Institute of Macroheterocyclic Compounds, Ivanovo State University of Chemistry and Technology, Sheremetevskiy Str. 7, Ivanovo, 153000, Russian Federation.
Laboratory of Clinical Biochemistry and Metabolism, Department for Pediatrics, Medical Center, University of Freiburg, Mathildenstr. 1, 79106, Freiburg, Germany.
J Biol Inorg Chem. 2018 Jul;23(5):725-738. doi: 10.1007/s00775-018-1562-8. Epub 2018 May 2.
Serum albumin binds to a variety of endogenous ligands and drugs. Human serum albumin (HSA) binds to heme via hydrophobic interactions and axial coordination of the iron center by protein residue Tyr161. Human serum albumin binds to another tetrapyrrole, cobalamin (Cbl), but the structural and functional properties of this complex are poorly understood. Herein, we investigate the reaction between aquacobalamin (HOCbl) and bovine serum albumin (BSA, the bovine counterpart of HSA) using Ultraviolet-Visible and fluorescent spectroscopy, and electron paramagnetic resonance. The reaction between HOCbl and BSA led to the formation of a BSA-Cbl(III) complex consistent with N-axial ligation (amino). Prior to the formation of this complex, the reactants participate in an additional binding event that has been examined by fluorescence spectroscopy. Binding of BSA to Cbl(III) reduced complex formation between the bound cobalamin and free cyanide to form cyanocobalamin (CNCbl), suggesting that the β-axial position of the cobalamin may be occupied by an amino acid residue from the protein. Reaction of BSA containing reduced disulfide bonds with HOCbl produces cob(II)alamin and disulfide with intermediate formation of thiolate Cbl(III)-BSA complex and its decomposition. Finally, in vitro studies showed that cobalamin binds to BSA only in the presence of an excess of protein, which is in contrast to heme binding to BSA that involves a 1:1 stoichiometry. In vitro formation of BSA-Cbl(III) complex does not preclude subsequent heme binding, which occurs without displacement of HOCbl bound to BSA. These data suggest that the two tetrapyrroles interact with BSA in different binding pockets.
血清白蛋白结合各种内源性配体和药物。人血清白蛋白（HSA）通过疏水相互作用和蛋白质残基 Tyr161 的轴向配位与血红素结合。人血清白蛋白结合另一种四吡咯，钴胺素（Cbl），但对该复合物的结构和功能特性知之甚少。本文使用紫外-可见和荧光光谱以及电子顺磁共振研究了水合钴胺素（HOCbl）与牛血清白蛋白（BSA，HSA 的牛对应物）之间的反应。HOCbl 与 BSA 的反应导致形成与 N-轴向配位（氨基）一致的 BSA-Cbl(III) 复合物。在形成这种复合物之前，反应物通过荧光光谱检查参与了另一个结合事件。BSA 与 Cbl(III) 的结合减少了结合的钴胺素与游离氰化物之间形成氰钴胺素（CNCbl）的复合物形成，这表明钴胺素的β-轴向位置可能被来自蛋白质的氨基酸残基占据。含还原二硫键的 BSA 与 HOCbl 的反应生成 cob(II)alamin 和二硫化物，同时形成硫代 Cbl(III)-BSA 复合物及其分解。最后，体外研究表明，只有在蛋白质过量存在的情况下，钴胺素才与 BSA 结合，这与血红素与 BSA 结合涉及 1:1 化学计量比形成鲜明对比。BSA-Cbl(III) 复合物的体外形成并不排除随后与 HOCbl 结合到 BSA 上的血红素结合，而不会置换 BSA 结合的 HOCbl。这些数据表明，两种四吡咯与 BSA 以不同的结合口袋相互作用。
