NacoleDonnell

EnzymeConcentrationEffect
Abstract
Enzymesspeedupreactionswhileloweringactivationenergy.Therearespecificfactorsthatcan
manipulatetheenzymecatalyzedreactionsuchaspH,temperatureandconcentrationoftheenzyme.In
thisparticularexperiment,temperaturewasthefactortested.Alowtemperaturewasmoreoptimalfor
theenzymesrateofreactionandtoohighofatemperaturewoulddenaturethesecondaryandtertiary
structureoftheenzyme,essentiallydestroyingtherateofreactiontheenzymespeedsup.Thistheoryis
testedandprovenbytakingtubeswiththesameamountofbufferandenzymeandputtingtheminto
differenttemperaturecontrolledwaterbaths,thusprovidingprooftoourtheoryanddisprovingthe
hypothesisbelow.
Introduction
Enzymesareproteinsthatcatalyzespecificreactionsdependingontheshapeoftheenzymesactive
site.Catalystsspeedupchemicalreactionswhileloweringactivationenergy.Eachenzymehasaunique
threedimensionalshapethathasasmallportionspecificallyusedastheactivesiteinwhichbindingto
specificreactantsorsubstratesoccurs.Whenthesubstrateorreactantbindstotheactivesiteofthe
enzyme,theenzymesubstratecomplexaremade.Duringtheprocessofcreatingthiscomplex,the
substratemoleculesarebroughttogetherandalignedinaspecificmanner.Thisreactionrequires
activationenergyandisreducedsothereactioncanproceedrapidly.Oncethereactioniscompletethe
substratethathasbeenconvertedintoitsnewproductisreleasedandfreetobindwithother
substrates.Specificconditionssuchasconcentrationoftheenzyme,pHandtemperatureaffecttherate
inwhichthechemicalreactionsorproteinsoccur.Whenasubstratefitsintotheactivesiteofan
enzymetheenzymesubstratecomplexisformed.Thereactionratebetweentheenzymeandchemical
reactiondependsontheenzymeconcentration.Ifthereisanexcessofthesubstratethereactionrate

willincrease.WhenpHisafactorinthereactionrate,optimalpHisthemostfavorableasthatiswhen
theenzymewillbemostactive.However,ifthereisanextremeofhighorlowpHvaluesdenaturation
occursandanenzymecanloseitsenzymeactivity.Therateofanenzymereactionincreasesasthe
temperatureincreases.However,ifthetemperaturereachesabove40degreesCelsius,thesecondaryor
tertiaryproteinstructurebecomesdenatured.Withoutenzymes,reactionsnecessarytolifewouldoccur
atamuchslowerrateandwouldntbeabletokeepupwiththebasicfunctionsoflife.Thepurposeof
theexperimentwastodeterminetheeffectofenzymeconcentrationontherateofreactionwhenthere
isasurplusofsubstratepresent.Myhypothesiswasthatthewarmerthetemperaturewasthefaster
thereactionwouldoccur.
MaterialsandMethods
Materials

1ContainerofParafilm
1Spectrophotometer
1BoxofKimwipes
1BoxofSalt
1Teaspoon
17RegularTestTubes
1SetofSpec20tubes
1SmallTestTubeRack
1RegularTestTubeRack
1WaxPencil
210mLGraduatedCylinders
2CelsiusThermometers
3400mLBeakers
1LargeBeakerLabelledUsedSpec20Tubes
pH7ONPG
1%Diastase
pH7Buffer
8%,6%,4%and2%BetaGalactosidase
.1%StarchSolution
1mLPipets
1mLPipetPumps
1TrayforUsedPipets
2DishpansofSoapyWater
SeveralTestTubeBrushes
37degreesCelsiusWaterBathwithTestTubeRacks
60degreesCelsiusWaterBathwithTestTubeRacks

1BagofIce

TheexperimentisstartedbylabelingfivetesttubesA,B,C,DandE.2mLofpH7bufferedONPGis
addedintoeachofthetubes.While5othertesttubesA1,B1,C1,D1andE1,had1mLof8%Beta
Galactosidaseadded.Theywereeachplacedintheirownwaterbathsatdifferenttemperatures.TubeA
andA1wasplacedina5degreeCelsiuswaterbath.TubeBandB1wasplacedinaroomtemperature
waterbath.TubeCandC1wasplacedina50degreeCelsiuswaterbath.AndTubeDandD1was
placedina60degreeCelsiuswaterbath.Thetimesthattheywereadministeredisasfollows:
TubeA&A1:5degreeCelsiusWaterBath

7:43pm

TubeB&B1:RoomTemperatureWaterBath

7:44pm

TubeC&C1:50degreeCelsiusWaterBath

7:44pm

TubeD&D1:60degreeCelsiusWaterBath

7:45pm

Eachtubewascoveredbyparafilmandplacedintheircorrespondingwaterbathfor5minuteseach.
Afterthe5minuteshadpassedthecontentsofcorrespondingtubeswerecombined;AandA1,Band
B1,CandC1,DandD1.TheeffectswereasfollowedandTubeswillnowonlybelabeledasA,B,C,
andD.
Results
EffectofEnzymeConcentrationonProductionofONitrophenol
Tube

Temperature

Intensityof

Absorbance

Yellow

UmolesO

UmolesO

Nitrophenol

Nitrophenol

Produced/5

produced/min

min
A8%

5degrees
Celsius

0.41

102.5

20.5

B8%

RoomTemp.

0.211

52.75

10.55

C8%

50degrees

0.02

Celsius
D%

60degrees
Celsius

10

The

RateofReaction

0
5

20

50

60

TemperatureinCelsius

resultsrecordedprovideevidencethatthecolderthetemperaturewasthentheenzymewasmore
effective.Thereactionwasbestperformedaround5degreesCelsius.However,whenthetemperature
wasabove50degreesCelsiustheenzymebrokeapartandwasineffectiveasitwasdenatured,proven
bytheloweringoftheabsorbancelevelasitclosedinonazeroresult.
Discussion
Myhypothesiswasthatthewarmerthetemperature,thefastertheenzymecatalyzedreactionwould
occur.However,myresultsdisputedmyhypothesisbyprovingthathighertemperaturesdestroythe
enzyme,whilethecoolertemperaturewastheoptimaltemperatureforthisexperiment.Themethods
usedforthisstudywereperfectforwhatwasneededaseachtubehadtheexactamountofbufferand

enzymeandinordertotesttheeffectsoftemperature,atemperaturecontrolledwaterbathwasused.
Anothermethodoftestingtemperaturecouldhavebeenwithheatfromflames.Whatisconcluded
fromtheexperimentisthattemperaturedoeseffectthereactionofanenzymecatalyzedsubstrate.The
coolertemperatureallowedtheenzymetobindtotheactivesitesmorefreely,however,toowarmand
thereactiondoesntoccur.Thetemperaturehastobeoptimalinorderfortheenzymetospeedupa
reaction.
Acknowledgements
IdliketothankmypartnerSchylerThomasforcomingoverandhelpingmegettheresultsnecessaryfor
thisexperimentasIwasabsentthedaywediditandhetooktimeoutofhisdaytomakesureIhad
everythingIneededinordertowriteupthislabreport.IdliketothanktheBiologydepartmentfor
providinguswiththelabroomandmaterialsinordertoconductourexperimentstobroadenour
biologyunderstanding.Last,butnotleast,Mr.BreedloveforbeingaccessiblewhenIneedquestions
answered.
Citation
BiologyDepartment,The.(2010).LaboratoryManuel.4thEdition.Glendale,AZ:Glendale
CommunityCollege.