We then tested Trxs, titrating them to physiological 10 µM in the presence of 10 µM probe. These assays require TrxR and NADPH to reduce Trx from the non-reactive, oxidised, stored state, and to rereduce it after each oxidation by probe or by molecular O2 in the non-degassed assay buffer. RX1 is already fully and rapidly activated by 20 nM TrxR1 alone (Fig. ), so, unsurprisingly, titrating in Trx1/2 or other Trx-fold proteins such as TRP14 did not reveal extra processing by those species (cf. TrxR1 data in Fig. ). To study direct probe reduction by Trx only, we instead used concentrations of TrxR1 and TrxR2 that were ineffective at directly activating RX1, observing half-maximal probe activation with ca. 0.1-0.2 µM of Trx1 or Trx2 regardless of which TrxR was chosen (TrxR2 data in Fig. , Fig. and Fig. ; TrxR1 data in Fig. and Fig. ). This represents a ca. 50-fold lower sensitivity to Trx than to TrxR1, under continually re-reduced conditions. The TrxR-resistant disulfide comparison probes S1-S3 were activated by Trx as expected, with half-maximal activation around 0.3 µM Trx (Fig. ).