Formate → Phosphate

Beteiligte Stoffe

Reaktionsschritte

Reagents: Lyophilized nicotinamide adenine dinucleotide-diaphorase (NAD-diaphorase), p-iodonitortetrazolium violet (INT), formate dehydrogenase, and sodium formate are all obtained from Sigma Chemical Co. Acetonitrile, Na2HPO4, and KH2PO4 are of analytic grade, and reverse osmosis-deionized water is used. Phosphate buffer (100 mmol/L, pH 6.0) is prepared by mixing 100 mmol/L KH2PO4 and 100 mmol/L Na2HPO4 (5:1). Buffered NAD-diaphorase is prepared by adding 150 ml of buffer to one bottle of lyophyhlized NAD-diaphorase (Sigma). Buffered NAD-diaphorase-INT is prepared by addition of 140 mg INT (Sigma) to 140 ml of buffered NAD-diaphorase, and recombinant P. pastoris FDH is lyophilized. Specific activity of the P. pastoris FDH is 0.4 to 1.0 unites/mg solid (0.4 to 1.8 units/mg protein). Portions (5 to 15 mg) are weighed into 1.5 ml glass vials and stored at −70° C. One vial is used for each batch analysis. Each is reconstituted with about 100 to 200 μl/mg buffered NAD-diaphorase (5° C.) and kept in an ice bath during analysis. Serum and aqueous standards (10 ml each) are supplemented with stock aqueous sodium formate solutions containing 10.0 or 1.0 g/L to produce concentrations ranging from 0 to 400 mg/L with less than 1% volumetric alteration of the matrix. Working reagent and final reaction (cuvette) concentrations are shown in the table, below.

Herkunft & Nachweise