Biochemistry Laboratory (BCM 362L), Experiment No.

6, © March 4, 2006
3nd Quarter A.Y. 2005-2006

Catalase: A Study of Enzyme Activity

Mr. *****1, *****2, *****2

1
Professor, School of CHE-Chm, Mapua Institute of Technology
2
Student, BCM 362L/ A31, School of CHE-Chm, Mapua Institute of Technology
______________________________________________________________________________________

ABSTRACT

Enzymes are special proteins that function to were computed using Lineweaver-Burke plot
lower the activation energy required for a and the Hanes-Woolf plot which was 2.116x10-05
reaction to occur, thus increasing the rate of mol/mL and 0.139 mL/s. The Eadie-Hofstee plot
reaction. One such enzyme studied in this was omitted since the actual plot varies from the
experiment is catalase. Catalase was extracted theoretical plot. From these parameters, the
from unripped papaya. Only the frist trial were enyme activity computed was 1313.3980 mol
used in the analysis since the second trial wasn’t H2O2 / sec · mol.
performed properly and increased in error might
occur. Five set-ups were made with varying Keywords: emzyme, catalase, activity, hydrogen
hydrogen peroxide and phosphate buffer peroxide, phosphate buffer.
solutions to obtain a relationship. Km and Vmax
______________________________________________________________________________________

INTRODUCTION product of metabolic processes - primarily that

Enzymes are very large and complex
organic molecules that are synthesized by the
cell to perform very specific functions. These
biological catalysts are important because they
speed up the rate of the reaction they catalyze
that would otherwise be too slow to support life.
Enzyme activity is controlled by a number of
factors, all of which affect the efficiency with
which the enzyme binds to the substrate: enzyme
concentration, substrate concentration,
temperature, and inhibitors. Catalase is an
enzyme present in the cells of plants, animals
and aerobic bacteria.
Catalase was one of the first enzymes to
be purified to homogeneity, and has been the
subject of intense study. The enzyme is among
the most efficient known, with rates approaching
200,000 catalytic events/second/subunit (near the
diffusion-controlled limit). Catalase structure
from many different species has been studied by
X-ray diffraction. Although it is clear that all
catalases share a general structure, some differ in
the number and identity of domains.
Catalase is nearly ubiquitous among
organisms that can grow in the presence of
oxygen. The major function of catalase within
cells is to prevent the accumulation of toxic
levels of hydrogen peroxide formed as a by-
of the electron transport pathway. The only
exceptions are the lactic acid bacteria, which
cannot synthesize the fundamental building
block porphyrin, and hence do not even possess
cytochromes that would otherwise make the
toxic H2O2.
Each molecule of catalase has four
polypeptide chains, each composed of more than
500 amino acids, and nested within this tetrad
are four porphyrin heme groups - very much like
the familiar hemoglobins, cytochromes,
chlorphylls and nitrogen-fixing enzymes in
legumes. Catalase, which normally accompanies
SOD, is found in almost all cells, but may be
found in its highest content in hydroponically
grown, concentrated wheat sprouts.
METHODOLOGY
Sample Preparation
Unripped papaya was peeled and cut
into small pieces. 100 g of cut papaya was
weighed and 100 mL of 0.1 M phosphate buffer
pH 7 was added. It was homogenized in a
blender until the texture was smooth. The crude
extract was filtered through cheesecloth. The
filtrates were collected and were centrifuged for
10 minutes. The supernatant were collected and
was stored in ice bath.

Gas Collector
An improvised gas collector was made
using a 50 mL Erlenmeyer flask covered with a
rubber stopper. A hole was bored into the
stopper to allow the insertion of small, straight 5
inches glass tubing. The glass tubing was
connected to a 12-inch rubber tubing. The end of
the rubber tubing was inserted into a water-filled
graduated cylinder inverted into a 400 mL
beaker.
Catalase Activity
The following set-ups were prepared in
a 50 mL Erlenmeyer flask according to the table
below.
graphical plot. The activity of the enzyme was
also calculated.
RESULTS AND DISCUSSION
Catalase speeds up the conversion of
hydrogen peroxide:
2H2O2  2 H2O + O2
In the absence of catalase, this reaction
occurs spontaneously, but very slowly. The
volume of oxygen with respect to time is shown
below for the five set-ups. The volume of
oxygen increases when hydrogen peroxide is
increased because more conversion of hydrogen
peroxide will be present when in large quantities,
which will form oxygen. Velocity is computed
by taking the slope of the most linear part of the
graph. The parameters calculated and data
obtained are shown on the following page.
60

50
Volume (mL) 40 A
Set-up A B C D E B
30
Sample extract 5 5 5 5 5
VO2

C
Phosphate buffer 0.8 0.6 0.4 0.2 -- 20 D
30% H2O2 0.2 0.4 0.6 0.8 1 10 E

0
0 100 200 300 400 500 600 700
Each of the flasks was immediately -10
Time
sealed. The amount of water displaced by the
oxygen gas was monitored. Volume readings
were listed in uniform intervals of 10 seconds.
Two trials were made.
The Lineweaver-Burke and Eadie-
Data Analysis Hofstee plot was made and both graph obtained a
Km value of 2.116x10-05 mol/mL and a Vmax of
Determination of Initial Velocity 0.139 mL/s.
For each reaction flask VO2 vs. time
were plotted. Velocity was calculated by taking Lineweaver-Burke

the slope of the linear part of the graph. 20

15

Calculating Substrate Concentration

1/v

10

The pressure of dry oxygen gas 5

produced was calculated. From the total volume 0

of O2 produced, the pressure of dry O2, and 0 10000 20000 30000 40000
1/[S]
50000 60000 70000 80000

temperature, the number of moles O2 was

calculated. The number of moles H2O2 was
determined based on the stoichiometry of the
reaction equation.

Determination of KM and Vmax

The [S] and V data for each reaction
flask were used to get the Lineweaver-Burke
plot, Hanes-Woolf plot and the Eadie-Hofstee
plot. KM and Vmax were determined from the
 Eadie-Hofstee

0.25

0.2

0.15
v

0.1

0.05

0
0 2000 4000 6000 8000 10000 12000
v/[S]

A B C D E
Time (s)
ht (mm) Vol (mL) ht (mm) Vol (mL) ht (mm) Vol (mL) ht (mm) Vol (mL) ht (mm) Vol (mL)
10 0 0 0 0 0 0 0 0 0 0
20 0 0 4 2.22 0 0 1 0.57 7 3.89
30 0 0 9 5 0 0 3 1.67 17 9.44
40 2 1.11 12 6.67 0 0 6 3.33 22 12.22
50 3 1.67 13 7.22 2 1.11 7 3.89 26 14.44
60 5 2.78 15 8.33 5 2.78 9 5 34 18.89
70 6 3.33 16 8.89 8 4.44 10 5.56 34 18.89
80 7 3.89 17 9.44 11 6.11 11 6.11 34 18.89
90 8 4.44 19 10.56 13 7.22 12 6.67 36 20
100 8 4.44 20 11.11 14 7.79 14 7.78 38 21.11
110 9 5 21 11.67 16 8.89 15 8.33 40 22.22
120 10 5.56 21 11.67 16 8.89 16 8.89 42 23.33
130 10 5.56 23 12.79 16 8.89 18 10 45 25
140 11 6.11 24 13.33 16 8.89 19 10.56 47 26.11
150 11 6.11 24 13.33 17 9.44 20 11.11 48 26.67
160 11 6.11 25 13.89 17 9.44 21 11.67 50 27.78
170 11 6.11 26 14.44 17 9.44 22 12.22 52 28.89
180 12 6.67 26 14.44 17 9.44 24 13.33 53 29.44
190 12 6.67 27 15 17 9.44 25 13.89 55 30.56
200 12 6.67 27 15 17 9.44 26 14.44 57 31.67
210 13 7.22 28 15.56 17 9.44 27 15 59 32.79
220 13 7.22 28 15.56 17 9.44 28 15.56 60 33.33
230 13 7.22 28 15.56 17 9.44 29 16.11 62 34.44
240 13 7.22 29 16.11 17 9.44 30 16.67 63 35
250 13 7.22 29 16.11 17 9.44 31 17.22 64 35.56
260 13 7.22 29 16.11 17 9.44 32 17.78 67 37.22
270 13 7.22 30 16.67 34 18.89 68 37.78
280 14 7.79 30 16.67 36 20 70 38.89
290 14 7.79 30 16.67 37 20.56 71 39.44
300 14 7.79 30 16.67 38 21.11 72 40
310 15 8.33 30 16.67 38 21.11 73 40.56
320 15 8.33 31 17.22 38 21.11 74 41.11
330 15 8.33 31 17.22 38 21.11 75 41.67
340 15 8.33 31 17.22 38 21.11 76 42.22
350 15 8.33 38 21.11 78 43.33
360 15 8.33 39 21.67 79 43.89
370 16 8.89 39 21.67 81 45
380 16 8.89 39 21.67 82 45.56
390 39 21.67 83 46.11
400 40 22.22 84 46.67
 410 40 22.22 85 47.22
420 40 22.22 85 47.22
430 41 22.78 86 47.78
440 41 22.78 87 48.33
450 42 23.33 88 48.89
460 42 23.33 88 48.89
470 42 23.33 89 49.44
480 43 23.89 90 50
490 43 23.89 90 50
500 43 23.89 91 50.56
510 43 23.89 91 50.56
520 44 24.44 92 51.11
530 44 24.44 92 51.11
540 93 51.67
550 93 51.67
560 93 51.67
570 94 52.22
580 95 52.78
590 95 52.78

Velocity (mL/s) Height (mm) PO2 (mmHg) PO2 (atm) VO2 (mL) V (L) Mole O2 [H2O2], mol/mL
A 0.0556 16 737.4065 0.9703 8.88 0.0089 3.5238x10-04 1.4095x10-05
B 0.2278 31 738.5094 0.9717 17.22 0.0172 6.8435x10-04 2.7374x10-05
C 0.1667 17 737.48 0.9704 9.44 0.0094 3.7464x10-04 1.4986x10-05
D 0.0556 44 739.4653 0.9730 24.44 0.0244 9.7255x10-04 3.8902x10-05
E 0.1111 95 743.2153 0.9779 52.78 0.0528 2.1109x10-03 8.4438x10-05

The enzyme activity was computed by

dividing kcat with KM, the activity was 1313.3980
mol H2O2 / sec · mol.

CONCLUSION

The more catalase available, the higher

the catalysis rate up to a certain amount of
enzyme.
The rate declines rapidly because there
are not enough substrates in enzyme molecules.
When catalase runs out of hydrogen peroxide to
convert, the reaction stops.

LITERATURE CITED

1. Biochemistry, Garrett and Grisham, 3rd Edition

2. © 1993-2003 Microsoft Corporation. All

rights reserved.

3.http://www.lsbu.ac.uk/biology/enzyme/practica
l1.html