Purification and partial amino acid sequence of the chloroplast cytochrome b-559

• Published in: The Journal of biological chemistry Vol. 259; no. 6; pp. 3870 - 3876
• Main Authors: Widger, W R, Cramer, W A, Hermodson, M, Meyer, D, Gullifor, M
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 25.03.1984; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Amino Acids - analysis; Biological and medical sciences; Cell structures and functions; Chloroplast, photosynthetic membrane and photosynthesis; Chloroplasts - metabolism; Cytochrome b Group - isolation & purification; Fundamental and applied biological sciences. Psychology; Intracellular Membranes - metabolism; Molecular and cellular biology; Molecular Weight; Photosystem II Protein Complex; plant biochemistry; plant physiology; Plants - metabolism; Spectrophotometry; Photosystem 2; Absorption spectrometry; Hemoprotein; Vegetals; Purification; Gel electrophoresis; Aminoacid; Edman degradation; Primary structure; Spinach; Column chromatography; Chloroplast
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)43178-4

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.