Fig. 6.

Elution profiles of individual assay components in gel filtration. The isolated components of the different insulin reduction assays were analyzed on gel filtration with measurement of absorbance at 280 nm. Here the resulting chromatograms are shown for (A) TXNL1 wildtype ("WT") protein, (B) TXNL1 C34, C37S active site mutant, (C) the TXNL1 CS completely cysteine-less mutant, (D) Trx1 with both reduced and oxidized species of the protein present, (E) a mix of Trx1, TrxR1 and NADPH resulting in purely reduced Trx1 and also seeing the NADPH/NADP⁺ peak, (F) intact insulin, (G) reduced insulin obtained after incubation with DTT (with the B chain being too small to reliably detect), H) NADPH, and I) NADP⁺. The chromatography peaks of the respective components are indicated with horizontal bars and the approximate elution volume, with the full volume of the elution given at the x-axis and absorbance at 280 nm at the y-axis given for each chromatogram. Concentrations of proteins and NADPH were as subsequently used in Fig. 7.