Purification and spectral characterization of a 121-kilodalton phytochrome from etiolated pea (Pisum sativum L.) seedlings

Procedures for the purification of native phytochrome from etiolated pea seedlings without the use of immuno-purification techniques are described. Phytochrome (in the P_FR form) was purified by polyethyleneglycol fractionation, adsorption to pentyl agarose and batch elution, chromatography on DEAE-Sepharose, adsorption to phenyl Toyopearl and batch elution, and chromatography on Red Toyopearl. The resulting phytochrome had specific absorbance ratios (SAR = A₆₆₆/A₂₈₀ of P_R) that ranged from 0.55 to 0.6. The subsequent chromatography on Sephacryl S-300 yielded very pure phytochrome with a SAR of 0.98. P_R and P_FR peaks in the difference spectrum of the phytochrome were centered at 665 and 730 nm, respectively. The spectral change ratio (ΔAr/ΔAfr) of the difference spectrum was unchanged after the chromatography on phenyl Toyopearl, and the value was 1.05-1.08, indicating that the spectral properties of this preparation were intact. The absorption spectra indicated that the peak absorbance of P_FR was at 728-730 nm and that of P_R was at 666-667 nm. These peak positions were essentially same as those obtained with the undegraded oat phytochrome. Incubation of the sample purified on Sephacryl S-300 at 25°C for 5 h in either the P_R or P_FR form did not result in degradation of the molecule. The rate of dark reversion of P_FR observed with the purified pea phytochrome was similar to that observed in vivo. The addition of dithionite had no effect on the reversion rate.