Analysis of Guanylate Cyclase Activity by High-Pressure Liquid Chromatography

Abstract

A method for the assay of guanylate cyclase which permits the correction of concurrent phosphatase and phosphodiesterase reactions has been developed using HPLC. The method, based on the conversion of tritium labelled guanosine triphosphate to tritium labelled cyclic guanosine monophosphate, uses [¹⁴C]-cGMP as the internal standard to account for the degradative and procedural-losses. Radiolabelled reaction products are isolated by high pressure liquid chromatography on a Partisil SAX column with a single step isocratic elution using 12.5 mM potassium phosphate buffer (pH 3.25). Since column recovery of the nucleotides is virtually quantitative and complete purification is achieved, the method possesses a high degree of accuracy and precision.