Factors Affecting Enzyme Activity: Lactose Intolerance

Goals
• To learn how enzyme concentration, pH, and substrate concentration affect enzyme activity
• To make measurements using a spectrophotometer
• To graph data to visually show the relationship between enzyme activity and enzyme
concentration, pH, and substrate concentration

Introduction
Lactose is the principal carbohydrate in milk. This accounts for its common name, milk sugar.
Lactose makes up 6–8% of human milk, almost double the 4–5% lactose found in cow’s milk.
Lactose is a disaccharide made up of a b-d-galactose unit and an a-d-glucose unit joined by a
b(1,4) glycosidic linkage. Lactose can by hydrolyzed by acid or by the enzyme lactase, forming an
equimolar mixture of galactose and glucose.
CH2OH
O CH2OH CH2OH
CH2OH OH HO O OH O
HO O O H+ or Lactase
OH OH + OH
OH OH H2O
HO OH
OH OH
OH
lactose galactose glucose

Many people have trouble digesting and absorbing lactose. This problem, called lactose
intolerance, is a condition in which people lack the enzyme lactase. Deficiency of lactase can be
caused by a genetic defect, by physiological decline with age, or by injuries to the mucosa lining
the intestines. When lactose molecules remain in the intestine undigested, they attract water
molecules to themselves, causing fullness, discomfort, cramping, nausea, and diarrhea. Bacterial
fermentation of the lactose further along the intestinal tract produces lactic acid and gas, adding
to the discomfort.
The level of the enzyme lactase in humans varies with age. Most children have sufficient lactase
during the early years of life when milk is a much-needed source of calcium in their diet. In
adulthood, the enzyme level decreases, and lactose intolerance develops. Some researchers
estimate that as many as one in three adult Americans exhibits a degree of lactose intolerance.
The level of the enzyme lactase in humans
varies widely among ethnic groups, 80% Asian Americans 60% Inuits
indicating that the trait is genetically 80% Native Americans 50% Hispanics
determined (inherited). The occurrence 75% African Americans 20% Caucasians
of lactose intolerance is lowest among 70% Mediterranean peoples 10% Northern Europeans
Scandinavians and other northern
Europeans and highest among native North
Americans, Southeast Asians, Africans, and Mediterranean people.
Some people who enjoy dairy products but also suffer from lactose intolerance find that the
painful symptoms can be relieved by taking tablets containing lactase, such as Lactaid®.
 Factors Affecting Enzyme Activity
Background to the Procedure
In this experiment, a substitute for lactose will be used, called o-nitrophenyl-b-d-
galactopyranoside (ONPG). ONPG is hydrolyzed by lactase to form the yellow-colored
o-nitrophenolate ion. The rate of the reaction can be studied by measuring the intensity of the
yellow color using a spectrophotometer.

O2N

CH2OH CH2OH O2N

HO O O HO O OH
Lactase
OH OH + H+ + O
H2O

OH OH

ONPG galactose o-nitrophenolate ion

(yellow)

If a liquid is colored, it is because some component of the liquid absorbs visible light. In a solution
the greater the concentration of the light-absorbing substance, the more light absorbed, and the
more intense the color. The quantity of light absorbed can be measured by a spectrophotometer.
The next page will give you more information about the spectrophotometer.
Without going into the derivation of the equations, the concentration of the light-absorbing
substance is directly proportional to the absorbance (one of the readings the spectrophotometer
gives). Therefore, the greater the reaction rate (enzyme activity), the more yellow-colored
o-nitrophenolate ion is produced. The more yellow-colored o-nitrophenolate ion produced, the
deeper the color and the greater the absorbance. Thus,

Absorbance ∝ enzyme activity

In this experiment, you will plot the absorbance on the y-axis and one of the factors affecting
enzyme activity (enzyme concentration, pH, or substrate concentration) on the
x-axis. The expected graphs of enzyme activity versus the various factors are shown below:
Enzyme Activity

Enzyme Activity

Enzyme Activity

Enzyme Concentration pH Substrate Concentration


 Factors Affecting Enzyme Activity
How the Spectrophotometer Works
The spectrophotometer has a light source that emits all wavelengths of light in the visible region
(wavelengths of ~400 to 700 nm); a monochromator, which filters out all the light except a given
wavelength; a sample holder for the solution being measured; and a detector.
A given compound will not absorb all wavelengths equally—that’s why things are different colors.
In a solution the greater the concentration of the light-absorbing substance, the more light
absorbed, and the more intense the color.
When using a spectrophotometer, you put the sample in a sample holder called a cuvette and
place it in the spectrophotometer. Cuvettes come in many shapes and sizes, but the two most
common shapes are round (looking like a test tube) and rectangular. Light of a particular
wavelength passes through the solution inside the cuvette and the amount of light passing
through the solution (transmittance) or absorbed (absorbance) by the solution is measured by
the detector. Because other compounds in a solution (or the solvent itself) may absorb the same
wavelengths as the compound being analyzed, we compare the absorbance of our test solution
to a reference blank. Ideally, the reference blank should contain everything found in the sample
solution except the substance you are trying to analyze or measure. For instance, in today’s lab the
blank will contain the buffer, the substrate, and water (everything except the enzyme).

How to Calibrate the Spectrophotometer

The spectrophotometer should already be on. If it’s not, turn it on and let it warm up for
15 minutes.
Before you use the spectrophotometer to make readings, you must calibrate it. Older style
spectrophotomers require a multistep process to calibrate the machine. First, set the wavelength
to read 420 nm. Next, make sure the spectrophotometer is reading transmittance (T). Turn the
0%T knob until the spectrophotometer reads 0.
Next, insert your reference blank into the sample holder. Turn the 100%T knob until the
spectrophotometer reads 100.
The machine is now calibrated. Finally, change the spectrophotometer from reading transmittance
to absorbance and you are ready to take readings of your sample.
There are two YouTube videos that show this process. Watch all of part 1 and the first two minutes
of part 2. The spectrophotometer you will use may not look exactly like the one in the video;
buttons or dials may be in different places, but the concept is the same. Your instructor will help
you if you have trouble calibrating the machine.

Part 1: http://www.youtube.com/watch?v=jmZomizSPxw
Part 2: http://www.youtube.com/watch?v=fY7Sy4OQ7ps

It’s much easier to calibrate modern spectrophotomers. Set the wavelength to read 420 nm, insert
the reference blank into the sample holder, press the calibrate button and you’re done.


 Factors Affecting Enzyme Activity
The spectrophotometer is a sensitive machine. There are several reasons why your results may not
be accurate:
• Air bubbles in the cuvette. You should always make sure that there are no air bubbles in the
cuvette.
• Solid particles or contaminants in the sample. Contaminants in the sample will scatter
light and result in absorbance readings that are significantly higher than the true reading.
Contaminants may absorb light and result in higher absorbance readings too.
• Incomplete mixing of samples can yield improper results. Although this seems like a no-
brainer, you must thoroughly mix your solution (three times by inversion) before placing the
sample in the sample holder.
• Dirty cuvettes. The cuvettes should be clean on both the outside (clean off fingerprints) and
the inside.
• Residual liquid in the cuvette before addition of the sample. This will cause either a) dilution
of the sample, resulting in an artificially low reading; or b) contamination of the sample with a
solution containing a “high absorbing” compound, resulting in an artificially high reading.

Experimental Procedures

Wear your safety goggles and gloves!

The material safety data sheet for o-nitrophenolate indicates that the yellow product of the
experiment is slightly toxic through skin absorption. Therefore, each student is required to wear
protective gloves during the experiment.
The following procedures assume you are using the round cuvettes (the ones that look like test
tubes). If you have rectangular cuvettes, you must mix your solutions in test tubes first and then
transfer as much as will fit into the rectangular cuvette.
Also, the solutions are air-sensitive. Keep your solutions on ice until you are ready to use them in
the spectrophotometer. Read all procedures before starting the experiment.
A. Effect of Enzyme Concentration
Materials: 6 cuvettes, about 4 mL of the substrate solution (ONPG), about 6 mL of the enzyme
solution (lactase), about 20 mL of pH 7 buffer solution, disposable pipettes (5 mL
and 1 mL), ice, test tubes
A.1 To make your reference blank, place 3 mL of the pH 7 buffer in a cuvette using a 5 mL
disposable pipette. Add 0.5 mL of the substrate (ONPG) to the cuvette using a 1 mL
disposable pipette. Add 0.5 mL of distilled water to the cuvette. Mix by inversion three
times.
A.2 Use the reference blank to calibrate the spectrophotometer (reread the section on How to
Calibrate the Spectrophotometer. Once calibrated, don’t turn the dials or you will lose your
calibration.


 Factors Affecting Enzyme Activity
A.3 Your next 5 cuvettes will contain different enzyme concentrations, but each contains the
same amount of buffer and substrate. Place 3 mL of the pH 7 buffer in each of the five
cuvettes using a 5 mL disposable pipette. Add 0.5 mL of the substrate (ONPG) to each of
the cuvettes using a 1 mL disposable pipette.

A summary of the dilution process is shown on the following page.

A.4 Cuvette #1 will contain the most dilute enzyme solution. In a separate test tube, add 1 mL
of the stock lactase solution and 2 mL of distilled water. Mix by inversion three times.
Adding the enzyme solution:
Add 0.5 mL of this diluted enzyme solution to one of the cuvettes with the buffer and
substrate and quickly mix by inversion three times. Start timing the solution. Wipe off any
fingerprints or spills on the outside of the cuvette and place it in the spectrophotometer.
Make sure the spectrophotometer is set to read absorbance. At exactly 2 minutes, record the
absorbance reading of this solution.
A.5 Cuvette #2 will contain the next dilute enzyme solution. Use the following dilution. In a
separate test tube, add 1 mL of the stock lactase solution and 1.5 mL of distilled water. Mix
by inversion three times. Follow the Adding the enzyme solution procedure.
A.6 Cuvette #3 will contain the next dilute enzyme solution. Use the following dilution. In a
separate test tube, add 1 mL of the stock lactase solution and 1 mL of distilled water. Mix by
inversion three times. Follow the Adding the enzyme solution procedure.
A.7 Cuvette #4 will contain the next dilute enzyme solution. Use the following dilution. In a
separate test tube, add 1 mL of the stock lactase solution and 0.5 mL of distilled water. Mix
by inversion three times. Follow the Adding the enzyme solution procedure.
A.8 Cuvette #5 will contain the most concentrated enzyme solution. No dilution is necessary.
Add 0.5 mL of the stock lactase solution to the last cuvette with the buffer and substrate
and quickly mix by inversion three times. Start timing the solution. Make the absorbance
measurement as before.
A.9 Make a graph of absorbance on the y-axis versus relative enzyme concentration on
the x‑axis. Using a ruler, draw a straight line through your data points. (Optional): use
Microsoft Excel to graph the data. A template will be provided.


 Dilution No Dilution

Start with stock lactase solution

1.0 mL 1.0 mL
1.0 mL Stock 1.0 mL Stock
lactase lactase
solution solution


2.0 mL 1.5 mL 1.0 mL 0.5 mL
H2O H2O H2O H2O

1 1 1 1
Dilution factor = 0. = 0.40 = 0.50 = 0.67
1+ 2 1 +1.5 1 +1 1 + 0.5

0.5 mL 0.5 mL 0.5 mL 0.5 mL 0.5 mL

Cuvette Cuvette Cuvette Cuvette Cuvette

#1 #2 # #4 #5
Factors Affecting Enzyme Activity
 Factors Affecting Enzyme Activity
B. Effect of pH
Materials: 10 cuvettes, about 6 mL of the substrate solution (ONPG), about 3 mL of the
enzyme solution (lactase), Buffer solutions: pH 4, pH 6, pH 7, pH 8, and either pH 9
or 10, disposable pipettes (5 mL and 1 mL), ice, test tubes
B.1 You must have a reference blank for each pH buffer. Start with the pH 4 buffer. To make
your reference blank, place 3 mL of the buffer solution in a cuvette using a 5 mL disposable
pipette. Add 0.5 mL of the substrate (ONPG) to the cuvette using a 1 mL disposable pipette.
Add 0.5 mL of distilled water to the cuvette. Mix by inversion three times.
B.2 Use the first reference blank to calibrate the spectrophotometer (reread the section on How
to Calibrate the Spectrophotometer. Once calibrated, don’t turn the dials or you will lose
your calibration.
B.3 Make the sample solution at the same pH that you calibrated the spectrophotometer. Place
3 mL of the buffer in an empty cuvette. Add 0.5 mL of the substrate (ONPG) to a cuvette
using a 1 mL disposable pipette.
Adding the enzyme solution:
Add 0.5 mL of the stock lactase solution to the cuvette with the buffer and substrate and
quickly mix by inversion three times. Start timing the solution. Wipe off any fingerprints
or spills on the outside of the cuvette and place it in the spectrophotometer. Make sure the
spectrophotometer is set to read absorbance. At exactly 2 minutes, record the absorbance
reading of this solution.
B.4 Make a new reference blank with the next pH buffer using the procedure in B.1.
Recalibrate the spectrophotometer with the new reference blank. Again, once calibrated,
don’t turn the dials or you will lose your calibration.
B.5 Make a new sample solution with the new pH buffer, following the procedure in B.3. Follow
the Adding the enzyme solution procedure.
B.6 Repeat until you have a reading for each pH buffer.
B.7 Make a graph of absorbance on the y-axis versus pH on the x‑axis. Draw a smooth curve
through your data points. (Optional): use Microsoft Excel to graph the data. A template will
be provided.


 Factors Affecting Enzyme Activity
C. Effect of Substrate Concentration
Materials: 7 cuvettes, about 7 mL of the substrate solution (ONPG), about 4 mL of the enzyme
solution (lactase), about 20 mL of pH 7 buffer solution, disposable pipettes (5 mL
and 1 mL), ice, test tubes
C.1 To make your reference blank, place 3 mL of the pH 7 buffer in a cuvette using a 5 mL
disposable pipette. Add 0.5 mL of the enzyme (lactase) solution to the cuvette using a 1 mL
disposable pipette. Add 0.5 mL of distilled water to the cuvette. Mix by inversion three
times.
C.2 Use the reference blank to calibrate the spectrophotometer (reread the section on How to
Calibrate the Spectrophotometer. Once calibrated, don’t turn the dials or you will lose your
calibration.
C.3 Your next 4 cuvettes will contain different substrate concentrations, but each contains the
same amount of buffer and enzyme. Place 3 mL of the pH 7 buffer in each of four cuvettes
using a 5 mL disposable pipette. Add 0.5 mL of the enzyme (lactase) to each of the cuvettes
using a 1 mL disposable pipette.

A summary of the dilution process is shown on page 10.

C.4 Cuvette #1 will contain the most dilute substrate solution. In a separate test tube, add 1 mL
of the stock substrate (ONPG) solution and 2 mL of distilled water. Mix by inversion three
times.
Adding the substrate solution:
Add 0.5 mL of this diluted substrate solution to one of the cuvettes with the buffer and
enzyme and quickly mix by inversion three times. Start timing the solution. Wipe off any
fingerprints or spills on the outside of the cuvette and place it in the spectrophotometer.
Make sure the spectrophotometer is set to read absorbance. At exactly 2 minutes, record the
absorbance reading of this solution.
C.5 Cuvette #2 will contain the next dilute substrate solution. Use the following dilution. In a
separate test tube, add 1 mL of the stock ONPG solution and 1 mL of distilled water. Mix
by inversion three times. Follow the Adding the substrate solution procedure.
C.6 Cuvette #3 will contain the next dilute substrate solution. Use the following dilution. In a
separate test tube, add 1 mL of the stock ONPG solution and 0.5 mL of distilled water. Mix
by inversion three times. Follow the Adding the substrate solution procedure.
C.7 Cuvette #4 will contain the stock substrate solution; no dilution is necessary. Add 0.5 mL
of the stock ONPG solution to the cuvette with the buffer and enzyme and quickly mix by
inversion three times. Make the absorbance measurement as before.
C.8 Cuvettes #5, and #6 contain different amounts of buffer and substrate than your first four
cuvettes! Read carefully how to make these last two samples.

In cuvette #5, place 2.5 mL of the pH 7 buffer in the cuvette using a 5 mL disposable pipette.
Add 0.5 mL of the enzyme (lactase) to the cuvette using a 1 mL disposable pipette.

Add 1.0 mL of the stock ONPG solution to the cuvette with the buffer and enzyme and


 Factors Affecting Enzyme Activity
quickly mix by inversion three times. Start timing the solution. Make the absorbance
measurement as before.
C.9 In cuvette #6, place 2 mL of the pH 7 buffer in the cuvette using a 5 mL disposable pipette.
Add 0.5 mL of the enzyme (lactase) to the cuvette using a 1 mL disposable pipette.

Add 1.5 mL of the stock ONPG solution to the cuvette with the buffer and enzyme and
quickly mix by inversion three times. Start timing the solution. Make the absorbance
measurement as before.
C.10 Make a graph of absorbance on the y-axis versus relative substrate concentration on the
x‑axis. Compare your graph with the expected graph of enzyme activity versus substrate
concentration. Draw a smooth curve through your data points that mimics the expected
curve. (Optional): use Microsoft Excel to graph the data. A template will be provided.


 Dilution No Dilution

Start with stock ONPG solution

1.0 mL
1.0 mL Stock 1.0 mL Stock Stock Stock
ONPG ONPG ONPG ONPG
solution solution solution solution

10
2.0 mL 1.0 mL 0.5 mL
H2O H2O H2O

1 1 1
Dilution factor = 0. = 0.50 = 0.67
1+ 2 1 +1 1 + 0.5

0.5 mL 0.5 mL 0.5 mL 0.5 mL 1.0 mL 1.5 mL

Cuvette Cuvette Cuvette Cuvette Cuvette Cuvette

#1 #2 # #4 #5 #6
Factors Affecting Enzyme Activity
 Factors Affecting Enzyme Activity
Report Sheet

Date Name

Section Team

Instructor

Pre-Lab Study Questions

1. What is the function of enzymes in the body?

2. Into what category of biological molecules (carbohydrates, lipids, proteins, or nucleic acids) do
enzymes belong?

. What is meant by the activity of an enzyme?

4. How can the activity of an enzyme be measured?

5. List at least four factors that affect enzyme activity.

11
 Factors Affecting Enzyme Activity
A. Effect of Enzyme Concentration

Relative Enzyme
Cuvette # Absorbance
Concentration
Blank 0 0

1 0.33

2 0.40

3 0.50

4 0.67

5 1

Enzyme Activity vs. Enzyme Concentration

2.000

1.500
Absorbance

1.000

0.500

0.000
0.00 0.20 0.40 0.60 0.80 1.00
Relative Enzyme Concentration

Q.1 Explain why the enzyme activity increased when the enzyme concentration increased.

12
 Factors Affecting Enzyme Activity
B. Effect of pH

Cuvette # pH Absorbance
1

Enzyme Activity vs. pH

2.0

1.5
Absorbance

1.0

0.5

0.0
4 5 6 7 8 9 10
pH

Q.2 What was the optimum pH for lactase?

13
 Factors Affecting Enzyme Activity
Q.3 There are many digestive enzymes in the human body, each with optimum reaction
conditions (pH, temperature, etc.). Use your data to explain why lactase most likely is not
found in the stomach (very acidic, pH = 1–2) or in the large intestine (slightly basic,
pH = 8).

Q.4 Lactase is produced in the small intestine. Use your data to predict the general pH of the
small intestine.

14
 Factors Affecting Enzyme Activity
C. Effect of Substrate Concentration

Relative Substrate
Cuvette # Absorbance
Concentration
Blank 0 0

1 0.33

2 0.50

3 0.67

4 1

5 2

6 3

Enzyme Activity vs. Substrate Concentration

2.0

1.5
Absorbance

1.0

0.5

0.0
0.00 0.50 1.00 1.50 2.00 2.50 3.00
Relative Substrate Concentration

Q.5 What happens to the rate of an enzyme-catalyzed reaction as substrate concentration is

raised beyond the saturation point?

15