Characterization of non-covalent oligomers of proteins treated with hypochlorous acid.
Hypochlorous acid (HOCl) is a potent oxidant produced by myeloperoxidase that causes aggregation of many proteins. Treatment of apohaemoglobin and apomyoglobin with HOCl produced a regular series of oligomer bands when the proteins were separated by SDS/PAGE under reducing conditions. Aggregation was detectable at a HOCl/protein molar ratio of 0.5:1 and was maximal at ratios of 10:1-20:1. Dimers formed within 1 min of adding HOCl, and further aggregation occurred over the next 30 min. No convincing evidence for covalent cross-linking was obtained by amino acid analysis, peptide analysis or electrospray ionization-MS of HOCl-modified apomyoglobin. The latter showed an increase in mass consistent with conversion of the two methionine residues into sulphoxides. A 5-fold excess of HOCl generated approximately three chloramines on the apomyoglobin. These underwent slow decay. Protein carbonyls were formed and were almost entirely located only on the polymer bands. Conversion of positively into negatively charged groups on the protein by succinylation caused preformed aggregates to dissociate. Treatment of apomyoglobin with taurine chloramine generated methionine sulphoxides but few protein carbonyls, and did not result in aggregation. We conclude that aggregation was due to strong, non-covalent interactions between protein chains. We propose that formation of protein carbonyls and possibly chloramines, along with methionine oxidation, alters protein folding to expose hydrophobic areas on neighbouring molecules that associate to form dimers and higher-molecular-mass aggregates. This process could lead to the formation of aggregated proteins at sites of myeloperoxidase activity and contribute to inflammatory tissue injury.
Chapman
Anna L P
AL
Free Radical Research Group, Department of Pathology, Christchurch School of Medicine and Health Sciences, P.O. Box 4345, Christchurch, New Zealand.
Winterbourn
Christine C
CC
Brennan
Stephen O
SO
Jordan
T William
TW
Kettle
Anthony J
AJ
D000596:Amino Acids / Q000187:drug effects; D000818:Animals; D001059:Apoproteins / Q000737:chemistry / Q000187:drug effects; D002700:Chloramines / Q000737:chemistry; D019281:Dimerization; D006454:Hemoglobins / Q000737:chemistry / Q000187:drug effects; D006801:Humans; D006997:Hypochlorous Acid / Q000494:pharmacology*; D007700:Kinetics; D009211:Myoglobin / Q000737:chemistry / Q000187:drug effects; D016877:Oxidants / Q000494:pharmacology*; D011506:Proteins / Q000187:drug effects*; D021241:Spectrometry, Mass, Electrospray Ionization; D013386:Succinates / Q000737:chemistry; D013654:Taurine / Q000031:analogs & derivatives* / Q000494:pharmacology
D000596:Amino Acids; D001059:Apoproteins; D002700:Chloramines; D006454:Hemoglobins; D009211:Myoglobin; D016877:Oxidants; D011506:Proteins; D013386:Succinates; C023478:apohemoglobin; C010857:apomyoglobin; D013654:Taurine; C043410:N-chlorotaurine; D006997:Hypochlorous Acid