College Biology: Cells and Molecules

Summer 2008
Laboratory Experiment #2: A Quantitative Enzyme Study
I. INTRO!"TION
Enzymes are biological catalysts that carry out the thousands of chemical reactions that occur in
living cells. hey are generally large !roteins made u! of several hundred amino acids and often
contain a non!roteinaceous grou! called the !rosthetic grou! that is im!ortant in the actual
catalysis.
"n an enzyme#catalyzed reaction$ the substance to be acted u!on %substrate& binds to the active
site of the enzyme. he enzyme and substrate are held together in an enzyme#substrate com!le'
by hydro!hobic bonds$ hydrogen bonds and ionic bonds.
he enzyme then converts the substrate to the reaction !roducts in a !rocess that often re(uires
several chemical ste!s and may involve covalent bonds. )inally$ the !roducts are released and
the enzyme is ready to form another enzyme#substrate com!le'. *s is true of any catalyst$ the
enzyme is not used u! as it carries out the reaction but is recycled again and again. +ne enzyme
molecule can carry out thousands of reaction cycles every minute.
Each enzyme is s!ecific for a certain reaction because its amino acid se(uence is uni(ue and
causes it to have a uni(ue three#dimensional structure %tertiary or (uaternary structure&. he
business end of the enzyme molecule$ the active site$ also has a s!ecific sha!e so that only one or
a fe, of the thousands of com!ounds in the cell can interact ,ith it. "f there is a !rosthetic grou!
on the enzyme$ it ,ill form !art of the active site. *ny substance that bloc-s or changes the
sha!e of the active site ,ill interfere ,ith the activity and efficient of the enzyme. "f these
changes are large enough$ the enzyme can no longer act at all and is said to be denatured. here
are several factors that are es!ecially im!ortant in determining the enzyme.s sha!e and these are
closely regulated both in the living organism and in laboratory e'!eriments to give the o!timum
or most efficient enzyme activity: salt concentration and tem!erature.
"n this e'ercise$ you ,ill study the enzyme catalase$ ,hich accelerates the brea-do,n of
hydrogen !ero'ide$ a common end !roduct of o'idative metabolism$ into ,ater and o'ygen
according to the summary reaction:
2/
2
+
2
2/
2
+ 0 +
2
his catalase#mediated reaction is e'tremely im!ortant in the cell because it !revents the
accumulation of hydrogen !ero'ide$ a strong o'idizing agent that tends to disru!t the delicate
balance of cell chemistry.
Catalase is found in animal and !lant tissues and is es!ecially abundant in !lant storage organs
such as !otato tuber$ corns and the fleshy !arts of fruits. Catalase has been isolated from !otato
tubers and you ,ill measure its rate of activity under different conditions. * glass fiber filter
,ill be immersed in the enzyme solution and then !laced in the hydrogen !ero'ide substrate.
Catalase
he o'ygen !roduced in the subse(uent reaction ,ill become tra!!ed in the dis-$ ma-ing it
buoyant. he time measured from the moment the disc touches the substrate to the time it
reaches the surface of the solution is a measure of the rate of the enzyme activity.
II. #RO"E!RE
A. Extra$tion o% "ata&a'e: T(i' )i&& be done %or you*
1. 2eel a fresh !otato tuber and cut the tissue into small cubes. 3eigh out 40 gm of tissue.
2. 2lace the tissue$ 40 m5 of cold ,ater and a small amount of crushed ice into a !rechilled
blender.
6. /omogenize for 60 seconds at high s!eed.
+rom t(i' point on, t(e enzyme preparation mu't be $arried out in an i$e bat(*
7. )ilter the !otato e'tract through cheesecloth and !our the filtrate into a 100 m5 graduated
cylinder. *dd cold distilled ,ater to bring u! the final volume to 100 ml. his e'tract
,ill be arbitrarily labeled 100 units of enzyme !er m5 %100 units8m5& and ,ill be used in
!arts B#E.
A. E%%e$t o% $ata&a'e $on$entration
Before considering the factors that affect enzyme reactions$ it is im!ortant to demonstrate that
the enzyme assay sho,s that the enzyme actually follo,s acce!ted chemical !rinci!les. +ne ,ay
to demonstrate this is by determining the effect of enzyme concentration on the rate of activity
,hile using a substrate concentration that is in e'cess.
5abel four 40#m5 bea-ers as follo,s: 100$ 94$ 40 and 0 units !er ml. 2re!are 70 m5 of enzyme
for each of the above concentrations in the follo,ing manner:
m5 original enzyme 0 m5 cold$ :" /
2
+ ; units8m5
70< 0 100
60 10 94
20 20 40
0 70 0
-SA.E T/IS !NIL!TE EN012E +OR #ARTS "3E*
=ee! your catalase !re!arations in the ice buc-et. 5abel an identical set of bea-ers for the
substrate. "nto each of these bea-ers$ measure out 70 m5 of 1> /ydrogen !ero'ide %/
2
+
2
&
solution.
?sing force!s$ immerse a 2.0 cm glass fiber filter disc to one#half its diameter in the catalase
solution you have !re!ared. *llo, the disc to absorb the enzyme solution for 4 seconds$ remove
and drain for 10 seconds on a !a!er to,el. :ro! the disc into the first substrate solution. he
disc ,ill ra!idly sin- into the solution. he o'ygen !roduced from the brea-do,n of the /
2
+
2

by catalase becomes tra!!ed in the fibers of the disc causing the disc to float to the surface of the
solution. he time %t& in seconds$ from the moment the disc touches the solution to the time it
again reaches the surface is used to determine the initial velocity$ %@
i
& of catalase$ ,here @
i
; 18t
%sec&. Each assay should be carried out t,ice. Re$ord and avera4e t(e re'u&t' in your
noteboo5'.
6. E%%e$t o% 'ub'trate $on$entration
o determine the effect of substrate concentration on enzyme activity$ obtain nine 40#m5
bea-ers and label them as follo,s:
0> /
2
+
2
& 0.8 > /
2
+
2
0.1> /
2
+
2
1.0 > /
2
+
2
0.2> /
2
+
2
4.0 > /
2
+
2
0.4> /
2
+
2
10.0 > /
2
+
2
14.0 > /
2
+
2
*dd 70 m5 of the !ro!er 0> /
2
+
2
solution to each bea-er. *dd 70 m5 of d/
2
+ for 0> /
2
+
2.

Ma-e sure that the substrate solutions reach room tem!erature before beginning your assay.
?sing the filter discs !rocedure described above$ and the undiluted enzyme from !art B$
determine the rate of the reaction at the various substrate concentrations. Aecord your results in
your lab noteboo-s. Each assay should be carried out t,ice and the results averaged.
". E%%e$t o% temperature
?sing 70 m5 of a 1> /
2
+
2
solution as the substrate and 4 m5 ali(uots of the 100#units8ml#
enzyme solution$ measure the enzyme activity %as outlined in B&. Aun the reactions in the
different tem!erature ,ater baths. he catalase and substrate %/
2
+
2
& should be brought to the
testing tem!erature before they are used. *llo, 10 minutes for e(uilibration. 2roceed to E
,hile the catalase and substrate are e(uilibrating. Aecord the e'act tem!erature and your data in
a table in your noteboo-. *lso test the activity of the enzyme that has been boiled. :+ B+
boil the /
2
+
2
. hese assays should be run in du!licate.
. E%%e$t o% p/
+btain 4 40#m5 bea-ers and label them as follo,s:
1. !/ 7 2. !/ C
3 6. !/ 9 7. !/ 8
4. !/ 10
"nto each bea-er$ !our 10 m5 of enzyme !re!aration and 60 m5 of buffer solution at the
a!!ro!riate !/. ?sing 70 m5 of a 1> /
2
+
2
as the substrate$ measure the enzyme activity in
the usual manner.
III. ANAL1SIS O+ RES!LTS
1. "n an a!!endi'$ include a table of ra, data you collected for each !art of the e'!eriment.
2. Aecord your results from B %the effect of catalase concentration& and !lot them on a gra!h
labeling the '#a'is catalase and the y#a'is @
i
. /o, does the enzyme activity vary ,ith
enzyme concentrationD
6. Aecord and !lot your results from C %the effect of substrate concentration on enzymatic
activity&. 2lot the Michaelis#Menten gra!h as ,ell as the 5ine,eaver#Bur-e gra!h ,ith this
data. /"B: use the 5ine,eaver#Bur-e !lot to determine @
ma'
and =
m
. /o, is the rate of
enzyme activity affected by increasing the concentration of the substrateD 3hat do you thin-
,ould ha!!en if you increased the substrate concentration to 20> /
2
+
2
. :oes changing the
substrate concentration e'hibit the same effect as changing the enzyme concentrationD
Comment on the use of the Michaelis#Menten versus the 5ine,eaver#Bur-e analysis.
7. 2lot the results of : %the effect of tem!erature on enzymatic activity& on a gra!h. 3hat can
you conclude about ho, tem!erature affects enzyme activityD /o, ,ould you e'!lain the
resultsD
4. /o, does !/ affect enzyme activityD Era!h your results. 3ould you e'!ect similar results
,ith salivary amylaseD 3ith 2e!sinD
*c-no,ledgements: * Modified 2E3 E'!eriment and :r. Susan Morgan