2] Separation and purification of cyclic nucleotides by ion-exchange resin column chromatography

Abstract

For determinations of cyclic nucleotides and related enzyme activities, ion-exchange column chromatography is the most widely used separation technique. Cation-exchange as well as anion-exchange resins can be used for cyclic nucleotide separations. Dowex-50 columns developed with acid solvents are especially useful if the samples contain large amounts of electrolytes (e.g., tissue constituents, salts of incubation medium or acid used for extraction) because such samples can directly be applied. The principles of cyclic nucleotide separations by thin-layer chromatography on polyethyleneimine (PEI)-cellulose have been adapted to column chromatography. PEI-cellulose columns as described in this chapter are especially useful and effective for assays of cyclase and phosphodiesterase activities. However, the sensitivity of this material to electrolytes is relatively high, so that columns of it have to be preceded by an electrolyte-removing step if they are used for cyclic nucleotide purification from tissue extracts. In addition to the ion-exchange column chromatography systems described in this chapter, the use of Dowex-1 developed with formic acid or hydrochloric acid for purification of tissue cyclic nucleotides has been reported.