A Versatile Chromatographic Procedure for Purifying PS II Reaction Center Complex from Digitonin Extracts of Spinach Thylakoids

摘要

A versatile, two-step chromatographic method using DEAE-Toyopearl (Toyo Soda, Japan) is described for purifying photosystem II reaction center complex from digitonin extracts of spinach thylakoid membranes. The method is very simple and brings about an approximate four-fold increase in the specific activity, on a chlorophyll basis, of 2,4-dichlorophenol-indophenol photoreduction with 1,5-diphenylcarbazide (to about 2,000μelectron equivalents per mg chlorophyll per h), with an approximate 40 percent recovery in chlorophyll. The SDS-polyacrylamide gel electrophoresis performed in the presence of 4 M urea in the analyzing gel shows four polypeptide bands of the photosystem II reaction center of about 47, 43, 30 and 9 kilodaltons.The absorption and fluorescence properties, as well as the pigment and chemical compositions and the above mentioned polypeptide profile of the purified complex are essentially identical with those of the preparations isolated by the previously described method (Satoh 1982).The digitonin solubilization of thylakoid membranes destroys the water splitting machinery, so that the purified complex shows no oxygen evolving activity, even although 0.6–0.7 atoms of manganese per 50 chlorophyll molecules still rema