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Anal Biochem. 1995 Nov 1;231(2):440-6.

Determination of 2-oxohistidine by amino acid analysis.

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Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-0320, USA.


Oxidative modification of proteins has been implicated in aging, ischemia reperfusion, carcinogenesis, and other phenomena. Oxidation of the C-2 position of the imidazole ring of histidine converts the residue to 2-oxohistidine, a novel amino acid which may serve as a marker of oxidative modification of proteins (K. Uchida and S. Kawakishi, J. Biol. Chem. 269, 2405-2410, 1994). It has been identified in oxidatively modified proteins by electrochemical detection during reverse-phase high-pressure liquid chromatography, by mass spectrometry, and as the phenylthiohydantoin after Edman degradation, but not by amino acid analysis of protein hydrolysates. We now describe procedures for stabilizing 2-oxohistidine which allow its quantification by routine methods of amino acid analysis. These include classical ion exchange chromatography with postcolumn derivatization by o-phthaldialdehyde, reverse-phase chromatography with precolumn derivatization by o-phthaldialdehyde, and reverse-phase chromatography with precolumn derivatization by 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. Using these techniques, a previously unidentified amino acid which appears during the oxidative inactivation of glutamine synthetase was shown to be 2-oxohistidine. One picomole of 2-oxohistidine was readily detected in a protein hydrolysate containing 1700 pmol total amino acids.

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