LAB EXERCISE: Enzymes II-Lactase
Laboratory Objectives:
After completing this lab topic, you should 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.
Introduction and Background:
Enzymes are protein molecules, which act to catalyze the chemical reactions in living things.
These chemical reactions 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 lactose (a
disaccharide) into the monomers, glucose and galactose (monosaccharides).
Figure 1. Hydrolysis of the disaccharide lactose to the monosaccharides galactose and glucose.
Lactose is the principle sugar found in milk and is found in all dairy products. People who are
lactose intolerant either have a primary or a secondary lactase deficiency. The primary deficiency
develops over time (mostly after the age of 2) whereby the body naturally produces less lactase.
Secondary deficiency is caused by some sort of damage to the small intestinal cells that produce
lactase (examples include: Crohn’s disease, AIDS, surgical removal of the small intestine, celiac
disease and inflammatory bowel disease.) Lactose intolerance can run in families as researchers
have found that there is a genetic link. Lactose intolerant people cannot digest lactose and suffer
various types of intestinal distress. People who are lactose intolerant commonly take a dietary
supplement that contains the enzyme lactase. Lactase is sold under the commercial names of
Lacteeze, Dairy Ease or Lactaid.
Since milk is an opaque liquid, it is difficult to examine lactase activity because there is no
noticeable change to indicate that hydrolysis of lactose has occurred. However, ONPG (orthonitrophenyl-β-galactoside), is a colorless liquid that can be used as a substitute for the milk to
examine lactase activity. Lactase splits (via hydrolysis) ONPG into ortho-nitrophenol (ONP) and
galactose. ONP is clear and yellow, so we can observe a color change from colorless to yellow
and use a spectrophotometer to measure how much product is being produced.
Figure 2. Hydrolysis of ONPG (ortho-nitrophenyl-β-galactoside) to galactose and ONP (orthonitrophenol).
In this laboratory you will examine the effects of changes in substrate and enzyme concentration,
pH, and temperature on the activity of lactase.
Spectrophotometry
The spectrophotometer is an instrument which measures the amount of light of a specified
wavelength which passes through a medium. According to Beer's law, the amount of light
absorbed by a medium is proportional to the concentration of the absorbing material or solute
present. Thus the concentration of a colored solute in a solution may be determined in the lab by
measuring the absorbency of light at a given wavelength. Wavelength (often abbreviated as
lambda) is measured in nm. The spectrophotometer allows selection of a wavelength to pass
through the solution. Usually, the wavelength chosen corresponds to the absorption maximum of
the solute. Absorbency is indicated with a capital A.
Safety:
This lab uses ortho-nitrophenyl-β-galactoside (ONPG) as a substitute for lactose. Since ONPG is
a phenolic compound, it should be handled with caution. Be sure to wash hands immediately
after coming into contact with the solution. Any spills should be wiped up immediately with
paper towels and the towels disposed of by placing them into the trash. The ONPG waste will be
collected.
Part 1: For all four experiments, write a hypothesis and prediction based on your understanding
of enzyme function and protein folding.
Part 2—Experimental procedure:
A. Effects of enzyme concentration –
1. Turn on the spectrophotometer and set it to 420 nm (Genesys20 should warm-up at least 10
minutes before using).
2. Prepare 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 to a test tube, mix and then place a portion in a cuvette.
3. Wipe the cuvette with a Kimwipe and place the “blank” cuvette into the spectrophotometer.
Make sure the triangle faces to the front of the holder and close the lid. Make sure that an
“A” is present on the screen indicating absorbance. If a T or C is present press the A/T/C button
until an A appears.
4. Press 0A/100%T button and wait for the blank to register “0.000”.
5. Set up eight clean test tubes. Label them 1-8.
6. Add 0.5 ml of ONPG to each, then add the following:
Cuvette
1
2
3
pH 7 buffer
4.5 ml
4.0 ml
3.5 ml
Cuvette
4
5
6
pH 7 buffer
3.0 ml
2.5 ml
2.0 ml
7. Add the following amounts of lactase to each test tube and time the reaction for two minutes:
Cuvette
1
2
3
Lactase
0.0 ml
0.5 ml
1.0 ml
Cuvette
4
5
6
Lactase
1.5 ml
2.0 ml
2.5 ml
8. 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.
9. Pour the solutions into the cuvettes. Place the cuvettes one at a time into the
spectrophotometer (remembering to wipe them with a Kimwipe and make sure the triangle faces
to the front of the holder). Read and record the absorbance. (Note: the greater the absorbance, the
greater the amount of product that was formed.) Record your results in data table A. Rinse the
cuvette with DI water between measurements. After every couple of measurements, re-verify
your blank still reads zero or very close to it.
10. Rinse cuvettes well with deionized water.
B. The effect of substrate concentration –
1. Use your blank from part A to re-zero the absorbance of the spectrophotometer.
2. Set up eight clean cuvettes and add the following:
Cuvette
1
2
3
pH 7 buffer
5.0 ml
4.5 ml
4.0 ml
ONPG
0.0 ml
0.5 ml
1.0 ml
Cuvette
4
5
6
pH 7 buffer
3.5 ml
3.0 ml
2.5 ml
ONPG
1.5 ml
2.0 ml
2.5 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. Place the solution into the cuvette one at a time and measure with the spectrophotometer. Read
and record the absorbance. Record your results in data table B.
6. Rinse cuvettes well with deionized water.
C. 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 in a cuvette.
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:
Cuvette
1
2
3
4
5
6
4.5 ml
pH 2 buffer
pH 4 buffer
pH 6 buffer
pH 8 buffer
pH 10 buffer
pH 12 buffer
0.5 ml
ONPG
ONPG
ONPG
ONPG
ONPG
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. Place the solution into cuvettes and measure with the spectrophotometer. Read and record the
absorbance. Record your results in data table C.
9. Rinse cuvettes well with deionized water.
D. The effect of temperature on enzyme activity
1. Use the blank from part C to re-zero 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 tubes into the racks and into the following:
Test tube
1
2
3
Water bath temp
0oC (ice bath)
23oC (room temp)
37oC
Test tube
4
5
Water bath Temp
60oC
90oC
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. Pour the solutions into the cuvettes one at a time and measure with the spectrophotometer.
Read and record the absorbance. Record your results in data table D.
8. Rinse the cuvettes well with deionized water.
A. Effect of enzyme concentration
Tube # Concentration Absorbance
1
0.0 ml lactase
2
0.5 ml lactase
3
1.0 ml lactase
4
1.5 ml lactase
5
2.0 ml lactase
6
2.5 ml lactase
B. Effects of substrate concentration
Tube # Concentration Absorbance
1
0.0 ml ONPG
2
0.5 ml ONPG
3
1.0 ml ONPG
4
1.5 ml ONPG
5
2.0 ml ONPG
6
2.5 ml ONPG
C. Effect of pH
Tube #
pH
1
pH 2
2
pH 4
3
pH 6
4
pH 8
5
pH 10
6
pH 12
Absorbance
D. Effect of temperature
Tube #
1
Temperature
0oC
2
23oC
3
37oC
4
60oC
5
90oC
Absorbance
Homework:
Submit a hypothesis and prediction, properly labeled graph and an interpretation for each
of the 4 experiments. In addition, answer the following questions.
Questions:
1. What happens to the reaction as you increase the amount of enzyme?
2. What happens to the reaction as you increase the amount of substrate?
3. At which pH(s) did the enzyme 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?