Download Enzyme Lab

- - - Please do NOT write on the Pre-Lab - - -
Enzyme Pre-Lab
Using the Enzyme worksheet and Enzyme lab handout answer the Pre-Lab 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:
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.
Read the entire lab before answering the questions. USE COMPLETE SENTENCES!!!
1. In the reaction above, what is the substrate? What are the products?
2. What is the purpose of an enzyme?
3. Define the following:
a) Active site:
b) Substrate:
c) Enzyme-substrate complex:
4. Describe the “Lock and Key” model. (Use a picture and descriptions)
5. Based on the “Lock and Key” model, do enzymes get “used-up”?
6. What are enzymes made of?
7. What are the safety precautions for this lab?
8. Why do we need to use CLEAN test tubes for each part?
9. Briefly describe the purpose of each part of the Enzyme lab.
10. Write out the chemical question for this lab.
- - - Please do NOT write on this lab - - -
Enzyme Lab
Materials:
Raw liver
Enzyme solution on ice
Dropper
Graduated cylinder
Glass rod
Straw
Safety:
Safety goggles
Boiling water
Warm water bath
Ice bath
0.1 M sodium hydroxide
0.1 M hydrochloric acid
6 test tubes 13 x 100 mm
3% hydrogen peroxide
pH paper
forceps
test tube rack
hot plate
test tube holder
Keep lab area CLEAN and ORGANIZED
Procedure:
RECORD DETAILED LAB RESULTS
(temperature change, color change, if bubbles were produced, measure the height of the bubbles, time)
Complete each part entirely (including the questions) before continuing
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).
6. Answer questions.
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.
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. Dump solution in the waster container (NOT down the sink) and CLEAN the test tube (use test tube brushes)
5. Answer questions.
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
4. Add 2 ml of sodium hydroxide (NaOH) to test tube C. Gently swirl the test tube.
5. 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)
12. Answer questions.
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 (100 ºC) – USE TEST TUBE HOLDER
put tube B in warm water bath (about 37 ºC) – USE TEST TUBE HOLDER
put tube C in an ice bath (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
9. Answer questions.
Name: __________________
Period: ____
Enzyme Lab Results Sheet
RECORD DETAILED LAB RESULTS
(temperature change, color change, if bubbles were produced, measure the height of the bubbles, time)
PART 1: Mixing an enzyme and substrate
Observation notes:
1. What was the source of the temperature change?
2. What is a possible explanation for the bubbling during the reaction? (What chemical might be being
released?)
3. The stock enzyme solution was made by crushing a piece of liver in a small amount of water. This released
many molecules of enzyme (catalase) into the water. The reaction mixture in your test tube contained hydrogen
peroxide (the substrate for the reaction) and this enzyme catalase. Below are three possible explanations:
a) The enzyme, catalase, and the substrate, hydrogen peroxide, react together to form a new product and
both changed during the reaction.
b) The substrate, hydrogen peroxide, is changed during the reaction, but the enzyme, catalase, is
unchanged.
c) The enzyme, catalase, is changed during the reaction, but the substrate, hydrogen peroxide, is
unchanged.
Which of the above explanations do you think is correct? Explain why.
PART 2: Is an enzyme or substrate changed (“used up”) in reaction?
Step # Tube
4
A
6
B
Enzyme Source
Fresh piece of liver
Substrate Source
2 ml of peroxide
Fresh piece of liver
“Used” peroxide (A)
(reaction liquid from A)
7
A
“Used” liver (A)
2 ml fresh peroxide
Results
1. Does the peroxide substrate (used peroxide) change after the reaction? How do you know, use evidence
from the lab?
2. Does the enzyme (used liver) change after the reaction? How do you know, use evidence from the lab?
3. If left open in the sunlight, a bottle of hydrogen peroxide would slowly break down into water and oxygen.
Under these conditions, it takes a long time for the reaction to occur. On the other hand, when catalase is
present, this reaction takes place almost immediately. What does this tell you about the function of an enzyme?
PART 3: Is an enzyme specific for only one substrate?
Step # Tube
3
-
Enzyme Source
Small piece of liver
Substrate Source
Carbonic acid
Results
Using your data collected in parts 1-3 answer the questions below:
1. Write out the formula for the breakdown of hydrogen peroxide in the cell.
2. What role does catalase play in keeping the cell in a healthy environment?
3. Is this enzyme found only in the liver? (Hint: if you put hydrogen peroxide on a cut finger, it bubbles)
4. Compare your results for the action of the liver catalase on the two substrates (hydrogen peroxide in part 2
and carbonic acid) you have tested so far. Did the enzyme catalase have an effect on the carbonic acid (formed
when you blew bubbles into the water)? Based on your results, is an enzyme substrate specific? Explain.
Part 4: Effects of pH on enzyme activity
Tube
A
B
C
Contents
Distilled water +
1 ml enzyme
HCl +
1 ml enzyme
NaOH +
1 ml enzyme
pH
Results
1. Write out an explanation about the possible effects of pH on enzyme activity.
2. What effect does a high pH (base) have on the enzyme activity? Use evidence to support your answer.
3. What effect does a low pH (acid) have on the enzyme activity? Use evidence to support your answer.
4. What effect does a neutral pH have on the activity of the enzyme? Use evidence to support your answer.
5. Which pH is optimal for this reaction to occur? How does this relate to what we know about the body’s
normal pH level? Use evidence to support your answer.
6. Graph pH versus enzyme activity rate (slow, medium, fast). Label the acid, base and neutral pH ranges and
use an arrow to show the optimum rate and pH level for this reaction.
fast
Rate of
Reaction
medium
slow
0
2
4
6
8
pH
10
12
14
Part 5: Effects of temperature on enzyme activity
Tube
A
B
C
Contents
1 ml enzyme + 1 ml
hydrogen peroxide
1 ml enzyme + 1 ml
hydrogen peroxide
1 ml enzyme + 1 ml
hydrogen peroxide
Temp
100 ºC
Results
37 ºC
0 ºC
1. At which temperature was the optimal for enzyme activity? Use evidence to support your answer.
2. Graph the enzyme activity rate (slow, medium, fast) versus temperature. Use an arrow to point out the area
of the graph showing the optimal rate and temperature at which the reaction occurred.
fast
Rate of
Reaction
medium
slow
0
25
50
75
Temperature ºC
100
3. What is the effect of the temperature on enzyme activity and how does this relate to the normal body
temperature of mammals? Use evidence to support your answer.