OXIDATION AND REDUCTION OF THYMOSIN BETA(4) AND ITS INFLUENCE ON THE INTERACTION WITH G-ACTIN STUDIED BY REVERSE-PHASE HPLC AND POSTCOLUMN DERIVATIZATION WITH FLUORESCAMINE

Oxidation of methionine residues has been shown to provide a potential regulatory mechanism in protein function in vivo. Thymosin beta(4) wh ich forms a one-to-one complex with G-actin is the most abundant membe r of beta-thymosins in mammalian tissues and possesses a methionine re sidue at position 6. In preparations of mammalian tissues thymosin bet a(4) is, in most cases, accompanied by a small amount of its sulfoxide . Using reverse-phase HPLC we showed that the oxidation of the methion ine residue of thymosin beta(4) can be achieved by millimolar concentr ations of H2O2 in vitro and is accompanied by an 18-fold increase in t he apparent dissociation constant of its complex with G-actin. Peptide s were separated by reverse-phase HPLC using a RP-18 column applying a linear gradient of n-propanol in 20 mM pyridine - 0.11 M formic acid - 0.05 M lithiumperchlorate and were detected by fluorescence after po stcolumn derivatization with fluorescamine. 50% of thymosin P4 is oxid ized after 3.5 or 6 hours using 3 mM or 1 mM H2O2, respectively. In th e case of 0.5 mM H2O2, about 45% of the methionine residues are oxidiz ed after 18 hours. The resulting sulfoxide is reduced with aqueous sol utions of sodiumbisulfite. The reduction is accompanied by the recover y of the initial affinity to G-actin. With a solution of 90% saturated Na2S2O5 we find 50% reduction of the sulfoxide in about 5 hours and 8 0% after 12 hours while only 30% is reduced with dithiothreitol (0.81 M) after 25 hours. The large amount of sodiumbisulfite necessary for r eduction can be separated from the peptide by solid phase extraction. (C) 1997 Elsevier Science B.V.