Li S, Nguyen T H, Schöneich C, Borchardt R T
Department of Pharmaceutical Chemistry, University of Kansas, Lawrence 66045, USA.
Biochemistry. 1995 May 2;34(17):5762-72. doi: 10.1021/bi00017a008.
The interactions of proteins with reactive oxygen species may result in covalent modifications of amino acid residues in proteins and possible alterations of protein conformation. In an attempt to elucidate the mechanisms of the metal-catalyzed oxidation of human relaxin, we employed ascorbic acid/transition metal ion [Cu(II) or Fe(III)]/O2 as a model oxidizing system. Experimental results indicated selective oxidation of His and Met residues and rapid formation of aggregates (noncovalent, pH dependent) following the oxidation reaction. Amino acid analysis and LC/MS data following tryptic digestion demonstrated the oxidation of the His A(12) residue, which resulted in 2-oxohistidine and some other unidentified degradation products. The oxidation of both Met residues to Met-sulfoxides was also identified, and it was found that Met B(4) was more easily oxidized than Met B(25). The comparative kinetic studies of two Met-containing fragments of relaxin suggested that the preferred oxidation of Met B(4) is due to its close proximity to some metal-binding neighboring amino acid residues. These covalent alterations may lead to the modification of secondary and tertiary structure and increase the exposure of the hydrophobic surface of the protein which eventually induces aggregation of precipitation. The modification of relaxin by ascorbic acid/CuCl2 solution could be totally inhibited by the presence of EDTA. In contrast, catalase and superoxide dismutase showed no effects on the oxidation process.
蛋白质与活性氧的相互作用可能导致蛋白质中氨基酸残基的共价修饰以及蛋白质构象的可能改变。为了阐明人松弛素金属催化氧化的机制，我们采用抗坏血酸/过渡金属离子[铜（II）或铁（III）]/氧气作为模型氧化系统。实验结果表明，氧化反应后组氨酸和甲硫氨酸残基发生选择性氧化，并迅速形成聚集体（非共价，pH依赖性）。胰蛋白酶消化后的氨基酸分析和液相色谱/质谱数据表明，组氨酸A（12）残基发生氧化，产生2-氧代组氨酸和一些其他未鉴定的降解产物。还鉴定出两个甲硫氨酸残基均氧化为甲硫氨酸亚砜，并且发现甲硫氨酸B（4）比甲硫氨酸B（25）更容易被氧化。松弛素两个含甲硫氨酸片段的比较动力学研究表明，甲硫氨酸B（4）优先氧化是由于其与一些结合金属的相邻氨基酸残基距离较近。这些共价改变可能导致二级和三级结构的修饰，并增加蛋白质疏水表面的暴露，最终导致沉淀聚集。抗坏血酸/CuCl2溶液对松弛素的修饰可被EDTA完全抑制。相反，过氧化氢酶和超氧化物歧化酶对氧化过程没有影响。
