Transcript Enzyme Lab - Strive Studios

Enzyme Lab
Dr. Ippolito
BIO121 section MC, SD
Materials
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Test Tubes
Sulfuric Acid
3% Hydrogen peroxide
Liver homogenate
pH 3 buffer
pH 7 buffer
pH 11 buffer*
Goals
• Understand and quantify enzyme activity
• Practice the Principles of the scientific
method
• Develop laboratory skills
• Develop inductive / deductive reasoning
skills
Revised Data Table
please copy this exactly
Tube #
Conditions
1
Positive
Control
2
+ Strong Acid
3
pH 3
4
pH 7
5
pH 11
6
Temp 45 C
7
Temp 4 C
8
Temp 100 C
9
0.1x Substrate
Rel. Activity
(cm of bubbles)
Rx Time
(s)
NOTES
Background
September 2004 Molecule of the Month
Background
• Catalase: more accurately, hydrogen
peroxidase, this enzyme catalyzes the
breakdown of hydrogen peroxide to water
and molecular oxygen.
• Because it was the first enzyme truly
characterized, it has a historical name that
simply means “catalyst” enzyme.
• Reaction: 2 H2O2  2H2O + O2 (gas)
About Catalase
• Catalases are some of the most efficient enzymes found
in cells. Each catalase molecule can decompose millions
of hydrogen peroxide molecules every second.
• The cow catalase shown here (PDB entry 8cat) and our
own catalases use an iron ion to assist in this speedy
reaction.
• The enzyme is composed of four identical subunits, each
with its own active site buried deep inside.
• The iron ion, shown in green, is gripped at the center of
a disk-shaped heme group.
• Catalases, since they must fight against reactive
molecules, are also unusually stable enzymes. Notice
how the four chains interweave, locking the entire
complex into the proper shape.
Why the liver?
• The liver detoxifies dangerous molecules.
• What is a ‘dangerous’ molecule?
– Some are dangerous because they have a lot
of free energy.
– The liver carefully extracts this energy, like
diffusing a bomb.
– How? It oxidizes! It removes the high-energy
electrons.
Why the liver?
• One of the side-products includes a lot of
Hydrogen Peroxide, which while dangerous, is
much less dangerous than the toxic molecules
the liver processed.
• The final step in diffusing this energy is to
convert hydrogen peroxide into harmless water
and oxygen.
• So the liver has tons and tons of catalase,
because it has tons and tons of H202 to deal
with.
Get into groups and grab your
gear!
Control Reaction
•
Preparation
– 9 tubes labeled 1-9
– Mark them at 1 and 2 cm
•
Protocol
– Step 1: Substrate Preparation
• Add 1 ml water to Tube 1
• Add 1 ml sulfuric acid to Tube 2
• Add 1 ml of 3% H202 to each tube
– Step 2: Enzyme addition (and reaction start)
• Add 3 drops of catalase (from crude liver preparations) to Tube 1, then tap the bottom of
the tube GENTLY. Immediately after this, record initial time.
• Measure bubbles with ruler and measure time; we will be calculating reaction rate with
these numbers, so be as accurate as you can.
• Repeat this for Tube 2.
• Record Data.
Discussion
• What are some good reasons to do this
control?
• Why are we only doing a positive control?
• Why did I change the table in the book?
Experiment 1 Background
• Enzymes are proteins; what effect with
changing pH have on catalase activity?
– We have 3 buffers to work with, but pH 11 was
made incorrectly.
• State our Hypothesis
• State our Predictions
Experiment 1
• Setup
– Use tubes 3, 4, 5…
• Buffer Preperation
– Add 1 ml of pH 3 buffer to tube 3
– Add 1 ml of pH 7 buffer to tube 4
– Add 1 ml of pH 11 buffer to tube 5
• Substrate Addition
– Add 1ml of 3.0% H202 to each tube.
• Enzyme Addition (and reaction start)
– Add 3 drops of enzyme to tube 3 and mix. Quantitate reaction similar to
control experiment.
• REPEAT FOR TUBE 4, THEN 5. RECORD ALL RESULTS.
Experiment 1 Discussion
• Did you get your expected results? Why
or why not?
• Why isn’t everyone getting the exact same
data (or are they)?
• What would be a good next experiment to
conduct?
Experiment 2 Background
• Enzymes are proteins; what effect with
changing temperature have on catalase
activity?
– We will look at cold, warm, and hot conditions
(and compare these to our room temperature
control)
• State our Hypothesis
• State our Predictions
Experiment 2: Temperature
• Preparation
– Make sure there is an icebath, a 45 C waterbath, and a boiling
waterbath.
– Use tubes 6, 7, 8
• Add 1ml of water and 1 ml of H202 to each tube.
• Buffer Prep: 20 minutes of boiling will evaporate; let’s do
5 minutes in each with a gentle swirl every 2.5 minutes.
– Tube 6: 45 C
– Tube 7: ice
– Tube 8: boil
• Enyzme addition (and reaction start). You know the drill.
Experiment 2 Discussion
• Did you get your expected results? Why
or why not?
• Why would a weak buffer not work?
• What would be a good next experiment to
conduct?
Experiment 3 Background
• Another variable that alters enzyme reaction
rates is how much substrate the enzyme has to
work with.
• What effect will reducing substrate levels have
on the reaction rate?
• State your hypothesis
• State your prediction
Experiment 3
• Preparation and reaction start (this one’s
easy)
– We’ll be using tube 9 only.
– Add 1 ml of water to tube 9.
– Add 1 ml of 0.3% H202 (a dilution factor of
1:10)
– Add 3 drops of enzyme
– Quantitate the reaction as you’ve done
previously
Experiment 3 Discussion
• Did you get your expected results? Why or why
not?
• What would be a good next experiment to
conduct?
• What effect would adding 10x more substrate
have?
• What kinds of variables would you expect to
impact an enyzmatic reaction rate besides pH,
temperature, and substrate concentration?
– (we usually write concentration like this: [substrate])
Expectations
• In two weeks: Hand in a packet that contains:
– A completed data table from each group
– The answers to questions 1-13 on page 29 of your lab manual
– A written summary of the control and each of the 3 experiments. Do not
exceed 2 pages double-spaced. One paragraph / experiment is
sufficient.
– A brief discussion on your results
• Hand this in and you will receive a maximum of 20/20 points. Points
will be given based on effort and final presentation of the lab report.
– This means that well-thought out wrong answers will not hurt your
grade.
• For an additional 5 points, download this powerpoint and answer the
discussion questions for each “Discussion” section