Incertainfruits,suchaspineapplesandmangoes,thefleshcontainsprotein-digestingenzymes(proteases).Thesemayplayapartinhelpingtosoftenthefruittissuesasthefruitripens,makingitevenmoreattractivetoanimalsthatmightdispersetheseeds.Soperhapstheactivitiesoftheseproteasesenzymeswillincreaseduringtheripeningprocess.

Applicationsofplantproteases,suchas‘bromelain’fromthestemsandfruitsofpineapples,includeusesinthepharmaceuticalindustryasabloodanti-coagulant,andinthepreventionofproteinaceoushazesinchill-proofbeers!

Theassay

Preparationoftheeggalbumencolloidalsuspension(enzymesubstrate)

• Separatethewhiteofasingleeggintoa250cm³beakerandadd150cm³tapwater,stirringthemixturethoroughly.Themixturebecomesquitecloudyasaresultofthedenaturationofsomeoftheeggalbumenbythewater.
• Placethebeakeronatripodandgauzeandheatwithabunsenuntilthemixtureboils,stirringitregularly.
• Allowthemixturetocool,thendecantitthroughtwoorthreelayersofmuslinintoanotherbeaker.Thiscreatesahomogenousmilkycolloidalsolution.

Carryingoutthereaction

• Selectatest-tubethatfitsintothecolorimeter.Add2cm³ofanappropriatebuffer(e.g.pH7),followedby2cm³ofthealbumensubstratesolutionand1cm³offruitextract.
• Mixthecontentsofthecolorimetertube,andplaceitinthecolorimeter(previouslyzeroedusingatubewith4cm³bufferand1cm³offruitextract).Readtheabsorbanceandnotethetime.
• Placethereactionmixture(s)inawaterbathat30°Ctopromotetheactivityofanyproteaseenzymespresent.
• TakefurtherabsorbancereADIngsatregulartimeintervals(sayevery5or10minutes),untilnofurtherchange(decrease)inabsorbanceisdetected.
• Plotagraphofthechange(decline)inabsorbanceagainsttime.Measuringthetimetakenfora50%reductioninabsorbancevaluegivesanindicationoftheproteaseactivityoftheoriginalfruitextract.

Ideasforinvestigationswiththissystem(withfruitsandvegetables)

• Followthechangesinproteaseenzymesduringripening
• ComparetheeffectsofpHonproteasesfromdifferentfruits
• Comparetheeffectsoftemperatureonproteasesfromdifferentfruits
• Investigatethepresenceofprotease-inhibitorsintheseedsoflegumes
• Monitorthereleaseofaminoacidsfromthedigestedproteins,usingpaperchromatography

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