Methods for Analysis of Carbohydrate Metabolism in Photosynthetic Organisms. Plants, Green Algae and Cyanobacteria

224 Pages·2016·3.49 MB·English

by  Horacio G Pontis

Methods for Analysis of Carbohydrate
Metabolism in Photosynthetic Organisms
Methods for Analysis
of Carbohydrate Metabolism
in Photosynthetic Organisms
Plants, Green Algae, and Cyanobacteria
Horacio G. Pontis
Professor Emeritus of Biochemistry,
Universidad Nacional de Mar del Plata, and
Emeritus Senior Investigator and Vice-President,
Applied Biological Research Foundation — FIBA,
Buenos Aires, Argentina
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To Samantha, who accompanied me on my journey writing this book
This book is dedicated to Graciela, Sheila and Andre´
Biography
Horacio Guillermo Pontis was born in Mendoza, Argentina in 1928. He graduated from the
University of Buenos Aires chemistry school and obtained his doctorate working in organic
chemistry in 1951. He worked alongside Professor Luis F. Leloir for three years, who
influenced his interest in carbohydrate metabolism, sugar phosphates, and sugar nucleotides.
He spent a long stay at King’s College, Durham University (United Kingdom) and at
Karolinska Institutet, University of Stockholm (Sweden), where he started enzymology
studies with Prof. Peter Reichard. After his return to Argentina in 1960, he began his work
on the field of plant biochemistry, studying fructan and sucrose metabolism. In connection
with these studies, he synthesized fructose-2-phosphate, the first ketose that allowed other
researchers the chemical synthesis of fructose-2,6, diphosphate (a key glycolysis activator)
two decades later. Since 1961, he has been a member of the National Research Council of
Argentina and Professor of Biochemistry, firstly at University of Buenos Aires and then at
University of Mar del Plata. Between 1967 and 1977, Pontis was Director of the Biology
Department of Fundacio´n Bariloche, and also in 1973, he was President of the Sociedad
Argentina de Investigacio´n Bioqu´ımica (SAIB). In Mar del Plata (Argentina), he set up the
Institute for Biological Research at the University, and together with Prof. Leloir, they
established the Foundation for Biochemical Applied Research (FIBA), where as Head of its
Biological Research Center, he maintained an active research group, training graduate and
postgraduate students, and producing a steady flow of research publications.
At present, Pontis is Professor Emeritus of University of Mar del Plata, Emeritus
Researcher and vice-president of FIBA. His important contribution to plant biochemistry
was recognized by the Argentinean Society of Plant Physiology and by the American
Society of Plant Biologists, which awarded him as a corresponding member.
xvii
Preface
The idea of writing a book on the analytical methods for the study of carbohydrates,
essential components of the central metabolism of land plants, unicellular green algae, and
cyanobacteria, came to me at the insistent request of my best student who insisted that
I should pass on the experience I had gained over so many years devoted to this field.
At first, the request seemed simple to implement; however, I had to tell my experience in a
way that no other published texts had done so before, where general principles of separation
and purification of proteins or methods to quantify a given carbohydrate were provided.
It suddenly occurred to me that the best way to achieve this goal was to describe the
different methodologies that I had been extensively using in my laboratory as if I were
orally explaining them to my students or colleagues who were not familiar with them.
My intention was, to some extent, to show that carbohydrates and their metabolic pathways
could be studied by following quick and easily accessible methodologies.
Building on my idea, I decided to describe in detail each experiment to be performed,
starting from the technique’s fundamentals or principles, mentioning the necessary steps for
implementation, also including the biological starting material, and the choice of
methodology among different alternatives. To put it into a few words, I intended to provide
researchers with tools and information ranging from an overview of the extraction and
purification of the enzymes involved in carbohydrate metabolism from photosynthetic
organisms to different analytical techniques for the measurement of their activities, and
separation and determination of sugars and other compounds related to them. To me, the
clearest way to transmit this information was to apply it to the study of specific cases,
selecting the key sugar players. I trust readers will find this approach useful and that it will
assist those who are taking their first steps into the field of sugar metabolism.
Perhaps the most important aspect of this book, and what distinguishes it from the literature
available, is that it brings to the present a selection of reliable and tested methods used
decades ago but not widely applied these days, which do not require costly instruments and
specially trained personnel, together with some more modern techniques.
Regardingthebookorganization,ithasbeendividedintothreepartsforanorderlyand
systematicdescriptionofthegeneralanalyticalmethods.PartI(seeChapter1:Determination
ofCarbohydratesMetabolismMolecules)focusesonproceduresfordeterminingthemost
xix
xx Preface
relevantcarbohydratesandsomecompoundsrelatedtotheirmetabolism,including
colorimetric,spectrophotometric,andspectrofluorimetricmethods.Theapplicationsofthese
methodsarepresentedinlaterchapters.PartIIstartswiththedescriptionofgeneralmethods
fortheextractionofproteinswithenzymaticactivityinvolvedinsugarmetabolism(see
Chapter2:PreparationofProteinExtracts).Chapter3,ProteinandCarbohydrateSeparation
andPurificationdescribesthemostcommonmethodsforseparatingproteins(ionexchange,
gelfiltration,affinitychromatographicmethods,isoelectrofocusing,andfastproteinliquid
chromatography)andcarbohydratefractionation(chromatographicmethods,includingHPLC
(high-performanceliquidchromatography)andHPLCcoupledtomassspectrometry).The
determinationsofenzymeactivities,measuredaseithersubstrateconsumptionorasproduct
appearance,arereportedinChapter4,MeasurementofEnzymeActivity.Thenextchapter
dealswithageneraloverviewofthefundamentalsofthemethodologiesbasedonmass
spectrometry(MS)andnuclearmagneticresonance(NMR),whichdespitenotbeing
laboratorybenchmethodologiesaresometimesresortedtothroughexternalservices.Specific
casesarecoveredinPartIII,startingwithsucrose,themainplantsugar(seeChapter6:Case
Study:Sucrose),andfollowingwithtrehalose(seeChapter7:CaseStudy:Trehalose),
raffinose(seeChapter8:CaseStudy:Raffinose),fructosepolymersorfructans(seeChapter9:
CaseStudy:Fructans),polysaccharidesingeneral(seeChapter10:CaseStudy:
Polysaccharides),starch(seeChapter11:CaseStudy:Starch),andglycogen(seeChapter12:
CaseStudy:Glycogen),reservepolysaccharidesofplantsandcyanobacteria,respectively,
andstructuralpolysaccharidesascellulose(seeChapter13:CaseStudy:Cellulose).This
thirdpartendswiththedescriptionofthemainsugar-phosphates(seeChapter14:Case
Study:SugarPhosphates)andnucleosidediphosphate-sugars(seeChapter15:CaseStudy:
NucleotideSugars)involvedincarbohydratemetabolisminphotosyntheticorganisms.
Toclose,mydeepestgratitudetomywife,GracielaSalerno,whohasbeenofvitalsupport
throughoutthemonthsprecedingtheproductionofthebook,andalsooffundamentalhelp
withtheeditingandproofreadingofthemanuscript.Withoutherhelpthisbookwouldhave
notbeenpossible.Andtomydaughter,SheilaPontis,whohashelpedmethroughoutthe
writingofthismanuscriptwithherknowledgeofEnglish,design,andcommunication.In
addition,IwouldlikealsotoexpressmysincerethankstoAlejandroPariseforhisadviceand
councilonthewritingofChapter5,GeneralIntroductiontoMassSpectrometryandNuclear
MagneticResonance,andmyspecialgratitudetoValentinaMariscottiforhelpingmewiththe
translation,andtoGonzaloCalo´ andCintiaPereyrawhoassistedmetowarditscompletion.
Horacio G. Pontis, PhD
Professor Emeritus of Biochemistry,
Universidad Nacional de Mar del Plata, and
Emeritus Senior Investigator and Vice-President,
Applied Biological Research Foundation — FIBA,
Buenos Aires, Argentina
CHAPTER 1
Determination of Carbohydrates
Metabolism Molecules
Chapter Outline
1.1 Introduction 3
1.2 Determination of Reducing Sugars by the Somogyi(cid:1)Nelson Method 4
1.3 Determination of TotalSugars by the AnthroneMethod 5
1.4 Determination of TotalSugars by the Phenol(cid:1)Sulfuric Acid Assay 7
1.5 Enzymatic Determinationof Glucoseand Fructose 9
1.6 Enzymatic Determinationof Sucrose 10
1.6.1 SpectrophotometricMethod(MeasurementofNADPHAbsorption) 11
1.6.2 DirectFluorometricMethod(MeasurementbyNADPHNativeFluorescence) 12
1.6.3 AssayWithFluorescenceEnhancement 12
1.7 Determination of Glucose and Galactoseby an Enzymatic Colorimetric Method 13
1.8 Determination of Glucose byan Enzymatic Fluorometric Method 14
1.9 Determination of Amino Sugarsby the Elson(cid:1)MorganMethod 15
1.10 Determination of Acetyl Amino Sugars bythe Morgan(cid:1)Elson Method 16
1.11 Determination of Uronic Acids bythe Carbazole Assay 17
1.12 Determination of Pentoses bythe Orcinol Procedure 17
1.13 Determination of Fructose Derivatives by the Thiobarbituric Acid Assay 18
1.14 Determination of Inorganic Phosphateby the Fiske(cid:1)SubbarowAssay 19
1.15 Determination of Inorganic Phosphateby the Chifflet’s Method 21
1.16 Determination of Uridine Diphosphate by the Measurement of Pyruvate 22
1.16.1 ColorimetricMethodUsing2,4-Dinitrophenylhydrazine 23
1.16.2 SpectrophotometricMethodUsingLacticDehydrogenase 24
1.17 Determination of UDP-GlucoseUsingUDP-Glucose Dehydrogenase 25
1.18 Determination of UDP-GlucoseUsingUDP-Glucose Pyrophosphorylase 25
Further Reading and References 26
1.1 Introduction
Thepresentchapterdescribesaselectionofthemostcommonlyusedmethodsfortheanalysisof
differentmonosaccharides,suchasglucose,fructose,galactose,mannose,aminosugarspentoses,
anduronicacids,aswellasforthedeterminationofdisaccharides(sucroseandtrehalose).
MethodsforAnalysisofCarbohydrateMetabolisminPhotosyntheticOrganisms.
3
DOI:http://dx.doi.org/10.1016/B978-0-12-803396-8.00001-6
©2017ElsevierInc.Allrightsreserved.
4 Chapter 1
Finally, methods for the quantification of other compounds involved in the metabolism of
carbohydrates, such as inorganic phosphate, ADP, UDP, and UDP-glucose, are included.
The application of these methods is of value in any biochemical study dealing with
carbohydrate metabolism of photosynthetic organisms. Particularly, some applications to
enzyme studies are covered in Part III: Case Studies.
(cid:1)
1.2 Determination of Reducing Sugars by the Somogyi Nelson Method
Principle
This classical method is one of the most widely used for the quantitative determination of
reducing sugars. Sugars (aldoses or ketoses) containing a free carbonyl group in a slightly
alkaline medium are in equilibrium with their corresponding enediol, which has a high
reduction potential. Under these conditions, when heated with alkaline copper tartrate
(Somogyi’s reagent), these sugars reduce the copper from the cupric to cuprous state. Since
the cuprous ion can be reoxidized to cupric, arsenomolybdic acid (Nelson’s reagent) is
added to stabilize the cuprous oxide produced, which in turn, reduces the molybdic acid to
molybdenum blue (Nelson, 1944; Somogyi, 1952).
Reagents
Prepare the stock solutions of the two components of the Somogyi’s reagent (solutions
A and B) and of Nelson’s reagent (chromogenic reagent). Somogyi’s reagent (obtained by
mixing solutions A and B in the proportion indicated below) as well as the dilution of
Nelson’s reagent should be freshly prepared at the moment of use.
Somogyi’s reagent stocks
Solution A
Na CO (sodium carbonate anhydrous) 25g
2 3
KNaC H O (cid:3)4H O (potassium sodium tartrate tetrahydrate) 25g
4 4 6 2
NaHCO (sodium bicarbonate) 20g
3
Na SO (sodium sulfate anhydrous) 200g
2 4
Preparation of solution A: Add salts, one by one, to a volume of 800mL distilled
water, under continuous stirring. Allow each salt to be completely dissolved before
adding the next one. Make up to 1000mL.
Storage conditions: Dark glass bottle, at 20(cid:1)30(cid:4)C.
Solution B
CuSO (cid:3)5H O (copper sulfate pentahydrate) 15g
4 2
H SO (c) (sulfuric acid, δ: 1836kg(cid:3)L21) 2 drops
2 4
Preparation of solution B: Dissolve the salt in 50mL of distilled water, add the
drops of acid while stirring and make up to 100mL.
Storage conditions: Room temperature. Make up freshly every 2 months.