Determination of 2-oxohistidine by amino acid analysis.
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.
Lewisch
S A
SA
Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-0320, USA.
Levine
R L
RL
D000596:Amino Acids / Q000032:analysis*; D015415:Biomarkers / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange; D005974:Glutamate-Ammonia Ligase / Q000737:chemistry; D006639:Histidine / Q000031:analogs & derivatives* / Q000032:analysis; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000502:physiology*; D012680:Sensitivity and Specificity
D000596:Amino Acids; D015415:Biomarkers; C083725:2-oxohistidine; D006639:Histidine; D005974:Glutamate-Ammonia Ligase