Enzyme Catalysis

BioOverview:
In this laboratory you will use a chemical titration to measure and then calculate the rate of conversion of hydrogen peroxide (HO) to water and oxygen using the enzyme catalase.

Objectives:
Before doing this exercise, you should understand:
the general functions and activities of enzymes
the relationship between the structure and function of enzymes
the concept of initial reaction rates of enzymes
how the concept of free energy relates to enzyme activity
that changes in temperature, pH, enzyme concentration, and substrate concentration can affect the initial reaction rates of enzyme-catalyzed reactions.

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After doing the exercise, you should be able to:
measure the effects of changes of temperature, pH, enzyme concentration, and substrate concentration on rates of an enzyme-catalyzed reaction in a controlled experiment.

explain how environmental factors affect the rate of enzyme-catalyzed reactions.

Materials:
5mL and 10mL syringe
1mL pipet
60mL plastic cup
beaker
hot plate
test tubes
test tube tongs
marker (to label)
potassium promgemte
sulfuric acid
catalyze
hydrogen peroxide
bovine liver
distilled water
goggles
apron
gloves
Procedure:
Part A: Test of Catalase Activity
1.a) Transfer 10 mL of 1.5% (0.44 M) hydrogen peroxide
(H2O2) into a 50-mL glass beaker.

b) Add 1 mL of freshly made catalase solution.


2. a) Fill a glass beaker with 300 mL of water.

b) Place beaker on a burner and allow it to boil.

c) Transfer 5 mL of purified catalase extract to a test tube.

d)Put the test tube with the catalase extract in the beaker on
the burner and leave it for 5 minutes.

e) Allow the catalase to cool.

f)Transfer 10 mL of 1.5% hydrogen peroxide (H2O2)
into a 50-mL glass beaker.

g) Add 1 mL of the cooled, boiled catalase solution.


Part B: The Establishment of a Baseline
*These procedures must be performed without adding catalase
(enzyme) to the reaction mixture.


1.Accumulate 10 mL of 1.5% hydrogen peroxide (H2O2), two
50-mL glass beakers, 10 ml of 1M sulfuric acid (H2SO4),
approximately 6 mL of 2% potassium permanganate (KMnO4)
1 mL of water (H2O), two appropriately labeled syringes
(for contamination prevention), white paper, a pipette, aprons,
goggles, and gloves.
2. Put on aprons, protective goggles, and latex gloves.


3.a) Put 10 mL of 1.5% hydrogen peroxide (H2O2) into a clean
glass beaker.

b) Add 1 mL of water (H2O), in place of the enzyme solution.

c) Add 10 mL of 1M sulfuric acid (H2SO4),
USING EXTREME CAUTION WHEN HANDLING
ACIDS.

d) Mix well via a pipette.


4.Using a syringe remove a 5 mL sample from the resulting
solution.


5. Place this 5 mL sample in another beaker.


5.a) Assay, or examine, for the amount of hydrogen peroxide
(H2O2).

b) Place the beaker containing the sample over a white paper.

c)Use another syringe with a capacity of 5 mL to add potassium
permanganate (KMnO4) a drop at a time until a persistent
pink or brown color is obtained while gently shaking the
solution after each drop.

d) Record findings.


Part C: This portion of the lab was not able to be executed.


Part D: An Enzyme-Catalyzed Rate of H2O2 Decomposition
1.Gather 50 mL of 1M sulfuric acid (H2SO4), 50 mL of 1.5%
hydrogen peroxide (H2O2), 5 mL of catalase, 10 50-mL glass
beakers, 30 mL of potassium permanganate, 8 syringes, a white
sheet of paper, and a stop watch.


2.Use syringe to put 10 mL of 1.5% hydrogen peroxide (H2O2)
in a 50 mL glass beaker.
3. Add 1 mL of catalase.


4. At 10 seconds add 10 mL of sulfuric acid.
MAKE CERTAIN TO WEAR LATEX GLOVES AND
PROTECTIVE GOGGLES WHEN HANDLING DANGEROUS
CHEMICALS AND ACIDS.


6.Remove a 5 mL sample and use syringe to drop potassium
permanganate (KMnO4) into solution. Once the sample turns pink
or brown calculate the amount of hydrogen peroxide in the sample.
7.Repeat steps 1 through 5 above, making sure to change the 10
seconds to 30 seconds, the 30 seconds to 60 seconds, 60 seconds to
120 seconds, 120 seconds to 180 seconds, and 180 seconds to 360 seconds.
Conclusions:
Exercise 2A: Test of Catalase Activity
1) To observe the reaction to be studied, use the labeled syringe to transfer 10mL of 1.5% (0.44-MH2O2) into your unlabeled 60-mL plastic cup. Use a plastic transfer pipet to add 1mL of the catalase solution. The bubbles coming from the reaction mixture are the O2 resulting from the breakdown of H2O2 by catalase. How could you show that

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