Probing the molecular structure of phytochrome with immobilized Cibacron blue 3GA and blue dextran

Abstract

Phytochrome [extracted from Secale cereale cv. Cougar] binds to agarose-immobilized Cibacron blue 3GA. A higher affinity of the dye for the putative biologically active form (Pfr) than the inactive form (Pr) of phytochrome was observed. Effective general eluants of Pr included 40% (vol/vol) ethylene glycol, 1% Triton X-100, or 0.5 M potassium iodide. Increasing ionic strength (1 M KCl) did not effectively elute phytochrome. Of the natural cofactors that were reported to be analogs of the dye, NAD+, NADH, NADP+, cAMP, AMP, ADP, ATP and coenzyme A at a concentration of 10 mM would not elute phytochrome. At 10 mM, FMN eluted at least 65% of the bound Pr, FAD eluted 40%. Blue dextran/agarose bound to Pfr but exhibited essentially no affinity for P4. Phytochrome that was bound as Pfr could be subsequently released by photoconversion to Pr. Because of the high degree of selectivity that the blue dye and its dextran conjugate exhibit for the Pfr form of phytochrome, and because of the known property of the dye as an analog of natural ligands of proteins, the dye and its conjugate may be used as probes of a binding domain on the phytochrome protein that is important to its biochemical action.