BIOL 207: Lab 4
BIOLOGY 207 LABORATORY 4
MEASUREMENT OF ENZYME
ACTIVITY
OBJECTIVES
1. Define enzymes and explain how they act as catalysts.
2. Explain the Lock and Key model to describe enzyme specificity and the mechanism of action.
3. Examine the differences between chemical catalysts and biological catalysts.
4. Examine enzyme activity by determining the disappearance of substrate or appearance of
product. Establish which of these approaches is likely to be most convenient during experiments.
5. Examine the effects of time of incubation (time of exposure of substrate to enzyme) on
enzymatic reactions.
6. Examine the effects of enzyme concentration on the rate of a reaction.
7. Examine the effects of varying pH and temperature on enzyme activity.
8. Define the units of enzyme activity and the international unit.
GENERAL STATEMENT REGARDING SUBJECT MATTER
At any given time, thousands of chemical reactions are occurring in your body. These reactions are
catalyzed (reactions are speeded up or the rates increased) by specialized protein molecules called enzymes.
The enzymes exhibit great specificity, thus each reaction is catalyzed by a different enzyme. This enzyme
and substrate (molecule that is changed by the enzyme) specificity is frequently compared to the specificity
of locks for particular keys. Different enzymes can be detected by using their specific substrates and the
amount of enzyme activity can be determined by measuring the amount of substrate that disappears with
time, or the amount of product formed. Each enzyme has a pH and temperature optimum at which it
exhibits maximum activity. The temperature optimum for human enzymes is body temperature (37ºC).
As incubation temperature decreases below 37ºC, the enzyme activity will also decrease. If the temperature
increases far above 37ºC, the enzyme will denature, losing its three-dimensional shape and its activity.
Most often, the pH optimum of the enzyme is similar to the pH of the cellular or bodily compartment in
which the enzyme is found. Enzyme activity is also influenced by substrate concentration and the presence
of activators, inhibitors, cofactors and coenzymes. Saturation occurs at high substrate concentrations when
the active sites of all of the enzymes are occupied by substrate, and thus the enzyme is working at its
maximum rate. At this point, to increase the rate of product formation, the cell would need to produce more
of that particular enzyme by gene expression. In this laboratory, we will be investigating fundamental
properties of an enzyme. You should consider the experiments we do and the results obtained as a model for
most enzyme activities. When doing these assays, it is important to measure materials and time accurately in
order to obtain good results. Most often, when these assays don’t work properly, it is due to sloppy lab
procedures used by the students.
HAZARDS
Iodine is toxic and an irritant, and will stain clothing. You must handle the acids with care (Goggles are
required) and avoid contact of acids and bases (again, Goggles are required) with your skin or eyes. Your lab
instructor may suggest that he/she be the one to add acid or base to your test tubes.
Ex. 4.1: Ferric Oxide and Liver Catalase as Catalysts DEMO
Hydrogen peroxide is a strong oxidizing agent that is produced in many of our tissues as a result of some
biochemical reactions. Because it has adverse effects on our body cells, its conversion into water and
4.1
, BIOL 207: Lab 4
oxygen gas is constantly catalyzed by an enzyme called catalase that is also present in our tissues. This is a
rapidly acting enzyme and its activity can be demonstrated very easily. The breakdown of hydrogen
peroxide into water and oxygen gas is also catalyzed by chemical catalysts, such as ferric oxide present on
rusty nails. In both cases, the overall biochemical reaction is the same.
This exercise will be carried out as a demonstration. The instructor will pour about 50 ml of hydrogen
peroxide into two separate 100 ml beakers labeled beaker-1 and beaker-2. A rusty nail will be dropped
into beaker-1 and about a quarter teaspoon of minced chicken liver will be added to beaker-2. Observe the
demonstration and answer the questions on p. 4.4.
Ex. 4.2: Digestion of Starch by Amylase
Starch is an important nutrient. Chemically, it is a polysaccharide consisting of repeating units of glucose
molecules joined together by covalent bonds (see page 27 in textbook). Starch is digested (broken down) in
our digestive system into disaccharide (two glucose molecules joined together by covalent bond) molecules
called maltose by the action of an enzyme named amylase. This enzyme is present in our saliva and also in
pancreatic secretions. Salivary amylase has a pH optimum of 7.00 and temperature optimum of 370C. In
this exercise we will study amylase activity by adding this enzyme to starch solutions and examining both
the disappearance of starch (substrate) and the appearance of maltose (product). The effects of the four
parameters (incubation time, enzyme concentration, pH and temperature) will be examined.
Each group of students will prepare a set of eight tubes. Mark them 1-8 and identify them. Then, add the
following reagents in the order from left to right. The enzyme amylase MUST be added LAST.
Tube number Starch solution Water Other reagents Amylase
1 3.00 ml 3.00 ml none 0.00
2 3.00 ml 2.5 ml none 0.5 ml
3 3.00 ml 0.00 ml none 3.00 ml
4 3.00 ml 0.00 ml none 3.00 ml added
after 50 minutes
5 3.00 ml 0.00 ml none 3.00 ml
6 3.00 ml 0.00 ml 4 drops of 2.0 M 3.00 ml
HCl
7 (incubate on ice) 3.00 ml 0.00 ml None 3.00 ml
8 3.00 ml 0.00 ml None 3.00 ml of
boiled amylase
4.2
BIOLOGY 207 LABORATORY 4
MEASUREMENT OF ENZYME
ACTIVITY
OBJECTIVES
1. Define enzymes and explain how they act as catalysts.
2. Explain the Lock and Key model to describe enzyme specificity and the mechanism of action.
3. Examine the differences between chemical catalysts and biological catalysts.
4. Examine enzyme activity by determining the disappearance of substrate or appearance of
product. Establish which of these approaches is likely to be most convenient during experiments.
5. Examine the effects of time of incubation (time of exposure of substrate to enzyme) on
enzymatic reactions.
6. Examine the effects of enzyme concentration on the rate of a reaction.
7. Examine the effects of varying pH and temperature on enzyme activity.
8. Define the units of enzyme activity and the international unit.
GENERAL STATEMENT REGARDING SUBJECT MATTER
At any given time, thousands of chemical reactions are occurring in your body. These reactions are
catalyzed (reactions are speeded up or the rates increased) by specialized protein molecules called enzymes.
The enzymes exhibit great specificity, thus each reaction is catalyzed by a different enzyme. This enzyme
and substrate (molecule that is changed by the enzyme) specificity is frequently compared to the specificity
of locks for particular keys. Different enzymes can be detected by using their specific substrates and the
amount of enzyme activity can be determined by measuring the amount of substrate that disappears with
time, or the amount of product formed. Each enzyme has a pH and temperature optimum at which it
exhibits maximum activity. The temperature optimum for human enzymes is body temperature (37ºC).
As incubation temperature decreases below 37ºC, the enzyme activity will also decrease. If the temperature
increases far above 37ºC, the enzyme will denature, losing its three-dimensional shape and its activity.
Most often, the pH optimum of the enzyme is similar to the pH of the cellular or bodily compartment in
which the enzyme is found. Enzyme activity is also influenced by substrate concentration and the presence
of activators, inhibitors, cofactors and coenzymes. Saturation occurs at high substrate concentrations when
the active sites of all of the enzymes are occupied by substrate, and thus the enzyme is working at its
maximum rate. At this point, to increase the rate of product formation, the cell would need to produce more
of that particular enzyme by gene expression. In this laboratory, we will be investigating fundamental
properties of an enzyme. You should consider the experiments we do and the results obtained as a model for
most enzyme activities. When doing these assays, it is important to measure materials and time accurately in
order to obtain good results. Most often, when these assays don’t work properly, it is due to sloppy lab
procedures used by the students.
HAZARDS
Iodine is toxic and an irritant, and will stain clothing. You must handle the acids with care (Goggles are
required) and avoid contact of acids and bases (again, Goggles are required) with your skin or eyes. Your lab
instructor may suggest that he/she be the one to add acid or base to your test tubes.
Ex. 4.1: Ferric Oxide and Liver Catalase as Catalysts DEMO
Hydrogen peroxide is a strong oxidizing agent that is produced in many of our tissues as a result of some
biochemical reactions. Because it has adverse effects on our body cells, its conversion into water and
4.1
, BIOL 207: Lab 4
oxygen gas is constantly catalyzed by an enzyme called catalase that is also present in our tissues. This is a
rapidly acting enzyme and its activity can be demonstrated very easily. The breakdown of hydrogen
peroxide into water and oxygen gas is also catalyzed by chemical catalysts, such as ferric oxide present on
rusty nails. In both cases, the overall biochemical reaction is the same.
This exercise will be carried out as a demonstration. The instructor will pour about 50 ml of hydrogen
peroxide into two separate 100 ml beakers labeled beaker-1 and beaker-2. A rusty nail will be dropped
into beaker-1 and about a quarter teaspoon of minced chicken liver will be added to beaker-2. Observe the
demonstration and answer the questions on p. 4.4.
Ex. 4.2: Digestion of Starch by Amylase
Starch is an important nutrient. Chemically, it is a polysaccharide consisting of repeating units of glucose
molecules joined together by covalent bonds (see page 27 in textbook). Starch is digested (broken down) in
our digestive system into disaccharide (two glucose molecules joined together by covalent bond) molecules
called maltose by the action of an enzyme named amylase. This enzyme is present in our saliva and also in
pancreatic secretions. Salivary amylase has a pH optimum of 7.00 and temperature optimum of 370C. In
this exercise we will study amylase activity by adding this enzyme to starch solutions and examining both
the disappearance of starch (substrate) and the appearance of maltose (product). The effects of the four
parameters (incubation time, enzyme concentration, pH and temperature) will be examined.
Each group of students will prepare a set of eight tubes. Mark them 1-8 and identify them. Then, add the
following reagents in the order from left to right. The enzyme amylase MUST be added LAST.
Tube number Starch solution Water Other reagents Amylase
1 3.00 ml 3.00 ml none 0.00
2 3.00 ml 2.5 ml none 0.5 ml
3 3.00 ml 0.00 ml none 3.00 ml
4 3.00 ml 0.00 ml none 3.00 ml added
after 50 minutes
5 3.00 ml 0.00 ml none 3.00 ml
6 3.00 ml 0.00 ml 4 drops of 2.0 M 3.00 ml
HCl
7 (incubate on ice) 3.00 ml 0.00 ml None 3.00 ml
8 3.00 ml 0.00 ml None 3.00 ml of
boiled amylase
4.2
