NAD is used in the determination of amylase, creatine kinase, transaminases, lactate dehydrogenase, phosphohexose isomerase, ethanol, galactose, glucose, uric acid, L-lactate, triglycerides and other enzymes and metabolites.
Reagents
Sodiumdiphophate/semicarbazide/substrate, pH 8.7: 3.33 g Na4P2O7&10 H2O, 0.84 g semicarbazide&HCl, 0.17 glycine, 1.00 ml ethanol with 80 ml distilled water. Adjust pH to 8.7 with 2 M NaOH; adjust volume to 100 ml.
Alcohol dehydrogenase, from yeast (30 mg protein/ml): 300 U/ml.
Procedure
Dissolve 25 mg NAD in 25 ml distilled water in a volumetric flask.
Set spectrophotometer (equipped with strip chart recorder and temperature control) at 340 nm and 25°C.
Into a cuvette, pipette the following:
Buffer (1) 3.00 ml
sample 0.10 ml
Mix and read the absorbance A1
Start the reaction by adding 0.01 ml of Alcohol dehydrogenase (2). Mix and read absorbance A2.
Add another 0.01 ml Alcohol dehydrogenase. Mix and read absorbance A3.
