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RESEARCH PRODUCT

Methodical Studies in the Separation of Chlorophyll-Protein Complexes by Polyacrylamide Gel Electrophoresis

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Summary Chlorophyll-protein complexes from thylakoids of Sinapis alba plants were separated, using Tris-HCl and Tris-borate buffer in the sodium dodecyl sulphate acrylamide gel electrophoresis. In the electrophoretogram with Tris-borate buffer, 6 bands could be characterized in the gel pattern. In addition to the three major green zones (P-700-chlorophyll a-protein, light-harvesting chlorophyll a/b-protein, free pigment zone), which were also observed in experiments with the Tris-HC1 buffer, three further bands (LHCP 1 , LHCP 2 , CPa) were electrophoretically separated. An elongation of the electrophoretical period resulted especially in a decrease of the proportion of the light-harvesting chlorophyll a/b-protein, whereas the free pigment increased by a comparable proportion. In addition, three different techniques for the determination of the chlorophyll contents and the chlorophyll a/chlorophyll b ratios of the green gel bands were compared. It could be demonstrated that the analysis of the densitometric tracings by means of standard curves is the most suitable method. Bands migrating closely together can easily be characterized by this method although a separation by elution is impossible. The absorption coefficients of chlorophyll a and chlorophyll b were determined for the separated chlorophyll-proteins at 671 and 653 nm. A formula was developed by which one can calculate the chlorophyll a/chlorophyll b ratio by means of the absorbance in the densitometric tracing peaks.