FACULTY OF PRE-MEDICAL SCIENCES

DEPARTMENT OF CHEMISTRY
LABORATORY MANUAL BIOCHEMISTRY 2 CH2012
 EXPERIMENT 1: EFFECT OF CATALASE ON HYDROGEN PEROXIDE
Theory
Hydrogen peroxide is an oxidant that is produced by reactions that occur in living organisms
produced in small amounts. It is a toxic chemical and organisms detoxify it by breaking it into
water and oxygen that are not harmful to the cells. The enzyme catalase is useful in breaking
down this oxidant. Catalase is found in the peroxisomes in both animals and plants.
Catalase is an enzyme. Enzymes speed up the rate of chemical reactions and are involved in the
chemical reaction but remain unconsumed by the reaction. Enzymes catalyze biochemical
reactions by lowering the activation energy needed to break the chemical bonds in reactants
and form new chemical bonds in the products. They act by bringing reactants together in the
appropriate orientation and weakening bonds thus increasing the rates of reactions. Enzymes
are biological catalysts and without them reactions would proceed slowly in living organisms
and unable to sustain life. Enzymes contain an active site which fits onto a specific substrate.
They are therefore substrate specific. Enzyme activity is however affected by the environmental
factors such as temperature, pH, and concentration of the substrate.
In this experiment, potato catalase will be utilized to observe the effects of catalase on
hydrogen peroxide.
Materials/requirements
1. test tubes
2. ruler
3. hydrogen peroxide
4. distilled water
5. catalase (from the potato)
6. Markers
7. Stop watch
Procedure
Get 2 test tubes and label them A and B. Measure and mark each of the test tubes with a ruler 1 cm
from the bottom of the test tube clearly using a marker. Fill each of the test-tube with catalase from
potatoes. Add 10 drops of Hydrogen peroxide to test tube A and 10 drops of distilled water to test tube
B. Wait for a minute and measure the height of the bubbling observed in both test tube A and B.
Write down your observations in a table and a report discussing your observations.
Questions
1. What happened when H2O2 was added to the potato in test tube A and that in test tube
B?
2. Give reasons for the observations made and explain the purpose of water in test tube B.
3. Write down the reaction for the breakdown of hydrogen peroxide.
 Experiment 2: Effects of Temperature on Enzyme activity
Enzyme activity is however affected by the environmental factors such as temperature, pH, and
concentration of the substrate. In this experiment, potato catalase will be utilized to observe
the effects of temperature on activity of enzymes.
Materials and requirements.
1. Test tubes
2. Catalase (from potato)
3. Hydrogen peroxide
4. Distilled water
5. Hot plate or heating mantle (for boiling water)
6. Ice
7. Thermometer

Procedure
Get 2 test tubes and label them A and B. Measure and mark each of the test tubes with a ruler 1
cm from the bottom of the test tube clearly using a marker. Fill each of the test-tube with catalase
from potatoes. Add 10 drops of Hydrogen peroxide to test tube A and 10 drops of distilled water
to test tube B. Wait and report the time when you first observe the bubbles in both test tube A
and B. Repeat the same procedure at temperature of 30 degrees after by placing your test tubes
in a water bath at temperature of 30 degrees and record the time at which you first observe the
bubbles after adding hydrogen peroxide in the test tubes. Also repeat the same procedure by
placing your test tubes in ice and record the temperature and time at which you observe the
bubbles after adding hydrogen peroxide.
Write a report on your findings.
Questions
1. How does temperature affect the ability of enzymes to catalyze chemical
reactions?
 Experiment 3: Separation of amino acids using paper chromatography and
identification of amino acids in unknown solutions
THEORY
Chromatography can be performed in a variety of ways. Each specific
chromatography technique (paper, gas, liquid, column, etc.) has advantages but one
thing is common to all. In every type of chromatography there is a stationery phase
and a mobile phase. The molecules to be separated partition themselves between
the two phases depending upon their individual chemical constitution. If a stationery
phase happens to be more organic than the mobile phase, hydrophobic molecules
will spend less time in the mobile phase and migrate only a short distance. In the
same system, charged molecules will spend more time in the mobile phase and
migrate further. Mobile and stationery phases can be selected that will discriminate
between the molecules to be separated.
Paper chromatography requires little apparatus and can be used for separation of a
variety of small molecules such as amino acids, sugar and nucleic acids. The identity
of the unknown compounds can be determined by comparing their chromatographic
behavior to that of solutions of known compounds. To visualize colorless
compounds, various reagents have been developed to produce a characteristic
colored product after reaction with amino acids, sugars etc. Ninhydrin is one of
these reagents which react with amino acids.
MATERIALS
Chromatography paper (12.5 ꓫ 25 cm)
Chromatography tank (or 600ml beaker)
Capillary tubes
Paper clips
Unknown solutions: #1, #2 and #3
Solvent: n-propanol: water (2:1v/v)
Staining reagent: 0.1 % Ninhydrin in acetone
Oven
PROCEDURE
Place chromatography paper on a clean surface. Be careful to handle the paper as
little as possible. Using pencil, label the top of the paper with your name, date and
solvent. Draw a faint line 1.5 cm from the bottom of the paper and mark the line
every 1.5 cm starting and continuing to 21 cm. using a capillary tube, carefully apply
small samples of each amino acid and unknown solution to the line intervals
marked. The spots should be 3-5mm in diameter. Use a pencil to identify each spot.
Apply two samples of each solution. Allow spots to dry.
+ + + + + +
4cm Lys 1 Ala 2 Leu 3
 Fold the paper into a cylinder and fix it at the top with a paper clip. The cylinder
should fit inside a 600 ml beaker without touching any side. Be sure that the over
lapping edges touch only at the top of the paper. Add 20 ml of the solvent (n-
propanol: water) to the beaker and carefully insert the chromatogram. Do not
remove the beaker until the chromatogram is removed. Cover the top with a piece
of paper and allow at least one hour for development.
After one hour, remove the chromatogram and place on a clean surface. Draw a
faint line across the upper limit of the solvent development. Dry at room
temperature or a warm oven for 30 to 50 minutes making sure the chromatogram
does not char.
Using a sharp pencil, outline the spots on the chromatogram. Record the color of
each spot. Mark the darkest region of each spot (ignoring the trailing).Measure
accurately the distance from the origin of the spot to the centre of the developed
spot. Again measure the distance from the origin but this time up to the solvent
front. Maximum distance the solvent moves. Calculate the Rf values using the formula.
𝑅 𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡ℎ𝑒 𝑎𝑚𝑖𝑛𝑜 𝑎𝑐𝑖𝑑 𝑡𝑟𝑎𝑣𝑒𝑙𝑠 𝑓𝑟𝑜𝑚 𝑜𝑟𝑖𝑔𝑖𝑛
𝑓=
𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡ℎ𝑒 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝑓𝑟𝑜𝑛𝑡 𝑚𝑜𝑣𝑒𝑠 𝑓𝑟𝑜𝑚 𝑜𝑟𝑖𝑔𝑖𝑛
Report
In your report, make a drawing of your chromatogram, and on the drawing indicate
amino acid corresponds to each spot. Compare Rf values for the spots in the
unknown samples that you think represent the same amino acid. For each group of
4, the group leader should submit a signed chromatogram and attach names of
group members to the lab demonstrator. The rest should submit drawn
chromatograms.

Questions
1. Explain the Rf values observed for lysine, leucine and alanine. Explain why one amino
acid moves more than the other.