Purification of Protochlorophyllide Holochrome

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Abstract
Phototransformable protochlorophyllide holochrome was prepared from etiolated bean leaves. The detergent Triton X-100 in the presence of glycerol and tricine-KOH buffer (pH 8) enhanced the extractability, specific activity, and phototransformability of the holochrome. Purification was achieved by polyethylene glycol-6000 precipitation and hydroxyl-apatite, DEAE-cellulose, and agarose chromatography. The presence of Triton X-100 permitted removal of the carotenoid contamination from the holochrome. The 678-nm absorption maximum of newly formed chlorophyllide a holochrome shifts to 672 nm in a temperature-dependent manner. The purified holochrome contains 0.24 g of protein per μmole of protochlorophyllide. Estimation of the molecular weight of the holochrome by gel filtration on agarose revealed the presence of aggregates of approximately 550,000 and 300,000. There are at least 2 chromophores per 550,000 molecular weight.