NADP is used in the determination of amylase, creatine kinase, glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase, and glucose.
Reagents
0.1M Triethanolamine buffer/substrate, pH 7.6: 1.86 g TEA¿HCl, 210 mg glycerate-3-P, 125 mg MgSO4¿7H 2O and 50 mg EDTA with 80 ml distilled water. Adjust pH to 7.6 with 1M NaOH-Na 2 and adjust volume to 100 ml with distilled water.
0.1 M MgCl2: 2.03 g MgCl2¿6H 2O in 100 ml distilled water.
14 mM D,L-Isocitrate: 4 mg D,L-isocitrate-Na3 in 1 ml TEA buffer.
Isocitrate dehydrogenase, from porcine heart (5 mg protein/ml): 4 U/mg
Procedure
Dissolve 50 mg NADP in 50 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: sample 0.10 ml Mix and read the absorbance A1.
Buffer (1) 2.50 ml
MgCl 2 (2) 0.10 ml
D,L-isocitrate (3) 0.10 ml
Add 0.02 ml of the ICDH. Mix and read absorbance A2.
Add an additional 0.02 ml ICDH. Mix and read absorbance A 3 (absorbance due to enzyme).
