Protease assay
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Proteaseassay
Incertainfruits,suchaspineapplesandmangoes,thefleshcontainsprotein-digestingenzymes(proteases).Thesemayplayapartinhelpingtosoftenthefruittissuesasthefruitripens,makingitevenmoreattractivetoanimalsthatmightdispersetheseeds.Soperhapstheactivitiesoftheseproteasesenzymeswillincreaseduringtheripeningprocess.
Applicationsofplantproteases,suchas‘bromelain’fromthestemsandfruitsofpineapples,includeusesinthepharmaceuticalindustryasabloodanti-coagulant,andinthepreventionofproteinaceoushazesinchill-proofbeers!
Theassay
ProteaseenzymesareaddedtoamilkycolloidalsUSPensionofeggalbumen.Astheproteaseenzymesdigestthesuspendedparticlesofproteins,themixturebecomesmoretransparent.Theabsorbancechangesinthereactionmixturearefollowedwithacolorimeter.Theproteasecontent,forexampleinextractsoffruits,canbeassayedbymeasuringtherateatwhichthesolutionofeggalbumenandextractbecomesclearer. |
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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