Lab. 2 – Enzyme Catalysis
lab will observe the conversion of hydrogen peroxide to water and oxygen gas by
the enzyme catalysis. The amount of oxygen generated will be measured and used
to calculate the rate of the enzyme-catalized reaction. Enzymes are proteins
produced by living cells. Enzymes act as biochemical catalysts during a
reaction, meaning they lower the activation energy needed for that reaction to
occur. Through enzyme activity, cells gain the ability to carry out complex
chemical activities at relatively low temperatures. The substance in an
enzyme-catalyzed reaction that is to be acted upon is the substrate, which binds
reversibly to the active site of the enzyme. The active site is the portion of
the enzyme that interacts with the substrate. One result of this temporary union
between the substrate and the active site is a reduction in the activation
energy required to start the reaction of the substrate molecule so that products
are formed. In a mathematical equation of the substrate (S) binding with the
activation site (E) and forming products (P) is:
+ S ---> ES --> E + P
ways enzyme action may be affected include:
Salt Concentration -- For
example, if salt concentration is close to zero, the charged amino acid side
chains of the enzyme molecules will attract each other. The enzyme will then
denature and form an inactive precipitate. If salt concentration is extremely
high, the normal interaction of charged groups will be blocked, new interactions
will occur, and again the enzyme will precipitate.
An intermediate salt concentration such as that of human blood (0.9%) is
the optimum for many enzymes.
pH of the environment -- . The pH of a solution is a logarithmic scale
that measures the acidity or H+ concentration in a solution.
The scale begins at 0, being the highest in acidity, and ends at 14,
containing the least amount of acidity. As the pH is lowered an enzyme will tend
to gain H+ ions, disrupting the enzyme’s shape. In turn, if the pH is raised,
the enzyme will lose H+ ions and eventually lose its active shape.
Temperature -- Usually, chemical reactions speed up as the temperature is
raised. When the temperature is increased, more of the reacting molecules have
enough kinetic energy to undergo the reaction. However, if the temperature goes
past a temperature optimum, the conformation of the enzyme molecules is
Activations and Inhibitors -- Many molecules other than the substrate may
interact with an enzyme. If such a molecule speeds up the reaction it is an
activator, but if it slows the reaction down it is an inhibitor.
enzyme used in this lab is catalase, which has four polypeptide chains that are
composed of more than 500 amino acids each. One function of this enzyme is to
prevent the accumulation of toxic levels of hydrogen peroxide formed as a
by-product of metabolic processes. Catalase is also involved in some of the many
oxidation reactions that occur in the cells of all living things. The primary
reaction catalyzed by catalase is the decomposition of hydrogen peroxide to form
water and oxygen.
2 H2O +
the help of catalase, this reaction occurs spontaneously, but very slowly.
Catalase helps to speed up the reaction considerably. In this lab, a rate for
this reaction will be determined.
enzyme catalase, under optimum salt conditions, temperature, and pH level will
speed up the reaction as it denatures the hydrogen peroxide at a higher rate
the first part of the lab, 10 mL of 1.5% H2O2,
a 50-mL glass beaker, and 1 mL of fresh catalase are needed. At the second
stage a test tube, a hot water bath, 5 mL of catalase, 10 mL of 1.5% H2O2
are needed. Finally, in the third
part, a potato, and10 mL of 1.5% H2O2 are
this experiment, 10 mL of 1.5% H2O2, 1 mL of water, 10 mL of sulfuric acid,
two 25 mL beakers, 5-10 mL syringe, potassium permanganate, lab aprons and trays
this section of the experiment, 20 mL of 1.5% H2O2, two glass beakers, 1 mL of H2O, 10 mL of sulfuric acid, a 5 mL syringe, 5-10 mL of potassium
permanganate, and lab aprons and trays are used.
the final part of the lab, 6 plastic cups labeled 10, 30, 60, 120, 180, 360, 6
plastic cups labeled acid, 60 mL of 1.5% H2O2,
a 50-mL beaker, 6 mL of catalase extract, two 5-mL syringes, potassium
permanganate, a timer (clock), lab aprons and trays are needed.
10 mL of 1.5% H2O2
into a 50-mL glass beaker and add 1 mL of freshly made catalase solution. The
fresh catalase should be kept on ice until ready to be used. Observe the
reaction. Then transfer 5 mL of purified catalase extract to a test tube and
place it in a hot water bath for five minutes. Transfer 10 mL of 1.5% H2O2 into a 50-mL beaker and add 1 mL of the boiled catalase solution, after
it has cooled. Observe the changes in the reaction. To demonstrate the presence
of catalase in living tissue, cut 1 cubic cm of potato, macerate it, and
transfer it into a 50-mL glass beaker containing 10 mL of 1.5% H2O2.
Observe the results.
10 ml of 1.5% H2O2
into a clean glass beaker. Add 1
mL of H2O. Add 10 mL
of sulfuric acid (1.0 M). USE EXTREME CARE IN HANDLING ACIDS. Mix the
solution well. Remove a 5 mL sample. Place
this 5 mL sample in another beaker, and assay for the amount of H2O2
as follows: Place the beaker containing the sample over white paper. Use a
burette or 5 mL pipette to add potassium permanganate a drop at a time to the
solution until a persistent pink or brown color is obtained. Remember to gently
swirl the solution after adding each drop.
determine the rate of spontaneous conversion of H2O2 to H2O
and O2in an uncatalyzed reaction, put about 20 mL of 1.5% H2O2
in a beaker. Store it uncovered at room temperature for approximately 24
hours. Put 10 mL of 1.5% H2O2 into a clean glass beaker
(using the uncatalyzed H2O2 that set out). Add 1 mL of H2O2
and then add 10 mL of sulfuric acid (1.0 M). Be
careful when using acid. Mix this solution well. Remove a 5 mL sample and
place it into another beaker. Assay for the amount of H2O2 as
follows: Use a 5 mL syringe to add one drop of potassium permanganate at a time
to the solution until it becomes a persistent pink or brown color. Gently swirl
the solution after adding each drop. Record all results.
a day or more has passed since Exercise B was performed, a baseline must be
reestablish. Repeat the assay from Exercise B and record the results. Compare
with other groups to check that results are similar. To determine the course of
an enzymatic reaction, how much substrate is disappearing over time must be
measured. The first thing to be done is to set up the six cups labeled with
times, and the other six, one directly in front of each cup with a time on it.
Then put 10 mL of 1.5% H2O2 into the cup marked 10 sec.
Add 1 mL of catalase extract to this cup. Swirl gently for 10 seconds using a
timer or clock for help. At 10 seconds, add 10 mL of sulfuric acid. Remove 5 mL
and place in the cup directly in front of the cup marked 10 sec. Assay the 5 mL
sample by adding one drop of potassium permanganate at a time until the solution
turns a pink or brown. Repeat the previous steps, with clean cups using the
times 30, 60, 120, 180, and 360. Record all results and observations.