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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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