Purification and partial amino acid sequence of the chloroplast cytochrome b-559.
The hydrophobic cytochrome b-559, purified from unstacked, ethanol-washed spinach thylakoid membranes, using extraction with 2% Triton X-100 in 4 M urea and three chromatographic steps in the presence of protease inhibitors, has a dominant band on sodium dodecyl sulfate-urea gels corresponding to Mr = 10,000. The yield of this preparation is 30-50% (5-10 mg) starting with 600 mg of chlorophyll. The heme content yields a calculated molecular weight of no more than 17,500/heme, and perhaps somewhat smaller after correction for impurities. The Mr = 10,000 band is stained by the tetramethylbenzidine-H2O2 heme reagent on lithium dodecyl sulfate gels run at 0 degrees C. The Mr = 10,000 protein, further separated by high performance liquid chromatography, contains a unique NH2 terminus that is not blocked, and the amino acid sequence for the first 27 residues is NH2-Ser-Gly-Ser-Thr-Gly-Glu-Arg-Ser-Phe-Ala-Asp-Ile-Ile-Thr-Ser-Ile-Arg-Tyr-Trp -Val-Ile-X-Ser-Ile-Thr-Ile-Pro. . . COOH. Approximately 55% of the amino acids are hydrophobic, based on amino acid analysis of the Mr = 10,000 peptide, which also indicated the presence of at least one histidine. Only one cytochrome b-559 component could be identified, whose yield indicated that it arises from a single b-559 protein in chloroplasts corresponding to the in situ high potential cytochrome of the chloroplast photosystem II.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002736:Chloroplasts / Q000378:metabolism*; D003573:Cytochrome b Group / Q000302:isolation & purification*; D007425:Intracellular Membranes / Q000378:metabolism; D008970:Molecular Weight; D045332:Photosystem II Protein Complex; D010944:Plants / Q000378:metabolism; D013053:Spectrophotometry
D000596:Amino Acids; D003573:Cytochrome b Group; D045332:Photosystem II Protein Complex; C019313:cytochrome b559