Thioredoxins are a group of ca. 12 kDa redox proteins that mediate numerous cytosolic processes in all cells. Human thioredoxin can be exported out of the cell where it has additional functions including the ability to stimulate cell growth. A recent crystal structure determination of human thioredoxin revealed an inactive dimeric form of the protein covalently linked through a disulfide bond involving Gys 73 from each monomer [Weichsel et al. (1996) Structure 4, 735-751]. In the present study, apparent dissociation constants (K(app)) for the noncovalently linked dimers were determined at various pHs using a novel assay in which preformed dimers, but not monomers, were rapidly linked through oxidation (with diamide) of the Cys 73 disulfide bond, and the relative amounts of monomer and dimer were detected by gel filtration. The values obtained were pH-dependent, varying between 6.1 and 166 μM for the pH range of 3.8-8.0, and were consistent with the titration of a single ionizable group having a pK(a) of 6.5. A similar value was obtained using gel filtration at pH 3.8 (K(app) = 164 μM), and the crystal structure of the diamide-oxidized protein was determined to be nearly identical to that obtained in the absence of diamide. Asp 60 lies in the dimer interface and was found to be responsible for the pH dependence for dimer formation, and therefore must have a pKa elevated by ~2.5 pH units. Mutation of Asp 60 to asparagine abolished nearly all of the pH dependence for dimer formation. The crystal structure of the D60N mutant revealed a dimer nearly identical to the wild type, but, surprisingly, it had the Ash 60 side chain rotated out of the dimer interface and replaced with two water molecules. The values obtained for K(app) suggest human thioredoxin may dimerize in vivo and possible roles for such dimers are discussed.
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