. Author manuscript; available in PMC: 2013 Feb 11.

Published in final edited form as: Biochemistry. 2008 Sep 25;47(42):11144–11157. doi: 10.1021/bi800966v

Table 6.

hGrx1 and hGrx2 Exhibit Equal Rates of Protein Glutathionylation in the Presence of GS^•, GSNO, and GSSG^a

	protein–SSG formation
substrate	Grx1-mediated (s⁻¹)	Grx2-mediated (s⁻¹)
GS^• [≥10 µM (33)]	0.43 ± 0.03	0.38 ± 0.04
GSNO (500 µ M)	0.21 ± 0.07	0.24 ± 0.08
GSNO (250 µM)	0.09 ± 0.04	0.11 ± 0.07
GSSG (250 µM)	0.38 ± 0.01	0.12 ± 0.007
GSSG (100 µM)	0.24 ± 0.03	0.06 ± 0.003

Reaction mixtures contained Na/K phosphate buffer (0.1 M, pH 7.5), rabbit muscle GAPDH (25 µM), Grx (0.02–0.25 µM), S-carboxymethyl BSA (7 mg/mL), and [³⁵S]GSNO (500 µM), [³⁵S]GSSG (500 µM), or GSH (0.5 mM) and GS^• (approximately 10 µM; see ref 33). Reactions took place at room temperature and were quenched by addition of 20% ice-cold TCA. Precipitated proteins were pelleted by centrifugation, washed, and analyzed for bound radioactivity. Grx-mediated GAPDH–SSG formation was assessed by comparing the bound radioactivity in the presence and absence of the enzyme. Rates are expressed as nanomoles of protein–SSG per minute per nanomole of Grx (i.e., turnover, min⁻¹). Turnovers were determined within the linear range of Grx dependence for each substrate and are expressed as the mean ± the standard error (n = 3–12).