Polyphenoloxidase(catecholoxidases)assay

Browningofthecutsurfaceofsomefruitsandvegetablesisduethepresenceofagroupofenzymescalledpolyphenoloxidases.Theseenzymesarereleasedbythebrokencellsandtheycatalysethereactionbetweencolourlessmoleculescalledpolyphenolsandmolecularoxygen.Thisreactioncreatescolouredcompoundsandthesenewcompundscanspontaenouslycrossreactwithoneanothertoformblack-browncomplexescalledmelanins.

Oneexampleofasubstratefortheseenzymesiscatechol,hencethealternativename‘catecholoxidases’fortheseenzymes.Catecholisoxidisedinitiallytotheorangecompoundbenzoquinonewhichisthenconvertedtomelanins.Theconversiontomelaninisspontaneousbutslow.

Foodprocessingandcookingofteninvolveprocedureswhichareintendedtoinhibittheactionofpolyphenoloxidases.Whydoyouthinkacookimmediatelyplacesfreshlypeeledpotatoesintoapanofwater?Orwhydopeoplesqueezeafewdropsoflemonjuiceontoafreshlycutavocado?Mushroomscontainhighlevelsofpolyphenoloxidases,sohowdoyouthinkpre-slicedpackagedonescanbepreventedfromgoingbrown?

Theassay

• Makeanextractoffruitorvegetable,bygrindinginamortarorblendingwithanequivalentmassofwater.
• Straintheextractthroughmuslin,thencentrifugethefiltratetoremovetheremainingsolids.(Alternatively,thefiltratecouldbefilteredusingaBuchnerfunnel,orjustallowedtostand-sothesolidsformasedimentatthebottomofthevessel.Thendrawtheliquidoffintoanothervessel.)Notethatiftheplanttissuescontainmuchpolyphenoloxidaseactivity,thentheextractitselfwillbecomequitedarklycoloured—butthisneednotbeaproblemasonlyaverysmallvolumeisusedinthereactionmixture.Theenzymeactivityoftheextractlastsforatleastacoupleofdays,provideditisstoredinarefrigerator.
• Toprepareacolorimetertube,add2cm³standardpH7bufferand2cm³of0.1%catechol.Notethetimeandthenadd0.1cm³enzymeextract,quicklymixingthecontentsofthetube.Placeitintoacolorimeter(previouslyzeroedusingatubewith4cm³waterand0.1cm³oftheextract).TakereADIngsoftheabsorbanceatregularintervals(e.g.every10seconds).
• Plotagraphofthechangeinabsorbanceagainsttime.Anincreaseinabsorbanceisduetotheformationofbenzoquinone,theproductofthereaction.Theinitialslopeofthegraphgivesameasureofthepolyphenoloxidaseactvityofthefruitorvegetableextract.

Ideasforinvestigationsintotheactivityoftheseenzymesinplanttissues

• theeffectsofpH,orionicconcentration,ortemperature
• theeffectsofplantpathogeninfection
• theeffectsofenzymeinhibitors,suchasmetalions
• theeffectsofanti-oxidantchemicals

Alternativetechniquesformonitoringtheprogressofthereactionincludea‘low-tech’method,suchasfollowingthereactionbyallowingtheformationofmelaninsovernight,ora‘high-tech’method,byfollowingtheuptakeofdissolvedoxygenfromareactionmixtureusinganoxygenelectrode.

Formoreinformation,youwillfindsuggestionsforexperimentswithpotatopolyphenoloxidasesathttp://food.oregonstate.edu/ref/plant/weaver/.TypingthesearchtermpolyphenoloxidaseonWWWsearchengineswillprovideplentyofhits.

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