Properties of Protochlorophyllide and Chlorophyll(ide) Holochromes from Etiolated and Greening Leaves

Abstract

Protochlorophyllide and chlorophyll(ide) holochromes (Pchl-H and Chl-H) were extracted from dark-grown and greening seedlings with saponin and partly purified by ammonium sulfate fractionation. Sephadex gel filtration in the presence of saponin showed that the photoactive saponin Pchl-H from dark-grown leaves of bean (Phaseolus vulgaris L. cv. Redlands Pioneer) or pea (Pisum sativum L. cv. Greenfeast) has an apparent molecular weight of about 170,000, compared with 51,000 to 75,000 for the saponin Pchl-H from barley (Hordeum vulgare L. cv. Svalöfs Bonus). Photoconversion of saponin Pchl-H from dark-grown barley seedlings yields Chl-H with an absorption maximum at 678 nm, and with no change in apparent molecular weight. Above 0 C, a spectral shift from 678 to 672 nm follows, and a change in apparent molecular weight from about 63,000 to 29,000 is observed.Saponin Chl-H extracted from barley leaves illuminated for 15 minutes has an absorption maximum at 670 nm and an apparent molecular weight greater than 100,000. This chlorophyll holochrome has photosystem I activity and it is eluted together with the cytochromes. Saponin holochrome extracted from barley leaves returned to darkness after a light period, contains chlorophyll(ide) and protochlorophyllide complexes. Gel chromatography yields a complete separation of Chl-H (apparent molecular weight > 100,000) and photoactive Pchl-H (63,000).It is proposed that Chl-H dissociates into a chlorophyll(ide) a carrier protein complex and a photoenzyme, before the incorporation of chlorophyll into the lamellar membrane.Spectrofluorimetry on partially photoconverted preparations of saponin holochrome from barley, bean, and pea gave no indication for resonance energy transfer from protochlorophyllide to chlorophyllide. The saponin holochromes gave high polarization values, in contrast with bean holochrome extracted without the aid of detergents and bean leaves.