Department of Chemistry and Biochemistry, Florida International University, 11200 SW 8th St, Miami, FL, 33199, USA.
Marine Biochemical Sciences, Hollings Marine Laboratory, National Institute of Standards and Technology, 331 Fort Johnson Rd., Charleston, SC, 29412, USA.
Sci Rep. 2017 Nov 1;7(1):14794. doi: 10.1038/s41598-017-14653-w.
MS-based proteomic analysis was combined with in silico quantum mechanical calculations to improve understanding of protein adduction by N-phenylhydroxylamine (PhNHOH) and nitrosobenzene (NOB), metabolic products of aniline. In vitro adduction of model peptides containing nucleophilic sidechains (Cys, His, and Lys) and selected proteins (bovine and human hemoglobin and β-lactoglobulin-A) were characterized. Peptide studies identified the Cys thiolate as the most reactive nucleophile for these metabolites, a result consistent with in silico calculations of reactivity parameters. For PhNHOH, sulfinamides were identified as the primary adduction products, which were stable following tryptic digestion. Conversely, reactions with NOB yielded an additional oxidized adduct, the sulfonamide. In vitro exposure of human whole blood to PhNHOH and NOB demonstrated that only sulfinamides were formed. In addition to previously reported adduction of βCys of human Hb, two novel sites of adduction were found; αCys and βCys. We also report CD and UV-Vis spectroscopy studies of adducted human Hb that revealed loss of α-helical content and deoxygenation. The results provide additional understanding of the covalent interaction of aromatic amine metabolites with protein nucleophiles.
基于 MS 的蛋白质组学分析与计算机量子力学计算相结合，以提高对苯胺代谢产物 N-苯羟胺 (PhNHOH) 和亚硝基苯 (NOB) 与蛋白质加合作用的理解。对含有亲核侧链（半胱氨酸、组氨酸和赖氨酸）的模型肽和选定蛋白质（牛和人血红蛋白和β-乳球蛋白-A）进行了体外加合作用的表征。肽研究确定半胱氨酸硫醇盐是这些代谢物最具反应性的亲核试剂，这一结果与反应性参数的计算机计算一致。对于 PhNHOH，鉴定出亚磺酰胺是主要的加合产物，在胰蛋白酶消化后稳定。相反，与 NOB 的反应生成了另一种氧化加合物，即磺酰胺。人全血与 PhNHOH 和 NOB 的体外暴露表明仅形成亚磺酰胺。除了先前报道的人血红蛋白βCys 的加合作用外，还发现了两个新的加合部位；αCys 和βCys。我们还报告了加合人血红蛋白的 CD 和紫外可见光谱研究，发现α-螺旋含量丧失和脱氧。这些结果为芳香胺代谢物与蛋白质亲核试剂的共价相互作用提供了更多的了解。
