Oxidation of myosin by haem proteins generates myosin radicals and protein cross-links.
Previous studies have reported that myosin can be modified by oxidative stress and particularly by activated haem proteins. These reactions have been implicated in changes in the properties of this protein in food samples (changes in meat tenderness and palatability), in human physiology (alteration of myocyte function and force generation) and in disease (e.g. cardiomyopathy, chronic heart failure). The oxidant species, mechanisms of reaction and consequences of these reactions are incompletely characterized. In the present study, the nature of the transient species generated on myosin as a result of the reaction with activated haem proteins (horseradish peroxidase/H2O2) and met-myoglobin/H2O2) has been investigated by EPR spectroscopy and amino-acid consumption, product formation has been characterized by HPLC, and changes in protein integrity have been determined by SDS/PAGE. Multiple radical species have been detected by EPR in both the presence and the absence of spin traps. Evidence has been obtained for the presence of thiyl, tyrosyl and other unidentified radical species on myosin as a result of damage-transfer from oxidized myoglobin or horseradish peroxidase. The generation of thiyl and tyrosyl radicals is consistent with the observed consumption of cysteine and tyrosine residues, the detection of di-tyrosine by HPLC and the detection of both reducible (disulfide bond) and non-reducible cross-links between myosin molecules by SDS/PAGE. The time course of radical formation on myosin, product generation and cross-link induction are consistent with these processes being interlinked. These changes are consistent with the altered function and properties of myosin in muscle tissue exposed to oxidative stress arising from disease or from food processing.
Food Chemistry, Department of Food Science, Faculty of Life Sciences, University of Copenhagen, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark.
Marianne N
MN
Lund
Catherine
C
Luxford
Leif H
LH
Skibsted
Michael J
MJ
Davies
D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D006420:Hemeproteins / Q000737:chemistry / Q000378:metabolism*; D009218:Myosins / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D011487:Protein Conformation; D013113:Spin Labels; D013552:Swine
D000596:Amino Acids; D006420:Hemeproteins; D013113:Spin Labels; D009218:Myosins