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Oxidative damage to collagen and related substrates by metal ion/hydrogen peroxide systems: random attack or
1Department of Chemistry, University of York, UK.
Abstract:
Degradation of collagen by oxidant species may play an important role in the progression of rheumatoid arthritis. Whilst the overall effects of this process are reasonably well defined, little is known about the sites of attack, the nature of the intermediates, or the mechanism(s) of degradation. In this study electron paramagnetic resonance spectroscopy with spin trapping has been used to identify radicals formed on collagen and related materials by metal ion-H2O2 mixtures. Attack of the hydroxyl radical, from a Fe(II)-H2O2 redox couple, on collagen peptides gave signals from both side chain (.CHR'R"), and alpha-carbon[.C(R)(NH-)CO-,R = side-chain]radicals. Reaction with collagen gave both broad anisotropic signals, from high-molecular-weight protein-derived radicals, and isotropic signals from mobile species. The latter may be low-molecular-weight fragments, or mobile side-chain species; these signals are similar to those from the alpha-carbon site of peptides and the side-chain of lysine. Enzymatic digestion of the large, protein-derived, species releases similar low-molecular-weight adducts. The metal ion employed has a dramatic effect on the species observed. With Cu(I)-H2O2 or Cu(II)-H2O2 instead of Fe(II)-H2O2, evidence has been obtained for: i) altered sites of attack and fragmentation, ii) C-terminal decarboxylation, and iii) hydrogen abstraction at N-terminal alpha-carbon sites. This altered behaviour is believed to be due to the binding of copper ions to some substrates and hence site-specific damage. This has been confirmed in some cases by electron paramagnetic resonance studies of the Cu(II) ions.
Insights
This study used electron paramagnetic resonance spectroscopy to identify radicals formed during collagen degradation by metal ion-hydrogen peroxide mixtures, revealing distinct attack sites and mechanisms depending on the metal ion used.
Area of Science:
- Biochemistry
- Spectroscopy
- Rheumatology
Background:
- Collagen degradation by oxidants is implicated in rheumatoid arthritis progression.
- The specific sites, intermediates, and mechanisms of collagen degradation by oxidants are not well understood.
Purpose of the Study:
- To identify radicals formed on collagen and related materials using metal ion-hydrogen peroxide mixtures.
- To elucidate the mechanisms of collagen degradation by different metal ions.
Main Methods:
- Electron paramagnetic resonance (EPR) spectroscopy with spin trapping.
- Utilized Fe(II)-H2O2 and Cu(I)/Cu(II)-H2O2 redox couples to induce collagen degradation.
- Analyzed radical species formed on collagen peptides and intact collagen.
Main Results:
- Hydroxyl radical attack on collagen peptides generated side chain and alpha-carbon radicals.
- Degradation of intact collagen produced high-molecular-weight protein-derived radicals and mobile species.
- Copper ions (Cu(I)/Cu(II)) induced altered attack sites, fragmentation, C-terminal decarboxylation, and N-terminal alpha-carbon hydrogen abstraction compared to iron ions.
- Copper ion binding to substrates was suggested as the cause of site-specific damage.
Conclusions:
- EPR spectroscopy effectively identified radical species involved in collagen degradation.
- The metal ion plays a critical role in determining the sites and mechanisms of collagen attack.
- Findings provide insights into the molecular mechanisms of collagen degradation relevant to rheumatoid arthritis.