A method for chromatographic separation and fluorometric quantification of 2′,3′-cyclic nucleotide-3′-phosphodiesterase reaction products

Abstract

A sensitive method for separation and fluorometric quantification of 2′,3′-cyclic nucleotide-3′-phosphodiesterase (EC 3.1.4.37) (CNPase) reaction products with 2′,3′-cyclic adenosine monophosphate as substrate is presented. The 2′-AMP product was separated by cellulose thin-layer chromatography in 4 m MgSO 4-0.5 m sodium acetate-2-propanol (80:18:2, v/v/v). After reaction with glyoxal dihydrate, the amount of reaction product was determined fluorometrically using excitation at 328 nm and emission at 382 nm. These results correlated well with those obtained using; Kurihara and Tsukada's [(1967) J. Neurochem. 14: 1167–1174] paper chromatographic method ( r = 0.96). With this fluorometric method, amounts as low as 0.20 nmol of 2′-AMP can be determined, and its sensitivity is comparable to that of the radiochemical method. The method is easy to use and sensitive enough for measuring CNPase activity in tissue with low enzyme activity.