Aggregation and precipitation of human relaxin induced by metal-catalyzed oxidation.

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.