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   

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Plant & Cell Physiology
Aug 2015



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

  1. 50 ml polypropylene centrifuge tubes with conical bottom (Corning, Falcon®, catalog number: 352070 )
  2. 20 ml standard glass test tubes (Thermo Fisher Scientific, Fisher Scientific, catalog number: S63289 )
  3. 10 ml Oak Ridge high-speed PPCO centrifuge tubes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3119-0010 )
  4. 1.5 ml polypropylene microcentrifuge tubes (Eppendorf, catalog number: 0030125150 )
  5. 0.20 µm cellulose acetate membrane filter, sterile (Toyo Roshi Kaisha, catalog number: 25CS020AS )
  6. Micro spatula
  7. Synechococcus elongatus PCC 7942 (http://cyanobacteria.web.pasteur.fr/)
  8. BG11 as a culture medium (Rippka et al., 1979)
  9. 0.1 mm diameter Zirconia/Silica beads (BioSpec, catalog number: 11079101z )
  10. Sodium ascorbate (Sigma-Aldrich, catalog number: A7631 )
  11. Sodium hydrosulfite (Sigma-Aldrich, catalog number: 157953 )
  12. Methanol (Wako Pure Chemical Industries, catalog number: 139-13995 )
  13. HEPES (Dojindo Molecular Technologies, catalog number: GB10 )
  14. Sodium hydroxide (NaOH) (Wako Pure Chemical Industries, catalog number: 198-13765 )
  15. Sodium chloride (NaCl) (Wako Pure Chemical Industries, catalog number: 191-01665 )
  16. Tricine (Dojindo Molecular Technologies, catalog number: GB19 )
  17. Hydrochloric acid (HCl) (Wako Pure Chemical Industries, catalog number: 080-01066 )
  18. Potassium ferricyanide (III) (Sigma-Aldrich, catalog number: 702587 )
  19. Hydroquinone (Sigma-Aldrich, catalog number: H9003 )
  20. HN buffer (see Recipes)
  21. 50 mM tricine buffer (pH 7.5) (see Recipes)
  22. 100 mM potassium ferricyanide solution (see Recipes)


  1. Vortex mixer (Scientific Industries, model: Vortex-Genie 2 )
  2. Tabletop centrifuge (KUBOTA, model: 5220 ) equipped with ST-720M swing rotor (16 x 50 ml)
  3. High speed refrigerated centrifuge (KUBOTA, model: 7700 ) equipped with RA-150 rotor (12 x 12 ml)
  4. High speed refrigerated micro centrifuge (Tomy, model: MX-301 ) equipped with 1.5 ml/2.0 ml rotor
  5. Ultrasonic cell disruptor (Branson, model: Sonifier SFX250 ) equipped with tapered microtip
  6. Spectrophotometer (Beckman Coulter, model: DU 640 )
  7. 300 µl micro cuvette, 10 mm optical pathlength (Hellma, catalog number: 105-QS )
  8. PTFE coated nickel stainless steel micro spatula (Kokugo, catalog number: 101-378-51 )


  1. Culture the cyanobacterial cells at 30 °C in 200 ml of BG11 medium, with illumination (10 W m-2) and aeration, to the exponential phase of growth (optical density at 730 nm ca. 0.3 to 0.4).
  2. Harvest cells in 4 x 50 ml tubes by centrifuging at 3,000 x g for 5 min at room temperature (Tabletop centrifuge). Discard supernatant by decantation and collect precipitated cells in one of these 50 ml tubes by resuspending them in 50 ml of ice-chilled HN buffer. Repeat the cell-washing step, i.e., centrifugation-resuspension step, twice, with the cells finally resuspended in 3 ml of the same buffer. Transfer the cell suspension into a glass test tube. Work under a dim light on ice for all following steps.
  3. Agitate the cell suspension with 2 g of Zirconia/Silica beads by a vortex mixer to disrupt the cells. Agitation for 30 sec at the maximal speed is repeated 4 times with ice-cooling intervals of 60 sec. Transfer the whole cell extracts into a PPCO centrifuge tube.
  4. Centrifuge the cell extracts at 3,000 x g for 5 min at 4 °C for removal of cell debris (high speed refrigerated centrifuge). Transfer supernatant into a PPCO centrifuge tube.
  5. Centrifuge the supernatant at 30,000 x g for 20 min at 4 °C (high speed refrigerated centrifuge). Discard supernatant by pipetting and resuspend precipitate in 5 ml of pre-chilled HN buffer. Repeat this centrifugation-resuspension step twice, with the precipitate finally resuspended in a small volume (< 0.5 ml) of the same prechilled buffer.
  6. Transfer the resuspended membranes sample for cytochromes and P700 measurement into a micro centrifuge tube. Divide the sample into five aliquots to be stored at -80 °C until use.
  7. Dilute the sample with 50 mM Tricine buffer (pH 7.5) to a chlorophyll a concentration of 50 to 70 μM (see Note 1).
  8. Sonicate the diluted sample twice for 1 sec at 20 W with 10 sec interval on ice to disperse the membranes (Ultrasonic cell disruptor). Transfer 200 µl of the sonicated sample into a micro cuvette.
  9. Add 2 µl of 100 mM potassium ferricyanide solution (final concentration 1 mM) to the sample in the micro cuvette and mix it well with a micro spatula. Incubate the sample for several minutes at room temperature to completely oxidize cytochromes and P700.
  10. Set the micro cuvette in a spectrophotometer and record the absorbance spectra at the range of wavelengths from 500-750 nm at room temperature. The complete oxidation can be confirmed by observation of no further change in the spectrum (see Note 2).
  11. Add a few powder particles of hydroquinone to the sample in the micro cuvette and mix it well with a micro spatula to reduce Cyt f. Record the absorbance spectra at the same range of wavelengths in step 10 at room temperature. Check that Δλ maximum in difference spectra of this step minus step10 is exhibited at 556.5 nm. Repeat step 11 several times until Cyt f is completely reduced, i.e., until the spectrum shows no further change (the final amount of hydroquinone, ca. 0.02 to 0.09 mg) (see Note 2).
  12. To reduce Cyt b559 and P700 in the sample, add a few powder particles of sodium ascorbate to the sample in the micro cuvette and mix it well with a micro spatula. Record the absorbance spectra at room temperature. Check that Δλ maximum in Cyt b559 difference spectra of this step minus step 11 is exhibited at 559 nm. Also check that Δλ maximum in P700 difference spectra of this step minus step 10 is exhibited at 700 nm. Repeat step 12 several times until Cyt b559 and P700 are completely reduced, i.e., until the spectrum shows no further change (the final amount of sodium ascorbate, ca. 0.04 to 0.16 mg) (see Note 2).
  13. Add a few powder particles of sodium hydrosulfite to the sample in the micro cuvette and mix it well with a micro spatula to reduce Cyt b6. Record the absorbance spectra at room temperature. Check that Δλ maximum in Cyt b6 difference spectra of this step minus step 12 is exhibited at 563 nm. Repeat step 13 several times until Cyt b6 is completely reduced, i.e., until the spectrum shows no further change (the final amount of sodium hydrosulfite, ca. 0.03 to 0.14 mg) (see Note 2).
  14. Quantitate Cyt b559, Cyt b6, Cyt f and P700 by using following formulas, [1], [2], [3] and [4], respectively (see Note 3). The quantitative results should be presented by mean values ± standard deviations from at least three experiments.
    Cytochrome b559 [μM] = 64.5 x {(A559 - A580) reduced (step12) - (A559 - A580) oxidized (step 11)} [1]
    Cytochrome b6 [μM] = 71.4 x {(A563 - A580) reduced (step13) - (A563 - A580) oxidized (step 12)} [2]
    Cytochrome f [μM] = 46.5 x {(A556.5 - A544.5) reduced (step11) - (A556.5 - A544.5) oxidized (step 10)} [3]
    P700 [μM] = 15.6 x {(A700 - A730) reduced (step12) - (A700 - A730) oxidized (step 10)} [4]

Representative data

Figures 1 and 2 show representative examples of difference spectra with the use of thylakoid membranes of Synechocystis sp. PCC 6803.

Figure 1. A difference spectrum of P700 in Synechocystis sp. PCC 6803. The sample included thylakoid membranes equivalent to 60 µM chlorophyll a.

Figure 2. Difference spectra of the cytochromes in Synechocystis sp. PCC 6803. Each sample included thylakoid membranes equivalent to 60 µM chlorophyll a.


  1. Chlorophyll a concentration is determined according to the method of Porra et al. (1989). After extracting chlorophyll a in the membranes sample with 100% methanol, measure the absorbance at 650.0, 665.2 and 730 nm, and calculate the chlorophyll a concentration by using following formula [5].
    Chlorophyll a [μM] = 18.22 x (A665.2 - A730) – 9.55 x (A652.0 - A730) [5]
  2. Be careful to avoid addition of oxidizing or reducing reagents in excess, since it will cause aggregation and/or bleaching of the sample.
  3. Differential extinction coefficients used were Δε(700 - 730 nm) = 64 mM-1·cm-1 (P700), Δε(559 - 580 nm) = 15.5 mM-1·cm-1(Cyt b559), Δε(563 - 580 nm) = 14.0 mM-1·cm-1 (Cyt b6), and Δε(556.5 – 544.5 nm) = 21.5 mM-1·cm-1 (Cyt f), which has been reported by Hiyama and Ke (1972), Garewal and Wasserman (1974), Stuart and Wasserman (1973), and Böhme et al. (1980), respectively.


  1. HN buffer (5 mM HEPES-NaOH [pH 7.5], 10 mM NaCl)
    Dissolve 1.19 g of HEPES and 0.58 g of NaCl to ~800 ml of ddH2O
    Adjust pH to 7.5 with NaOH
    Add ddH2O to final volume of 1,000 ml
    Sterilized by filtration (0.20 µm cellulose acetate membrane filter)
    Store at room temperature
  2. 50 mM tricine buffer (pH 7.5)
    Dissolve 8.96 g of tricine to ~800 ml of ddH2O
    Adjust pH to 7.5 with HCl
    Add ddH2O to final volume of 1,000 ml
    Sterilized by filtration (0.20 µm cellulose acetate membrane filter)
    Store at room temperature
  3. 100 mM potassium ferricyanide solution
    Dissolve 0.329 g of potassium ferricyanide (III) to ddH2O in a final volume of 10 ml
    This solution should be prepared just before use and store in dark


We are deeply grateful to Drs. Fujita and Murakami who established this protocol (1987). This work was supported in part by Grants-in-Aid (CREST) from Japan Science and Technology Agency.


  1. Böhme, H., Pelzer, B. and Boger, P. (1980). Purification and characterization of cytochrome f-556.5 from the blue-green alga Spirulina platensis. Biochim Biophys Acta 592(3): 528-535.
  2. Fujita, Y. and Murakami, A. (1987). Regulation of electron-transport composition in cyanobacterial photosynthetic system-stoichiometry among photosystem-I and photosystem-II complexes and their light-harvesting antennae and cytochrome-b6 cytochrome-f complex. Plant Cell Physiol 28(8): 1547-1553.
  3. Garewal, H. S. and Wasserman, A. R. (1974). Triton X-100-4 M urea as an extraction medium for membrane proteins. I. Purification of chloroplast cytochrome b559. Biochemistry 13(20): 4063-4071.
  4. Hiyama, T. and Ke, B. (1972). Difference spectra and extinction coefficients of P700. Biochim Biophys Acta 267(1): 160-171.
  5. Porra, R. J., Thompson, W. A. and Kriedemann, P. E. (1989). Determination of accurate extinction coefficients and simultaneous-equations for assaying chlorophyll a and b extracted with 4 different solvents - verification of the concentration of chlorophyll standards by atomic-absorption spectroscopy. Biochim Biophys Acta 975(3): 384-394.
  6. Rippka, R., Deruelles, J., Waterbury, J. B., Herdman M. and Stainer R. Y. (1979). Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol 111(1): 1-61.
  7. Stuart, A. L. and Wasserman, A. R. (1973). Purification of cytochrome b6. A tightly bound protein in chloroplast membranes. Biochim Biophys Acta 314(3): 284-297.


dA 6m DNA免疫沉淀随后深度测序(DIP-Seq)是鉴定和研究N 6 - 甲基脱氧腺苷(dA 6)的全基因组分布的关键工具, 6m )。这种新的DNA修饰的精确功能仍然需要完全阐明,但是已知转录起始位点不存在并且从外显子中排除,表明在转录调节中的作用(Koziol等人,2015 )。重要的是,其存在表明DNA可能比先前认为的更多样化,因为进一步的DNA修饰可能存在于真核DNA中(Koziol等人,2015)。该方案描述了进行dA 6m DNA免疫沉淀(DIP)的方法,其用于表征高等真核生物中的第一dA 6m甲基化酶分析(Koziol等人。,2015)。在该协议中,我们描述了如何基因组DNA被分离,片段化,然后用识别基因组DNA中的dA 6m 的抗体拉下含有dA 6m 的DNA。在随后的洗涤之后,消除不含dA 6m的DNA片段,并且从抗体洗脱含有dA 6m的片段,以便进一步处理用于随后的分析。

[背景] 此协议是为了识别基因组中包含dA 6m 的区域而开发的。它可以用于检测不同基因组中的dA 6m 。作为指导,本方案从用于检测RNA中腺苷甲基化的现有方法建立(Dominissini等人,2013)。我们开发这个协议,并适应它的dA 6 m 在DNA中的检测,而不是检测腺苷甲基化RNA。这是必需的,因为当时没有方案可用于允许在真核DNA中dA 6m 的全基因组鉴定。...


  1. 50ml具有圆锥形底部的聚丙烯离心管(Corning,Falcon ,目录号:352070)
  2. 20ml标准玻璃试管(Thermo Fisher Scientific,Fisher Scientific,目录号:S63289)
  3. 10ml Oak Ridge高速PPCO离心管(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:3119-0010)
  4. 1.5ml聚丙烯微量离心管(Eppendorf,目录号:0030125150)
  5. 0.20μm乙酸纤维素膜过滤器,无菌(Toyo Roshi Kaisha,目录号:25CS020AS)
  6. 微铲
  7. 细长聚球藻 PCC 7942( http://cyanobacteria.web .pasteur.fr/
  8. BG11作为培养基(Rippka等人,1979)
  9. 直径为0.1mm的氧化锆/二氧化硅珠(BioSpec,目录号:11079101z)
  10. 抗坏血酸钠(Sigma-Aldrich,目录号:A7631)
  11. 连二亚硫酸钠(Sigma-Aldrich,目录号:157953)
  12. 甲醇(Wako Pure Chemical Industries,目录号:139-13995)
  13. HEPES(Dojindo Molecular Techologies,目录号:GB10)
  14. 氢氧化钠(NaOH)(Wako Pure Chemical Industries,目录号:198-13765)
  15. 氯化钠(NaCl)(Wako Pure Chemical Industries,目录号:191-01665)
  16. Tricine(Dojindo Molecular Techologies,目录号:GB19)
  17. 盐酸(HCl)(Wako Pure Chemical Industries,目录号:080-01066)
  18. 铁氰化钾(III)(Sigma-Aldrich,目录号:702587)
  19. 氢醌(Sigma-Aldrich,目录号:H9003)
  20. HN缓冲区(参见配方)
  21. 50mM三羟甲基氨基甲烷缓冲液(pH7.5)(见Recipes)
  22. 100 mM铁氰化钾溶液(参见配方)


  1. 涡旋混合器(Scientific Industries,型号:Vortex-Genie 2)
  2. 装备有ST-720M摆动转子(16×50ml)的台式离心机(KUBOTA,型号:5220)
  3. 装备有RA-150转子(12×12ml)的高速冷冻离心机(KUBOTA,型号:7700)
  4. 装有1.5ml/2.0ml转子的高速冷冻微量离心机(Tomy,型号:MX-301)
  5. 装有锥形微尖的超声波细胞破碎仪(Branson,型号:Sonifier SFX250)
  6. 分光光度计(Beckman Coulter,型号:DU 640)
  7. 300μl微比色皿,10mm光程(Hellma,目录号:105-QS)
  8. PTFE涂覆的镍不锈钢微量刮刀(Kokugo,目录号:101-378-51)


  1. 将蓝细菌细胞在30℃下在200ml BG11培养基中培养至具有生长的指数期(在730nm处的光密度)的照明(10Wm -2 -2s)和通气。0.3 to 0.4)。
  2. 通过在室温下以3,000xg离心5分钟(台式离心机)在4×50ml管中收获细胞。通过倾析弃去上清液,并通过将其重悬浮于50ml冰冷的HN缓冲液中,在这些50ml管中的一个中收集沉淀的细胞。重复细胞洗涤步骤,即,离心 - 再悬浮步骤,两次,细胞最终重悬浮在3ml的相同缓冲液中。将细胞悬液转移到玻璃试管中。在冰暗的灯光下工作以进行所有后续步骤。
  3. 通过涡流混合器搅拌细胞悬浮液与2克氧化锆/硅胶珠破裂细胞。以最大速度搅拌30秒,以60秒的冰冷间隔重复4次。将全细胞提取物转移到PPCO离心管中
  4. 离心细胞提取物在3,000×g离心5分钟,在4℃下除去细胞碎片(高速冷冻离心机)。将上清液转移到PPCO离心管中
  5. 在4℃下将上清液以30,000×g离心20分钟(高速冷冻离心机)。通过吸取弃上清液和重悬在5ml预冷的HN缓冲液中的沉淀。重复该离心 - 再悬浮步骤两次,最后将沉淀物重悬浮在小体积(<0.5ml)的相同预冷缓冲液中。
  6. 将重悬的膜样品转移到微量离心管中以进行细胞色素和P 700的测量。将样品分成五份,待储存于-80℃直至使用。
  7. 用50mM Tricine缓冲液(pH7.5)稀释样品至50至70μM的叶绿素浓度(参见注释1)。
  8. 超声处理稀释的样品两次,在20瓦下1秒,在冰上间隔10秒,以分散膜(超声波细胞破碎器)。将200μl超声处理的样品转移到微量比色皿中。
  9. 加入2微升100 mM铁氰化钾溶液(终浓度1 mM)到微量比色杯中的样品,并用微量刮刀充分混合。在室温下孵育样品几分钟以完全氧化细胞色素和P 700。
  10. 在分光光度计中设置微量比色皿,并在室温下记录在500-750nm波长范围内的吸收光谱。完全氧化可以通过观察光谱没有进一步变化来确认(见注2)
  11. 向微量比色皿中的样品中加入一些氢醌的粉末颗粒,并用微量刮刀将其充分混合以减少Cyt em。在室温下在步骤10中记录相同波长范围的吸收光谱。检查在该步骤减去step10的差光谱中的Δλ最大在556.5nm处呈现。重复步骤11几次,直到Cyt f 完全减少,即,直到光谱没有进一步变化(氢醌的最终量, ca 0.02?0.09mg)(见注2)。
  12. 要减少样品中的Cyt b 559 和P 700 ,请将几个抗坏血酸钠粉末颗粒样品在微量比色皿中,并用微量刮勺充分混合。记录在室温下的吸收光谱。检查在559nm处显示该步骤减去步骤11的Cyt 559 差异光谱中的Δλ最大值。还检查在700nm处显示该步骤减去步骤10的P <700>差光谱中的Δλ最大值。重复步骤12几次直到Cyt 和 完全减少,没有进一步的变化(抗坏血酸钠的最终量,ca ,0.04?0.16mg)(见注2)。
  13. 向微量比色杯中的样品中加入几个连二亚硫酸钠的粉末颗粒,并用微量刮刀将其充分混合以减少Cyt 6 b 6。记录在室温下的吸收光谱。检查在该步骤减去步骤12的Cyt bb 6+差异光谱中的Δλ最大值展现在563nm。重复步骤13几次,直到Cyt b 6 完全缩减,即变化(亚硫酸氢钠的最终量, <0.03>)。(见注2)。
  14. 定量Cyt b , 分别使用以下公式[1],[2],[3]和[4](参见注释3)定量结果应以至少三次实验的平均值±标准偏差表示 细胞色素b [sub] 559 [μM] = 64.5x {(A 559 Sub A 580)步骤12) - (A <559 - A <580> )氧化(步骤11) 细胞色素b [sub] 6 [μM] = 71.4x {(a 563 Sub A 580)步骤13) - (A <563 - 580 )氧化(步骤12) 细胞色素 [μM] = 46.5×{(556.5 - 子544.5 )减少(步骤11) A556.5 - A 544.5 氧化(步骤10)


图1和2显示了使用集胞藻属的类囊体膜的差异光谱的代表性实例。 PCC 6803.

图1.在集胞藻中的P <700> 的差异谱。 PCC 6803。样品包括相当于60μM叶绿素的类囊体膜。。

图2.细胞色素在集胞藻中的差异光谱。 PCC 6803。每个样品包括相当于60μM叶绿素a的类囊体膜。


  1. 叶绿素a浓度根据Porra等人的方法测定。 (1989)。在用100%甲醇提取膜样品中的叶绿素a后,测量在650.0,665.2和730nm的吸光度,并通过使用下式计算叶绿素a浓度[5] ]。
    叶绿素a [μM] = 18.22×(A 665.2 -A 730)-9.55×(A 652.0- A 730 )[5]
  2. 小心避免加入过量的氧化剂或还原剂,因为它会引起样品的聚集和/或漂白。
  3. 使用的差分消光系数为Δε(700-730nm)= 64mM ·cm -1 -1 (P < ),Δε(559-580nm)= 15.5mM cm -1 (Cyt 559 ),Δε(563-580nm)= 14.0mM -1 ·cm <-1> (556.5-544.5nm)= 21.5mM - 1 ),其中, -1 (细胞色素的?F的),已经由桧山和柯(1972年),Garewal和沃瑟曼(1974年),斯图尔特和沃瑟曼(1973),和报道的B?hme的等al 。 (1980)。


  1. HN缓冲液(5mM HEPES-NaOH [pH7.5],10mM NaCl) 将1.19g的HEPES和0.58g的NaCl溶解在?800ml的ddH 2 O中。
    用NaOH调节pH至7.5 将ddH <2> O加入到最终体积为1000ml
  2. 50mM三羟甲基氨基甲烷缓冲液(pH7.5) 将8.96g三羟甲基丙氨酸溶解至?800ml ddH 2 O 2 / 用HCl
    调节pH至7.5 将ddH <2> O加入到最终体积为1000ml
  3. 100mM铁氰化钾溶液 将0.329g铁氰化钾(III)溶解于ddH 2 O中,最终体积为10ml


我们非常感谢博士。 Fujita和Murakami谁建立了这个协议(1987)。这项工作得到日本科学技术局的Grants-in-Aid(CREST)的部分支持。


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引用: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.