Figure 5. Sodium selenite and dehydroascorbic acid as substrates for CLIC1.

(A) The oxidoreductase enzymatic reaction using sodium selenite as a substrate was performed in 0.1 mM Tris-HCl (pH 7.5) with 1 mM EDTA containing 200 uM NADPH, 50 nM GR,15 uM sodium selenite, 0.1 mg/mL BSA and 5 uM CLIC1(WT) reduced monomer or 5 uM Grx-1 as a control. The reaction was initiated by the addition of 50 uM GSH at 20°C with consumption of NADPH measured at A_{340 nm}. Error bars represent the S.E. of at least three experimental repeats. (B) The reaction was performed in 0.1 mM Tris-HCl (pH 7.5) with 1 mM EDTA containing 200 uM NADPH, 50 nM GR, 5 uM CLIC1 (WT) reduced monomer and sodium selenite (0, 1, 2, 4, 8 or 16 uM). The initiation of the reaction was achieved by adding 50 uM GSH at 20°C where the consumption of NADPH was measured at A_{340 nm}. (C) The oxidoreductase enzymatic reaction using DHAR as a substrate was performed in 137 mM sodium phosphate buffer (pH 7.5) containing 2 mM EDTA, 0.35 mM NADPH, 50 nM GR, 2 mM GSH and 1 mM DHA. The reaction was initiated after addition of 5 uM reduced CLIC1, CLIC4 or HcTrx-5 (as control). Consumption of NADPH was measured at A_{340 nm}. Error bars represent the S.E. of at least three experimental repeats. (D) DHAR activity of the CLIC proteins was determined using 137 mM sodium phosphate buffer (pH 7.5) with 2 mM EDTA, 0.35 mM NADPH, 50 nM GR, 2 mM GSH and DHA (0, 0.25, 0.5,1, 2, 4 or 6 uM). The reaction was initiated after the addition of 5 uM CLIC1 (WT) protein and the NADPH consumption was monitored at A_{340 nm}.