Introduction: Neutrophils are recruited to heart tissues after acute myocardial infarction. Activated neutrophils release myeloperoxidase that produces the potent oxidant hypochlorous acid (HOCl). Oxidation of the oxygen transport protein myoglobin (Mb) by HOCl decreases the efficiency of the Fe(III) haem reduction by cytochrome b5 to the oxygen-binding by Fe(II)Mb form. Mb is also a pseudo-peroxidase that can regulate levels of hydrogen peroxide (H2O2) in the heart.
Methods: Horse heart Mb was exposed to increasing doses of HOCl and were then assessed by determining changes in mobility within agarose gels, mass using MALDI mass spectrometry, ultraviolet-visible (UV/vis) absorbance. The reaction of Mb with H2O2 was monitored by electron paramagnetic resonance (EPR) spectroscopy with spin trapping and by assessing ABTS oxidation by the Mb/H2O2 pseudo-peroxidase system.
Results: Pathological concentrations of HOCl produced myoglobin oxidation products of increased electrophoretic mobility and markedly different UV/vis absorbance. Mass analysis indicated protein mass increases by multiples of 16 a.m.u., consistent addition of molecular oxygen. Parallel analysis of protein chlorination by quantitative mass spectrometry revealed an increase in the 3-chrlorotyrosine/tyrosine ratio in Mb exposed to HOCl. Pre-treatment of Mb with HOCl affected the reaction between the haem protein and H2O2 as judged by a decrease in the level of spin-trapped tyrosyl radicals detected by EPR spectroscopy. In addition, the rate constant The ABTS oxidation study showed a HOCl dose-dependent reduction in rate constant, indicating reduced ability to peroxide.
Conclusion: Exposure to HOCl causes in myoglobin structural changes, including methionine oxidation and tyrosine chlorination. These modifications alter Mb catalase-like activity, inhibiting the rate of reaction with H2O2. Decreased Mb catalase-like activity may promote oxidative stress in heart tissue following myocardial infarction.