The Activity of Lactase

Standards:
PA S. T. & E.:

3.1.B.A2 Explain the importance of enzymes as catalysts in cell reactions.

Identify how factors such as pH and temperature may affect enzyme function.
3.1.B.A7 Explain the consequences of extreme changes in pH and temperature on cell proteins.

Introduction:
Enzymes are protein molecules, which act to catalyze the chemical reactions in living
things. These chemical reacddddddtions make up the metabolism of the organism. There are
two basic types of reactions that occur in living organisms: catabolic reactions and anabolic
reactions. Catabolic reactions break larger molecules down into smaller molecules; anabolic
reactions build larger molecules from smaller molecules.
Lactase is a digestive enzyme found in the small intestine. This enzyme breaks down the
sugar lactase into its two component sugars, glucose and galactose. Lactose is the principle
sugar found in milk and is found in all dairy products. People who are lactose intolerant either
lack lactase or have a defective version of the enzyme. These people cannot digest lactose and
suffer various types of intestinal distress. People who are lactose intolerant commonly take a
dietary supplement that contains lactase. Lactase is sold under the commercial names of Dairy
Ease or Lactaid.
Since milk is an opaque liquid, it is difficult to work with because there is no noticeable
change. There is a substance called ONPG, which can be used as a substitute for the milk.
ONPG is a colorless liquid. Lactase splits ONPG into ONP and galactose. ONP is a clear
yellow material, so we can observe a color change from clear to yellow and use a
spectrophotometer to measure how much product is being produced.
In this laboratory you will examine the effects of concentration, pH, temperature, and
competing substrates on the activity of lactase.

Guiding Questions:

1. What are some of the main factors that are involved with enzyme function?
2. What do you think will happen when you keep adding more and more of an enzyme to a
chemical reaction?
3. What do you think will happen if you keep the amount of enzyme the same but add more
of the reacting chemicals?

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Vocabulary:
Catalyst-A compound that speeds up a chemical reaction
Disaccharide-A carbohydrate made of two monosaccharides, lactose is an example
Enzyme- A protein catalyst that controls a chemical reaction in living organisms.
Lactase- An enzyme that digests lactose into one molecule of glucose and one molecule of
galactose
Lactose- The sugar found in dairy products. Lactose is a disaccharide made of glucose and
galactose.
Monosacchardide- A simple sugar. The three common monosaccharides are glucose, fructose,
and galactose.
Polysaccharide- A carbohydrate composed of many monosaccharides, such as starch in animals
or cellulose in plants

Materials:

Lactase solution 0.5 Molar Lactose solution

5 milliMolar ONPG solution Stop watch or clock
Buffer solutions 2, 4, 6, 7, 8, 10, 12 Spectrophotometer
1M Sodium carbonate solution Test tubes
1 ml pipettes Distilled water
10 ml pipettes/Pipumps Cuvettes for spectrophotometer
Test tube rack (if available)
Water baths 0oC, 4 oC, 23 oC, 37 oC, 60 oC Graph paper
Ice Thermometer
Hot plate

Safety Notes:
This lab uses Ortho-NitroPhenol-beta-Galactopyranoside (ONPG) as a substitute for
lactose. Since ONPG is a phenolic compound, it should be handled with caution. Do not
dispose of the ONPG by dumping it into a sink drain. Instead, collect the used materials into a
waste bottle and return it with your lab materials. The concentration of ONPG that we are
working with is small (5 milliMolar) and should not pose a problem to your students, however, if
any gets onto the skin it is probably a good idea to make sure the student washes the affected
area immediately. Any spills should be wiped up immediately with paper towels and the towels
disposed of by placing them into the trash.
Care should be taken when working with the pH buffer solutions and water baths in order
to avoid skin burns. Students should wear protective goggles and possibly latex rubber gloves.
Wash your hand at the end of the lab period.

Procedure: PART 1 -- Effects of Concentration

A. Observing the reaction – If time is a factor due to a short lab period, skip and go to B.
1. Into a test tube place 4.5 ml of the pH 7 buffer solution and 0.5 ml of the ONPG. You can
use a 10 ml pipette with a pi-pump or disposable 1 ml pipette. The 1 ml pipette is marked
in 1.0, .75, .5 and .25 increments. Near the top of the narrow tube = 1ml marking. You
will need to use a different pipette for each substance to avoid contamination.
2. Add 0.5 ml of the Lactase and observe the tube for several minutes.
3. Describe the appearance of the tube as the reaction occurs.
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B. Effects of enzyme concentration --

1. Turn on the spectrophotometer and set it to 420 nm by pushing gently on the correct up
or down nm arrow button. Create a blank by adding 4.5 ml of pH 7 buffer, 0.5 ml of
ONPG and 1 ml of the 1M sodium carbonate solution into a small test tube.
2. Place the solution for the blank into a cuvette (if available, if not use original test tube)
and insert it into the spectrophotometer. Always wipe the outside of each tube with a
Kimwipe to prevent corrosion and remove fingerprints. Push gently on the 0ABS/100% T
button to set the absorbance to 0 A.
3. Being careful to use a different pipette for each liquid throughout the experiment,
Set up eight clean test tubes and add 0.5 ml of ONPG to each, then add the following:

Test Tube pH 7 buffer Test tube pH 7 buffer

1 4.5 ml 5 2.5 ml

2 4.0 ml 6 2.0 ml

3 3.5 ml 7 1.5 ml

4 3.0 ml 8 1.0 ml

6. Add the following amounts of lactase to each test tube and time the reaction for two
minutes:
Test tube Lactase Test tube Lactase

1 0.0 ml 5 2.0 ml

2 0.5 ml 6 2.5 ml

3 1.0 ml 7 3.0 ml

4 1.5 ml 8 3.5 ml
7.

7. After timing the reaction for 2 minutes with a stop watch, add 1 ml of the 1M sodium
carbonate solution to each test tube. This should stop the reaction.
8. Fill a cuvette with the solution from your reaction or use the actual test tube; use a
Kimwipe to wipe any moisture, fingerprints from each tube and place it into the
spectrophotometer. Read and record the absorbance on your table. (Note: the greater the
absorbance, the greater the amount of product that was formed.)
9. Collect the class data and draw a graph to show the effect of enzyme concentration on the
reaction.

C. The effect of substrate concentration --

1. Use your blank from part B to rezero the absorbance of the spectrophotometer.
2. Set up eight clean test tubes and add the following:

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Test tube pH 7 buffer ONPG Test tube pH 7 buffer ONPG

1 5.0 ml 0.0 ml 5 3.0 ml 2.0 ml

2 4.5 ml 0.5 ml 6 2.5 ml 2.5 ml

3 4.0 ml 1.0 ml 7 2.0 ml 3.0 ml

4 3.5 ml 1.5 ml 8 1.5 ml 3.5 ml

3. Add 0.5 ml of Lactase to each test tube and time the reaction for two minutes.
4. After timing the reaction for 2 minutes, add 1 ml of the 1M sodium carbonate solution to
each test tube. This should stop the reaction.
5. Fill a cuvette with the solution, or use the actual test tube from your reaction and place it
into the spectrophotometer. Read and record the absorbance.
6. Collect the class data and draw a graph to show the effect of substrate concentration on
the reaction.

Procedure: PART 2 -- Effects of pH and temperature

D. The effect of pH on enzyme activity --

1. Create a new blank for the spectrophotometer by combining 4.5 ml pH 7 buffer, 0.5 ml
ONPG and 1 ml 1M sodium carbonate.
2. Set the spectrophotometer at 420nm and use the blank to zero the absorbance.
3. Each group will set up six test tubes as follows:

Test tube 4.5 ml 0.5 ml

1 Tube 1 pH 2 buffer ONPG
2 Tube 1 pH 4 buffer ONPG
3 Tube 1 pH 6 buffer ONPG
4 Tube 1 pH 8 buffer ONPG
5 Tube 1 pH 10 buffer ONPG
6 Tube 1 pH 12 buffer ONPG

4. Add 0.5 ml of Lactase to each of your test tubes and time the reaction for two minutes.
5. After timing the reaction for 2 minutes, add 1 ml of the 1M sodium carbonate solution to
each test tube in order to stop the reaction.
6. Fill a cuvette with the solution from the reaction or use the actual test tube and place it
into the spectrophotometer. Read and record the absorbance.
7. Collect class data and draw a graph to show the effect of pH on the activity of the
enzyme.

E. The effect of temperature on enzyme activity --

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1. Use the blank from part D to rezero the spectrophotometer.
2. Set up six test tubes by placing 4.5 ml of pH 7 buffer and 0.5 ml Lactase in each tube.
3. Place the test tube into any of the following temperatures that are available:

Test tube Water bath temp Test tube Water bath Temp

1 0oC (ice bath) 4 37oC

2 4oC (ice water) 5 60oC

3 23oC (room temp) 6 100oC

4. Allow the test tubes to remain at the temperature for at least 5 minutes.
5. Add 0.5 ml of ONPG to each test tube and keep the tubes in the assigned temperature.
Time the reaction for two minutes.
6. After timing the reaction for 2 minutes, add 1 ml of the 1M sodium carbonate solution to
stop the reaction.
7. Fill a cuvette with the solution from your reaction or use the actual tube and place it into
the spectrophotometer. Read and record the absorbance.
8. Collect class data and draw a graph to show the effect of temperature on the activity of
the enzyme.

Procedure: PART 3 -- Other factors (optional)

F. Effect of a competitive substrate

1. Set up a new blank containing 4.5 ml pH 7 buffer, 0.5 ml ONPG and 1 ml 1M sodium
carbonate solution. Use the blank to rezero the absorbance of the spectrophotometer at
420nm.
2. Label two test tubes.
3. To test tube one add 4.0 ml pH 7 buffer and 1.0 ml ONPG.
4. To test tube two add 3.0 ml pH 7 buffer, 1.0 ml ONPG and 1.0 ml lactose solution.
5. Add 0.5 ml of Lactase to each test tube and time for two minutes.
6. After timing the reaction for 2 minutes, add 1 ml of 1M sodium carbonate solution to
each test tube in order to stop the reaction.
7. Fill a cuvette with the solution from test tube one or use the actual tube and place it into
the spectrophotometer. Read and record the absorbance.
8. Fill a second cuvette with the solution from test tube two or use the actual test tube and
place it into the spectrophotometer. Read and record the absorbance.

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The Activity of Lactase
Name:

EVALUATION

Objectives:

The student will be able to:

1. Define the terms enzyme and substrate.
2. Explain the result of increasing the enzyme concentration in a reaction.
3. Explain the result of increasing the substrate concentration in a reaction.
4. Discuss the effect of pH on the activity of lactase.
5. Discuss the effect of temperature on the activity of lactase.
6. Define the term competing substrate.
7. Explain the effect of a competing substrate on the activity of lactase.

Data:

A. Observing the reaction -- Describe the change in the appearance of the reaction
mixture as the reaction occurs.

B. Effect of enzyme concentration -- C. Effects of substrate concentration - -

Concentration Absorbance Concentration Absorbance

0.0 ml lactase 0 ml ONPG

0.5 ml lactase 0.5 ml ONPG

1.0 ml lactase 1.0 ml ONPG

1.5 ml lactase 1.5 ml ONPG

2.0 ml lactase 2.0 ml ONPG

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2.5 ml lactase 2.5 ml ONPG

3.0 ml lactase 3.0 ml ONPG

3.5 ml lactase 3.5 ml ONPG

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D. Effect of pH -- E. Effect of Temperature

pH Absorbance Temperature Absorbance

pH 2 0oC

pH 4 4oC

pH 6 23oC

pH 8 37oC

pH 10 60oC

pH 12 100oC

F. Competitive substrate --

Substrate(s) Absorbance

0.5 ml ONPG

0.5 ml ONPG and

0.5 ml lactose

Analysis and Conclusions:

1. What happens to the reaction as you increase the amount of enzyme (Lactase)?

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2. What happens to the reaction as you increase the amount of substrate (ONPG)?

3. At which pH(s) did the enzyme (Lactase) work the best?

4. At which pH(s) did the enzyme work the least?

5. How does pH affect enzymes?

6. At which temperature(s) did the enzyme work best?

7. At which temperature(s) did the enzyme work the least?

8. How does temperature affect enzymes?

9. What happened when you added lactose to the reaction mixture?

10. Why did the lactose affect the reaction?

11. Write a paragraph summarizing the main concepts you have learned in conducting these
lab activities.

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