MULTIPLE CHROMOPHORE SPECIES IN PHYTOCHROME*,†,‡

Abstract— Buffered aqueous solutions of pure phytochrome, when irradiated at 730 nm, had a main absorption band at about 660 nm and a shoulder or secondary band at 580–600 nm. When irradiated at 660 nm, these absorption bands bleached and a pair of bands at 670 and 725–730 nm appeared. When 660 nm irradiated samples were placed in the dark the 730 nm absorption slowly bleached and the 670 nm absorption band shifted to 660 nm. The kinetics of the bleaching indicated that two populations of P_FR existed initially. These two populations decayed by first order kinetics with k's of 4.8 × 10^-4 sec^-1 and 3.1 × 10^--⁵ sec^-1at 25°. While the bleaching of P_FR was occumng, the appearance of the 660 nm and 580–600 nm absorption bands characteristic of P_R took place. The kinetics of the increase in the 580 and 660 nm absorption bands indicated that it was arising from two populations of reactants by two first order reactions with k's of 6.4 × 10^-4 sec^-1 and 3.1 × 10^-5sec^-1 at 25°. When the sodium chloride concentration of the solvent was changed the proportions of the kinetically different populations were altered. In some conditions, especially in the presence of air. reversible but non-reciprocal changes in the four absorption bands were observed. These effects were evident after the lapse of many hours in the dark. When native phytochrome was treated with sodium dodecyl sulfate all absorption bands but the 580–600 nm absorption band were bleached and photoreversibility was lost. When native phytochrome was treated with glutaraldehyde, the 730 nm absorption band was bleached but photoreversibility was retained. It was concluded that at least four species of chromophore exist in phytochrome with absorption maxima at 580, 660 , 670 and 730 nm. Each chromophore is capable of being bleached by appropriate irradiation or in the dark by chemical reactions rather than photochemical reactions. The reactions are probably coupled redox reactions between the 580–660 nm pair and the 670–730 pair of chromophores. Discrepancies observed in the reciprocity of the absorption changes in these paired bands are probably due to various degrees of uncoupling and secondarily to the redox potential of the solvent when such uncoupling occurs.