Oxidation and reduction of thymosin beta(4) and its influence on the interaction with G-actin studied by reverse-phase HPLC and post-column derivatization with fluorescamine

Oxidation of methionine residues has been shown to provide a potential regulatory mechanism in protein function in vivo. Thymosin beta(4) which forms a one-to-one complex with G-actin is the most abundant member of beta-thymosins in mammalian tissues and possesses a methionine residue at position 6. In preparations of mammalian tissues thymosin beta(4) is, in most cases, accompanied by a small amount of its sulfoxide. Using reverse-phase HPLC we showed that the oxidation of the methionine residue of thymosin beta(4) can be achieved by millimolar concentrations of H2O2 in vitro and is accompanied by an 18-fold increase in the apparent dissociation constant of its complex with G-actin. Peptides 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 postcolumn derivatization with fluorescamine. 50% of thymosin P4 is oxidized after 3.5 or 6 hours using 3 mM or 1 mM H2O2, respectively. In the case of 0.5 mM H2O2, about 45% of the methionine residues are oxidized after 18 hours. The resulting sulfoxide is reduced with aqueous solutions of sodiumbisulfite. The reduction is accompanied by the recovery 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 80% after 12 hours while only 30% is reduced with dithiothreitol (0.81 M) after 25 hours. The large amount of sodiumbisulfite necessary for reduction can be separated from the peptide by solid phase extraction. (C) 1997 Elsevier Science B.V.