An enzymatic cycling procedure for beta-NADP+ generated by 3'-phosphodiesterase, 2':3'-cyclic nucleotide.
An enzymatic cycling procedure for beta-NADP+ generated by the enzyme 3'-phosphodiesterase, 2':3'-cyclic nucleotide (EC 3.1.4.37) from its substrate 2':3'-cyclic NADP+ is described. The enzymes glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and diaphorase (EC 1.8.1.4) are used to cycle the cofactor between its oxidized and reduced forms in the presence of glucose-6-phosphate and p-iodonitrotetrazolium violet (INT) with the concomitant production of colored INT-formazan, monitored at 492 nm. The amplification is about 400-fold per hour and is sensitive enough to detect 6 x 10(-13) mol of NADP(H). A simple procedure for the optimization of this cycling assay is also described. Conjugates to 3'-phosphodiesterase, 2':3'-cyclic nucleotide may be used in heterogeneous enzyme immunoassays for the detection of small quantities of haptens or proteins in biological fluids.
Stephon
R L
RL
Center for Health Sciences, Lehigh University, Bethlehem, Pennsylvania 18015.
Niedbala
R S
RS
Schray
K J
KJ
Heindel
N D
ND
D063309:2',3'-Cyclic Nucleotide 3'-Phosphodiesterase; D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases / Q000378:metabolism*; D008058:Dihydrolipoamide Dehydrogenase / Q000378:metabolism; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism; D007700:Kinetics; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D010727:Phosphoric Diester Hydrolases; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet; D013778:Tetrazolium Salts / Q000737:chemistry
D013778:Tetrazolium Salts; C013574:iodonitrotetrazolium; D009249:NADP; D005954:Glucosephosphate Dehydrogenase; D008058:Dihydrolipoamide Dehydrogenase; D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases; D010727:Phosphoric Diester Hydrolases; D063309:2',3'-Cyclic Nucleotide 3'-Phosphodiesterase