ANALYSIS OF THE CHLOROPLAST PROTEIN COMPLEXES BY BLUE-NATIVE POLYACRYLAMIDE-GEL ELECTROPHORESIS (BN-PAGE)

Blue-native polyacrylamide gel electrophoresis (BN-PAGE) is a powerful procedure for the separation and characterization of the protein comp lexes from mitochondria. Membrane proteins are solubilized in the pres ence of aminocaproic acid and n-dodecylmaltoside and Coomassie-dyes ar e utilized before electrophoresis to introduce a charge shift on prote ins. Here, we report a modification of the procedure for the analysis of chloroplast protein complexes. The two photosystems, the light-harv esting complexes, the ATP synthase, the cytochrome b(6)f complex and t he ribulose-bisphosphate carboxylase/oxygenase are well resolved. Anal ysis of the protein complexes on a second gel dimension under denaturi ng conditions allows separation of more than 50 different proteins whi ch are part of chloroplast multi-subunit enzymes. The resolution capac ity of the blue-native gels is very high if compared to 'native green gel systems' published previously. N-terminal amino acid sequences of single subunits can be directly determined by cyclic Edman degradation as demonstrated for eight proteins. Analysis of chloroplast protein c omplexes by blue-native gel electrophoresis will allow the generation of 'protein maps' from different species, tissues and developmental st ages or from mutant organelles. Further applications of blue-native ge l electrophoresis are discussed.