A fluorometric assay for cyclic guanosine 3 ',5 '-monophosphate incorporating a Sep-Pak cartridge and enzymatic cycling

We developed a sensitive and nonradioactive fluorometric assay for cyclic g uanosine 3',5'-monophosphate (cGMP). Guanine nucleotides except cGMP were e nzymatically phosphorylated to GTP. cGMP, absorbed into a Sep-Pak amino pro pyl cartridge, was eluted separately from GTP. Purified cGMP was enzymatica lly converted to GTP, which was applied to the GTP-GDP cycle using succinic thiokinase and pyruvate kinase. When pyruvic acid produced by the GTP-GDP cycle was reduced by lactate dehydrogenase, a reduced form of nicotinamide adenine dinucleotide (NADH) was equivalently oxidized to NAD(+). NAD(+) was further converted into fluorescent compound, which was excited at 370 nm a nd emitted fluorescence at 460 nm, by a strong alkali. When 20 nmol NADH wa s used for this assay, the calibration curve over 50 to 500 fmol cGMP becam e sufficiently linear. The detection limit for cGMP was ca. 5 fmol (signal to noise ratio >3). Using this assay, we confirmed that the cGMP content in the left atrial strip of dog was changed from 11.4 +/- 3.8 to 19.3 +/- 2.6 fmol/mg wet wt of tissue (mean +/- SE, n = 6) by electrical driving at 1 H z. Carbachol (1 mu M) further increased the cGMP to 45.6 +/- 9.2 fmol/mg we t wt of tissue. From these results, it is suggested that this novel assay f or cGMP is highly sensitive and can be applied to various biological sample s. (C) 1999 Academic Press.