A rapid direct assay of 3'-5' cyclic nucleotide phosphodiesterase activity using chromatography on immobilized...

A chromatographic method using immobilized acriflavin has been developed for the separation of unreacted cyclic nucleotides from their corresponding 5'-nucleotides, in a direct assay of 3'-5' cyclic nucleotide phosphodiesterase using [3H]-cyclic nucleotides as substrate. The method based on the so-called charge-transfer overlap recognition between flavin and indol rings, provides a rapid (15-20 min) and sensitive elution of [3H]-5'nucleotides with high recovery (up to 98%) and low blanks, while [3H]-cyclic nucleotides are retarded on the column. By this method, the formation of some secondary products by purine metabolizing enzymes such as 5'-nucleotidases, nucleosidases and/or deaminases is taken into account, using [14C]-5'-AMP thus allowing an accurate determination of phosphodiesterase activity in any preparations.