量化蓝藻细胞中的细胞色素b559、b6和f以及光和体系的核心成分I P700

Motohide  Aoki; Mikio  Tsuzuki; Norihiro  Sato

doi:10.21769/BioProtoc.1991

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Quantitation of Cytochromes b₅₅₉, b₆, and f, and the Core Component of Photosystem I P₇₀₀ in Cyanobacterial Cells

量化蓝藻细胞中的细胞色素b559、b6和f以及光和体系的核心成分I P700

Motohide Aoki

Mikio Tsuzuki

Norihiro Sato email

发布: 2016年11月05日第6卷第21期 DOI: 10.21769/BioProtoc.1991 浏览次数: 7960

评审: Maria Sinetovalina YinFanglian He

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Abstract

Cytochrome (Cyt) b₅₅₉, an important and essential core component of photosystem II in the photosynthetic electron transport system, is a heme-bridged heterodimer protein composed of an alpha subunit (PsbE) and a beta subunit (PsbE), and its reduced form has an absorption maximum in the α-band at 559 nm. The amounts of Cyt b₅₅₉ can be determined by spectrophotometrical measurement of reduced minus oxidized difference spectra that are normalized with absorbance of isosbestic point at 580 nm. The authors use differential extinction coefficients of Cyt b₅₅₉ [Δε_{(559-580 nm)} = 15.5 mM^-1·cm^-1], which have been reported by Garewal and Wasserman (1974). In addition to the Cyt b559, this procedure can be used for quantitation of Cyt b₆ and Cyt f, the subunits of the Cyt b₆/f complex, and P₇₀₀, one of the core components of photosystem I. This protocol, which is adapted from Fujita and Murakami (1987), is used in a cyanobacterium, Synechococcus elongatus PCC 7942, and also in other cyanobacterial strains including Synechocystis sp. PCC 6803.

Materials and Reagents

50 ml polypropylene centrifuge tubes with conical bottom (Corning, Falcon^®, catalog number: 352070 )
20 ml standard glass test tubes (Thermo Fisher Scientific, Fisher Scientific, catalog number: S63289 )
10 ml Oak Ridge high-speed PPCO centrifuge tubes (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 3119-0010 )
1.5 ml polypropylene microcentrifuge tubes (Eppendorf, catalog number: 0030125150 )
0.20 µm cellulose acetate membrane filter, sterile (Toyo Roshi Kaisha, catalog number: 25CS020AS )
Micro spatula
Synechococcus elongatus PCC 7942 (http://cyanobacteria.web.pasteur.fr/)
BG11 as a culture medium (Rippka et al., 1979)
0.1 mm diameter Zirconia/Silica beads (BioSpec, catalog number: 11079101z )
Sodium ascorbate (Sigma-Aldrich, catalog number: A7631 )
Sodium hydrosulfite (Sigma-Aldrich, catalog number: 157953 )
Methanol (Wako Pure Chemical Industries, catalog number: 139-13995 )
HEPES (Dojindo Molecular Technologies, catalog number: GB10 )
Sodium hydroxide (NaOH) (Wako Pure Chemical Industries, catalog number: 198-13765 )
Sodium chloride (NaCl) (Wako Pure Chemical Industries, catalog number: 191-01665 )
Tricine (Dojindo Molecular Technologies, catalog number: GB19 )
Hydrochloric acid (HCl) (Wako Pure Chemical Industries, catalog number: 080-01066 )
Potassium ferricyanide (III) (Sigma-Aldrich, catalog number: 702587 )
Hydroquinone (Sigma-Aldrich, catalog number: H9003 )
HN buffer (see Recipes)
50 mM tricine buffer (pH 7.5) (see Recipes)
100 mM potassium ferricyanide solution (see Recipes)

Equipment

Vortex mixer (Scientific Industries, model: Vortex-Genie 2 )
Tabletop centrifuge (KUBOTA, model: 5220 ) equipped with ST-720M swing rotor (16 x 50 ml)
High speed refrigerated centrifuge (KUBOTA, model: 7700 ) equipped with RA-150 rotor (12 x 12 ml)
High speed refrigerated micro centrifuge (Tomy, model: MX-301 ) equipped with 1.5 ml/2.0 ml rotor
Ultrasonic cell disruptor (Branson, model: Sonifier SFX250 ) equipped with tapered microtip
Spectrophotometer (Beckman Coulter, model: DU 640 )
300 µl micro cuvette, 10 mm optical pathlength (Hellma, catalog number: 105-QS )
PTFE coated nickel stainless steel micro spatula (Kokugo, catalog number: 101-378-51 )

Procedure

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Aoki, M., Tsuzuki, M. and Sato, N. (2016). Quantitation of Cytochromes b₅₅₉, b₆, and f, and the Core Component of Photosystem I P₇₀₀ in Cyanobacterial Cells. Bio-protocol 6(21): e1991. DOI: 10.21769/BioProtoc.1991.