Table 1. Enzymatic activity of apo and holo Grx2.
| Substrate, kcat, s-1
|
||
|---|---|---|
| Enzyme | HED assay | RNase-SG |
| Apo Grx2 | 0.52 ± 0.05 | 0.67 ± 0.07 |
| Holo [2Fe-2S] Grx2 | No activity* | No activity* |
| Holo Grx2 (dithionite-treated) | 0.48 ± 0.04 | 0.59 ± 0.08 |
The reduction of 2-mercaptoethanol GSSG (HED assay) and glutathionylated RNase (RNase-SG) were assayed directly after purification of the proteins by gel filtration at pH 7 in a mixture containing GSH reductase, 200 μM NADPH, 1 mM GSH, and either 0.7 mM HED or 1.5 μM RNase-SG (equivalent to 12 μM GSH moieties). The specific activities were calculated from four experiments using two different enzyme concentrations.
*
Up to 5 μM Grx2 were assayed