Effect of chloride ions on rate of - amylase hydrolysis · 2018-01-18 · humans and many other mammals e.g. ptyalin is produced by the salivary glands , whereas ... amylase. In this

BiologyInternalAssessment

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Effectofchlorideionsonrateof∝-amylasehydrolysis

By:ChristinaWong

ALL RIGHTS BELONG TO OWNERTAKEN FROM WWW.INTERNALASSESSMENTS.WORDPRESS.COM


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ExplorationIntroductionAmylaseisanymemberofaclassofenzymesthatcatalysethehydrolysisofstarchintosmallercarbohydratemolecules such as maltose.∝-amylase is widespread among living organisms. In the digestive systems ofhumans and many other mammals e.g. ptyalin is produced by the salivary glands, whereas the pancreassecretespancreaticamylaseintotheduodenum.(TheEditorsofEncyclopædiaBritannica,1998)Allostericactivators, suchaschloride ions, can increasereactionrates foracertainenzyme.Theybind toanallostericsiteandinduceaconformationalchangethatincreasestheaffinityoftheenzyme’sactivesiteforitssubstrate,whichincreasesthereactionrate.(Boundless,2017)

Activatorscanplayanimportantrole inmaintainingtheconformationoftheprotein,forminganessentialcomponentoftheactivesite,ortheymaybepartof thesubstrateof theenzyme. Theamountofenzymeactivatorformedisequaltotheamountofactivatorpresentinthemixture.An example of an activator of an enzyme is the chloride ion with∝-amylase. In this case,∝-amylase has some activity in the absence ofchloride. With saturating levels of chloride, the ∝ -amylase activityincreases about fourfold. Other anions, including F-, Br- and I- (halogenions)alsoactivate∝-amylase.TheideaforthisinvestigationcameaboutwhenIwasstudyingMolecularBiology,unit7.5Proteinsand7.6Enzymes.Enzymeinhibitionwaspartofthesyllables,yetitexcludesactivation.Thus,Iwasintriguedtoinvestigatesuch mechanism.∝- Amylase was specially selected, as it is such animportant enzyme in the body, and sodium chloride (salt) is ingestedfrequently; hence it sparks my interest to see if chloride ions wouldactuallyaffect the rateofhydrolysisof amylase,whichcould in termaidwithdigestioniftherateofcatabolismincreases.(Aghilan,Chaeroseshinie,AishahDewi)Concentrationofsodiumchloride ions(0.0,0.3,0.6,0.9,1.2,1.5,1.8)/%

ResearchQuestion

Howdoestheconcentrationofsodiumchlorideions(0.0,0.3,0.6,0.9,1.2,1.5,1.8)%affecttherateinwhichenzyme∝-amylasehydrolysesstarch,measuredastherateofchangeofabsorbancevalues/Aus-1(±0.001)ofstarchiniodinesolutionusingaspectrophotometer(lightwavelengthsetat430nm)in300s?

Independentvariable

Concentrationofsodiumchlorideions(0.0,0.3,0.6,0.9,1.2,1.5,1.8)/%(±0.2)

Justification of range selected: extreme high concentration of sodium chloride ions is unnecessary as thesubstrate is a limiting factor. Moreover, highly concentrated ions may alter the electrostatic interactionsbetweenchargedaminoacids,causingconformationalchangeswhich in turndestroys theactivesites,whichmaydecreaserateofreaction.Thus,suchlowconcentrationisselectedasthemostsuitable,toinvestigatetheactivatingpropertyofCl-ions.Anintervalof0.3%intermsofsodiumchlorideconcentrationisselectedasitgivesconsiderablechange inrateofabsorbancevalueofstarch in iodinesolution,yetnotasignificant jump.Thisistheappropriateincrementformeasurementsasitisabletoshowacleartrend,correlationbetweenCl-concentrationandrateofchange inabsorbancevaluesofstarch in iodinesolution.Moreover, this isalso therangeandincrementofmeasurementselectedbyotherScientistconductingsimilarexperiments.Referencetootherscientists’research:(Aghilan,Chaeroseshinie,AishahDewi)

Figure1illustratingthemechanismofallostericactivation



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Dependentvariable

Rateofchangeofabsorbancevaluesofstarchiniodinesolutionwithaspectrophotometer (lightwavelength set at 430nm) in 300s /Au s-1(±0.00100) in different concentration of sodium chloride ions (0.0,0.3,0.6,0.9,1.2,1.5,1.8)/%

Whenstarchismixedwithiodine,thecoilsofbetaamylosemoleculesfoundinstarchtrapiodine,causingthemixture to turn into a shade of blue black. (Senese, 2015)When starch is broken down intomaltose/ othersugar,thedisaccharide/monosaccharidedoesnotreactwithiodine.Therefore,iodinedoesnotchangecolour(appearorange).Correspondingly,whendropsofamylaseareinputtedintoablue-blackmixtureofstarchandiodine,thestarchmoleculeswillbebrokendowncausingthemixturetoturncolourlessappearorange).Thus,the rate of reaction of amylase correlates to the absolute value of the rate of change in absorbance of thesolution.Arapiddecreaseintheabsorbancevalueoftheblue-blackcolourequatestoahighrateofreactionofamylase. In the experiment, an external variable-activator of sodium chloride will be manipulated into theamylaseenzymetodeterminetheeffectofCl-ontherateofreactionofamylase.(Lee,2011)

RateofReaction= ∆ !"#$%"&'() !"#$%∆ !"#$

Unit=Arbitraryunitpersecond(Aus-1)

Methodofmeasuring:Using a spectrophotometer set at 430nm(wavelength of dark blue colour) and Logger Pro, linked to Maccomputer. Rate of reaction calculated using above equation, absorbance v.s. time graph can be used forreferenceandconfirmresults.ControlledVariablesControlledvariable Reasonsforcontrolling MethodstocontrolConcentration of enzyme ∝ -amylaseused

Increased concentration of ∝ -amylasewill increase rate of reaction, thusaffectingtheresults.

Use 2.0 % bacterial amylase,measured usingmeasuring cylinder(± 0.2)

Volume of enzyme ∝- amylaseused

Increased volume of ∝ -amylase willincrease rate of reaction, thus affectingtheresults.

Use 1.0 cm3 of bacterial amylase,measured usingmeasuring cylinder(± 0.2)

Timelimitforreaction Rate of reaction would slow down astimeprogressesasthesubstrateiodineisused up, thus longer time span wouldyieldunfairresults.

Timelimitsetat300s,settimelimitincomputer/datalogger

Temperatureofsurrounding

Temperatureisafactorwhichwillaffectrateofenzymeactivity.Askineticenergyof enzymes increases, rate of reactionwould increase, but extreme hightemperatures could cause enzymedenaturation.Soneedtobecontrolledtokeepexperiments fair (alteringonlyonevariable)

Turnonairconditionerinlab,setat24℃

ExplanationofdependentvariableEquationshowinghydrolysisofstarchusingamylase:

∝ 𝑎𝑚𝑦𝑙𝑎𝑠𝑒Starch+water--------------->maltose

(appearblue-blackiniodine)(appearorangeiniodine)

Figure2Diagramshowinghydrolysisofstarchintomaltoseatmolecularlevel



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Temperature of amylase andstarch

Amylase like all enzymes work at anoptimumtemperature, toowarmorcoldwillleadtodifferenceinrateofreaction.Thus need to be constant to keepexperimentfair.

Place starch and amylase in 37℃water bath for 10 minutes beforeuse.

Samesourceof∝-amylase

∝-amylase fromdifferent sourcesmayhavedifferentrateofreaction.

Use amylase from bacteria Bacillusspp.B.amyloliquefaciens

Concentrationofsubstrate Increasedconcentrationofsubstratewillincreaseproduct(oxygen)produced.

Use 1.0% starch solution,measuredusingmeasuringcylinder(± 0.2)

Volumeofsubstrate Increased volume of substrate willincreaseproduct(oxygen)produced.

Use 1.0 cm3 starch solution,measured usingmeasuring cylinder(± 0.2)

pHlevel pHlevelisalsoafactorwhichaffectstherateof enzymeactivity.Thusneed tobeconstant to keep experiment fair.Amylase works at a pH optimum ofaround7.

Usewaterasbufferstomaintaintheneutralcondition

Volumeofiodine More drops of iodine will darken thecolour of the mixture, leading toinaccuratemeasureofabsorbancevalue

Use3dropsviaaplasticdropper

Volumeofsodiumchlorideused Concentration of sodium chloride is theindependent variable, increased volumewould increase activation of amylase,leadingtounfairexperiment.

Use 1.0 cm3 measured usingmeasuringcylinder(± 0.2)

Wavelength of light ofspectrophotometer

Lightofaparticularwavelengthwillpassthrough the cuvette in thespectrophotometer, then theabsorbancevalue recorded. If the wavelength isdifferent, the absorbance value may bealtered,leadingtoanunfairresult.

Setat430nm

HypothesisAsconcentrationofsodiumchlorideincreases,therateofreaction/Aus-1(hydrolysisofstarchby∝-amylase)shouldincrease,absorbancevaluewilldecreaseatfasterrateduetotheincreaseinsubstrateactivator,whichcouldcatalysethereactionbetter.Materials/ApparatusMaterials/Apparatus Justificationforchoiceofuse100cm3of2.0%∝-amylasefrombacteriaBacillusspp.B.amyloliquefaciens

2%amylase is a suitable concentrationas if too concentratedwill lead toa rateofreaction which may be too fast for record of results. It may also be dangerous/hazardoustousehighlyconcentratedenzyme.TheamylasesourcesfromBacillusspp.B.amyloliquefaciens isusedas it isthemostcommoncommercialised microbial amylaseproduced, thus canbeobtainedeasilyandatlowprice.

100cm3of1.0%starchsolution

Substrate for hydrolysis by amylase1% starch is a suitable concentration as if tooconcentratedwillleadtoveryslowrateofreaction,inefficient.

Abottleofiodine Iodine test is used to determine concentration of starch in solution, important formeasuringdependentvariable.

100cm3of2.0%Sodiumchloridesolution

Activatorfor∝-amylase

ExcessDistilledwater Used as buffers to dilute sodium chloride solution to alter concentration forindependentvariables.

LoggerPro+computer Adigitalwayofrecordingabsorbancevaluemoreaccurately,avoidshumanerrors.



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Spectrophotometer/Aus-1(±0.00100)

Tomeasurechangeinabsorbancevalueofblue-blackcolourinmixture,importantasitistheequipmentusedtomeasurethedependentvariable.Chosenovercolorimeteras it has a wider range of wavelength, more suited for dark blue colour of starchsolutioninpresenceofiodine.

3Cuvette Tocontainthemixtureandplaceinsidespectrophotometer.Itstransparentpropertyallowslighttopassthroughinordertoaccuratelymeasuretheabsorbancevalue.

Dropper Toobtainthesolutionsinsmallvolumeeasily10cm3measuringcylinder/cm3(±0.2)

To measure 1 cm3 of starch solution, amylase, sodium chloride, better for smallvolumeastheuncertaintyislower.

50cm3measuringcylinder/cm3(±0.4)

Tomeasurevolumeofwaterandsodiumchlorideforserialdilution.

250cm3Conicalflask/cm3(±0.2),withbung

Tocontain/storethesodiumchloridesolutionofvariousconcentration.Usebungtopreventevaporationorcontaminationfromair.

Waterbathat37℃ To warm starch solution and amylase before experiment to enhance optimumtemperatureof∝-amylase.

1glassrod Tostirthestarchandamylasesolutionsomoleculesaremoreevenlyspreadinthesolution.Ensurestheabsorbancevaluesrecordedareaccurate.

Safety,ethical,environmentalprecautions

- ∝-amylase frommicrobial sources is chosenoverhuman/animals sources forethical andeconomicalissues.

- Be careful to dispose left over chemicals such as enzyme∝-amylase, pour into sink and flush withexcesswatertopreventenvironmentalhazards/pollution.

- Donotingestanychemicalsfromthelab, includingsodiumchlorideastheremaybetoxicsubstanceswhichmaycausecontamination.

- Becarefulwhenwarmingsolutions inwaterbath,donot touch theelectricityplugwithwethands ifsettingupwaterbath.

- Weargogglesatalltimestoensuresafety,avoidcontactofeyeswithanychemicals, ifoccurred,flushwithcleanwaterimmediatelyandseekmedicalattention.

PreliminarytrialConducted2 times to ensureLoggerPro andMac computer areworking.Experimentwas then conducted3times with 0.0 % NaCl, then 0.3% and 0.6% NaCl respectively to ensure the increment for measurementselected is suitable. The concentration range selected for independent variables corresponds to publishedresearchandtheresultsshowatrendduringpreliminarytrial,thusdonotrequireanyalterations.Methodology*Makesureexperimentiscarriedinalabwithuniformtemperature,airconditionerturnedonto24℃.I.PreparesodiumchloridesolutionwithvaryingconcentrationConductserialdilutionusingtablebelow:

Concentration/%(±𝟎.𝟐)ofsodiumchloridesolution

Volumeof2%NaCl/cm3(±𝟎.𝟒)

VolumeofH2O/cm3(±𝟎.𝟒)

0.0 0 200.3 3 170.6 6 140.9 9 111.2 12 81.5 15 51.8 18 2

Volumeratiocalculatedusingformula:M1V1=M2V2e.g.tomake0.3%sodiumchloridesolution1.Measure3.0cm3of2%NaCl,using50.0cm3measuringcylinder.2.Measure17.0cm3ofdistilledwaterusing50.0cm3measuringcylinder.3.Pourtheliquidintoaconicalflaskandswirl.4.Repeatforallconcentration.



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II.Conductexperiment1. Setupspectrophotometer,setlightto430nmwavelength.2. Calibratespectrophotometerusingdistilledwaterincuvetteandwaitforabsorbancetodropto0.3. Connect loggerPro to computer and spectrophotometer, set the time limit at 300s and record an

absorbancevalueat5secinterval.4. Measure1.0cm3ofstarchsolution,amylase,0.3%sodiumchlorideusingdifferent10.0cm3measuring

cylinder.Makesuretoreadthemeniscusateyeleveltoavoiderrorofparallax.5. Pourthestarchsolutionandsodiumchlorideintoacuvetteandadd3dropsofiodinesolutionusinga

plasticdropper.6. Placethecuvetteintothespectrophotometer.7. Pourinthe∝-amylaseandimmediatelyclosethelidofspectrophotometerandstartthe loggerProon

computer.Datarecordingcommences.8. Wait for 300s and loggerPro will automatically stop and record the final value. Observe the curve

drawn.9. Repeatstep1-7,15times.Observeifthecurvesoverlap,suggestsaccurateresults.*Importanttoensure

resultsarereliable.10. Thenrepeatstep1-8usingsodiumchlorideofdifferentconcentration.

III.Analyseresults- Compare finalabsorbancevalueand initialabsorbancevalue/Au (±𝟎.𝟎𝟎𝟏𝟎0)andcalculate therateof

changeofabsorbancevalue/Aus-1- Calculatetheaveragerateofchangeofabsorbancevalue/Aus-1andobservethecorrelationbetween

concentrationofNaCl/%andaveragerateofchangeofabsorbancevalue.Note:averagereferstomean.- Statisticaltest(Spearman’srankcoefficient)istobecarriedout(moredetailbelow)

Analysis

Table#1:Rawdatatableshowinginitialandfinalabsorbancevalue/Au(±𝟎.𝟎𝟎𝟏𝟎𝟎)within300secformixtureinvariousconcentrationofNaCl(0.0,0.3,0.6,0.9,1.2,1.5,1.8)/%(±𝟎.𝟐)

ConcentrationofNaCl/%(±𝟎.𝟐)

Trialnumber

Initialabsorbancevalue/Au

(±𝟎.𝟎𝟎𝟏𝟎𝟎)

Finalabsorbancevalue/Au

(±𝟎.𝟎𝟎𝟏𝟎𝟎)

0

1 1.26200 0.670002 1.02100 0.780003 0.98700 0.560004 1.25900 0.830005 1.10700 0.750006 1.34000 0.980007 1.23000 0.880008 1.45000 0.960009 1.26000 0.8900010 0.98000 0.6500011 1.13000 0.7700012 0.87000 0.6400013 0.96000 0.7400014 1.03000 0.5900015 1.23000 0.96000

0.6

1 0.91900 0.340002 0.95400 0.250003 1.11800 0.370004 1.21200 0.390005 1.12600 0.240006 1.14000 0.450007 1.25000 0.560008 0.98000 0.320009 0.86000 0.2900010 1.05000 0.4300011 0.94000 0.3400012 1.02000 0.4700013 1.09000 0.3200014 0.78000 0.4500015 1.13000 0.49000


Trialnumber


(±𝟎.𝟎𝟎𝟏𝟎𝟎)


(±𝟎.𝟎𝟎𝟏𝟎𝟎)

0.3

1 1.04800 0.760002 0.83500 0.590003 0.98600 0.660004 1.07600 0.470005 1.05400 0.910006 0.85000 0.430007 0.98000 0.440008 0.87000 0.370009 1.09000 0.3400010 1.23000 0.5800011 1.12000 0.5400012 1.32000 0.7800013 1.24000 0.8800014 0.89000 0.4300015 0.97000 0.47000

0.9

1 0.32755 0.670002 0.26224 0.780003 0.32765 0.560004 0.30405 0.830005 0.30495 0.750006 0.28273 0.980007 0.27669 0.880008 0.25987 0.960009 0.39894 0.8900010 0.30090 0.6500011 0.32882 0.7700012 0.32882 0.6400013 0.45000 0.7400014 0.34000 0.5900015 0.29000 0.96000



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Qualitativeobservation:

- Thecolourofmixtureincuvettewasinitiallyblack-darkbluecoloured.- Aftermixingamylaseandwaited300seconds,thecolourofmixtureislightyellow,andithasbecomea

transparentsolution.- Thereissomeblackishsedimentsatthebottomofthecuvetteafter300seconds.

Table#2:ProcessedDatashowingrateofchangeofabsorbancevalue/Aus-1anditsaveragewithin300secforvariousconcentrationofNaCl(0.0,0.3,0.6,0.9,1.2,1.5,1.8)/%(±𝟎.𝟐)

Concentrationof

NaCl/%(±𝟎.𝟐)

Trialnumber


(±𝟎.𝟎𝟎𝟏𝟎𝟎)


(±𝟎.𝟎𝟎𝟏𝟎𝟎)

1.2

1 1.39394 0.760002 1.59600 0.590003 1.61159 0.660004 1.05574 0.470005 1.71171 0.910006 0.97343 0.430007 1.42454 0.440008 1.22990 0.370009 1.35602 0.3400010 1.27616 0.5800011 1.48003 0.5400012 1.58096 0.7800013 1.58933 0.8800014 1.45557 0.4300015 1.49338 0.47000

1.8

1 1.65128 0.386852 1.35925 0.160003 1.46624 0.230004 1.41410 0.170005 1.50564 0.180006 1.66245 0.371577 1.31956 0.321748 1.65555 0.200009 1.04711 0.2100010 1.51329 0.1400011 1.38352 0.2400012 1.72157 0.2900013 1.51267 0.1900014 1.40032 0.2300015 1.47917 0.12000

Concentrationof

NaCl/%(±𝟎.𝟐)

Trialnumber


(±𝟎.𝟎𝟎𝟏𝟎𝟎)


(±𝟎.𝟎𝟎𝟏𝟎𝟎)

1.5

1 1.13473 0.340002 1.57239 0.250003 1.58933 0.370004 1.55099 0.390005 1.61509 0.240006 1.66376 0.450007 1.49397 0.560008 1.64247 0.320009 1.63464 0.2900010 1.37761 0.4300011 1.43370 0.3400012 1.54134 0.4700013 1.80365 0.3200014 1.42505 0.4500015 1.48636 0.49000

Figure3showingthechangeincolourofstarchsolution,300secafteramylaseisadded

Concentrationof

NaCl/%(±𝟎.𝟐)

Trialnumber Rateofchange

ofabsorbancevalue/Aus-1

AverageRateofchangeofabsorbancevalue/Aus-1

0.3

1 0.00096

0.00154

2 0.000823 0.001094 0.002025 0.000486 0.001407 0.001808 0.001679 0.0025010 0.0021711 0.0019312 0.0018013 0.0012014 0.0015315 0.00167

Concentrationof

NaCl/%(±𝟎.𝟐)

Trialnumber Rateofchangeof

absorbancevalue/Aus-1

Average Rateof change ofabsorbancevalue/Aus-1

0.0

1 0.00197

0.00121

2 0.000803 0.001424 0.001435 0.001196 0.001207 0.001178 0.001639 0.0012310 0.0011011 0.0012012 0.0007713 0.0007314 0.0014715 0.00090



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*datainredindicatesanomalousresultNote:Dataarerecordedto5decimalplacesbecausetherateofchangeofabsorbancevalue/Aus-1istooaccurate,thevaluesareverysmall,ifroundedupto3decimalplaces,allthedatawouldjustbe0.001or0.002whichisnotusefulforgraphing(onlyahorizontallinewouldbeseen).Thus,Rawdataarealsorecordedto5decimalplacestokeepthedecimalplacesintablesconsistent.





0.9

1 0.00022

*0.00026

2 0.000163 0.000294 0.000155 0.000306 0.000207 0.000228 0.000249 0.0003610 0.0002411 0.0003012 0.0003313 0.0003714 0.0002315 0.00033

1.5

1 0.00246

0.00384

2 0.003873 0.003974 0.003735 0.004046 0.004407 0.003778 0.004289 0.0043210 0.0034011 0.0034912 0.0038013 0.0048514 0.0035115 0.00372





0.6

1 0.00193

0.00219

2 0.002353 0.002494 0.002745 0.002956 0.002307 0.002308 0.002209 0.0019010 0.0020711 0.0020012 0.0018313 0.0025714 0.0011015 0.00213

1.2

1 0.00390

0.00362

2 0.004493 0.004164 0.002765 0.004586 0.001857 0.003698 0.002929 0.0032910 0.0031911 0.0035412 0.0041013 0.0042314 0.0037115 0.00395

1.8

1 0.00421

0.00414

2 0.004003 0.004124 0.004155 0.004426 0.004307 0.003338 0.004859 0.0027910 0.0045811 0.0038112 0.0047713 0.0044114 0.0039015 0.00453



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Table#3:ProcessedDataTableshowingconcentrationofNaCl/%(±𝟎.𝟐)andtheaveragerateofchangeofabsorbancevalue/Aus-1

ConcentrationofNaCl/%(±𝟎.𝟐) Averagerateofchangeofabsorbancevalue/Aus-1

0 0.001210.3 0.001540.6 0.002190.9 0.000261.2 0.003621.5 0.003841.8 0.00414

*rednumbersindicateanomalousdatawhichwouldbeeliminatedwhendrawinglineartrendline.

Calculationsforprocesseddata:Calculation Formula ExampleRateofchangeofabsorbancevalueAus-1

!"!#!$%!!"#$% !"#$%"!&'( !"#$%

!""

Trialnumber1for0.3%NaCl:(1.04800-0.76000)÷300=0.00096

Standarddeviation Calculatedusingexcel𝑒.𝑔 for0.0%NaCl,SD≈0.00121

Average(mean)rateofchangeofabsorbancevalue/Aus-1

Calculatedusingexcel

Screenshotfromexcelshowingcalculations---e.g.0.0%NaCl:



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GraphshowingcorrelationbetweenconcentrationofNaCl(0,0.3,0.6,0.9,1.2,1.5,1.8)/%andrateof

changeofabsorbancevalue/Aus-1

Analysisofgraph:Thebluediamondpointindicatesanomalousdatawhichisfaroffthelinearregressiontrend,thusisexcludedfromtheresultswhendrawingthelinearregressionlinebecauseitisevidentthattheremaybeasystematicerrorintheexperimentatione.g.wrongconcentrationofstarch/amylaseusedsinceforall15repeats,whenadded0.9%NaCl,theinitialabsorbancevalue/Auwasverylow,belowanyotherexperiments.When other initial value usually ranges from 0.80000 to 1.32000, all the 15 data using 0.9%NaCl ranges inbetween0.25987to0.45000which issignificantly lower thanother initialabsorbancevalues/Au. (thiswillbediscussedfurtherlaterinconclusion)Errorbarsshowthestandarddeviationof therateofchangeofabsorbancevalue/Aus-1 foreachdatapoint.There is a different standarddeviation for each concentrationofNaCl (0.0, 0.3, 0.6, 0.9, 1.2, 1.5, 1.8 )%, thestandard deviations are 0.00034, 0.00055, 0.00044, 0.00073, 0.00055 and 0.0054 (in increasing order ofconcentration,standarddeviationfordatausing0.9%NaCliseliminatedfromcalculationasitisananomaly.)Theprocesseddataandgraphshowsthatthehypothesisiscorrect.AsconcentrationofNaClincreases,rateofabsorbancevalueincreases.Thereisapositive,linearcorrelationbetweenconcentrationofNaCl/%andrateofchangeofabsorbancevalue/Aus-1.StatisticalTest-Spearman’srankcorrelationcoefficientJustificationforuse: Spearman’sRankcorrelationcoefficient tests if twovariablesareconnected (correlated)witheachother.Itwillalsomeasurethestrengthofanycorrelation,i.e.howclosetoaperfectlystraightlineitis. (Ecofieldtrips Pte Ltd, 2015 )My experiment is testing if there is a correlation between concentration ofchloride ions/%and∝-amylaseactivitymeasuredby rateof changeof absorbancevalue/Aus-1.Having twovariablespredictedtocorrelateeachother,Spearman’sRankcorrelationseemsthemostsuitable.Also, fromthegraph,itisevidentthatthereisamonotoniccorrelation(Laerdstatistics)insteadofotherrelationshipssuch

y=0.0018x+0.0012R²=0.97731

0.00000

0.00050

0.00100

0.00150

0.00200

0.00250

0.00300

0.00350

0.00400

0.00450

0.00500

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2

Rateofchangeofabsorbancevalue/Aus-1

ConcentrationofNaCl/%(+/-0.2)



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asnon-monotonic, andSpearman’s rank correlation is specifically suited tomonotonic relationshipbetweentwovariables.NullHypothesis:ThereisnosignificantcorrelationbetweenconcentrationofNaClandaveragerateofchangeinabsorbancevalue.Alternative Hypothesis: There is significant correlation between concentration of NaCl and average rate ofchangeinabsorbancevalue.ConcentrationofNaCl/%(±𝟎.𝟐)(A) RankA

Averagerateofchangeabsorbancevalue/Aus-1(B) RankB Difference Differencesquared

0 1 0.00121 1 0 00.3 2 0.00153 2 0 00.6 3 0.00219 3 0 00.9 - 0.000263 - - -1.2 4 0.00362 4 0 01.5 5 0.00384 5 0 01.8 6 0.00414 6 0 0

Note:anomalousdataisexcludedfromthestatisticaltestFormulatofindthersvalue,thecloseritisto1,thestrongerthecorrelation

rs=1– !(!)!(!!!!)

=1ConclusionThefindinganswersmyresearchquestionwhichis:“Howdoestheconcentrationofsodiumchlorideions(0.0, 0.3, 0.6, 0.9, 1.2, 1.5, 1.8 )% affect the rate in which enzyme∝- amylase hydrolyses starch,measuredastherateofchangeofabsorbancevalues/Aus-1(±0.001)ofstarchiniodinesolutionusingaspectrophotometer (lightwavelength setat430nm) in300s?”Asconcentrationofsodiumchlorideions(0.0, 0.3, 0.6, 0.9, 1.2, 1.5, 1.8 )/%(±𝟎.𝟐)increases, rate in which enzyme ∝ - amylase hydrolyses starchincreases,ascanbeseenfromtheincreaseinrateofchangeofabsorbancevalues/Aus-1(±0.001)ofstarchiniodinesolution.Thisalsosupportsthehypothesis,suggestingthatthehypothesisiscorrect,canbeaccepted.AsconcentrationofNaClincreasesfrom0.0%to1.8%,theaveragerateofchangeofabsorbancevaluesincreasesfrom0.00121to0.00414Aus-1.Thereisa0.00293Aus-1increaseintermsofrate,thatmeanswhenusing1.8%NaCl,therateofchangeofabsorbancevalueisalmost3timesfastercomparedtostarchsolutionwith0.0%NaCladded.Thissuggests,increasechlorideionsconcentrationincreasesenzyme∝-amylase’sactivitysoitisabletohydrolysestarchfaster,allowingthecolourofstarchtoturnfromblack/bluetoyellowfaster,causingafasterrateindecreaseofabsorbancevalue/Aus-1.Thequalitativeobservationsalsosupportsthetheoryasthecolourofstarchsolutionisinitiallyblack/darkbluebutchangestolightyellowafteramylaseisadded,indicatingthathydrolysisofstarchhasoccurred.After300s,solutionwith1.8%NaCladdedappearslightestyellowcomparedto0.0%NaCladded,evidently,addingchlorideionswhichareactivatorsspeedupthisprocess.

Thus,apositivecorrelationisobservedfromthelinegraphplottedbetweenconcentrationofNaCl/%andrateof change of absorbance value/ Au s-1. The equation of the linear line y= 0.0018x +0.0012 has a positivegradient of 0.0018, suggesting that as concentration of NaCl increases by every 0.1%, the rate of change ofabsorbancevaluewould increaseby0.0018Aus-1.Datashows thatasconcentrationofNaCl increases from0.3%to0.6%,theaveragerateofchangeofabsorbancevalueincreasesfrom0.00154to0.00219Aus-1,thatis0.00065Aus-1increaseinrateofchangeofabsorbancevaluefor0.3%increaseinconcentrationofHCl.Whendivide0.00065by3(someaning,every0.1%increaseinNaClconcentration),theincreaseinrateofchangeofabsorbance value is approximately 0.00022. This is quite close to the gradient of the equation, which is

Result:Rsvalueisexactlyone,whichmeansthereisaverystrongcorrelationbetweenthetwovariables.NullHypothesisisrejected,AlternativeHypothesisisaccepted,thereissignificantpositiveandnegativecorrelationbetweenconcentrationofNaClandaveragerateofchangeinabsorbancevalue.



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0.00018. This shows that the equation generated is quite accurate and is a good model of the correlation,further confirming the strong correlation and affirms that there is indeed an effect (positive correlation)betweenthetwovariables.DetailedBiologicaltheory:∝-amylaseisanallostericenzymewhichmeansitmostlyhastwoormoresubunitsandcanoscillatefromactiveformtoinactiveform.Thepresenceofanallostericeffectorcanchangetheaffinityoftheenzymeforitssubstrate,oralterthemaximalcatalyticactivityoftheenzyme,orboth.Moreover,thebindingofanactivatoratregulatorsitecanstabilizetheactiveformofthatallostericenzyme.T-stateisknowntotenseupthemoleculeandislessactive.TheR-stateisknowntobemorerelaxedanddecreasestheKm.Chlorideionissuspectedtobeanallostericeffector-activator,whichaidsinthetransformationof∝-amylasefromT-statetoR–state.Thus,increasingtheenzyme∝-amylaseactivity.(Berg,2015)

Reliabilityofdata:Theequationofthelinearliney=0.0018x+0.0012hasaR2valueof0.977whichsuggestsastrongcorrelation.Thedatapointsfittothelineartrendlinemorecloselyaftereliminatingtheanomaly.Theresultsobtainedfromtheexperimentusing0.9%NaCliseliminatedasitisanoutlierwhichisfaroffthelineartrend observed from other results. It is thought that the outlier is caused by a systematic error whenconducting the experiment. The initial absorbance value/Au of starch in iodine solution in addition of 0.9%NaCl,wasverylow.Whilstotherinitialvaluesrangefrom0.80000to1.32000,allthe15datausing0.9%NaClrangeinbetween0.25987to0.45000only,whichissignificantlylowerthanotherinitialabsorbancevalues/Au.All procedureswere conducted identically, following themethodology, thus the source of errormay be thematerialsorequipment. Astheexperimentwasconductedoveraspanofaweek, thestarchsolutionand∝-amylaseusedweredifferenteachday.Hence,thestarchsolutionusedthatdaymaybeofwrongconcentration,below 1%, or it may be that it has been stored for too long that all the starch sinks to the bottom of thecontainerandwhatispouredoutisonlyverydilutedstarchsolution.The∝-amylasemaybeasourceoferroraswell,itmayhavebeenstoredfortoolongthatithasbecomeinactive.However,IfeelthespectrophotometerortheLoggerPromayhavebeenthemaincontributortothesystematicerror,thespecificoneusedtoconductthe experimentwith 0.9%NaClmay have been faulty, thus yieldingwrong results. To avoid such problemsagain,equipmentshouldbecheckedthoroughly,runtrialsandcompareresultswithotherdatasetsobtained,beforeexperimentsnexttime.Eliminatingtheanomalousdata,when:calculatingaverage,standarddeviation,plottinglinearregressionline,allows the trend tobemoreaccurateandhasabetter fit. In combination to theSpearman’s rankcoefficientstatisticaltest,thedataproducedfromthisexperimentisreliableandstatisticallyvalid.Sincethersvalue=1,suggesting perfect positive correlation. However, only 6 data points are ranked, instead of the ideal 8(datapoints), thismay cause the statistical test to be less applicable to these results.On the other hand,R2 valuegeneratedbyexcelfromthegraphis0.977,thisalsosuggeststhedatapointsareveryclosetothetrendline,notscattered,soagainreaffirmingthestrongcorrelationbetweenthetwovariables.The error bars onmy graph further show the data obtained are quite precise. The standard deviations arerelativelysmall.Thissuggeststheresultsarereliableasitshowsthe‘goodnessoffit’tothelinearfunction(y=0.0018x+0.0012)isrelativelyhigh,i.e.thefunctiondescribesthedataverywell.

Reference from published research: it isstated that Cl- is essential in the activity of∝ −𝑎𝑚𝑦𝑙𝑎𝑠𝑒 , suggesting that thisexperiment is true. “Activation experimentsof A. haloplanctis α-amylase by severalmonovalent anions show that a negativecharge, not restricted to that of Cl−, isessential for the amylolytic reaction.Engineering of the chloride binding sitereveals that a basic residue is an essentialcomponentofthesite.”(GeorgesFeller‡) Asconcentrationofstarchincreases,velocityof∝ −amylaseactivityincreases.Thegraphalsoshowsalineartrendjustasmyresultshaveshown.

Figure4GraphshowingActivation of starch and EPS hydrolysis by chloride obtained from published data.



13

EvaluationStrengthofinvestigation

- LoggerPro is used to recorddata, this eliminates human error anduncertainty of using timewatch,whichgreatlyincreasetheaccuracyoftheresults.

- Spectrophotometer isused,whichenables calibrationof460nm, this is thewavelength fordarkbluewavelength,makingtheabsorbancevaluemoreaccurate.

- There are 7 data points for independent variables (0.0, 0.3, 0.6, 0.9, 1.2, 1.5, 1.8 )%which suggestssufficientdatatotesttheresearchquestion.

- Spectrophotometeriscalibratedusingdistilledwaterbeforeexperimenttocorrectforsystematicerror.Limitations

Limitation Reasons/significance PossibleimprovementsSmallsamplesize,notenoughrepeats,only15times

Dataunreliable/notreliableenough Repeatmethodology50timesmoretoimprovereliability

Starchsinktobottominbeakerwhenstored

Starchresiduesmaynotbepouredintothecuvette,thesolutiondoesnotcontainasmuchstarch,leadingtolowerabsorbancevalues.

Stirthestarchsolutionwithaglassrod,andswirlthebeakereverytimebeforepouringandmeasuring.

Overallrateiscalculated Notasaccuratebecauseconcentrationofamylaseandstarchwouldhavedecreasedprogressivelyasthereactantsareusedup

Initialrateofreactionshouldbecalculated,drawcurvesusingLoggerProsoftware(recorddataevery10s)andfindgradientoftangenttotheinitialpartofthecurve.

Temperatureofamylaseandstarchmayhavecooleddown

Theamylasewouldnotbeworkingatitsoptimumcondition,whichmaycauseslowerrateofreaction.

Placetheamylaseandstarchinwaterbathat36℃,whenconductingtheexperiments.Onlyextractthesmallvolumeneededforeachrepeatatatime.

Starchsinktobottomofcuvettebeforeamylaseisevenadded

Absorbancevaluewilldecreaseevenbeforethehydrolysisreactionastheblack/bluestarchresiduessinktothebottomofthecuvette.

Shakethecuvettebycoveringthumboveropeningitandinvert5times,beforeplacingintothespectrophotometer.Thenstartthetimerstraightawayassooncuvetteisinspectrophotometer.

Notfreshenzymeamylase/notstoredproperly

Chemicalreactionsmayoccurwhenamylasearestoredinrefrigeratorovernight,affectingamylaseactivity

Usefresh/new(unopened)amylaseeachday,orcompleteexperimentsinaday.

Extensions- Conductexperimentusingsameprocedure/methodologybutsubstitute∝-amylasewith 𝛽-Amylase,.- Amylase fromdifferent sources suchas fungioranimals canbeused to investigate, as thiswouldbe

evenclosertotherealconditioninthebody.- Conduct experiment using same procedure/methodology but substitute Cl-with other halogen ions

suchasF-,I-,Br-andseeiftheyhavesimilaractivatingeffectasCl-.- Experiment with other allosteric protein/ enzyme such as e.g. animal hemoglobin, the binding of

oxygenmay induce a conformational change in that subunit that interactswith the remaining activesitestoenhancetheiroxygenaffinity.

BibliographyTheEditorsofEncyclopædiaBritannica.(1998,July20).AmylaseBIOCHEMISTRY.RetrievedJanuary2017,fromENCYCLOPAEDIABRITANNICA:https://global.britannica.com/science/amylaseBoundless.(2017,May26).ControlofMetablosimThroughEnzymeRegulation.RetrievedJanuary16,2017,fromBoundless.com:https://www.boundless.com/biology/textbooks/boundless-biology-textbook/metabolism-6/enzymes-72/control-of-metabolism-through-enzyme-regulation-351-11577/Senese,F.(2015,August17).Howdoesstarchindicateiodine?RetrievedJanuary16,2017,fromGeneralChemistryOnline!:http://antoine.frostburg.edu/chem/senese/101/redox/faq/starch-as-redox-indicator.shtmlLee,S.S.(2011,March31).Slideshare.RetrievedJanuary16,2017,fromSlideshare:http://www.slideshare.net/wkkok1957/effect-of-on-the-activity-of-amylase-using-visible-spectrophtometerGeorgesFeller‡,O.l.(n.d.).StructuralandFunctionalAspectsofChlorideBindingtoAlteromonashaloplanctisα-Amylase*.RetrievedJanuary2017,fromibcTheJournalofBiologicalChemistry:http://www.jbc.org/content/271/39/23836.longAghilan,Chaeroseshinie,AishahDewi.Effectofsaltconcentrationonsalivaryamylaseonthebreakingdownofstarch.Present.me.EcofieldtripsPteLtd.(2015).MANGROVE-STATISTICS.Singapore:ECOFIELDTRIPS.Laerdstatistics.(n.d.).Spearman'sRank-Ordercorrelation.RetrievedFebruary9,2017,fromLaerdstatistics:https://statistics.laerd.com/statistical-guides/spearmans-rank-order-correlation-statistical-guide.php


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