Department of Biology, Faculty of Sciences, Mashhad Branch, Islamic Azad University, Mashhad, Iran.
J Biomol Struct Dyn. 2012;29(5):1013-50. doi: 10.1080/073911012010525029.
Here, we report on the effect of aspirin (ASA), on the binding parameters with regard to bilirubin (BR) to human serum albumin (HSA). Two different classes of binding sites were detected. Binding to the first and second classes of the binding sites was dominated by hydrophobic forces in the case of HSA-BR, whereas in the case of the ternary system, binding to the first and second classes of the binding sites was achieved by electrostatic interaction. The binding constant (K(a)) and number of binding site (n) obtained were 1.6 × 10(6)M(-1) and 0.98, respectively, for the primary binding site in the case of HSA-BR, and 3.7 × 10(6)M(-1) and 0.84, respectively, in the presence of ASA (ternary complex) at λ(ex)= 280 nm. The progressive quenching of the protein fluorescence as the BR concentration increased indicated an arrangement of the domain IIA in HSA. Changes in the environment of the aromatic residues were also observed by synchronous fluorescence spectroscopy (SFS). Changes of the secondary structure of HSA involving a decrease of α-helical and β-sheet contents and increased amounts of turns and unordered conformations were mainly found at high concentrations of BR. For the first time, the relationship between the structural parameters of HSA-BR by RLS for determining the critical induced aggregation concentration (C(CIAC)) of BR in the absence and presence of ASA was investigated, and there was a more significant enhancement in the case of the ternary mixture as opposed to the binary one. Changes in the zeta potential of HSA and the HSA-ASA complex in the presence of BR demonstrated a hydrophobic adsorption of this anionic ligand onto the surface of HSA in the binary system as well as both electrostatic and hydrophobic adsorption in the case of the ternary complex. By performing docking experiments, it was found that the acting forces between BR and HSA were mainly hydrophobic > hydrogen bonding > electrostatic interactions, and consequently BR had a long storage time in blood plasma, especially in the presence of ASA. This was due to the electrostatic interaction force between the BR and HSA being stronger in (HSA-ASA) BR than in the HSA-BR complex. In addition, it was demonstrated that, in the presence of ASA, the first binding site of BR on HSA was altered, but the parameters of binding did not become significantly modified, and thus the affinity of BR barely changed with and without ASA.
在这里，我们报告了阿司匹林（ASA）对人血清白蛋白（HSA）与胆红素（BR）结合参数的影响。检测到两种不同类别的结合位点。对于 HSA-BR，结合到第一类和第二类结合位点主要由疏水作用力主导，而对于三元体系，结合到第一类和第二类结合位点则通过静电相互作用实现。对于 HSA-BR，在 λ(ex)=280nm 时，在存在 ASA 的情况下（三元复合物），获得的结合常数（K(a)）和结合位点数（n）分别为 1.6×10(6)M(-1)和 0.98，对于初级结合位点。对于 HSA-BR，在 λ(ex)=280nm 时，在存在 ASA 的情况下（三元复合物），获得的结合常数（K(a)）和结合位点数（n）分别为 3.7×10(6)M(-1)和 0.84，对于初级结合位点。当 BR 浓度增加时，蛋白质荧光的渐进猝灭表明 HSA 中域 IIA 的排列。通过同步荧光光谱法（SFS）也观察到芳香族残基环境的变化。HSA 的二级结构发生变化，涉及α-螺旋和β-折叠含量的减少，以及更多的转角和无序构象的增加，主要发生在 BR 的高浓度下。首次研究了通过 RLS 确定 BR 在不存在和存在 ASA 时的临界诱导聚集浓度（C(CIAC)）的 HSA-BR 结构参数之间的关系，并且在三元混合物的情况下存在更显著的增强，而不是二元混合物。BR 存在时 HSA 和 HSA-ASA 复合物的 ζ 电位变化表明，在二元体系中，这种阴离子配体主要通过疏水吸附在 HSA 表面，而在三元复合物中则通过静电和疏水吸附。通过进行对接实验，发现 BR 与人血清白蛋白之间的作用力主要为疏水>氢键>静电相互作用，因此 BR 在血液中的储存时间较长，特别是在存在 ASA 的情况下。这是由于 BR 与人血清白蛋白之间的静电相互作用力在（HSA-ASA）BR 中比在 HSA-BR 复合物中更强。此外，结果表明，在存在 ASA 的情况下，BR 与人血清白蛋白的第一结合位点发生了改变，但结合参数没有显著改变，因此 BR 的亲和力在有无 ASA 的情况下几乎没有变化。
