NADPH is used in the determination of leucine aminopeptidase, creatine, ammonia and urea.
Reagents
0.1M Triethanolamine buffer/substrate, pH 7.6: 1.86 g TEA&HCl, 210 mg glycerate-3-P, 125 mg MgSO4&7H2O and 50 mg EDTA with 80 ml distilled water. Adjust pH to 7.6 with 1M NaOH-Na2 and adjust volume to 100 ml with distilled water.
3 M Ammoniumcholride:.16.5 g NH4Cl in distilled water, adjust volume to 100 ml.
0.2 M a-Ketoglutarate: 45 mg a-Ketoglutarate-Na2&2H2O in 1 ml distilled water.
Glutamate dehydrogenase, from bovine liver (20 mg protein/ml): 120 U/mg.
Procedure
Dissolve 25 mg NADP 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) 2.50 ml
NH4Cl (2) 0.15 ml
a-KG (3) 0.10 ml
Mix and read the absorbance A1.
Add 0.10 ml of the sample. Mix and read absorbance A2.
Start reaction by adding 0.01 ml GLDH. Mix and read absorbance A3.
Add an additional 0.01 ml GLDH. Mix and read absorbance A4 (absorbance due to enzyme).
