Efects o pH, Temperature and Concentration on Enzyme Properties
Niyanthesh Reddy Dr. D’ Aessio Cass! DA" Date! #$%%'$#(
Abstract
This e)periment is meant to understand the efects o *arious interactions on the acti*ity o enzymes in the en*ironment +in *i*o and in *itro. -n this study, the efects o pH, temperature and concentration e*es on enzymes are measured and potentia data is e)istent. Amonst pH, the reatest acti*ity occurred at neutra +pH / e*e and started approachin 0aseine e*e at more acidic +pH ' and 0asic +pH #$ e*es. 1ith reater temperature, the num0er o su0strates coidin 2ith the enzymes increased +mosty at &' C, ho2e*er ater optima e*e, it started droppin due to reater num0er o interna 0onds 0ein damaed and the o*era enzyme endin in a denatured state. 1ith concentration, the reater the amount o enzyme, the reater the reaction rate due to num0er o su0strates coidin 2ith more acti*e sites. The data coected to support this study 2as done 2ith a spectrophotometer +measurin the a0sor0ance e*es and conducted usin the enzyme A3P +A4aine Phosphatase.
Introduction -n nature, enzymes are *ery important as they hep decrease the acti*ation enery, there0y increasin the rate o a chemica reaction. E*ery chemica reaction occurs usin an enzyme, and 2ithout it, ie 2oud 0e simpy impossi0e. Enzymes or instance hep to hydroyze macromoecues in our ood to tiny su0units 2hich can 0e used or nutrients and enery needed or the ro2th o the 0ody. Enzymes are aso used in medicine, especiay 2hen dianosin diseases. -n this e)periment, one 2i 0e e)aminin the efects o pH, temperature, and concentration on the unction o enzymes usin a spectrophotometer. This is done to understand ho2 the roe o enzymes is afected 0y *arious conditions. -n this e)periment particuary, the enzyme A3P +A4aine Phosphatase, 2i 0e used. This enzyme hydroyzes the phosphate roups on proteins and ipids, necessary in many in *i*o chemica reactions in our 0ody. 5nderstandin the efect o pH on enzymatic unction 2i 0e done usin acidic, neutra and 0asic soutions. Conductin this part o the e)periment is important, as one can understand 2hat the optima pH o an enzyme is, 2ithout ma4in denaturation occur +E). 0rea4in interna 0onds. The efect o concentration on enzymatic acti*ity is *ery important to measure, 2hether increasin or decreasin su0strate concentration. -n scienti6c iterature, enzyme acti*ity increases as the concentration o su0strates increases as 2ith more su0strates 0ondin to enzymes, a acti*e sites are 6ed up. To a point ho2e*er, 2hen a the acti*e sites are 6ed up, enzymatic acti*ity drops. Thereore, the optima concentration at 2hich enzyme acti*ity reaches its hihest 2i 0e necessary or speedin a reaction. 3ast, in this e)periment, temperature 2i 0e measured. The ma7or reason or the denaturation o an enzyme occurs due to temperature. -n this e)periment, one 2i
0e measurin the temperature o enzyme A3P at *arious temperatures o*er a period o time. 8y nu hypothesis or the pH section o the a0 is that pH 2i not ha*e much o an efect o enzyme concentration as the 0onds hodin the enzyme toether are tron. 8y nu hypothesis or the concentration section is that concentration 2i ha*e ony a partia efect on enzymatic acti*ity. 9or temperature, the nu hypothesis is that enzyme acti*ity 2i not chane sini6canty e*en i partia structura chane occurs 2ith temperature. The aternati*e hypothesis or the efect o pH on enzyme acti*ity is that an im0aance o H: ion concentration may ha*e some efect on enzymatic acti*ity, and thereore the reatest rate o reaction may 0e around / +neutra pH. The aternati*e hypothesis or the efect o temperature on enzymatic acti*ity is that since temperature increases the re;uency o coisions +4inetic enery 0et2een the su0strates and enzyme +enzyme
Materials/ Procedure Part One- Measuring pH levels for Enzyme activity of ALP
+1ison, Properties of Enzymes, $
Table 1. Mixing Instructions for pH Experiment
Tube
Relative pH
Solution E
0.2 M HCl
0.1 M Na2CO3
Distilled water
Solution D Hi! "on". en#$%e
1 contro l
---
3 mL
---
---
2 mL
---
5 mL
---
---
1"" µL
5 mL
Total &olu%e
2
Aciic
3 mL
1.! mL
3
#eutral
3 mL
---
---
1.! mL
1"" µL
5 mL
$
%asic
3 mL
---
1.! mL
---
1"" µL
5 mL
Add =oution >, ?.( m3, +pNPP
ein procedures #<@ 2ith cu*ette ', & and @ and add =oution D to it +riht 0eore doin the readin. 8i) careuy, usin para6m paper to in*ert +cu*ette and pace the cu*ettes in the spectrophotometer. Record the a0sor0ance readin at Time $, and henceorth e*ery &$ seconds or 6*e minutes. Each *aue is to 0e recorded on Data Ta0e '. Repeat procedures or cu*ettes & and @ +steps ' Notes! =ince Tu0e # is the contro, the spectrophotometer shoud 0e set to 0an4 at @$( nm. Do not add soution D to any cu*ette unti ready or insertion into the spectrophotometer. Pre in a ($ m3 0ea4er and a0e it =oution 9. This soution 2i 0e used 2hen measurin the efects o concentration and temperature on enzymatic acti*ity in reactions +A3P enzyme. 3a0e eiht cu*ettes #a< @a, #0< @0. -n this pre
able !- Mi"ing table for #uvettes $-%a& 'e E(ect of #oncentration on Enzymatic )ate
Tube
&elati'e En()me *onc.
+ol * Lo AL En()me0 conc.
"
+ol , comprise of +ol A% %uffersubstrate stoc/ solution0 3 mL
---
+ol Hig4 AL En()me0 conc. ---
1 blan/ no en()me0 1a blan/ plus en()me 2a 3a
Loest
3 mL
1"" µL
---
Meium Hig4er meium
3 mL 3 mL
$"" µL ---
--2"" µL
$a
Hig4est
3 mL
---
5"" µL
+1ison, Properties o Enzymes
able %- Mi"ing able for #uvette $b-%b& 'e E(ect of emperature on Enzymatic )ate
Tube
Temperature *0
1b 2b 3b $b
$ 23 32 6"
+ol , %uffersubstrate stoc/ solution0 3 mL 3 mL 3 mL 3 mL
+ol * Lo AL En()me0 conc. 1"" µL 1"" µL 1"" µL 1"" µL
+1ison, Properties o Enzymes
Part *o& +etermining t'e E(ect of Enzyme #oncentration on )eaction )ate +1ison, Properties o Enzymes 5sin a micropipette +preera0y #$$<#$$$ 3, add #$$ 3 o soution C +contains o2 concentration o A3P to cu*ette #a and put immediatey into spectrophotometer ater mi)in thorouhy +pace a para6m and in*ert the cu*ette or 0est resuts. Ater, pace the cu*ette into the spectrophotometer, startin 2ith the 6rst readin at $ seconds. Read the a0sor0ance e*es e*ery &$ seconds or ( minutes in Ta0e (. Ne)t, usin a micropipette, add @$$ 3 o =oution C +contains o2 A3P concentrations to cu*ette 'a, mi) it thorouhy and insert into the spectrophotometer. Read the a0sor0ance e*ery &$ seconds or ( minutes ater insertion into the spectrophotometer. 5sin a micropipette, add '$$ 3 o =oution D +hih A3P enzyme concentration to cu*ette &a, co*er 2ith a para6m paper, in*ert the cu*ette to mi), and immediatey pace it into the spectrophotometer. Record the a0sor0ance e*e e*ery &$ second or 6*e minutes post
µ
3 o =oution D to cu*ette @a, co*er 2ith
para6m, in*ert to mi), and pace it into a spectrophotometer. Read the a0sor0ance e*e at $ seconds, and read the a0sor0ance e*ery &$ seconds or ( minutes.
,ote& )efer to ables ! and % for detailed mi"ing instructions
Part 'ree& +etermining t'e E(ect of emperature on Enzyme )eaction )ate +1ison, Properties o Enzymes 9irst, o0tain cu*ette #0 stored in the ride and usin a micropipette, add #$$ 3 o soution C +containin o2 A3P concentration to cu*ette #0, in*ertin the mi)ture or 0etter resuts. -mmediatey pace the cu*ette into the spectrophotometer and record 6rst measurement at $ seconds, and ater2ards, e*ery &$ seconds or ( minutes post< insertion. =econd, use a micropipette, addin #$$ 3 o soution C to cu*ette '0 and co*er the cu*ette 2ith para6m or mi)in. Pace immediatey into the spectrophotometer and record the a0sor0ance e*es e*ery &$ minutes or a ( minute period in Ta0e (. Third, o0tain cu*ette &0 +in the 2ater 0ath at &'C and add #$$ 3 o soution C +o2 A3P to the cu*ette. 8i) the soution in the cu*ette thorouhy 0y in*ertin, and immediatey pace into the spectrophotometer. >ein recordin the a0sor0ance e*es e*ery &$ seconds or ( minutes and record in Ta0e (. 9ourth, et cu*ette @0, paced in the 2ater 0ath heated at ?$C, and add #$$ 3 o =oution C +o2 A3P. 8i) thorouhy and pace in the spectrophotometer. Record the a0sor0ance at &$ second inter*as or ( minutes. Record in Ta0e (. Note! Reer to Ta0es & and @ or detaied mi)in instructions
)esults- Part $ Efect o pH on the Enzyme Controed Reaction Ta0e #. Efect o pH on the Acti*ity o the Enzyme A3P time +sec
pH ' $ &$ ?$ $ #'$ #($ #"$ '#$ '@$ '/$ &$$
pH / $.$&" $.$&? $.$&( $.$&@ $.$&& $.$&& $.$&& $.$&& $.$&' $.$&' $.$&'
pH #$ $.$/? $.$"& $.$"@ $.$" $.$@ $.$" $.#$# $.#$( $.##' $.##( $.##"
$.$/ $.$/' $.$/( $.$/? $.$// $.$" $.$"' $.$"@ $.$"? $.$"" $.$
-igure $& E(ect of pH on t'e Activity of Enzyme ALP over ime $.#@ $.#' $.#
pH '
f+), E $) : $.$"
3inear +pH ',
$.$" f+), E $) : $.$/
ph /
Absorbance Levels $.$?
3inear +ph /, pH #$
$.$@ $.$' $ $
3inear +pH #$,
f+), E < $) : $.$@ ($ #$$ #($ '$$ '($ &$$ &($ ime .sec/
-n Ta0e #, pH ' sho2ed a decrease in the acti*ity o the enzyme, 0y the a0sor0ance e*es oin rom $.$&" to $.$&'. The pH / e*e sho2ed an increase rom $.$/? to $.##" and pH#$ sho2ed an increase rom $.$/ to $.$. 9iure # sho2ed a inear decrease or pH ' data points, rom the e;uation y< $.$$$$') :$.$&?&. The pH / e*e sho2ed a inear increase, as indiciated 0y the e;uation, y$.$$$$/:$.$/$#. 3ast, pH #$ sho2ed the same inear increase 0y the e;uation, y $.$$$#):$.$/?"
Ta0e '. Efect o Diferent pH e*es on the Rate o Enzyme Reaction pH
Rate o Reaction '
<$.$$$$'
/
$.$$$#
#$
$.$$$$/
-igure 0& E(ect of +i(erent pH levels on t'e )ate of Enzyme )eaction over ime $ $ $ $
)ate of Enzyme )eaction
$ $ $ '
/
#$
$ $
pH level
-n ta0e ' and 6ure ', the rate o reaction 2as most or pH /, as indiciated 0y the rate o the reaction 0ein y$.$$$#. The rate o the reaction 2as then the reatest or pH #$, at y$.$$$$/), and ast or pH ', at a decreasin rate o y<$.$$$$')
)esults- Part 0 E(ect of #oncentration on Enzyme Activity Ta0e &! Efect o enzyme concentration on the Acti*ity o Enzyme A3P
Time +s
#a +o2
'a +med
&a +med
@a +hih
$
$.##/
$.@"(
$.'?&
$.('"
&$
$.#'?
$.(#'
$./
$.?@(
?$
$.#&'
$.(&?
$.&?
$./??
$
$.#@$
$.(("
$.@'&
$."/"
#'$
$.#@"
$.("@
$.@/(
$.""
#($
$.#(@
$.?#$
$.(@
#.$"
#"$
$.#?#
$.?&@
$.(/"
#.'$
'#$
$.#?"
$.??$
$.?&$
#.@
'@$
$.#/(
$.?"#
$.?"'
#.@'"
'/$
$.#"'
$./$@
$./'
#.('(
&$$
$.#"
$./&$
$.//"
#.?'
-igure !& E(ect of Enzyme #oncentration on Enzyme Activity over #."$$ #.?$$ f+), E $) : $.(@ #.@$$
#a +o2, 3inear +#a +o2,,
#.'$$
'a +med, #.$$$ Absorbnace Levels
3inear +'a +med,, &a +med
$."$$ $.?$$
3inear +&a +med
f+), E $) : $.'/ f+), E $) : $.@D
@a +hih, 3inear +@a +hih,,
$.@$$ $.'$$ f+), E $) : $.#' $.$$$ $
($
#$$
#($
'$$
'($
&$$
&($
ime .sec/
Ta0e @. Efect o Enzyme Concentration on the Rate o Reaction Enzyme concentration
Rate o Reaction
3o2
$.$$$'
8ed
$.$$$"
med hih
$.$$#/
Hih
$.$$&/
-igure %& E(ect of +i(erent Enzyme #oncentration on t'e )ate of Enzyme )eaction over ime $ $ $ $ )ate of )eaction $ $ $ $ $
#a +o2,
'a +med,
&a +med
@a +hi.h,
Enzyme #oncentration
-n Ta0e & and 9iure &, it is indicated that the enzyme concentration had the reatest efect on the Acti*ity o Enzyme A3P indicated 0y cu*ette @a +hih concentration. The a0sor0ance e*e or @a rose rom $.('" to #.?'. The a0sor0ance e*e or &a +med
-n Ta0e @ and 9iure @, a enzyme concentrations sho2ed an increase in enzymatic acti*ity. The rate o the reaction, in efect to e*e o concentration, ho2e*er 2as most or @a +hih concentration at y$.$$&/). Ne)t hihest 2as &a +med
)esults-Part 'ree E(ect of emperature on Enzymatic Activity in )eactions Ta0e (! Efect o temperature on the rate o an enzyme controed reaction @oC
Time +s $ &$ ?$ $ #'$ #($ #"$ '#$ '@$ '/$ &$$
'$oC $.$(& $.$(/ $.$?# $.$?' $.$?? $.$? $.$/' $.$/( $.$/" $.$"' $.$"@
&'oC $.$// $.$"# $.$"( $.$' $.$/ $.#$' $.#$/ $.### $.##? $.#'# $.#'?
?$oC $.$"@ $.$# $.#$# $.## $.#' $.#'" $.#&/ $.#@@ $.#(# $.#(" $.#??
$.#'' $.#'( $.#& $.#&@ $.#&/ $.#@ $.#@& $.#@? $.#@ $.#(# $.#(@
-igure 1& E(ect of emperature on an Enzyme )eaction over i $.#" $.#? f+), E $) : $.$" f+), E $) : $.#' $.#@ @C
$.#' f+), E $) : $.$"
3inear +@ C, '$ C
$.#
3inear +'$ C,
Absorbance Levels
&' C $.$" f+), E $) : $.$(
3inear +&' C, ?$ C 3inear +?$ C,
$.$?
$.$@
$.$'
$ $
($
#$$
#($
'$$
'($
&$$
&($
ime .sec/
Ta0e ?. Efect o Temperature on the Rate o an Enzymatic Reaction Temperature +oC
Rate o Reaction
@
$.$$$#
'$
$.$$$'
&'
$.$$$&
?$
$.$$$#
-igure 2& E(ect of emperature on t'e )ate of an Enzymatic )eaction over ime $
$
$
$
)ate of )eaction $
$
$
$ @
'$
&'
?$
emperature .#/
-n ta0e ( and 9iure (, the efect o temperature on the rate o the enzyme controed reaction had a 0i impact. The a0sor0ance e*e rose rom $.$(& to $.$"@ or @oC temperature. The a0sor0ance e*e rose rom $.$// to $.#'? or '$ oC. At &' o C, the hihest increase in a0sor0ance e*e occurred, risin rom $.$"@ to $.#??. 3ast, at ?$ oC, the a0sor0ance e*e rose rom $.#'' to $.#(@. -n 9iure (, the efect o temperature on enzyme acti*ity 2as pued in and or @ o C, y$.$$$#) :$.$(?&. 9or '$ oC, y$.$$$'):$.$/?( 2as the inear trend ine. 9or &' oC, the trend ine inear e;uation 2as $.$$$&) :$.$"@. 3ast, the trend ine at ?$ o C 2as $.$$$#):$.#'&'. -n Ta0e ?, the rate o the reaction 2as cacuated or each Temperature, 0ased of o ym), These *aues 2ere paced in a ta0uar orm, in Fraph ?. The reatest rate o reaction, as indicated in the coumn raph, 2as at &' oC, 2here y$.$$$#). The second hihest rate 2as at '$ oC, 2here the rate o reaction 2as y$.$$$'). The
east rate o reaction occurred at temperatures @ oC and ?$ oC 0oth o 2hich had y$.$$$#) rates. Discussion -n the resut sections, or pH, temperature and concentration, the resuts a sho2ed patterns and my hypothesis 2as statisticay supported. 9or pH e*es, the reatest enzyme reaction occurred at / +neutra in comparison to pH ' +acidic and pH #$ +a4aine. The reason 0ein that chanes in pH, either too o2 or too hih can ater an enzyme’s o0uar or 60rous state. -n other 2ords, pH has an efect on the undamenta 0asis o enzymes, amino acids. Acidic amino acids contain a car0o)y unctiona roup +iooy #$th Edition te)t0oo4, enzymes ha*e a pH at 2hich the enzyme 2or4s 0est. The most efecti*e pH is 0et2een ?<" +optimum, ho2e*er there are some e)ceptions i4e the enzyme pepsin 2hich 2or4s in the stomach at pH '. 9or temperature, indicated 0y Ta0es ( and ? and 9iures ( and ?, the reatest efect on the rate o enzyme acti*ity occurred at room temperature +&' oC. At @ oC, the temperature does not ha*e enouh 4inetic enery or the enzymes to *i0rate and 0rea4, thereore ma4in it impossi0e or the su0strates to 6t into the acti*e site. At '$ C, since 4inetic enery increased, more su0strates 2ere interactin 2ith the enzyme acti*e site. At optimum temperature +&' derees C, the enzyme had the reatest acti*ity o su0strates 0indin to acti*e sites, and thereore had the reatest increase in the rate o reaction. At ?$ oC ho2e*er, the enzyme most pro0a0y 2ent into a denatured state, osin its speci6c shape so the acti*e site 0ecomes atered and the hihy eneretic su0strate moecues cannot 6t into the enzyme’s acti*e site. Thereore, the reater 4inetic enery +Temperature, the reater the enzyme reaction rate, unti ater the optimum temperature has 0een reached .Accordin to the Camp0e >iooy #$ th Edition te)t0oo4, most enzymes ha*e optimum temperatures 0et2een &(<@$ Deress Cecius, 2hereas some ha*e optima temperatures at /$ C.
+Reece, Camp0e >iooy
9or concentration e*es, as the concentration increases, the rate o the reaction increases unti a o the su0strate moecues ha*e 0inded to their compementary acti*e site on the enzyme. As indicated in Ta0es & and @, our cu*ettes 2ith diferent concentrations o A3P enzyme 2as tested or. The ones containin the most enzymes had the hihest reaction rate as more enzymes 2ere a0e to react 2ith the su0strates, and the ones containin the east concentration o enzymes had the east reaction rate. This 2as indicated 0y Ta0es &, throuh a0sor0ance e*es in the spectrophotometer, and Ta0e @, 0y measurin the rate o reaction. Thereore, the reater the concentration o enzyme, the reater the reaction rate as more su0strates can 0ond to the enzymes. This increase occurs unti the acti*e sites o a the enzymes are u. -n pH and Temperature, 0ufers 2ere used as ma7or diferences occurred. As demonstrated in the theoretica raph +Concusion , the 0eufers 2ere thereore used to e;uaize the point o interaction 0et2een enzymes and the correspondin actors +pH and temperature. -n concusion, pH, temperature, and concentration ha*e a reat efect on the unction and shape o enzymes. Temperature, pH and concentration, i too hih or too o2 can damae the interna and e)terna structures o enzymes, there0y causin reactions to so2 or not e*en 2or4, as demonstrated 0y the enzyme< su0strate compe). Thereore, to o0tain statisticay more correct resuts, urther e)perimentation must 0e done.
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