1 Biology pH Protocol

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Biology pHProtocol

ExperimentalGroup(circleone) pHInhibitorTemperatureGroup#_______

Eachgroupwillrun2trials.

Writethenameofthemixer,reader,timerandrecorderforeachtrialinatableinyournotebook.

Mixer Timer Reader RecorderTrial1(control)

Trial2(experimental)

Central questionWhatareenzymesandhowcanwecausechangesinenzymefunction?

Overview of experimentInthislabyouwillobservechangesinenzymeactivitywhenyouchangetheenvironmentofthereaction.Youwillfirstper-formacontroltrial,followedbyanexperimentaltrial.Inyourexperimentaltrial,youwillalterthepHofthesurroundings.Inthisexperiment,theenzymetyrosinasecatalyzestheconversionofanon-coloredmoleculeintoareddish-brownmolecule.Youwilldeterminetheactivityoftheenzymebyrecordingthecolorchangeofthereactionsolutionandgraphingyourdata.

How will the change in pH affect enzyme activity? - Hypothesis

Student pre-lab questions1. Whatdoesanenzymedo?

2. Whatdoestheenzymetyrosinasedo?

3. Whatisthepurposeofgrindingthemushroom?

4. WhatisthefunctionofL-DOPAinthereaction?

BIOLOGYPHPROTOCOL

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Material Checklist___(1)“CTRL”label

___(1)“EXP”label

___(2)“ENZ”labels

___(1)“ACID”label

___(1)“GB”label

___(1)“SUB”label

___(1)pink0.6mLtubecontainingacid(HCl)

___(2)piecesofpHpaper

___(1)pHcolorchart

___(3)glasstesttubes

___(1)weighingboat

___(4)disposabletransferpipettes

ENZYMESUBSTRATEREACTION

Label your test tubes and pipettes as described___(1)TestTube:“CTRL”forcontroltrial

___(1)TestTube:“EXP”orforexperimentaltrial

___(1)TestTube“ENZ”forenzymeextract

___(1)Pipette:“GB”forusewithgrindingbuffer

___(1)Pipette:“SUB”forusewithL-DOPAsubstrate

___(1)Pipette:“ENZ”forusewithenzyme

___(1)Pipette:“ACID”fortheHCl

5. Describethedifferencebetweenthecontrolreactionandtheexperimentalreaction.

___(1)filterpaper(cutcoffeefilter)

___(1)testtuberack

___(1)1cm3mushroomcube

___(1)colorimetricchart

___(1)liquidwastecontainer

___(1)stopwatch/timer

___SafetyGogglesforeachperson

___(1)Styrofoamcupwithice

on ice

___(1)15mLtubewithGrindingBuffer

___(1)15mLtubewithL-DOPA

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Preparing enzyme extract1. Obtainamushroompieceandplaceitin

theweighingboat.

2. Usingthe“GB”pipette,add4mLofcoldgrindingbufferontopofthemushroom.

3. GrindupthemushroomusingthecapendoftheGBtubefor3minutesoruntilitismixedwell.

GB

GB

4 mL

BIOLOGYPHPROTOCOL

GB

Lab Procedure

4. Makeafilteringconefromthecoffeefilterbyfoldingitthreetimes,creatingaconeshape.Opentheconetocre-ateafunnel.Placethefunnelintothetesttubelabeled“ENZ”forenzyme.

ENZ

Why are you grinding the mushroom?

4 |

Asateam,youwilldetermineandrecordthecolorchangeofyoursolutionforaTOTALofSEVENMINUTESforeachtrial.

Perform control trial (Trial 1)1. Usingthepipettelabeled“SUB”,pipette2mLofthe

L-DOPAsubstratesolutionintothetesttubelabeled“CTRL”.

5. Slowlypourtheliquidfromtheweighingboatthroughthefilterandintothe“ENZ”testtube.Thisisyourty-rosinaseenzymeextract!Placeitinyourtest-tubeholderatroomtemperature.Thefunnelcanbethrownaway.

ENZ

L-DOPA

2 mL

CTRL

SUB


2. Usingthetransferpipettelabeled“ENZ”,add0.1mLofthetyrosinaseenzymeextracttothe“CTRL”tubeandswirlthetesttubecontinuouslytomix.

D E F G H I J

A B C

Colorimetric Chart

0 .0625 .125 .375 .5 1.0 1.5 2.0 3.0 4.0

mM of DOPA chrome produced

ENZ

0.1 mL

CTRL

ENZ

swirl continuously

What are you filtering? Why does it flow

through the filter?

Why do we perform a control trial?

What is L-DOPA in this reaction?

Hint: What does “SUB” stand for?

Note:youmaynotseeallcolorsinthespectrum.Yourreactionmayproceedpastsomevalues.Youmustswirlcontinuouslyforbestresults.Holdyourtesttubeoverawhitesheetofpapernearyourcolori-metricchartsoyoucanobservesubtlechanges.

3. Onceyouhavecompletedthetrial,emptythecontentsofthetubeintoyourliquidwastecontainerandplaceyourtesttubeinthedrywastecontainer.

Immediately start your interval recordings. At each time interval have the timer call out “time”, write the letter that is reported. Make sure you are recording data for each time point in your data table.

Important! Get your data chart, timer and colori-metric chart ready before you add the enzyme in the next step. Cross out the line of numbers you are not using. !

!

Each team member should be prepared to do his or her job immediately. The reaction moves very quickly! !

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L-DOPA

2 mL

EXP

SUB

SUB

HCl

EXP

ACID

Determine the effect of pH on tyrosinase activity

3. Usingthe“ACID”pipette,transfertheentirecontentsofthepinkHCl(acid)tubeintothesame“EXP”testtubeandmix.DONOTDISCARDPIPETTE.

BIOLOGYPHPROTOCOL

0 1 2 3 4 5 6

7 8 9 10 11 12 13

pH

pH

4. Withthe“ACID”pipette,addonedropfromthe“EXP”testtubeontoaNEWpieceofpHpaper.UsingthepHcolorchart,determinepHofthesubstrateandrecordonthedatasheet. EXP

ACID

0 1 2 3 4 5 6

7 8 9 10 11 12 13

pH

pH

1. Usingthepipettelabeled“SUB”,transfer2mLoftheL-DOPAsubstratesolutionintothetesttubelabeled“EXP”.

2. Fromthesametesttubelabeled“EXP”,usethe“SUB”pipettetodrawuponesmalldropofsubstrateandplaceitonpHpaper.UsingthepHcolorchart,determinepHofsubstrateandrecordpHonthedatasheet.

Why do you need to know the pH of the

substrate?

Important! Get your data chart, timer and colori-metric chart ready before you add the enzyme in the next step.!

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5. Usingthetransferpipettelabeled“ENZ”,add0.1mLofthetyrosinaseenzymeextracttothe“EXP”tubeandswirlthetesttubetomix.

D E F G H I J

A B C

Colorimetric Chart

0 .0625 .125 .375 .5 1.0 1.5 2.0 3.0 4.0


ENZ

0.1 mL

EXP

ENZ


swirl continuously

Post-Lab1. Whatdoescolorchangetellyouaboutenzymeactivity?

2. Whatdifferencesdidyouobservebetweenthecontrolandtheexperimentaltrials?

3. Foryourexperimentalcondition(temperature,pHorinhibitor),explainhowthatconditionaffectedenzymeactivity.

4. Howdoesthecontrolreactionhelpyouinterpretyourexperimentalresults?Howwouldyouknowifenzymeactivitywaschangedintheexperimentalreactionifyouhadnocontrolreactiondata?

5. ANSWERTHESEQUESTIONSFOREACHEXPERIMENTALOPTION…IFYOUDIDN’TPERFORMTHATPART,THENMAKESURETOTALKWITHOTHERGROUPS…

a. ForthepHexperimentdidthepHincreaseordecrease?(ifyoudidn’tdothisone,thencheckwiththeothergroups)?Whywasthis?Howdidthisaffecttheexperimentalresults?


Immediately start your interval recordings. At each time interval, write the letter that is reported. Make sure you are recording data for each time point in your data table. !

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b. Howwasthetemperaturechanged?Howdidthisaffecttheexperimentalresults?

c. Whatwastheinhibitorused?Howdidthisaffecttheexperimentalresults?Whywouldsodiumbenzoatebeusedasafoodpreservative?

Conclusion / summary (revisit hypothesis)

BIOLOGYPHPROTOCOL

8 | ENZYMESUBSTRATEREACTION

Aftercompletingalltrials,convertcolorimetricletterstomMconcentrationofsubstrateandgraphyourdata.

Trial#1Control

Trial#2Experimental:______

Time(Minutes:Seconds)

Converttimetoseconds

ColorChartLetter

DOPA-chrome(mM)

ColorChartLetter

DOPA-chrome(mM)

10secondintervals

6:500:10

6:400:20

6:300:30

6:200:40

6:100:50

6:001:00

5:501:10

5:401:20

5:301:30

5:201:40

5:101:50

5:002:00

20secondintervals

4:402:20

4:202:40

4:003:00

3:403:20

3:203:40

3:004:00

1minuteintervals

2:005:00

1:006:00

07:00

SubstratepH:____

Substrate+acidpH:_____

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Dopachrome product formed (mM)

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60

120

180

240

300

360

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Elap

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time

(sec

onds

)

0.5

0.25

0.75

1.75

1.25

2.53

2.75

2.25

Grap

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Use

adi

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each

cond

ition

and

listt

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Color

key:

Cont

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____

____

Your

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BIOLOGYPHPROTOCOL

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| 11BIOLOGYTEMPERATUREPROTOCOL

Biology TemperatureProtocol







Overview of experimentInthislabyouwillobservechangesinenzymeactivitywhenyouchangetheenvironmentofthereaction.Youwillfirstperformacontroltrial,followedbyanexperimentaltrial.Inyourexperimentaltrial,youwillalterthetemperatureofthesurroundings.Inthisexperiment,theenzymetyrosinasecatalyzestheconversionofanon-coloredmoleculeintoareddish-brownmolecule.Youwilldeterminetheactivityoftheenzymebyrecordingthecolorchangeofthereactionsolutionandgraphingyourdata.

How will increasing the temperature affect enzyme activity? - Hypothesis





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___(1)“GB”label


___(1)weighingboat




___(1)testtuberack














on ice




Lab ProcedurePreparing enzyme extract1. Obtainamushroompieceandplaceitintheweighing

boat.

2. Usingthe“GB”pipette,add4mLofcoldgrindingbuf-ferontopofthemushroom.


GB

4 mL

GB

GB


ENZ


14 |





ENZ

L-DOPA

2 mL

CTRL

SUB





D E F G H I J

A B C

Colorimetric Chart

0 .0625 .125 .375 .5 1.0 1.5 2.0 3.0 4.0


ENZ

0.1 mL

CTRL

ENZ

swirl continuously


Important! Get your data chart, timer and colori-metric chart ready before you add the enzyme in the next step. Cross out the line of numbers you are not using.



through the filter?



!

!

Each team member should be prepared to do his or her job immediately. The reaction moves very quickly! !


Determine the effect of temperature on tyrosinase activity

ENZ 0.1 mL EXP

ENZ

1. Usingthepipettelabeled“ENZ”,add0.1mLofthetyrosinaseenzymeextracttothe“EXP”tube.

2. Placethe“EXP”tubecontainingtheenzymeinhot(90°C)waterfor1minute.

EXP90oC

1 minute

3. Usingthepipettelabeled“SUB”,transfer2mLoftheL-DOPAsubstratesolutionintoyourtesttubelabeled“EXP”andswirlthetesttubetomix.


D E F G H I J

A B C

Colorimetric Chart

0 .0625 .125 .375 .5 1.0 1.5 2.0 3.0 4.0


L-DOPA

2 mL

EXP

SUB

swirl continuously

What is the importance of this step?

Important! Get your data chart, timer and colo-rimetric chart ready before you add the substrate in the next step.

Immediately start your interval recordings. At each time interval, write the letter that is reported. Make sure you are recording data for each time point in your data table.

!

!

16 |


Trial#1Control




ColorChartLetter

DOPA-chrome(mM)

ColorChartLetter

DOPA-chrome(mM)

10secondintervals

6:500:10

6:400:20

6:300:30

6:200:40

6:100:50

6:001:00

5:501:10

5:401:20

5:301:30

5:201:40

5:101:50

5:002:00

20secondintervals

4:402:20

4:202:40

4:003:00

3:403:20

3:203:40

3:004:00

1minuteintervals

2:005:00

1:006:00

07:00












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11.52

Dop

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prod

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00

60

120

180

240

300

360

420

Elap

sed

time

(sec

onds

)

0.5

0.25

0.75

1.75

1.25

2.53

2.75

2.25

Grap

hing d

ata

Use

adi

ffere

ntco

lort

ogr

aph

each

cond

ition

and

listt

heco

lorb

elow.

Color

key:

Cont

rol:_

____

____

Your

expe

rim

enta

l gro

up: T

empe

ratu

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Biology InhibitorProtocol







Overview of experimentInthislabyouwillobservechangesinenzymeactivitywhenyouchangetheenvironmentofthereaction.Youwillfirstperformacontroltrial,followedbyanexperimentaltrial.Inyourexperimentaltrial,youwillintroduceaninhibitortothesurroundings.Inthisexperiment,theenzymetyrosinasecatalyzestheconversionofanon-coloredmoleculeintoareddish-brownmolecule.Youwilldeterminetheactivityoftheenzymebyrecordingthecolorchangeofthereactionsolutionandgraphingyourdata.

How will the introduction of an inhibitor affect enzyme activity? - Hypothesis





BIOLOGYINHIBITORPROTOCOL

20 |



___(1)testtuberack







on ice







___(1)“INH”label

___(1)“GB”label


___(1)blue0.6mLtubeofsodiumbenzoate/inhibitor(INH)

___(1)weighingboat









___(1)Pipette:“INH”fortheSodiumBenzoateinhibitor

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Lab Procedure

Preparing enzyme extract1. Obtainamushroompieceandplaceitin

theweighingboat.

2. Usingthe“GB”pipette,add4mLofcoldgrindingbuf-ferontopofthemushroom.

GB

4 mL


GB

GB

3. GrindupthemushroomusingthecapeendoftheGBtubefor3minutesoruntilitismixedwell.

ENZ



22 |






ENZ

L-DOPA

2 mL

CTRL

SUB


D E F G H I J

A B C

Colorimetric Chart

0 .0625 .125 .375 .5 1.0 1.5 2.0 3.0 4.0





ENZ

0.1 mL

CTRL

ENZ

swirl continuously


through the filter?




Each team member should be prepared to do his or her job immediately. The reaction moves very quickly!


!

Important! Get your data chart, timer and colori-metric chart ready before you add the enzyme in the next step. Cross out the line of numbers you are not using. !

!

| 23

L-DOPA

2 mL

EXP

SUBDetermine the effect of an inhibitor on tyrosinase activity


2. Withthe“INH”pipette,placetheentirecontentsoftheblueinhibitortube(sodiumbenzoate“INH”)intothe“EXP”testtubeandswirlthetesttubetomix.

3. Usingthepipettelabeled“ENZ”,add0.1mLofthetyrosinaseenzymeextracttothe“EXP”tubeandmix.


D E F G H I J

A B C

Colorimetric Chart

0 .0625 .125 .375 .5 1.0 1.5 2.0 3.0 4.0


ENZ

0.1 mL

EXP

ENZ

INH

EXP

INH

swirl continuously

1. Usingthepipettelabeled“SUB”,transfer2mLoftheL-DOPAsubstratesolutionintoyourfirsttesttubelabeled“EXP”.

Important! Get your data chart, timer and colo-rimetric chart ready before you add the substrate in the next step.!

Immediately start your interval recordings. At each time interval, write the letter that is reported. Make sure you are recording data for each time point in your data table.

!

What is the importance of this step?

24 |

Trial#1Control




ColorChartLetter

DOPA-chrome(mM)

ColorChartLetter

DOPA-chrome(mM)

10secondintervals

6:500:10

6:400:20

6:300:30

6:200:40

6:100:50

6:001:00

5:501:10

5:401:20

5:301:30

5:201:40

5:101:50

5:002:00

20secondintervals

4:402:20

4:202:40

4:003:00

3:403:20

3:203:40

3:004:00

1minuteintervals

2:005:00

1:006:00

07:00


| 25BIOLOGYINHIBITORPROTOCOL










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11.52

Dop

achr

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prod

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00

60

120

180

240

300

360

420

Elap

sed

time

(sec

onds

)

0.5

0.25

0.75

1.75

1.25

2.53

2.75

2.25

Grap

hing d

ata

Use

adi

ffere

ntco

lort

ogr

aph

each

cond

ition

and

listt

heco

lorb

elow.

Color

key:

Cont

rol:_

____

____

Your

expe

rim

enta

l gro

up: In

hibito

r


| 27

Biology

Protocol2-textonlyExperimentalGroup(circleone) pHInhibitorTemperature

Group#______

Eachgroupwillrun4trials.Eachstudentshoulddoeachjobatleastonce.





Overview of experimentInthislabyouwillobservechangesinenzymeactivitywhenyouchangetheenvironmentofthereaction.Youwillfirstper-formacontroltrial,followedbyanexperimentaltrial.Inyourexperimentaltrial,youwillalterthepHof,temperatureof,orpresenceofaninhibitorwithinthesurroundings.Inthisexperiment,theenzymetyrosinasecatalyzestheconversionofanon-coloredmoleculeintoareddish-brownmolecule.Youwilldeterminetheactivityoftheenzymebyrecordingthecolorchangeofthereactionsolutionandgraphingyourdata.

How will the change in (pH, temperature,inhibitor) affect enzyme activity? - Hypothesis






BIOLOGYPROTOCOL2

28 |

Material Checklist - pH Groups___(1)“CTRL”label



___(1)“ACID”label

___(1)“GB”label


___(1)pink0.6mLtubecontainingacid(HCl)

___(2)piecesofpHpaper

___(1)pHcolorchart


___(1)weighingboat



___(1)testtuberack







on ice









___(1)Pipette:“ACID”fortheHCl

Lab Procedure Preparing enzyme extract1. Obtainamushroompieceandplaceitintheweighing

boat.





Eachteammembershouldbepreparedtodohisorherjobimmediately.Thereactionmovesveryquickly!Asateam,youwilldetermineandrecordthecolorchangeofyoursolutionforaTOTALofSEVENMINUTESforeachtrial.



Important!Getyourdatachart,timerandcolorimetricchartreadybeforeyouaddtheenzymeinthenextstep.Crossoutthelineofnumbersyouarenotusing.


Immediatelystartyourintervalrecordings.Ateachtimeintervalhavethetimercallout“time”,writetheletterthatisreported.Makesureyouarerecordingdataforeachtimepointinyourdatatable.

Note:youmaynotseeallcolorsinthespectrum.Yourreac-tionmayproceedpastsomevalues.Youmustswirlcontinu-ouslyforbestresults.Holdyourtesttubeoverawhitesheetofpapernearyourcolorimetricchartsoyoucanobservesubtlechanges.


| 29


Determine the effect of pH on tyrosinase activity1. Usingthepipettelabeled“SUB”,transfer2mLofthe

L-DOPAsubstratesolutionintothetesttubelabeled“EXP”.

2. Fromthesametesttubelabeled“EXP”,usethe“SUB”pipettetodrawuponesmalldropofsubstrateandplaceitonpHpaper.UsingthepHcolorchart,determinepHofsubstrateandrecordpHonthedatasheet.

3. Usingthe“ACID”pipette,transfertheentirecontentsofthepinkHCl(acid)tubeintothesame“EXP”testtubeandmix.DONOTDISCARDPIPETTE.

4. Withthe“ACID”pipette,addonedropfromthe“EXP”testtubeontoaNEWpieceofpHpaper.UsingthepHcolorchart,determinepHofthesubstrateandrecordonthedatasheet.

Important!Getyourdatachart,timerandcolorimetricchartreadybeforeyouaddtheenzymeinthenextstep.

5. Usingthetransferpipettelabeled“ENZ”,add0.1mLofthetyrosinaseenzymeextracttothe“EXP”tubeandswirlthetesttubetomix.


BIOLOGYPROTOCOL2

30 |

Material Checklist - Temperature Groups




___(1)“GB”label


___(1)weighingboat




___(1)testtuberack







on ice



Label your test tubes and pipettesas described___(1)TestTube:“CTRL”forcontroltrial







boat.













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Determine the effect of temperature on tyrosinase activity1. Usingthepipettelabeled“ENZ”,add0.1mLofthe

tyrosinaseenzymeextracttothe“EXP”tube.

2. Placethe“EXP”tubecontainingtheenzymeinhot(90°C)waterfor1minute.

Important!Getyourdatachart,timerandcolorimetricchartreadybeforeyouaddthesubstrateinthenextstep.

3. Usingthepipettelabeled“SUB”,transfer2mLoftheL-DOPAsubstratesolutionintoyourtesttubelabeled“EXP”andswirlthetesttubetomix.

Immediatelystartyourintervalrecordings.Ateachtimeinterval,writetheletterthatisreported.Makesureyouarerecordingdataforeachtimepointinyourdatatable.


BIOLOGYPROTOCOL2

32 |


boat.


















on ice









___(1)Pipette:“INH”fortheSodiumBenzoateinhibitor

Material Checklist - Inhibitor Groups___(1)“CTRL”label



___(1)“INH”label

___(1)“GB”label


___(1)blue0.6mLtubeofsodiumbenzoate/inhibitor(INH)

___(1)weighingboat




___(1)testtuberack


| 33


Determine the effect of an inhibitor on tyrosinase activity1. Usingthepipettelabeled“SUB”,transfer2mLofthe

L-DOPAsubstratesolutionintoyourfirsttesttubelabeled“EXP”.

2. Withthe“INH”pipette,placetheentirecontentsoftheblueinhibitortube(sodiumbenzoate“INH”)intothe“EXP”testtubeandswirlthetesttubetomix.

Important!Getyourdatachart,timerandcolorimetricchartreadybeforeyouaddthesubstrateinthenextstep.

3. Usingthepipettelabeled“ENZ”,add0.1mLofthetyrosinaseenzymeextracttothe“EXP”tubeandmix.

Immediatelystartyourintervalrecordings.Ateachtimeinterval,writetheletterthatisreported.Makesureyouarerecordingdataforeachtimepointinyourdatatable.


BIOLOGYPROTOCOL2

34 |

Trial#1Control




ColorChartLetter

DOPA-chrome(mM)

ColorChartLetter

DOPA-chrome(mM)

10secondintervals

6:500:10

6:400:20

6:300:30

6:200:40

6:100:50

6:001:00

5:501:10

5:401:20

5:301:30

5:201:40

5:101:50

5:002:00

20secondintervals

4:402:20

4:202:40

4:003:00

3:403:20

3:203:40

3:004:00

1minuteintervals2:005:00

1:006:00

07:00



| 35










BIOLOGYPROTOCOL2

36 |

11.52

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60

120

180

240

300

360

420

Elap

sed

time

(sec

onds

)

0.5

0.25

0.75

1.75

1.25

2.53

2.75

2.25


Grap

hing d

ata

Use

adi

ffere

ntco

lorp

encil

fore

ach

cond

ition

.

Color

key:

Cont

rol:_

____

____

CirCl

e You

r exp

erim

enta

l gro

up: p

H, Te

mpe

ratu

re, In

hibito

r

| 37

Appendix 1 – Enzymes are special proteins

AlllivingthingsareconstructedofproteinsthatareproducedbycellsfollowingtheDNA-RNA-proteinpathway.Proteinsmakeupourfleshandblood,carryoxygentoourlungs,andregulateourbodilyfunctions.Enzymesareproteins,too.Theyaidindigestingourfood,creatingpigmentinourskin,andarerequiredforDNAreplication,eliminationoftoxins,andothervitalbodilyfunctions.Fromvirusestomammals,enzymesarerequiredbyeverylivingthingtoexistandsurvive.

Enzymesaremacromoleculescreatedfromachainofaminoacidsfoldedintoaunique3-dimensionalshape.Enzymesdifferfromotherproteinsduetothepresenceofan“activesite”—thelocationwhereinteractionsandreactionsbetweenmoleculestakesplace.Thisuniquecharacteristicallowsenzymestoaidinbiochemicalreactionsbyspeedinguptheprocessofbuildingorbreakingdownsubstances.Theenzymeactsonmoleculesbyprovidingtheconditionsforchemicalbondcreationorbreakage.SeeAppendix2

Often,enzymesarenamedforthesubstratetheyinteractwith(i.e.protein)andaddingthesuffix–ase(“prote-ase”).

A few examples of enzymes are:

LysozymeAnaturallyoccurringenzyme(partoftheinnateimmunesystem),foundinoursalivaandnasaldrippings,lysozymeactslikescissorstocutpeptidebondsthatmakeupthecellwallofbacteria,thusdestroyingandkillingpotentiallyharmfulorganisms.Virusesalsocreateanduselysozymetobreakintoandinfectbacte-rialcells.

LactaseTheenzymefoundinthedigestivesystemofmammals,includinghumansthatdrinkmilkand/oreatmilkproducts.Ithelpstobreakdownmilkproteinsintosimplesug-arsthatcanbeutilizedbythebody.SeeAppendix3

Proteinfolding

Lysozyme

ProteaseAgroupofenzymesthatbreakdownproteinsinthebodyintosmallermoleculesthroughthehydrolysisofpeptidebonds(be-longingtothelargerenzymegrouphydro-lases).Forthisreason,proteasesareutilizedindetergentsandsoaps.

Lactase

ProteaseofhumanmalariaparasitePlasmodium falciparum

Similartoprotease,therearemanydigestiveenzymeswhichbreakdownlargermolecules(e.g.amylasebreaksstarchintosugars,lipasebreaksdownlipidsintofattyacids)intosmallercounterpartsthroughhydrolysis.

APPENDIX1

38 |

Appendix 2 – Enzymes act as a Biological Catalyst to assist and speed up biochemical reactions.

Acatalystspeedsupachemicalreactionwithoutbeingconsumedinthereaction.Enzymesarebiologicalcatalysts—theyarerequiredforspecificbiochemicalreactionstooccurwithinlivingorganisms.Enzymeshelptocreateproductsbyeitherbreak-ingdownpre-existingchemicalbondsandmoleculesorbuildingnewones.Whenproductsarecreated,thesubstrateisusedup,buttheenzymeisstillpresent.

Enzyme+Substrate—>Enzyme-Substrate—>Enzyme+Product

Enzymeslowertheactivationenergyofareactionbycreatinganenvironmentthatallowsforreactionstooccurfasterandwithlessrequiredenergy.Theysometimesachievethisstatebyalteringtheshapeofthesubstratetoallowforeasierbreakageorconnectionofchemicalbonds.

Somechemicalreactionswilloccurwithoutenzymes,butbenefitfromthecatalyticnatureofthemolecule.Ananalogywouldbeahumanbeingattemptingtogoupandoveramountainpass.Walkingmaytakedays,butthejourneycouldbemade.Byusingabicycle,thetimeittakestocompletethejourneyisreducedsignificantly.Astrongercatalyst,suchasacar,canspeedtheprocessevenfurther.Someenzymesareweakcatalysts(likethebicycle)andsomearestrong(suchasthecar).

Anotheranalogycouldbemaderegardingthebuildingofaproductfromtwoormoreexistingmolecules(anabolicreaction)andthebreakingdownofamoleculeintotwoormoresub-units(catabolicreaction).Iftherearetwopeoplewhowouldbeagreatcouple,buttheydonotknoweachother,thechancesofthemmeetingarelowandthetimeitmaytakeforthatmeetingcouldbelong.Amatchmakeractsasacatalysttobringtwopeopletogetherfasterandmoreefficientlythanifthematchwasmadeonitsown.Alongthesameline,arelationshipcouldbebrokenupbyarumororeventthatwaspresentedtothecouplebyanoutsideentity.Thispersonorentityisacatalystforabreakup,asitweakensthebondbetweenthetwopeople.

Anexampleofananabolicbiochemicalreactionisthecreationofgranulatedsugarfromfruitsugarthroughbondformation.Theenzymesucrosesynthetasehelpstoformsucrosesugar(whitegranularsugar)fromglucoseandfructose(fruitsugars).SeeAppendix3

Activation energy without enzyme

Activation energy with enzyme

Energy of reactants

Free

ene

rgy

Change in free energy

Progress of Reaction (time)

Without enzyme

With enzyme

Energy of products


| 39APPENDIX3

Appendix 3 – Enzymes act as biological catalysts to assist and speed up biochemical reactions.

Enzymescatalyzeparticularreactionsandonlyworkwithspecificsubstrates.Forexample,lactaseisanenzymethatbreaksdownmilkprotein.Lactasedoesnotreactwithanyotherproteinorsubstanceotherthanmilkprotein.(and,viceversa--lactose,themilkprotein,isunabletobebrokendown/digestedwithouttheenzymelactasebeingpresent).

OH

OHOH

OH

HO

HO

HO

HO

HO

OO

A common catabolic reaction is the digestion or breakdown of milk protein (lactose) by the enzyme lactase into simple sugars (glucose and galactose).

Lactase

SUBSTRATE

Active Site

Active Site

Lactose(Glucose + Galactose)

+

PRODUCTS

Glucose Galactose

COMPLEX

OHHO

HO

HO

OH

O

OHOH

OH

HO

HHO

O+

Thespecificityofenzymesisduetodifferentcharacteristicsoftheenzymesandsubstratesincludingshape,charge,andhydro-phobic/phyllicproperties.

Theshapeoftheactivesiteandthefitofthesubstratehasbeenreferredtoasalockandkeymodel,where,likeajigsawpuzzle,thesubstrateandenzymeconnect.However,manyscientistsbelievethattheenzymewillchangeshapetofitthesub-strateintotheactivesite.Thisisknownastheinducedfitmodel.

40 |

Theenzymesucrosesynthetasecombinestwoseparatesubstrates(glucoseandfructose)intoasinglemolecule(sucrose).Thistypeofreactionisrepresentativeofananabolicreaction.Glucoseandfructosearebothsimplesugars(monosaccharides)andsucrosesythetasecombinesthemintoapolysaccharide(sucrose).

SUBSTRATE SUBSTRATE

Active Site

Active Site

Sucrose Synthetase

Sucrose (white granular sugar)

PRODUCT

GlucoseFructose

ENZYME-SUBSTRATE COMPLEX

OH

OHO

HO

OH

OH

OHHO

HO

HO

OOH

OHO

HO OH

O

OH OH

OH

OHO

OH


| 41APPENDIX4

Appendix 4 – Enzymes are sensitive to changes in pH and temperature.Mostenzymesareonlyfunctionalwithinaspecific,narrowrangeofpHandtemperature.Enzymesinhumansandothermammalswillnotfunctionifthebodytemperaturerisesordropstoomuch.Digestiveenzymesinourstomachsfunctioninahighlyacidicenvironment,andwillnotworkoutsideofthestomachwherethepHishigherandlessacidic.

Enzymescanbedenatured(rendereduseless)byheatingorotherfactorsthatchangetheshape,thusthespecificityandfunc-tion,oftheenzyme.Theycanbe“unfolded”andcansometimesre-foldtofunctionproperlyundertherightconditions.

Tyrosinase,theenzymeusedinthelabactivity,isanexampleofanenzymethatistemperaturesensitive.Itisoptimallyfunc-tionalwithinaspecifictemperaturerangeinmostorganisms(e.g.humansatbodytemperature),butisuniquelydependantontemperatureinSiamesecats.Forexample,thecolorpatternobservedmostfrequentlyisblackface,blacktail,blackpaws,withabrownorblondemid-section(likethecatontheleft).

Theblackpigmentisaresultoftyrosinaseactivityattheextremities,wheretemperaturesaresignificantlycoolerthantherestofthebody.Attheselocations,tyrosinaseisactivelyworkingtoproducemelanin,resultingindarkfur.Theoretically,ifsocksweretobeappliedtothecatforalongperiodoftime(enoughtimeforthepreviousfurtofalloutandnewfurtogrowin),thenowwarmerfrontlegswouldreducetheactivityoftyrosinaseinthatarea,resultinginlighterfur(likethecatontheright).

42 |

Appendix 5 – Enzymes activity can be slowed or prevented by the presence of inhibitors.Inhibitorsarenaturalorsyntheticmoleculesthatdonotallowtheenzymetoreactwiththesubstrate.In“competitive”inhibi-tion,theinhibitorandthesubstratecompeteforthesameenzyme.Iftheinhibitorbindstotheactivesite,theenzymecannotfunctionproperlyandcannotreactwiththesubstrate.

Inhibitorsmayslowdownorcompletelypreventenzymefunction.Byunderstandinginhibitors,scientistscanapplywhattheyknowtocreatingandutilizinginhibitorsforthebenefitofhealthandmedicine,economicgrowth,andotherhumanconcerns.Bothnaturallyoccurringandsyntheticallyproducedinhibitorsareutilizedinoureverydaylives.

Examples of inhibitorsCommonpreservativesusedinfoodproductsareactuallyenzymeinhibitors.Sodiumbenzoateisacommonpreservativeusedtopreserveflavor,preventbrowning,andtogivealongershelflifetotheproduct.

Enzymeinhibitorsarecommonlyusedasinsecticidesandchemicalwarfareagents—regularenzymefunctionwithinthenervoussystemcanbeblockedorslowed,resultingindeath.Acetylcholinesterase(AChE)istheenzymetargetedbyinhibi-torsfoundininsecticidesandchemicalssuchasserum(nerve)gas.AChEisfoundinmanyanimalsanditfunctionstobreakdowntheneurostransmitteracetylcholineinthesynapticcleft(formoreinfooncholinesterases,seeAppendix6).Revers-iblecompetitiveinhibitorshavebeenidentifiedandmanufacturedintoantidotesusedtotreatpeopleexposedtonervegasorothersimilaragents.

SomecommondrugsthatactasenzymeinhibitorsareViagra,HIVproteaseinhibitors,andsomecancerdrugtreatments(chemotherapy).

Tyrosinase,theenzymeusedinthislab,hasmanyinhibitors,bothnaturalandsynthetic.

In“non-competitive”orallostericinhibitioniswhentheinhibitorbindstotheenzymesatanotherlocationotherthantheactivesite,thuschangingtheshapeoftheproteinstructureandalteringtheactivesiteenoughthatthesubstratecannotbind.Anallostericactivatorcreatesthecorrectshapeforthesubstratetobindtotheenzyme.

Enzyme

Active Site (where substrate binds)

Substrate

Allosteric Site- any site other than the active site

EnzymeSubstrate

Competitive InhibitorInhibitor- competes with substrate for active site

Non-competitive InhibitorEnzyme

Substrate

Inhibitor- binds to allosteric site and changes enzyme’s conformation (shape), so substrate is unable to bind

Allosteric Activator (or facilitator)

Enzyme

Activator- binds to allosteric site and stabilizesactive conformation of enzyme, making it easier for the substrate to bind

Substrate


| 43APPENDIX6

Appendix 6– CholinesterasesCholinesteraseisanenzymefoundinthecirculatoryandnervoussystemsofmanyanimals,includinghumans.Thisenzymeactstometabolizeaneurotransmitter,acetylcholine,thatfunctionsinboththeperipheralnervoussystemandcentralnervoussystem.Acetylcholineactivatesmuscles,stimulatesthebody(whenexcited)toreleasesubstancessuchassweatandepineph-rine(adrenaline),andplaysasignificantroleinaddiction.

Twosimilaryetdistinctcholinesterasesexist:“acetylcholinesterase”and“butyrylcholinesterase”.Bothareequallyeffective,butworkondifferentsizesofcholinesubstrates.Acetylcholinesteraseconvertsacetylcholineintotheinactivemetabolitescholineandacetate.Thisenzymeisabundantinthesynapticcleft,anditsroleinrapidlyclearingfreeacetylcholinefromthesynapseisessentialforpropermusclefunction.Certainneurotoxinsworkbyinhibitingaceylcholinesterase,thusleadingtoexcessacetylcholineattheneuromuscularjunction,causingparalysisofthemusclesneededforbreathingandstoppingthebeatingoftheheart.Thebuildupofacetylcholineallowsforanincreaseinthelevelanddurationofactivityinthejunction.

Thecholinesteraseactivesiteisburieddeepintheglobularprotein,andthemolecularconstructoftheenzymecreatesastreamlinedgorgestraighttotheactivesite,allowingforveryquickin-and-outactiononthesubstrate.

Inhibitorsofacetylcholinesteraseoccurnaturallyasvenomandpoisons,andareusedasnerveagentsinterrorismattacksandwarfare(e.g.nervegasorserumgas).Humanshavealsotakenadvantageoftheworkofcholinesteraseinhibitorsbycreatingpesticidesandinsecticidesthatactonthenervoussystemofants,ticks,aphids,andotherinvertebrates.

ReadmoreaboutcholinesterasesandtheresearchbeingconductedatUCSD’sSchoolofPharmacy

http://pharmacy.ucsd.edu/faculty/TaylorLab/

1906? Discoveryofcholinesterases

1960’s-1970’s Inhibitorsandsubstratesfoundthatbindandinterferewithcatalyticreactionofcholinesterase

1980’s ProteinPurificationtechniquesallowedforidentificationofcholinesteraseactivesiteandgenesequenceto allowforcloningofgenethatencodeforcholinesterase

1990’s Three-dimensionalstructureofacholinesterasewassolved

2000’s Determinationof>125primarystructuresand>60three-dimensionalstructuresofcholinesterase

Fun fact• Acetylcholinewasthefirstneurotransmittertobeidentified.

Humanrecombinantacetylcholinesterase

Enzyme

Active Site (where substrate binds)

Substrate

Allosteric Site- any site other than the active site

EnzymeSubstrate

Competitive InhibitorInhibitor- competes with substrate for active site

Non-competitive InhibitorEnzyme

Substrate

Inhibitor- binds to allosteric site and changes enzyme’s conformation (shape), so substrate is unable to bind

Allosteric Activator (or facilitator)

Enzyme

Activator- binds to allosteric site and stabilizesactive conformation of enzyme, making it easier for the substrate to bind

Substrate

44 |

Appendix 7 – Albinism and TyrosinaseAlbinismisanautosomal,recessivegeneticconditionwhichischaracterizedbyalackofpigmentation(melanin)infish,am-phibians,reptiles,birds,andmammals—includinghumanbeings.Thelackofpigmentationisduetoamutationinthegenethatcodesfortyrosinase.Withouttyrosinase,theproductsneededtoformmelanincannotbeproduced.

Becausemelanincreatescolorinskin,eyes,andhair,anyalbinisticorganismwillappearwhiteorverypalewithlightblueeyes(oreyesappearingreddishduetotheretinalbloodvesselsshiningthrough).

Approximately1in70peopleisacarrierofthealbinismgene,leadingtoanincidenceof1in17,000amonghumanbeings.Whiletherearedifferentformsofalbinismwithvariouscauses,onetypewithafrequencyof1:40,000personsisoculocuta-neousType1(OCA1)whichiscausedbyamutationinthetyrosinasegene,TYR.AlbinisticpersonswithOCA1producelittleornomelaninleadingtowhite,almosttranslucenthairandskin.Theytendtohavepooreyesight.

Sincealbinisticpersonsproducenomelaninasaresultofthetyrosinasemutation,theycannottanandmustbecarefulaboutlongexposuretosunlight.Theyareparticularlysusceptibletomelanoma,averydeadlyformofskincancer.

Twoheterozygotesforalbinismwillhavea1:4chanceofhavingalbino

offspring.


| 45APPENDIX8

Appendix 8– Protein Data BankEnzymesandotherproteinsarecomplexstructureswhichcanbedifficulttovisualize.TheProteinDataBankcontainsimagesofmanyproteinstohelpillustratethestructureandfunctionofindividualproteins.Programsforvisualizingtheseproteinsarefreeandavailabetoteachers.Thefollowingguideshowshowtodownloadaproteinandsomeideasforuseintheclassroom.Thisprocesstakesyouthroughtheproteindatabank,butifyoujustwishtodownloadthevisualizationsoftware,gotowww.pymol.organdfollowtheinstructionsonpg.122ofthisguide.Ifyoujustwantaquickviewofproteinswithoutdownloadingsoftware,simplytogowww.rcsb.organdtypeinaproteintypeinthetopsearchbar.Seeinstructionsforthisonpg.123ofthisguide.

To access the Protein Data Bank

Goonlinetowww.rcsb.organdthiswilltakeyoutothehomepagefortheproteindatabank.Thispagecontainsawealthofinformationonmolecularstructures.

Scrolldownthepage,lookingattheleftsidemenuandclickonEducationalResources(picturedabove).ThistakesyoutoapageofeducationalresourcesbasedonmoleculesinthePDB.Inthefirstparagraphonthispage,youwillseealinktoLook-ingatStructures.ClickingonthislinktakesyoutoinformationaboutthePDBandhowitcanbeusedtovisualizeavarietyofstructures,alongwithprovidingabriefintroduction.

46 |

ClickthelinkforPyMOLonthetop,right-handsideofthepage.Thisisthevisualizationsoftwareyouwillneedtodown-loadtolookatproteins.

OntheUnderstandingPDBpage,thereareseveralsectionstohelpyoumakesenseofthisdatabase.Downloadingavisu-alizationprogramtoyourcomputerallowsyoutobypassanyinternetissuesyoumayhaveatyourschoolduringclasstime.UndertheVisualizingStructuresheading,youwillseeabluelinktoVisualizationPrograms.Clickonthislinktoseewhatvisualizationprogramscanhelpyouillustrateaboutvariousproteins,includingoptionsforshapes,colors,andstructures.

TowardthetopofthenewpageyouwillseeMolecularGraphicsSoftwareLinks.Thiswilltakeyoutoalargelistofmolec-ulargraphicssoftwareoptions.Dependingonyourpotentiallevelofuse,thispagecontainssomeexcellentlinkstoadditionalmaterialsavailableonline.Duringthetraining,youwereshownproteinsinaprogramcalledPyMOL.Followthesedirectionstodownloadandusethisprogram.


| 47APPENDIX8

Fillinthenecessaryinformationintheregistration.*Warning*Theregistrationletterstheformasksyoutofillintoconfirmyourregistrationcanbeveryhardtoread!Youwillreceiveyourdownloadcredentialsviayoure-mailaddress,somakesurethiswebsitewillnotbeblockedbyyoure-mail.Clickonthelinksentinthee-mailtodownloadPyMOL.Ausernameandpasswordwillbeprovidedinthee-mailandwillbeaskedforwhenyouclickonthedownloadlink.

Onceyouhaveenteredyourusernameandpassword,downloadinformationwillcomeup.Rightclickonthedownloadinfor-mationforyourappropriatesystemanddownloadtheprogram.Onceyouhavetheprogramdownloadedandopened,youwillneedsomeproteinstolookat!Todothis,returntowww.rcsb.orgtofindsomeproteinsofinterest.

DownloadthePyMOLprogram.ThiscanrunonLinux,MacOSXoronWindows.Whenyouclickthislinkitwilltakeyoutoaregistrationpage.Onthispage,thereisalinkforstudentsandinstructors.Clickpymol.org/educationaltogotothefreeeducationversion.


48 |

Tostartviewingproteins,therearetwoapproaches.Thesimplestapproachistotypeinaproteininthesearchbaronrcsb.orgtoviewthatprotein.Inthisexample,wehavetypedintheenzymetyrisonase.

Awidevarietyofarticlesontheproteinyouchosemayappear.Somewillbemorereleventthanothers,soyoumayneedtoscrolldownandfindtheparticularproteinyouarelookingfor.Inthisexample,wewilllookatthefirsthitfortyrosinase.Clickonthepictureofthetyrosinaseproteintochosethismodel.

Anewpagewillcomeupwithalargeversionoftheprotein,aspicturedbelow.BelowthatpicturethereisalinkwhichsaysViewinJmol.Clickthislinktoviewtheprotein.Theproteincanberolledaroundviamouseclicktobeviewedinanyorien-tation.


| 49APPENDIX8

PyMOLTodownloadtheproteinyouwanttoviewinPyMOL,lookforthe4digitalphanumericcodeinthetoprightcornerofthepage.ClickonDownloadFilesandthefollowingmenuwillappear.ClickonPDBfile(gz)todownloadthecorrectfiletypetoberuninPyMOL.Whenthisfileisfinisheddownloading,openitusingPyMOLtoviewyourchosenprotein.

OnceyouhaveopenedyourmoleculeinPyMOL,itshouldresemblethescreenbelow.Clickonthemoleculeandmoveyourmousearoundtochangetheorientationofthemolecule.

50 |

Tochangetheappearanceofyourprotein,clickontheletter“S”(forShow)besideyourmolecule’sID.Themenupicturebelowwillappear.Clickonthedifferentappearancestoseewhichversionoftheproteinyouwanttouse.Toremovetheseoptions,clickonthe“H”button(forHide)andchoosewhichoptionsyouwishtohide.Showandhideoptionsuntilyouhavereachedthedesiredappearanceofyourprotein.

To customize your proteinIfyouclickon“C”forColoryoucanchooseColorbyChainandchooseacolorschemetoaddacolortoeachsubunitiftheproteinconsistsofmorethanonesubunit.Youcanalsoselectthe“S”inthebottomrightcornerforsequence.Thiswillshowyouthesequenceoftheproteinatthetop.Youcanclickonanysingleaminoacidormanyaminoacidsandchoose“S”forshowandchoosestickstoshowtheaminoacidsonthestructure.

Ifyouwanttocreatehotkeysforcertainsnapshotsoftheprotein,placetheproteininthedesiredorientation,gotothetopmenuandselect“Scene”-->Store-->F1.NowF1willalwaystakeyoutothisspecificsnapshot.YoucandothisforothersnapshotsandselectF2,F3,etc.tostoremultiplesnapshots.

Atwo-buttonmousewithawheelwillallowfortheeasiestmovementinPyMOL.Leftclickallowsyoutoselectaminoacidsandholdingleftclickwillrotatetheprotein.Holdingrightclickwillzoominandout,whileholdingscrollwillallowyoutomovetheproteinonthescreen.

SpecialthankstoSocratesFellowalumniJohnYamauchiforhisguidanceincreatingthistutorial.


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