。
The interaction of Coomassie brilliant blue G-250(CBBG-250) with bovine serum albumin(BSA) was investigated by the methods of ultraviolet-visible(UV-Vis) spectroscopy, Fourier transform infrared spectroscopy(FT-IR), circular dichroism(CD), and molecular modeling technique. The UV-Vis results showed that BSA could interact with CBBG-250 to form a CBBG-BSA complex in a molar ratio of 1:1 with increasing BSA concentrations. At different temperatures, the apparent binding constant(Ka) values were 5.03 × 104(298 K), 3.04 × 104(303 K), 2.84 × 104(308 K), and 1.99 × 104 L/mol(313 K), respectively, which decreased with increasing temperatures. The whole process was enthalpically and entropically driven, and the enthalpy(△H) and entropy changes(△S) were 45.32 k J/mol and 139.18 J/(mol·K), respectively, indicating that the hydrogen bonds and Van der Waals forces played a dominant role in the molecular interaction. FT-IR was used to study the changes in characteristic amide bands(I and II) of α-helix in BSA before and after the interaction. The results showed that amide band I shifted from 1650 cm-1 to 1710 cm-1, amide band II shifted from 1573 cm-1 to 1544 cm-1, and the α-helical structure content was reduced. CD measurements and molecular docking technique further verified the changes in BSA structure and the content of α-helical structure decreased from 42.15% to 1.27%.