Solved by verified expert:The rubric for the lab report will be attached along with all my collected data. My group and I did the “Effect of Enzyme Concentration on Reaction Rate”. This is important because this lab requires you to only describe the experiment that I did, UNTIL #5 (RESULTS) & #6 (DISCUSSION), then the other experiments can be individually discussed.For the results, there has to be 4 tables and/or graphs (1 for each experiment). Appropriate software must be used for this. (This will all be attached below, but feel free to ask if you need anything)Also, where you see a “Trial 2” data table, is the data that another group collected from doing the same experiment. So in the results there is only 1 table/ graph per experiment. There are 4 experiments (2 of them have a second trial). So for those with a second trial, you have to take both trials & collect the avg. With that create 1 table/ graph.Page 100 of the lab must be completed (The bottom 3 questions) and attached with the report. So I have attached it below for you to complete.Please also be careful with the references, because this professor is very picky!! She recommended us to use our school’s database, however I’m sure any other database sources will work just as fine as well!This is due April 4th, so it would be great to get it by or before tomorrow morning (before 9am central time)Thank you!!! 🙂
formal_lab_report1.doc
catalase_article_1.pdf
effect_on_sub___ph0000.pdf
effect_on_enzyme0000.pdf
effect_on_temp0000.pdf
bio_lab_pg_1000000.pdf
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RUBRIC FOR FORMAL LABORATORY REPORTS
Biology 151-405 Spring 2018
EVALUATING THE FACTORS THAT AFFECT ENZYME CATALYZED REACTIONS (CATALASE LAB)
Papers are due April 4, 2018 (no exceptions will be made. Late papers will not
be accepted.
Scientific reports usually follow a standard format especially if it is published in a peer reviewed
scientific journal. The different section(s) of a scientific paper is outlined below. This is a
general guideline for preparing high quality scientific research papers. It should help you
prepare the research paper on factors that affect enzyme catalyzed reactions. (Excuse all
typos)
Guidelines for Preparing a HIGH QUALITY REPORT:
•
Prepare all the sections of the report on a computer (word processor). The accepted
standardized format includes a readable letter style, font size of 12 and 1 inch (2.54 cm)
margins all the way around.
•
Double-space the text of the report. This makes the report easier to read and edit.
•
Get ideas and suggestions from other classmates and tutors. Proofread each section for
grammar, punctuation and spelling errors. Use the spell check feature on the computer.
PLEASE USE COMPLETE SENTENCES AND WRITE THE REPORT IN PAST TENSE.
Grading Rubric for the lab write up. (I reserve the right to make changes to assigned grades).
The paper should have the following
1. Running Title
A reasonable title and one that makes sense should be given.
2. Abstract (no more than ¾ three quarters of a page)
This should have a brief introduction followed by a brief description of your experiments and
result. Give a conclusion at the end.
3. Introduction (no more than 1½ one and a half pages)
Introduction is a history (scientific inquiry) of the experiment you have done. Research papers
that you have identified and read should be quoted here. Talk about experiments that have
been done before, how their experiments, results etc are similar or different from yours. Be
sure to site your references. Talk specifically about the experiment and why it is important for
the reactions to take place and how it relates health.
4. Materials and Methods (No more than one page)
If you have to list materials, only list those that are important. Do not list the obvious
materials everyone knows you have to use, such as pipettes, water, measuring cylinder etc.
Please note: you should only describe the experiment your group did. You will be penalized
for writing up an experiment you did not do.
Your method should not be in bullet points (THIS MEANS ABSOLUTELY NO BULLET POINTS).
You should refrain from using, I, me, we. You should use complete sentences
5. Result (As many pages you need to use)*This is the longest of all sections.
Your result should include all the four experiments that was done by the class
Explain the results for each experiment. Include your tables and graphs and please refer to
your tables and graphs in your results.
Tables: should be properly labeled and have a title
Graphs (Figures) should be properly labeled and have a title
ALL TABLES AND GRAPHS SHOULD BE OF JOURNAL QUALITY PRODUCED USING APPROPRAITE
SOFTWARE (WORD, XCEL OR ANY OTHER SOFTWARE OF YOUR CHOICE THAT DOES THE JOB)
6. Discussion (No more than 1½ , one and a half page)
Discussion is not your result. It is where you explain why you got the results you have. You
should discuss each experiment individually. Compare results with experiments that have
been done by other authors and discuss any similarities or differences.
7. References
You should have five references in all.
1. 3 should be from a peer reviewed journal no earlier than 2010. Samples of peer review
journals will be on the course page.
2. The other two should be ONE online article, the website should be cited properly AND A
TEXT BOOK OTHER THAN YOUR BIOLOGY TEXT.
RUBRIC FOR WRITE UP (I RESERVE THE RIGHT TO CHANGE IF NECESSARY).
1. Title: 2 points
2. Abstract: 3 points
3. Introduction: 5 points
4. Materials and Methods: 10 points
5. Results: 10 points
2
6. Discussion: 5 points
7. References: 3 points
8. Page 100 of your lab book: 3 points. The three questions should be answered
and attached to your paper
10. Overall organization of the paper: 4 points
11. Present for the lab exercise and took part in the lab: 5 points
Below is how your references should be cited.
JOURNAL: Alexander, Helen M., C.L. Cummings, L. Kahn and A.A. Snow 2001. Seed size
variation and predation of seeds produced by wild and crop-wild sunflowers.
American Journal of Botany, 88(4), 623-627.
BOOK: Raven, Peter H. and Linda R. Berg. 2000. Environment, 3rd Edition, New York:
Harcourt College Publishers.
WEBSITE: Parmentier H, Golding S, Ashworth M, Rowlands G. 2004. Community
pharmacy treatment of minor ailments in refugees. Journal of Clinical Pharmacy
and Therapeutics [Internet]. [cited 2007 Jul 24]; 29(5):465-469. Available from:
http://journals.ohiolink.edu/
3
A SPECTROPHOTOMETRIC
METHOD
FOR
BREAKDOWN
OF HYDROGEN
PEROXIDE
BY ROLAND
(From
the
Department
F. BEERS,
of Biology,
JR.,
Massachusetts
Massachusetts)
(Received for publication,
AND
MEASURING
THE
BY CATALASE*
IRWIN
W. SIZER
of Technology,
Institute
September
Cambridge,
24, 1951)
EXPERIMENTAL
Spectrophotonwtric Studies
Since the decrease in ultraviolet absorption by hydrogen peroxide as a
function of time is to be used to follow the catalase-peroxide reaction, a
* This work was done under a fellowship of the American Cancer Society, recommended by the Committee on Growth of the National Research Council.
133
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Several methods have been developed for following the breakdown of
hydrogen peroxide catalyzed by catalase, but these either have not been
sufficiently quantitative or have not proved rapid enough to yield reliable
data during the critical 1st or 2nd minute of the reaction. Chemical procedures in which residual peroxide is titrated with permanganate (l-3) or
an excess of permanganate is measured calorimetrically (4) are accurate
except for reaction times of less than a minute, although Lemberg and
Foulkes (5) developed a micromethod for obtaining data every 10 seconds
(see also Ogura et al. (6)). Considerable variability is unavoidable, however, when samplesmust-be taken at such short intervals. The manometric
method for measuring oxygen evolved from the system proved in detailed
studies to be unsuited for following the rapid breakdown of peroxide in
which a diffusion process across the liquid-air interface becomes limiting.
This is manifested by changes in both the order of the reaction and the
rate of evolution of oxygen with variations in the rate of agitation of the
reaction mixture (7). Direct measurement of hydrogen peroxide by polarography provides good quantitative data during the 1st minute of the
reaction which fit first order kinetics (8). However, an elaborate, special,
electronic circuit is needed for such measurements. Furthermore, as
pointed out by Bonmschen, Chance, and Theorell (8), this method appears
to give lower values for catalase activity than do titration techniques.
Preliminary experiments for following the breakdown of hydrogen peroxide by observing the decrease in light absorption of peroxide solutions
in the ultraviolet were reported by Chance (9) and Chance and Herbert
(10). The potentialities of this method have been investigated and a
quantitative, spectrophotometric technique for following the breakdown
of hydrogen peroxide has been developed for routine studies of catalase
kinetics.
134
BREAKDOWN
OF
HYDROGEN
PEROXIDE
knowledge of the absorption characteristic
of hydrogen peroxide is essential. The absorption spectrum of hydrogen peroxide, measured from 200
to 400 rnD, is shown in Fig. 1. At any wave-length
in this range it is
possible to use optical density as a measure of peroxide concentration,
since the optical density increases linearly with peroxide concentration
in
accordance with the Beer-Lambert
law. The reaction products, oxygen
and water, do not absorb light in this spectral region nor does catalase at
Downloaded from www.jbc.org by guest, on May 15, 2012
A
FIG. 1. Ultraviolet
absorption
curve
o? hydrogen
peroxide
in distilled
e = extinction
coefficient
= (optical
density)/(concentration)
; optical
density
IO/I, where 10 is the incident
light and I is the transmitted
light intensity.
water.
= log
the concentration employed (usually 10WgM) ; hence, the ultraviolet, absorption is a direct measure of the peroxide concentration in the catalaseperoxide system. If the system contains other substances that absorb in
the ultraviolet, the resulting error may be minimized by utilizing a wavelength at which the absorption of the foreign components is a minimum
in the region from 200 to 300 rnp, or by preparing a new calibration curve
for peroxide to which an appropriate amount of the absorbing foreign
substanceshas been added. An unknown absorbing impurity in the catalase-peroxide system will be revealed not only by an increase in the initial
optical density but also by an apparent change in the velocity and order
of the reaction.
R.
F.
BEERS,
JR.,
AND
I.
W.
SIZER
135
For computation of velocity constants optical density readings obtained
at successive time intervals suffice, and, in a particular experiment, any
convenient wave-length
can be used, since the velocity constant of a first
order reaction is independent of the type of units employed.
A convenient
initial optical density is between 0.5 and 1.0. In the spect.ral range from
2100 to 2400 A it is possible to use peroxide concentrations
ranging from
5 x lo-4 t0 3 x lo-2 M.
Materials
and Apparatus
Method
Into each of four cuvettes are pipetted 2 ml. of buffered catalase solution; to one, which serves as control, is added 1 ml. of buffer.
The slide
wire coil is set at the optical density near the initial value for the hydrogen
peroxide and the slit width is adjusted to the particular
wave-length
selected.
For routine assays 2400 A has been found to be the most useful
wave-length.
The selector switch is set at 1.0.
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Catalase Preparation-Crystals
of beef liver catalase (Worthington)
suspended in a saturated ammonium sulfate solution are dissolved by dialyzuffer, pH 7.0. The amber solution is then
ing against 0.05 M phosphal
centrifuged and aliquots of the supernatant are removed for kinetic studies.
of low9 M
Provided the buffer is not less than 0.05 M, final concentrations
catalase are sufficiently stable at room temperature to permit six to eight
successive kinetic trials before appreciable inactivation
occurs.
Substrate Preparation-An
approximately
5 X 10e3 M solution of hydrogen peroxide is prepared by diluting 0.15 ml. of superoxol (Merck)
with
25 ml. of 0.05 M phosphate buffer, pH 7.0.
A Beckman ultraviolet
spectrophotometer
(model DU) with a temperature-controlled
mounting block and three sets of cooling coils, as
supplied by the National Technical Laboratories,
is used. The mounting
block and cooling coil adjacent to the photometer are kept close to room
temperature with tap water.
The central coils adjacent to the cuvettes
are regulated to f0.1’
by a thermostatically
controlled water bath.
Four quartz 1 cm. cuvettes are used in each set of three kinetic trials.
The cuvett,es are cleaned with a suspension of magnesium oxide in distilled
water by means of a cotton swab, soaked in concentrated nitric acid for
12 hours, then rinsed thoroughly with distilled water, followed by drying
with a jet of filtered air or nitrogen.
Drying with alcohol is ineffective.
Unless adequate precautions are taken to prevent bubbles of oxygen from
forming on the cuvette walls during the reaction, serious errors are introduced. Bubbles are especially prone to form if the breakdown of hydrogen
peroxide lasts for more than 3 minutes or if any trace of dirt or grease
remains on the quartz surfaces of the cuvettes.
136
BREAKDOWN
OF
HYDROGEN
PEROXIDE
At zero time, 1.0 ml. of buffered hydrogen peroxide is blown from a
pipette in a continuous stream directly into the enzyme solution in the
This insures early and complete mixing of the two solutions in
cuvette.
2 or 3 seconds.
The cover is quickly replaced on the cuvette housing
unit and optical density readings are taken every 10 seconds (designated
TABLE
Comparison
of Per
Cent
I
Standard
Deviations
of Chronometric
for Measuring
Catalase
Activity*
Method
Standard
kot
and
Tracking
Methods
deviation
Per cent deviation
Sec.-’
Average…………………
Tracking………………….
Average
1.260
1.275
1.098
9.67
1.392
6.90
4.54
4.12
x
X
X
X
x
X
x
X
10-Z
1O-2
1OF
1O-3
10-a
1O-3
10-s
lo-
1.05
3.24
1.6
1.25
5.07
2.7
1.36
4.33
x
X
X
X
x
X
X
x
10-b
1O-4
1O-4
1O-4
10-4
10~~
1O-4
10-s
-I/-
ho.84
zt2.54
f1.46
f1.29
rt3.67
f3.90
rt3.00
zko.95
f2.2
8.77
8.14
8.62
1.325
3.54
1.355
1.355
X
X
X
X
x
x
x
10-a
1OP
lo+
lO-“
10-a
10-Z
10-Z
2.04
1.32
1.44
1.16
5.0
1.83
2.43
X
X
x
X
x
X
X
lO+
1O-4
10-d
1O-4
10-S
IOIO-
zh2.33
f1.63
f1.67
f0.88
f1.40
f1.35
fl.80
fl.6
* Temperature
20”; pH 7.0; 0.05 M phosphate
buffer.
t The velocity
constants
were determined
from
an average
of six consecutive
kinet,ic
trials
for each experiment.
In three
instances
one kinetic
trial
was eliminated
from the average
because
its deviation
was more than twice
the standard
deviation.
The catalytic process should be followed until
the tracking technique).
at least one-half of the substrate is destroyed in order to facilitate calcuThe same procedure is repeated
lating the order and velocity constant.
for the other two cuvettes.
Two modifications of the above spectrophotometric
technique have been
employed which are adapted to special types of catalase studies.
In the
chronometric modification a first order velocity constant is assumed without consideration of possible changes in the order of the reaction produced
by inhibiting processes.
The half time of the reaction may be determined
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Chronometric.
R.
F.
BEERS,
JR.,
AND
I.
W.
137
SIZER
easily with a stop-watch.
h specific optical density value is chosen, and,
when the gdvanometcr
pointer passes ~luough 1hc 11~11point, t,hc stopwatch is stnrtecl.
‘l’hc opt icxl tlc~sily SCYI~Cis than set, at, one-Mf
Ihe
initial value and 1hr: ~a~i.c~hstopped I-hen the null l~oint, is again pxwtl.
The velocity constant is tlctcrmincd
from 111~ quotitl11t (111 Z)/(time
required to halve the initial density).l
X IO-“M.
CATALAS
I
IO
I
20
I
30
I
40
TIME
I
I
50
60
70
IN SECONDS
I
80
I
90
I
100
I_
110
FIG.
2. First
order
rate curves
of destruction
of hydrogen
peroxide
by varying
concentrations
of wtalnse
(J)II 7.0, 0.01 II l)hosllhnte
buffer,
tcmlxrnturc
25.5”,
w:avo-length
210 mp, initi:tl
cof~c~~~~l,~.:~i ion ol lrytlro~cu
lwroitlc
:1pproxim:ltcly
0.015
M).
Table I shows a comparison of the results obtained by the chronomctric
and tracking methods.
The wide variations in stand:xd deviations observed in the former method (0.88 to 3.90 per cent) arc attributable to (1)
fewer points for determining the velocity constants,
(2) deviations from
1 The second modification
utilizes
fwturcs
of both the tracking
and chronometric
techniques.
In viex
of the uncertainty
of the exact zero time of the reaction
it is
often desirable
to select a particular
ol)ticnl
density
ns an arbitrary
starting
point
in order to permit
comparison
of similar
points
at a pxticul:w
time from scvernl
curves.
This mny be accomplished
by- setting
the optical
d(~usil,y initially
in thcl
sxne manner
as above,
but, instead
of recording
the half time,
record
the optical
densities
every
10 seconds.
Downloaded from www.jbc.org by guest, on May 15, 2012
.2-
138
BREAKDOWN
OF
HYDROGEN
PEROXIDE
first order rates caused by inhibitors, and (3) fluctuation in the spectrophotometer.
All three sources of error are taken into consideration in the
tracking method, thereby permitting
calculation of more accurate velocity
TABLE
Experimental
and
Enzyme concentration
ko,
experimental
M
x
x
x
X
x
ko,
sec.-’
10-10
10-10
10-10
lo-lo
10-g
3.1
5.9
7.8
1.4
2.3
x
x
x
x
X
3.1
6.0
8.6
1.34
2.3
mole-l
TABLE
Concentration
of enzyme
M x L-1
24.0
8.85
4.6
8.85
4.75
9.3
9.3
17.95
17.95
9.3
9.3
9.3
9.3
9.3
9.3
9.3
Velocity
X
x
X
x
x
x
x
x
x
x
x
x
x
x
x
x
Average………………
lo-lo
IO-10
10-l”
10-10
10-10
10-10
10-10
10-l”
10-10
10-10
10-10
10-10
10-10
10-10
10-10
10-10
Constant
3bserved velocity
l-
Varying
with
theory
Per cent deviation
sec.-’
10-d
10-d
10-h
10-a
lo-$
* k. = k, X E; k, = 2 X IO7 liters
of enzyme.
Standard
Constants*
sec.-l.
10-d
1OW
1OP
10-a
1O-3
0.00
1.65
9.30
0.45
0.00
E is the
molar
concentration
III
of Beef
Liver
constant, ko
sec.0.046
0.0145
0.0113
0.0187
0.0086
0.0173
0.0165
0.035
0.0324
0.0198
0.021
0.0198
0.021
0.0182
0.0192
0.0182
x
X
X
x
X
Catalase
Standard velocity constant, ks
z. x mole-’
–
1.9
1.64
2.4
2.1
1.81
1.86
1.77
1.95
1.805
2.13
2.26
2.13
2.26
1.96
2.06
1.96
2.0
sec.-
x
X
X
x
x
x
x
x
X
X
X
X
X
X
X
X
f
107
lo7
lo7
107
107
107
107
10’
lo7
10’
lo7
10’
lo7
lo7
10’
lo7
0.17
x
107
constants. Hence, the average standard deviations are lower (1.6 per cent)
and show less fluctuation than in the chronometric method.
For crystalline beef liver catalase the reaction follows first order kinetics
over a wide range of enzyme and peroxide concentrations (Fig. 2, Table
II) and the reaction remains first order for longer than its half life if the
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1.55
3.0
4.3
6.7
1.15
II
Theoretical
First Order
Velocity
Concentrations
of Catalase
R.
F.
BEERS,
JR.,
AND
I.
W.
SIZER
139
SUMMARY
A simple, rapid, quantitative spectrophotometric method for following
the action of catalase on hydrogen peroxide, based upon the measurement
of the ultraviolet absorption of peroxide, is suited to studies of catalase
kinetics. By this method the breakdown of hydrogen peroxide was found
to follow first order kinetics under a variety of conditions and to increase
linearly with catalase concentration. The velocity of this reaction, at low
ionic strength, increasesonly slightly with temperature; the corresponding
activation energy is 600 calories.
BIBLIOGRAPHY
1.
2.
3.
4.
Bach, A.,
Hennicks,
von Euler,
Goldblith,
and Zubkowa,
S., Biochem.
Z., 126, 283 (1921).
S., Biochem.
Z., 146, 286 (1924).
H., and Josephson,
K., Ann. Chem., 452, 158 (1927).
S. A., and Proctor,
B. E., J. Biol.
Chem.,
187, 705 (1950).
Downloaded from www.jbc.org by guest, on May 15, 2012
molarity of the buffer is 0.05 M. The kinetics obtained by the spectrophotometric method are the same as those obtained by other workers (5,8).
Table III presents values of the first order velocity constant, IGO,obtained by the spectrophotometric
method, for a series of concentrations
of catalase.
The molarity of the catalase was calculated from the extinction coefficient at 4050 A by using the value of 340 cm.-’ X rn& for horse
liver catalase reported by Bonnischen et al. (8). The rather wide fluctuations in the catalase activity shown in Table III are attributable to variations in stability of the enzyme in different preparations. The average
standard velocity constant, Ic,, calculated for 1 M catalase is 2:O f 0.17
X 10’ liters mole-’ sec.-’ and may be compared with 3.0 X 10’ liters
mole-’ sec.-l reported by Bonnischen et al. (8) for horse liver catalase.
Several experiments, performed at different temperatures over the range
ll-35”, were used to compute the energy of activation for the catalasehydrogen peroxide system by means of the Arrhenius equation. The
activation energy is quite variable and is sensitive to change in ionic
strength and to the presence of small amounts of inhibitors (7, 11). In
0.005 M phosphate buffer, pH 7.0, the experimental activation energy in
one seriesof experim …
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