Cell-free oxidoreductase profiling. We next tested the probes against a range of purified recombinant reducing enzymes in cell-free assays, including TrxRs as well as non-targeted Trxs, Grxs, GR, TRP14, and GPx1. The source and production methods are relevant for TrxRs, since the key selenocysteine (U498) residue in their active sites that is predominant in native enzymes, is typically not incorporated using standard recombinant expression systems. Therefore, we used human cytosolic TrxR1 and human mitochondrial TrxR2 produced with recent recombinant production methodologies that approach 100% CU active site content. To stringently test the validity of our design for selenolthiol-dependent activation, we also used the U498C point mutant of rat TrxR1 (rTrxR1 U498C ). The selenenylsulfide probes showed strong signal responses to TrxR1. RX1 at 10 µM was strongly activated by 20 nM TrxR1, with halftime to maximal signal of ca. 30 min (Fig. ): contrasting with its total stability to 1 million times higher concentrations of GSH over 6 hours. RX1 responded well to TrxR1 down to the subphysiological concentration of 5 nM (Fig. ). As this cell-free assay underestimates probe sensitivity to low concentrations of TrxR (see Supporting Note 5), this was encouraging for cellular use. TrxR1turn-on kinetics and response profiles were similarly good for all other cyclic selenenylsulfide probes (A2-A3, G1-G3) (Fig. ). This unity of TrxR1-selective performance despite the structural diversity of the probes matched our hypothesis that the cyclic selenenylsulfide is responsible for reactivity, and that the solubilising motif can be exchanged without altering the triggers' redox activity. An important result for selectivity, which completes the krel-H discussion (see section "Design"), is that the rates of probe activation by TrxR1 were essentially identical between the A-type and G-type triggers (Fig. ). If on-TrxR cyclisation (Fig. ) were significant for probe release, the G-type probes would be significantly faster than the A-type in releasing cargo. However, the data rather support that TrxR1 indeed fully reduces the cyclic selenenylsulfide probes to C/J, before they cyclise: full reduction by TrxR is faster than cyclisation.