Name/Group#
CANOHilA
Date
StudentGuide
AP'Biology Laboratory2
EnzymeCatalysis
Objectives
.
Observe the action of an enzyme
.
Determine the rate of an enzyme-catalyzed reaction
.
Study the characteristics of an enzyme-mediated reaction
.
Observe the effect of heat on enzyme activiry
Background to Activity A
In this lab, you will study somecharacteristicsof enzymeaction.The specificreactionyou will
investigateis the decompositionof hydrogenperoxideby the enzyme,catalase.At room temperature,
hydrogenperoxidevery slowlydecomposes
into water and oxygen.The addition of catalaselowersthe
activationenergyof the reactionuntil it proceedsswiftly at room temperature.At the end of the
reaction,the catalaseis unchangedand is availableto catalyzethe reactionof more hydrogenperoxide.
Catalase,like most enzymes,is a protein. Its abiliry to form a complexwith hydrogenperoxideis based
on its molecularshape.Any factor that can alter the shapeof a protein moleculecan be expectedto
impact the ability of catalaseto facilitatethe decompositionof hydrogenperoxide.
Activity A: Observing the Reaction
Materials
H2O2in labeledcup, catalaseon ice, unlabeled60-mL cup, test tube, test tube holder,test tube rack,
syringelabeledHtOr, transferpipets,glassrod, scalpelor other sharpinstrument,pieceof potato or liver,
boiling water bath, WashWatercup, Wasrecup.
Caution: Youwill be workingwith cmdaroundhot liquids.YouwiII usea sharpscalpelor otherinstrumentto
cut the cubeof potatoor lioxr.
Introduction
In Activity A, you will makeyour first observationsof the interactionof catalaseand hydrogenperoxide.
@20 05 Car olina Biologic al
Supply C o m p a n y
9-1
Procedure
Testingfor enzymeactiuity
1. Usethe labeledH2O2sy:irnge
to transfer10mL of H2O2into the unlabeled
6O-mLcup.Usea
transferpipetto add 1 mL of catalase
solutionto the unlabeled
cup.Observefor 30 to 60 seconds.
Do youobserve
anythingthat indicatesthat a chemicalchangeis takingplace?If so,explain.
2. On the basisof your observationof the reactionand your knowledgeof chemistrywrite a balanced
equationfor the reaction.
3. Give the followingfor this reacrion:
a. Substrate:
c. Intermediate complex that formed (not observed):
d. Product(s):
The effect of boiling on enzyme actiuity
1' Use the sametransferpipet (not theH2O2 syringe)to transfer3-5 mL of catalaseto a test tube.
Placethe test tube in a boiling warerbath for five minures.
2. While waiting,rinseyour unlabeledcup. Use theH2O2 syringeto transfer10 mL of H2O2into rhe
rinsedunlabeledcup. Use a clean,unusedtransferpipet to add 1 mL of the boiled caralaseto the
unlabeledcup. Observethe results.What is the effectof boiling the caralase?
Explain how boiling
affectscafaIase
activirV.
3. List at least three other factors that could affect the activiry of the catalase and explain why they
would have an effect.
O20 05 Car olina Biologic al
Supply C o m p a n y
5-2
Testing for catalase inliving
tissue
1. Rinseyour unlabeled6O-mLcup. Use a scalpelor other sharpinstrument to cur a cube about one
cm on a sidefrom a pieceof potato or liver. Placethe cube in the unlabeledcup and macerarewith a
glassrod. Caution: Do not usesomuchforcethatyou breaktherod.
2. Use the H2O2syringeto add 10 mL of H2O7to the cup of tissue,and then observe.Doesthe tissue
contain catalase?
Explainvour answer.
Background to Activities B, C, and D
In Activity B, you will determinethe amount of HrO, initially presentin solution.In Activity C, you will
determinethe rate at which H2O, spontaneouslydecomposes
at room temperature,and in Activity D, you
will determinethe rate at which catalasedecomposes
HrO2. In all of theseactivities,you will need to
determinethe amount (concentration)of HrO2 left in solution.Youwill make this determinarionby usinga
titration procedure.In a titration, you usea solution of known concentrationto determinethe
concentrationof an unknown. An examplewould be usinga soludonof 0.1 N O.Jormal)NaOH to
determinethe concentrationof an unknown acid.To do the dtration, you might put 5 mL of the acid in a
beakerand add a pH indicator.The indicator is yellow in an acid but becomesgreenar a pH of 7 (neutral).
Then you add 0.1 N NaOH drop-by-drop,stirring betweendropsuntil the solution in the beakerturns
greenand remainsgreen.This is the endpoint of the dffation. The NaOH hasreactedwith (neurralized)all
of the acid in solution;therefore,the amount of NaOH usedto reachthe endpoint is directly proportionalto
the amount of acid that wasin solution.Let us saythat it took 25 mL of 0.1 N NaOH ro rirrate to the
endpoint.Then the concentrationof acid in solution is equivalentto 25 mL of 0.1 N NaOH.
In the followingactivities,you will use2o/opotassiumpermanganate(KMnOo) ro tirrare H2O2in
solution.As potassiumpermanganate(darkpurple) is addedto the H202 solution,the permanganate
ion (MnO*-) is reducedand becomescolorless.When all the H2O2in solutionhas reacted,the solution
becomesa persistentpink or pale brown. This is the endpoint of the dffation. Youwill usethe volume of
KMnOa neededto titrate to the endpoint as a measureof the amount of HrO, presentin the solution.
Titration
Protocol
The titrations can be done with a 5- or 6-mL titration syringe(a syringewith a pieceof Tygon@
tubing
attached)or with a buret. If you are usinga buret, fill it with KMnOa beforebeginning.If usinga
titration syringe,draw in about 0.2 mL of air, then about 5 mL of KMnOa. If you do not draw in the air,
it will be almostimpossibleto read the level of KMnO* from the scaleon the syringe.
1. Recordthe initial volume readingfor the titration syringeor burer.
2. Add one drop of KMnOa to the solution in your ritration cup. Gently swirl the cup until the purple
color disappears.
3. RepeatStep 2 until the solution in the cup becomesa persistentpink or brown; that is, continue
until the pink or brown color persistsafter swirlingthe cup. This is the endpoint of the titration.
4. Once you have reachedthe endpoinr,record the final volume of KMnO*.
5. Rinsethe titration syringeby drawingup water from your WashWatercup and expellingit into the
Wastecup.Rinseyour other equipment,also.
@2 00 5 Car olina Biologic al
Supply C o m p a n y
5-3
Activity B: Determining the Initial (Baseline)Amount
of H,O, in Solution
Materials
H2O, in labeledcup, H2SOain labeledcup, KMnOa in labeledcup, cup labeledBaseline,
cup labeled
Titration,syringelabeledHtOr, syringelabeledHrSO+,syringelabeled'Iiansfer,rransferpiper, titration
syringe,WashWatercup,Wastecup, distilledwarerin cup labeleddH2O.
Caution: Use Extreme Care When Handling Acids. H2SO4can irritate or bum the eyes,skin,and mouth.
Auoid all skincontactwith thisand other chemicals.
YourteacherwiII instructyou abouttheproper safety
procedures
handlinghazardous
materials.KMnOo can stain skinand clothes.H2O2can discolorclothes.
for
Introduction
In this activity, you will use the Titration Protocol to determine the initial amount of H,O, present in
solution. You will use this amount as a baseline for activities C and D.
Procedure
Preparation of Baseline Sample
1. Usethe syringelabeledH2O2to put 10mL of H2Orin the 60-mLplasticcuplabeledBaseline.
2. Usethe plastictransferpipetto add 1 mL of distilledwaterfrom thedHrO cupto the cuplabeled
(Distilledwateris usedto replacethe 1 mL of catalase
Baseline.
that is addedto rheHrOr in
Activities A and D.)
3. Use the syringelabeledH2SO1to add 10 mL of 1 M H2SO4from the H2SO4cup ro the cup labeled
Baseline.
4. Gently swirl the Baseline
cup to mix the conrenrs.
5. Use the 5- or 6-mL syringelabeledTiansferto remove5 mL of solution from the cup labeledBaseline
and put it in the cup labeled Titration. Rinse the Tiansfersyringeby drawing up somewater from the
WashWatercup and expellingit into the Wasrecup.
6. Titrate your 5-mL sample(referto the Titration Protocol) to determinethe baselineamount of
HrOr. Recordyour data in Thble 1 below.
Table 1: Baseline
InitialVolume
mL
F i n a l V ol ume
mL
A Volume
mL
A Volume= changein volume
(lnitial- Final)for titrationsyringes;(Final- lnitial)for burets
7. You will usethe value of A Volumefrom Table 1 asthe baselineamount of H2O2presenrin soludon.
Recordthe baselinebelow.
Baseline=
0 20 05
mL
Car olina Bjologic al
Supply C o m p a n y
9-4
Activity C: The Uncatalyzed Rate of Decomposition of HrO,
Materials
H2O2in labeledcup, H2SO4in labeledcup, KMnOa in labeledcup, cup labeledUncatalyzed
Decomposition,
cup labeledTitration,syringelabeledHrOr, syringelabeledH2SOa,syringelabeled
'Iiansfer,titration syringe,transferpipet,
WashWater cup,Wastecup,distilledwater in cup labeled dH2O,
cup labeledH2O, Overnight.
Introduction
In this activity,you will determinethe rate at which HrO2 spontaneously
decomposes
when exposedto
room temperaturesand ambientlight {or a 24-hourperiod.
Preparation
Use the H2O2 syringeto transfer15 mL of H2O, from the H2O2 cupto rhe 60-mL plasticcup labeled
H2O2Ouernrght.
Store the cup uncoveredat room temperaturefor approximately24 hours.
Wait24 hoursbeforeproceedingwith ProcedureSrep 1, below.
Procedure
1. Use the syringelabeledHrO, to transfer10 mL of H2O2from the H2O2C)vemrghr
cup to the 6O-mL
plastic cup labeled Uncatalyzed D ecompo sition.
2. Use the transferpipet to add 1 mL of distilledH2O from the dH2O cup ro the cup labeied
(Distilledwater is usedto replacethe 1 mL of caralasethat is addedto
Uncatalyzed
Decomposition.
the H2O2in activitiesA and D.)
3. Use the 10- or 17-mLsyringelabeledH1SO4to add 10 mL of 1 M H2SO4from the labeledH2SO4
cup to the cup labeledl)ncata\zedDecomposition.
4. Gently swirl the UncatalyzedDecomposition
cup ro mix the conrenrs.
5. Use the 5. or 6-mL syringelabeledTransferto remove5 mL of the reactionmixture and put it in the
cup labeled Titration. Rinse the 'Iiansfer syringeby drawing up warer from the WashWatercup and
expellingit into the \X/asre
cup.
6. Titrate your sample(referto the Titration Protocol) to determinerhe amount of H2O, left in
solution after24 hours.Recordvour dara in Table2 below
InitialVolume
mL
FinalVolume
mL
A Volume
mL
Table 2: Uncatalyzed
Decomposition
The amount o{H2O2 that hasdecomposedis equalto the Baselinedeterminedin Activiry B minus
A Volumefrom Table2. The amount of H2O2that spontaneously
decomposedat room temperarure
mLl24 hours.
0 20 05
Car olina Biologic ai
Supply C o m p a n y
5-5
Activity D: The CatalyzedRate of Decomposition of HrO,
Materials
H2O2in labeledcup,H2SOa
in labeledcup,KMnOoin labeledcup,catalase
on ice,cuplabeledTitration,
syringe
labeledHrOr, syringe
labeledH2SOa,
syringelabeledTiansfer,
titrationsyringe,
piper,
rransfer
WashWater
cup,Waste
cup,andcupslabeled
respectively
10 sec,30
sec,60sec,120sec,
andIBOsec.
Introduction
In this activity,youwill determinethe rateat whichcatalase
decomposes
HrOz.To do this,youwill add
catalase to a measured amount of H2O2, and after a predetermined lapse of time (10 sec, 30 sec, 60 sec,
etc.) you will add H2SO4 to halt the reaction. Once the reaction is stopped, you will titrare ro derermine
the amount of H2O2 left in solution. You will subtract this amount from the baseline that you
determined in Activity B to determine the amount of H2O2 decomposed over the timed interval. You
will graph the resulting data and calculate the rate at which catalase was decomposed by catalase.
Read the following directions thoroughly before beginning.
Procedure
Lineup the 60-mLplasticcupswith the labels10 sec,30sec,60 sec,120 sec,and lB0 sec.Usethe syringe
labeledHtC),to add 10mL of H2O2to eachcup.
Beforebeginningeachtest, premeasure10 mL of H2SOain the H2SO4syringe,so you can stop the
reactionpreciselywhen required.
One studentshouldadd the reagentswhile another studentkeepsthe dme.
For the lO-secondtime trial:
1. Use the transferpipet to add 1 mL of catalaseextract to the 1Oseccup.
2. Gently swirl the l0 seccup to mix the contenrs.
3. At 10 seconds,
add 10 mL of H2SO4to rhe cup.
For each time trial, repeatstepsI through 3, asabove,but allow the reactionsto proceedfor 30, 60, l2O,
or 180 seconds,as assigned,
beforeaddingthe 10 mL of H2SOa.
Titrating
the Time Trials
Use the rinsedsyringeto remove5 mL of solution from the l0 secreactioncup and placeit in the plastic
cup labeled Titration. Rinse the Tiansfersyringeby drawing in water from the WashWatercup and
expellingit into theWastecup. Titrate accordingto the Titration Protocol.If you overshootan
endpoint,you will have enoughsolutionfor a secondtitration. Recordyour resultsin Thble3. Repeatfor
each assignedtime trial.
02 00 5
Car olina Biologic al
Supply C o m p a n y
9-6
Table 3: CatalyzedDecomposition
10 Sec
30 Sec
60 Sec
120Sec
180Sec
lnitial
Volume
Final
Volume
A Volume
HzO,
Decomposed
(BaselineA Volume)
Analysis of Results,Activity D: The CatalyzedRate of Decomposition of Hror.
1. Vhy wasit necessary
to determinea baselinefor the HrO,?
2. Why did the addition of H2SOastop rhe reacrion?
3. Graph your data from Table3 for H2O2decomposedby catalase.Title the graph and supplythe
following information:
a. The independentvariableis
b. The dependentvariableir
.
Plot the independentvariableon the x-axis,and the dependentvariableon the y-axis.
4. \Uhat doesthe line on your graph represenr?
@2 00 5 Car olina Biologic al
Supply C o m p a n y
5-7
5. What does your graph tell you about the rate of the reaction over time?
6. When an enzymeis mixed with an excessof its substrate,it will react with the substratear a
constantrate. The reactionwill continue at the sameconstantrate as long as the substrateis
presentin excess.As the substrateis consumedby the reaction,there will be lesssubstrateavailable
to react with the enzyme,and the rate of the reactionwill slow.The rate of the reactionwhile the
substrateis presentin excessis calledthe initial rate. At a given temperatureand pH, the initial rate
of a given enzyme/substrate
reactionwill alwaysbe the same.
Look at your graph.The initial rate of the catalaseft1r.O,reacrionis represenredby the linear
(straight)portion of the line on your graph.Usingyour graphand dara,calculatethe rate of the
catalaseA{rO2reactionfor the following time intervals:O-10 sec,1O-30sec,30-60 sec,60-120 sec,
and 120-180sec.This is easilydone by referencingyour graph.For example,supposeyour graph
showsthat 0.1 mL of H2O2had beendecomposed
at 10 seconos.
Reaction Rate (ml/sec)
:
Yz - Yt
or
X z-X l
Ay
Ax
\7here Yr : 0.0mL
Yz: 0.1mL
:Osec
^1
x2 : 10sec
then
:
ReactionKate
0.1mL
10sec
0.01mlisec
Calculatethe resultsfor your data. Recordyour resultsin a Data Thblethat you constructbelow.
7. On the basis of your results, what is the initial rate of rhe catalaseftfrO, reacrionl
0 20 05
Car olina Biologic al
Supply C o m p a n y
5-O
Tiile:
O20 05 Car olina Biologic al
Supply C o m p a n y