Chemical Pathways of Peptide Degradation. X: Effect of Metal-Catalyzed Oxidation on the Solution Structure of a Histidine-Containing Peptide Fragment of Human Relaxin

Abstract

Purpose. To elucidate the major degradation products of the metal-catalyzedoxidation of (cyclo S-S) AcCys-Ala-X-Val-Gly-CysNH₂(X = His, cyclic-His peptide), which is a fragment of the proteinrelaxin, and the effect of this oxidation on its solution structure.

Methods. The cyclic-His peptide and its potential oxidative degradationproducts, cyclic-Asp peptide (X = Asp) and cyclic-Asn peptide(X = Asn), were prepared by using solid phase peptide synthesisand purified by preparative HPLC. The degradation of the cyclic-Hispeptide was investigated at pH 5.3 and 7.4 in an ascorbate/cupricchloride/oxygen [ascorbate/Cu(II)/O₂] system in the absence or presenceof catalase (CAT), superoxide dismutase (SOD), isopropanol, andthiourea. The oxidation of the cyclic-His peptide was also studied in thepresence of hydrogen peroxide (H₂O₂). All reactions were monitored byreversed-phase HPLC. The main degradation product of the cyclic-Hispeptide formed at pH 7.4 in the presence of ascorbate/Cu(II)/O₂was isolated by preparative HPLC and identified by ¹H NMR andelectrospray mass spectrometry. The complexation of Cu(II) with thecyclic-His peptide was determined with ¹H NMR. The solution structureof the cyclic-His peptide in the presence and absence of Cu(II) at pH5.3 and 7.4 and the solution structure of the main degradation productwere determined using circular dichroism (CD).

Results. CAT and thiourea were effective in stabilizing the cyclic-Hispeptide to oxidation by ascorbate/Cu(II)/O₂, while SOD and isopropanolwere ineffective. Cyclic-Asp and cyclic-Asn peptides were notobserved as degradation products of the cyclic-His peptide oxidized atpH 5.3 and 7.4 in an ascorbate/Cu(II)/O₂ system. The main degradationproduct 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 inlow yields, including the cyclic 2-oxo-His peptide. The cyclic 2-oxo-Hispeptide appeared to have a different secondary structure than didthe cyclic-His peptide as determined by CD. ¹H NMR results indicatecomplexation between the cyclic-His peptide and Cu(II). CD resultsindicated that the solution structure of the cyclic-His peptide in thepresence of Cu(II) at pH 5.3 was different than the solution structureobserved at pH 7.4.

Conclusions. H₂O₂ and superoxide anion radical ($\left( {\operatorname{O} _2^{\cdot - } } \right)$) were deducedto be the intermediates involved in the ascorbate/Cu(II)/O₂-inducedoxidation of cyclic-His peptide. H₂O₂ degradation by a Fenton-typereaction appears to form secondary reactive-oxygen species (i.e.,hydroxyl radical generated within complex forms or metal-bound formsof hydroxyl radical) that react with the peptide before they diffuse intothe bulk solution. CD results indicate that different complexes areformed between the cyclic-His peptide and Cu(II) at pH 5.3 and pH7.4. These different complexes may favor the formation of differentdegradation products. The apparent structural differences between thecyclic-His peptide and the cyclic 2-oxo-His peptide indicate that conformationof the cyclic-His peptide was impacted by metal-catalyzedoxidation.