Sequential extraction of chlorophyll from chlorophyll-protein complexes in lyophilized pea thylakoids with solvents of different polarity

Lyophilized chloroplasts of Pisum sativum (pea) have been extracted with petroleum ether of different polarity (obtained by adding varying amounts of ethanol to the petroleum ether). Extracted thylakoids have then been solubilized by sodium dodecyl sulphate (SDS) and chlorophyll-protein complexes have been isolated by polyacrylamide gel electrophoresis (PAGE). Absorption- and low temperature fluorescence emission spectro-scopy have been used to characterize thylakoids and purified chlorophyll-protein complexes. Weakly polar solvents extracted mainly chlorophyll a. SDS-PAGE scan profiles of similarly extracted thylakoids contained no photosystem II chlorophyll a reaction center antennae (CP-a_n) and the amount of photosystem I chlorophyll a reaction center antennae (CP-a₁) was reduced as compared with an unextracted control. This was due partly to the extraction of chlorophyll a prior to SDS-PAGE, and partly to the increased solubilization of chlorophyll a by SDS as a result of β-carotene extraction. By increasing the polarity of the solvent CP-a₁ also disappeared in the scan profile, leaving only the light-harvesting chlorophyll a/b-protein complex (CP-a/b) and SDS complexed chlorophyll. From these results we conclude that the chlorophyll molecules in the reaction center antennae are relatively more hydrophobically associated than the molecules in the light-harvesting CP-a/b complex. The chlorophyll a of CP-a_u and the far red absorbing chlorophyll a fraction of CP-a₁ appear to be the most hydrophobically associated chlorophyll molecules.