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TheEffectofpHontheRateofEnzymeCatalysisofCatalase

Objectives:
Theobjectiveofthislabwastodevelopa protocoltoinvestigatethee ffectofa ne nvironmental
variableonthec atalyticfunctionofa ne nzyme.Morespecifically,theobjectivewastoperforma n
experimentinordertotestthee ffectofpHonthefunctionofthee nzymec atalase.

Introduction:
Enzymesa reproteinsthata cta sc atalystsforreactions.Thissimplymeansthate nzymeslower
thea ctivatione nergyrequiredfora reactiontotakeplace,a llowinga particularreactiontotakeplace
muchquickera nde asier.Specifice nzymesonlylowerthea ctivatione nergyforspecificreactions,a nd
enzymesa reshapespecific.Theuniquefoldsofthea minoa cidc hainsthatmakeupa ne nzymeresultin
theformationofa specificallyshapeda ctivesite.Whenthereactantsofa reactions,c alledsubstrates,fit
perfectlyintothea ctivesiteofa ne nzyme,thee nzymeisa bletoc atalyzethereaction.Thea ctivityof
enzymesisa ffectedbyboththec oncentrationsofe nzymespresenta ndthec oncentrationofsubstrate
present.Asthea mountofe nzymepresentincreases,therateofreactionincreases.Furthermore,a sthe
amountofsubstrateincreases,therateofreactionwillinitiallyincreases.Moste nzymesrequirespecific
environmentalc onditionstobemetinorderforthemtofunctionproperlya nde fficiently.These
conditionsincludetemperature,thenc oncentrationofsalt,a ndthepHlevel.Iftheoptimumc onditions
fora ne nzymea rea ltered,thee nzymemaydenature,orc hangeitsshape,a nddeactivate.Asa result,the
enzymewouldnolongertobea bletoc atalyzethereaction,a ndthereactionratewouldsignificantly
decrease("WorthingtonBiochemicalCorporation").
Catalaseisfoundina llorganismsthatuseoxygenfortheirmetabolism.Thee nzymeisfoundin
highc oncentrationsina organelleinc ellsc alledtheperoxisome.Oneofthefunctionsofc atalaseisto
preventa toxica ccumulationofhydrogenperoxide(H2O2)inc ells.Itc atalysesthec onversionof
hydrogenperoxidetowatera ndmolecularoxygen.Hydrogenperoxideisa byproductofmetabolic
processes.Itisusuallyproducedinperoxisomeswhentheypartiallyoxidizefattya cids.Whenc atalase
isa bsent,thereactionitc atalyzesisspontaneous,buta tverylowratesthata renota bletoreducethe
harmfule ffectsofhydrogenperoxide(Crook).

ResearchQuestionandHypothesis:
Question:Howwilla lteringthepHlevelofa solutiona ffecttherateofe nzymec atalysisofthee nzyme
catalase?

Hypothesis:Ifthee nzymec atalaseisplacedindifferentsolutionsc ontainingdifferentpHlevels,the


enzymewillfunctionmoste ffectivelya ta neutralpH,ora pHof7.AsthepHofthesolutionsa re
lowered,c atalasewillbegintoworklesse ffectively,a ndtherateofreactionwillbegintodecrease.
Catalase,likemoste nzymes,worksbestinspecifice nvironmentalc onditions.Thereasonthatc atalase
willbegintoworklesse ffectivelyisbecausea sthepHdecreases,a ndthee nvironmentalc onditionsa re
changed,thec hangeinpHwillbegintodenaturea nddeactivatethee nzymes.Asmorea ndmore
enzymesbecomedeactivated,therateofreactiondecreases.
Materials:

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HydrogenPeroxide(1%H2O2solution)
Catalase(wellblended/strainedpotatoe xtract)
SulfuricAcid
Buffers(pHof7,6,5,3)
PotassiumPermanganate
Water
10mLSyringes
PlasticCups
Beakers
10mLgraduatedc ylinders
Gloves
Goggles

Procedure:
1. Beforethee xperimentbegan,a baselinewasdetermined.
2. Five50m LbeakerswerelabelledwiththepHofthebufferthatwasbeingtested(Control,6,5,
3).ThepHofthesolutionwastheindependentvariable.
3. 10mLof1%hydrogenperoxidesolutionwasa ddedtoe achbeaker.
4. 10mLofthec orrectbuffersolutionwasa ddedtothefirstc orrespondingbeaker.
5. 10mLofthee xtractedc atalasewasa ddedtothesamebeaker,whichinitiatedthereaction.
6. Thereactionwastimedfor180seconds,ortwominutes.Oncethetwominuteshadpassed,it
wastimetostopthereaction.
7. Inordertostopthereaction,10mlofhydrochlorica cidwasa ddedtothebeaker.Witha pHof2,
thesulfurica cidisverya cidica ndwassuretodenaturethee nzyme.
8. Afterthereactionwasstopped,thea mountofsubstrate(H2O2)remaininginthebeakerhadtobe
measured.Tomeasurethisquantity,potassiumpermanganate(KMnO4)wasused.
9. 5mLofthesolutionwasremovedfromthebeaker.This5mLsamplewasplacedintoa nother
smallplasticc upa ndthea mountofhydrogenperoxidewasdetermineda sfollows.Asyringewas
usedtoa ddpotassiumpermanganate,onedropa ta time,tothesolutionuntila persistentpinkish
brownc olorwasobtained.Thesolutionwasgentlyswirleda sdropswerea dded.Alldatawas
recorded(theinitialreadingonsyringea ndfinalreadingonthesyringewasrecorded).
10. Steps38wererepeatedfore achpHlevelsothattherewasdatafora pHof6,5,4,a ndthe
control.
11. Oncethea mountofH2O2usedinthereactionhadbeenc alculated,thereactionratea te achpH
levelwasc alculatedbydividingthetotala mountofH2O2usedbythetime(twominutes).The
rateofreactionwasthedependentvariableofthee xperiment.

DeterminingtheBaseline:
1. 10mLofhydrogenperoxidewasputintoa smallplasticc up.
2. 1mLofwaterwasa dded(insteadofe nzymesolution).
3. 10mLof1.0Msulfurica cidwasa dded.
4. Thesolutionwasmixedwell.
5. A5mLsamplewasremoved.This5mLsamplewasplacedintoa nothersmallplasticc upa nd

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thea mountofhydrogenperoxidewasdetermineda sfollows.Asyringewasusedtoa dd


potassiumpermanganate,onedropa ta time,tothesolutionuntila persistentpinkishbrownc olor
wasobtained.Thesolutionwasgentlyswirleda sdropswerea dded.Thea mountofpotassium
permanganateusedisproportionaltothea mountofhydrogenperoxidepresent,a ndwastherefore
useda sthebaseline.

Data:
Potassium
pHLevel pHLevel pHLevel pHLevel
Permanganate(ml)

Control(7) 6 5 3

A.Baseline 7ml 7ml 7ml 7ml

B.FinalReading 4ml 3.6ml 3.0ml 2.1ml

C.InitialReading 8ml 8ml 8ml 8ml

D.Amountof
4ml 4.3ml 5ml 5.9ml
KMnO4used(CB)

E.AmountofH2O2
3ml 2.6ml 2ml 1.1ml
consumed(AD)
Table1:Determiningthea mountofH2O2c onsumedine achreaction.


Figure1:AmountofH2O2c onsumeda te achpHlevel.

pHLevel Control(7) 6 5 3

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ReactionRate
1.5ml/min 1.3ml/min 1ml/min .55ml/min
(ml/minute)
Table2:Thereactionratefore achpHlevel.


Figure2:Reactionratewhenc atalaseispresenta te achpHlevel.

Conclusion:
Thedatafromthee xperimentsupportsthehypothesisthatc atalasefunctionsthemoste fficiently
ata neutralpHof7.Table1showsthatc atalasehelpedc onsume3mLofhydrogenperoxideinthe
solutionwitha pHof7,morethana nyothersolution.AsthepHofthesolutionsdecreased,sodidthe
amountofhydrogenperoxidec onsumed.Furthermore,a fterc alculatingthereactionrates,itisshownthat
thereactionrateinthesolutionwitha pHof7(1.5ml/min)washigherthana nyothersolution(Table2).
Bylookinga tfigure2,onec anseethata sthepHofthesolutionrosetoa pHof7,c atalasebecamemore
efficienta ndwasa bletobetterc arryoutitsfunction.Theseresultshelpsupporttheideathata sa solution
becomesmorea cidicthantheoptimumpHofa ne nzyme,thee nzymespresentinthesolutionwill
denature,a ndinturnwillnotbea bletofunctionproperly.Thiswillresultinlowerreactionrates,which
isshowninfigure2.
Theinformationgatheredthroughoutthise xperimentisveryusefulforthefuture.This
experimenthasshownthate nzymesmusthavec ertaine nvironmentalc onditionspresentinorderforthem
tofunctionproperly.Withthisknowledge,onec ansuccessfullyperforme xperimentsusinge nzymesin
thefuturebymakingsurethatthee nvironmentalc onditionspresenta reoptimumforthee nzymethatis
beingused.
Alimitationoftheprocedurewasthatwewereunabletotestforthepresenceofc atalaseinthe
extractbeforebeginningthee xperiment.Ifwewerea bletotestforthepresenceofc atalaseinthee xtract,
wec ouldhavee nsuredthatthedecompositionofhydrogenperoxideresultedfrome nzymec atalysisa nd

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notfromthenaturalspontaneousdecompositionofthec hemical.Instead,wewereforcedtoa ssumethat


catalasewaspresentinthee xtract,a na ssumptionthatmay,ormaynothave,beenc orrect.
Onesourceofe rrorwasthattherewasnote noughpotatoe xtractpresenta tthebeginningofthe
experimentinordertoc ompleteit.Inordertogetthea mountofc atalaseneeded,wewereforcedto
manuallyc rusha ppleslicesa nde xtractthejuicefromtheslices.Withouttheuseofa blendera nda filter
inordertoc orrectlye xtractthec atalase,thisprocessmaynothaveprovidedthenecessarya mountof
catalaseneededinorderforthee xperimenttorunproperly.Ifthiswasthec ase,thenthedatawouldnot
bevalid,a sthec hangeinhydrogenperoxidewouldhavebeenduetothenaturalspontaneousreactionthat
alreadytakesplace.Anothersourceofe rrorwasthatitwasdifficulttotellwhena persistentpinkish
brownc olorhadbeenobtainedduringthepotassiumpermanganatetitration.Asa result,wemayhave
addedtoomuch,ortoolittle,potassiumpermanganatetothehydrogenperoxidesolution.Ifthisdid
happen,thenthiswouldhavea lteredthea mountofhydrogenperoxidec onsumedduringthereaction,
affectingthec alculatedrateofreaction.Athirdsourceofe rrorwasthata nunequala mountofc atalase
mayhavebeendistributedtoe achsolution.Sincewewereforcedtoe xtractthec atalasefromthea pple
sliceswhilewewereperformingthee xperiment,morec atalasemayhavebeenputintothesolutionswith
apHof7a nd6,thanthesolutionswitha pHof5a nd3.Ifthishappened,thenthehigherc oncentration
ofc atalasewouldhaveresultedintherateofreactionincreasing.This,notthec hangeinpH,would
accountforthedifferencesinthea mountofhydrogenperoxidec onsumed.

EvaluationofProcedure:
AlthoughtheprocedurebasicallyworkedlikeIthoughtitwould,thereweresometimesthatIfelt
stressedorunderpressure.Whileperformingthee xperiment,Ihadtomakesuretoa lwaysbeonestep
aheadofwhatIwasa ctuallydoing,otherwiseIwouldriskfallingbehinda ndpotentiallyc ausinginvalid
resultstobereceived.Ina ddition,itwasstressfultohavetotrytoe xtractc atalasefromthea ppleslices
andperformthee xperimenta tthesametime.Inordertosolvethisproblem,itwillhavetobee nsured
thatthereisa ne xcessofpotatoe xtractpresentbeforethee xperimentisstarted.Otherthanthesefew
challenges,theproceduredidrunrelativelysmoothly,a ndthee xperimentwasa success.
Onethingthatc ouldbea ddedtotheprocedureinordertoimproveitistotestthefunctionof
catalaseinsolutionsthata rebasic,withpHlevelsof8a ndhigher.Thisc ouldbedoneinordertoshow
thata neutralpHof7isreallytheoptimumpHforthefunctionofc atalase,a ndnota pHthatismore
basicthan7.Thiswoulda lsofurthershowhowa lteringthepHofa solution,e itherloweringitorraising
it,willa ffectthefunctionofa ne nzyme.Anotherthingthatc ouldbedoneinordertoimprovethe
procedurewouldbetotestmultiplesolutionswiththesamepHlevel.Thisc ouldbedoneinorderto
ensurethatthedatareceivedisa ccuratea ndisnotflawed.Ifc onflictingresultsa rereceived,however,
thenthatmeansthatsomethingmayhavegonewrongduringthee xperiment.Inordertohavea betterlab
experience,wec ouldbemoreprepared.Fore xample,wec oulde nsurethate noughpotatoe xtractwas
presentbeforethee xperimentbegan.Ina ddition,wec ouldpracticetitratingsolutionssothatwehave
experiencebeforewebegin.

WorksCited:
1. Crook,James."CatalaseAnExtraordinaryEnzyme."CatalaseWebsite.N.p.,05Jul2003.Web.
3Mar2012.
2. "IntroductiontoEnzymes."WorthingtonBiochemicalCorporation.N.p.,27Feb2012.Web.3

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Mar2012.