The synthesis and characterisation of a cyclic AMP-Sepharose 4B matrix with affinity for cyclic AMP-dependent...

A novel synthesis route for the preparation of a cyclic AMP-containing affinity adsorbent is described. This involves the use of a simple single-vessel, two-step, solid-phase reaction in which 5'-AMP, originally coupled to the matrix in the form of 5'-AMP-Sepharose 4B, is cyclised by a sequence involving activation of the phosphate moiety, followed by intramolecular condensation to give a 3',5'-phosphodiester. Physicochemical evidence, including 31P NMR studies, shows that this cyclisation takes place leaving no trace of original phosphomonoesters. The gel retains its excellent chromatographic properties, but now its specificity is directed towards cAMP-dependent proteins. Protein kinase (EC 2.7.1.37) binds to the gel, the catalytic subunit only being eluted when a simple buffer 0.2 M phosphate/1 mM theophylline is applied to column, whilst the regulatory unit is eluted using 20% ethylene glycol of 2% Pharmalyte, pH 5-8. This gel should find a use in the purification of protein kinase subunits and, by inference, of other cAMP-dependent proteins.