Oxidation and reduction of thymosin β4 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 β4 which forms a one-to-one complex with G-actin is the most abundant member of β-thymosins in mammalian tissues and possesses a methionine residue at position 6. In preparations of mammalian tissues thymosin β4 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 β4 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 β4 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.