Large-scale isolation of phytochrome from oat seedlings

Abstract Phytochrome, the photomorphogenetic pigment in plants, was isolated on a large scale from oat seedlings. The most important step in the isolation was the selective batch adsorption of the pigment on calcium phosphate gel in the neutral hydroxylapatite form. Adsorption was best performed at pH 6.2 and low ionic strength, while elution was most effective at pH 7.8 with 0.1 M potassium phosphate buffer. Subsequent gradient elution on DEAE-Sephadex produced two peaks, the first a sharp phytochrome peak and the second a broader complex protein peak with low phytochrome activity. A minor proportion of the phytochrome in the main fraction was present as a high-molecular-weight compound which could be separated by Sephadex G-200 gel filtration. The molecular weight of the purified phytochrome was estimated to be 55 500 by analytical gel filtration on Sephadex G-200.