To determine whether kinetic trapping can be applied to human Trx1, we created recombinant wild-type and mutant Trx proteins, each equipped with a C-terminal dual affinity tag composed of a streptavidin-binding peptide (SBP) and a hexahistidine tag. To create a trapping mutant, the second cysteine of the 32 CXXC 35 motif was replaced by serine (C35S). Trx1 harbors three additional cysteine residues distal to the active site (cysteines 62, 69 and 73). As these residues are dispensable for catalytic activity but may cause oxidative inactivation by either intra-or intermolecular disulfide bond formation (Casagrande et al, 2002; Watson et al, 2003) , we also created mutants containing amino-acid substitutions for those additional cysteines ( Figure 1B ; CCCCC, CCAAA, CSCCC, CSAAA and SSAAA annotate the identity of residues 32, 35, 62, 69 and 73).