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Objectives:
Introduction: As you know, enzymes represent the class of proteins that catalyze
reactions. Catalase is an enzyme found in most living cells. It is abundant
in blood, liver, and kidney. It has been isolated in crystalline form and it
has a molecular weight of about 250,000 daltons. Catalase is a conjugated
enzyme in that it has a heme group. This heme-containing enzyme has
Fe3+ at the center of the heme group. Beef liver catalase has 4 heme
groups per molecule.
The International Union of Biochemists has assigned numbers to all
characterized enzymes as an aid in classifying their functions. The first
digit in this number identifies the class to which an enzyme belongs. The
enzyme commission number for catalase is E.C. 1.11.1.6. This number
indicates that catalase is an oxidoreductase meaning that it catalyzes an
oxidation-reduction reaction.
Catalase is very specific in its reaction, which is the conversion of
hydrogen peroxide to water and oxygen:
H2O2
Catalase
H2O + 1/2 O2
Procedure:
1. Transfer 0.25mL of a catalase solution to a single eppendorf tube. Place this tube in a
boiling water bath and boil for two minutes. Note: The sample must be kept hot until
you are ready to add it to Tube 3 (below) at the appropriate time.
2. Set up five test tubes as indicated in the following table. Make additions in the
precise order given in the following table.
Reagent
Phosphate buffer (mL)
H2O2 (mL)
Water (mL)
5 x 10-5 M KCN (mL)
6 N H2SO4 (mL)
Catalase Soln. (mL)
Catalase previously boiled for 2 min. (mL)
Tube 1
5.0
1.0
0.5
--1.0
0.25
---
Tube 2
5.0
1.0
0.5
----0.25
---
Tube 3
5.0
1.0
0.5
------0.25
Tube 4
5.0
1.0
0.25
0.25
--0.25
---
Tube 5
5.0
1.0
--0.5
--0.25
---
---
1.0
1.0
1.0
3. Fill a buret with 0.005M KMnO4 and use this reagent to titrate all reaction tubes (1
5) to the pink endpoint. Record these data (mL KMnO4 used) in the following table.
Tube 1
Tube 2
Tube 3
Tube 4
Tube 5
mL KMnO4
When this relationship is multiplied by the volume of KMnO4 (mL) used, the resulting
number will indicate the mols of substrate present in the tube either initially (molsi) or
at the end of the 5-minute incubation (molsf).
The quantity of substrate converted (decomposed) to product in five minutes defines
molsdecomp.
The catalase activity will be calculated by dividing the quantity of substrate converted to
product in five minutes by the volume of enzyme used (0.25mL).
1.0
Parameter
Calculation
molsi H2O2
mL KMnO4
molsf H2O2
mL KMnO4 x 12.5
molsdecomp H2O2
molsi - molsf
(Tube 1)
Tube 1
x 12.5
Inhibition (%)
Activity T2 Activity Tx
Activity T2
x 100
Tube 2
Tube 3
Tube 4
Tube 5
______> 1/ H O + O
2 2 2
2
d. H2O2 + 1/2 O2
______> H O + O
2 2
2
4. In the "Catalase" experiment, titration of the solution in Tube 1 (the Control) tells
you __________.
a. how many micromoles of hydrogen peroxide are decomposed in 5 minutes
b. how many micromoles of hydrogen peroxide are decomposed in 5 minutes in all
tubes (Tubes 1 through 5)
c. how many micromoles of hydrogen peroxide are initially present in all tubes
d. how many milliliters of KMnO4 are used in all the tubes
5.
131.25
56.25
318.75
187.5
None of the above.