Probing the molecular structure of phytochrome with immobilized Cibacron blue 3GA and blue dextran.
Phytochrome was shown to bind to agarose-immobilized Cibacron blue 3GA. A higher affinity of the dye for the putative biologically active form (P(fr)) than the inactive form (P(r)) of phytochrome was observed. Effective general eluants of P(r) 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 have been reported to be analogues of the dye, NAD(+), NADH, NADP(+), cyclic AMP, 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 P(r), whereas FAD eluted 40%. Blue dextran/agarose was found to bind P(fr) but exhibited essentially no affinity for P(r). Phytochrome that was bound as P(fr) could be subsequently released by photoconversion to P(r). Because of the high degree of selectivity that the blue dye and its dextran conjugate exhibit for the P(fr) form of phytochrome and the known property of the dye as an analogue of natural ligands of proteins, it is proposed that 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.
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Smithsonian Institution, Radiation Biology Laboratory, 12441 Parklawn Drive, Rockville, Maryland 20852.