ENZYME LAB

Introduction-
Enzymes are proteins that speed up the rate of reactions in living things. In this lab, we will
perform four experiments exploring the way enzymes work.
PART A: pH SPECIFICITY
Every enzyme has a specific pH at which it works best. In this section, you will determine which
pH is best for the enzyme, catalase.
iving tissues produce the enzyme catalase, which is able to break down hydrogen peroxide into
oxygen gas and water. !he reaction is shown below"
H
#
$
#
$
#
% H
#
$
&ells make hydrogen peroxide as a normal product of metabolism. 'ut it is toxic to the cell if it
builds up. !herefore, all cells produce catalase to get ride of hydrogen peroxide. In the following
experiment, we will use liver as a source of catalase.
'efore you do the procedures below, set up a lab paper with overall lab title, sections lab title,
propose and hypothesis.
(rocedures"
). *et + test tubes and label them ) through + with a pen. (lace all tubes in a rack..
#. *et + pieces of raw liver ,how gross- and place ) piece in each test tube.
+. .sing a graduated cylinder, add / ml of distilled water to tube ).
0. (ut on your goggles. .sing the graduated cylinder labeled acid, add /ml of hydrochloric
acid ,H&l- to tube #. 'e careful.
/. .sing the graduated cylinder label base, add /ml of sodium hydroxide ,1a$H- to tube +.
2. 3ecord the contents of all tubes in a data chart on your lab paper.
4. 5ait +min.
6. 7ou will now use a graduated cylinder labeled H
#
$
#
to add /ml of hydrogen peroxide to
each tube. 'e sure to bring the hydrogen peroxide to your tube in the rack. 1ever hold your
test tube in your hand while pouring in the hydrogen peroxide. 'e prepared for overflow.
8. &heck for the production of gas in the data charts for each tube, using the terms none, fair,
or many.
)9. &lean up procedures"
-!hrow all liver in the trashcans.
- Empty li:uids down the drain, rinse, and place tubes in dirty bin in front of the room.
;nswer the :uestions below on your lab paper"
). 5hat is the substrate in this experiment< 5hat are the products of reaction< 5hat is the
name of the enzyme< 5hat is the source of the enzyme<
#. 5hy did we look for bubbling in the test tube< 5hat does bubbling tell you<
+. 'ased on your data. ;t what pH does catalase enzyme work the best< Explain how you
know.
0. 5hat affects does too much acid or too much base have on an enzyme< 'e specific.
PART B: TEMPERATURE SPECIFICITY
=ost enzymes have a certain temperature range to which they work best. $utside of the temperature
range, an enzyme>s structure may be destroyed, rendering it ineffective. In this experiment, we will
determine the effect that boiling temperatures have on the enzyme catalase. 5e will compare the
gas production with boiled liver versus raw, room temperature liver.
'efore you start the lab, write the section title, purpose and a hypothesis on your lab paper.
(rocedures"
). *et # clean test tubes and label them 0 and /.
#. !o tube 0 add a piece of raw liver.
+. !o tube / add a piece of cooked liver.
0. ;dd /ml of distilled water to each tube.
/. ;dd /ml of hydrogen peroxide to each tube.
2. 3ecord the contents of each tube in your data chart, as well as the relative amounts of
bubble production.
4. &lean up as instructed in section ;.
;nswer"
). 'ased on your data, what effect does boiling have on the effectiveness of catalase enzyme<
Explain how you know.
#. ?uppose you had prepared another test tube with frozen raw liver, distilled water, and
hydrogen peroxide. 5hat affects do you think very low temperatures might have on the
catalase enzyme<
+. ;t what approximate temperatures does catalase enzyme work the best<
0. 5hat affects does temperature outside the normal optimum have on the catalase enzyme< 'e
specific.
PART C: SURFACE AREA AND ENZYME ACTION
In order of an enzyme to be most effective, it most be exposed to its substrate. In this experiment,
you well use a potato as a source of catalase. ) test tube will contain the enzyme within a solid
potato cub. !he other test tube will contain the enzyme in a masked raw potato.
'efore toy start this section, write the section title, purpose and a hypothesis on your lab paper.
(roduces"
). *et # new test tubes and label them 2 and 4.
#. to tube 2, add a whole raw potato cube.
+. !o tube 4, add an e:ual amount of raw, mashed potatoes.
0. ;dd /ml of distilled water to each tube.
/. ;dd /ml of hydrogen peroxide to each tube.
2. 3ecord the contents of the tubes in your data chart.
4. 3ecord the relative amounts of bubble production in each tube.
;nswer"
).5hich form of the potato, raw or mashed, had more surface area to make catalase enzyme more
available<
#. 5hich form of the potato showed more enzyme action-taking place< 5hy<
PART D: SUBSTRATE SPECIFICITY
Enzymes are specific to respect to the substrate they will react with. !he active site of an enzyme
fits its substrate like a key fits only a specific lock.. 7our body has ),999 of different enzymes, each
capable of catalyzing ) specific reaction. In his section you will compare the effect # different
enzymes, amylase and pepsin, have on the substrate, starch.
'efore you set up the lab, write the section title and purpose on your lab paper, then answer these
:uestions.
). ook up amylase and pepsin in the digestion chapter of your text. 5hat substrate does
amylase react with< 5hat substrata does pepsin react with<
#. ;fter reading the procedures below, write a hypothesis stating which test tube you think will
show the digestion of starch by enzymes< 5hy<
+. 5hy do we incubate the test tubes at body temperature<
0. 5hat is the purpose of test tube )9< Have you set up a controlled experiment< 5hat is the
variable<
(rocedure"
@ay )"
). *et + test tubes and label them 6, 8 and )9. (ut ) person initials on each tube.
#. .sing a clean graduated cylinder, add / ml of starch suspension to each tube. 3inse the
graduated cylinder several times with water.
+. ;dd # ml of pepsin enzyme to tube 6. 3inse the cylinder
0. ;dd # ml of amylase enzyme to tube 8. 3inse the cylinder.
/. ;dd # ml of distilled water to tube )9.
2. 3ecord the contents of all tubes in your data chart.
4. (ut all test tubes in test tube rack in front of room for incubation at body temperature.
@ay #"
). ?et up a boiling water bath.
#. *et your test tubes from the test tube rack in the front of the room.
+. ;dd )9 drops of 'enedict>s solution to each tube,.
0. (lace all tubes in a boiling water bath and observe.
/. In you data table, note the number of the tube that shows a color change to yellow orange
first. !his color change indicates the presence of simple sugars.
2. ;s soon as the first tube changes color, unplug the water bath. @isregard further color
changes
;nswer the following :uestions on your paper"
/. 'enedict>s reagent is use to test for the presence of simple sugars. 5hich test tube showed
the presence of simple sugars<
2. 5hy does a positive test for sugar indicate starch digestion<
4. 5hat do your results indicate about substrate specificity<
&$1&.?I$1"
$n separate paper, type or write a conclusion summarizing the factors necessary for optimum
enzyme action. !he conclusion should begin with a brief introductory paragraph. 7our first
sentence should be an overall statement naming the four factors investigated in the lab, followed by
a brief explanation of how enzymes work. !he introductory paragraph should be followed by four
paragraphs explaining what you learned about each aspect of enzyme action. ?tart each of the
paragraphs with an overall statement about that aspect of enzyme action, then support the statement
with data from the lab. @$ 1$! restate procedures in your conclusion. ; final summary paragraph
is not necessary. It is not necessary to discuss sources of error in this lab.
Four Factors Affect!"
Enzyme ;ctivity
#$ pH Specfct%

E!&%'e Su(strate Source of E!&%'e
Co!ta!s pH A'ou!t of Reacto!
!est tube A)
!est tube A#
!est tube A+
). 'ased on your data, at what pH does catalase enzyme work best<
#. 5hat effect does too much acid or too much base have on an enzyme< 'e specific.
)$ Te'perature
E!&%'e Su(strate Source of e!&%'e
Co!ta!s A'ou!t of Reacto!
!est tube A0
!est tube A/
). 'ased on your data, what effect does boiling have on the effectiveness of catalase< Explain
how you know.
#. 5hat effect do you think very low temperatures ,frozen raw liver- will have on the catalase
enzyme<
+. ;t what approximate temperature does catalase enzyme work best< 5hat effect does
extreme temperatures have on the catalase<
*$ Surface Area a!+ E!&%'e Acto!
E!&%'e Su(strate Source of E!&%'e
Co!ta!s A'ou!t of reacto!
!est tube A2
Test tu(e ,-
). 5hich form of potato, raw, or mashes had more surface area to make catalase enzyme more
available<
#. 5hich form of the potato showed more enzyme action-taking place< 5hy<
*$ Su(strate Specfct%

E!&%'e Su(strate Source of E!&%'e
Co!ta!s A'ou!t of reacto!
Test tu(e ,.
!est tube A8
!est tube A)9
). 5hat do the results indicate about substrate specificity<