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PhotosynthesisResearch Protocols

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Robert CarpentierDépartement de Chimie-Biologie (GREIB),

Université du Québec à Trois-Rivières,Trois-Rivières, Québec, Canada

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Library of Congress Cataloging in Publication DataPhotosynthesis research protocols / edited by Robert Carpentier. p. ; cm. -- (Methods in molecular biology, 1064-3745 ; v. 274)Includes bibliographical references and index. ISBN 1-58829-232-0 (alk. paper) 1. Photosynthesis--Laboratory manuals. [DNLM: 1. Photosynthesis. 2. Plant Proteins. ] I. Carpentier,Robert. II. Series: Methods in molecular biology (Clifton, N.J.) ; v.274. QK882.P539 2004 572'.46--dc22

2003023725

v

Preface

Photosynthesis is one of the most important biological phenomena on earth.The conversion of sunlight by photosynthetic organisms supplies most of theenergy required to develop and sustain life on the planet. Photosynthesis is notonly at the heart of plant bioenergetics, it is also fundamental to plant produc-tivity and biomass. Photosynthetic carbon fixation and oxygen evolution di-rectly intervene in many environmental, including the global atmospheric CO2level and global climate. Therefore, it is not surprising that a large effort isdevoted to photosynthesis research.

Several biochemical methods of isolation, treatment, and analysis have beendeveloped to fulfill the needs of photosynthesis research. PhotosynthesisResearch Protocols contains a broad range of general and fundamental meth-ods that are commonly used by plant biochemists, physiologists, and molecu-lar biologists. This book is thus intended as a source of information forscientists working on any of the multiple aspects of photosynthesis, and shouldbe of great interest to a multidisciplinary field of research involving agricul-ture, biochemistry, biotechnology, botany, cell biology, environmental sci-ences, forestry, plant genetics, plant molecular biology, photobiology,photophysics, photoprotection, plant physiology, plant stress, etc.

Each technique is described by an expert, and the methods presented shouldserve as basic protocols for new photosynthesis researchers as well as for ex-perienced ones needing to use a new type of preparation or method. The bookis especially valuable to the beginner in the field of photosynthesis becauseeach technique is described in simple terms, requiring no previous knowledgeof the method. The “Notes” section of each chapter contains some further hintsand tips that are not provided in regular research papers.

I would like to acknowledge and congratulate our series editor John Walkerfor suggesting a book on the methods used in photosynthesis; such a book hadbeen badly missing from our shelves. I also want to thank my wife Johanne forher great help in preparing the final layout and arrangement of the chapters.Finally, I wish to express my deep gratitude to all the contributors for agreeingto participate. Thanks to their considerable effort, Photosynthesis ResearchProtocols should become a valuable reference book in many laboratories.

Robert Carpentier

vii

Contents

Preface ..............................................................................................................vContributors .....................................................................................................xi

1 Fractionation of Thylakoid Membranes Into Grana and StromaThylakoids

Juan Cuello and María José Quiles ....................................................... 12 Isolation of Photosystem I Particles From Spinach

Tetsuo Hiyama .................................................................................... 113 Rapid Isolation and Purification of Photosystem I

Chlorophyll-Binding Protein From Chlamydomonas reinhardtiiVelupillai M. Ramesh and Andrew N. Webber .................................. 19

4 Isolation of Photosystem II-Enriched Membranesand the Oxygen-Evolving Complex Subunit ProteinsFrom Higher Plants

Yasusi Yamamoto, Shinsuke Sakuma, and Jian-Ren Shen .................. 295 Isolation of Functional Photosystem II Core Particles

From the Cyanobacterium Synechocystis sp. PCC 6803Dmitrii V. Vavilin ................................................................................ 37

6 Isolation of Photosystem I Reaction Center PreparationFrom Spinach

Tetsuo Hiyama .................................................................................... 497 Isolation of Photosystem II Reaction Center Complexes

From PlantsMichael Seibert, Inmaculada Yruela, and Rafael Picorel ................... 53

8 Isolation of Photosystem I Reaction Center Subunit PolypeptidesFrom Spinach

Tetsuo Hiyama .................................................................................... 639 Purification and Crystallization of the Cytochrome b6f Complex

in Oxygenic PhotosynthesisHuamin Zhang and William A. Cramer .............................................. 67

10 Purification of Plastocyanin and Cytochrome c6 From Plants,Green Algae, and Cyanobacteria

José A. Navarro, Manuel Hervás, and Miguel A. De la Rosa ............. 79

viii Contents

12 Isolation and Characterization of Lamellar Aggregates of LHCIIand LHCII-Lipid Macro-Assemblies With Light-InducibleStructural Transitions

Ilian Simidjiev, Zsuzsanna Várkonyi, and Gyozo Garab .................. 10513 Separation, Purification, and Characterization of Polypeptide

Composition of Subcomplexes of the Main Light-HarvestingChlorophyll a/b–Protein Complex of Photosystem II

Grzegorz Jackowski .......................................................................... 11514 Isolation of CP43 and CP47 Photosystem II Proximal Antenna

Complexes From PlantsRafael Picorel, Miguel Alfonso, and Michael Seibert ....................... 129

15 The Determination and Quantification of Photosynthetic Pigmentsby Reverse Phase High-Performance Liquid Chromatography,Thin-Layer Chromatography, and Spectrophotometry

Tessa Pocock, Marianna Król, and Norman P. A. Huner ................. 13716 Isolation and Identification of Chloroplast Lipids

Norihiro Sato and Mikio Tsuzuki ...................................................... 14917 DNA Adducts With Chlorophyll and Chlorophyllin

As Antimutagenic Agents: Synthesis, Stability,and Structural Features

Heidar-Ali Tajmir-Riahi, Jean-Francois Neault,and Stavroula Diamantoglou ........................................................ 159

18 Incorporation and Analysis of LHCII in Model SystemsWieslaw I. Gruszecki ........................................................................ 173

19 Photosystem II Reconstitution Into Proteoliposomes:Structure–Function Characterization

Mário Fragata .................................................................................... 18320 Extraction of the Functional Manganese and Calcium

From Photosystem IIJoel Freeman, Garth Hendry, and Tom Wydrzynski ........................ 205

21 Assay of Photoinhibition of Photosystem II and ProteaseActivity

Yasusi Yamamoto, Yoji Nishi, Hitoshi Yamasaki, Suguru Uchida,and Satoshi Ohira ......................................................................... 217

22 Thermoluminescence: A Technique for Probing Photosystem IIPrafullachandra V. Sane.................................................................... 229

11 Preparation of Native and Recombinant Light-HarvestingChlorophyll-a/b Complex

Wolfgang Rühle and Harald Paulsen .................................................. 93

""

Contents ix

23 Detection of Free Radicals and Reactive Oxygen SpeciesÉva Hideg .......................................................................................... 249

24 Stabilization of Photosynthetic MaterialsRégis Rouillon, Pierre Euzet, and Robert Carpentier ....................... 261

25 Determination of Phosphoproteins in Higher Plant ThylakoidsEva-Mari Aro, Anne Rokka, and Alexander V. Vener ....................... 271

26 Identifying Photoprotection Mutants in Arabidopsis thalianaJan Bart Rossel, Abby Cuttriss, and Barry J. Pogson ........................ 287

27 A Simple Method for Chloroplast Transformationin Chlamydomonas reinhardtii

Velupillai M. Ramesh, Scott E. Bingham, and Andrew N. Webber .. 30128 The Construction of Gene Knockouts in the Cyanobacterium

Synechocystis sp. PCC 6803Julian J. Eaton-Rye ............................................................................ 309

29 Gene Inactivation in the Cyanobacterium Synechococcus sp.PCC 7002 and the Green Sulfur Bacterium Chlorobiumtepidum Using In Vitro-Made DNA Constructs and NaturalTransformation

Niels-Ulrik Frigaard, Yumiko Sakuragi, and Donald A. Bryant ........ 325Index ............................................................................................................ 341

Contributors

MIGUEL ALFONSO • Estación Experimental de Aula Dei, ConsejoSuperior de Investigaciones Científicas, Apdo. Zaragoza, Spain

EVA-MARI ARO • Department of Biology, University of Turku, Turku,Finland

SCOTT E. BINGHAM • Department of Plant Biology and Center for the Studyof Early Events in Photosynthesis, Tempe, AZ

DONALD A. BRYANT • Department of Biochemistry and Molecular Biology,The Pennsylvania State University, University Park, PA

ROBERT CARPENTIER • Département de Chimie-Biologie (GREIB), Universitédu Québec à Trois-Rivières, Trois-Rivières, Québec, Canada

WILLIAM A. CRAMER • Department of Biological Sciences, Lilly Hall of LifeSciences, Purdue University, West Lafayette, IN

JUAN CUELLO • Departamento de Biología Vegetal, Facultad de Biología,Universidad de Murcia, Campus de Espinardo, Murcia, Spain

ABBY CUTTRISS • School of Biochemistry and Molecular Biology, TheAustralian National University, Canberra, Australia

MIGUEL A. DE LA ROSA • Instituto de Bioquímica Vegetal y Fotosíntesis,Universidad de Sevilla y Consejo Superior de Investigaciones Científicas,Américo Vespucio s/n, Sevilla, Spain

STAVROULA DIAMANTOGLOU • Département de Chimie-Biologie (GREIB),Université du Québec a Trois-Rivières, Québec, Canada

JULIAN J. EATON-RYE • Biochemistry Department, University of Otago,Dunedin, New Zealand

PIERRE EUZET • Centre de Phytopharmacie, Université de Perpignan,Perpignan, France

MÁRIO FRAGATA • Département de Chimie-Biologie (GREIB), Universitédu Québec a Trois-Rivières, Québec, Canada

JOEL FREEMAN • Photobioenergetics, Research School of BiologicalSciences, The Australian National University, Canberra, Australia

NIELS-ULRIK FRIGAARD • Department of Biochemistry and MolecularBiology, The Pennsylvania State University, University Park, PA

GYOZO GARAB • Institute of Plant Biology, Biological Research Center,Hungarian Academy of Sciences, Szeged, Hungary

WIESLAW I. GRUSZECKI • Department of Biophysics, Institute of Physics,Maria Curie, Sklodowska University, Lublin, Poland

xi

""

GARTH HENDRY • Photobioenergetics, Research School of BiologicalSciences, The Australian National University, Canberra, Australia

MANUEL HERVÁS • Instituto de Bioquímica Vegetal y Fotosíntesis,Universidad de Sevilla y Consejo Superior de Investigaciones Científicas,Américo Vespucio s/n, Sevilla, Spain

ÉVA HIDEG • Institute of Plant Biology, Biological Research Center, Szeged, HungaryTETSUO HIYAMA • Department of Biochemistry and Molecular Biology,

Saitama University, JapanNORMAN P. A. HUNER • Department of Biology, The University of Western

Ontario, London, CanadaGRZEGORZ JACKOWSKI • Adam Mickiewicz University, Department of Plant

Physiology, Poznan, PolandMARIANNA KRÓL • Department of Biology, The University of Western

Ontario, London, CanadaJOSÉ A. NAVARRO • Instituto de Bioquímica Vegetal y Fotosíntesis,

Universidad de Sevilla y Consejo Superior de Investigaciones Científicas,Américo Vespucio s/n, Sevilla, Spain

JEAN-FRANCOIS NEAULT • Département de Chimie-Biologie (GREIB),Université du Québec a Trois-Rivières, Québec, Canada

YOJI NISHI • Graduate School of Natural Science and Technology, OkayamaUniversity, Okayama, Japan

SATOSHI OHIRA • Graduate School of Natural Science and Technology,Okayama University, Okayama, Japan

HARALD PAULSEN • Institut f. Allgemeine Botanik der Johannes-Gutenberg-Universität, Mainz, Germany

RAFAEL PICOREL • Estación Experimental de Aula Dei, Consejo Superior deInvestigaciones Científicas, Apdo. Zaragoza, Spain

TESSA POCOCK • Department of Biology, The University of Western Ontario,London, Canada

BARRY J. POGSON • School of Biochemistry and Molecular Biology, TheAustralian National University, Canberra, Australia

MARÍA JOSÉ QUILES • Departamento de Biología Vegetal, Facultad deBiología, Universidad de Murcia, Campus de Espinardo, Murcia, Spain

VELUPILLAI M. RAMESH • Department of Plant Biology and Center for theStudy of Early Events in Photosynthesis, Arizona State University,Tempe, AZ

ANNE ROKKA • Department of Biology, University of Turku, Turku, FinlandJAN BART ROSSEL • School of Biochemistry and Molecular Biology, The

Australian National University, The Australian National University,Canberra, Australia

xii Contributors

RÉGIS ROUILLON • Centre de Phytopharmacie, Université de Perpignan,Perpignan, France

WOLFGANG RÜHLE • Institut f. Allgemeine Botanik der Johannes-Gutenberg-Universität, Mainz, Germany

SHINSUKE SAKUMA • Graduate School of Natural Science and Technology,Okayama University, Okayama, Japan

YUMIKO SAKURAGI • Department of Biochemistry and Molecular Biology,The Pennsylvania State University, University Park, PA

PRAFULLACHANDRA V. SANE • National Botanical Research Institute, RanaPratap Marg, Lucknow, India

NORIHIRO SATO • School of Life Science, Tokyo University of Pharmacy andLife Science, Horinouchi, Hachioji, Tokyo, Japan

MICHAEL SEIBERT • Basic Sciences Center, National Renewable EnergyLaboratory, Golden, CO

JIAN-REN SHEN • Riken Harima Institute, Sayo-gun, Mikazuki-cho, Hyogo, JapanILIAN SIMIDJIEV • Institute of Plant Biology, Biological Research Center,

Hungarian Academy of Sciences, Szeged, HungaryHEIDAR-ALI TAJMIR-RIAHI • Département de Chimie-Biologie (GREIB),

Université du Québec a Trois-Rivières, Québec, CanadaMIKIO TSUZUKI • School of Life Science, Tokyo University of Pharmacy and

Life Science, Horinouchi, Hachioji, Tokyo, JapanSUGURU UCHIDA • Graduate School of Natural Science and Technology,

Okayama University, Okayama, JapanZSUZSANNA VÁRKONYI • Institute of Plant Biology, Biological Research

Center, Hungarian Academy of Sciences, Szeged, HungaryDMITRII V. VAVILIN • School of Life Sciences, Arizona State University,

Tempe, AZALEXANDER V. VENER • Division of Cell Biology, Linköping University,

Linköping, SwedenANDREW N. WEBBER • Department of Plant Biology, Arizona State

University, Tempe, AZTOM WYDRZYNSKI • Photobioenergetics, Research School of Biological

Sciences, The Australian National University, Canberra, AustraliaYASUSI YAMAMOTO • Graduate School of Natural Science and Technology,

Okayama University, Okayama, JapanHITOSHI YAMASAKI • Graduate School of Natural Science and Technology,

Okayama University, Okayama, JapanINMACULADA YRUELA • Estación Experimental de Aula Dei, Consejo Superior

de Investigaciones Científicas, Apdo, Zaragoza, SpainHUAMIN ZHANG • Department of Biological Sciences, Lilly Hall of Life

Sciences, Purdue University, West Lafayette, IN

Contributors xiii

Photosystem I Particles From Spinach 11

11

From: Methods in Molecular Biology, Vol. 274: Photosynthesis Research ProtocolsEdited by: R. Carpentier © Humana Press Inc., Totowa, NJ

2

Isolation of Photosystem I Particles From Spinach

Tetsuo Hiyama

SummaryA method to prepare photosystem I (PSI) particles is described. Spinach leaves are used to

prepare broken chloroplasts that are then solubilized by using a detergent (Triton X-100). Solu-

bilized chloroplasts are then applied on an ion-exchange column. Eluted by a linear concentra-

tion gradient of NaCl, fractions enriched in PSI particles are collected and applied on a small

hydroxyapatite column. By eluting with phosphate buffer, a concentrated preparation of PSI

particles is obtained. The particles consist of PsaA, PsaB, PsaC, PsaD, PsaE, and PsaY. Assay

methods that involve SDS-PAGE and P700 determination are also presented.

Key Words: Photosystem I; preparation; reaction center.

1. IntroductionFunctionally, photosystem I (PSI) is defined as “a pigment–protein com-

plex” embedded in thylakoid membranes that can photoreduce ferredoxin by

electrons from photosystem II (PSII) fed through plastocyanin (1). In short, it

may also be called a “light-driven plastocyanin: ferredoxin oxidoreductase”,

although its inherently irreversible nature might not fit well the word “oxido-

reductase” in its enzymological sense. The core of the complex is a heterodimer

of two, 80 kDa polypeptides (PsaA and PsaB). This core binds a P700 (the

photochemical reaction center pigment: a heterodimer of chlorophylls a and

a'), two phylloquinones, an iron-sulfur cluster and a number of light-harvest-

ing chlorophyll a molecules. Thus far, as many as 15 other subunits smaller

than 20 kDa have been proposed to be members of the PSI complex.

Efforts to isolate PSI activity in a form of a complex date back to the 1960s.

Currently, a variety of different preparations have been reported from numer-

ous photosynthetic organisms. Their subunit compositions vary widely even

within the same plant species. Those complexes are categorized into three types:

12 Hiyama

Types I, II, and III (1). One of the most common types of PSI complex, catego-

rized as Type II, consists of PsaA, PsaB, PsaC, PsaD, PsaE, and occasionally a

few other small polypeptides. Core complexes that consist only of large sub-

units (PsaA and PsaB) are Type III. This chapter discusses Type II prepara-

tion; Type III will be discussed in Chapter 6.

2. Materials1. Spinach leaves (see Note 1).

2. Kitchen blender.

3. Cheese cloth on a funnel (for filtration).

4. Chloroplast preparation buffer: 50 mM sodium phosphate buffer, pH 7.0, and

10 mM NaCl.

5. Centrifuge, refrigerated type.

6. Spectrophotometer.

7. Temperature-controlled water bath (45°C and 37°C).

8. Solubilization medium: 50 mM Tris-HCl, pH 8.8, and 3% Triton X-100 (see Note 2).

9. Column chromatography apparatus equipped with a peristaltic pump, a gradient

maker (Note 3), a three-way valve, and a fraction collector (see Note 4).

10. Anion-exchange column (BioRad Econo-Pac HighQ, 5-mL type).

11. Starting buffer: 10 mM Tris-HCl, pH 8.8, 0.2% Triton X-100, and 20% sucrose.

12. Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) appara-

tus with a gradient former. Less time-consuming mini-size (10 cm � 10 cm) equip-

ment is preferred for quick analysis.

13. Pretreatment medium: 90 mM Tris-HCl, pH 6.8, 10% 2-mercaptoethanol, and 0.6 M

sucrose.

14. Upper and lower electrode buffer: 25 mM Tris-hydroxyaminomethane (Tris), 192

mM glycine, and adjust 0.1% SDS to pH 8.3 at room temperature. Supplement the

upper buffer with 0.1 mL/100 mL of 0.1% ethanol solution of bromophenolblue

(BPB).

15. Stacking gel: 4.87% acrylamide, 0.13% methylene-bis-acrylamide, 0.125 M Tris-

HCl buffer, pH 6.8, and 7.5 M urea.

16. Separating gel: linear gradient of the following monomer solutions (a and b): a,

16% acrylamide, 0.27% methylene-bis-acrylamide, 7.5 M urea and 600 mM Tris-

HCl, pH 8.8; b, 22% acrylamide, 0.37% methylene-bis-acrylamide, 6% sucrose,

7.5 M urea and 600 mM Tris-HCl, pH 8.8.

17. Staining solution: 0.23% Coomasie brilliant blue (CBB) in 50% methanol and 10%

acetic acid.

18. Hydroxyapatite medium: 10 mM Tris-HCl, pH 7.5, and 0.6 mM CaCl2.

19. Hydroxyapatite column (BioRad Econo-Pac CHT-II, 1-mL type).

20. Equilibration buffer: 10 mM Tris-HCl, pH 8.8, 0.3 mM CaCl2, and 0.05% Triton

X-100.

21. Elution buffer: 50 mM sodium phosphate buffer, pH 8.0 and 0.05% Triton X-100.

22. Deep freezer (below �50°C, preferably �80°C).

Photosystem I Particles From Spinach 13

3. Methods

3.1. Preparation

Preparation should be conducted at low temperatures (approx 4°C). The

methods described below outline (1) the preparation of broken chloroplasts,

solubilization, and column chromatographies, (2) assay methods including SDS-

PAGE, optical measurement of chlorophyll concentration, and optional P700

determination.

3.1.1. Preparation of Broken Chloroplasts

1. Spinach leaves, remove ribs and wash in ice water, loosely packing in plastic

bags, and leave overnight in a cold room (see Note 5).

2. Blend leaves (about 100 g wet weight) in a kitchen mixer with 500 mL of chloro-

plast preparation buffer. Thirty seconds is usually adequate.

3. Combine two batches (about 1000 mL from 200 g leaves) and filter through four

layers of cheesecloth.

4. Centrifuge the filtrate 10,000g for 5 min. Resuspend the precipitate in the same

buffer.

5. Adjust the chlorophyll concentration by dilution to 2 mg/mL. For this purpose, a

rough estimation of total chlorophylls is adequate (see Note 6).

3.1.2. Solubilization

1. Mix one volume of the suspension with two volumes of preheated (45°C) solubi-

lization medium at the chlorophyll concentration of 2 mg/mL and incubate for 30

min at 45°C (see Note 7).

2. Chill the suspension (approx 50 mg chlorophylls) in an ice bath and centrifuge at

12,000g for 30 min to remove debris.

3. Collect the supernatant and use in the next step.

3.1.3. Column Chromatography

1. Wash a newly purchased HighQ column with 100 mL of the starting buffer (see

Note 8).

2. Keep the flow rate constant at 1 mL/min throughout the procedure.

3. The supernatant (approx 50 mg chlorophylls) is loaded on the column by inject-

ing it through the three-way valve (see Note 4).

4. Wash the loaded column with 1000 mL of the starting buffer supplemented with

10 mM NaCl, then with 300 mL of the starting medium supplemented with 50 mM

NaCl, and finally with 400 mL of linear gradient NaCl (50–200 mM, see Note 3).

The concentration gradient is formed by filling a mixing chamber of the gradient

apparatus with 200 mL of the starting buffer supplemented with 50 mM NaCl and

the bottom-connected chamber with an equal volume of the same buffer supple-

mented with 200 mM NaCl.

14 Hiyama

5. Subject collected fractions (1 mL each) to SDS-PAGE for PSI assay (see Note 8).

Collect and dilute fractions that show a typical PSI pattern (Fig. 1) with an equal

volume of the hydroxyapatite medium, and then load on an Econo-Pac CHT-II

column equilibrated with the equilibration buffer. To load, inject the combined

fractions through a 50-mL syringe directly fitted on the column.

6. Wash the column with 20 mL of the above medium, then elute with a small volume

(1–2 mL) of the elution buffer to obtain a concentrated preparation.

7. Immediately supplement this final preparation with 20% sucrose for stabilization

and store in a deep freezer for months.

3.2. Assays

3.2.1. SDS-PAGE

For SDS-PAGE, a linear gradient gel supplemented with urea gives a favor-

able result. We routinely use a mini-size slab gel (10 cm wide and 7 cm high

for separating gel and 1.5 cm high for stacking gel on top; 1 mm thick).

Fig. 1. SDS-PAGE of Type II PSI particles from spinach. Electrophoresis was per-

formed as described in the text.

Photosystem I Particles From Spinach 15

1. Mix a sample suspension with an equal volume of a pretreatment medium, and

incubate at 37°C for 20 min before applying on the gel.

2. Prepare the electrophoresis gel using the stacking and separating gel solutions.

3. Perform electrophoresis at room temperature using upper and lower buffers.

4. Apply 70 volt until the BPB front line reaches the stacking gel, then raise the volt-

age to 170 volt.

5. Stop electrophoresis when the BPB front line comes near the end.

6. Stain the gel for 30 min with the staining solution, and then rinse in 7% acetic acid

for destaining.

3.2.2. Optical Measurements

Absorbance is measured by using a spectrophotometer for determination of

chlorophyll concentration (see Note 6). Any type of spectrophotometer can be

used for this purpose.

3.2.3. P700 Determination

This step is optional because the method requires specialized equipment

(see Note 9).

4. Notes1. Spinach (Spinacia oleracea) available at markets comes in a wide variety of cul-

tivated strains. They not only differ from locality to locality but also depend on

seasons. Fortunately for broken chloroplast preparation, most varieties of spinach,

in any season, can be successfully used for this purpose. Preparation yields may

fluctuate somewhat.

2. The pH values of the media and buffers stated in this chapter are all measured and

adjusted at room temperatures.

3. Any type gradient maker can be used. An apparatus routinely used in our labora-

tory consists of a mixing chamber (cylinder) with two spouts at the bottom. One

spout is connected to another chamber of the same size and shape, with a short

silicon rubber tubing pinched by a pinch cock. The mixing chamber is filled with

a medium with a lower salt concentration and the other with a higher concentra-

tion medium. The mixing chamber with a stirring bar on the bottom is placed on a

magnetic stirrer. Before starting the flow, the pinch cock is removed and the stir-

ring is initiated. A device of the similar but smaller type is used for gradient gel

making.

4. Prepacked columns with an assembly based on luer fittings are recommended.

Utilizing a peristaltic pump between the column and a buffer reservoir, flow rate

can be kept constant. A simple three-way valve (a disposable luer fitting type) is

inserted between the pump and the column for sample injection.

5. This overnight cold storage is rather optional; washed leaves can be used immedi-

ately without much trouble.

16 Hiyama

6. A rough molar extinction coefficient (90 mg�1 mL1 cm�1 or 100 mM�1 cm�1) at

665 nm for chlorophyll a in 80% acetone can be used for this calculation. Add 3

mL of the chloroplast suspension to 3 mL of 80% acetone and mix well. Measure

the absorbance at 665 nm (A665). A rough chlorophyll a concentration is calcu-

lated as A665 � 1000/90 (mg/mL). Here, chlorophyll means chlorophyll a, as chlor-

ophyll b content in PSI is low.

7. Heat treatment is quite effective to remove PSII and other heat-labile components.

8. The column can be rejuvenated by washing with the starting buffer supplemented

with 1.0 M NaCl, and used repeatedly several times.

9. The best way to prepare photochemically sound particles is to collect fractions

enriched in the reaction center pigment P700. P700 can be determined in several

ways: light-induced oxidation, chemical oxidation, and determination of chloro-

phyll a'. For the oxidation methods, either light induced or chemically induced, it

is essential to use a spectrophotometer highly sensitive and stable enough to mea-

sure absorbance changes as small as 0.001. Preferably, a photodetector (e.g., pho-

tomultiplier) is set near the cuvet so that scattering interference may be minimal.

Such instruments currently available include a Shimadzu MPS-2400 spectropho-

tometer. I recommend the chemical method, because a custom-made accessory

for either flash or continuous illumination is needed for measurement of light-

induced changes (2,3). The chemical procedure is as follows:

a. Add 10 µL each of 0.1 mM TMPD (N,N,N',N'-tetramethyl-p-phenylenedia-

mine) and 0.1 mM potassium ferricyanide to 3 mL of the reaction buffer (50 mM

Tris-HCl, pH 8.8 supplemented with 0.05% Triton X-100) in a standard cuvet

(1-cm light path), and mix well.

b. Scan and record the absorbance spectrum from 650 nm to 750 nm. It is essen-

tial to store the data in a computer memory.

c. Then, add 10 µL of 2 mM ascorbic acid to the same cuvet using a disposable

tiny-headed coffee spoon, and stir it well without disturbing the cuvet position.

d. Scan again, and subtract the spectrum recorded prior to obtain a difference

spectrum (oxidized-minus-reduced). From the trough size around 700 nm, the

concentration of P700 can be calculated by using a molar extinction coeffi-

cient for P700, 64 mM�1 cm�1 (4).

The 3-D structure analysis of the PSI reaction center (5) determined that the

molecular nature of P700 is a heterodimer of chlorophyll a and chlorophyll

a' (6,7). The determination of chlorophyll a' involves a special extraction proce-

dure followed by a high-performance liquid chromatography (HPLC) analysis,

beyond the scope of this chapter. Refer to Maeda and colleagues for details (8).

References1. Hiyama, T. (1996) Photosystem I: structures and functions, in Handbook of Pho-

tosynthesis (Pessarakli, M. ed.), Marcel Dekker, New York, pp. 195–217.

Photosystem I Particles From Spinach 17

2. Hiyama, T., Ohinata, A., and Kobayashi, S. (1993) Paraquat (methyl viologen): Its

interaction with primary photochemical reactions. Z. Naturforsch. 48c, 374–378.

3. Hiyama, T. (1985) Quantum yield and requirement for the photoreduction of P700.

Physiol. Veg. 23, 605–610.

4. Hiyama, T. and Ke, B. (1972) Difference spectra and extinction coefficients of P

700. Biochim. Biophys. Acta 267, 160–171.

5. Jordan, P., Fromme, P., Witt, H. T., Klukas, O., Saenger, W., and Krauss, N. (2001)

Three-dimensional structure of cyanobacterial photosystem I at 2.5 A resolution.

Nature 411, 909–917.

6. Kobayashi, M., Watanabe, T., Nakazato, M., et al. (1988) Chlorophyll a'/P-700

stoichiometries in higher plants and cyanobacteria determined by HPLC analysis.

Biochim. Biopys. Acta 936, 81–89.

7. Hiyama, T., Watanabe, T., Kobayashi, M., and Nakazato, M. (1987) Interaction

of chlorophyll a' with the 65 kDa subunit protein of photosystem I reaction cen-

ter. FEBS. Lett. 214, 97–100.

8. Maeda, H., Watanabe, T., Kobayashi, S., and Hiyama, T. (1993) Normal-phase

HPLC quantitation of chlorophyll a' and phylloquinone in photosystem I particles.

Photosynthesis Res. 35, 179–184.

18 Hiyama