BIOL 1406
Properties of Enzymes: Peroxidase, A Case Study
Objectives
Name the class of macromolecules to which peroxidase belongs and the monomers that make it up.
Name the substrates and products of the peroxidase catalyzed reaction.
Explain the role of guaiacol in this experiment.
Define enzyme, activation energy, active site, pH, and denaturation.
Distinguish between oxidation/reduction, activation energy/catalysis, substrate/product, and
hydrogen peroxide/peroxidase.
Describe how temperature, pH, enzyme concentration, and substrate concentration affect the
reaction rate.
Explain why peroxidase is a necessary enzyme for all aerobic or oxygen-using cells.
Background Information
The thousands of chemical reactions that occur in a cell at any given time do not occur randomly, but
are highly under the control of biological catalysts called enzymes. A catalyst is a substance that
speeds up a chemical reaction without being consumed by the reaction. Enzymes speed up reactions by
lowering the activation energy of a reaction, which is that initial amount of energy necessary to bring
reactants together with the proper amount of energy and in the proper orientation so that the products
can be formed.
Most enzymes are proteins with particular primary structures dictated by genes. As proteins, upon their
synthesis, enzymes assume particular shapes. This shape, especially in its “active site”, determines its
catalytic effects. The active site of each enzyme binds to specific molecules – for example, the enzyme
sucrase binds to sucrose but not to lactose, even though both are disaccharides. The reactant molecule
that binds with to the active site of an enzyme and undergoes chemical modification is called the
substrate of that enzyme. Some enzymes bind to two substrates to form products. Certain enzymes
have metallic ions (such as Cu2+, Fe2+, Mn2+ ) as part of their active site These metal ions are called
cofactors. Sometimes a cofactor is organic in nature and not an ion (e.g. some vitamins); in that case
the cofactor is properly called a coenzyme.
The binding between enzyme and substrate(s) consists of weak, non-covalent chemical bonds, forming
and enzyme-substrate complex that exists for only a few milliseconds. During this instant, the covalent
bonds of the substrate(s) either come under stress or are oriented in such a way that facilitates the
formation of a different molecule or molecules. This results in the formation of a product. The product
leaves the enzyme’s active site and is used by the cell. The enzyme is unchanged by the reaction and
may enter the catalytic cycle again if more substrate molecules are available.
Enzyme + Substrate
Enzyme-Substrate
Complex
Enzyme + Products
Fig. 7-1 A pictorial representation of an enzyme-catalyzed reaction.
1
An individual enzyme may convert about a thousand substrate molecules in one second.
Their fast rates combined with the fact that they are not used up during the reaction means that
enzymes are needed in small amounts. Eventually enzymes wear out, break apart and lose their
catalytic capacity. Cellular enzymes called “proteases” degrade inactive enzymes to amino acids,
which are used by the cell to make other enzymes or other proteins.
The amount of a particular enzyme found in a cell is determined by the balance between the processes
that degrade the enzyme and those that synthesize it. When no enzyme is present, the chemical reaction
catalyzed by the enzyme does not occur at an appreciable rate. Generally, if the concentration of the
enzyme increases, the rate of the reaction will also increase. This is true up to a certain point. At some
point the amount of substrate also plays a role in the rate of the reaction: adding more enzyme will
not speed of a reaction if there is not sufficient substrate.
The pH (which is a measure of the concentration of H+ in an aqueous solution) and high salt
concentrations affect the shape of enzyme molecules by interfering with ionic bonds that are necessary
to maintain their tertiary structure. This, in turn, affects the substrate-binding efficiency of the
enzyme. Temperature, within the physiological limits of 0 to 40oC, affects the frequency with which
the enzyme and its substrates collide and, hence, also affects binding. Extremely high temperatures
typically cause enzyme denaturation, rendering the enzyme completely inactive. All factors that
influence binding obviously affect the rate of enzyme-catalyzed reactions. Some of these factors will
be investigated during this laboratory.
During this experiment you will study a type of enzyme called peroxidase, which is a large protein
containing an iron ion in its active site, acting as a cofactor. Peroxidase is a good experimental enzyme
because it is easily prepared and assayed. Turnips, horseradish roots, and potatoes contain a large
amount of this enzyme. For this experiment, we extracted the peroxidase from turnips.
The normal function of peroxidases is to rid the cell of toxic hydrogen peroxide (H2O2), which is
produced as by-product in certain metabolic reactions. It is critical to remove this H2O2 before the cells
become damaged by it.
We will carry out the peroxidase reaction “in vitro”, and will monitor its activity by observing the
production of an oxidized product. The dye guaiacol (extracted from the guaiacum tree of Central,
South America and the Caribbean) binds to peroxidase and becomes oxidized as the hydrogen peroxide
is reduced to water. The complete reaction is as follows:
peroxidase
4 H202 + 4 guaiacol (reduced)
COLORLESS
8 H2O
+ tetraguaiacol (oxidized)
BROWN
To quantitatively measure the amount of brown color in the final product, the enzyme and substrates
can be mixed in a tube and immediately placed in a spectophotometer. As color accumulates, the
absorbance at 500 nm will increase. [The procedure for using a spectrophotometer was explained in
the previous lab and you should take a few minutes to review those instructions before proceeding.]
For each particular run, plots of absorbance versus time can be constructed. The data points of each run
are connected by drawing a best-fit line that goes through (0, 0). The slope of each plot is directly
related to the enzyme reaction rate for that run.
2
Factors Affecting Enzyme Activity
Your laboratory instructor may decide to divide your class up into teams; each team will tackle one or
more of the following experimental variables (A – E). The results will then be shared at the end of the
lab period and should be included in your lab report.
To perform each of the tests on the experimental variables, you will be following the same basic
procedure. Please read over the instructions for A, Effect of Enzyme Concentration, whether or not
this is the variable that your group is experimenting with and make sure you understand the
instructions before you continue on with the experiment. This is the same basic procedure that you
used in the Enzyme Lab Practice (Lab. 6)
For each of the following experimental variables (A – E), use the Mixing Table for each variable to set
up the tubes. After you have all of the tubes set up, with the appropriate volumes of solutions, you are
ready to begin the experiment. In each test, tube 1 is always the control and is used to blank the
spectrophotometer. You will always be mixing tubes 2 & 3 together, tubes 4 &5 together, tubes 6 & 7
together, and tubes 8 & 9 together (when applicable). Remember to mix the solutions in the test tubes
and then pour the contents of the tube into the cuvette.
The peroxidase extract has been prepared for you. The extract contains hundreds of different types of enzymes, including
peroxidase.
The Effect of Enzyme Concentration:
Changing the amount of turnip extract changes the amount of peroxidase!
1. Obtain a beaker or cup of each of the following solutions: Turnip Extract, pH 5 buffer, 10mM
H2O2, and 25 mM guaiacol. You ONLY need to pour about 20-mL of each solution. Make sure
the beakers (or cups) are properly labeled.
2. Obtain two 1-mL pipets and two 5- or 10-mL pipets. Use of the wrong pipet will cross
contaminate your reagents and introduce errors into your subsequent experiments in this
exercise. DO NOT CROSS CONTAMINATE!
3. Using the china marker on your trays, label seven test tubes from 1 to 7. Prepare these tubes
according to Table 7-1. Amounts given are in mL. These tubes will be used for the following
purposes:
Tube 1: Control without hydrogen peroxide; to be used in calibrating the spectrophotometer
Tube 2: Substrates.
Tube 3: Low concentration of peroxidase.
Tube 4: Substrates (same as tube 2)
Tube 5: Medium concentration of peroxidase.
Tube 6: Substrates (same as tube 2)
Tube 7: High concentration of peroxidase.
3
Table 7-1 Mixing table for effect of enzyme concentration test. (All volumes in milliters)
Tube
1(Blank)
2
3
4
5
6
7
Buffer (pH5)
6.0
0
4.5
0
4.0
0
3.0
H2O2
0
2.0
0
2.0
0
2.0
0
Extract
1.0
0
0.5
0
1.0
0
2.0
Guaiacol
1.0
1.0
0
1.0
0
1.0
0
Total Volume
8
3
5
3
5
3
5
4. Refer to the handout from the previous lab for the instructions on how to use the
spectrophotometer. Adjust the wavelength to 500 nm for this experiment.
5. Pour the contents of Tube 1 into a cuvette. This tube is used to “blank” the spectrophotometer, so
that any color caused by contaminants in the reagents will not influence subsequent measurements.
6. In your lab groups, one person should be the Timer, another the Spectophotometer Reader, and
another the Data Recorder. Note the time to the nearest second and mix the contents of tubes 2 and
3 by pouring the contents of tube 3 into tube 2 two or three times. Mixing should be completed
within ten seconds. The contents of this tube will now be referred to as the reaction mixture.
7. Immediately after mixing, add the reaction mixture to a cuvette, wipe the outside of the cuvette
with a Kimwipe, and place the cuvette in the spectrophotometer. Read the absorbance at 20 sec
intervals from the start of mixing. Record your measurements in
Table 7-2. After two minutes (six readings) remove the tube from the spectrophotometer and
visually note the color change. Discard the solution.
8. Mix the contents of tubes 4 and 5, pour into a cuvette, and repeat your measurements for two
minutes at 20-second intervals. Record the results in Table 7-2.
9. Mix the contents of tubes 6 and 7, pour into a cuvette, and record the absorbance measurements in
Table 7-2.
Table 7-2 Results from effects of enzyme concentration
(entries should be given as absorbance units at 500nm).
Time (sec)
Tubes 2 & 3
0.5 ml extract
0
0
Tubes 4 & 5
1.0 ml extract
0
20
40
4
Tubes 6 & 7
2.0 ml extract
0
60
80
100
120
Activity 7-2 Temperature Effects on Peroxidase Activity:
To determine the optimum temperature for peroxidase activity, perform the following experiment.
1. Set up four water baths:
1 In an ice bath at approximately 0oC
2. At room temperature at about 23oC
3. At 40oC
4. At 60oC
5. At 100oC (boiled extract)
2. Number nine test tubes in sequence 1 through 11. Refer to Table 7-3 for the volumes of
reagents to be added to each tube.
Table 7-3 Mixing table for temperature experiment (all values in milliliters)
Temperature
Tube
Buffer
H2O2
Extract Guaiacol Total Volume
(pH5)
1(blank)
6.0
0
1.0
1.0
8
2
0
2.0
0
1.0
3
0C
3
4.0
0
1.0
0
5
0C
4
0
2.0
0
1.0
3
23 C
5
4.0
0
1.0
0
5
23 C
6
0
2.0
0
1.0
3
40 C
7
4.0
0
1.0
0
5
40 C
8
0
2.0
0
1.0
3
60 C
9
4.0
0
1.0
0
5
60 C
o
100 C
10
0
2.0
0
1.0
3
o
100 C
11
4.0
0
1.0**
0
5
3. **For the 100oC sample, take 2 mL of the turnip extract and place it in a boiling water bath.
After five minutes, remove the tube, let it cool and add 1 mL of this boiled extract in tube 11.
4. Pre-incubate the sample solutions at 0oC, 40oC and 60oC for at least 15 minutes before
running the enzyme reaction.
5
5. After reaching temperature equilibrium and calibrating the spectrophotometer with the
contents of test tube 1, mix tubes 4 and 5 (room temperature), and measure changes in
absorbance every 40 seconds for two minutes. Record the changes in absorbance in Table 7-4.
6. Repeat Step 5 for tubes 10, 11 (boiled extract).
7. For more accurate results run the experiments at 0oC, 40oC and 60oC in the following way:
After incubating in the proper temperature for at least 15 minutes, mix tube pairs (one pair at
a time, e.g. tubes 2 & 3), pour the mix in a cuvette, and place the cuvette back in its
appropriate temperature. You will be measuring absorbance every 40 seconds, so leave the
cuvette in its bath until 10 seconds before it is time to read the spectrophotometer.
After reading the absorbance, return the cuvette to the temperature bath and leave it there until
10 seconds before the next reading. Using this procedure, the temperature will remain more or
less constant during the experiment.
Table 7-4 Results from temperature effects on peroxidase activity
(entries should be given as absorbance units at 500nm).
Time (sec)
0
Tubes 2 & 3
0 C
Tubes 4 & 5
23 C
0
Tubes 6 & 7
40 C
0
0
Tubes 8 & 9
60 C
0
40
80
120
Activity 7-3 pH Effects on Peroxidase Activity
To determine the optimum pH for peroxidase, perform the following experiment.
1. Number nine test tubes 1 through 9.
2. Obtain two 1-mL pipets and two 5- or 10 mL pipets.
6
Tubes 10 & 11
100 oC
0
3. Set up the test tubes by adding the reagents to these tubes as described in Table 7-5. Amounts
given are in mL. The pH buffers have been provided for you in the laboratory.
4. After calibrating the spectrophotometer using the contents of Tube 1 (in a cuvette), mix pairs
of tubes one pair at a time (2 and 3, 4 and 5, 6 and 7, 8 and 9) and measure absorbance
changes at 20-second intervals for two minutes for each mixed pair. Record the results in
Table 7-5.
Table 7-5 Mixing table for pH experiment (all values in milliliters).
Amount
Buffer
Tube
of
H2O2
Extract
Guaiacol Total Volume
Buffer
pH 5
1 (blank)
6.0
0
1.0
1.0
8
pH 3
2
0
2.0
0
1.0
3
pH 3
3
4.0
0
1.0
0
5
pH 5
4
0
2.0
0
1.0
3
pH 5
5
4.0
0
1.0
0
5
pH 7
6
0
2.0
0
1.0
3
pH 7
7
4.0
0
1.0
0
5
pH 9
8
0
2.0
0
1.0
3
pH 9
9
4.0
0
1.0
0
5
Table 7-6 Results from effects of pH on peroxidase activity
(entries should be given as absorbance units at 500nm).
Time (sec)
Tubes 2 & 3
pH 3
Tubes 4 & 5
pH 5
Tubes 6 & 7
pH 7
Tubes 8 & 9
pH 9
0
0
0
0
0
20
40
60
80
100
120
7
The Effect of an Inhibitor on Peroxidase
Hydroxylamine (HONH2) has a structure similar to hydrogen peroxide (HOOH). This molecule binds
with the iron atom at the active site of peroxidase and prevents hydrogen peroxide from entering the
site. What will be the effect on enzyme activity?
To test this hypothesis follow this procedure:
1. Mix five drops of 1% hydroxylamine and 2 ml of enzyme extract in a test tube, letting the mixture
stand for at least five minutes.
2. Prepare 5 test tubes as indicated in Table 7-7.
Table 7-7 Mixing table for inhibitor experiment (all volumes in milliliters).
Inhibitor
Total
Tube
Buffer (pH5) H2O2 Extract Guaiacol
and Extract
Volume
1 (Blank)
6.0
0
1.0
1.0
0
8
2
0
2.0
0
1.0
0
3
3
4.0
0
1.0
0
0
5
4
0
2.0
0
1.0
0
3
5
4.0
0
0
0
1.0
5
3. After calibrating the spectrophotometer with the contents of Tube 1, mix pairs one at a time
(2 and 3; 4 and 5,) and measure the changes at 20-second intervals for two minutes. Record
your measurements in Table 7-8.
Table 7-8 Results for the enzyme inhibition experiment
(entries should be given as absorbance units at 500nm)
Tubes 2 & 3
Tubes 4 & 5
Time (sec)
Normal extract
Inhibitor-treated extract
0
0
0
20
40
60
80
100
120
8
Data Analysis
NOTE: Appendix 1 has instructions for plotting graphs in the computer using
Microsoft Excel.
QUESTIONS
1.The data of absorbances and time obtained for each experiment should be graphed as part of the lab
report. The x-axis should be the independent variable (time in seconds) and the
y-axis should be the dependent variable (absorbance units). Explain why absorbance is
considered the dependent variable.
2.
The slopes of the linear portions of the curves are a measure of enzyme reaction rate.
What are the units?
3.
Explain why the slopes are different.
4. Does the activity of peroxidase vary with temperature? What is the optimum temperature?
How do you know this?
5. Does the activity of peroxidase vary with pH? What is the optimum pH?
6. How did boiling affect the activity of peroxidase?
7. Was hydroxylamine a strong, moderate or weak inhibitor? Give the basis for your answer.
References:
1. Dolphin, W.D., Exercise 4 (Properties of Enzymes) in: Biology Laboratory Manual, 4th edition,
1997, WCB Publishers
2. Eberhard,C., General Biology Lab Manual, 1996, Harcourt
3. MadScientist network: www.madsci.org
9
Appendix 1 Plotting Enzyme Activity Data Using Microsoft Excel
Purpose: This document provides information for the use of Microsoft Excel to plot best-fit lines with the enzyme activity
data obtained in the lab, and to calculate the slope of each best-fit. It also provides information for plotting bar graphs that
represent “differential plots” of enzyme reaction rates under various conditions.
This document will show you how to manipulate the data of the “Effect of Enzyme Concentration” experiment ONLY.
After you learn this, you can follow the same basic procedure to plot your data on the effect of pH, inhibitor and
Temperature on peroxidase (see addendum at the end of this handout).
Laboratory 8 Plotting Best-Fit Lines of Absorbance vs. Time data
1.
Double click on the Microsoft Excel icon to open the program. You will see a grid. Each box in the grid is called a
cell. Each cell has an “address” made up of a letter indicating the column the cell is in and a number indicating the
row the cell is in, e.g. the upper left cell is A1.
Note:
a. To save paper, I will show only the whole screen view on the first picture; future diagrams will be simplified,
showing only the data area.
b. Unless specified otherwise, when you are asked to “click”, use the left mouse button (LMB).
2.
3.
Double click on cell A1 to start entering information. Using the diagram on the next page as a reference, enter the data
of time (in seconds) and absorbance readings of YOUR enzyme concentration experiment (numerical data will be on
cells A3 through D9).
Click on cell A3 with the left mouse button (LMB) and drag the mouse to highlight the area
containing all the data points (go down to cell D9). The screen should look like this:
10
Time
1/2 x
0
20
40
60
80
100
120
4.
Absorbance
1x
2x
0
0
0.01
0.02
0.02
0.05
0.05
0.11
0.06
0.12
0.08
0.16
0.11
0.23
0
0.04
0.1
0.22
0.24
0.32
0.42
Once the data is highlighted, look on the upper toolbar and click on “Insert”, then on “Chart”. A box with graph
options will appear. Select “XY(Scatter)” and click on “Next”.
Your data will appear already plotted on an x-y axis system. Now you will learn how to set-up
the axes write the title of the graph and write the legend. You will also command the
computer to draw best-fit lines that go through (0,0), and to calculate the slope of each plot.
You can also choose to make some cosmetic touches (changing the size of fonts, colors of the
plot etc.…) on your graph before printing.
5.
Find “Series” towards the top of the graph and click on it. “Series1” should appear highlighted. To the right of this
there is an empty space for you to write the name of this plot: click on this empty box and type 1/2x [enzyme] . Now
highlight “Series2” by clicking on it with the LMB. Just as you did for series1, name this series: 1x [enzyme]. Repeat
the procedure for Series3 (name it 2x [enzyme]).
6.
Click on “Next”. Another box will appear with options to modify titles on the graph.
a.
b.
Click on the blank space below “Chart Title” and name your graph
(e.g. The effect of Enzyme Concentration on Peroxidase Activity”).
Click on space below Value(X)Axis and type: Time (seconds)
c. Click on space below Value(Y)Axis and type: Absorbance
d. Click on “Axes” (upper left of graph) and make sure that Value(X)Axis and
Value(Y)Axis have a checkmark √ .
e. Click on “Gridlines” and alter the appearance of these as you choose. (I chose to show
only “Major Gridlines” on the y-axis.)
f. Click on “Legend” and choose the location of the legend (I chose bottom).
7.
Click on “Next” and then on “Finish”.
11
Your graph will appear on the screen as shown below. You can alter its size by clicking the LMB on the black squares
around the graph and dragging the mouse with the LMB pressed down.
Time
1/2 x
0
0.01
0.02
0.05
0.06
0.08
0.11
0
0.04
0.1
0.22
0.24
The Effect of Enzyme Concentration on
0.32
0.42
Peroxidase Activity
Absorban
ce
0
20
40
60
80
100
120
Absorbance
1x
2x
0
0.02
0.05
0.11
0.12
0.16
0.23
0.5
0
0
50
100
150
Time (sec)
1/2x [enzyme]
1x [enzyme]
2x [enzyme]
You can move the whole graph by clicking the LMB (and holding it down) on the white of area of the graph until the cross
symbol appears. With the LMB down, drag the graph below the area containing your data points. The graph should look
something like this:
Time
1/2 x
0
20
40
60
80
100
120
0
0.01
0.02
0.05
0.06
0.08
0.11
Absorbance
1x
2x
0
0.02
0.05
0.11
0.12
0.16
0.23
0
0.04
0.1
0.22
0.24
0.32
0.42
Absorbance
The Effect of Enzyme Concentration on
Peroxidase Activity
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
0
20
40
60
80
100
120
Time (sec)
1/2x [enzyme]
1x [enzyme]
12
2x [enzyme]
140
8.
Now you are going to add the line that goes through the data points (trendline). Click on the
graph to make sure that the black squares around the graph are there.
Move the pointer to any of the data points of a particular series (any Δ, for example). As you do this,
information about this data point will appear on the screen. Press the
RIGHT mouse button. A box with choices appears. Select and click on “Add
Trendline”. Another box will appear. Select Linear (should already be highlighted).
Before clicking OK, go to “Options” on the top of the box. Click on the box “Set
intercept = 0” ( √ should appear) and on “Display equation on chart”. Click on
“OK”. The graph will show a best-fit line that goes through 0,0 for the data points of
that series. Also on the graph you should see an equation (e.g. y = 0.0033x) which
gives you the value of the slope for that series).
a.
b.
Repeat the steps outlined in “8a” to add the trendline through the two other Series.
The graph should look something like this:
Time
1/2 x
0
20
40
60
80
100
120
0
0.01
0.02
0.05
0.06
0.08
0.11
Absorbance
1x
2x
0
0.02
0.05
0.11
0.12
0.16
0.23
0
0.04
0.1
0.22
0.24
0.32
0.42
The Effect of Enzyme Concentration on
Peroxidase Activity
Absorbance
0.5
0.4
y = 0.0033x
0.3
0.2
y = 0.0017x
0.1
y = 0.0008x
0
0
20
40
60
80
100
120
Time (sec)
1/2x [enzyme]
1x [enzyme]
2x [enzyme]
Linear (2x [enzyme])
Linear (1x [enzyme])
Linear (1/2x [enzyme])
Cosmetic changes for graphs:
13
140
After cosmetic changes, your graph may look like the one shown on the next page.
a.
b.
c.
Changing the size of the font used for the equation: Click on the equation to be changed. Click the RIGHT
mouse button. Select and click on “Format Data Labels”. Click on “Font” and reduce the font size (I chose 8).
Click on “OK”.
Changing the position of the equation: Click on the equation and drag that box to the desired position.
Changing the size of the axis label: Click on the desired label to change (e.g. the x axis label that says “Time in
seconds”). Click the RIGHT mouse button. Select and click on “Format Axis Title”. Click on “Font” and reduce
the font size (I chose 10). Click on “OK”. Do the same for the other axis.
You may also want to alter the size of the numbers on the scale. Click the RIGHT
mouse button on top of the scale numbers. Click on “Format Axis”, then set the font to
the desired size (I chose 8). Do this for the scale on the other axis also.
d.
To edit the Legend box: Click on the Legend box. Click on
____linear 2x [enzyme] until you see black squares around its box. Press the RIGHT mouse
button, select and click on “Clear”. Do the same for the other items like these
(___linear …) on the legend box.
You can also alter the size of the font for the legend items. Click on top of 1/2x [enzyme](for
example) with the RIGHT mouse button until black squares around it appear. Select “Format
Legend”, and change the font size as desired (I chose 10). Do this for all the items in the
legend.
e.
9.
To change the color of the graph background: Press the RIGHT mouse button while the cursor is on the gray
area. Select and click on “Format Plot Area”. You may want to choose a white background if your printer is
black and white.
Go to the top toolbar. Click on “File”, “Print Preview” and check if the graph is ready to
print. If it’s not ready, try more cosmetic changes as desired, until you are satisfied with the
product. When it is ready, SAVE your data: go to “File”, “Save As” and choose the proper
destination. (If working at home, use your hard drive, if working at school, choose
drive A: after inserting a formatted 3 ½ “ diskette.
10. Click on “Print” and “OK”.
14
The Effect of Enz yme Concentration on Peroxidase
Activity
0.45
0.4
y = 0.0033x
Ab sor bance
0.35
0.3
0.25
0.2
y = 0.0017x
0.15
0.1
y = 0.0008x
0.05
0
0
20
40
60
80
100
120
140
Tim e (se c)
1/2x [enzyme]
1x [enzyme]
2x [enzyme]
II. Obtaining the “differential plots” (bar graphs)
Once you have the values of the slopes, you can build the bar graphs of enzyme reaction rate versus the various parameters
tested.
15
Under your previous graph, use 6 cells to enter the relative enzyme concentrations used and the values for the three
slopes obtained with the previous graph:
Relative
[Enzyme]
1/2 x
1x
2x
2.
4.
0.0008
0.0017
0.0033
Highlight the data as you learned before (down LMB, dragging mouse over data points).
Rel.[Enz]
1/2 x
1x
2x
3.
RxnRate
RxnRate
0.0008
0.0017
0.0033
Click on “Insert Chart”. Select “Column” (=bar graph). Click on “Next”.
a.
Click on “Titles”.
1. Name the chart (Example: Peroxidase reaction rates at different enzyme concentrations)
2. Name the x-axis: Relative concentrations of peroxidase
3. Name y-axis: Reaction rate (absorbance units/sec)
b.
c.
Click on Legend to change the position of the legend box if desired.
Click on “Finish”.
Drag the chart below your data points and enlarge it by dragging the black squares with the
LMB. At this point your graph should look like the graph on the next page.
Peroxidase Reaction Rates at
Different Enzyme Concentrations
Reaction Rate (absorbance
units/sec)
1.
0.0035
0.003
0.0025
0.002
RxnRate
0.0015
0.001
0.0005
0
1/2 x
1x
2x
Relative Concentrations of
Peroxidase
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4. You can make any other cosmetic changes as desired.
[I chose a white plot area; axes and legend labels with a size 8 font, and a chart title with a size 12 font, boldface. I
also chose a white background for the plot area and deleted the legend box completely]
Note: For the color of the bars, I clicked with the RIGHT mouse button when the pointer was inside the bar. That
activates the “Format Data Series” box, which allows you to change the color after clicking on it. I chose light gray.]
5. To check your graph, click on “File”, “Print Preview”.
6.
Save your data (click on “File”, “Save As”. Type “concentr2.xls”
after selecting the “a” drive as your destination)
7.
The final graph may look like the one on next page.
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Peroxidase Reaction Rates at Different Enz yme
Concentrations
Reaction Rate ( absorb ance units /s ec)
0.0035
0.003
0.0025
0.002
0.0015
0.001
0.0005
0
1/2 x
1x
Relative Concentrations of Peroxidase
Laboratory 7 ADDENDUM
Notes for plotting Temperature, pH and Inhibitor graphs:
1.
a.
Temperature:
Absorbance vs Time graphs
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2x
Enter your data with the following format:
Time
(sec)
Temp.
(oC)
0
0
0
40 data
80 data
120 data
23
0
data
data
data
40
0
data
data
data
60
0
data
data
data
100
0
add this row of 0’s
data
data
data
When ready to “Insert” “Chart”, make sure to highlight ONLY the cells that contain data and the added
row of 0’s:
0
40 data
80 data
120 data
0
0
data
data
data
0
data
data
data
0
data
data
data
0
data
data
data
After selecting “Insert”, “Chart”, “XY (Scatter)”, and clicking on“NEXT”, make sure to select: Series in
COLUMNS, NOT IN ROWS!
Label each series according to which particular temperature they represent (0 oC, 23oC, …)
b.
Bar graphs of temperature experiment
Make sure that the data is entered with this specific format:
T=0
T=23
T=40
T=60
T=100
slope
slope
slope
slope
slope
The slopes were obtained from the
best-fit plots
Later on, make sure to label the X-axis: Temperature (oC).
2.
pH experiment
Absorbance vs. Time graphs: Use same procedure as for
“Enzyme concentration”. Don’t forget to add the row of zero’s!
Time
pH 3
00
20 data
40 data
60 data
80 data
100 data
120 data
pH 5
0
data
data
data
data
data
data
pH 7
0
data
data
data
data
data
data
pH 9
0
data
data
data
data
data
data
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Bar graphs: Make sure to follow this specific format:
pH
pH
pH
pH
3.
3
5
7
9
slope
slope
slope
slope
The slopes were obtained from the
best-fit plots
Inhibitor experiment
Absorbance vs. Time graphs: Enter data as follows. Proceed as for pH or enzyme concentration experiment:
Time
without
0
20
40
60
80
100
120
with inh
0
data
data
data
data
data
data
0
data
data
data
data
data
data
Bar graphs:
Enter data with this format:
with inhib slope
no inhib. slope
The slopes were obtained from the
best-fit plots
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Appendix 2
Writing the Enzyme Lab Report
[ NOT CREATIVE WRITING! ]
{ALL SECTIONS, EXCEPT THE TITLE, MUST BE LABELED ON YOUR REPORT}
TITLE
Should be short, formal, complete and specific. (E.g. “Enzymes, the Goodies of Life” IS NOT an appropriate title). The title
should mention “peroxidase” since that is the enzyme under study.
Your name goes below the title.
I. ABSTRACT:
This is a short paragraph that summarizes the whole experiment. It is the part of a scientific paper that most likely will determine
whether the reader will read the whole paper. Give the reader as MUCH information as possible (including purpose, procedure
and RESULTS!!) in a very concise way. Usually the Abstract is written in boldface and with a smaller font than the rest of the
paper.
II. INTRODUCTION
In this section you want to give background information on the experiment. Since we want to verify your knowledge on this
material, assume that your readers will be people that are not acquainted with enzymes and want to know accurate information
about how they operate.
-Use this section to define: enzyme, substrate, active site, enzyme-substrate complex, competitive inhibitor
and make connections to the particular reaction that you are performing (what enzyme? from what source?
what is the substrate? inhibitor?…). You should show the specific reaction under study and its coupling to
the oxidation of guaiacol.
-You should explain briefly what a spectrophotometer is, and why you can use this instrument to monitor the
progress of this particular reaction.
-In this section you can also explain how the data is analyzed (explain how are the enzyme reaction rates determined).
-After all the background information, you should state what your hypotheses were concerning the effect of
enzyme concentration, pH, temperature, and inhibitor on the peroxidase reaction, taking in consideration that
hypotheses are educated guesses.
III. MATERIALS AND METHODS
You may want to start this section by stating that “we followed the procedure given in our Laboratory Manual”. Then, try to
summarize the procedure as much as possible (no need to say that 4.7 mL of this was mixed with 0.1 mL of that, or “we mixed
tubes 4 and 5”, etc.).
IV. RESULTS:
The results should be presented in a neat and organized manner. For this experiment you will show:
1. Four graphs of ABSORBANCE vs. TIME , one for each of the experiments done: pH, temperature, enzyme concentration and
presence of inhibitor. All graphs have a title and a legend for each plot. All plots should be BEST-FIT lines that go through 0,0
(because at time zero there is no product present).
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On each graph you will show the slope of each plot, which represents the enzyme reaction rate for that particular run. When
using MSExcel to plot the graphs, the computer will be commanded to show the slope of the best-fits, and the slopes will be in
units of absorbance units/second.
2. Four bar graphs, EACH showing data on REACTION RATE vs. the particular parameter studied (whether pH’s,
presence/absence of inhibitor, enzyme concentration or temperatures). Recall that the reaction rates were the slopes previously
determined.
The bar graphs facilitate the visualization of optimum conditions for the enzyme and, therefore, make it easier to write the
conclusions.
V. DISCUSSION (Conclusions):
Indicate your findings in terms of optimum pH and Temperature for the enzyme, and the effect of enzyme concentration and
the presence of an inhibitor on enzyme activity. (Was it a strong, moderate or weak inhibition?) Overall, you may want to postulate
reasons for your findings (make connections with the results and your ideas of how enzymes operate). Were your hypotheses
supported? How could you improve or expand the experiment?
VI. REFERENCES: List ALL the sources used for the experiment and the writing of the report. The
reference should include author, year paper was published, publisher, etc.
Lab manuals:
1) Perez-Ramos S., Couvillon, S., Laboratory 7: Properties of Enzymes, in: Laboratory Manual
for Biology 1406: Molecular and Cellular Aspects of Life 2/e. McGraw-Hill, 2005
Textbooks:
2) Solomon, E.P., Berg, L.R., Martin, D.W., Biology, 7th edition, Thomson Learning, 2005
Articles in journal and magazines:
3) Power, P. and P. J. Fonteyn. 1995. Effects of Acidity and Temperature on Salivary Amylase, Journal of
Bionatural Stuff 45: 1-4.
Websites:
4) www.peroxidaseiscool.com
5) www.enzymesunderstress.com
Note: these are not real
websites
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