Structural identification of valine hydroperoxides and hydroxides on radical-damaged amino acid, peptide, and protein molecules

We have previously demonstrated the formation of two reactive moieties on proteins during free radical attack: hydroperoxides, and 3,4-dihydroxyphenylalanine (DOPA). Here we have undertaken the structural elucidation of the hydroperoxides of valine, the amino acid which is most susceptible to peroxidation. Exposure of L-valine to free radicals generated by radiolysis in an oxygen-saturated system produced three valine hydroperoxides. Upon treatment with sodium borohydride these were reduced to their corresponding hydroxides, which can be separated and purified by high performance liquid chromatography (HPLC). Based on spectroscopic data from high resolution chemical ionization (CI) mass spectrometry (MS), electrospray (ES) MS, electron impact (EI) MS, proton (1H) nuclear magnetic resonance (NMR) and carbon-13 (13C) NMR studies, the three valine hydroxides have been identified as β-hydroxyvaline [(2S)-2-amino-3-hydroxy-3-methyl-butanoic acid], (2S,3S)-γ-hydroxyvaline [(2S,3S)-2-amino-3-hydroxymethyl-butanoic acid], and (2S,3R)-γ-hydroxyvaline [(2S,3R)-2-amino-3-hydroxymethyl-butanoic acid]. HPLC analysis of O-phthaldialdehyde (OPA) derivatives of the hydroxyvalines provides a sensitive and accurate method for quantitative measurement. This method enabled hydroxyvalines to be detected in the hydrolysates of a tripeptide (glutamylvalinyl-phenylalanine) and a protein (bovine serum albumin) that had been gamma-radiolysed and treated with sodium borohydride. Hydroxyvaline may be useful as a marker in studying protein oxidation in some biological systems under oxidative stress.