Dojindo Oxidative Stress Products - Dojindo Molecular … · · 2013-02-21Dojindo Oxidative Stress Products Oxidative Stress Research Products ... Dojindo Oxidative Stress Products

Dojindo Oxidative Stress Products

1


Oxidative Stress Research Products - Index

Purpose Product Page

1. Anti Oxidant Detection Measurementofsuperoxidedismutaseactivity SODAssayKit-WST 1

Quantificationoftotalglutathione TotalGlutathioneQuantificationKit 4 Separatequantificationofreduced(GSH)and oxidized(GSSG)glutathione GSSG/GSHQuantificationKit 7

2. DNA Damage Detection QuantificationofdamagedbaseingenomicDNA DNADamageQuantificationKit-APSiteCounting- 8

NitratedbaseofDNAandRNAdetection Anti-Nitroguanosineantibodies 11

Standardagentof8-nitroguaninedetection 8-Nitroguanine(Iyophilized) 12

3. Lipid Peroxide Detection Detectionoflipidperoxidebyfluorescent microscopyandflowcytometry

Liperfluo 13

DetectionoflipidperoxidebyHPLC DPPP 14

4. Radical Detection DetectionofproteinorDNAradicalbyWB,ELISA, andimaging

DMPO 15

DetectionofradicalbyEPR DMPO 16

DetectionofsuperoxideradicalbyEPR BMPO 17

5. Nitric Oxide(NO) Detection NOscavenger,NOdetectionbyEPR Carboxy-PTIO 18

DetectionofNO2-byfluorometricanalysis 2,3-Diaminonaphthalene(forNOdetection) 19

DetectionofNObyEPR DTCSNa 20

DetectionofNObyEPR MGD 21

6. Nitric Oxide(NO) Donation NOdonor(stableinacidiccondition) NORs 22

NOdonor(stableinalkalinecondition) NOCs 23

NOdonorfromnitrosothiolcompound S-Nitrosoglutathione 25

ONOO-(peroxynitrite)donor SIN-1 26

7. AGEs Research StandardsubstanceforAGEprecursor 3-Deoxyglucosone 27

Detectionof3-DGbyHPLC(fluorometric) 3-DeoxyglucosoneDetectionReagent 27


O2

hypoxanthine

Carbamylation

Protein

HCitProteinMPO

Smoking/Diet

Xanthine

XO

O2•-

e-

DMPOBMPO

H2O2•OH H2O

e- e- e-

H2O

O2•- O2

SODSOD Assay Kit-WST

LOH+H2O

GPx

LOOH GSH

GSSG

Total GlutathioneQuantification Kit

GRGSSG/GSH

Quantification Kit

NADP+

NADPH

TR

redTRX

oxTRX

NAD+

NADH

GPx,Cat

HOCl

MPOCl-

O2

MDA,HNEetcLOOH

Carbonyl-protein

DNAdamage(8-OhdG,APsiteetc)

DPPP

DPPP

Liperfluo

Liperfluo

DNA Damage Quantification Kit

sugarlipid

LH

Protein

DNA

Cat:catalaseGPx:glutathioneperoxideGR:glutathionereductaseMPO:myeloperoxideTR:thioredoxinreductaseTRX:thioredoxinLOOH:lipidperoxideXO:xanthineoxidase

hypoxanthine Xanthine

NAD+NADHG6PD

gluconolactone-6-P G6-P

glucose

NAD+NADHAR

sorbitol

fructose-6-phosphate

glyceraldehyde-3-phosphate

1,3-diphosphoglycerate

pyruvicacid

TCAcycle

mitochondrial electron transfer system

fructose

3-DGmethylglyoxalglycolytic

pathway

Amadorirearrangement

NAD+

NADHProtein-NH2

SDH

AGE

O2 ROS Protein

polyol pathway

glycation

O2

ROS

dehydrationcondensation

3-DeoxyglucosoneDetection Reagent

autoxidation

Dojindo Products

Dojindo Products

DMPOBMPO

H2O2RelatedOxidativeStress

DiabetesandOxidativeStress

Oxidative Stress Map

ROS:reactiveoxygenspeciesAR:aldosereductaseSDH:sorbetoldehydrogenaseAGE:advancedglycationendproduct


NO2-,NO3

-,H+

N2O4N2O3

H2O H2O

•NO2•NO

ONOO-

SIN-1

H+

•NO

R-SNO

R-SS-R

•NO Fe(II)-NO+

Fe(III)

R-SH

R-SH

O21/2

•OH

NO donor(NOCs, NORs)

Carboxy-PTIO

MGD

L-citruline

L-arginine

leukocyte

•NO

O2

ApoptosisDevelopmentReproduction

GrowthControl

iNOSNOSinhibitor

NAD(P)H NAD(P)+

LeukocyteNAD(P)Hoxidase

O2•-

Pathogens

Defencecontrol

resistance artery(artery)

bloodvessel

Hb-O2

bloodflowoxygentransportNO-Hb HbNO3

-

urinecontraction-relaxation

FlowControl

O2L-arginine

L-citruline

eNOS

dietary(extrinsic)Nitrate(NO3

-)

Nitrite(NO2-)

erythrocyte

mammals Nox family oxidasegenelocus(human)

aminoacidnumber(human)

tissueofhighexpression(function)

Nox1 Xq22 564 colonicepithelium,vascularsmoothmuscle(bloodpressureelevationofAngII)

Nox2/gp91phox Xp21.1 570 phagocyte,Blymphocyte(phylaxis)

Nox3 6q25.1-26 568 innerear,fetalkidneys(otolithformation)

Nox4 11q14.2-21 578 renaltublar,vascularendothelial

Nox5 15q22.31 737 spermatozoon,spleen,lymphocyte

Duox1 15q21 1,551 thyroid,bronchial

Duox2 15q21 1,548 thyroid,largeintestine(syntheticthyroidhormone)

NitricOxide(NO)RelatedStress

PropertyofNADPHOxide(NOX)Family


Dojindo Products

DTCS Na


5

*SignalingandmetabolicpathwayswereeditedunderthesupervisionofDr.KeizoSato/KyushuUniversityofHealthandWelfare.

VI IV

senescencecarcinogenesisneuropathydiabetesmellitussterilityrheumatismetc.

ADP ATP ATP

ROS+mitochondrialdamage

Citricacidcycle

Acetyl-CoA

NADHNAD+

e-

e-

H2O

*I - V:Complex I - V

CoenzymeQ

IIICytochromeC

H+ H+ H+H+

H+H+

H+

H+

H+O2

O2•- O2

•-

mitochondrion

MitochondrialOxidativeStress

ReactiveOxygenSpeciesRelatedinAtherosclerosis



6

Introduction

OxygenisaveryimportantmoleculeforthesynthesisofbiologicallyactivematerialssuchashormonesandATP. Acquisition of the ability to utilize oxygenwas a significant driving force for the evolutionof life.Oxygenactivates various enzymes in cells andactivatedoxygen species are involved in theoperation of cell functions.Although oxygen itself is an essential element of life,molecules in cells,

suchasDNAandproteins,aresometimesdamagedbyreactiveoxygenspecies(ROS)inwhatiscalledoxidativestress.ROScanbecreatedbymetabolism,ionizingradiation,andcarcinogeniccompoundsthatdirectlyinteractwithDNA.Duringmetabolism,asmallportionofoxygenisconvertedtosuperoxideanionbyoneelectronreduction;superoxideanionisthenconvertedtooxygenandhydrogenperoxidebysuperoxidedismutase(SOD).Hydrogenperoxideisreducedtowaterbycatalaseorglutathioneperoxidase.However,ifhydrogenperoxideisnotcompletelyreducedbytheseenzymes, itcangenerateanextremelyreactivehydroxyradicalwhenoxidizedby iron(Fentonreaction).HydroxyradicalisalsogeneratedbyUVirradiationordirectlyfromwaterbyionizingradiation.Hydroxyradicalreactswithlipidtogeneratelipidperoxide.However,notallROSareunwanted.Hypochloriteion,anROSderivedfromhydrogenperoxidebymyeloperoxidaseinneutrophils,hasgermicidalactivity.Nitricoxide,alsoknownasendothelial-derivedrelaxationfactor,isgeneratedbyNOsynthetase.However,NOandsuperoxideanionmayreacttogenerateperoxynitrite,whichiscytotoxic.

TheROSandreactivenitrogencompoundshavemanydifferentactivitiesinbiologicalsystems.Inresponse,aerobicorganismscreateddefensemechanismstoavoidoxidativestress.Oxidativestresshasrecentlybecomethefocusofmanystudiesseekingtounderstandthesedefensemechanismsandtherelationshipsbetweenoxidativedamageanddiseaseoragingprocesses.Tothisend,manyassaymethodshavebeendevelopedforthedetectionofROS-relatedorROS-derivedsubstancessuchassuperoxideanion,superoxidedismutase,glutathione,glutathionereductase,glutathioneperoxidase,DNAlesions,8-oxoguanine,8-nitroguanosine,andproteincarbonyl.

Nitricoxide(NO)hasbeenidentifiedasanendothelial-derivedrelaxationfactorandantiplateletsubstance.Itservesasaneurotransmitterwhenderivedfromaneutrophil,andasacytotoxicsubstancewhenderivedfromanactivatedmacrophage.NOreactswithsuperoxideaniontogeneratehighlytoxicperoxynitrite.ThereactionrateofNOwithsuperoxideisthreetimesthatofSOD.Insomecases,NOalsoactivatescyclooxygenase.ThemostimportantroleofNO is thought tobe theactivationofguanylatecyclase.Recently,publishedNOresearchhas reportedmanycontradictoryresults,whichareduetoNO’suniquechemicalproperties.SinceNOisafreeradical,itisveryreactiveandunstable.NOchangesitsforminacomplexmannerimmediatelyafterappearinginabiologicalenvironment.EachofNO’smetabolitesmighthavedifferentbioactivitiesfromNOitself.Forthisreason,it isvitaltoseparatelyinvestigateeachfunctionoftheNO-relatedmetabolites.


1

Measurementofsuperoxidedismutaseactivity

SOD Assay Kit-WST

Superoxidedismutase(SOD),whichcatalyzesthedismutationofthesuperoxidean-ion(O2

.-)intohydrogenperoxideandmolecularoxygen,isoneofthemostimpor-tantantioxidativeenzymes.InordertodeterminetheSODactivity,severaldirectandindirectmethodshavebeendeveloped.Amongthesemethods,anindirectmethodus-ingnitrobluetetrazolium(NBT)iscommonlyusedduetoitsconvenienceandeaseofuse.However,thereareseveraldisadvantagestotheNBTmethod,suchaspoorwa-tersolubilityoftheformazandye,andtheinteractionwiththereducedformofxanthineoxidase.SODAssayKit-WSTallowsaveryconvenientandhighlysensitiveSODassaybyuti-lizingDojindo’shighlywater-soluble tetrazoliumsalt,WST-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfo-phenyl)-2H-tetrazolium,monosodiumsalt),whichproducesawater-solubleformazandyeuponreductionwithasuperoxideanion(Fig.1).TherateofWST-1reductionbysuperoxideanionislinearlyrelatedtothexanthineoxidaseactivityandisinhibitedbySOD(seefigurebelow).Therefore,theIC50(50%inhibitionconcentra-tion)ofSODorSOD-likematerialscanbedeterminedusingcolorimetricmethods.

1. Preparation of Sample Solutions► ErythrocytesorPlasma1. Centrifuge2-3mlofanticoagulant-treatedblood(suchasheparin10U/mlwith

finalconcentration)at600xgfor10minutesat4°C.2. Removethesupernatantanddiluteitwithsalinetouseasaplasmasample.

Addsalinetothepellettopreparethesamevolume,andsuspendthepellet.3. Centrifugethepelletsuspensionat600xgfor10minutesat4°C,anddiscard

thesupernatant.4. Addthesamevolumeofsaline,andrepeatStep3twice.5. Suspendthepelletwith4mldistilledwater,thenadd1mlethanoland0.6ml

chloroform.6. Shakethemixturevigorouslywithashakerfor15minutesat4°C.7. Centrifugethemixtureat600xgfor10minutesat4°Candtransfertheupper

water-ethanolphasetoanewtube.8. Mix0.1mloftheupperphasewith0.7mlofdistilledwater,anddilutewith0.25%

ethanoltopreparesamplesolution.

► Tissue(100mg)1. Wash the tissuewith saline to removeasmuchbloodaspossible.Blot the

tissuewithpapertowelsandthenmeasureitsweight.2. Add400-900µl sucrosebuffer (0.25Msucrose, 10mMTris, 1mMEDTA,

pH7.4)andhomogenizethesampleusingTeflonhomogenizer.Ifnecessary,sonicate thehomogenized sampleonan icebath (60Wwith0.5 secondintervalsfor15minutes).

3. Centrifugethehomogenizedsampleat10,000xgfor60minutesat4°C,andtransferthesupernatanttoanewtube.

4. Dilutethesupernatantwithdistilledwatertopreparesamplesolution.

2. Preparations of Solutions (for one 96-well plate)► WSTworkingsolution

Dilute1mlofWSTSolutionwith19mlofBufferSolution► Enzymeworkingsolution

CentrifugetheEnzymeSolutiontubefor5seconds.Mixbypipettinganddilute15μlofEnzymeSolutionwith2.5mlofDilutionBuffer.

► SamplesolutionDilutesamplesolutionpreparedwithDilutionBufferorSaline.e.g.)dilutionrate:x1(nodilution),x1/5,x1/52,x1/53,x1/54,x1/55,x1/56

Contents of the Kit 500testsWSTSolution 5mlx1EnzymeSolution 100μlx1BufferSolution 100mlx1DilutionBuffer 50mlx1

Required Equipment & MaterialsMicroplateReader(450nmfilter)96-wellmicroplate2-20μl&20-200μlmulti-channelpipettesIncubator(37°C)

Fig.1 SOD inhibition assay mechanism

Fig. 2 Absorption spectrum of WST-1 formazan

Fig. 3 Serial dilution process

Product Code: S311

Fig. 4 Assay procedure

4)

xanthineoxidase

Samplesolution

50μl

50μl50μl 50μl 50μl 50μl

Dilutionbuffer200μl






1/51/521/531/541/551/56

1) 2)

3)

300400500600700Wavelength/nm

2.0

1.0

0

Absorbance

1. Anti O

xidant D

etection2. D

NA D

amage

Detection

3. Lipid Peroxide D

etection4. R

adical Detection

6. NO

Donor

5. Nitric O

xide D

etection7. A

GEs R

esearch


2

3. General Protocol (refer to Table 1, Fig. 4 and Fig. 5)1. Add20μlofsamplesolutiontoeachsamplewellandblank2well,andadd20

μlofddH2O(double-distilledwater)toeachblank1andblank3well.2. Add200μlofWSTWorkingSolutiontoeachwell,andmixbypipetting.3. Add20μlofDilutionBuffertoeachblank2andblank3well.4. Add20μlofEnzymeWorkingSolutiontoeachsampleandblank1well.5. Incubatetheplateat37ºCfor20minutes.6. Readtheabsorbanceat450nmusingamicroplatereader.7. CalculatetheSODactivity(inhibitionrate%)usingthefollowingequation.

SODactivity(inhibitionrate%)={[(Ablank1-Ablank3)-(Asample-Ablank2)]/(Ablank1-Ablank3)}x100

4. Inhibition CurveAsFig.6shows,SODAssayKit-WSTcanmeasure100%inhibitionbecauseWST-1doesnotreactwiththereducedformofxanthineoxidase(XO).

5. Definition of Unit(U)One unit is defined as a point where a 20 μl of sample solution gives 50% inhibition of a colorimetric reaction between WST-1 and superoxide anion.*UnitdefinitiondifferfromtheunitdefinitionofCytochromeCassay.

6. Calculate Unit(U)1. Calculateadilutionratiowheretheinhibitioncurvegives50%inhibition.2. SODunitinoriginalsamplecanbecalculatedbymultiplyingthedilutionrate.

7. Example of Calculating Unit(U): Erythrocytes(x108 dilution sample)1. Calculateadilution ratio from thepointof IC50 in the inhibitioncurve.Fig.7

givesthedilutionrateatIC50of1/1.8.2. Accordingtothedefinitionofunit,20μlofthissampleiscalculated1.8U.3. SODunitper1mlof thissamplesolutioncanbecalculatedby the following

equation,1.8/0.02=90.0U/ml.4. Original erythrocytes samplewasdiluted108 timesduring the sample

preparation.To calculate theSODunit in theoriginal,multiply 90.0U/ml by108.TheSODunitintheoriginalsampleis9,720U/mlofblood.

*SODunitcanbecalculatedasU/gramorU/mg.

8. Distinguish Mn-SOD from Cu/Zn-SOD and EC-SODMn-SODcanbemeasuredbyblocking theCu/Zn-SODandEC-SODactivity usingpotassiumcyanide(KCN)orDiethyldithiocarbamate(DDC).

9. InterferenceReducingagents,suchasascorbicacidsandreducedformsofglutathiones,interferewith theSODassay.Table2 shows the concentrationsofmaterials that cause10%increaseintheO.D.value.Ifsamplecontainsthesematerials,pleasedilutethesampletoavoidtheinterfere.

10. References1. J.M.McCord,et al.,AnEnzymicFunctionforErythrocuprein(hemocuprein).J Biol Chem.1969;244:6049-6055.2. B.L.Geller,et al.,AMethodforDistinguishingCu,Zn-andMn-ContainingSuperoxideDismutases.Anal Biochem.1983;128:86-

92.3. S.Goldstein,et al.,ComparisonBetweenDifferentAssaysforSuperoxideDismutase-likeActivity.Free Rad Res Commun.

1991;12:5-10.4. R.H.Burdon,et al.,ReductionofaTetrazoliumSaltandSuperoxideGenerationinHumanTumorCells(HeLa).Free Rad

Res Commun.1993;18:369-380.5. M.W.Sutherland,et al.,TheTetrazoliumDyesMTSandXTTProvideNewQuantitativeAssaysforSuperoxideandSuperoxide

Dismutase.Free Radic Res.1997;27:283-289.6. H.Ukeda,et al.,Flow-InjectionAssayofSuperoxideDismutaseBasedontheReductionofHighlyWater-SolubleTetrazolium.

Anal Sci.1999;15:353-357.7. H.Ukeda,et al.,SpectrophotometricAssayforSuperoxideDismutaseBasedontheReductionofHighlyWater-solubleTet-

razoliumSaltsbyXanthine-XanthineOxidase.Biosci Biotechnol Biochem.1999;63:485-488.8. H.Ukeda,et al.,SpectrophotometricAssayofSuperoxideAnionFormedinMaillardReactionBasedonHighlyWater-soluble

TetrazoliumSalt.Anal Sci.2002;18:1151-1154.9. N.Tsuji,et al.,EnhancementofTolerance toHeavyMetalsandOxidativeStress inDunaliella TertiolectabyZn-induced

PhytochelatinSynthesis.Biochem Biophys Res Commun.2002;293:653-659.

Fig. 5 Sample and blank arrangement on a 96-well microplate

Fig. 6 Inhibition curve of Cu/Zn-SOD

Fig. 7 Inhibition curve on erythrocytes sample

Table 2 Minimum Concentrations of Interfering Substances

Table 3 Measurement Examples

sample blank1 blank2 blank3SampleSolution 20μl - 20μl -

ddH2O - 20μl - 20μlWSTWorkingSolution 200μl 200μl 200μl 200μl

DilutionBuffer - - 20μl 20μlEnzymeWorkingSolution 20μl 20μl - -

TotalSODerythrocyte 9,720U/mlofbloodserum 355U/mlofblood

heart(rat) 15,712U/g(wet)liver(rat) 142,907U/g(wet)HeLacell 73U/1x107cellsHL60cell 226U/1x108cells

Detergents

SDS 0.05%Tween20 0.5%NP-40 0.5%TritonX-100 0.2%

SolventsEthanol 25%DMSO 5%

Reducingagents

Glutathione,reducedform

1.25mmol/l

Ascorbicacid 0.1mmol/l

OtherEDTA 2mmol/lBSA 1%w/v

Table 1 Solution and buffer volumes in each well

1 2 3 4 5 6 7 8 9 10 11 12

A sample1,1

B sample1,1/5

C sample1,1/52

D sample1,1/53 sample2 sample3 sample4

E sample1,1/54

F sample1,1/55

G sample1,1/56

H blank1 blank2 blank3

1/1041/1031/1021/101Dilutionratio

100

90

80

70

60

50

40

30

20

10

0

Inhibition(%)

▲ ▲ ▲▲

▲

▲

1/1.8

1/1051/1041/1031/1021/101

Dilutionratio

100

90

80

70

60

50

40

30

20

10

0

Inhibition(%)

▲▲ ▲ ▲ ▲▲

▲

▲▲

▲▲

1. A

nti O

xida

nt

Det

ectio

n2.

DN

A D

amag

e D

etec

tion

3. L

ipid

Per

oxid

e D

etec

tion

4. R

adic

al D

etec

tion

6. N

O D

onor

5. N

itric

Oxi

de

Det

ectio

n7.

AG

Es R

esea

rch


3

Title ReferenceAmyotrophicLateralSclerosisModelDerivedfromHumanEmbryonicStemCellsOverexpressingMutantSuperoxideDismutase1

T.Wada,et al.,Stem Cells Trans Med.2012;1:396

Copperchelationbytetrathiomolybdateinhibitslipopolysaccharide-inducedinflammatoryresponsesinvivo

H.Wei,et al., Am J Physiol Heart Circ Physiol.2011;301:H712

ANewEnteralDiet,MHN-02,WhichContainsAbundantAntioxidantsandWheyPeptide,ProtectsAgainstCarbonTetrachloride–InducedHepatitis

T.Takayanagi,et al., JPEN J Parenter Enteral Nutr.2011;35:516

EffectsofdietarysupplementationofmethionineanditshydroxyanalogDL-2-hydroxy-4-methylthiobutanoicacidongrowthperformance,plasmahormonelevels,andtheredoxstatusofbroilerchickensexposedtohightemperatures

H.Willemsen,et al., Poult. Sci.,2011;90:2311

RafKinaseInhibitorProteinRKIPEnhancesSignalingbyGlycogenSynthaseKinase-3β

F.Al-Mulla,et al., Cancer Res.2011;71:1334

TemporalchangesintheexpressionofmRNAofNADPHoxidasesubunitsinrenalepithelialcellsexposedtooxalateorcalciumoxalatecrystals

SaeedR.Khan,et al., Nephrol. Dial. Transplant.2011;26:1778

Profilingofsuperoxidedismutaseisoenzymesincompartmentsofthedevelopingbovineantralfollicles

C.Combelles,et al., Reproduction,2010;139:871

Deletionofnuclearfactor-E2-relatedfactor-2leadstorapidonsetandprogressionofnutritionalsteatohepatitisinmice

H.Sugimoto,et al., Am J Physiol Gastrointest Liver Physiol.2010;298:G283

GalliumDisruptsIronUptakebyIntracellularandExtracellularFrancisellaStrainsandExhibitsTherapeuticEfficacyinaMurinePulmonaryInfectionModel

O.Olakanmi,et al., Antimicrob. Agents Chemother.2010;54:244

Ironrestrictionimprovestype2diabetesmellitusinOtsukaLong-EvansTokushimafattyrats

Y.Minamiyama, et al., Am J Physiol Endocrinol Metab,2010;298:E1140

12. FAQ►WhatisthedefinitionofaUnit?

Oneunitisdefinedasapointwhereasamplegives50%inhibitionofacolorimetricreactionbetweenreactivedye(suchascytochromeC,WST-1,nitro-tetrazoliumblueorXTT)andsuperoxideanion.Forexample,iftheO.D.of"Blank1"thatdoesnotcontainanySODis1.0,thesamplethatgives0.5O.D.isdefinedashaving1unitofSODactivity.YoucanusethisunittodeterminetheSODactivityofyoursample.Therefore,SODactivitiesdeterminedusingdifferentdyesormethodsarenotcomparablewitheachother.

►CanIusestandardSODtodetermineSODactivityinsamplesolutions?Yes,youcan.Prepareainhibitioncurve(typicalinhibitioncurve,anddetermineSODactivityinthesamplesolution.SODbovineerythrocytes(CAS#9054-89-1,EC1.15.1.1)canbepurchasedfromSigma(catalog#S7571).

►CanIuseakineticmethodtodetermineSODactivity?Yes,youcanuseakineticmethodforSODassay.Sincetherateofthecolordevelopmentremainsthesameforupto20minutes,measuretheslopefor5minutesduringthislinearphase.

►Thesamplehascolor.CanIstillusethissample?Yes,youcanstillusethesample.Dilutingthesamplewillminimizetheinterference.SubtracttheO.D.ofblank2fromtheO.D.of thesampletocancelout thebackgroundcolor.However, if theSODactivity in thesample is low, itmaynotbemeasurable.

►HowdoIpreparemoreDilutionBuffer?DilutionBuffer isPBS.PleasepreparetheDilutionbufferwithfollowingconcentrations;137mMNaCl,2.7mMKCl,1.47mMKH2PO4,8.1mMNa2HPO4,pH7.4.

►CanIdetermineMn-SODandCu/Zn-SODindependentlyusingthiskit?Yes.InordertomeasureMn-SODactivity,itisnecessarytoblocktheCu/Zn-SODactivityusingpotassiumcyanide(KCN).Adding1mMKCNtosamplescanblockCu/Zn-SODactivitycompletely.TomeasureCu/Zn-SODactivity,measurethetotalSODactivitywithandwithoutKCN,andthensubtracttheMn-SODactivityfromtotalSODactivity.

►HowlongcanIstorethesample?Asamplestoredinafreezerat-80ºCisstablefor1month.

►CanImeasurethelevelsofsuperoxideanionusingthiskit?No.However,youcouldsimplyuseWST-1, insteadof thiskit, tomeasuresuperoxide.Youwouldneedastandard todeterminetheamountofsuperoxideinsamplesolution.Sincesuperoxideisnotstableandreactswithvariousmaterials,itmightbedifficult todeterminethetotalamountofsuperoxidegenerated in thesystem.Thexanthine-xanthineoxidasesysteminthiskitcanbeusedasastandardformeasuringtherelativeamountofsuperoxideproductionineachsample.

11. Recent Publications

1. Anti O

xidant D

etection2. D

NA D

amage

Detection

3. Lipid Peroxide D

etection4. R

adical Detection

6. NO

Donor

5. Nitric O

xide D

etection7. A

GEs R

esearch


4

Glutathione(GSH) is themostabundant thiolcompound inanimal tissues,planttissues,bacteria,andyeast.GSHhasmanydifferentroles, includingprotection

againstreactiveoxygenspeciesandthemaintenanceofproteinthiolgroups.Duringtheseprocesses,GSHisconvertedintoitsoxidizedform,glutathionedisulfide(GSSG).SinceGSSG is thenenzymatically reducedbyglutathione reductase,GSH is thedominantforminorganisms.DTNB(5,5’-Dithiobis(2-nitrobenzoicacid)),knownasEllman’sReagent,wasdevelopedtodetectthiolcompounds.In1985,Dr.M.E.AndersonsuggestedthattheglutathionerecyclingsysteminvolvingDTNBandglutathionereductasecouldbeusedasahighlysensitiveglutathionedetectionmethod.DTNBandGSHreact togenerate2-nitro-5-thiobenzoicacidandGSSG(Fig.1).Since2-nitro-5-thiobenzoicacid isyellow, theGSHconcentration inasamplesolutioncanbedeterminedbyO.D.measurementat412nmabsorbance(Fig.2).GSHisregeneratedfromGSSGbyglutathionereductaseandwillagainreactwithDTNBtoproduce2-nitro-5-thiobenzoicacid.Thisrecyclingreactionimprovesthesensitivityoftotalglutathionedetection.

1. Preparation of Sample Solutions► Cell(adhesivecell:5x105cells,leukocytecell:1x106cells)1.Collectcellsbycentrifugationat200g for10minutesat4°C.Discard thesupernatant.

2.Washthecellswith300μlPBSandcentrifugeat200gfor10minutesat4°C.Discardthesupernatant.

3.Add80μl10mMHCl,andlysethecellsbyfreezingandthawingtwice.4.Add20μl5%SSAandcentrifugeat8,000gfor10minutes.5.Transfer thesupernatant toanewtube,anduse it for theassay. If the finalconcentrationofSSAisover1%,addddH2OtoreducetheconcentrationofSSAfrom0.5to1%.

► Tissue(100mg)1.Homogenizethetissuein0.5-1.0ml5%SSA.2.Centrifugethehomogenizedtissuesampleat8,000gfor10minutes.3.Transfer the supernatant to a new tube and add ddH2O to reduce theconcentrationofSSAfrom0.5to1%.Useitfortheassay.

► Plasma1.Centrifugeanticoagulant-treatedbloodat1,000gfor10minutesat4°C.2.Transferthetopplasmalayertoanewtubeandadd5%SSAequivalenttohalfofthevolumeoftheplasma.

3.Centrifugeat8,000gfor10minutesat4°C4.Transfer the supernatant to a new tube, and add ddH2O to reduce theconcentrationofSSAfrom0.5to1%.Useitfortheassay.

► Erythrocyte1.Centrifugeanticoagulant-treatedbloodat1,000gfor10minutesat4°C.2.Discardthesupernatantandthewhitebuffylayer.3.Lyse theerythrocyteswith5%SSAequivalent to4 times thevolumeof theerythrocytes.

4.Centrifugeat8,000gfor10minutesat4°C.5.Transfer the supernatant to a new tube, and add ddH2O to reduce theconcentrationofSSAfrom0.5to1%.Useitfortheassay.Erythrocytescanbeisolatedfromtheremainingsamplesolutionaftertheplasmasampleisolation.

*Preparationof5%5-SulfosalicylicAcid(SSA)SolutionNote:SSAisnotincludedinthiskit.1.Dissolve1gSSAin19mlwater.2.Storethesolutionat4°C(stablefor6monthsat4°C).

Fig.1 Principal of total glutathione detection

Fig. 2 Absorption spectrum of 5-Mercapto-2-nitrobenzoic acid

Quantificationoftotalglutathione

Total Glutathione Quantification Kit Product Code: T419

Contents of the KitSubstrate(DTNB) 2vialsEnzymeSolution 50μlx1Coenzyme(lyophilized) 2vialsStandardGSH(lyophilized) 1vialBufferSolution 50mlx1

Required Equipment & MaterialsMicroplateReader(405or415nmfilter)96-wellmicroplate20-200μlmulti-channelpipettesIncubator(37°C)5-sulfosalicylicacid(SSA)

300400500600Wavelength/nm

1.5

1.0

0.5

0

Absorbance

1. A

nti O

xida

nt

Det

ectio

n2.

DN

A D

amag

e D

etec

tion

3. L

ipid

Per

oxid

e D

etec

tion

4. R

adic

al D

etec

tion

6. N

O D

onor

5. N

itric

Oxi

de

Det

ectio

n7.

AG

Es R

esea

rch


5

2. Preparations of Solutions► Substrateworkingsolution

Add1.2mlofBufferSolutiontoonevialofSubstrate,anddissolve.Storethesolutionat-20°C(stablefor2months).

► EnzymeworkingsolutionMixEnzymeSolutionusingpipette.Takeout20µlofEnzymeSolution,andmixitwith4mlofBufferSolution.Storethesolutionat4°C(stablefor2months).

► CoenzymeworkingsolutionAdd1.2mlofddH2OtotheCoenzymevialanddissolve.TheCoenzymevialisdecompressed.UseasyringetoaddddH2O,thenopenthevial.Storethesolutionat-20°C(stablefor2months).

► GSHstandardsolutionAdd2mlof0.5%SSAtoStandardGSHvial,anddissolvetoprepare200µMofGSHstandardsolution.TheStandardGSHvialisdecompressed.Useasyringetoadd0.5%SSA,thenopenthevial.Storethesolutionat-20°C(stablefor2months).Dilute100µlof200µMGSHstandardsolutionbyserialdilutionwith100ulof0.5%SSAinplastictubesasindicatedFig.4.

3. General Protocol (refer to Fig. 3)1.Add20µlofEnzymeworkingsolution,20µlofCoenzymeworkingsolutionand

120µlofBufferSolutiontoeachwell.2. Incubatetheplateat37°Cfor5minutes.3.Add20µlofGSHstandardsolutionand20µlofsamplesolutiontoeachwell.4. Incubatetheplateat37°Cfor10minutes.5.Add20µl ofSubstrateworking solution, and incubate theplate at room

temperaturefor10minutes.6.Readtheabsorbanceat405nmor415nmusingamicroplatereader.7.DeterminconcentrationsofGSHinthesamplesolutionsusingacalibrationcurve.

Sincethecolorimetricreaction isstableandtheO.D. increases linearlyover30min.AtimecourseofthecolorimetricreactionisshownFig.5.Typicalcalibrationcurvespreparedusingthepseudo-endpointmethodisindicatedinFig.6.

4. Calculation of total glutathione (GSH and GSSG) concentrationDeterminethetotalglutathioneconcentrationa)inasamplesolutionusingthefollowingequations.► pseudo-endpointmethod

Total glutathione (GSH+GSSG)=(O.D.sample - O.D.blank)/slopeb)

► kineticmethodTotal glutathione (GSH+GSSG)=(Slopec)

sample - Slopec)blank)/slopeb)

a) Sincethevaluesobtainedbytheseequationsaretheamountof totalglutathionein treated sample solutions, further calculationsarenecessary if theactualconcentrationsofglutathioneinsamplesneedtobedetermined.

b) slopeofthecalibrationcurvec) slopeofthekineticreaction

5. InterferenceReducingagents suchasascorbic acid,β-mercaptoethanol, dithiothreitol (DTT)andcysteine, or thiol reactive compounds suchasmaleimide compounds, interferewiththeglutathioneassay. Therefore,SHcompounds, reducingagentsandSH reactivematerialsshouldbeavoidedduringthesamplepreparation.

6. Notes• Thekitisstablefor6monthsat0to5°C.• Usethereagentsinthekitafterthereagentstemperatureareequilibratedtotheroomtemperature.

• Triplicatemeasurementspersampleisrecommendedtoobtainaccuratedata.• SincethecolorimetricreactionstartsimmediatelyaftertheadditionofSubstrateworkingsolutiontoawell,useamultichannelpipettetoavoidthereactiontimelagofeachwell.

• If theconcentration rangeof totalglutathione inasample isunknown,preparemulti-dilutedsamplesolutions.

• This kit is not forGSSG/GSH ratio determination. ForGSSG/GSH ratiodetermination,pleaserefertoGSSG/GSHQuantificationKit.

7. References1. G.L.Ellman,Tissuesulfhydrylgroups.Arch Biochem Biophys.1959;82:70-77.2. O.W.Griffith,DeterminationofGlutathioneandGlutathioneDisulfideUsingGlutathioneReductaseand2-Vinylpyridine.Anal

Biochem.1980;106:207-212.3. M.E.Anderson,DeterminationofGlutathioneandGlutathioneDisulfideinBiologicalSamples.Methods Enzymol.1985;113:548-555.4. M.A.Baker,et al.,MicrotiterPlateAssayfortheMeasurementofGlutathioneandGlutathioneDisulfideinLargeNumbersof

BiologicalSamples.Anal Biochem.1990;190:360-365.


Fig. 5 Time-dependent Abs. increase on eight diferent samples

Fig. 6 Calibration curve prepared using pseudo-endpoint method(10 min

incubation at room temperature)

Fig. 4 Serial dilution process

4)3)

200μMGSH100μl

100μl100μl 100μl 100μl 100μl

0.5%SSA100μl

0.5%SSA100μl

0.5%SSA100μl

0.5%SSA100μl

0.5%SSA100μl

0.5%SSA100μl

051015202530Time(min)

3.0

2.5

2.0

1.5

1.0

0.5

0Absorbanceat405nm

▲▲

▲▲

▲▲

▲ ▲

●

■♦

●

● ■

■

■

■

♦♦

♦♦

♦♦

♦

020406080100ConcentrationofGSH(μmol/l)

2.5

2.0

1.5

1.0

0.5

0

Absorbanceat405nm

▲▲▲

▲

▲

▲

1. Anti O

xidant D

etection2. D

NA D

amage

Detection

3. Lipid Peroxide D

etection4. R

adical Detection

6. NO

Donor

5. Nitric O

xide D

etection7. A

GEs R

esearch

1) 2)


6

Title ReferenceCopperChaperone-Dependentand-IndependentActivationofThreeCopper-ZincSuperoxideDismutaseHomologsLocalizedinDifferentCellularCompartmentsinArabidopsis

C.Huang,et al. Plant Physiology.2012;158:737-746

IRE1αactivationprotectsmiceagainstacetaminophen-inducedhepatotoxicity K.Yeon,et al., J. Exp. Med.2012;209:307-318

TheNitricOxideProdrugJS-KIsEffectiveagainstNon–Small-CellLungCancerCellsInVitroandInVivo:InvolvementofReactiveOxygenSpecies

A..Maciag,et al., J. Pharmacol. Exp. Ther.,2011;336:313-320

MutationsinPNKDcausingparoxysmaldyskinesiaaltersproteincleavageandstability

Y.Shen,et al., Hum. Mol. Genet.,2011;20:2322-2332

Phosphate-activatedglutaminase(GLS2),ap53-inducibleregulatorofglutaminemetabolismandreactiveoxygenspecies

S.Suzuki,et al., PNAS,2010;107:7461-7466

OxidativeStressandSodiumMethyldithiocarbamate–InducedModulationoftheMacrophageResponsetoLipopolysaccharideInVivo

S.Pruett,et al., Toxicol. Sci.2009;109:237-246

Nrf2EnhancesCellProliferationandResistancetoAnticancerDrugsinHumanLungCancer

S.Homma,et al., Clin. Cancer Res.2009;15:3423-3432

InhibitionofhepaticNiemann-PickC1-like1improveshepaticinsulinresistance K.Irie,et al., Journal of Dental Research.2008;87:456-460

3-Morpholinopropylisothiocyanateisanovelsyntheticisothiocyanatethatstronglyinducestheantioxidantresponseelement-dependentNrf2-mediateddetoxifying/antioxidantenzymesinvitroandinvivo

Y.Keum,et al., Carcinogenesis.2008;29:594-599

CellsDeficientintheFANC/BRCAPathwayAreHypersensitivetoPlasmaLevelsofFormaldehyde

J.Ridpath,et al., Cancer Res.2007;67:11117-11122

9. FAQ►DoIhavetodilutethesamplesolutionpriortotheassay?

Ifyoudonotknowthetotalglutathionelevelofyoursample,multipledilutionsmaybenecessary. If thetotalglutathionelevelofyoursampleislessthan100μM,nodilutionisnecessary.

►Whatinterfereswiththeassay?Reducingagents(suchasascorbicacid,beta-mercaptoethanol,dithiothreitol,andcysteine)andthiolreactivecompounds(suchasmaleimides)interferewiththeglutathioneassay.Therefore,reducingagentsandthiolreactivecompoundsshouldbeavoidedduringthesamplepreparation.


1. A

nti O

xida

nt

Det

ectio

n2.

DN

A D

amag

e D

etec

tion

3. L

ipid

Per

oxid

e D

etec

tion

4. R

adic

al D

etec

tion

6. N

O D

onor

5. N

itric

Oxi

de

Det

ectio

n7.

AG

Es R

esea

rch


7

DistinguishMeasurementofGlutathione

GSSG/GSH Quantification Kit Product Code: G257

Glutathione(γ-L-glutamyl-L-cysteinylglycine)isatripeptidepresentinthebody,andit is involved inantioxidation, drugmetabolism,andother asenzymesubstrate

of glutathioneperoxidase, glutathioneS-transferase, and thiol transferase, etc.Glutathioneisusuallypresentasreducedform(GSH),butGSHisconvertedinto itsoxidizedform(GSSG)bystimulationsuchasoxidativestress.Therefore,theratioofGSHandGSSGhasbeennotedasindexofoxidativestress.TheGSSG/GSHQuantificationkitcontainsMaskingReagentofGSH.TheGSHcanbedeactivated in the samplebyadding theMaskingReagent.Therefore, only theGSSGisdetectedbymeasuringtheabsorption(λmax=412nm)ofDTNB(5,5’-dithiobis(2-nitrobenzoic acid) using theenzymatic recycling system.Also,GSHcanbedeterminedthequantitybysubtractingGSSGfromthetotalamountofglutathione.The kit canbe limited to quantifyGSH/GSSGconcentration from0.5µmol/l to 50µmol/landGSSGconcentrationfrom0.5µmol/lto25µmol/l.

Contents of the KitEnzymeSolution 50μlx1Coenzyme 2vialsBufferSolution 60mlx1Substrate(DTNB) 4vialsStandardGSH 1vialStandardGSSG 1vialMaskingReagent 20μlx1

Required Equipment & MaterialsMicroplateReader(405or415nmfilter)96-wellmicroplate20-200μlmulti-channelpipettesIncubator(37°C)5-sulfosalicylicacid(SSA)Ethanol

Glutathionereductase

GSH

GSSG 5-Mercapto-2-nitrobenzoicacid

(λmax=412nm)

GSSGGSHGSHMasking

GSSGMeasurement

Maskingreagent+Sample

DTNBFig.1 Principal of GSSG/GSH detection

1. General Protocol► PreparationofSampleSolution

PleaserefertoTotalGlutathioneQuantification,page5.► DeterminationofGSSGconcentration1. Add4µlofMaskingSolutiontosamplesolutionand200µlofGSSGstandard

solutiondilutedwith0.5%SSArespectively,thentransfer40µlofthesolutiontoeachwell.

2. Add120µlofBufferSolutiontoeachwellandincubatefor1hourat37°C.3. Add20µlofSubstrateworkingsolutiontoeachwell,thenadd20µlofCoen-

zymeworkingsolutionandEnzymeworkingsolutiontoeachwellrespectively.4. Incubatefor10minutesat37°Candreadtheabsorbanceat405or415nmus-

ingamicroplatereader.5. DetermineconcentrationsofGSSGinthesamplesolutionusingaGSSGcali-

brationcurve(Fig.2).

► Determinationoftotalglutathioneconcentration1. Addsamplesolutionand40µlofGSHstandardsolutiondilutedwith0.5%

SSAtoeachwell.2. Add120µlofBuffersolutiontoeachwellandincubatefor1hourat37°C.3. Add20µlofSubstrateworkingsolutiontoeachwell,thenadd20µlofCoen-

zymeworkingsolutionandEnzymeworkingsolutiontoeachwellrespectively.4. Incubatefor10minutesat37°Candreadtheabsorbanceat405or415nmus-

ingamicroplatereader.5. Determineconcentrationsof totalglutathione in thesamplesolutionusinga

GSHcalibrationcurve(Fig.3).

► CalculatingtheconcentrationofGSHGSH(conc.)=totalGlutathione(conc.)-2xGSSG(conc.)


Fig. 2 Determination of the concentration of GSSG

Fig. 3 Determination of the concentration of total glutathione

0510152025ConcentrationofGSSG(μmol/l)

2.0

1.5

1.0

0.5

0

Absorbanceat405nm

●

●●●●

●

●

01020304050ConcentrationofGSH(μmol/l)

2.0

1.5

1.0

0.5

0

Absorbanceat405nm ●

●

●●●●

●

Title Reference

DiurnalVariationofcadmium-inducedmortalityinmice N.Miura,andT.Hasegawa,et al., J. Toxicol. Sci.2012;37:191

1. Anti O

xidant D

etection2. D

NA D

amage

Detection

3. Lipid Peroxide D

etection4. R

adical Detection

6. NO

Donor

5. Nitric O

xide D

etection7. A

GEs R

esearch


8

1. Purification of genomic DNASeveraldifferentmethodsandproductsareavailablefortheisolationofgenomicDNAfromsamplessuchasmembranebindingmethod,guanidine/detergentlysismethod,andpolyelectrolyteprecipitationmethod.Amongthesemethods,theguanidine/detergentlysismethodissimple,anditgiveshighlypurifiedgenomicDNAfortheARP-basedabasicsitesdetection.Duringthepurificationprocess,avoidheatingoftheDNAsolution.DeterminetheconcentrationandpurityofthepurifiedgenomicDNAusingthespectrophotometer*andagarosegelelectrophoresis.DissolvethegenomicDNAinTEattheconcentrationof100μg/ml.ItisimportantforanaccurateassaythattheDNAconcentrationisadjustedexactlyto100μg/ml.

*1OD260nm=50μg/ml.TheratioofOD260nm/OD280nmofhighlypurifiedDNAsolutionis1.8orhigher.Proteincontaminationinthesamplesolutionmaycauseapositiveerror.

2. General Protocol► ARPreaction1. Mix10µlofpurifiedgenomicDNAsolution(100µg/ml)and10µlofARP

Solutionina0.5mltube,andincubateat37°Cfor1hour.2. WashtheinsideoftheFiltrationTubewith100µlofTEtwice.3. Add380µlofTEtothereactionsolution,andtransferthesolutiontothe

FiltrationTube.4. CentrifugetheFiltrationTubeat2,500xgfor15minutes,anddiscardthe

filtratesolution.5. Add400µlofTEtotheFiltrationTubeandresuspendtheDNAonthefilter

withapipette.6. CentrifugetheFiltrationTubeat2,500xgfor15minutes.a)7. Add200µlofTEtotheFiltrationTubetoresuspendtheDNAonthefilterwith

apipette.8. TransfertheDNAsolutiontothe1.5mltube,andadd200µlofTEagainto

theFiltrationTubetotransfertheARP-labeledDNAonthefiltercompletelytothe1.5mltube.b)

9. StoretheARP-labeledgenomicDNAsolutionat0to5°C.a) IftheDNAsolutionstillremainsonthefilterafterthecentrifugation,spinforanother

5minutes.b) RecoveryrateofDNAusingthefiltrationtubeis90%,sotheconcentrationofthe

ARP-labeledDNAis2.25µg/ml.FormoreaccuratedeterminationofthenumberofabasicsitesinthesampleDNA,werecommendmeasuringtheDNAconcentration.

Contents of the Kit5samplesARPSolution(10mMARP) 100μlx1ARP-DNAStandardSoln.* 250μlea.(0,2.5,5,10,20,40APsites/100,000bp)DNABindingSolution 10mlx1SubstrateSolution 10mlx1TEBuffer 15mlx1HRP-Streptavidin 25μlx1WashingBuffer 1packFiltrationTube 5tubes96-wellMicroplate/Ubottom 1plate

20samplesARPSolution(10mMARP) 250μlx1ARP-DNAStandardSoln.* 250μlea.(0,2.5,5,10,20,40APsites/100,000bp)DNABindingSolution 10mlx1SubstrateSolution 10mlx1TEBuffer 40mlx1HRP-Streptavidin 25μlx1WashingBuffer 1packFiltrationTube 20tubes96-wellMicroplate/Ubottom 1plate

Required Equipment & MaterialsMicroplateReader(650nmfilter)10μl,100-200μland1mlpipettes50-250μlmulti-channelpipettesIncubator(37°C)0.5mland1.5mltubeCentrifugePaperTowel

Fig. 1 Reaction of AP site with ARP

QuantificationofdamagedbaseingenomicDNA

DNA Damage Quantification Kit-AP Site Counting- Product Code: DK02

OxidativedamagetoDNAisaresultofitsinteractionwithreactiveoxygenspecies(ROS), in particular, thehydroxy radical.Hydroxy radicals,whichareproduced

fromsuperoxideanionandhydrogenperoxidebytheFentonreaction,producemultiplemodificationsinDNA.OxidativeattacksbyhydroxyradicalsonthedeoxyribosemoietywillleadtothereleaseoffreebasesfromDNA,generatingstrandbreakswithvarioussugarmodificationsandsimpleabasicsites(APsites).Infact,APsitesareoneofthemajortypesofDNAdamagegeneratedbyROS.AldehydeReactiveProbe (ARP;N’-aminooxymethylcarbonylhydrazin-D-biotin) reactsspecificallywithanaldehydegrouppresentontheopenringformoftheAPsites(Fig.1).ThisreactionmakesitpossibletodetectDNAmodificationsthatresultintheformationofanaldehydegroup.AftertreatmentwithexcessARPreagent,alloftheAPsitesonDNAaretaggedwithabiotinresidue.Thesebiotin-taggedAPsitescanbequantifiedusingtheavidin-biotinassay,followedbycolorimetricdetectionwitheitherperoxidaseor alkalinephosphatase conjugated to theavidin.DNADamageQuantificationKitcontainsallthenecessarysolutionsfordetectingbetween1to40APsitesper1x105basepairs.

1. A

nti O

xida

nt

Det

ectio

n2.

DN

A D

amag

e D

etec

tion

3. L

ipid

Per

oxid

e D

etec

tion

4. R

adic

al D

etec

tion

6. N

O D

onor

5. N

itric

Oxi

de

Det

ectio

n7.

AG

Es R

esea

rch


9

► DeterminationofthenumberofAPsiteinDNADay1

1. Dilute90µloftheARP-labeledgenomicDNAwith310µlofTE.2. Add60µlofARP-DNAStandardSolutionperwell.Usethreewellsper1

standardsolution.3. Add60µlofthedilutedARP-labeledgenomicDNAsolutionperwell.Useat

leastthreewellsper1sample.4. Add100µloftheDNABindingSolutiontoeachwell,thenallowtheplateto

remainatroomtemperatureovernight.Day2

1. Preparestocksolutions» WashingBuffer:DissolvethecontentsoftheWashingBufferpacketin

1Lofdeionizedordistilledwater.StorethisWashingBuffersolutionatroomtemperature.

» HRP-Streptavidinsolution:DiluteHRP-StreptavidinwithWashingBuffertoprepare1/4000dilutedworkingsolution.*

» 1/4000dilutionpreparation:CentrifugeHRP-Streptavidintubefor30seconds.Add10µlofHRP-Streptavidininto40mlofWashingBuffersolution,andmixwell.

*Sincethisworkingsolutionisnotstable,alwaysusefreshlypreparedsolution.

2. DiscardtheDNABindingSolutioninthewells,andwashthewellwith250µlWashingBuffer5times.

3. Add150µlofdilutedHRP-Streptavidinsolutiontoeachwell,andincubatetheplateat37°Cfor1hour.

4. Discardthesolutioninthewell,andwashthewellwith250µlWashingBuffer5times.b)

5. Add100µlofSubstrateSolutiontoeachwell,andincubateat37°Cfor1hour.6. MeasuretheO.D.at650nmwithin1houraftertheincubationisfinished,and

prepareacalibrationcurveusingthedataobtainedwithARP-DNAStandardsolutions.

7. DeterminethenumberofabasicsitesinthegenomicDNAusingthecalibrationcurve.

3. Notes1. Pleasestorethekitat0-5°C.Donotfreeze.StoreWashingBuffersolutionat

roomtemperature.2. AP-DNAisnotstable.PleasetreatitwithARPandpurifywithFiltrationTube

aftertheisolationofgenomicDNAfromasample.3. PurifiedARP-DNAsolutioninTEbufferisstableoveroneyearat0-5°C

storage.4. AfterthespinningofFiltrationTubeforARP-labeledDNApurification,add200

µlTEimmediately.IftheDNAstaysinFiltrationTubeformorethan30minutesafterthespinning,theDNArecoveryratiomaydecline.

5. γ-Ray-sterilizedtubesmaycauseDNAbindingonthesurfaceofthetubeduringthemixingoftheDNAsolutionwithDNABindingSolution.IfyouprefertomixARP-DNAsolutionwithDNABindingSolutioninatuberatherthanmixingtheminawell,pleaseavoidusingg-ray-sterilizedtubes.

6. Ifthe650nmfilterisnotavailableforthemeasurementofO.D.afterthecolordevelopment,transfer50µlofthesolutionineachwelltoawellofanewplate(notprovided).Then,add50µlof1Msulfuricacid,andmeasuretheO.D.at450nm.

7. Remainingsolutioninawellmaycauseerror,sopleaseremovethesolutionthoroughlybytappingtheplateonapapertowelineachstep.

4. References1. T.Lindahl,et al.,RateofDepurinationofNativeDeoxyribonucleicAcid.Biochemistry.1972;11:3610-3618.2. M.Liuzzi,et al.,ANewApproachtotheStudyoftheBase-excisionRepairPathwayUsingMethoxyamine.J Biol Chem.

1985;260:5252-5258.3. A.Sancar,et al.,DNARepairEnzymes.Annu Rev Biochem.1988;57:29-67.4. M.Weinfeld,et al.,ResponseofPhageT4PolynucleotideKinaseTowardDinucleotidesContainingApurinicSites:Design

ofa32P-postlabelingAssayforApurinicSitesinDNA.Biochemistry.1990;29:1737-1743.5. B.X.Chen,et al.,PropertiesofaMonoclonalAntibodyfortheDetectionofAbasicSites,aCommonDNALesion.Mutat

Res.1992;273:253-261.6. J.A.Gralnick,et al.,TheYggXProteinofSalmonellaentericaIsInvolovedinFe(II)TraffickingandMinimizestheDNA

DamageCausebyHydroxylRadicals:ResidueCYS-7isEssentialforYggXFunction.J Biol Chem.2003;278:20708-20715.


Fig. 3 Typical calibration curve of DNA Damage Quantification Kit

1. Anti O

xidant D

etection2. D

NA D

amage

Detection

3. Lipid Peroxide D

etection4. R

adical Detection

6. NO

Donor

5. Nitric O

xide D

etection7. A

GEs R

esearch

010203040NumberofAPsites/100,000bp

1.0

0.8

0.6

0.4

0.2

0

Absorbanceat650nm

●

●

●

●●●

4)

1) 2)

3)

8)

5) 6)

7)


10

Title Reference

NovelRoleofBaseExcisionRepairinMediatingCisplatinCytotoxicity A.Kothandapani,andS.M.Patrick,et al.,J. Biol. Chem.2011;286:14564-14574

Cadmiumaffectsmetabolicresponsestoprolongedanoxiaandreoxygenationineasternoysters(Crassostreavirginica)

I.O.Kurochkin,andI.M.Sokolova,et al.,Am J Physiol Regulatory Integrative Comp Physiol.2009;297:R1262-R1272

ImpairmentofAPE1FunctionEnhancesCellularSensitivitytoClinicallyRelevantAlkylatorsandAntimetabolites

DanielR.,andD.M.Wilson,et al., IIIMol. Cancer Res.2009;7:897-906

TemporaryPretreatmentWiththeAngiotensinIIType1ReceptorBlocker,Valsartan,PreventsIschemicBrainDamageThroughanIncreaseinCapillaryDensity

Jian-MeiLi,andM.Horiuchi,et al.,Stroke.2008;39:2029-2036

Bcl2InhibitsAbasicSiteRepairbyDown-regulatingAPE1EndonucleaseActivity J.Zhao,andX.Deng,et al., J. Biol. Chem.2008;283:9925-9932

CockaynesyndromeBproteinstimulatesapurinicendonuclease1activityandprotectsagainstagentsthatintroducebaseexcisionrepairintermediates

H.Wong,andD.M.Wilson,et al., IIINucleic Acids Res.2007;35:4103-4113

AngiotensinII-InducedNeuralDifferentiationviaAngiotensinIIType2(AT2)Receptor-MMS2CascadeInvolvingInteractionbetweenAT2Receptor-InteractingProteinandSrcHomology2Domain-ContainingProtein-TyrosinePhosphatase1

Jian-MeiLi,andM.Horiuchi,et al., Mol. Endocrinol.2007;21:499-511

ADominant-NegativeFormoftheMajorHumanAbasicEndonucleaseEnhancesCellularSensitivitytoLaboratoryandClinicalDNA-DamagingAgents

DanielR.McNeillandDavidM.Wilson,IIIMol. Cancer Res.2007;5:61-70

Bcl2SuppressesDNARepairbyEnhancingc-MycTranscriptionalActivity Z.Jin,andX.Deng,et al., J. Biol. Chem.2006;281:14446-14456

FolateDeficiencyIncreasesPostischemicBrainInjury M.Endres,andK.Gertz,et al., Stroke.2005;36:321-325

6. FAQ►CanIusesingle-strandedDNAorRNA?

No,youcannotusethiskit todeterminethenumberofabasicsitesinsingle-strandedDNAorRNA.TheO.D.readingofsingle-strandedDNAwillbenearlytwicethatofdouble-strandedDNAbecauseofthebindingefficiencyonthemicroplate.

►HowshouldgenomicDNAbestored?PrepareaDNApelletandstoreat-20°Cor-80°CiftheDNAcannotbelabeledwithARPimmediatelyafterisolation.AfterARPlabeling,thesamplecanbestoredat4°CinTEBufferforseveralmonths.

►HowshouldIpreparetheDNA?YoucanusegeneralprotocolsorcommerciallyavailableDNAisolationkits.Between2to4abasicsitesper1x105basepairswillbecreatedduringtheDNAisolationprocess.Therefore,usethesameisolationmethodtoprepareeachDNAsample.

►HowcanIdeterminethenumberofabasicsitesiftherearemorethan40per1x105basepairs?Simplydilute theARP-labeledsampleDNAwith0.5μgpermldouble-strandedgenomicDNA,suchascalf thymusorsalmonspermDNA,usingTEBuffer.

►WhatshouldIdoifthesampleDNAconcentrationislessthan100μgperml?YoucaneitheruseafiltrationtubetoconcentrateyoursampleDNAorethanolprecipitationtorecoverDNAasapelletandthenre-dissolveittopreparea100μgpermlsolution.

►WhatshouldIdoifthesampleDNAislessthan1μg?AddthesamevolumeofARPSolutionandfollowthemanual.TherecoveryoftheARP-labeledDNAmaybelowerthantheusualreactions,someasuretheARP-labeledDNAsolution.Theaveragerecoveryrateofthe0.5μgDNAand0.25μgDNAis70%and50%,respectively.


1. A

nti O

xida

nt

Det

ectio

n2.

DN

A D

amag

e D

etec

tion

3. L

ipid

Per

oxid

e D

etec

tion

4. R

adic

al D

etec

tion

6. N

O D

onor

5. N

itric

Oxi

de

Det

ectio

n7.

AG

Es R

esea

rch


11

Fig. 1 Structure of 8-Nitroguanosine

8-Nitroguanine is a nitratedbaseofDNAandRNA. It is formedbyperoxynitrite,which is generated fromnitric oxideand superoxide anion radical. It is known

that a largeamount of nitric oxidemoleculesand superoxideanion, generatedbyinflammation, causesnitrationof guanosine.Since chemicallymodifiednucleotidescausemutationduringDNA replication, 8-Nitroguanine is thought to beamarker ofDNAdamagerelatedtomutationandcancer.Anti-NitroguanosineantibodieshavebeendevelopedjointlybyDr.AkaikeatKumamotoUniversityandDojindoLaboratories.

stronglyreact(10μmol/l)8-NO2-guanosine 8-NO2-guanine 8-NO2-cGMP 8-NO2-Xanthine

slightlyreact(>1mmol/l)8-Br-guanosine 8-Br-guanine 8-Br-cGMP 8-Cl-guanine

noreactionguanosine guanine 8-OH-guaninecytosine xanthine adenineadenosine thymine deoxythymidineuracil uridine 3-NO2-tyrosine

2-NO2-imidazole 8-OH-deoxyguanosineFig. 2 Reactivity of Anti-Nitroguanosine

monoclonal antibodyNO2G52(IC50)

Anti-Nitroguanosinepolyclonal antibodyalso recognizes8-nitroguanineand8-nitroguanosine,but it doesnot cross-reactwithnormal guanosine, guanine,

8-hydroxyguanine,or3-nitrotyrosine.Sincethisantibodywaspreparedusingrabbits,itcanbeusedforimmuno-histostainingofrodenttissuessuchasmiceorrats.

NitratedbaseofDNAandRNAdetection

Anti-Nitroguanosine Antibodies


Anti-Nitroguanosine monoclonal antibody (Clone# NO2G52) Product Code: AB02

Becauseof its veryhigh specificity,monoclonal antibodyNO2G52 recognizes8-nitroguanineand8-nitroguanosine,but it doesnot cross-reactwithnormal

nucleotidebases, 8-hydroxyguanine8-hydroxydeoxy-guanosine, 3-nitrotyrosine,xanthine,or2-nitroimidazole.The specificity ofNO2G52wasdeterminedby a competitiveELISAusing an8-nitroguanosine-BSA-coatedplate.As shown in the figuresbelow,NO2G52has veryhighaffinity for 8-nitroguanineand8-nitroguanosine, and it slightly cross-reactswith8-bromoguanosine,8-bromoguanine,and8-chloroguanine.

1. Specification► Species:mouse(BALB/c)► Clonality:monoclonal► Isotype:IgG1► Concentration:1mg/mlinPBS,contains0.1%ProClinasapreservative


Anti-Nitroguanosine polyclonal antibody Product Code: AB01

stronglyreact(10μmol/l)8-NO2-guanosine 8-NO2-guanine

noreactionguanosine guanine 8-OH-guanine

3-NO2-tyrosine

Table 1 Reactivity of monoclonal antibody

Table 2 Reactivity of polyclonal antibody

Fig. 3 Reactivity of Anti-Nitroguanosine polyclonal antibody

10-210-1110102

conc.(μmol/l)

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0

Absorbanceat490nm ● ● ●

●

●

●

●●

▲ ▲ ▲

▲

▲

▲

▲▲

■

■

■

■

■

■■ ■

♦ ♦ ♦♦

♦

♦

♦

♦

○

○ ○ ○ ○

○

○

○

□ □ □ □ □ □ □ □◊

◊ ◊ ◊ ◊ ◊ ◊◊X X X X X X X X* * *

* * * * *

▼

▼

▼

▼▼▼▼▼

■

NO2-Guanine Br-Guanosine

NO2-cGMP Guanosine

NO2-Xanthine Xanthine

NO2-Guanosine cGMP

Br-cGMP Control

●▲

♦○

▼

□◊

*X

10-310-210-1110102103

conc.(μmol/l)

3.0

2.5

2.0

1.5

1.0

0.5

0

Absorbanceat490nm

▲▲▲▲▲

▲▲

▲▲▲▲▲▲

▲▲ ■■■■■■■■■

■■■■■■

●●

●

●

●●●

●●

●

●

●●

●

● NO2-GuanineNO2-GuanosineGuanineGuanosineOH-GuanineNO2-Tyrosine

●▲

■▲

■

1. Anti O

xidant D

etection2. D

NA D

amage

Detection

3. Lipid Peroxide D

etection4. R

adical Detection

6. NO

Donor

5. Nitric O

xide D

etection7. A

GEs R

esearch

Type:IgGUnit:50μg

Type:IgGUnit:50μg


12

8-Nitroguanine(lyophilized)C5H4N6O3=196.12Unit:100μg

Fig. 4 Tissue staining with Anti-Nitroguanosine antibody

8-Nitroguanine(lyophilized) ismadebythe lyophilizationof itsphosphatebufferedsalinesolution,and isused in immunohistochemistry forabsorption testing.

Adding0.4mlofdistilledwatertothe8-Nitroguaninepowderproducesa1.2mmol/lof8-Nitroguaninesolution.8-Nitroguanine/PBSsolutionisstableforonemonthat4°C.Ifanantibodypre-treatedwithexcessive8-Nitroguanineshowsnegativestaining, thenthesubsequentpositivestainingwiththisantibodywillbespecificfor8-nitroguanineor8-nitroguanosineformedinDNAorRNA.

1. Example of Immunostaining(Fig. 4)► Sample

Influenzavirus-infectedmouselung► Immunostaining1. Fixthemouselungwith2%periodate-lysine-paraformaldehyde.2. Addanti-nitroguanosineantibody(10µg/ml)tothelungsample.3. Addalkalinephosphatase-conjugatedsecondaryantibody.4. StainthesamplewithVectorredsubstratekitI.

2. Notes1. Freezeandthawcyclescancausedegradationoftheantibody.Afteropening,

storeintherefrigerator.2. If8-Nitroguanosinestainingwasobservedwithpolyclonalantibody,itis

reccomendedtoconfirmtheexperimentalverificationasfollows,» nostainingisobservedbythecompetitionwith8-Nitroguaninestandard» nostainingisobservedbytreatingthesamplewithreducingagent,such

asSodiumhydrosulfite3. Monoclonalantibodycanbeusedonhumansamplesduetothehighactivity

andselectivity.

Standardagentof8-Nitroguaninedetection

8-Nitroguanine(lyophilized) Product Code: N455

3. Specification► Species:mouse(BALB/c)► Clonality:monoclonal► Isotype:IgG1► Concentration:1mg/mlinPBS,contains0.1%ProClinasapreservative

4. References1. T.Akaike,et al.,8-nitroguanosineformationinviralpneumoniaanditsimplicationforpathogenesis,PNAS.2003;100:685-

690.2. J.Yoshitake,et al.,NitricoxideasanendogenousmutagenforSendaiviruswithoutantiviralactivity.J Virol.2004;78:8709-

8719.3. T.Sawa,et al.,ProteinS-guanylationbythebiologicalsignal8-nitroguanosine3’,5’-cyclicmonophosphate.Nat Chem Biol.

2007;3:727-735.4. M.H.Zaki,et al.,Cytoprotectivefunctionofhemeoxygenase1inducedbyanitratedcyclicnucleotideformedduring

murinesalmonellosis.J Immunol.2009;182:3746-3756.5. Y.Terasaki,et al.,Guaninenitrationinidiopathicpulmonaryfibrosisanditsimplicationforcarcinogenesis.Am J Respir Crit

Care Med.2006;174:665-673.6. T.Sawa,et al.,Analysisofurinary8-nitroguanine,amarkerofnitrativenucleicaciddamage,byhigh-performanceliquid

chromatography-electrochemicaldetectioncoupledwithimmunoaffinitypurification:associationwithcigarettesmoking.Free Radic Biol Med.2006;40:711-720.

Fig. 5 Detection of guanine nitration on RAW264.7 cell

non-stimulation

LPS + INF-γ

+ L-NMMA

1. A

nti O

xida

nt

Det

ectio

n2.

DN

A D

amag

e D

etec

tion

3. L

ipid

Per

oxid

e D

etec

tion

4. R

adic

al D

etec

tion

6. N

O D

onor

5. N

itric

Oxi

de

Det

ectio

n7.

AG

Es R

esea

rch


13

Liperfluo,aperylenederivative containingoligooxyethylene, isdesignedandexclusivelydevelopedbyDojindoforadetectionoflipidperoxidesandemitsintense

fluorescencebyalipidperoxidespecificoxidationinorganicsolventssuchasethanol.AmongfluorescentprobesthatdetectReactiveOxigenSpecies(ROS),Liperfluoistheonlycompoundthatcanspecificallydetect lipidperoxides.Since theexcitationandemissionwavelengthsoftheoxidizedLiperfluoare524nmand535nm,respectively,bothaphoto-damageagainstasampleandanauto-fluorescencefromthesamplecanbeminimized.Thetetraethyleneglycolgroup linkedtooneendofdiisoquinolineringhelpsitssolubilityanddispersibilitytoaqueousbuffer.ThoughLiperfluooxidizedformisalmostnonfluorescentinanaqueousmedia,itemitsfluorescenceinlipophilicsitessuchasincellmembranes.Thereforeitcaneasilybeappliedtolipidperoxideimagingbyafluorescencemicroscopyandaflowcytometricanalysisforlivingcells.

LiperfluoN-(4-Diphenylphosphinophenyl)-N'-(3,6,9,12-tetraoxatridecyl)perylene-3,4,9,10-tetracarboxydiimideC51H41N2O8P=840.85,Unit:50μgx5

Detectionoflipidperoxidebyfluorescentmicroscopyandflowcytometry

Liperfluo Product Code: L248

Fig. 1 Reaction of Liperfluo with lipid peroxide

80

60

40

20

0

Fluorescenceintensity/A.U

.

400 450 500 550 600 650Wavelength(nm)

Excitation Emission

withoutlipidperoxide

withlipidperoxide

Fig. 3 Reaction selectivity of Liperfluo against the various reactive oxygen species.

15

12

9

6

3

0

F/F 0

1 2 3 4 5 6 7 8

1. Blank2. H2O2

3. •OH4. O2•-

5. NO6. ONOO-

7. ROO•

8. MeLOOH

ROS

Fig. 2 Excitation and emission spectra of Liperfluo with or without lipid per-oxide in ethanol.

1. General Protocol1.Add60μlofDMSOtoavialcontainingLiperfluo(50μg)anddissolvetheproduct(concentration:1mmol/l).-Ifitishardtodissolveitbypipetting,usevortexmixer,sonicatororwarmthesolution.-Coverthesolutionwithanaluminumfoilanduseitwithinadayafterthereconstition.

2.Add10μloftheLiperfluosolutionto1mlofcellculturecontaining1.0x105cells.-HigherfinalconcentrationofDMSOmaycausedamagetocells.-Sincethebackgroundfluorescencemaybeincreasedinculturemedium,replacingthemediumwithanaqueousbuffersuchasPBSisrecommendedbeforetheadditionofLiperfluosolution.

3.Incubatethecellsuspensionat37°Cfor30minutes.4.Analysethecellswithafluorescencemicroscopeoraflowcytometer.-AlthoughtheLiperfluooxidizedformisalmostnon-fluorescentinanaqueoussolution,washthecellswithPBSasnecessaryifthebackgroundfluorescenceishigh.

Ex: 524 nmEm: 535 nm

Liperfluooxidizedformfluorescenceunderlipophiliccondition

2. Live cell imaging of lipid peroxide (Fig. 4)1.InnocurateSH-SY5Ycells(6.0x105cells/well)toa6-wellplate.2.Incubatetheplateat37ºCforovernight.3.AddLiperfluo,DMSOsolution(finalconc.20μM)andincubateat37ºCfor15minutes.

4.AddeitherCumeneHydroperoxide(finalconc.100μM)orAIPH*(finalconc.6mM).5.Incubateat37ºCfor2hours.6.Observefluorescentbymicroscope**(Ex.524nm,Em.535nm).

*AIPH:2,2’-azobis-[2-(2-imidazolin-2-yl)propane]dihydrochloride

**OlympusIX-71epifluorescentmicroscope,mirrorunit:U-MNIBA3,exposuretime:10sec,ISO:800

Fluorescentimage Bright-fieldimage

Fig. 4 Reaction selectivity of Liperfluo against the various reactive oxygen species.

3. References1. K.Yamanaka,et al.,Anovelfluorescentprobewithhighsensitivityandselectivedetectionoflipidhydroperoxidesincells,

RSCAdvances,2012,2,7894.2. N.Soh,et al.,Novelfluorescentprobefordetectinghydroperoxideswithstrongemissioninthevisiblerange.Bioorg Med

Chem Lett.2006;16:2943-2946.3. N.Soh,et al.,Swallow-tailedperylenederivative:anew tool forfluorescent imagingof lipidhydroperoxides.Org Biomol

Chem.2007;5:3762-3768.

4. Specification► Appearance:reddishblackcrystallinepowder► Purity:≥90.0%(HPLC)

DatawaskindlyprovidedfromDr.N.Noguchi,Doshi-shaUniversity,SystemLifeScienceLaboratory.

1. Anti O

xidant D

etection2. D

NA D

amage

Detection

3. Lipid Peroxide D

etection4. R

adical Detection

6. NO

Donor

5. Nitric O

xide D

etection7. A

GEs R

esearch


14

Lipidperoxidesarederivedfromunsaturated lipids,phospholipids,glycolipidsandcholesterolesters.Infoodindustry,lipidperoxideshavebeenconsideredasoneof

amajorcauseoffooddeterioration.Meanwhile,therearemanyongoingstudiestodaythat investigate themechanismof lipidoxidation inhumandiseases,disorders,andaging.Hence,measuringamountof lipidperoxideinbiologicalsamplesissignificant,andanaccuratemethodfordetectinglowleveloflipidperoxidesiseagerlyanticipated.TBARS(Thiobarbituricacidreactivesubstances)assay isawell-establishedmethodandwidelyusedformeasuring lipidperoxidation.However,MDA(Malondialdehyde),oneofaend-productgeneratedinthelipidperoxidation,isnotreflectedinactuallevelofperoxidationbecausethereareothersourceofMDA.DPPP,Diphenyl-1-pyrenylphosphine,wasdevelopedbyDr.Meguro,et al. asafluorescentprobefordetectinglipidperoxide.ItselectivelyreactswithhydroperoxidestogenerateDPPPoxidethatemitsfluorescenceat380nm(ex:352nm),andmakesitpossible toquantify0.1 to7nmolofhydroperoxide. Inaddition, therangeof1 to2pmolof lipidperoxidecanbeselectivelydetectedwith thecombinationofHPLCseparationandthepost-columnreactionwithDPPP.

DPPPDiphenyl-1-pyrenylphosphineC28H19P=386.42CASNo.[110954-36-4]Unit:10mg

1. General Protocol► HPLCdetectionofhydroperoxidesinplasmasample1. Prepare10mg/mlBHT(Butylhydroxytoluene)/CHCl3-Methanol(2:1)solution.2. Dissolveasamplein100µlofthesolutionabove.3. Add50µlofDPPPsolution(1mg/10mlinCHCl3:Methanol=1:1)tothesolution

preparedinstep2.4. Incubatethesolutionunderdarkconditionfor60minutesat60°C.5. CooldownthesolutionandmeasurethefluorescenceintensitybyHPLC.

► Determinationofhydroperoxidesoncellmembrane(in vivo)1. DisolveDPPPinDMSOandprepare5mMDPPP/DMSOsolution.2. Add thesolutionabove to cell suspension(1 x107 cells/ml) to the final

concentrationof50μMDPPP.3. Incubatethecellsat37°Cfor10minutes.4. WashthecellstwicewithHank'ssolution.5. Stimulate thecellsbyaddingH2O2orMethyl linoleatehydroperoxideand

measureflueorescenceintensity.

2. References1. K.Akasaka,et al.,AnAromaticPhosphineReagentfortheHPLC-fluorescenceDeterminationofHydroperoxides-Determi-

nationofPhosphatidylcholineHydroperoxidesinHumanPlasma.Anal Lett.1988;21:965-975.2. K.Akasaka,et al.,Normal-phaseHigh-performanceLiquidChromatograohywithaFluorimetricPostcolumnDetection

SystemforLipidHydroperoxides.J Chromatogr A.1993;628:31-35.3. Y.Okimoto,et al.,ANovelFluoresceintProbeDiphenyl-pyrenylphosphinetoFollowLipidPeroxidationinCellMembranes.

FEBS Lett.2000;474:137-140.

3. Specification► Appearance:slightlyyellowpowder► Purity:≥97.0%(HPLC)


DetectionoflipidperoxidebyHPLC

DPPP Product Code: D350

Fig. 1 Reaction scheme of DPPP with hydroperoxide

Title ReferenceDysregulationofverylongchainacyl-CoAdehydrogenasecoupledwithlipidperoxidation Y.Kabuyama,et al., Am J Physiol Cell Physiol.2010;298:

C107-C113.

CellDeathCausedbySeleniumDeficiencyandProtectiveEffectofAntioxidants Y.Saito,et al., J. Biol. Chem.2003;278:39428-39434.

Inductionof1-cysperoxiredoxinexpressionbyoxidativestressinlungepithelialcells Han-SukKim,et al., Am J Physiol Lung Cell Mol Physiol.2003;285:L363-L369.

AnAntisenseOligonucleotide to1-cysPeroxiredoxinCausesLipidPeroxidationandApoptosisinLungEpithelialCells

JhangHoPak,et al., J. Biol. Chem.2002;277:49927 -49934.

1-Cysperoxiredoxinoverexpressionprotectscellsagainstphospholipidperoxidation-mediatedmembranedamage YefimManevich,et al., PNAS.2002;99:11599-11604.

1. A

nti O

xida

nt

Det

ectio

n2.

DN

A D

amag

e D

etec

tion

3. L

ipid

Per

oxid

e D

etec

tion

4. R

adic

al D

etec

tion

6. N

O D

onor

5. N

itric

Oxi

de

Det

ectio

n7.

AG

Es R

esea

rch

L=Lipid


15

DetectionofproteinorDNAradicalbyWB,ELISA,andimaging

DMPO Product Code: D048

Immuno-spinTrappingmethodwasdevelopedfordetectingDNAandProteinradicalsinbiologicalanalysis.ROS(ReactiveOxygenSpecies)producesmodificationofthestructureandfunctionofbiomoleculesthatrelateonthecauseofvarietydiseases.Tounderstandthemechanismofoxidativereactions,itisveryimportanttoanalyzewhichmoleculesareinvolvedintheoxidationprocess.DMPOisthemostpopularspin-trappingreagentthattrapsradicalsinproteinandDNAsamples.TheDMPO-ProteinorDMPO-DNAnitroneadductsaredeterminedusingaELISA,WesternBlotting,MassSpectorometry,Imaging,andsoon.ThepurityofDojindo’sDMPOishigherthananothercommercializedDMPO.Sinceitdoesnotcontainimpuritiesthatmightcausehighbackground.Dojindo’sDMPOdoesnotrequireanypre-purificationsteps.

DMPO5,5-Dimethyl-1-pyrrolineN-oxideC6H11NO=113.16CASNo.[3317-61-1]Unit:1ml

Fig. 1 Principle of Immuno-spin Trapping Method

1. Protocol Example : Radical DNA DetectionReferred PublicationDetectionandimagingofthefreeradicalDNAincells--site-specificradicalformationinducedbyFentonchemistryanditsrepairincellularDNAasseenbyelectronspinresonance,immuno-spintrappingandconfocalmicroscopy.BhattacharjeeS,ChatterjeeS,JiangJ,SinhaBK,MasonRP.,Nucleic Acids Res. 2012,40,5477-86

► EvaluationofradicalDNAbyELISA1. TreateRAWcells(1-3x106cells)with100µMCuCl2,100µMH2O2,and100

mMDMPOandincubatefor12-15hoursat5%CO2incubator.2. ExtractDNAfromRAWcellsanddiluteDNAto5μg/mlinPBS.3. Add25μlofDNAsolutionand25μlofReacti-BindDNAcoatingsolutionineach

welloftheplateandincubatefor2–4hoursat37°C.4. Washthewellsoncewithwashingbuffer(PBScontaining0.05%non-fatdry

milkand0.1%Tween-20).5. Blockwithblockingbuffer(PBScontaining3%non-fatdrymilk)for2hoursat37°C.6. DetectDMPO-DNAradicaladductwithanti-DMPOantibodyandHRP-conjugated

secondaryantibody.7. Afterthreewashes,addtheImmobilonchemiluminescencesubstrateeachwell

andmeasuretheintensityofluminesscense.► Anotherapplicationinthispaper

CellImaging

2. Protocol Example : Radical Protein DetectionReferred PublicationSuperoxideinducesendothelialnitric-oxidesynthaseproteinthiylradicalformation,anovelmechanismregulatingeNOSfunctionandcoupling.ChenCA,LinCH,DruhanLJ,WangTY,ChenYR,ZweierJL.,J Biol Chem.2011,286,

► Evaluationofradicalproteinbycellimaging1. PrepareBovineaorticendothelialcells(104cells)in35-mmdishes.2. Add10μMMenadioneand50mMDMPOandincubatecells.3. WashthecellswithPBSandfixthemwith3.7%paraformaldehydefor10minutes.4. Permeabilizethecellswith0.25%TritonX-100inTBST(Trisbufferedsalinewith

Tween)for10minutesandblockthecellswith5%goatseruminTBST.5. VisualizeDMPO-proteinradicaladductwithanti-DMPOantibodyandfluorescein

labeledsecondaryantibody.6. Analyzeproteinradicalsbyfluorescentmicroscopy.► Otherapplicationsinthispaper

Immunoblotting,MassSpectrometry,Immunoprecipitation

3. Specification► Appearance:colorlessliquid► Purity:≥99.0%(GC)

Fig. 2 Purity comparison in HPLC spectra

(*:impurities)

Dojindo

supplierS.

supplierE.

Fig. 3 Radical Detection Scheme

Radical

DNAorProtein

anti-DMPOPrimaryAntibody

FluoresceinorEnzymeLabeledSecondaryAntibody

DNA,Protein

DNARadicalProteinRadicallifetime:μsec.tosec.

DMPO-DNARadicalAdductsDMPO-ProteinRadicalAdductslifetime:min.tohr.

DMPO-DNAnitroneAdductsDMPO-ProteinnitroneAdductslifetime:monthstoyear

Oxidants

DMPO

-e-,-H+

DetectionMethod

EPR

EPR-spinTrapping

Immuno-spinTrapping• ELISA• CellImaging• Immunoblotting• Massspectrometry

1. Anti O

xidant D

etection2. D

NA D

amage

Detection

3. Lipid Peroxide D

etection4. R

adical Detection

6. NO

Donor

5. Nitric O

xide D

etection7. A

GEs R

esearch► ESRspectrum:topasstest► IRspectrum:authentic


16

DetectionofradicalbyEPR

DMPO Product Code: D048

Becauseofpotentialcancerrisksandtheirage-promotingeffects,freeradicalsinliv-ingbodieshavebecomeafrequentlystudiedsubject.DMPOisthemostfrequent-

lyusedspin-trapping reagent for thestudyof free radicals. It issuitable for trappingoxygenradicals,especiallysuperoxides,andforproducingadductswithcharacteristicEPR(ESR)patterns.However,mostcommerciallyavailableDMPOcontainsimpuritiesthatcausehighbackgrounds.Thus,DMPOrequiresfurtherpurificationwhenrunningexperimentsonEPR.ThequalityofDojindo’sDMPOiswellcontrolledandDojindo’sDMPOdoesnotrequireanypre-purificationprocess.Therearenoimpuritiestocauseabackgroundproblem.

DMPO5,5-Dimethyl-1-pyrrolineN-oxideC6H11NO=113.16CASNo.[3317-61-1]Unit:1ml

Fig. 1 ESR Spectera of DMPO Adducts

1. Protocol► Evaluationofsuperoxidescavengingactivities1. Add15µlofDMPOand50µlof5mMhypoxanthineto35µlof0.1MPhosphate

buffer(pH7.8).2. Add50µlofSODstandardorsamplestobetestedandvoltexfor1-2seconds.3. Add50µlof0.4U/mlxanthineoxidaseandvolteximmediately.4. TransferthesolutiontoESRsampletubeandmeasureESRspectraaftercertain

timeofperiod,e.g.1minute.5. Calculaterelativeintensity(DMPO-O2

-/Mn2+)fromthepeakheight.

► C-,N-,andS-centeredradicalsDetection1.Prepareasolutionof100mMphosphatebuffer(pH7.4)containing25µMDTPA.2.Makeupasolutionofthefollowingperoxidasesubstrates:(A)100mMsodiumformate

(HCOONa);(B)100mMpotassiumcyanide(KCN);(C)100mMsodiumazide(NaN3);(D)100mMsodiumsulfite(Na2SO3)in100mMphosphatebuffer,pH7.4.

3.Makeupasolutionofhorseradishperoxidasewithconcentrationof4.0mg/ml(~100µM)and1mMsolutionofhydrogenperoxide(H2O2).

4.MakeupasolutionofDMPOwithconcentrationof1M.5.Prepareyourreactionmixturetoatotalreactionvolumeof200µlandadd130µlof

buffertoanEppendorftube.6.Add20µlDMPOofyour1MDMPOsolution,20µlofoneofthesubstrates’stock

solutions,10µlof1mMH2O2,andinitiatethereactionwith20µlHRP.7.Vortexthetube,transferthesolutiontoaflatcell,andacquirethespectrum.8.Thefinalconcentrationsofthecomponentsare:100mMDMPO,10mMsubstrate

(formate,cyanide,azide,sulfite),50µMH2O2,and10µMHRP. *ThisprotocolwaskindlyprovidedbyBrukerCorporation.

2. SpecificationRefertopreviouspage(Page15)


Fig. 2 Purity comparison in HPLC spectra

(*:impurities)

Dojindo

supplierS.

supplierE.

Dojindo

supplierS.

supplierE.

Fig. 3 Purity comparison of ESR spectra

(black:fentonreaction,blue:blank)

Title ReferenceNonenzymaticdisplacementofchlorineandformationoffreeradicalsuponthereactionofglutathionewithPCBquinones

YangSong,BrettA.,andGarryR.Buettner,et al., PNAS.2009;106:9725-9730.

ManganeseSuperoxideDismutaseModulatesHypoxia-InducibleFactor-1InductionviaSuperoxide SuwimolKaewpila,andLarryW.Oberley,et al., Cancer Res.2008;68:2781-2788.

Hyperglycemia-InducedReactiveOxygenSpeciesToxicity toEndothelialCells IsDependentonParacrineMediators

JuliaV.Busik,andMariaB.Grant,et al., Diabetes,2008;57:1952-1965.

OverexpressionofExtracellularSuperoxideDismutaseAttenuatesHeparanaseExpressionandInhibitsBreastCarcinomaCellGrowthandInvasion.

MelissaL.T.,andFrederickE.Domann,et al., Cancer Res.,2009;69:6355-6363

SmokingInducesBimodalDNADamageinMouseLung "ShunjiUenoandKyosukeTemma,Toxicol. Sci.2011;120:322-330."

CardiacMyocyte–SpecificExpressionofInducibleNitricOxideSynthaseProtectsAgainstIschemia/ReperfusionInjurybyPreventingMitochondrialPermeabilityTransition

MatthewB.andAruniBhatnagar,et al., Circulation.2008;118:1970-1978.

1. A

nti O

xida

nt

Det

ectio

n2.

DN

A D

amag

e D

etec

tion

3. L

ipid

Per

oxid

e D

etec

tion

4. R

adic

al D

etec

tion

6. N

O D

onor

5. N

itric

Oxi

de

Det

ectio

n7.

AG

Es R

esea

rch


17

DetectionofsuperoxideradicalbyEPR

BMPO Product Code: B568

Spintrappinganalysisisoneofthemostreliabletechniquesfordetectingandiden-tifying short-lived free radicals.TheEPR (ESR) spin trap reagent detects both

superoxideandhydroxyl radicals producedby systems in vitro and in vivo. BMPOwasdevelopedasaspintrappingreagentthatadductssuperoxideandshowsamuchlongerhalf-life(t1/2=24min)thanotherspintrapreagents.Itgivesusreproducibleandsteadyresults.BecauseBMPOishighlysolubleinwater,hydrophilicsampleisappli-cableforanalyzingthefreeradicals.

BMPO5-tert-Butoxycarbonyl-5-methyl-1-pyrrolineN-oxideC10H17NO3=199.25Unit:50mg

1. General Protocol► MeasuringhydroxyradicalfromFentonreaction1. Add15µlofBMPOsolution,75µlof1mMH2O2and75µlof100µMFeSO4to

50µlofddH2O.2. TransferthesolutiontoESRsampletubeandmeasureESRspectraaftercer-

taintimeofperiod,e.g.1minute.3. Calculaterelativeintensityfromthepeakheight.

► Measuringsuperoxideradicalfromxanthineoxidase(XO)reaction1. Dissolve1mgofBMPOwith1mlof50mMPhosphatebuffer(pH7.4)(solutionA).2. Prepare50mMPhosphatebuffer(pH7.4)containing1mMDTPAand0.4mM

Xanthine(solutionB).3. Prepare50mMPhosphatebuffer(pH7.4) containing0.1U/mlxanthine

oxidase(solutionC).4. Mix15µlofsolutionA,135µlofsolutionBand10µlofsolutionC.5. TransferthesolutiontoESRsampletubeandmeasureESRspectraaftercer-

taintimeofperiod,e.g.8minutes.6. Calculaterelativeintensityfromthepeakheight.

2. References1. H.Zhao,J.Joseph,H.Zhang,H.KarouiandB.Kalyanaraman,Free Radic Biol Med.2001;31:599-606.2. G.M.Rosen,P.Tsai,J.Weaver,S.Porasuphatana,L.J.Roman,A.A.Starkov,G.FiskumandS.Pou,J Biol Chem.

2002;277:40275-40280.

3. Specification► Appearance:whitecrystalorcrystallinepowder► Purity:≥99.0%(HPLC)

Fig. 1 ESR Spectra of hydroxy radical adduct(black:fentonreaction,blue:blank)

Fig. 2 ESR Spectra of superoxide radical adduct(black:XOreaction,blue:blank)

Fig. 3 Half-life of BMPO-superoxide radical adduct

1591317212529333739Times(minutes)

1700

1500

1300

1100

900

700

500

300

100

PeakHeight

t1/2=24min

1. Anti O

xidant D

etection2. D

NA D

amage

Detection

3. Lipid Peroxide D

etection4. R

adical Detection

6. NO

Donor

5. Nitric O

xide D

etection7. A

GEs R

esearch


18

2. References1. E.F.Ullman,et al.,StudiesofStableFreeRadicals.X.NitronylNitroxideMonoradicalsandBiradicalsasPossibleSmall

MoleculeSpinLabels.J Am Chem Soc.1972;94:7049-7059.2. Y.Miura,et al.,PolymersContainingStableFreeRaficals,5.PreparationofaPolymerContainingImidazoline3-Oxide

1-OxylGroups.Macromol Chem Phys.1973;172:233-236.3. K.Inoue,et al.,MagneticPropertiesoftheCrystalsofp-(1-Oxyl-3-Oxide-4,4,5,5-Tetramethyl-2-Imidazolin-2-Yl)Benzoic

acidandItsAlkaliMetalSalts.Chem Phys Lett.1993;207:551-555.4. T.Akaike,et al.,AntagonisticActionofImidazolineoxylN-OxidesAgainstEndothelium-DerivedRelaxingFactor/NOThrough

aRadicalReaction.Biochemistry.1993;32:827-832.5. J.Joseph,et al.,TrappingofNitricOxidebyNitronylNitroxides:anElectronSpinResonanceInvestigation.Biochem

Biophys Res Commun.1993;192:926-934.6. M.Yoshida,et al.,TherapeuticEffectsofImidazolineoxylN-OxideAgainstEndotoxinShockThroughItsDirectNitricOxide-

scavengingActivity.Biochem Biophys Res Commun.1994;202:923-930.7. T.Az-Ma,et al.,ReactionBetweenImidazolineoxilN-Oxide(Carboxy-PTIO)andNitricOxideReleasedfromCulturedEndo-

thelialCells:QuantitativeMeasurementofNitricOxidebyESRSpectrometry.Life Sci.1994;54:PL185-PL190.8. H.Maeda,et al.,MultipleFunctionsofNitricOxideinPathophysiologyandMicrobiology:AnalysisbyaNewNitricOxide

Scavenger.J Leukoc Biol.1994;56:588-592.

3. Specification► Appearance:darkbluepowder► Purity:≥97.0%(TLC)


Fig. 2 ESR Spectra of Carboxy-PTIO and Carboxy-PTI

Carboxy-PTIOisastable,water-solubleorganicradicalthatreactswithNOtoformNO2·Thisreactioncanbemonitoredbyelectronspinresonance(ESR).NOisan

unstablemoleculeandhasacomplexreactioncascadeforitsmetabolisminbiologicalsystems.Rapidly generatedNO-relatedmetabolites carryout variousphysiologicalactivities.Commonly usedNOscavengers suchashemoglobin trapNO; theyalsotrapNOS inhibitorssuchasargininederivatives.TheseNOscavengersalsoquenchallotherNO-relatedmetabolitesatthesametime.Incontrast,Carboxy-PTIOdoesnotdramaticallyaffectotherNO-relatedproductsystemsbecauseittransformsNOtoNO2,whichisametaboliteofNO.Thus,Carboxy-PTIOcanbeusedtoinvestigatetheeffectsofNOseparately from its downstreammetabolites.Dr.Akaikeandothers showedthatCarboxy-PTIOsuppresses relaxationof the rat aorta ring,which is inducedbyacetylcholine,twiceaseffectivelyasNG-nitroarginine.Dr.Yoshidaandothersreportedthat downstreammetabolites ofNO,generatedby treatmentwithCarboxy-PTIO,havean increasedantiviralactivitycompared toNOalone.TheNOmetabolitesplayimportantrolesinbiologicalsystems;therefore,theyshouldbeinvestigatedseparatelyfromNO.

Carboxy-PTIO2 - ( 4 -Ca rboxypheny l ) - 4 , 4 , 5 , 5 - t e t r ame thy -limidazoline-1-oxyl-3-oxide,sodiumsaltC14H16N2NaO4=299.28CASNo.[148819-93-6]Unit:10mg

NOScavenger,NOdetectionbyEPR

Carboxy-PTIO Product Code: C348

Fig. 1 Reaction of NO trapping

Carboxy-PTIO

Carboxy-PTI

Title Reference

EffectofHypoxiaonSusceptibilityofRGC-5CellstoNitricOxideTakakiSato andTsunehiko Ikeda,et al., Invest. Ophthalmol. Vis. Sci.2010;51:2575-2586.

UpregulationofNitricOxideProductioninVascularEndothelialCellsbyAll-transRetinoicAcidThroughthePhosphoinositide3-Kinase/AktPathway

Akira Uruno and Sadayoshi Ito, et al., Circulation.2005;112:727-736.

TheNitricOxide–cGMPPathwayControls theDirectionalPolarityofGrowthConeGuidanceviaModulatingCytosolicCa2+Signals

TakuroTojimaandHiroyukiKamiguchi,et al., J. Neurosci.2009;29:7886-7897.

NitricOxideTransientlyConvertsSynapticInhibitiontoExcitationinRetinalAmacrineCells BrianHoffpauirandEvannaGleason,et al., J Neurophysiol.2006;95:2866-2877.

1. A

nti O

xida

nt

Det

ectio

n2.

DN

A D

amag

e D

etec

tion

3. L

ipid

Per

oxid

e D

etec

tion

4. R

adic

al D

etec

tion

6. N

O D

onor

5. N

itric

Oxi

de

Det

ectio

n7.

AG

Es R

esea

rch


19

TheGriessassay isasimpleandpopularmethod fordetectingNOconcentration.2,3-Diaminonaphthalene (DAN) is a highly sensitivealternative to theGriess

assay.TheDANmethod is 50-100 timesmore sensitive than theGriessassay:While thedetection limit of theGriessassay is 1mM, the limit of theDANmethodis 10-50nM.DAN reactswithNO2

- in acidic conditions to produce fluorescentnaphthalenetriazole.Thewavelengthoftheemissionmaximumofnaphthalenetriazoleis410nm.However,detectionat450nmisrecommendedtoavoidfluorescentblanksand increase sensitivity.The fluorescent backgroundofDAN is low formaximumsensitivity.Theoptimal reactionconditionsofDANwithNO2

-havebeendetermined.The reaction shouldproceedat pH2at room temperature for 5minutes, and theresultingfluorescenceofnaphthalenetriazoleshouldbedeterminedatapHof10ormore.DANisaphotosensitivereagentandsometimesbecomesdarkbrowncoloredcrystals.Since this brownproduct cannot beutilized for the fluorescent detection,recrystallizationisnecessary.

2,3-Diaminonaphthalene(for NO detection)2,3-DiaminonaphthaleneC10H10N2=158.20CASNo.[771-97-1]Unit:10mg

DetectionofNO2-byfluorometricanalysis

2,3-Diaminonaphthalene (for NO detection) Product Code: D418

1. General Protocol1. Dissolve50µgDANin1ml0.62MHCltoprepare0.31mMDANsolution.a)2. Mix10µlDANsolutionwith100µlNaNO2solution(0-10mM)orsamplesolu-

tion.3. Incubatethemixtureatroomtemperaturefor10-15minutes.4. Add5µl2.8MNaOHsolutiontothereactionsolution.b)5. Dilute100µlofthissolutionwith4mlwater,followedbyfluorescentmeasure-

mentwithexcitationwavelengthat365nmandemissionwavelengthat450nm.

6. PrepareacalibrationcurveusingthisdatawheretheX-axisisNaNO2concen-trationandtheY-axisisfluorescenceintensity.ThenusethiscalibrationcurvetodeterminetheNO2concentrationofthesamplesolution.a) Acidicconditionsarerequiredforarapidreaction.b) Basic conditions (pH10or higher) are required for a high fluorescence

signal.

2. References1. W.R.Tracey,et al.,Comparisonofspectrophotometricandbiologicalassaysfornitricoxide(NO)andendothelium-

derivedrelaxing factor (EDRF):nonspecificityof thediazotizationreaction forNOand failure todetectEDRF.J Pharmacol Exp Ther.1990;252:922-928.

2. J.S.Pollock,et al.,Purificationandcharacterizationofparticulateendothelium-derivedrelaxingfactorsynthasefromculturedandnativebovineaorticendothelialcells.PNAS.1991;88:10480-10484.

3. S.Archer,Measurementofnitricoxideinbiologicalmodels.FASEB J.1993;7:349-360.4. J.H.Wiersma,2,3-DiaminonaphthaleneasaSpectrophotometricandFluorometricReagentfortheDeterminationof

NitriteIon.Anal Lett.1970;3:123-132.5. C.R.Sawicki,FluorimetricDeterminationofNitrate.Anal Lett.1971;4:761-775.6. P.Damiani,et al.,Fluorometricdeterminationofnitrite.Talanta.1986;8:649-652.7. T.P.Misko,et al.,A fluorometricassay for themeasurementofnitrite inbiological samples.Anal Biochem.

1993;214:11-16.8. G.L.Wheeler,etal.,Rapiddeterminationoftraceamountsofselenium(IV),nitrite,andnitratebyhigh-pressureliquid

chromatographyusing2,3-diaminonaphthalene.Microchem J.1974;19:390-405.

3. Specification► Appearance:whiteorpaleyellowish-brownpowder► MeltingPoint:185°Cto200°C

4. Recent PublicationsTitle Reference

RoleoftheSphingosineRheostatintheRegulationofCnidarian-DinoflagellateSymbioses OlivierDetournay,Biol. Bull.2011;221:261-269.

TAK-242(Resatorvid),aSmall-MoleculeInhibitorofToll-LikeReceptor(TLR)4Signaling,BindsSelectivelytoTLR4andInterfereswithInteractionsbetweenTLR4andItsAdaptorMolecules

N.Matsunaga,et al., Mol. Pharmacol.2011;79:34-41.

ANovelCyclohexeneDerivative,Ethyl (6R)-6-[N-(2-Chloro-4-fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate (TAK-242), Selectively InhibitsToll-LikeReceptor4-MediatedCytokineProductionthroughSuppressionofIntracellularSignaling

MIi,et al., Mol. Pharmacol.2006;69:1288-1295.

1. Anti O

xidant D

etection2. D

NA D

amage

Detection

3. Lipid Peroxide D

etection4. R

adical Detection

6. NO

Donor

5. Nitric O

xide D

etection7. A

GEs R

esearch


20

Diethyldithiocarbamate (DETC) is a good spin-trapping reagent for nitric oxide invivo.However,DETChasnotbeenwidelyutilized forNOdetection inbiological

samplesdue to its poorwater solubility.DTCS,ananalogofDETC, formsawater-solubleiron(II)complex(Fe-DTCS).TheFe-DTCScomplexthenformsacomplexwithNO(NO-Fe-DTCS).Dr.Yoshimurasuccessfullyobtainedtwo-dimensionalESRimagesofNO,inducedbylipopolysaccharideinmouseperitoneum.DTCSsodiumsalt(DTCSNa)wasusedforthisexperimentbecauseitislesstoxicthanammoniumsalt(sodiumsaltLD50:1942mg/kg;ammoniumsaltLD50:765mg/kg).SincetheFe-DTCScomplexismore stable than theother dithiocarbamate complexes in theair or in aqueoussolutions,itcouldbeausefulspintrappingreagentforbiochemicalresearch.TheFe-DTCScomplexshouldbeusedimmediatelyafterpreparation.AnexcessiveamountofDTCSNa(usually5equivalentsDTCSNatoFeSO4)isrequiredtomakeamorestablesolution.Dithiocarbamatestendtodecomposeunderphysiologicalconditionstoformtoxiccarbondisulfide.

DTCS NaN-(Dithiocarboxy)sarcosine,disodiumsalt,dihydrateC4H5NNa2O2S2•2H2O=245.23CASNo.[13442-87-0Unit:100mg,500mg

2. References1. T.Yoshimura,et al.,InvivoEPRDetectionandImagingofEndogenousNitricOxideinLipopolysaccharide-treatedMice.

Nat Biotechnol.1996;14:992-994.2. B.Kalyanaraman,DetectionofNitricOxidebyElectronSpinResonanceinChemical,Photochemical,Cellular,Physiologi-

cal,andPathophysiologicalSystems.Methods Enzymol.1996;268:168-187.3. H.Yokoyama,et al., InvivoESR-CTImagingoftheLiver inMiceReceivingSubcutenousInjectionofNitricOxide-Bound

IronComplex.Magn Reson Imaging.1997;15:249-253.

3. Specification► Appearance:whiteorpaleyellowpowder

DetectionofNObyEPR

DTCS Na Product Code: D465

1. General Protocol► PreparationofFe(II)-DTCSComplex1. Dissolve278mgFeSO4,7H2O(ferroussulfateheptahydrate)with20mlwa-

tera)toprepare50mMFeSO4solution.b)

2. Dissolve123mgDTCSNawith10mlwatera)toprepare50mMDTCSsolu-tion.

3. Mix1mlDTCSNasolutionwith8.8mlbuffersolutiona)(pH7orhigher).Add200µlFeSO4solutionjustpriortouse.

c)

a) Purgeanydissolvedoxygen in thewater or thebuffer bynitrogengasbubblingforatleast30minutespriortodissolvingFeSO4.

b) TheFeSO4solutioncanbestoredat-20°Cforatleast2months.c) Fe(II)-DTCScomplex is colorless. If the solution isbrown,Fe(III)-DTCS

mayhave formedbydissolvedoxygen in the solution.However, thebrownsolutioncanstillbeusedforNOtrapping.

► PreparationofNO-Fe(II)-DTCSComplex1. Underargongasflow,add200µlofFeSO4solutionto9.4mlbuffersolution(pH

7orhigher).ThenintroduceNOintothesolutionbybubblingNOgasthroughaglasscapillaryfor15minutes.

2. Add400mlofDTCSNasolutiontotheFeSO4solution,andcontinuetointro-duceNObybubblingforanother5minutes.

3. RemoveexcessNOwithargongasbubblingfor5minutes,andstoreat-20°C.TheNO-Fe(II)-DTCSsolutioncanbestoredat-20°Cforatleast2monthsinoxygen-freeconditions.

1. A

nti O

xida

nt

Det

ectio

n2.

DN

A D

amag

e D

etec

tion

3. L

ipid

Per

oxid

e D

etec

tion

4. R

adic

al D

etec

tion

6. N

O D

onor

5. N

itric

Oxi

de

Det

ectio

n7.

AG

Es R

esea

rch


21

DetectionofNObyEPR

MGD Product Code: M323

MGD isahighlywater-solubledithiocarbamate-typechelator that generateswi th complexes many t rans i t iona l method such as Fe and Cu. The

diethyldithiocarbamate-Fe2+ complexhasbeenused forNOdetectionbyelectronspin resonance (ESR).However, thepoorsolubilityof thiscarbamate inanaqueoussolutionlimitsitsapplication.Dr.Laiandothersimprovedthetechniqueusingawater-solubledithiocarbamate-Fe2+complex,MGD-Fe2+.TheysuccessfullydetectedinvivoNOof a nitroprusside-injectedmouseandNOgeneratedbyanLPS injectionusinginvivoESR.TheMGD-Fe2+complex iscapableofNOdetectionunderphysiologicalconditions,anddissolvedoxygeninthesolutiondoesnotinterferewithNOdetection.

MGDN-(Dithiocarbamoyl)-N-methyl-D-gluca-mine,sodiumsaltC8H16NNa2O5S2=293.34CASNo.[94161-07-6(freeacid)]Unit:500mg

1. General Protocol► PreparationofFe(II)-MGDComplex1. Dissolve278mgFeSO4,7H2O(ferroussulfateheptahydrate)with20mlwa-

tera)toprepare50mMFeSO4solution.b)

2. Dissolve147mgMGDwith10mlwatera)toprepare50mMMGDsolution.3. Mix1mlMGDsolutionwith8.8mlbuffersolutiona)(pH7orhigher)andthen

add200µlFeSO4solutionpriortouse.c)

a) Purgeanydissolvedoxygen in thewaterorbufferbynitrogengasbub-blingforatleast30minutespriortodissolvingFeSO4.

b) TheFeSO4solutioncanbestoredat-20°Cforatleast2months.c) Fe(II)-DTCSiscolorless.Ifthesolutionisbrown,Fe(III)-DTCSmayhave

formedbecauseofdissolvedoxygeninthesolution.However,thebrownsolutioncanstillbeusedforNOtrapping.

► PreparationofNO-Fe(II)-MGDComplex1. Underargongasflow,add200µlFeSO4solutionto9.4mlbuffersolution(pH

7orhigher).ThenintroduceNOintothesolutionbybubblingNOgasthroughaglasscapillaryfor15minutes.

2. Add400µlMGDsolutiontotheFeSO4solutionandcontinuetointroduceNObybubblingforanother5minutes.

3. RemoveexcessNOwithargongasbubblingfor5minutesandstoreat-20°C.TheNO-Fe(II)-MGDsolutioncanbestoredat -20°Cforat least2months inoxygen-free conditions.RemoveexcessNOwith argongasbubbling for 5minutes, andstoreat -20°C.TheNO-Fe(II)-DTCSsolution canbestoredat-20°Cforatleast2monthsinoxygen-freeconditions.

2. Specification► Appearance:whitecrystallinepowder► Purity:≥98.0%(HPLC)

1. Anti O

xidant D

etection2. D

NA D

amage

Detection

3. Lipid Peroxide D

etection4. R

adical Detection

6. NO

Donor

5. Nitric O

xide D

etection7. A

GEs R

esearch


22

Fig. 1 Time course of releasing NO from NOR compounds

NORsare idealNOdonorswith completely different chemical structures from theotherNOdonors.AlthoughNORsdonot haveanyONO2orONOmoiety, they

spontaneouslyreleaseNOatasteadyrate.EventhoughtheNOreleasemechanismofNORhasnot been completely determined, it is confirmed that thebyproductsdonot possessany significant bioactivities.NOR3, isolated fromStreptomycesgenseosporeus,isreportedtohavestrongvasodilatoryeffectsonratandrabbitaortasanddog coronaryarteries. Its activity (ED50=1nM) is 300 times that of isosorbidedinitrate(ISDN).NOR3alsoincreasestheplasmacyclicGMPlevels,whereasISDNdoesnot.NOR is a potent inhibitor of platelet aggregationand thrombus formation.NOR3 (IC50=0-7mM)effectively inhibits 100%ofADP-initiatedhumanplateletAggregation,whereasISDNinhibitsonly32%ofthetotalaggregation,evenat100mMconcentrations.NOR3hasalsobeenreportedtohaveantianginalandcardioprotectiveeffects in the ischemia/reperfusion system. In the ratmethacholin-induced coronaryvasospasmmodel,NOR3 suppressed theelevationof theST segment dose-dependentlyandsignificantlyat1mgperkg.Ontheotherhand,ISDNsuppresseditsignificantlyat3.2mgperkg.ThedifferenceintheNOreleaserateofNORreagentswasreflectedevenontheinvivohypotensiveeffects.NORmayalsobeusedorallyina0.5%methylcellulosesuspension.NORisrelativelystable inDMSOsolution.NOR1,whichhas the shortest half-life, is a promising reagent formakingNOstandardsolutionsforcalibration.Forthepreparationofthestandardsolution,apreciselydilutedNOR1/DMSOsolutionisaddedtothebuffersolutions.

NOR 1 (Product code: N388)(±)-(E)-4-Methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexenamideC8H13N3O5=231.21CASNo.[163032-70-0]Unit:10mg

NOR 3 (Product code: N390)(±)-(E)-4-Ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamideC8H13N3O4=215.21CASNo.[163180-49-2]Unit:10mg

NOR 4 (Product code: N391)(±)-N-[(E)-4-Ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexene-1-yl]-3-pyridinecarboxamideC14H18N4O4=306.32CASNo.[162626-99-5]Unit:10mg

NOR 5 (Product code: N448)(±)-N-[(E)-4-ethyl-3-[(Z)-hydroxyimino]-6-methyl-5-nitro-3-heptenyl]-3-pyridine-carboxamideC16H22N4O4=334.37Unit:10mg

1. References1. S.Shibata,et al.,CharacteristicsoftheVasorelaxingActionof(3E)-4-Ethyl-2-hydroxyimino-5-nitro-3-hexamideFK409,a

NewVasodilatorIsolatedfromMicrobialSources,inIsolatedRabbitArteries.J Cardiovasc Pharmacol.1991;17:508-518.2. Y.Kita,et al.,AntianginalEffectsofFK409,aNewSpontaneousNOReleaser.Br J Pharmacol.1994;113:1137-1140.3. Y.Kita,et al.,AntiplateletActivitiesofFK409,aNewSpontaneousNOReleaser.Br J Pharmacol.1994;113:385-388.4. Y.Kita,et al.,SpontaneousNitricOxideReleaseAccountsforthePotentPharmacologicalActionsofFK409.Eur J Pharma-

col.1994;257:123-130.5. M.Hino,et al.,FK409,aNovelVasodilatorIsolatedfromtheAcid-treatedFermentationBrothofStreptomyces Griseo-

sporeusI.Taxonomy,Fermentation,Isolation,andPhysico-chemicalandBiologicalCharacteristics.J Antibiot.1989;42:1578-1583.

6. J.Decout,et al.,DecompositionofFK409,aNewVasodilator:IdentificationofNitricOxideasMetaborite.Bioorg Med Chem Lett.1995;5:973-978.

7. S.Fukuyama,et al.,ANewNitricOxide(NO)Releaser:SpontaneousNOReleasefromFK409.Free Radic Res.1995;23:443-452.

8. Y.Kita,et al.,FR144420,aNovel,Slow,NitricOxide-releasingAgent.Eur J Pharmacol.1995;275:125-130.9. M.Kato,et al.,NewReagentsforControlledReleaseofNitricOxide.Structure-stabilityRelationships.Bioorg Med Chem

Lett.1996;6:33-38.10. Y.Kita,et al.,FK409,aNovelSpontaneousNOReleaser:ComparativePharmacologicalStudieswithISDN.Cardiovasc

Drug Rev.1996;14:148-165.11. Y.Hirasawa,et al.,AntianginalEffectsofFR144420,aNovelNitricOxide-releasingAgent.Eur J Pharmacol.1996;303:55-

59.12. M.Sato,et al.,NitricOxideRaisesCytosolicConcentrationsofCa2+inCulturedNodoseGanglionNeuronsfromRabbits.

Neurosci Lett.1996;206:69-72.13. Y.Kita,et al.,OralBiologicalActivitiesofSpontaneousNitricOxideReleaserareAccountedforbytheirNitricOxide-

releaseingRatesandOralAbsorptionManners.J Pharmacol Exp Ther.1996;276:421-425.

2. Specification► Appearance:Whiteorslightlyyellowpowder(NOR1,NOR4,NOR5),Whitecrystallinepowder(NOR3)

► Purity:≥98.0%(HPLC)

NOdonor(Stableinacidiccondition)

NOR Compounds

NODonorsNOR1 NOR3 NOR4 NOR51.8min 30min 60min 20hrs

Table 1 Half-life of NOR donors(pH 7.4)

▲ ▲

0 15 30Time(min)

2.0

1.5

1.0

0.5

0

Conc.ofreleasedNO(mM)

▲

▲

▲▲

▲

▲

●

●

●●●

060120180240Time(min)

1.0

0.8

0.6

0.4

0.2

0

Conc.ofreleasedNO(mM)

▲

▲

▲

▲

▲

●●

●

●

●

1. A

nti O

xida

nt

Det

ectio

n2.

DN

A D

amag

e D

etec

tion

3. L

ipid

Per

oxid

e D

etec

tion

4. R

adic

al D

etec

tion

6. N

O D

onor

5. N

itric

Oxi

de

Det

ectio

n7.

AG

Es R

esea

rch


23

NOCsare stableNO-amine complexes that spontaneously releaseNO,withoutcofactors, under physiological conditions.The rate ofNO releasedependson

the chemical structureofNOC.Themechanismof spontaneousNOgenerationbyNOCs is very simple compared to other classicalNOdonors, suchasnitroglycerinandnitropurusside, and theby-products donot interferewith cell activities.A singleNOCmolecule releases twoNOmolecules (as indicated in the reaction scheme);thereleaserateof thesecondNOmolecule isveryslow.NOCscanbeused toaddcontrolled amounts of pureNO toexperimental systemsat controlled rateswithminimalsideeffects.TheamountofNOreleasedcanbeeasilymanipulatedbyalteringtheconcentrationandselectionofNOCreagents.Dojindooffers fourdifferentNOCs(NOC5, 7, 12, and18)with different halflifes.Stock solutionsofNOCprepared inalkaline solutions, suchasaqueousNaOH,are relatively stable.However,theNOCstocksolutionshouldbeusedwithinonedaybecauseitdegradesabout5%perday,evenat-20ºC.ThereleaseofNObeginsimmediatelyafteraddingthestocksolutiontoasamplesolution.

NOC 5 (Product code: N380)1-Hydroxy-2-oxo-3-(3-aminopropyl)-3-isopropyl-1-triazeneC6H16N4O2=176.22CASNo.[146724-82-5]Unit:10mg,50mg

NOC 7 (Product code: N377)1-Hydroxy-2-oxo-3-(N-methyl-3-amino-propyl)-3-methyl-1-triazeneC5H14N4O2=162.19CASNo.[146724-84-7]Unit:10mg,50mg

NOC 12 (Product code: N378)1-Hydroxy-2-oxo-3-(N-ethyl-2-aminoethyl)-3-ethyl-1-triazeneC6H16N4O2=176.22CASNo.[146724-89-2]Unit:10mg,50mg

NOC 18 (Product code: N379)1-Hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-tria-zeneC4H13N5O2=163.18CASNo.[146724-94-9]Unit:10mg,50mg

NOdonor(Stableinalkalinecondition)

NOC Compounds

NODonorsNOC5 NOC7 NOC12 NOC18

pH

7.0 12min 2.2min 40min 13hrs7.2 20min 3.8min 1.2hrs 18hrs7.4 25min 5min 100min 21hrs7.6 42min 8.2min 3hrs 34hrs7.8 66min 12.4min 4.6hrs 45hrs

Table 1 Half-life of NOC donors

1. General Protocol1. Prepare10mMNOCstocksolutionusing0.1MNaOH.SincetheNOCstock

solutionisnotstable,keepitonanicebathanduseitinoneday.2. AddanappropriatevolumeoftheNOCstocksolutiontothesamplesolution

inwhichNOistobereleased.TomaintainthepHofthesamplesolution,thevolumeoftheNOCstocksolutionshouldnotexceed1/50ofthesamplevolume.Thesamplesolutionshouldhavesufficientbufferingaction.NOwillbereleasedimmediatelyaftertheadditionoftheNOCstocksolution.

2. References1. K.Hayashi,et al.,ActionofNitricOxideasaAntioxidantAgainstOxidationofSoybeanPhosphatidylcholineLiposomal

Membrane.FEBS Lett.1995;370:37-40.(Noc12)2. S.Shibuta,et al.,IntracerebroventricularAdministrationofaNitricOxide-releasingCompound,NOC-18,ProducesThermal

HyperalgesiainRats.Neurosci Lett.1995;187:103-106.(NOC18)3. S.Shibuta,et al.,Anewnitricoxidedonor,NOC-18,exhibitsanociceptiveeffectintheratformalinmodel.J Neurol Sci.

1996;141:1-5.(NOC18)4. N.Yamanaka,et al.,NitricOxideReleasedfromZwitterionicPolyamine/NOAdductsInhibitsCu2+-inducedLowDensity

LipoproteinOxidation.FEBS Lett.1996;398:53-56.(NOC5,NOC7)5. D.Berendji,et al.,NitricOxideMediatesIntracytoplasmicandIntranuclearZincRelease.FEBS Lett.1997;405:37-41.6. T.Ohnishi,et al.,TheEffectofCu2+onRatPulmonaryArterialRings.Eur J Pharmacol.1997;319:49-55.(Noc7)7. Y.Adachi,et al.,RenalEffectofaNitricOxideDonor,NOC7,inAnethetizedRabbits.Eur J Pharmacol.1997;324:223-226.

(Noc7)8. Y.Minamiyama,et al.,Effect ofThiolStatusonNitricOxideMetabolism in theCirculation.Arch Biochem Biophys.

1997;341:186-192.(NOC7)

3. Specification► Appearance:Whitepowder► Purity:≥90.0%(HPLC)

1. Anti O

xidant D

etection2. D

NA D

amage

Detection

3. Lipid Peroxide D

etection4. R

adical Detection

6. NO

Donor

5. Nitric O

xide D

etection7. A

GEs R

esearch


24

Fig. 1 Time course of NOC degradation(100 µM NOCs in 100 mM PBS, pH7.0, 37°C)

Fig. 2 Half life of NOC compounds in 100mM PBS(37°C) under the various pH conditions

Fig. 3 pH dependency of releasing NO from NOC 5(0.1 mM NOC 5, in 100 mM PBS at 37°C)

050100150200Time(min)

NO(μ

mol/l)

200

100

0

pH7.0(NO)pH7.2(NO)pH7.4(NO)pH7.6(NO)

○○○

○○○

○

○○○○

●

●●

●

●●●

●●●●

● ●

●

▲

▲

▲

▲

▲

▲

▲▲

▲▲ ▲

▲

□

□□

□□□□□

□□□□ □

□□

□

○●

▲□

ˡ ˡ ˡ ˡ ˡ ˡ ˡ ˡ ˡ ˡ

‒

‒

‒

‒

‒

NOC5 NOC7

NOC12 NOC18

t 1/2(m

in)

t 1/2(m

in)

t 1/2(hrs)

t 1/2(m

in)

80

60

40

20

0

12

8

4

0

300

250

200

150

100

50

0

50

40

30

20

10

077.27.47.67.8

●●

●

●

●

●●

●

●

●

●●

●

●

●

●●

●

●

●

ˡ ˡ ˡ ˡ ˡ ˡ ˡ ˡ ˡ77.27.47.67.8

ˡ ˡ ˡ ˡ ˡ ˡ ˡ ˡ ˡ

77.27.47.67.8ˡ ˡ ˡ ˡ ˡ ˡ ˡ ˡ ˡ

77.27.47.67.8ˡ ˡ ˡ ˡ ˡ ˡ ˡ ˡ ˡ

‒

‒

‒

‒

‒

‒

‒

‒

‒

‒

‒

‒

‒

‒

‒

‒

‒

‒

‒

‒

NOC(μ

mol/l)

NOC(μ

mol/l)

time(min) time(hrs)020406080100120 05101520253035404550

100

90

80

70

60

50

40

30

20

10

0

100

90

80

70

60

50

40

30

20

10

0

NOC18(UV250nm)NOC12(UV250nm)

ˡ ˡ ˡ ˡ ˡ ˡ ˡ ˡ ˡ ˡ ˡ ˡ ˡ ˡ

NOC5(UV250nm)NOC7(UV250nm)

1. A

nti O

xida

nt

Det

ectio

n2.

DN

A D

amag

e D

etec

tion

3. L

ipid

Per

oxid

e D

etec

tion

4. R

adic

al D

etec

tion

6. N

O D

onor

5. N

itric

Oxi

de

Det

ectio

n7.

AG

Es R

esea

rch


25

NNitrosothiol compounds releaseNOandbecomedisulfidesunder specificphysiologicalconditions.WhilemostoftheS-nitrosothiolcompoundsareunstable,

S-Nitrosoglutathione is exceptionally stable.Furthermore,S-Nitrosoglutathione iswater-soluble.AlthoughS-nitrosothiol is a goodNOdonorwith nonitrate tolerance,thereisevidencethatS-nitrosothiolitselfhasNO-likeactivityduringguanylatecyclaseactivation.Another important reactionof nitrosothiol isNO transfer to other thiolcompounds.Since itdependson thepKaof thiols, this transfer reactionproceedsatphysiologicalpHlevels.Previous research found that the vasorelaxant propertiesof endothelium-derived re-laxation factor (EDRF)aremore similar toS-nitrosocysteine thanNO;however, thisdoesnotseemtobethecurrentmajorityview.Thoughnitrosothiolisoneofthemostimportant factors for the studyof theNOpathway, only a fewnitrosothiols, suchasSNAPandS-nitrosoglutathione(GSNO),arestableenoughforuseasNOdonors.Un-fortunately,SNAPisinsolubleinwater.Thus,GSNOandS-nitrosocysteine(SNC)aretheonlycommerciallyavailablewater-solublenitrosothiols.Nitrosothiolsreleasenitricoxide,andformdisulfidesasshownbelow. 2RSNO→RSSR+2NO

This reaction isacceleratedby lightandheat. IfGSNO is incubatedat37°Cwithoutlight,NOwill not be released spontaneously.Metal ions, suchasCu(II),Cu(I), andHg(II), alsoaccelerate the reaction.Thus,masking reagents suchasEDTApreventthe releasing reaction.Another important characteristic of nitrosothiols is their abilitytocarryoutnitrosation.ThisreactionisfasterthanthedecompositionofRSNOitself,andproceedsreadilyatphysiologicalpHlevels.ThereactionratedependsonthepKaofthethiol.Thevasorelaxantactivitiesofnitrosothiolsinrataorticringshavebeenre-portedasfollows: SNAP>GSNO=SNAC(S-Nitroso-N-acetylcysteine) >CoAsNO(S-Nitroso-coenzymeA)>CYCNO(S-Nitrosocysteine)

Theinhibitorypotenciesofnitrosothiolsfortheplateletaggregationhavebeenreportedasfollows: GSNO>NO>SNAP>SIN-1

DenitrosationofS-nitrosothiolisnotspontaneous,anditneedstobecatalyzedonthesurfaceof external vascularmembranes.S-nitroso-L-cysteine raises the intracellularcalciumlevelofaPC12cellbymodifyingthethiolgroupofacaffeine-sensitivemoietyof the calcium-induced calcium release (CICR) channel.GSNOhasbeen shown toreducethebloodpressureofanesthetizeddogs(0.2mg/kg)andmonkeys(10mg/kg)throughtheinhibitionoftheplateletaggregation.

S-Nitrosoglutathione N-(N-L-γ-Glutamyl-S-nitroso-L-cysteinyl)glycineC10H16N4O7S=336.32CASNo.[57564-91-7]Unit:25mg,100mg

1. References1. A.Gibson,et al.,AnInvestigationofSomeS-Nitrosothiols,andofHydroxy-arginine,ontheMouseAnococcygeus.Br J

Pharmacol.1992;107:715-721.2. M.W.Radomski,et al.,S-Nitroso-glutathioneInhibitsPlateletActivationinVitroandinVivo.Br J Pharmacol.1992;107:745-

749.3. R.M.Clancy,et al.,NovelSynthesisofS-NitrosoglutathioneandDegradationbyHumanNeutrophils.Anal Biochem.

1992;204:365-371.4. J.W.Park,et al.,TransnitrosationasaPredominantMechanismintheHypotensiveEffectofS-Nitrosoglutathione.Bio-

chem Mol Biol Int.1993;30:885-891.5. D.Barrachina,et al.,NitricOxideDonorsPreferenciallyInhibitNeuronallyMediatedRatGastricAcidSecretion.Eur J

Pharmacol.1994;262:181-183.6. E.A.Konorev,et al.,S-NitrosoglutathioneImprovesFunctionalRecoveryintheIsolatedRatHeartAfterCardioplegicIsch-

emicArrest-evidenceforaCardioprotectiveEffectofNitricOxide.J Pharmacol Exp Ther.1995;274:200-2006.7. S.C.Askew,et al.,CatalysisbyCu2+ofNitricOxideReleasefromS-Nitrosothiols(RSNO).J Chem Soc Perkin Trans 2.

1995;741-745.8. D.J.Banett,et al.,NO-groupTransfer(Transnitrosation)betweenS-NitrosothiolsandThiols.Part2.J Chem Soc Perkin

Trans 2.1995;1279-1282.9. J.G.DeMan,et al.,EffectofCu2+onRelaxationstotheNitrergicNeurotransmitter,NOandS-NitrosothiolsintheRat

GastricFundus.Br J Pharmacol.1996;119:990-996.10. A.P.Dicks,et al.,GenerationofNitricOxidefromS-NitrosothiolsUsingProtein-boundCu2+Sources.Chem Biol.

1996;3:655-659.11. J.A.Cook,et al.,ConvenientColorimetricandFluorometricAssaysforS-Nitrosothiols.Anal Biochem.1996;238:150-158.12. S.X.Liu,et al.,NitricOxideDonors:EffectsofS-Nitrosoglutathioneand4-Phenyl-3-furoxancarbonitrileonOcularBlood

FlowandRetinalFunctionRecovery.J Ocul Pharmacol Ther.1997;13:105-114.13. C.Alpert,et al.,DetectionofS-NitrosothiolsandOtherNitricOxideDerivativesbyPhotolysis-chemiluminescenceSpec-

trometry.Anal Biochem.1997;245:1-7.14. T.Akaike,et al.,NanomolarQuantificationandIdentificationofVariousNitrosothiolsbyHighPerformanceLiquidChroma-

tographyCoupledwithFlowReactorsofMetalsandGriessReagent.J Biochem.1997;122:459-466.

2. Specification► Appearance:Pinkpowder► Purity:≥90.0%(HPLC)

NOdonorfromnitrosothiolcompound

S-Nitrosoglutathione Product Code: N415

1. Anti O

xidant D

etection2. D

NA D

amage

Detection

3. Lipid Peroxide D

etection4. R

adical Detection

6. NO

Donor

5. Nitric O

xide D

etection7. A

GEs R

esearch


26

SIN-13-(4-Morpholinyl)sydnonimine,hydrochlorideC6H11ClN4O2=206.63CASNo.[16142-27-1]Unit:25mg

1. References1. M.Feelisch,et al.,OntheMechanismofNOReleasefromSydnonimines.J Cardiovasc Pharmacol.1989;14:S13-S22.2. M.Feelisch,TheBiochemicalPathwaysofNitricOxideFormationfromNitrovasodilators:AppropriateChoiceofExogenous

NODonorsandAspectsofPreparationandHandlingofAqueousNOSolutions.J Cardiovasc Pharmacol.1991;17:S25-S33.

3. N.Hogg,et al.,ProductionofHydroxylRadicalsfromtheSimultaneousGenerationofSuperoxideandNitricOxide.Bio-chem J.1992;281:419-424.

4. M.E.Murphy,et al.,NitricOxideHyperpolarizesRabbitMesentericArteriesviaATP-sensitivePotassiumChannels.J Physiol.1995;486:47-58.

5. H.Kankaanranta,et al.,3-Morpholino-sydnonimine-inducedSuppressionofHumanNeutrophilDegranulationinNotMedi-atedbyCyclicGMP,NitricOxideorPeroxynitrite:InhibitionoftheIncreaseinIntracellularFreeCalciumConcentrationbyN-Morpholinoiminoacetoni.Mol Pharmacol.1997;51:882-888.

6. S.Yamamoto,et al.,SubarachnoidHemorrhageImpairsCerebralBloodFlowResponsetoNitricOxidebutNottoCyclicGMPinLargeCerebralArteries.Brain Res.1997;757:1-9.

7. S.Pfeiffer,et al.,Interferenceofcarboxy-PTIOwithNitric-oxideandPeroxynitrite-mediatedReactions.Free Radic Biol Med.1997;22:787-794.

8. M.B.Herrero,et al.,TyrosineNitrationinHumanSpermatozoa:APhysiologicalFunctionofPeroxynitrite,theReactionProductofNitricOxideandSuperoxide.Mol Hum Reprod.2001;7:913-921.

9. P.D. Lu,et al.,CytoprotectionbyPre-emptiveConditionalPhosphorylationofTranslation InitiationFactor 2.EMBO J.2004;23:169-179.

2. Specification► Appearance:Whiteneedlesorslightlyyellowish-whitecrystallinepowder► Purity:passtest(TLC)► MeltingPoint:180°Cto190°C

SIN-1, ametabolite of the vasodilatormolsidomine, is utilized to separatelyestimate theeffectivenessofNOandperoxynitritewith otherNOdonors.SIN-

1 spontaneously decomposes in thepresenceofmolecular oxygen to generateNOandsuperoxide.Bothproductsbindveryrapidlytoformperoxynitrite(rateconstantk:3.7x10-7M-1s-1).Therefore,SIN-1isausefulcompoundthatgeneratesperoxynitriteinanefficientmanner.Peroxynitriteisaverystrongoxidantthatgenerateshydroxylandnitrosyldioxylradicalsunderphysiologicalconditions.Peroxynitritealsodecomposestogeneratenitrate ionquickly inacidicconditionsandslowly inbasicconditions.ThosespecieshaveadifferentbioactivityfromNO.

ONOO-(peroxynitrite)donor

SIN-1 Product Code: S264

1. A

nti O

xida

nt

Det

ectio

n2.

DN

A D

amag

e D

etec

tion

3. L

ipid

Per

oxid

e D

etec

tion

4. R

adic

al D

etec

tion

6. N

O D

onor

5. N

itric

Oxi

de

Det

ectio

n7.

AG

Es R

esea

rch


27

Advancedglycationend-products(AGEs)havebeenstudiedasoneofthecausesofdiabeticcomplications.SeveralcompoundshavebeenidentifiedasAGEs,including

pyralline,pentosidine, imidazolone,andpyropyridine.Glyoxalandmethylglyoxalarereactivedicarbonylcompoundsgeneratedbyglucoseself-oxidation thatareknowntobeAGEprecursors.Anotherdicarbonylcompound,3-Deoxyglucosone (3-DG),isalsoknowntobeoneof theAGEprecursors.3-DG isderived fromtheAmadorirearrangementproductsofproteinsandsugarsinearlystagesoftheMaillardreaction.3-DGisalsoderivedfromfructose,whichispresentinhighlevelsindiabeticpatients,byaselfcondensation reaction.Fructose-3-phosphatehasbeen found toenhancecross-linkingreactionsoflensproteinsinadiabeticratmodel.Therefore,3-DGderivedfromfructose-3-phosphatehasbeenstudiedasapossiblecauseofcataracts.Thereare twomethods fordetermining3-DGlevels:HPLCandmassspectrometry(MS).However,thereissomediscrepancybetweentheHPLCandMSmethodswhenmeasuring3-DGlevels invivo.HPLCanalysis isbasedonafluorescentcompound,2-(2,3,4-trihydroxybutyl)-benzo[g]quinoxaline,generatedbyacoupling reactionbetween3-DGand2,3-diaminonaphthalene.Analogsof2,3-diaminonaphthalene,suchas1,2-diamino-4,5-dimethoxy-benzeneand1,2-diamino-4,5-methylenedioxybenzene,canalsobeused.

Contents of the KitDAN[2,3-Diaminonaphthalene] 10mgx13-DG/DANadduct[2-(2,3,4-Trihydroxybutyl)benzo[g]quinoxaline] 1mgx1

3-DGcanbeutilizedforAGEproductionorasastandardfor3-DGleveldetectioninplasmaorserumsamples. 3-Deoxyglucosone

3-Deoxy-D-erythro-hexos-2-uloseC6H10O5=162.14CASNo.[4084-27-9]Unit:1mg

1. Specification► Appearance:whiteorwhitepaleyellowsolid► Purity:≥99.0%(HPLC)


Detectionof3-DGbyHPLC(Fluorometric)

3-Deoxyglucosone Detection Reagents Product Code: D536

Fig. 1 Principal of 3-DG detection2. General Protocol► HPLCMethod:HumanSerum1. Add60%perchloricacidsolutionto1mlhumanserumandspinat3,000xg

for20minutesat4°C.2. Dilutethesupernatantwithbicarbonatebuffer,thenadd0.1mlof2,3-Diaminonaphthalene

/methanolsolutionand25µlof1ppm3,4-hexanedioneasaninternalstandard.3. Incubatethemixtureat4°Covernight.4. Extractthemixturewith4mlethylacetate,andadd4mlmethanoltotheextract.5. Analyzethemixturewithreverse-phaseHPLCat267nmexcitationand503

nmemissionforfluorescentdetectionorat268nmforUVdetection.DatacorrelateswellwithHbA1clevel.

*Normalserum3-DGlevel:12.8±5.2ng/ml *Serum3-DGlevelofdiabeticpatient:31.8±11.3ng/ml

3. References1. K.J.Knecht,et al.,Detectionof3-Deoxyfructoseand3-DeoxyglucosoneinHumanUrinendPlasma:EvidenceforInterme-

diateStagesoftheMaillardReactioninVivo.Arch Biochem Biophys.1992;294:130-137.2. T.Niwa,et al.,Presenceof3-Deoxyglucosone,aPotentProteinCrosslinkingIntermediateofMaillardReaction,inDiabetic

Serum.Biochem Biophys Res Commun.1993;196:837-843.3. H.Yamada,et al.,Increasein3-deoxyglucosonelevelsindiabeticratplasma.Specificinvivodeterminationofintermediate

inadvancedMaillardreaction.J Biol Chem.1994;269:20275-20280.

StandardsubstrateforAGEprecursor

3-Deoxyglucosone Product Code: D535

Title ReferenceGlutathionedepletionasamechanismof3,4-dideoxyglucosone-3-ene-inducedcytotoxicityinhumanperitonealmesothelialcells:roleinbiocompatibilityofperitonealdialysisfluids

T.Yamamoto,et al., Dial. Transplant.2009;24:1436-1442.

Anovelclassofadvancedglycation inhibitorsameliorates renalandcardiovasculardamageinexperimentalratmodels

Y. Izuhara,et al, Nephrol. Dial. Transplant.2008;23:497-509.

1. Anti O

xidant D

etection2. D

NA D

amage

Detection

3. Lipid Peroxide D

etection4. R

adical Detection

6. NO

Donor

5. Nitric O

xide D

etection7. A

GEs R

esearch

Documents

Dojindo Oxidative Stress Products - Dojindo Molecular … · · 2013-02-21Dojindo Oxidative Stress Products Oxidative Stress Research Products ... Dojindo Oxidative Stress Products