The role of aromatic amino acid oxidation, protein unfolding, and aggregation in the hypobromous acid-induced inactivation of trypsin inhibitor and lysozyme.
Hypobromous acid (HOBr) generated by activated eosinophils has been implicated in tissue injury observed in asthma, allergic reactions, and some infections. Proteins are major targets for this oxidant, but the mechanisms by which HOBr induces loss of function are not well-established. In this study, we have examined the effect of HOBr on protein structure (as assessed by amino acid loss, side chain oxidation, fragmentation, aggregation, and unfolding) and activity of a model protease inhibitor, soybean trypsin inhibitor (STI), and the protective enzyme lysozyme. Exposure of both proteins to low oxidant concentrations (< or = 5-fold molar excess) results in loss of function. In each case, loss of activity is associated with the selective oxidation of His, Trp, and Tyr residues, which results in protein unfolding (with lysozyme) and protein aggregation (with STI). Reaction with these residues accounts for 25 and 50% of the HOBr with STI (25-fold excess) and lysozyme (4-fold excess), respectively. These processes are believed to lead to changes in the structure of the proteins, which disrupts substrate binding. With both proteins, the oxidation of other residues, including Met, does not appear to play a major role. Bromamines, formed by reaction with amine groups, are major products, which account for 45 and 35% of the HOBr with STI (25-fold excess) and lysozyme (4-fold excess), respectively. Decomposition of these species correlates with secondary oxidation reactions, and with lysozyme, a time-dependent loss in activity. Overall, 70% of the HOBr can be accounted for with STI and 95% with lysozyme.
Hawkins
Clare L
CL
The Heart Research Institute, Camperdown, Sydney, NSW 2050, Australia. hawkinsc@hri.org.au
Davies
Michael J
MJ
D024322:Amino Acids, Aromatic / Q000737:chemistry*; D001959:Bromates / Q000633:toxicity*; D009113:Muramidase / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D017510:Protein Folding; D014361:Trypsin Inhibitors / Q000737:chemistry* / Q000378:metabolism
D024322:Amino Acids, Aromatic; D001959:Bromates; D014361:Trypsin Inhibitors; D009113:Muramidase; C027664:hypobromous acid