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

PURPOSE: 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.
METHODS: 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).
RESULTS: 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.
CONCLUSIONS: 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 secondary reactive-oxygen species (i.e., hydroxyl radical generated within complex forms or metal-bound forms of hydroxyl radical) that react with the peptide before they diffuse into the bulk solution. CD results indicate that different complexes are formed between the cyclic-His peptide and Cu(II) at pH 5.3 and pH 7.4. These different complexes may favor the formation of different degradation products. The apparent structural differences between the cyclic-His peptide and the cyclic 2-oxo-His peptide indicate that conformation of the cyclic-His peptide was impacted by metal-catalyzed oxidation.