Abstract
Myoglobin (Mb), an oxygen-binding heme protein highly expressed in heart and skeletal muscle, has been shown to undergo oxidative modifications on both an inter- and intramolecular level when exposed to hydrogen peroxide (H2O2) in vitro. Here, we show that exposure to H2O2 increases the peroxidase activity of Mb. Reaction of Mb with H2O2 causes covalent binding of heme to the Mb protein (Mb-X), corresponding to an increase in peroxidase activity when ascorbic acid is the reducing co-substrate. Treatment of H2O2-reacted Mb with ascorbic acid reverses the Mb-X crosslink. Reaction with H2O2 causes Mb to form dimers, trimers, and larger molecular weight Mb aggregates, and treatment with ascorbic acid regenerates Mb monomers. Reaction of Mb with H2O2 causes formation of dityrosine crosslinks, though the labile nature of the crosslinks broken by treatment with ascorbic acid suggests that the reversible aggregation of Mb is mediated by crosslinks other than dityrosine. Disappearance of a peptide containing a tryptophan residue when Mb is treated with H2O2 and the peptide’s reappearance after subsequent treatment with ascorbic acid suggest that tryptophan side chains might participate in the labile crosslinking. Taken together, these data suggest that while exposure to H2O2 causes Mb-X formation, increases Mb peroxidase activity, and causes Mb aggregation, these oxidative modifications are reversible by treatment with ascorbic acid. A caveat is that future studies should demonstrate that these and other in vitro findings regarding properties of Mb have relevance in the intracellular milieu, especially in regard to actual concentrations of metMb, H2O2, and ascorbate that would be found in vivo.
Highlights
One of the major mechanisms of toxicity from reactive oxygen species (ROS) is the direct oxidation of protein side chains [1]
To assess the effects of heme-protein cross-links on metMb peroxidase activity, we measured the activity of H2 O2 -reacted metMb with
When TMB was used as a substrate, pre-treatment had no effect on Mb peroxidase activity (Figure 1C)
Summary
One of the major mechanisms of toxicity from reactive oxygen species (ROS) is the direct oxidation of protein side chains [1]. Some oxidations are reversible, such as oxidation of the cysteine thiol to sulfenic acid, the majority are considered to be irreversible and to promote the destabilization of tertiary structure as well as the eventual loss of protein function [2,3]. One group of proteins that are susceptible to oxidative damage is heme proteins [4,5,6]. This is likely due to the redox activity of the porphyrin-centered iron. When present in the ferric state (III), heme proteins are prone to oxidation by endogenously produced H2 O2 , resulting in a highly unstable oxoferryl form, which can oxidize protein side chains either internally or on another protein [7].
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
