Overoxidized HSA polysulfide and thiol samples prepared as described above were first mixed with 1 mM DTT and reduced for 2 hours at 4°C. In the case of the DTT reduced samples (represented in Fig. 2C), the band position of HSA is higher than for the untreated samples, which is likely because of concomitant DTT-induced reduction of intramolecular disulfide bonds of the protein. Similar bands were previously identified as HSA by LC-MS proteomics, as discussed in (28).

Next, the different members of the Trx system were tested as reducing agents, in the following combinations: 500 μM NADPH and 200 nM TrxR1 WT or mutants (see Results and the Supplementary Materials) with or without the addition of 5 or 1 μM Trx1 or 5 or 1 μM TRP14. Rat TrxR1 WT and mutants, hTrx1, and hTRP14 were purified as described below. A control sample without any reducing equivalent was included into each group of samples. The time resolution of the reaction was monitored by fluorescent labeling of the reduced thiol product and subsequent SDS-PAGE. A sample of equal volume was taken from the reaction mixture at the indicated time points, and 0.5 mM 5-IAF (10 mM stock solution prepared in DMSO) was added to alkylate the thiol product and provide it with a fluorescent moiety. The proteins were heat-denatured at 100°C for 3 min and then separated on 10% nonreducing polyacrylamide gels.

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