Chemical pathways of peptide degradation. X: Effect of metal-catalyzed oxidation on the solution structure of a histidine-containing peptide fragment of human relaxin

To elucidate the major degradation products of the metal-catalyzed oxidation of (cyclo S-S) AcCys-Ala-X-Val-Gly-CysNH2 (X = His, cyclic-His peptide), which is a fragment of the protein relaxin, and the effect of this oxidation on its solution structure. The cyclic-His peptide and its potential oxidative degradation products, cyclic-Asp peptide (X = Asp) and cyclic-Asn peptide (X = Asn), were prepared by using solid phase peptide synthesis and purified by preparative HPLC. The degradation of the cyclic-His peptide was investigated at pH 5.3 and 7.4 in an ascorbate/cupric chloride/oxygen [ascorbate/Cu(II)/O2] system in the absence or presence of catalase (CAT), superoxide dismutase (SOD), isopropanol, and thiourea. The oxidation of the cyclic-His peptide was also studied in the presence of hydrogen peroxide (H2O2). All reactions were monitored by reversed-phase HPLC. The main degradation product of the cyclic-His peptide formed at pH 7.4 in the presence of ascorbate/Cu(II)/O2 was isolated by preparative HPLC and identified by 1H NMR and electrospray mass spectrometry. The complexation of Cu(II) with the cyclic-His peptide was determined with 1H NMR. The solution structure of the cyclic-His peptide in the presence and absence of Cu(II) at pH 5.3 and 7.4 and the solution structure of the main degradation product were determined using circular dichroism (CD). CAT and thiourea were effective in stabilizing the cyclic-His peptide to oxidation by ascorbate/Cu(II)/O2, while SOD and isopropanol were ineffective. Cyclic-Asp and cyclic-Asn peptides were not observed as degradation products of the cyclic-His peptide oxidized at pH 5.3 and 7.4 in an ascorbate/Cu(II)/O2 system. The main degradation product formed at pH 7.4 was the cyclic 2-oxo-His peptide (X = 2-oxo-His). At pH 5.3, numerous degradation products were formed in low yields, including the cyclic 2-oxo-His peptide. The cyclic 2-oxo-His peptide appeared to have a different secondary structure than did the cyclic-His peptide as determined by CD. 1H NMR results indicate complexation between the cyclic-His peptide and Cu(II). CD results indicated that the solution structure of the cyclic-His peptide in the presence of Cu(II) at pH 5.3 was different than the solution structure observed at pH 7.4. H2O2 and superoxide anion radical (O(*-)2) were deduced to be the intermediates involved in the ascorbate/Cu(II)/O2-induced oxidation of cyclic-His peptide. H2O2 degradation by a Fenton-type reaction appears to form seco