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Protein binding of alpha-hydroxyethyl free radicals
1Center for Gene Therapy, Department of Pharmacology and Toxicology, Faculty of Chemical and Pharmaceutical Sciences and Millennium Institute for Advanced Studies in Cell Biology and Biotechnology, University of Chile, Santiago, Chile.
Background:
The auto-oxidation of ethanol is likely to proceed via the initial formation of hydroxyethyl radicals (HERs), the one-electron oxidation product. In the laboratory, HERs can be generated by the Fenton reaction (H2O2+ Fe+2) in the presence of ethanol. We report studies on the binding of HERs to serum albumin, generated under Fenton and non-Fenton conditions.
Methods:
The generation of HER was determined by electron paramagnetic resonance spectroscopy. The formation of ethanol-derived protein adducts was determined by 14C-ethanol incorporation into serum albumin and by the binding of antibodies raised against HER adducts.
Results:
We report that serum albumin, used as a model protein, is an effective trapping agent of HERs. In addition, HER radicals covalently bind to albumin to form acid stable adducts. Unexpectedly, we found that under aerobic conditions, the incubation of 50 mM ethanol and phosphate buffer (which contains iron traces) in the absence of the Fenton reagent yields HER radicals as shown by electron paramagnetic resonance spectroscopy and the formation of acid stable protein adducts that are recognized by antibodies raised against HER radical adducts.
Conclusions:
Proteins (serum albumin used as a model) are avid trapping agents of HER. There are minimal requirements for the generation of HER, because in the presence of oxygen and a phosphate buffer that contains traces of iron, ethanol readily generates HERs. Thus, HER production is likely to occur in many tissues. The ability of proteins to bind this ethanol radical should be valuable in the diagnosis of alcohol abuse and may be relevant to some of the chronic effects of ethanol.