Institute for the Application of Nuclear Energy, INEP, Banatska 31b, 11000, Belgrade, Serbia.
Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, 11000, Belgrade, Serbia.
J Biol Inorg Chem. 2019 Feb;24(1):61-70. doi: 10.1007/s00775-018-1628-7. Epub 2018 Nov 19.
Cys34 thiol group of human serum albumin (HSA) represents major plasma antioxidant. Its reactivity is influenced by multiple factors. The influence of fatty acids (FA; saturated, mono, and poly unsaturated acids from fish oil) binding to HSA, on copper(II) binding affinity and Cys34 thiol group accessibility/reactivity, in the presence of carbonylation agent (methylglyoxal, MG) was examined. HSA-copper(II) content, thiol group reactivity, and HSA carbonylation level were monitored spectrophotometrically. Changes in HSA were followed by fluorescence spectroscopy and native PAG electrophoresis. FA/HSA molar ratio was screened by GC. Together, binding of copper(II) ions and FA to HSA increase the reactivity of Cys34 thiol group (depending on the type of FA), with constant contribution of copper(II) ions of one-third. Carbonylation of FA-HSA-Cu(II) complexes caused a decrease in the Cys34 thiol group content, accompanied by a decrease in the content of HSA-bound copper. The carbonylation level of guanidine groups was not affected by FAs and copper(II) binding. Fluorescent emission spectra of FA-HSA-Cu(II)-MG complexes showed conformational changes in HSA molecule. Although binding of fatty acids and copper ions caused a significant increase in the thiol group reactivity, Cys34 thiol from FA-HSA-Cu(II) complexes reacted with MG in smaller extent than expected, probably as a consequence of conformational changes introduced by carbonylation. Increase in the percentage of reacted-free thiol groups with MG (due to FA and copper binding) may not seem to be very significant, but it is very important in complex biological systems, where catalytic metal is present.
人血清白蛋白（HSA）的 Cys34 巯基代表主要的血浆抗氧化剂。其反应性受多种因素影响。本研究考察了脂肪酸（FA；来自鱼油的饱和、单不饱和和多不饱和酸）与 HSA 结合，对铜（II）结合亲和力和 Cys34 巯基可及性/反应性的影响，存在羰基化剂（甲基乙二醛，MG）。通过分光光度法监测 HSA-铜（II）含量、巯基反应性和 HSA 羰基化水平。通过荧光光谱法和天然聚丙烯酰胺凝胶电泳法跟踪 HSA 的变化。通过 GC 筛选 FA/HSA 摩尔比。铜（II）离子和 FA 与 HSA 的结合共同增加 Cys34 巯基的反应性（取决于 FA 的类型），而铜（II）离子的贡献恒定为三分之一。FA-HSA-Cu(II) 配合物的羰基化导致 Cys34 巯基含量降低，同时结合的铜减少。胍基的羰基化水平不受 FAs 和铜（II）结合的影响。FA-HSA-Cu(II)-MG 配合物的荧光发射光谱显示 HSA 分子构象发生变化。尽管脂肪酸和铜离子的结合导致巯基反应性显著增加，但 FA-HSA-Cu(II) 配合物中的 Cys34 巯基与 MG 的反应程度低于预期，可能是由于羰基化引起的构象变化所致。由于 FA 和铜结合，与 MG 反应的游离巯基百分比增加（due to FA and copper binding）可能看起来并不非常显著，但在存在催化金属的复杂生物系统中，这是非常重要的。
