First Lab Assignment

Enzyme Kinetics

Assignment Requirement (in this order)

Title Page (see format in the Marking file in the Lab Manual folder in Moodle
Results (see format in either the Marking file or the Statistics file of the Moodle Lab Manual
folder)
Letter of Understanding (see the Moodle Lab Manual folder)
Proof of R (see format in the Statistics file in the Moodle Lab Manual folder)

Due Date: See page 1 of the Intro file in the Moodle Lab Manual folder.

IMPORTANT NOTE: Unlike the group sessions, all lab assignments are to be completed
and handed in individually.

Enzyme Lab Objectives

1. The three main Lab Objectives for the course are outlined in the Introduction file of the
Moodle Lab Manual. The ultimate goal is to produce a full research paper at the end of Biology
103 in the winter term, similar to the type found in research journals. We will be undertaking this
task in incremental steps and in this first lab assignment we will focus on the production of a
Results section. To do this, we will measure the rate activity of the enzyme catalase.
2. Learn some aspects of enzyme kinetics.
3. Develop some pipetting skills.

The Enzyme

Catalase is present in the peroxisome of most aerobic cells and catalyzes the decomposition of
hydrogen peroxide (H2O2) to oxygen and water. This helps protect the cell from hydrogen
peroxide induced oxidative stress, which has been linked to many conditions such as asthma,
arthritis, heart disease, diabetes, cancer, and aging. Catalase is also involved in the oxidation of
various metabolites, and toxins such as phenols and alcohol.

2 H2O2 2 H2O + O2

The Assay

We will assess the catalyzed breakdown of H2O2 by catalase indirectly by using a pressure sensor
to measure the released oxygen gas in a sealed Erlenmeyer flask.

Hazards

There is a risk of the reaction flask breaking during the experiment so lab glasses are required if
you are in the vicinity of the reaction flask. The hydrogen peroxide is diluted from a 3% solution
that is normally used in regular household applications and poses little risk. Because chemicals
are in use, food or drink is NOT allowed in the lab rooms. Place coats and books along the floor
at the back wall, or on the floor under the chalkboard.

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Methods

Equipment at each bench:

Pressure sensor with tubing and cork attached to computer with GoLink (Figure 1)
125 mL Erlenmeyer flask

Equipment at the side bench:

A brown bottle containing 3% H2O2 (Your T.A. will transfer about 100 ml to a stock bottle
at the start of the lab)
Labeled stock bottle containing H2O
Labeled stock bottle containing H2O2
Labeled stock bottle containing the enzyme catalase
10 mL micropipette in Rm. 2320 and 2326 for transferring H2O2
5 mL micropipette in Rm. 2320 and 2326 for transferring H2O2
5 ml serological pipet with green pump for transferring water
1 mL micropipettes for transferring catalase
Pipet tips in labeled containers for H2O2 and catalase.

Figure 1. Experimental set up at bench with GoLink, pressure sensor, pressure cork and tubing.

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Micropipette Procedure:

1. Setting the Volume: Get the 125 Erlenmeyer flask from your bench and take it to the side
bench where the pipets are located. The pipet volume can be set by turning the plunger dial on
newer models and a black nut on older models. On the newer models IT IS IMPORTANT to
unlock the dial before turning, otherwise you risk breaking the (very expensive) pipet. For our
first trial (see Table 1), we want to get 5 ml of 3% H2O2, which is 500 on the volume dial of the
white topped pipets (range 1 to 10 mL), and 500 on the volume dial of the older purple topped
pipets (range 500 to 5000 L).

Helpful Youtube Video:

https://www.youtube.com/watch?v=TFXX8yCWjMo

2. Applying the tip: If the pipet does not have a tip already applied, get one from the H2O2 tip
container. Pick up an appropriate tip at its thicker base and gently push it onto the end of the
pipet. If you need to change a tip, you can return used tips to the container for re-use. This is not
sterile technique, but we cant apply sterile conditions in the busy first year lab room.

3. Getting the liquid: The pipet plunger stops at two different positions when it is depressed. Try
depressing to get a feel of these two positions. Now depress the plunger until you feel that first
resistance and insert the tip into the H2O2 flask to just below the surface of the liquid. Now slowly
release plunger all the way to suck up the required volume. DO NOT HOLD THE PIPET
SIDEWAYS, ALWAYS KEEP IT POINTING DOWN

4. Transferring the liquid: Insert the tip of the pipet to the bottom of your 125 Erlenmeyer flask
and slowly depress the plunger, only this time depress it to the point of initial resistance, wait
second, and then continue pressing the plunger as far as it will go in order to discharge the entire
volume of solution. Make sure to NOT immerse the pipet tip at any time in the liquid. If there
is any solution beaded at the pipet tip, touch it to the side of the flask before withdrawing the
pipet. Take your flask to your bench.

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Serological Pipet Procedure

At the request of the second year genetics course, we are also going to use a serological pipet to
transfer water, as directed by Table 1, to get some practice in its use. The green pump should
already be snugly applied when you go to use it.

1. Insert the pipet tip just beneath the surface of the water in the water
stock bottle and turn the dial downwards to suck up water. The smaller
numbers on the side of the pipet represent the volume of fluid you are
taking up. For example, next to the large 1 you will see a smaller 4,
which represents 4 ml. Remember to bring the fluid level up to the
meniscus (see example).

2. When you remove the pipet to

transfer water to your Erlenmeyer flask make sure no water
dribbles out the pipet, which would change your volume. If it
does dribble, it likely means the pump has come loose, and needs
to be more firmly applied. To do so, grasp the pipet near the
pump to push it on, AND NOT near the tip end as this could break
the pipet.

3. Turn the dial upwards to dispense the fluid to your 125 Erlenmeyer flask. If there are any
drops left on the tip, touch them to the side of the flask.

Table 1: Volumes and activity for H2O2/catalase experiment.

Trial 3% H2O [H2O2 ] Catalase Activity Run time Initial Rate
H2O2 mol/l (seconds) (kPa/s)
1 5 mL 0 0.88 450 L Swirl flask 100
2 4 mL 1 mL 0.70 450 L Swirl flask 100
3 3 mL 2 mL 0.52 450 L Swirl flask 100
4 3 mL 2 mL 0.52 450 L Do not swirl 100
5 2 mL 3 mL 0.36 450 L Swirl flask 100
6 1 mL 4 mL 0.22 450 L Swirl flask 300

The Assay

1. Make sure the spigot handle on the pressure sensor cork is in the off position (90 degrees to
the tubing), and the pressure sensor is plugged in to your computer via the GoLink USB cable.

2. Turn on the bench computer and click on the Logger Pro icon on the desktop. This will bring
up a logger pro screen and in some computers it will take a few minutes for the software to detect
the pressure sensor. When you see the word Pressure in red letters at the bottom left of the
screen you can proceed (Figure 2).

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 Figure 2. Screen capture of the Logger Pro
pressure program, showing Pressure reading,
the x,y coordinates, and the green Collect
button.

3. By now you have added 5 mL of the H2O2. Now go back to the side bench and obtain 450 L
(450 on the dial) of the catalase with one of the blue topped pipets (100 to 1000 l range) and
take it back to your bench (REMEMBER TO HOLD THE PIPET DOWNWARDS) and add to
the flask (REMEMBER NOT TO IMMERSE THE PIPET TIP IN THE LIQUID WHEN
DISPENSING). Immediately put the cork on, and immediately click on the green Collect button.
Return the pipet for others to use. Continue to gently swirl the flask on the desk, while also
keeping a gentle downward pressure on the cork with your finger. You will see the pressure
begin to increase on the screen. If the cork pops off that is okay, you can continue onto step 4. It
usually pops off around 125 - 130 kPa/s, which is equivalent to about 18 psi. For comparison, the
average recommended psi for a car tire is 30 psi.

4. After 100 seconds (or sooner if your cork popped off) press the red STOP button at the top of
the screen and remove the cork.

5. Click and drag the mouse to highlight the first 30 seconds of the slope (see t at the bottom
left of screen). In the pull down menu at the top of the screen, click on Analyze and then Linear
fit and copy down the subsequent slope (m) value. This represents the initial rate of reaction. For
example, the value in Figure 3 is 0.15 kPa/s. Write your reading in your table. If your cork pops
off earlier than 30 seconds, measure the steepest part of the slope over a lesser time interval. Why
do you think we are using a common value of 30 seconds?

6. Take your Erlenmeyer flask to the sink to give it a good rinse. Empty out the water and repeat
the experiment using the other volumes presented in Table 1. When you click on Collect the
second time it will bring up a choice box. Choose Erase and continue.

Note how the reaction rate differs with lesser concentrations of substrate. Any ideas as to why?

Note how you are not to swirl the flask for Trial 4. Any ideas as to why?

Note the longer run time for Trial 6. What happens to the curve over time? Why do you think
this is happening? You dont need to answer these questions for this assignment; they are just
something for you to think about during Dr. Sneddens lecture on enzyme kinetics.

7. Write the results for Trial 3 and 4 on the chalkboard comparing flask swirling and non-
swirling. In order to complete your first lab assignment, you need at least 8 readings. If there is
time repeat Trials 3 and 4 to get eight readings for the class, or go to the other lab rooms and copy
down their values.

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Figure 3. Slope of the line highlighted to measure the initial rate of reaction.

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Required Results in Assignment

1. A sentence describing your observation on substrate concentration and initial rate of oxygen
production as measured in kPa/s.
Example: When catalase was combined with higher . the initial rate of oxygen production as
measured in kPa/s was.. etc.
Note: The results section is merely for reporting results; do not make conclusions or
assessments.
2. A sentence describing your box plot figure as described in either the Marking file or the
Statistics file of the Moodle Lab Manual folder.
3. A statistical sentence comparing the eight readings for the swirling and non-swirling flask.
The statistical method for the required independent t-test is provided in the Statistics file of the
Moodle Lab Manual. If the results are not statistically significant, complete the sentence as
described in the lab manual, and simply include the word not in front of the word significant.
4. A box plot comparing the eight readings for the swirling and non-swirling flask. The method
for drawing the box plot is provided in the Statistics file of the Moodle Lab Manual.
Note: Most of the subsequent Biol 102 and Biol 103 assignments will require steps 2 to 4 to be
completed for the Results section.

Interesting Bits: Hydrogen peroxide is a product of redox reactions in the mitochondria, and is
used in various cellular processes. Hydrogen peroxide levels need to be regulated as it can cause
cellular damage, and stressed cells in particular produce greater amounts. For example, Nagaraja
et al., (2012) reported the H2O2 released in exhaled breath for smokers and people with
pulmonary disease to be as high as 2.2 mol/L and 3.0 mol/L respectively, whereas levels in
healthy individuals living in rural areas not exposed to high levels of air pollution were as low as
0.02 mol/L.

Exercise can increase H2O2 levels as much as three times higher than resting rates in exhaled
breath (Marek et al. 2012), which makes sense given the demands put on the mitochondria during
exercise. Given the damaging effects of H2O2, does it mean that exercise is bad for us?

Cytochrome C oxidase can also catalyze the conversion of H2O2. We wont be talking about that
conversion next week, but this particular enzyme is relevant to next weeks group session. See
you then.

Literature Cited

Marek, E., J. Volke, W. Marek, and P. Platen. 2012. Exercise increases the hydrogen peroxide
release in exhaled breath during exercise. European Respiratory Journal. 40: suppl. 56.

Nagaraja, C., B. Shashibhushan, M. Sagar, and P. Manjunath. 2012. Hydrogen peroxide in

exhaled breath condensate: A clinical study. Lung India. 29:123-127.

Student clean-up Checklist

Rinse out the 125 Erlenmeyer flask and return it to the bench.
Return all pipets to the side bench
If you are a 4:00 to 5:30 lab, turn off the computer