Download Enzyme Pre-Lab

Enzyme Pre-Lab
Using the Enzyme worksheet and Enzyme lab handout answer the PreLab questions – the pre-lab must be complete before beginning the
lab.
Background:
In this investigation, you will study several factors that effect the
activity of enzymes. Enzymes are made up of amino acids and have
optimal working conditions. The enzyme you will use is catalase, which
is present in most cells and found in high concentrations in liver and
blood cells. You will use liver as the source of catalase. Catalase
promotes the decomposition of hydrogen peroxide (H2O2) in the
following reaction:
After reading the instructions for each section make a drawing of
the test tubes used in the experiment.
PART 1: Mixing an enzyme and substrate
1. Obtain supplies: 2 test tubes and test tube rack.
2. Put 20 drops (1ml) of hydrogen peroxide in one test tube (you will
use the other test tube in part 2). (CLEAN ALL SPILLS)
3. Add 5 drops of stock enzyme solution (crushed liver) to the
peroxide. (BE SURE TO KEEP THE ENZYME SOLUTION ON ICE)
Note what happens inside the test tube.
4. Feel the bottom of the test tube and note your observation.
5. Dump solution in the waste container (NOT down the sink) and
CLEAN the test tube (use test tube brushes).
Drawing:
2H2O2
2H2O + O2
Hydrogen peroxide is formed as a by-product of chemical reactions in
cells. It is toxic and would kill cells if not immediately removed or
broken down.
Materials:
Raw liver
Enzyme solution on ice
Graduated cylinder
Straw
Warm water bath
0.1 M sodium hydroxide
6 test tubes 13 x 100 mm
pH paper
test tube rack
Safety:
Safety goggles
test tube holder
Dropper
Glass rod
Boiling water
Ice bath
0.1 M hydrochloric acid
3% hydrogen peroxide
forceps
hot plate
Keep lab area CLEAN and ORGANIZED
PART 2: Is an enzyme or substrate changed (“used up”) in
reaction?
1. Obtain supplies: Glass stirring rod (make sure it does not roll off
the table).
2. Label the test tubes A and B.
3. Put a piece of liver (the size of a rice grain) into the bottom of
test tube A, using a glass stirring rod to push it to the bottom of the
test tube.
4. Put the tube in a rack and add 40 drops (2 ml) of hydrogen
peroxide. Note what happens and record in the table in part 2.
5. Allow the reaction to continue until the bubbling stops, stirring
gently.
6. Pour only the reaction liquid into test tube B, do not transfer over
the left over liver pieces – use the glass rod to assist you.
7. Put another small piece of liver (the size of a rice grain) into test
tube B. Note whether or not a reaction occurs when using the “used
peroxide” (Is it bubbling?). Record your results in the table in part 2.
8. Pour another 2ml of hydrogen peroxide in test tube A to see if the
“used liver” is still reactive.
Record your results in table in part 2.
9. Dump solution in the waste container (NOT down the sink) and
CLEAN the test tube (use test tube brushes)
10. Using the results and answer questions.
Drawing:
PART 3: Is an enzyme specific for only one substrate?
1. Obtain supplies: straw
2. Put 3 ml of distilled water in a test tube and carefully blow into
the water with a straw for 1 minute. Carbon dioxide (CO2) is
produced in reactions in living cells. Blowing into the tube will add
carbon dioxide to the water which will form carbonic acid.
3. Add a small piece of liver to the test tube to see what effects the
enzyme catalase (from the liver) has on carbon dioxide. Record your
results in the table in part 3.
4. Repeat the above experiment, however use 3 ml of hydrogen
peroxide, do not blow through the solution, add the liver and record
the results.
5. Dump solution in the waster container (NOT down the sink) and
CLEAN the test tube (use test tube brushes)
Drawing:
Part 4: Effects of pH on enzyme activity
1. Obtain a total of 6 test tubes, label test tubes A, B, C. Do not
label the other 3
2. Add 2 ml of distilled water to test tube A. Gently swirl the test
tube.
3. Add 2 ml of hydrochloric acid (HCl) to test tube B. Gently swirl
the test tube
Add 2 ml of sodium hydroxide (NaOH) to test tube C. Gently swirl the
test tube.
Add 1 ml of stock enzyme solution to test tubes A, B, C.
6. CLEAN-UP ALL SPILLS. Be sure not to splash any chemicals in
your face.
7. Use a small piece of pH paper and tweezers, tilt the test tube
slightly to test the pH of test tube A, B and C (use a new piece for
each test tube). Record results.
8. To each of the three unmarked tubes add 1 ml of hydrogen
peroxide.
9. AT THE SAME TIME, pour the contents of the unmarked tubes
into each of the labeled test tubes.
10. Record your results. Note the relative rates of each individual
reaction.
11. Dump solution in the waster container (NOT down the sink) and
CLEAN the test tube (use test tube brushes)
Drawing:
Part 5: Effects of temperature on enzyme activity
1. Using your cleaned test tubes, label three “A”, “B”, “C”. Do not
mark the other three.
2. To each of the labeled tubes, add 1 ml of stock enzyme solution
(liver catalase).
3. AT THE SAME TIME:
put tube A in boiling water bath or heat source (100 ºC) – USE
TEST TUBE HOLDER
put tube B in warm water bath or heat source (about 37 ºC) –
USE TEST TUBE HOLDER
put tube C in an ice bath or refrigerator (0 ºC)
4. Wait 4 minutes. While you wait, add 1 ml of hydrogen peroxide
solution to each of the three unmarked test tubes.
5. After 4 minutes, remove the tubes from their baths and put them
in a test tube rack.
6. AT THE SAME TIME, add the contents of the unmarked tubes to
each tube. A, B, C.
7. Observe the results and BE SURE TO NOTE THE RATE OF EACH
REACTION! Record your results.
8. CLEAN AND RETURN ALL SUPPLIES.
Drawing: