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Bio H - Biochemistry
Enzymes: Activity of Catalase
Enzymes are specialized proteins that make it possible for our cells to perform all of the chemical
reactions that they need to. Simply put, each reaction that occurs in the body has a cost. The currency
for the cell, however, is not dollars and cents, but rather energy. The more energy a reaction requires,
the harder it is for the cell to perform. The energy required to complete a chemical reaction is called the
activation energy. Enzymes make it easier for the cell to perform a chemical reaction by lowering the
required amount of activation energy. You should also recall that since enzymes are proteins, they have
a complicated folded structure that provides an active site that is a perfect match to the enzyme’s
substrate. Like all proteins, the final structure of an enzyme can be disrupted by environmental
conditions, like temperature and pH. Each enzyme has a range of temperatures over which it will work,
but one temperature is best. In general, adding heat increases the rate of chemical reactions. This makes
the molecules of enzyme and substrate move around faster and increases the chances of a substrate
molecule finding the active site of the enzyme. Conversely, at cooler temperatures, everything is
moving more slowly. Enzyme-assisted reactions follow this rule up to a point when the level of heat
causes the protein to denature. Similarly, each enzyme has a range of pH values over which it will work
and one pH level will represent the optimum for that enzyme. Adding more substrate will also increase
the rate of the reaction, up to the point at which all enzyme molecules are working as fast as they
physically can. Beyond this concentration, adding more substrate has no effect on the rate of the
reaction. You can determine the rate at which an enzyme is doing its job by either looking at how fast a
product appears or by how fast the substrate disappears.
One of the most common enzymes in living things is catalase. Catalase breaks down its substrate, H2O2
(hydrogen peroxide) a potentially harmful chemical formed in cells. Catalase is found in large quantities
in the liver of an organism and can obtained by simply blending some fresh beef liver into liver puree!
There is also plenty of catalase in potatoes. Don’t worry, we’ve bought purified catalase enzyme to
reduce the “ick” factor. Your job is to use the catalase to breakdown hydrogen peroxide into its
products: water and oxygen gas. So how do we know that the reaction is happening? Well, what forms
when a gas is put into a liquid? (think soda!) The progress of the breakdown reaction can be monitored
by looking for bubbles of O2 gas. In this exercise you will measure how long it takes a piece of filter
paper soaked in catalase to be carried to the top of a beaker by the O2 bubbles produced. Because the
filter paper is porous, bubbles of O2 will be trapped in the paper and when enough are present, they will
life the piece of paper. This is a little different from just counting bubbles, but if you use circular pieces
of filter paper of the same diameter and thickness, the results will be very consistent. As a class, you
will test the reaction under several different conditions in order to determine the optimal conditions for
this enzyme to catalyze its substrate.
Procedure:
Your group will investigate the rate of catalase reaction over a range of values for one of the
environmental conditions described in the introduction. You should complete three trials at each of
three levels of your chosen variable. One of your conditions will be the basic control group procedure
that everyone will follow:
1) Pour 20 mL of 1% H2O2 at room temperature into a 50 mL beaker.
Bio H - Biochemistry
2) Use forceps to pick up ONE (make sure they aren’t stuck together) filter paper dot and dip it
into the standard concentration of catalase. Wipe the filter paper on the edge of the catalase
dish to get rid of the excess enzyme.
3) Quickly lower filter paper to the bottom of the beaker and release it. Start the timer as soon
as the filter dot is released. Stop timing when the dot reaches the top OR when time reaches
2 minutes, whichever comes first.
4) Record your data.
5) Discard used filter paper into receptacle
6) Repeat the experiment 2 more times under the same conditions.
7) After trial 3, pour your sample of H2O2 down the sink and repeat the procedure using a
different level of you chosen variable.
Your task: Design the experiment that your group will be doing on your next lab day. In you lab
journal, provide the following:
 Purpose, including what variable you are testing and a clear statement of your hypothesis. Your
hypothesis should express how you think the independent variable will affect the dependent variable.
What will you actually record as your dependent variable?
 a list of Materials
 describe your Procedures
 create a Data Table in which you will record your Results.
 We will deal with the Analysis and Conclusions later. Keep in mind, however that we are
interested in the reaction rate, and a rate is specified as something happening over a specified period
of time. In this case, we will be looking for something about the amount of O2 produced over time.
Does counting how long it takes a piece of filter paper to rise give us that information? Not directly.
Remember that when you graph the data, you will want a faster rate of reaction to be represented as
a higher number. In this procedure, a faster rate will be seen in a less time for the paper to rise.
What to do???
Possibilities…
Vary temperature You can have water available at temperatures ranging from about 100C to 1000C.
Vary pH You can adjust the pH of the substrate by adding drops of HCl or NaOH, OR by adding a
buffer solution of the desired pH from the ranges of 3, 7 and 11. If you choose to use the buffers, you
will need to use equal parts of 2% H2O2 and the buffer.
Vary substrate concentration H2O2 comes as a 3% solution, so that represents the maximum
concentration available. Dilutions of 1.5%, 1% (the control) and 0.5% will be available.
Vary enzyme concentration Catalase will be available as 2X, as the standard dilution, 0.5X.