Table 1

Catalytic activities of Trx Rs

SubstrateSpecific activities of TrxR enzymes, μmol per min per mg
Control TrxR1(B)CAM-TrxR1SeCys498 → Cys498 TrxR1
DTNB420.62.7
TrxS2130.071.5
Oxidized insulin, TrxS215Not detectableNot detectable
  • DTNB reduction was monitored spectrophotometrically at 412 nm in a 1-ml reaction mixture containing 100 mM potassium phosphate (pH 7.4), 2 mM EDTA, 1.5 mM DTNB, 0.2 mg of BSA, 0.2 mM NADPH, and 100–500 ng of native, 1–5 μg of (B)CAM-modified, or 1–5 μg of SeCys → Cys mutant TrxR1. NADPH-dependent reduction of TrxS2 was monitored spectrophotometrically at 340 nm in reaction mixtures containing 120 μM TrxS2, 50 mM potassium phosphate (pH 7.0), 1 mM EDTA, and 0.2 mM NADPH. Activities of TrxR were also measured by coupling the NADPH-dependent reduction of Trx and oxidized insulin in reaction mixtures containing 7.5 μM TrxS2, 80 μM oxidized insulin, 50 mM potassium phosphate (pH 7.0), 1 mM EDTA, 0.2 mM NADPH, and 1 μg of TrxR1.