Species-specific effects on non-enzymatic metmyoglobin reduction in vitro

• Non-enzymatic metmyoglobin reduction is species-specific.
• Number of histidine residues determines non-enzymatic metmyoglobin reduction.
• pH 5.6 resulted in greater metmyoglobin reduction compared with pH 7.4.
• Covalent binding of HNE decreased metmyoglobin reduction.

Our objectives were to determine the non-enzymatic metmyoglobin reduction properties of bovine, porcine, and equine myoglobins and to characterize the effects of pre-incubation of 4-hydroxy-2-nonenal (HNE) with myoglobins on non-enzymatic metmyoglobin reduction in vitro. Purified bovine, porcine, and equine metmyoglobins (0.05 mM) were reduced at pH 5.6 and 7.4 in the presence or absence of HNE. Rates of metmyoglobin reduction were monitored by spectrophotometry, and myoglobin adducts were characterized by high-resolution mass-spectrometry. Results showed that the species origins of individual myoglobins determined rates of non-enzymatic reduction (beef > equine > pork; P < 0.05). Irrespective of species, pre-incubation of HNE myoglobin decreased non-enzymatic metmyoglobin reduction compared with control at both pH 5.6 and 7.4 (P < 0.05). Mass spectrometric analysis revealed adducts of HNE with bovine, porcine, and equine myoglobins. The results indicate that the amino acid composition and the covalent binding of HNE with myoglobin can significantly decrease the ability of heme to accept electrons.