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Banerjee

TheEffectofSalicinonEnzymeElastase
By:SumonaBanerjee
NilesNorthHighSchool

Banerjee

TableofContents

Title.....1
TableofContents....2
Acknowledgements.........3
Purpose....4
Hypothesis...5
Variables......6
ReviewofLiterature....7
Materials........18
Procedure...19
Results....
.
..25
DataAnalysis....33
Discussion.....36
ExperimentalError....38
Conclusion.40
Impact....42
ReferenceList.......43

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Acknowledgments

IwouldliketothankMrs.Camel,Mr.Gaynes,&Mr.Margolisforsupportingandmentoringme
extensivelythroughoutthisproject.IwouldalsoliketothankMrs.Camel,Mrs.PosnockandMr.
ThielsenforstayingmanyhoursinthelabsothatIcouldfinishconductingresearch.Also,I
wouldliketothankMrs.Franceforhelpingmestatisticallyanalyzemydata.

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Purpose

Thepurposeofthisprojectistodetermineifsalicin,producedfromwillowtreebark,hasthe
abilitytoinhibitenzymaticelastaseactivityandeventuallybedevelopedintoanothermethodof
treatmentforemphysema.

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Hypothesis
Ifelastase(enzyme)reactedwithdifferentconcentrationsofelastin(substrate)istreatedwith
salicin,thentheconcentrationofelastinwithnoenzymewillbethesameaselastinreactingwith
boththeenzymeandsalicin.Thisisbecausesalicinhasantiinflammatorypropertiesthatwillbe
abletoinhibitelastasefrombreakingdownelastinandchangingitsconcentration.

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Variables

IndependentVariable
:Concentrationofsubstrateputintoeachassaywellatthebeginningof
theexperiment.

DependentVariable
:Absorbanceat405nm

ControlGroup
:Wellswithinhibitorincludedinthekit(elastatinal)

NegativeControl:
Wellswithoutanyinhibitororenzyme

Constants
:Amountofsalicin,amountofdistilledwater,amountofelastase,etc.

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ReviewofLiterature

Heartdiseaseandcancerareknowntobetwooftheleadingcausesofdeatharoundthe
world.(CDC,2014)ButwhataboutCOPD?ManypeopledonotknowthatChronicObstructive
PulmonaryDiseaseisthethirdleadingcauseofdeathinAmerica.Approximately12million
peopleintheUnitedStateshavebeendiagnosedwithCOPD,andmanymoremaybeaffected
butunaware.(AmericanLungAssociationCOPDFactSheet,2014)Butwhydoesthecommon
mannotknowaboutCOPD?Itisequallyasdeadlyasknowndiseasessuchascancer,yetit
remainsrelativelyunstudied.Inordertodirectthepublicsattentiontothistopic,itisextremely
importanttoresearchandstudythisconditiontofindaneffectivemethodfortreatment.
ChronicObstructivePulmonaryDisease:EmphysemaandChronicBronchitis
ChronicObstructivePulmonaryDisease,otherwiseknownasCOPD,consistsoftwo
separatediseases:emphysemaandchronicbronchitis.Chronicbronchitisiscausedbythe
buildupofdustinthebronchialtubes.Whensomeonebreathesin,theytakeindustparticlesand
otherthingsalongwiththeair.Theseparticlescauseirritationwithinthelungs.Fortunately,the
airwaysofthelungshaveaspecialmucusliningtocapturethisdustandfilteritout.However,
whenapersonsbronchialtubesarechronicallyirritated,theystayinflamedandthedustdoes
notfilterout,makingithardtoexhale.Thisirritationalsoimpairsthecilia,hairsinthebronchial
tube,thusresultinginthethickeningofthemucus.(COPD,n.d.)Symptomsofchronicbronchitis
arewheezing,cough,dyspneaandphlegm.Phlegmistheliquidhypersecretionbythemucous
membrane.Emphysemaoccurswhenthealveolarsacsloseelasticity.Thealveolarsacsaretiny
sacswithinthelungsthatallowoxygenandcarbondioxidetomovewithinthelungsand
bloodstream.(AlveoliFunction,n.d.)Whensomeonenormallybreathestheairgoesdownthe
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lungsandendsupinalveoli.Thealveolihelpsmakethelargestpossiblesurfaceareasothat
oxygencanmovethroughthelungsandintothebloodstream.Whentheairsacksbeginto
inflametheybecomeweakandlessflexible,thuscausingtheirsurfaceareatoreduce.Thisalso
resultsintheinadequateexchangeoffreshoxygen.Acollapsedlung,heartproblemsandlarge
holesinthelungsaresomecomplicationsthatcanbearesultofemphysema.Thesymptomsof
thisdiseasearegreatlysimilartochronicbronchitis.(COPD,n.d.)

Figure1:SpirometerDiagram
Furthermore,thereareseveralwaystotestforCOPD.Spirometryisthemostcommon
andeffectivemethodtodiagnoseemphysema,chronicbronchitisandotherlungdiseasessuchas
asthma,pulmonaryfibrosisetc.Thistestmeasuresthevolumeandthespeedoftheairthatcan
beexhaled.Whenoneisbeingtestedbyspirometry,theirnoseisclippedandtheyareaskedto
takeinafullbreathandthensealtheirlipsaroundthemouthpieceofthespirometer.Noseclip
andmouthpieceareshowninfigure1.Theyarethentoldtoblowoutasfastandasmuchairas
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theycanuntiltheirlungsareempty.(Seheult,2014)Theamountoftotalairexhaledintothe
spirometerchamber,ortheforcedvitalcapacity(FVC),isthenmeasured.SinceCOPDisan
obstructivediseaseitwillimpedethepersonsabilitytoreleaseairinstantaneously.So,afterthis
stephasfinished,thepatientisthenaskedtotakeanotherdeepbreathandblowintothe
spirometeroneagain.However,thistime,theamountofairexhaledwillonlybemeasuredfor
onesecond.Thiswilldeterminetheforcedexpiratoryvolumeinonesecond(FEV
).Finallyin
1
ordertodeterminethepersonspreciselungfunctionality,theFEV
isdividedbytheFVCto
1
findtheFEV
/FVCratio.Ifthemeasurementdeterminedislessthan0.7,thenthisisan
1
indicationofalungcomplicationandpossiblyCOPD.Spirometrycanalsoassessiftreatmentis
openinguptheairways.Thisiscalledreversibility.Thesereadingstendtoimproveifthe
airwaysbecomewideraftermedication.Afterthepersonhasbeengivenabronchodilator,the
severityofCOPDisdeterminedbyreversibilitystrictlybasedontheFEV
.
1
A. MildCOPDFEV1is80%ormoreofthepredictedvalue.Thiseffectivelymeansthat
someonewithmildCOPDcanhavenormalspirometryafterbronchodilatormedication.
B. ModerateCOPDFEV1is5079%ofthepredictedvalueafterabronchodilator.
C. SevereCOPDFEV1is3049%ofthepredictedvalueafterabronchodilator.
D. VerysevereCOPDFEV1islessthan30%ofthepredictedvalueafterabronchodilator.
(SpirometryNWMedicine,n.d.)
AdiagramofthespirometerisshownaboveinFigure1.(NuffieldFoundation,n.d.)
AnotherwaytodiagnoseCOPDisthroughthearterialbloodgastest.Thistestmeasures
thelevelsofoxygenandcarbondioxideinthebloodfromanartery.Toperformthistest,blood
samplesaretakenfromeithertheradialarteryinthewrist,femoralarteryinthegroin,orthe

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brachialarteryinthearm.
AbnormalresultsmaybeduetoCOPD.(
Seheult,2014)

Normal
resultsofthistestinclude:

A. Partialpressureofoxygen(PaO2):75100mmHg
B. Partialpressureofcarbondioxide(PaCO2):3842mmHg
C. ArterialbloodpH:7.387.42
D. Oxygensaturation(SaO2):94100%
E. Bicarbonate(HCO3):2228mEq/L
Note:mEq/L=milliequivalentsperlitermmHg=millimetersofmercury(Hadjiliadis,2014)
ToelaborateonthecausesofCOPD,accordingtoalongtermstudydoneby
researchersinSweden,smokingisrecognisedasthemostimportantcausativefactorofit.
Fromthisstudy,itwasreportedthatapproximat
elyfiftypercentofsmokerseventually
developCOPD.Cigarettesmokecontainsseveralharmfultoxinssuchastar,arsenic,benzene
andmanymorethataffectlungfunctionality.Longtermexposuretothesetoxinscanleadto
highlevelsofabnormallungirritation,causingCOPD.Thealveoliinthelungsalsoinflame
asaresponsetothetoxinsincigarettesmoke(EurRespir,2006).TotaldeathsfromCOPD
areprojectedtoincreasebymorethan30%inthenext10yearswithoutinterventionstocut
risks,particularlyexposuretotobaccosmoke(WHO,2013).
Unfortunately,thereisnoeffectivecureforbothchronicbronchitisoremphysema,
buttorelievesymptoms,doctorsprescribebronchodilatormedications,steroidsorantibiotics.
Theyalsosuggestthatthepatienttolookintooxygentherapy,surgeryorpulmonary
rehabilitation.ABiPaPoranendotrachealtubeareoftenalsogiventoimproveventilation

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whilesleeping.Thesemanagementoptionsarebeneficialbecausetheyalsoimproveexercise
ability,sleepandthecognitiveperformanceofthepatient.(Celli,2004)
PathophysiologyofEmphysema
Toexpandonemphysema,thelungsarealargepartoftherespiratorysystem.Theirmain
jobistomaintainfreshairandgetridofwastegasesinthebody.Oxygenisneededforevery
cellinthebody.Whenwebreatheinair,oxygentravelsthroughthewindpipe,orthetrachea,
andintothelungs.Thetracheadividesintotwomainpartscalledthebronchialtubes.The
bronchialtubessubdivideintoeachlobeofthelungsandconsistofbronchiolesandalveoli.The
alveolusisasmallanatomicalstructurethatregulatesgasexchange.Thealveoliaremadeupof
elasticfiberscalledelastin.Elastinallowthealveolitostretchastheyfillwithairwhenbreathing
in.Theythenspringbackduringbreathingoutinordertodischargeofcarbondioxide.Every
humancontainsaboutfourhundredandeightymillionalveoli.Thealveolushasahugesurface
area.Ifallthealveoliinthehumanbodyweretobestretchedout,theywouldtakeupasmuch
spaceasatenniscourt.(Ochs,2003)However,duringCOPD,theinflammationofthealveoli
occursandtheythenshrinktohavingthesurfaceareasimilartothesizeofatabletenniscourt.
Thiscausestheairwaytobecomecollapsibleandtheaircannotgetoutofthelungs.Thelungs
becomeextremelyunventilated.Inflammationmayalsocausewallsbetweentheairsacsto
deteriorate.(Khan,2010)Asshowninfigure2,alveolilosethedifferentchamberswithinthem.
Thewallsarenotthickandwithoutshape.(Unknown,n.d.)Also,alveolithathavebeendistorted
byemphysemacontainalargeamountofdeadspacewhichfillupwithCO
.
2

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Figure2:NormalvsAlveoliwithEmphysema

Furthermore,elastin,whichmaintainsthesurfaceareaofthealveoli,isconstantlybeing
brokendownbyelastase,aprotease.Aproteaseisanenzymethatregulatesmanybodily
functions.Alpha1antitrypsin,whichisaproteaseinhibitor,inhibitselastaseandallowsformore
elastin,thereforeregulatingtheshapeofthealveoli.Wheninflammationoccurs,elastaseis
hypersecreted.Neutrophils,whichareatypeofwhitebloodcell,producetoomuchelastasefor
thealpha1antitrypsininhibitortomanage.Thisresultsinthebreakdownofelastinand
consequently,theimpairmentofthealveoli.Also,elastinmaintainsequilibriumofthechests
tendencytoexpandandthelungstendencytocollapsein.Whenelastinisbrokendown,this
equilibriumissetoffandthechestbeginstobecomeabnormallybiggerthus,thetotallung
capacityriseseventhoughlungsarenotwellventilated.(Khan,2010)

InastudydonebyCecileOnclinxetal.,itwasdeterminedthatdistancesbetween
alveolarductwallsinratsincreasedwheninjectedwithelastase,displayingthattheyhadalarge
areaofdeadspace.Inthisexperiment,therewerethreegroupsofratsthatwerecategorizedby

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eitherhavingonetrachealinjectionof300IUofelastase,twotrachealinjectionsofthesame
amount,orhavingatrachealinjectionof200Lofsterilenormalsaline.Thegroupwiththe
highestamountofelastaseinducedhadasignificantlyhighermeaninterwalldistance(MIWD)
thantheothertwogroups.Theresearchersconcludedthatthiscouldhavebeencausedbythe
inflammationofthealveoli.Whentheinflammationoccurred,theelastinbrokedownandthis
resultedintheincreasedamountofdeadspace.Toaddtothisstudy,theyalsotestedthestatic
anddynamiccomplianceofthesameratlungs.Staticanddynamiccompliancemeasurethe
lungsabilitytostretchandexpand.Agreatincreaseordecreaseincomplianceequatesstiffer
lungs.Emphysemamaybeassociatedwithanincreaseinthecompliance,duetothelossof
alveolarandelastictissue.(Martin,n.d.)Theresultsofthisexperimentshowedthatthegroups
injectedwithelastasehadasignificantlyhigherdynamicandstaticcompliance,buttherewasnot
anydifferencebetweenthegroupinjectedonceandthegroupinjectedtwice.(Onclinx,2006)

EnzymaticInhibition

Asstatedbeforeinthispaper,anenzymeisaproteinthatspeedsupareactionby
providinganalternativepathtoovercomingactivationenergy.Thesubstanceinwhichthe
enzymeactsuponiscalledthesubstrate.Theselectivequalitiesofanenzymeiscalled
specificityandthespecificareainwhichtheenzymeandsubstratebindsandreactsatiscalled
theactivesite.Enzymesbindtemporarilytooneormorereactantandwhendoingsotheylower
theamountofenergyneededthusspeedupthereaction.(Pal,n.d.)Infigure3,theblackline(E
)
a
indicatestherouteareactionwouldtakewithoutthehelpofanenzyme.Theyellowline(E
)
a1
indicatestherouteofareactioninthepresenceofanenzyme.Theamountofenergyneededfor

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E
ismuchmoreantheenergyneededforE
.Thisshowsthesignificanceofanenzymeina
a
a1
reaction.However,sometimesthepresenceofanenzymecanbebeneficialandothertimesitcan
bedetrimental.Inthecaseofemphysema,theenzymeelastasespeedsuptheamountofelastin
beingbrokendown,thusitcausestheweakventilationinthealveoli.Tostoptheactionofan
enzyme,aninhibitorisneeded.

Figure3:ImportanceofEnzymeDiagram

Anenzymeinhibitorisasubstancethatinteractsinsomewaywiththeenzymeto
preventitfromworkinginthenormalmanner.(Ophardt,2003)Thereareavarietyofinhibitors
whichinclude:nonspecific,irreversible,reversiblecompetitiveandnoncompetitive.
Nonspecificinhibitionincludesanyphysicalorchemicalactionthatcandenaturetheentire
enzyme,andthusitisirreversible.Anexampleofanonspecificinhibitorcanbeextremelyhigh
orlowtemperatures,excessamountsofacidsorbasesorastrongsubstancethathadtheability
todrasticallyaffectanenzyme.Aspecificinhibitoraffectsaspecificenzyme.Theinhibitoronly
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hastheabilitytoinhibitorslowdownoneenzyme.Asstatedearlierinthispaper,alpha1
antitrypsinonlyhastheabilitytoinhibitelastase,thusitisaspecificinhibitor.Acompetitive
inhibitorisacompoundwhichhasaclosechemicalstructuretotheenzymessubstrate.The
inhibitorcompetesforthesameactivesiteasthesubstrate.Furthermore,theinhibitormay
interactwiththeenzymeattheactivesiteandpreventanysubstrateenzymeinteraction.The
competitiveinhibitoris,however,reversibleifthesubstrateconcentrationishigherthanthe
inhibitorconcentration.Lastly,anoncompetitiveinhibitorisasubstancethatinteractswithan
enzymeinalocationotherthanitsactivesite.Thegoalofanoncompetitiveinhibitoristo
changetheshapeoftheactivesitebychangingtheshapeoftheenzyme,sothatconsequently,
thesubstratecannolongerinteractwiththeenzymetogiveareaction.Noncompetitive
inhibitorsareusuallyreversibleforthesamereasonsasacompetitiveinhibitor.(Ophardt,2003)

WillowTreeBark:Salicin

TheuseofwillowbarkdatesbackthousandsofyearstowhenHippocratesandother
risingphilosopherswerecomingupwithnewideasandconcepts.In400BC,patientswere
advisedtochewonthebarktoreducefeverandinflammation.Willowbarkhasalsobeenused
throughouthistoryinChinaandEuropetotreatpain,headachesandinflammatorydiseases.
(Ehrlich,2013)PhysiciansofancientGreece,includingDioscorides,whowrotetheprecursorto
allmodernpharmacopeias,prescribedwillowforitsanalgesicandantiinflammatoryproperties.
(Hedner,1998)Today,wehavedeterminedthatthechemicalthatgiveswillowbarkthese
propertiesissalicin.

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Salicinwasfirstusedtoproduceacetylsalicylicacid,whichismostcommonlyknownas
aspirin.Aspirinhastheabilitytoactasananticoagulant,apainkilleranditcanbecharacterized
asanonsteroidalantiinflammatorydrug(NSAID).(Nordqvist,2014)NSAIDshelptoreduce
feverandrelievepaincausedbyheadaches,muscleachesandstiffness.Theyalsoreduce
inflammationandswelling.(PainRelievers,n.d.)Salicinisverysimilartoaspirinandsome
studiesshowsalicinbeingcomparedtoaspirinforreducingpainandinflammationbutatamuch
lowerlevel.Salicinalsodoesnotcreateunwantedsideeffectsassociatedwithaspirin,suchas
gastricupset.(WhatisSalicin?,n.d.)

Moreover,frommuscleandtendontoboneandligamentinflammation,thebasic
characteristicsofitremainsthesame.Itcanbedefinedbypain,heat,rednessandswelling.
(Maroonetal,2010)Osteoarthritishasrecentlybeencharacterizedasaninflammatorydisease
duetothepresenceofsynovitis.(Sokolove,2013)Synovitisistheinflammationofthejoint
liningcalledthesynovium.(Synovitis,n.d.)InastudydonebySchmidB,theefficiencyof
salicinsantiinflammatoryandanalgesicpropertiesweretestedonpatientswithosteoarthritis.
Seventyeightpatientsweredividedintotwogroups,placebooractivetreatment.Thedimension
ofpain,amountofmusclestiffnessandphysicalfunctionsweremeasuredpriortotreatment.The
activetreatmentgroupreceived240mgofsalicinperdayfortwoweeks.Afterthistimeperiod,
thedimensionofpain,amountofmusclestiffnessandphysicalfunctionsweremeasuredagain
andcomparedtotheinitialscores.Thepainscoreintheactivetreatmentgroupwasreducedby
14%fromthebaselinelevelaftertwoweekscomparedtoanincreaseof2%intheplacebo
group.Theamountofmusclestiffnessalsochanged.Duetosalicinpreviouslyproducinga
NSAIDandbecausesalicinischaracterizedasantiinflammatory,scientistsconcludedthis
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changewasbecausetheamountofinflammationinthejointsdecreasedintheactivetreatment
group.(Schmid,2001)

Furthermore,theantiinflammatorypropertiesofsalicinwereshowninastudydoneby
NirmalVermaetal.Micesufferingfrominflammatoryboweldisease(IBD)wereobservedafter
sevendaysofsalicinanddextransulfatesodiumadministrations.Gutfloraisavital
microorganismthatliveinthedigestivetractsofanimalsandhumans.WhenIBDoccursthe
amountofgutfloradecreasesandwithnotreatment,thebacteriacancompletelywipeout.
(Sisson,2010)Throughhistologicalexaminations,itwasindicatedthatsalicinhadallowedfor
thesuppressionofedemaandmucosaldamage.Also,itwasobservedthatsalicinhadprevented
thelossofgutflorainthemice.Therefore,scientistsconcludedthatsalicinhasananti
inflammatoryeffectatthecolorectalsiteanditmayhaveatherapeuticvalueinameliorating
inflammationduringIBD.(Vermaetal,2013)Ifsalicinhasbeensuccessfulinreducing
inflammationinthecolon,itwouldbebeneficialtotestifithasthesameabilitywith
emphysema.Furthermore,ifanexperimentisconductedandsalicinisproventohavetheability
toinhibitelastasethenitwouldalsobeimportanttofindout,specifically,whattypeof
enzymaticinhibitiondoesitperform.

Inconclusion,emphysemaisaprogressivedisease,mainlycausedbysmoking.Although
ithasbeenstudiedbyresearchers,itstillhasveryfeweffectivemethodsfortreatment.The
hypersecretionofelastasecanbeverydeadlyandcancausetheinflammationoftheelasticfibers
inthealveoli.Consequently,thelungsbecomeextremelyunventilatedandclogged.However,
withthesuccessofseveralantiinflammatorysubstancesinhibitingelastase,thefutureof

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inhibitionofelastaseasatreatmentlooksbright.Basedonpreviousstudies,ithasbeenshown
thatthewillowbarksalicinhasmanyantiinflammatoryproperties,thusitmayhavetheability
ofinhibitingelastase,andthereforetreatingemphysema.

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Materials:
Neutrophilelastasecolorimetricdrugdiscoverykit(Containselastase,substrate,
calibrationstandard,controlinhibitor,buffer,96WellMicroplate.)
D()Salicin5g
Pipetscapableofpipetting.1200ulaccuracy
PipetTipscapableofpipetting.1200ulaccuracy
Pipetscapableofpipetting.0220mlaccuracy
Icebucket
Glasstesttubes
DistilledWater
Gloves
Beaker
GraduatedCylinder
Scoopula
Glassstirrod
Waxpaper
Goggles

Equipment:

Incubator
AbsorbanceMicroplatereader
AbsorbanceMicroplatereaderfilter405nm
Scale
Centrifuge
ElectronicPipet

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Procedure
FreeTrial:FindingMostEffectiveConcentrationofSalicin:
1) Make1000uMstocksolutionwith0.028gofSalicindilutedin100mLofdistilledwater.
2) Preparethefollowingdilutionsforthefollowingconcentrationsofsalicinwiththestock
solution.Prepareeachinaseparatetesttube.
SalicinConcentration(uM)

Volumeof1mMStock(ml)

VolumeofWater(ml)

500

10

10

250

15

100

18

50

19

10

.2

19.8

.02

19.98

3)DefrostNeutrophilElastaseColorimetricDrugDiscoveryKitcomponentsandholdonice
untiluse.Brieflycentrifugeallvials.Minimizethetimethatanykitcomponentisthawed.
4)Dilute8uLofsubstrateBMLP2139090with72uLofassaybufferinatesttube.Label
o
testtubesubstrate.Afewminutespriortostartofassay,warmtoreactiontemperature(37
C).

5)Dilute1.6uLofneutrophilelastaseenzymewith158.4uLofassaybufferinatesttube.
Labeltesttubeenzyme.Afewminutespriortostartofassay,warmtoreactiontemperature
o
(37
C).

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6)Dilute1uLofinhibitorelastatinalBMLPI1039090with39uLofassaybufferinatest
tube.LabeltesttubeKI(kitinhibitor).Afewminutespriortostartofassay,warmtoreaction
o
temperature(37
C).

7)96WellPlateDiagramShownBelow.Addthespecifiedamountsofeachsolutiontothe
correspondingwell.Eachwellstotalvolumeis95uL.

10

11

20uLofSalicinwithconcentrationof
500uM
10uLofelastasesolution
65uLofassaybuffer

20uLofSalicinwith
concentrationof500uM
10uLofelastasesolution
65uLofassaybuffer

20uLofSalicinwithconcentrationof
250uM
10uLofelastasesolution
65uLofassaybuffer

20uLofSalicinwith
concentrationof250uM
10uLofelastasesolution
65uLofassaybuffer

20uLofSalicinwithconcentrationof
100uM
10uLofelastasesolution
65uLofassaybuffer

20uLofSalicinwith
concentrationof100uM
10uLofelastasesolution
65uLofassaybuffer

20uLofSalicinwithconcentrationof
50uM
10uLofelastasesolution
65uLofassaybuffer

20uLofSalicinwith
concentrationof50uM
10uLofelastasesolution
65uLofassaybuffer

20uLofSalicinwithconcentrationof
10uM
10uLofelastasesolution
65uLofassaybuffer

20uLofSalicinwith
concentrationof10uM
10uLofelastasesolution
65uLofassaybuffer

20uLofSalicinwithconcentrationof
1uM
10uLofelastasesolution
65uLofassaybuffer

20uLofSalicinwith
concentrationof1uM
10uLofelastasesolution
65uLofassaybuffer

Addthe20uLofKIsolutionmade
instep4
10uLofelastasesolution
65uLofassaybuffer

Addthe20uLofKIsolution
madeinstep4
10uLofelastasesolution
65uLofassaybuffer
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10uLofelastasesolution
85uLofassaybuffer

10uLofelastasesolution
85uLofassaybuffer

o
8)Incubateplatefor30minutesatreactiontemperature(37
C)toallowinhibitor\enzyme

interaction.
9)Startassaybytheadditionof5uLBMLP2139090substrate(dilutedandequilibratedto
reactiontemperatureinstep4).
10)ContinuouslyreadplatesatA
inabsorbancemicroplatereader.Recorddataat1min.
405nm
timeintervalsfor10mins.

11)Removeassayplatefromabsorbancemicroplatereader.Cleanwellplateandtesttubes.

TestingEffectivenessofSalicinonDifferentConcentrationsofSubstrate:
1)DefrostNeutrophilElastaseColorimetricDrugDiscoveryKitcomponentsandholdonice
untiluse.Brieflycentrifugeallvials.Minimizethetimethatanykitcomponentisthawed.
2)PreparethefollowingsolutionswithsubstrateBMLP2139090andtheassaybuffer
includedinthekit.Afewminutespriortostartofassay,warmallsolutionstoreaction
o
temperature(37
C).

Substrate
Concentration(M)

Substrate(uL)

Buffer(uL)

TotalVolume(uL)

0.00173(A)

2.6

27.4

30

0.00186(B)

2.8

27.2

30

0.00199(C)

27

30

0.00213(D)

3.2

26.8

30

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3)Dilute2uLofinhibitor(elastatinalBMLPI1039090)with78uLofassaybuffer
BMLKI581inseparatetesttube.LabeltesttubeKIforkitinhibitor.Warmtoreaction
o
temperature(37
C)

4)Dilute2.2ulneutrophilelastaseenzymein197.8ulassaybuffer.10ulperwell.(Makesure
nottoaddenzymetoblanks!Followwelldiagram.)Warmtoreactiontemperature.
5)Make1000uMstocksolutionwith0.028gramsofSalicindilutedin100mLofdistilled
water.

6)Fromstocksolutionpreparethefollowingdilution
SalicinConcentration

Volumeof1mMStock(ml)

VolumeofWater(ml)

1uM

0.02

19.98

7)96WellPlateDiagramShownBelow.Pipetthefollowingamountsofthepreparedsolutions
tothecorrespondingwells.(Columns912arenotshownbecausetheyarenotused.)
Key:BBlank
CControl
IEInhibitorElastatinal
TITestInhibitor(Salicin)
XWellNotUsed

A B
95uLassaybuffer
0uLneutrophil
elastase
0uLelastatinal
0uLsalicin

B
95uLassaybuffer
0uLneutrophil
elastase
0uLelastatinal
0uLsalicin

B
95uLassaybuffer
0uLneutrophil
elastase
0uLelastatinal
0uLsalicin

B
95uLassaybuffer
0uLneutrophil
elastase
0uLelastatinal
0uLsalicin

B C
85uLassaybuffer
10uLelastase
0uLelastatinal
0uLsalicin

C
85uLassaybuffer
10uLelastase
0uLelastatinal
0uLsalicin

C
85uLassaybuffer
10uLelastase
0uLelastatinal
0uLsalicin

C
85uLassaybuffer
10uLelastase
0uLelastatinal
0uLsalicin
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C TI
65uLassaybuffer
10uLelastase
0uLelastatinal
20uLsalicin

TI
65uLassaybuffer
10uLelastase
0uLelastatinal
20uLsalicin

TI
65uLassaybuffer
10uLelastase
0uLelastatinal
20uLsalicin

TI
65uLassaybuffer
10uLelastase
0uLelastatinal
20uLsalicin

D TI
65uLassaybuffer
10uLelastase
0uLelastatinal
20uLsalicin

TI
65uLassaybuffer
10uLelastase
0uLelastatinal
20uLsalicin

TI
65uLassaybuffer
10uLelastase
0uLelastatinal
20uLsalicin

TI
65uLassaybuffer
10uLelastase
0uLelastatinal
20uLsalicin

E TI
65uLassaybuffer
10uLelastase
0uLelastatinal
20uLsalicin

TI
65uLassaybuffer
10uLelastase
0uLelastatinal
20uLsalicin

TI
65uLassaybuffer
10uLelastase
0uLelastatinal
20uLsalicin

TI
65uLassaybuffer
10uLelastase
0uLelastatinal
20uLsalicin

F IE
65uLassaybuffer
10uLelastase
20uLelastatinal
0uLsalicin

IE
65uLassaybuffer
10uLelastase
20uLelastatinal
0uLsalicin

IE
65uLassaybuffer
10uLelastase
20uLelastatinal
0uLsalicin

IE
65uLassaybuffer
10uLelastase
20uLelastatinal
0uLsalicin

G X

H X

o
8)Incubateplatefor30minutesatreactiontemperature(37
C)toallowinhibitor\enzyme

interaction
9)Startassaybytheadditionof5uL(variousconcentrationsdilutedandequilibratedinstep2)
ofthecorrectconcentrationsofsubstratetothecorrespondingwells.Followwelldiagrambelow.
Totalvolumeofallwellsshouldbe100uL.

A 5uLofsubstrate
concentrationA

5uLofsubstrate
concentrationB

5uLofsubstrate
concentrationC

5uLofsubstrate
concentrationD
24

Banerjee

B 5uLofsubstrate
concentrationA

5uLofsubstrate
concentrationB

5uLofsubstrate
concentrationC

5uLofsubstrate
concentrationD

C 5uLofsubstrate
concentrationA

5uLofsubstrate
concentrationB

5uLofsubstrate
concentrationC

5uLofsubstrate
concentrationD

D 5uLofsubstrate
concentrationA

5uLofsubstrate
concentrationB

5uLofsubstrate
concentrationC

5uLofsubstrate
concentrationD

E 5uLofsubstrate
concentrationA

5uLofsubstrate
concentrationB

5uLofsubstrate
concentrationC

5uLofsubstrate
concentrationD

F 5uLofsubstrate
concentrationA

5uLofsubstrate
concentrationB

5uLofsubstrate
concentrationC

5uLofsubstrate
concentrationD

G X

H X

10)ContinuouslyreadplatesatA
inabsorbancemicroplatereader.Recorddataat1min.
405nm
timeintervalsfor10minutes.
11)Cleanlabareaandmaterialsanddisposeofanyusedpipets.
12)Performdataanalysis

25

Banerjee

Results

Neg
Control

Elastatinal 500uM

250uM

Trial1

1.081818 0.87272727 0.554545 0.790909

0.618181 0.690909 0.72727 0.95091

Trial2

2.254545 1.82727273

1.063636 0.645455 1.19091 1.69091

0.6 0.872727

100uM

Average 1.668182 1.34999979 0.577273 0.831818 0.840909

50uM

10uM

1uM

0.668182 0.95909 1.32091

Units:OD\min

26

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27

Banerjee

Substrate
Concentratio
n

Blank

Control

0.39090909

Trial1with Trial2with Trial3with Elastatinal


Salicin
Salicin
Salicin
Trial

.00173(A)

0.52818182

0.509090909

0.509090909

0.518181818

0.518181818

.00186(B)

0.536363636 0.450505051 0.5363636364 0.5354545455 0.5181818182 0.5354545454

.00199(C)

0.554545455 0.495959596 0.5545454545 0.5436363636 0.5545454545 0.5323232323

.00213(D)

0.681818182 0.532323232 0.6747474747 0.6818181818 0.6818181818 0.6272727273

Units:OD\min
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Banerjee

AverageBlanksvs.AverageElastatinal(AllSubstrateConcentrations)

29

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AverageBlanksvs.AverageSalicin(AllSubstrateConcentrations)

30

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AverageControlvs.AverageElastatinal(AllSubstrateConcentrations)

31

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AverageControlvs.AverageSalicin(AllSubstrateConcentrations)

32

Banerjee

DataAnalysis
Fordeterminingthemosteffectiveconcentrationofsalicinonconstantconcentrationsof
salicin,itwasnecessarytocomparethemtotwocontrolgroups,thenegativecontrol,which
containednoinhibitor,andtheelastatinaltrial.Duringtrialone,500uMshowedtheleast
absorbanceofcolor,while1uMshowedthemost.The250uMtrialabsorbed.2363636OD\min
morethan500uM.100uMabsorbedmorethan250uMand10uMabsorbedmorethan100uM.
50uMabsorbedlessthan100uM,250uM,10uM,and1uMbutitabsorbedsimilarlyto500
uM.1uMshowedthemostsimilaritytotheelastatinaltrial.Therewasanincreaseinabsorbance
from500uMto1uM,exceptforthedatafor50uM.Tovalidatetheseresults,anothertrialwas
conducted.Thedataintrial2showedthesametrendastrialone.Dueto1uMofsalicinhavinga
similarabsorbanceaselastaseinhibitingelastatinal,itwaschosenasthemosteffective
concentration.
Afterestablishing1uMtobethemosteffectiveconcentrationofsalicin,itwasusedto
determineitsefficiencyininhibitingelastasefrombreakingdowndifferentconcentrationsofthe
substrate.For.00173M(A)concentrationofthesubstratethecontrolshoweda0.39091OD\min
absorbanceandtheblankshoweda0.52818absorbance.Theaverageabsorbancewithsalicin
was0.51212andforelastatinalitwas0.51818.Bothsalicinandelastatinalabsorbedmorethan
thecontrolandsimilarlytotheblank.Onaverage,salicinabsorbedabout0.00970lessthanthe
blankand0.17576morethanthecontrol.Elastatinalabsorbed0.00364lessthantheblankand
0.12424morethanthecontrol.Theaveragedifferenceinabsorbancebetweensalicinand
elastatinalwas0.00606OD\min.

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Banerjee

For0.00186M(B)concentrationofthesubstratethecontrolshoweda0.53636OD\min
absorbanceandtheblankshoweda0.45051absorbance.Wellstreatedwithsalicinshowedan
averageabsorbanceof0.52999andtheabsorbancewithelastatinalwas0.53545.Forthis
concentrationtoo,bothsalicinandelastatinalabsorbedmorethanthecontrolandsimilarlytothe
blank.Wellswithelastatinalabsorbed0.08495morethanthecontroland0.00009lessthanthe
blank.Onaverage,wellswithsalicinabsorbed0.07798morethanthecontroland0.00609less
thantheblank.Theaveragedifferenceinabsorbancebetweensalicinandelastatinalwas0.00546
OD\min.
For0.00199M(C)concentrationofthesubstrate,thecontrolabsorbed0.49596OD\min
andtheblankabsorbed0.55455.Forthisconcentration,aswell,bothsalicinandelastatinal
absorbedmorethanthecontrolandsimilarlytotheblank.Wellstreatedwithsalicinabsorbed
0.55091OD\minonaverage,andwithelastatinal0.53232wasabsorbed.Wellswithelastatinal
absorbed0.03636morethanthecontroland0.02222lessthantheblank.Onaverage,wellswith
salicinabsorbed0.05494morethanthecontroland0.01091lessthantheblank.Theaverage
differenceinabsorbancebetweensalicinandelastatinalwas0.01859OD\min.
Lastly,for0.00213M(D)concentrationofthesubstrate,thecontrolabsorbed0.53232
OD\minandtheblankabsorbed0.68182.Onaverage,wellstreatedwithsalicinabsorbed
0.67946OD\minandwellswithelastatinalabsorbed0.62727.Forthisconcentration,aswell,
bothsalicinandelastatinalabsorbedmorethanthecontrolandsimilarlytotheblank.Wells
treatedwithsalicinabsorbed,onaverage,0.14713morethanthecontroland0.00236lessthan
theblankWellswithelastatinalabsorbed0.09495morethanthecontroland0.05455lessthan

34

Banerjee

theblank.Theaveragedifferenceinabsorbancebetweensalicinandelastatinalwas0.05219
OD\min.
Basedoffofthegraphsindicatingthecorrelationbetweentheaveragesofallfour
substrateconcentrations,itwasshownthattheaverageoftheblanksversustheaverageofthe
elastatinaltrialshadacorrelationcoefficientof0.98.Theblankswithsalicinhadacorrelation
coefficientof1.00.Thecontrolsandelastatinalhada0.62correlationcoefficientandthe
controlswithsalicinhasa0.70correlationcoefficient.

35

Banerjee

Discussion
Severalthingscanbeconcludedfromthedatacollected.Inordertodeterminethe
efficiencyofsalicininhibitingelastase,itwasfirstnecessarytodeterminewhichconcentration
ofsalicinwasthemosteffective,ifany,ininhibitingelastase.Sincethewellswith500uMand
50uMofsalicinshowedaboutthesameabsorbance,itwasnotcleariftheinconsistencywasdue
tobecausetheconcentrationsfrom500to50uMofsalicinweredenaturingtheenzyme.
Nevertheless,theresultsalsoshowedthattheabsorbanceof1uMandthecontrolinhibitor,
elastatinal,wereaboutthesame.Thismeantthatsalicinof1uMwasinhibitingelastaseasmuch
aselastatinalwas.Sinceelastatinalhasalreadybeenprovenaselastaseinhibiting,the1uMof
salicinwaschosentobemosteffective.Thisconcentrationwasusedtotesttheefficiencyof
salicinondifferentconcentrationsofthesubstrate.
Forthistest,thedatashowedthatsalicinwasabletoinhibitelastaseaswellaselastatinal
was.Theblanksonlycontainedthesubstrateandbufferwithnoenzyme.Thus,thedataforthe
blanksshowedtheabsorbanceofthesubstratebeforeitwasbrokendownbytheenzyme.The
controlwellscontainedtheenzyme,substrateandbuffer.Thus,thisdatashowedtheabsorbance
ofthesubstrateafterbeingbrokendownbytheenzyme.Alowerabsorbancemeantthatthere
wasmoresubstrateinthewellandmoresubstratecanbeexplainedbytheenzymebreaking
downthesubstrate.Wellstreatedwithsalicinabsorbedsimilarlytotheblank.Thismeantthat
salicinwasabletomaintainthesubstratesconcentrationforallfourconcentrationstested.The
dataalsoshowedthatsalicinwasabletoinhibitelastasefrombreakingdownthesubstrateand
consequentlydecreasingtheamountofabsorbance.Thecorrelationalgraphsshowedthatthere
wasacorrelationcoefficientof1.00fortheblanksandsalicin.Thecloserthecorrelationisto1,
36

Banerjee

thestrongertherelationshipis.Thismeantthatsalicinandtheblankswereveryclosely
correlatedandotherpointscaneasilybepredictedusingthegraphsbestfitline.Salicinandthe
blanksweremorecorrelatedthanelastatinalthetheblanks.Thismeantthattheabsorbanceofthe
wellswithsalicinandtheblanksweremoresimilarthanelastatinalandtheblanks.Comparedto
elastatinal,salicininhibitedelastasebetterforthesubstrateconcentrationsCandD,thehigher
twoconcentrations.Onaverage,however,salicinandelastatinalinhibitedelastaseverysimilarly.

37

Banerjee

ExperimentalError
Therewereseveralareasthaterrorcouldhaveoccurred.Itwashardtoseeandverifyif
thecorrectamountsweregoingintoeachwellsincemicrolitersissuchasmallunit.Itwas
difficulttoverifyifsmallamountssuchas0.4uLwereproperlytransferringfromthepipettips
intothewells.Ifthesesmallmeasurementswerenottransferringcorrectly,thenthismayhave
causedanoveralldifferenceintheamountofabsorbance.Forexample,if0.1uLofthesubstrate
didnottransferproperlyfromthepipettiptothewell,thenalowerabsorbancewouldhave
showedupforthatwell.Toavoidthiserrorinthefuture,theuseofapipetthatsayshowmuchit
haspipettedandreleasedwouldbehelpfultoverifythecorrecttransferofmaterial.
Also,whenmeasuring0.028gramsofsalcinforthestocksolution,theactualamounts
deviatedasmallamountfrom0.028.Thismayhavecausedthesolutionstobeeitherahigheror
lowermolarconcentrationthanindicated.Inaddition,thesalicinsolutionsweremade8hours
priortotesting.Although,itisnotknownforsure,this8hourwaitingperiodmayhave
diminishedtheeffectsofsalicin.Thus,ifthesolutionsweremadeimmediatelypriortotesting,
thentheeffectsmayhavebeenabitstronger.Inordertoeliminatethiserrorinthefuture,
solutionsshouldbemadeimmediatelybeforetestingtomakesurethattheeffectsofsalicinare
notbeingdiminishedbyawaitingperiod.
AnothersourceoferrormayhaveoccurredwhenthevialsintheNeutrophilElastase
ColorimetricDrugDiscoveryKitwerebeingdefrosted.Foreachtrial,therewasntasettime
indicatedthatthecomponentswoulddefrostuntil.Thecomponentswereplacedintheincubator
o
at37
Candtheywerecheckedonperiodicallytoseeiftheyfeltwarmenoughtotestwith.

Consequently,foreachtrialtheamountofdefrostingtimemayhavebeendifferent.Thismay
38

Banerjee

haveresultedinthecomponentsbeingmoreactiveinsometrialsthanothers.Inordertomake
surethatallthecomponentsarefunctioningatthesamelevelforalltrials,inthefutureitwould
benecessarytoindicatehowmuchtimethecomponentsshoulddefrostfor.

39

Banerjee

Conclusion
Theworldtodayisfilledwithabundantsourcesoftechnology,scienceandideas.
However,evenwithsomanyresources,somanyproblemsareleftunattended.Chronic
ObstructivePulmonaryDiseaseisthethirdleadingcauseofdeathinAmerica,yetitisrelatively
leftunstudied.Emphysema,abranchofCOPD,isaprogressive,inflammatorydisease,mainly
causedbysmoking.With1inevery5adultssmoking,itisnecessarytofindeffectivemethodsof
treatmentforemphysema.(ACS,2014)Thepurposeofthisexperimentwastodetermineif
salicin,anaturalantiinflammatorysubstance,hastheabilitytoinhibitelastaseandeventuallybe
usedasanothermethodoftreatmentforemphysema.Thehypothesiswasthatifelastasereacted
withdifferentconcentrationsofelastin,istreatedwithsalicin,thentheconcentrationofelastin
withnoenzymewillbethesameaselastinreactingwithboththeenzymeandsalicin.Thisis
becausesalicinhasantiinflammatorypropertiesthatshouldbeabletoinhibitelastasefrom
breakingdownelastinandchangingitsconcentration.
Inordertoconductthisexperiment,themosteffectiveconcentrationofsalicin,ifany,
hadtobedetermined.Forthispartoftheexperiment,concentrationsofsalicinrangingfrom500
uMto1uMweretestedonconstantamountsofthesubstrateandcomparedtoanegativecontrol
andacontrolinhibitorofelastase.Thedatashowedthatthewellswith1uMofsalicinhada
similarabsorbancetothewellswiththecontrolinhibitor,elastatinal.Duetothisfinding,the
1uMconcentrationofsalicinwaschosentobethemosteffectiveanditwasusedonthenextpart
oftheexperiment.Thesecondpartofthisexperimentconsistedof1uMofsalicinbeingtestedon
differentconcentrationsofthesubstrate.Thedifferentconcentrationssignifiedhowthesubstrate
wouldbebrokendownindifferentstagesofemphysemainreallife.Inahigherstageof
40

Banerjee

emphysema,therewouldbeahigherconcentrationofthesubstrate.Forthispart,thedata
showedthatsalicinhadmaintainedtheconcentrationofthesubstrate.Thewellswithoutthe
enzymeandjustthesubstratehadverysimilarabsorbancesincomparisontothewellswiththe
enzymetreatedwithsalicin.Thisappliedforallfourconcentrationsofthesubstrate.In
comparisontoelastatinal,salicininhibitedelastasebetterinthehighertwoconcentrationsofthe
substrate.Overall,however,salicinandelastatinalinhibitedelastaseverysimilarly.
Inconclusion,thehypothesiswassupported.Itwaspredictedthatsalicinwouldbeable
toinhibitelastaseandkeeptheconcentrationofelastinwithnoenzymeandelastinreactingwith
boththeenzymeandsalicinthesame.Theaveragedifferencebetweentheblanksandthewells
treatedwithsalicinwas0.00727OD\min.Thisinhibitioncanbeexplainedbysalicinsanti
inflammatoryproperties.Itwasabletoactasaspecificinhibitorandpreventany
substrateenzymeinteraction.Duetothisfinding,salicinwouldbeeffectiveintreating
emphysema.Althoughfutureresearchmustbeconducted,itisshownthatsalicininhibited
elastasebetterthanelastatinalinthehighertwoconcentrationsofthesubstrate.Sincethehigher
concentrationssignifiedhigherdegreesofemphysema,itmaybeconcludedthatsalicinwouldbe
moreeffectiveintreatinghigherdegreesofemphysema.

41

Banerjee

Impact

Wecanusethisinformationthatwasconcludedfromthisexperimentbytestingsalicins
effectsonthelungsofaCOPDpatient.Ifsuccessful,salicinwouldbeaneffective,cheapand
abundantsourcetotreatemphysemawith.Unlikeotherformsoftreatment,salicinwouldbe
natural.

42

Banerjee

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