Table 3.

Determination of free thiols (-SH) after insulin reduction using either Trx1 or TXNL1. Insulin reduction assays were performed under conditions to ensure that all insulin (180 μM) would be possible to reduce, i.e. using higher concentrations of NADPH (1 mM), TrxR1 (200 nM) and Trx1 (20 μM) or TXNL1 (20 μM) with a long duration of assay (2 h). Reduction of 180 μM insulin would maximally result in 1080 μM thiols, which was in good agreement with the determined thiol concentrations found using either Trx1-or TXNL1-driven reduction assays (bold, first line of the table). Omitting components of the assay gave low background values, confirming validity of the assay, while omitting insulin resulted in the expected number of thiols in reduced Trx1 with five Cys residues that would yield 100 μM thiols and seven Cys residues in TXNL1 that would yield 140 μM thiols (fifth line, bold and italics). Values represent average ± S.D. from three independent experiments each run with triplicates. See text for further details.

NADPH	Insulin	Trx1-driven insulin reduction assay			TXNL1-driven insulin reduction assay
		TrxR1 + Trx1	SH (μM)	SH/Insulin	TrxR1 + TXNL1	SH (μM)	SH/Insulin
+	+	+	1087.2 ± 50.3	6.0	+	1056.7 ± 39.2	6.0
+	+	–	25.5 ± 5.4	0.1
-	+	+	50.8 ± 24.5	0.3	+	27.4 ± 26.6	0.3
-	+	–	23.5 ± 5	0.1
+	–	+	104.7 ± 25.5	n.a.	+	150.4 ± 18	n.a.
+	–	–	31.9 ± 6.8	n.a.

n.a. = not applicable.