|Extraction of Chlorophyll from Plants|
In plants chlorophyll is associated with specific proteins, for example, chlorophyll-a binding proteins are referred to as CP I, CP 47 and CP 43. With improving biochemical techniques for use on the membrane systems there has been an ever increasing success in the isolation and characterisation of these proteins.
Initially, detergents are used to break down the membrane into fragments, and these fragments are further broken down by the use of different detergents. These detergents work by replacing the membrane lipids which surround integral membrane proteins. The resulting particles are separated by polyacrylamide gel electrophoresis (a standard biochemical method) in the presence of sufficient detergent to keep them 'solubilised'. The activity and polypeptide composition can then be assayed as the particle is purified. The detergents work by substituting lipids at different spots in the membrane, this is also affected by the concentration of the detergent. One such detergent that is very commonly used is SDS-PAGE (sodium dodecyl sulfate-polyacrylamide). This is generally used as it has several advantages over other detergents: the separation can be carried out fairly rapidly and it also gives a good overall picture of the distribution of chlorophyll.
Photosystem I - Fig. 2
Fig. 2 - Photosystem I showing the constituents of PS I-110 particles.
This figure shows a schematic representation of the major subfractions that can be isolated from thylakoid membranes. In PS I (photosystem I) an initial solubilisation produces large particles (called PS I-110). These particles contain two chlorophyll-protein complexes: the reaction centre chlorophyll-a protein (CP I) and a chlorophyll a+b complex (LHC I, light-harvesting complex). PS I-110 also contains 6 to 8 polypeptides of lower molecular weight (8 to 25kDa, where 1 dalton=1 a.m.u.) that do not bind to chlorophyll, called Subunits II-VII. CP I, the reaction centre P700 chlorophyll-a protein, can be isolated from any of these mixtures by treatment with SDS (sodium dodecyl sulfate) or LiDS (lithium dodecyl sulfate) followed by electrophoresis.
Initial experiments done by Ogawa et al. and Thornber isolated two complexes by SDS-PAGE from SDS-solubilised membranes. One of these complexes, CP I, had a high apparent molecular weight and contained only chlorophyll-a. CP I is the most stable of the complexes and retained the photochemical activity of P700, the reaction centre of chlorophyll in PS I. It has a chlorophyll to P700 ratio of ~45 and a beta-carotene to P700 ratio of ~8.
The nature of the reaction centre of chlorophyll, P700, is still unknown, as there is conflicting evidence. It has been suggested that this could be explained if there is a pair of electronically interacting chlorophyll-a molecules in the ground (reduced) state (P700), and that the unpaired electron of the P700+ (oxidised) state is localised on only one of the chlorophyll's . The other 40 to 50 chlorophyll-a molecules of CP I act as antennas, and are thought to be responsible for the 721nm fluorescence emission maximum.
Photosystem II - Fig. 3