A plasmid pACA/Trx was constructed containing the human thioredoxingene under T7 promoter control. To examine the role of the conservd amino acidresidues in the active site of human thioredoxin (-Cys31-Gly32-Pro33-Cys34-), G32H,P33H and G32H/P33H human thioredoxin -mutants were created by site-directedmutagenesis. The wild type thiorbdoxin and the mutants were expressed in highyield and purified to homogeneity. All thioredoxin proteins were characterized foractivity with thioredoxin reductase, their redox potential and tryptophan fluores-cence.properties, Protein disulfide reductfon coupling to NADPH and thioredoxin re-ductase was studied using insulin as a substrate. At pH 7.0, the Km, kcat and kcat/Km of wild type hTrx fot tbe human placenta thioredoxin reductasee (HP-TR) were2. 0 μM, 2, 800 min-1 and 1,400 μM-1 min-1, respectively. Km of P33H and G32H/P33H hTrx was to 4-5 fold bigher and kcat/Km2. 5 fold lower than for wild typethioredoxin. The redox potentials of wild type hTrx was - 247 mV, compared towhich, the redox potential of P33H and G32H/P33H hTrx were significantly high-er. The tryptophan fluoresence spectrum of human tnioredoxin showed only a smalldifference between oxidized and reduced forms. The folding properties and stabilityof the mutant proteins were similar to those of wild type thioredoxin,which showedthat the replacement of Gly.32 and Pro 33 residues with histidine had small effectson overall tertiary structure. A plasmid pACA / Trx was constructed containing the human thioredoxin gene under T7 promoter control. To examine the role of the conservd amino acid residues in the active site of human thioredoxin (-Cys31-Gly32-Pro33-Cys34-), G32H, P33H and G32H / P33H human thioredoxin-mutants were created by site-directed mutagenesis. The wild type thiorbdoxin and the mutants were expressed in highyield and purified to homogeneity. All thioredoxin proteins were characterized foractivity with thioredoxin reductase, their redox potential and tryptophan fluorescence. Properties, Protein disulfide reductfon coupling to NADPH and thioredoxin re-ductase was studied using insulin as a substrate. At pH 7.0, the Km, kcat and kcat / Km of wild type hTrx fot tbe human placenta thioredoxin reductase (HP-TR) were 2.0 μM, 2, 800 min-1 and 1,400 μM-1 min-1, respectively. Km of P33H and G32H / P33H hTrx was to 4-5 fold bigher and kcat / Km2.5 fold lower than for wild type thioredoxin. The redox potentials of wild type hTrx w as-247 mV, compared towhich, the redox potential of P33H and G32H / P33H hTrx were significantly high-er. The tryptophan fluoresence spectrum of human tnioredoxin showed only a small difference between oxidized and reduced forms. The folding properties and stability of the mutant proteins were similar to those of wild type thioredoxin, which showed that the replacement of Gly.32 and Pro 33 residues with histidine had small effectson overall tertiary structure.