Irradiation in the Frozen State: A Technique for Direct Photoaffinity Labeling

UV irradiation of rabbit muscle phosphofructokinase (PFK) in the presence of adenosine 3′,5′‐cyclic phosphate (cAMP) resulted in the covalent attachment of this ligand molecule to the enzyme protein. Irradiation in the frozen ice state enhanced the rate of this incorporation more than 10‐fold above that achieved in aqueous solution, without significantly affecting the rate of photodestruction of the protein. [ 3 H]‐cAMP and [ 32 P]‐cAMP were each incorporated into PFK at identical rates in the frozen state. Rates of photoincorporation in the frozen and liquid states were both half‐maximal at a free ligand concentration approximately equal to the dissociation constant of cAMP and PFK. Adenosine diphosphate (ADP) and adenosine monophosphate (AMP), both of which are known to compete for cAMP binding to PFK, inhibited photoincorporation of cAMP. Guanosine monophosphate (GMP), inosine monophosphate (IMP), and guanosine 3′,5′‐cyclic phosphate (cGMP), which do not compete for cAMP binding, had no effect on photoincorporation of cAMP. Irradiation of [ 3 H]‐AMP or [ 3 H]‐ADP resulted in photoincorporation into PFK at 0°C, with enhancement at — 77°C similar to that noted with cAMP.