Figure 1. Activity assay using pyruvate, coenzyme A and methyl viologen (Ctrl+). In the absence of pyruvate, no methyl viologen reduction occurs (Ctr-).

Materials and Reagents

         Note: All Reagents are dissolved freshly in an anaerobic tent.

1. Purified pyruvate: ferredoxin oxidoreductase (PFR1)
   
2. Sodium pyruvate (≥ 99%, Stock 500 mM) (Sigma-Aldrich)
   
3. Sodium coenzyme A (≥ 85%, Stock 20 mM) (Sigma-Aldrich)
   
4. Thiamine pyrophosphate (≥ 95%, Stock 250 mM) (Sigma-Aldrich)
   
5. Methyl viologen (98%, Stock 1 M) (Sigma-Aldrich)
   
6. Dithioerythriol (≥ 99%, Stock 100 mM) (Carl Roth)
   
7. 0.1 M Tris-HCl buffer (pH 8.0) (see Recipes)
   

Equipment

1. Anaerobic tent (1% H₂, 99% N₂) (Toepffer Lab Systems)
   
2. 96 well plate reader (Beckman Coulter, catalog number: Paradigm1113 )
   
3. PC running Multimode analysis software (Beckman Coulter)
   
4. NanoDrop (Paqlab)
   

Procedure