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-dihyd roxyphenylalanine (DOPA). Here we have undertaken the structural eluci dation of the hydroperoxides of valine, the amino acid which is most s usceptible to peroxidation. Exposure of L-valine to free radicals gene rated by radiolysis in an oxygen-saturated system produced three valin e hydroperoxides. Upon treatment with sodium borohydride these were re duced to their corresponding hydroxides, which can be separated and pu rified by high performance liquid chromatography (HPLC). Based on spec troscopic data from high resolution chemical ionization (CI) mass spec trometry (MS), electrospray (ES) MS, electron impact (EI) MS, proton ( H-1) nuclear magnetic resonance (NMR) and carbon-13 (C-13) NMR studies , the three valine hydroxides have been identified as beta-hydroxyvali ne [(2S)-2-amino-3-hydroxy-3-methyl-butanoic acid], (2S,3S)-gamma-hydr oxyvaline [(2S,3S)-2-amino-3-hydroxymethyl-butanoic acid], and (2S,3R) -gamma-hydroxyvaline [(2S,3R)-2-amino-3-hydroxymethyl-butanoic acid]. HPLC analysis of O-phthaldialdehyde (OPA) derivatives of the hydroxyva lines provides a sensitive and accurate method for quantitative measur ement. This method enabled hydroxyvalines to be detected in the hydrol ysates of a tripeptide (glutamyl-valinyl-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 studyi ng protein oxidation in some biological systems under oxidative stress .