Réaction #7441

Équation de réaction

NAD

KH2PO4

sodium formate

formate

nicotinamide adenine dinucleotide

NAD

NAD

NAD

sodium formate

KH2PO4

→

Phosphate

NAD

Solvants

Acetonitrile

water

Conditions de réaction

Traitement

1. 1
   AutreBuffered NAD-diaphorase is prepared
2. 2
   Autrestored at −70° C
3. 3
   Autrekept in an ice bath during analysis
4. 4
   workup.ADDITIONcontaining 10.0 or 1.0 g/L
5. 5
   Autreto produce
6. 6
   Concentrationconcentrations
7. 7
   AutreWorking reagent and final reaction (cuvette) concentrations

Mode opératoire

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.

Source