1. Nathan Cash 1065621 Page 1
Enzyme Kinetics of Polyphenol oxidase
Author: NathanCash
Experimenters:NathanCash& Jacob Doughan
Introduction
Enzymesare specialisedproteinsthatincrease the rate of a reaction,withoutbeingconsumed.
Polyphenol Oxidase isanenzyme thatcatalysesthe oxidationof phenolssuchascatechol.This
enzyme isthe majorcause of brownpigmentationanddiscolouration infruits,includingbanana[1].
Whenmolecularoxygenispresent,PolyphenolOxidase catalysesthe o-hydroxylationof o-diphenols
intocorrespondingo-quinones.Inbananas,catechol formsbenzoquinone,ayellow compound,after
whichbenzoquinone isoxidisedtomelaninasa resultof oxygenexposure.Consequently,the fruit
presentswithabrowncolourationseeninthe bruisingof fruits[2].Inthisexperiment,the kinetics
of Polyphenol Oxidasewere testedbycombiningenzymeextractwithcatechol.Reactionrateswere
measuredthroughspectrophotometry,atechnique whichwasutilisedtodemonstrate absorption
changesinthe solutionascatechol wascatalysedandunderwentcolourchange toyellow,which
was comparedtoa control.Itwas hypothesisedthatif the concentrationof catechol increased,the
absorptionof the solutionwouldalsoincrease.The aimof thisexperimentwastoobtainthe
Michaelisconstant(Km) andthe maximal velocityof enzymereaction(Vmax) of Polyphenol Oxidase.
Methods
1. Polyphenol Oxidase wasextractedfromapiece of bananaby 10g of the fruitbeingground
intoa paste consistencyusingamortar and pestle onice andwas dilutedusing30mLof ice
water
2. The homogenate (PolyphenolOxidase)solutionwasseparatedfromanysolidsand this
solutionwasthentransferredintoa10mL centrifuge tube.Attentiontodetail wasensured
to minimise oxidationof the sample.
3. The centrifuge tube containingthe enzyme extractwasthenplacedina refrigerated
centrifuge (4˚C) at10, 000 x g for 5 minutes.
4. The enzyme wasdilutedtoa 1:4 ratiowithice coldwater,whichresultedina5mL solution.
5. A 96-well microtiterplate wasusedtomeasure the enzymereactions.A 0.1M phosphate
buffer(pH6.8) was addedtowellsA1-A7usinga P200 air displacementmicropipette with
volumesof 178, 176, 172, 165, 150, 140 & 130µL addedinascendingorder(A1-A7).
6. Step6 wasrepeatedforwellsH1-H7.
7. A 200mM catechol stockwas addedtowellsA1-A7usingairdisplacementpipettesaccording
to volume added,eitherP20or P200. Volumesaddedare asfollowsinorderfromA1-A7:
2.0, 4.0, 8.0, 15.0, 30.0, 40.0 & 50.0µL.
8. Step7 wasrepeatedforwellsH1-H7.
9. Simultaneously,one experimenteradded20µL of water (H2O) towellsA1-A7andthe other
experimenteradded20µL of 1:4 enzyme extracttowellsH1-H7.Thisstepwas completedin
undera minute toensure accurate readingsforenzyme reactions.

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10. The microtiterplate wasthenimmediatelyplacedinthe plate readerandthe solutionswere
mixedusingthe shakerfunction(highsettingfor5 seconds).The measurementfilterwasset
to 405nm and absorptionreadingsforall wellswere printed.Resultsindicatedthe
absorptionunitsat1 minute.
11. Resultswere recordedinatable andthe differenceswere calculatedandwells withwater
(A1-A7) versuswellswiththe enzyme extract(H1-H7) were compared.
Results
The resultsshowedinTable 1 that the oxidationof catechol occurredmuchfasterandhence a
greaterabsorbance,whenPolyphenol Oxidase wasadded(wellsH1-H7) comparedtothat of wells
A1-A7 whenonlywaterwasaddedwithoutenzymeextract.WellsA1-A7were utilisedasthe control
inorder to measure the differenceinabsorbance whenenzyme extractisadded(tube H-tube A).
Thismethodgivesanaccurate measurementof absorptionforthe wellscontainingenzyme extract,
rather usinga straightreadingof absorption.Figure 1showedabsorptionunitsof Polyphenol
Oxidase catalysingcatechol inthe firstminutewithincreasingconcentrationsof catechol from
2.0mM to 50.0mM, withthe maximal absorptionbeing0.335Abs/minatsubstrate concentration
(catechol) of 50.0mM. A hyperboliccurve of the data showedthe velocityof the reactionoccurring.
The trend of the reaction beganto plateauwhenPolyphenol Oxidase iscompletelysaturatedin
substrate (Figure 2).Km isdemonstratedinFigure 2, at the half maximal response (0.1675Abs
units/min), the Km value can be estimatedtobe 8mM of catechol substrate.Fora more accurate
readingof Km and Vmax a linearLineweaverBurkplotwasconstructedusingvalues1/V0against1/[S].
Figure 3 showsa lineartrendline forenzyme substrate reaction,the y-interceptwhichis3.74Abs
units(V0) givesanindicationof 1/Vmax therefore Vmax being0.26Absunits/min.Furthermore Km was
calculatedbythe 1/x-interceptwhich wasKm wasfoundto be 3.07mM, whichshowedthe substrate
concentrationatthe half maximal reactionvelocity.
Table 1. Absorption unitsfor solutions
1 minute
Tube Enzyme Catechol
[S] (mM)
Absorption units
(Abs)
Tube H-Tube A
(Difference in Abs
units)
H1 + 2.0 0.175 0.143
A1 - 2.0 0.032
H2 + 4.0 0.122 0.083
A2 - 4.0 0.039
H3 + 8.0 0.260 0.221
A3 - 8.0 0.039
H4 + 15.0 0.296 0.224
A4 - 15.0 0.052
H5 + 30.0 0.351 0.292
A5 - 30.0 0.059
H6 + 40.0 0.334 0.270
A6 - 40.0 0.064
H7 + 50.0 0.403 0.335
A7 - 50.0 0.068

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Figure 1. Absorptionof enzyme extractin1 minute.Linearrepresentationof reactants
Polyphenol Oxidase andcatechol atvarying substrate concentrations 2.0mM-50.0mM
.
Figure 2. V0 (absorptionunitsperminute) plottedagainstsubstrate (catechol) concentration
[S]. Data representedwithahyperboliccurve of enzyme andsubstrate reaction.
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0 10 20 30 40 50 60
V0(Abs/min)
[S]

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Figure 3. LineweaverBurke Plotof Polyphenoloxidase. Thischartgivesa linearprojectionof
V0 plottedagainst[S] togive a more accurate value forVmax and Km.
Discussion
The activityof Polyphenol Oxidase increasedwiththe amountof substrate itreactedwith,by
increasingthe concentrationof catechol the activityof Polyphenol Oxidaseincreased.Thisbecame
evidentwhenthe dataobtainedfromthe spectrophotometryof the solutionshowedanincrease in
absorptionwhenthe catechol concentrationwasincreased,whichreflectsenzyme activity(Figure
2). Like all enzymes,PolyphenolOxidase isaffectedbytemperature andpH,factorswhichwere
accountedforusinga 0.1M phosphate bufferwithapHof 6.8 inall solutionstomaximiseenzyme
activity.The volume of phosphate bufferaddedincreasedwithincreasedvolumesof catechol
substrate,at50.0mM catechol substrate,130µl of 0.1M phosphate bufferwasaddedtothe solution.
The optimumpH level forPolyphenol OxidasevariesindifferentfruitsrangingfrompH4.0-8.0 [2],
usinga 0.1M phosphate bufferwithapH of 6.8 coincideswithotherstudiesshowingoptimumpH
for BananaPolyphenol OxidasebetweenpH6.5 - 7.0 [2][5] [4].
In bananatissue,PolyphenolOxidase isfoundinthe cytoplasmof cellsandcatechol iscontained
withinvacuoles[3].Whentissue damage occursasa resultof bruisingorinvasionof other
organisms,o-quinone productionservestoseal off the areaand acts on enzymesproducedby
pathogenstoultimatelyinactivatethemandpreventinfectionof the fruit[3].Preparationof the
enzyme extractatcold temperatureswasusedtokeepPolyphenol Oxidase fromreactingwith
catechol whenthe vegetativetissueisdisturbed.The optimumtemperature atwhichPolyphenol
Oxidase catalysescatechol is30˚C[2].Obtainingthe enzyme extractwascarriedoutwithbanana
groundin a mortar on ice and thenspunina centrifuge at4˚C to preventanyreactionof catechol
that may have beenpresentinthe enzymeextract.
Errors in the experimentwere made whichcanbe seeninFigure 1. Theoretically,the absorption
shouldincrease withincreasingconcentrationof catechol,althoughat2.0mM and30.0mM
substrate concentrationthe absorptionvaluesdidnotcorrespondwiththe trendof the data.This
couldpossiblybe due to incorrectmeasurementsof substrate beingaddedtowellsandthenhaving
y = 11.331x + 3.7474
0
2
4
6
8
10
12
14
-0.5 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6
1/V0
(Absunits/min)
1/[S] (mM)

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to be removed,polluting wellsH1& H5. These valuescanbe overall seenasoutliersasthe majority
of the data followedatrend.The foremostmethodof obtainthe Kmvalue istouse the data from
Figure 3. Thisshowedthatthe Km of polyphenol oxidaseoccuredat3.07mM of substrate andthat
the maximumvelocityof enzyme reactionwas0.26 Absunits/min.Thisvalueof Vmax contradicted
data, as at 50.0mM substrate concentration, the absorptioncanbe seentobe 0.335Abs (Table 1).
Therefore,amore accurate value of Vmax was obtainedthroughanalysisof Figure 2,wherebythe
enzyme reactionbegantoplateau at0.35 Abs units/min(Vmax).Inastudyof Polyphenol Oxidase
frombanana pulpbyYang, C.P etal [4] oxidation of substrate byPolyphenol Oxidasewasmeasured
inthe same wayonlyusinga varietyof substrates.Thisstudyshowedthatdopamine wasrapidly
oxidisedbyPolyphenolOxidase withaKmof 2.8mM [S],and uponadditionof catechol,the
oxidationrate washalf thatof dopamine.The studyconducted,showedthatthe oxidationrate of
catechol wasnot quite half thatof dopamine inthe literature[4] buthad a lowerspecificityfor
catechol witha Km of 3.07mM [S].These resultsdiffersubstantiallyfromthatobtainedin[5] where
avocadoPolyphenol Oxidase wasmeasuredandcatechol wasshowntohave a higherenzyme
activitythanthat of DL-DOPA substrate.
In conclusion,techniquessuchasspectrophotometrywere utilisedtomeasure the enzyme kinetics
of polyphenol oxidaseiseffective,asthe catalysisof catechol intoo-quinoneschangesthe solution
fromtransparentto yellowwhichcanbe measuredusingasetwavelength(405nm).Overall,the
data collectedinthisexperimentshowedthatanincrease incatechol substrate concentration
resultedinincreasedenzymeactivityof Polyphenol Oxidase untilPolyphenol Oxidaseisfully
saturatedinsubstrate at whichVmax occurs (0.35abs units/min)withahalf maximal velocity(Km)of
3.07mM [S],valueswhichcoincidewithsimilarstudies.

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1. Gooding PS, Robinson SP, Bird C. Molecular cloning and characterisation of banana
fruit Polyphenol oxidase. Planta.September2001;213:5-748-757
2. Umit Unal M. Properties of polyphenol oxidase from Anamur banana (Musa cavendishii).
Food Chemistry 2007; 100:3-909–913
3. Wuyts N, Waele DD, Swennen R. Extraction and partial characterization of polyphenol
oxidase frombanana(Musa acuminata Grande naine) roots.Plantphysiol andbiochemistry.
2007; 44:5–6:308–314
4. Yang CP, FujitaS, AshrafuzzamanM,NakamuraN, Hayashi N.J AgricFood Chem.
2000;48:2732-5.
5. Gomez-LopezVM. Some biochemical propertiesof polyphenoloxidase fromtwovarieties of
avocado. Food Chemistry. 2002; 77:2:163–169