IMPROVED ENZYMATIC CYCLE FOR NICOTINAMIDE-ADENINE DINUCLEOTIDE PHOSPHATE

An enzymatic cycling reagent for NADP is described which is capable of amplifying 100,000-fold in 4 hr at 38°C or 350,000-fold in 3 days at 15°C. The improvement over a previously described reagent results from several changes, the most important of which is substitution of glucose-6-P dehydrogenase from Leuconostoc mesenteroides for the baker's yeast enzyme. Tests of the same enzyme from Torula yeast showed it not to be well suited for this purpose. Procedures are given for final reading in either the fluorometer or spectrophotometer. The sensitivity limit for good precision would be about 50 fmol in the spectrophotometer and 0.5 fmol in the fluorometer (each with a 1-mi final volume). This sensitivity is available for measuring any metabolite or enzyme which can be induced to react directly or indirectly with an NADP enzyme. The cycle is also effective for NAD, although the rate is much slower than with NADP. Therefore, it would not in general be suitable for measuring tissue NADP. © 1978.