GLUTAREDOXIN-1 E. coli, Recombinant

Product Code G 3531 Storage Temperature −20 °C

Synonyms: Grx1; Thioltransferase

Product Description This product is an E. coli recombinant protein, Unit definition: 1 unit oxidizes 1 µmole NADPH per N-terminal histidine-tagged, expressed in E. coli. minute at pH 8.0 at 25 °C in a coupled reaction with glutathione reductase. E. coli glutaredoxins (Grx) participate in thiol-disulfide reactions via an active site which contains cysteine Storage/Stability residues in the sequence -Cys-Pro-Tyr-Cys-. The active Store dessicated at −20 °C. The product is stable for at site may exist as a disulfide (oxidized form) or a dithiol least 3 years as supplied. (reduced form). The oxidation-reduction (redox) reac- tion of the cysteines in the active site is responsible for Reconstitution the catalytic activity. The enzyme is involved in Dissolve in 1 ml deionized water to give a solution of electron transport (ribonucleotide reduction, sulfate 1 mg/ml protein (Bradford) in 20 mM Tris, pH 7.5, reduction, and methionine sulfoxide reduction), forma- 100 mM NaCl. After reconstitution store at 2-8 °C for tion of disulfide linkages in protein folding, and protein short term storage and at –20 °C for long term storage. regulation by thiol redox control.1 Procedure Glutaredoxins and thioredoxins are families of enzymes that catalyze thio-disulfide exchange reactions. Principle of assay Representatives of these protein families have been Glutaredoxin-1 has GSH-disulfide oxidoreductase or found in all organisms tested, indicating these proteins transhydrogenase activity.4 When the are essential for cellular function. In the cell, thioredoxin transhydrogenase activity between glutathione (GSH) and glutaredoxin differ in the manner they are reduced. and 2-hydroxyethyl disulfide (HED) is coupled to In the glutaredoxin system, glutaredoxins are reduced glutathione reductase, the reaction rate can be followed via the tripeptide glutathione (GSH). The oxidized spectrophotometrically. The decrease in absorbance at glutathione (GSSG) formed is then reduced by NADPH 340 nm, which accompanies the oxidation of NADPH, is and glutathione reductase. Thioredoxins are reduced monitored. The coupled reactions are as follows: directly by the specific flavoenzyme thioredoxin reductase and NADPH. Glutaredoxin 2 GSH + X-S-S-X > GSSG + 2 XSH Three different glutaredoxins were identified in E. coli, 1 2 3 Grx1, Grx2, and Grx3. Grx1 and Grx3 are small Glutathione Reductase proteins (about 10 kDa) whereas Grx2 is a 27 kDa GSSG + NADPH + H+ > 2 GSH + protein. Both Grx1 and Grx3 are hydrogen donors for NADP+ ribonucleotide reductase while Grx2 is not. Net Reaction The product is histidine-tagged at the N-terminus to give a protein of 12 kDa. NADPH + H+ + X-S-S-X > NADP+ + 2 XSH

Vial contents: Lyophilized powder containing approxi- GSH is reduced glutathione. mately 5-20% protein (Bradford). The balance is X-S-S-X is 2-hydroxyethyl disulfide (HED). sodium chloride and Tris buffer salt. GSSG is oxidized glutathione. Purity: > 90% by SDS-PAGE. Specific activity: 50-250 units per mg protein Assay buffer References 0.1 M Tris-Cl, pH 8.0 (25 Û& ÃÃmM EDTA, 0.1 mg/ml 1. Sandberg, V.A., et al., Biochemistry, 30, 5475-5484 BSA, 1 mM reduced glutathione (Product Code (1991). G 4251), 0.4 mM NADPH (Product Code N 6505), 2. Vlamis-Gardikas, A., et al., J. Biol. Chem., 272, 6 µg/ml glutathione reductase from Bakers yeast 11236-11243 (1997). (Product Code G 3664), 0.7 mM 2-hydroxyethyl 3. Aslund, F., et al., Proc. Natl. Acad., USA, 91, 9813- disulfide (Product Code 38,047-4). 9817 (1994). 4. Holmgren, A., J Biol. Chem., 254, 3664-3671 Assay procedure (1979). Record A340 for one minute or more. Calculate activity according to the formula below: lpg/mam 1/01

∆A340 x 1 ml 1 unit = = µmole NADPH reduced per minute(s) x 6.22 minute

6.22 is the millimolar extinction coefficient for NADPH and 1 ml is the reaction volume.

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