Lab 2

Macromolecules I : Carbohydrates and Enzymes

OBJECTIVES AND LAB ACTIVITIES

1. Learn about carbohydrates and their breakdown

2. Learn about enzyme activity and specificity with carbohydrates

Lab structure
Each student pair will test glucose and a second carbohydrate, as assigned by their TA. For all the
essays, one partner tests glucose and the other partner, tests the assigned second carbohydrate.
The results for all the carbohydrates provided will be shared and discussed with the whole lab
group at the end.

Safety Notes for Lab 2

This is the first lab in which you will be using a concentrated acid (sulfuric acid (H2SO4) and
alkali (sodium hydroxide (NaOH), which are corrosive. Hydrogen peroxide (H2O2) is an
irritant. Be cautious and keep the following safety guidelines in mind (e.g. wear gloves and
goggles).
• Do not use cracked or chipped glassware and dispose of it in the “sharps” container.
• To dispose of acids or alkalis, use the specially-prepared containers. Only after the tube
has been emptied should you rinse the tube out with tap water at the sink.
• Immediately advise your TA of any spill, incident or accident.
• Do not take more of a reagent than is necessary and return reagent bottles to the supply
bench after using them. If you sampled too much of one reagent, dispose of it in the
special waste container, never return it to the original bottle.
• Do not use the same pipette for different reagents – which would lead to cross
contamination of reagents and can be a hazard.
• Pipetting by mouth is absolutely forbidden (not just in this lab but in all other BIOL 112
labs).
• Consult the online MSDS for information about all the chemicals you are using

Introduction
Biological macromolecules fall into four main classes: carbohydrates, lipids, proteins and
nucleic acids. Each of these classes of macromolecules consists of a limited number of
repeating sub-units built up into long chains. The sub-units also exist as free molecules in cells
and are joined together to form a macromolecule. Each of these types of molecules play a crucial
role in any living organism, ensuring structural support, information transmission, energy storage
and metabolism (all of the different chemical reactions occurring in a living cell), and as such are
subjects in the science of biochemistry (the study of chemical composition of cells, and how
substances are synthesized and broken down). This week we will focus on carbohydrates and
enzymes (proteins).

1) Carbohydrates are organic compounds of carbon, hydrogen and oxygen derived in a series
of reactions during photosynthesis from CO2 and H2O. Sugar, starch and cellulose are common
examples of carbohydrates important to life.
2) Lipids are composed mainly of carbon, hydrogen and oxygen. They differ from carbohydrates
because their structure makes them insoluble in water and other polar solvents. Oils,
cholesterols and hormones are often composed mainly of lipids.
3) Proteins are macromolecules composed of chains of amino acids. These amino acids contain
carbon, hydrogen, oxygen and nitrogen. Proteins include enzymes, also called enzymatic
proteins, which are the main catalyst in cells to facilitate and accelerate reactions. Other proteins,
called structural proteins, provide structural support and many other roles.
4) Nucleic acids are chains of nucleotides. Nucleotides themselves are composed of three
components which includes a purine or pyrimidine base (sometimes termed nitrogenous base),
a pentose sugar, and a phosphate group. Nucleic acids will be covered in lab 9.

Carbohydrates
Carbohydrates are divided into three classes:
• Monosaccharides, which are simple sugars containing 3 to 7 carbon atoms. Glucose, the
most abundant monosaccharide, contains 6 carbon atoms.
• Disaccharides, which consist of 2 single sugar molecules linked together (cane sugar or
sucrose is made up of glucose linked to fructose).
• Polysaccharides, which consist of three or more sugar molecules linked together. Three
notable polysaccharides are cellulose and starch. Cellulose is the principal constituent of
wood (and paper made from wood pulp). Chitin, with a structure very similar to cellulose, is
the chief constituent of arthropods’ (insects and crustaceans) exoskeletons, among other
things. We do not study chitin in this lab as it is a common severe allergen. Starch is the
chief constituent of the grains and other seeds as energy reserve, while glycogen is a polymer
playing a similar role in animals.

Proteins
There are 2 main types of proteins, structural proteins and enzymatic proteins, also called
enzymes. Almost every chemical reaction that occurs in living cells is catalyzed by an enzyme.
Enzymes are catalysts, that means they are substances that influence the rate of a reaction
and can be recovered unchanged at the end of the reaction.

The substance upon which an enzyme acts as a catalyst is called the substrate. To be effective,
an enzyme must come into contact with its own particular substrate. Enzymes and their
substrates are specific to each other. The enzyme has an active site that is arranged
geometrically in such a way as to recognize and bind only specific types of molecules. It is this
active site that gives the properties of specificity and catalysis to an enzyme. Changing the
structure of an enzyme will cause it to lose its catalytic properties. Boiling for example coagulates
proteins, and therefore alters their structure.

2
Investigating the breakdown of carbohydrates
Objective
Learn about the breakdown of carbohydrates

Benedict’s test to detect reducing sugar

Benedict’s test is performed to detect the presence of a reducing sugar. The term reduction is
applied to a chemical reaction involving a gain of electrons, and substances which have the
characteristic of giving up electrons to other substances are called reducing agents.

All monosaccharides (e.g. glucose, Fig. 1), with either an aldehyde or ketone functional group,
are reducing sugars. Sucrose is a disaccharide made up of one glucose and one fructose
molecule joined together (Fig. 3). Do you expect it to be a reducing sugar?

Figure 1. Molecular structure of glucose (C6H12O6). Figure 2. Molecular structure of fructose (C6H12O6).

α-Glucose β-Fructose Sucrose: the carbon 1 of glucose is joined by an α-1,2

linkage to the carbon 2 of fructose
Figure 3. Molecular structure of sucrose.

Iodine to detect starch

Starch and glycogen are polysaccharides made of many glucose molecules attached to each
other through α 1,4 links (Fig. 4). Cellulose (made of glucose units) and chitin (made of N-acetyl-
glucosamine derived from glucose) are also polysaccharides, but they are linked through β 1,4
bonds (Figs. 5 and 6). Because the bonds between the glucose units are geometrically different,
many biological differences exist between these molecules. Furthermore, the specific geometry
of the link in starch and glycogen causes a reaction in the presence of iodine, not found in other
polysaccharides like chitin and cellulose.
Polysaccharides are chains of various monosaccharides linked together repeatedly.
Depending on the links between monomers the structure of the polysaccharides is either strong
or weak. That is why starch and glucose have different properties even though they are both
made up of glucose alone. Cellulose is the main component in plant cell walls and therefore one
of the most abundant organic molecules on earth.
Chitin is made up of a glucose derivate and it is also quite abundant in nature. It is present in
the cuticles of insects and various crustaceans and in the cell wall of mushrooms.
Figure 4. Molecular structure of starch (C6H12O6)n. The repeating unit, glucose (C6H12O6), is between brackets.
Note the difference in bonds linking the glucose molecules relative to cellulose (C6H12O6)n.

Figure 5. Molecular structure of cellulose (C6H12O6)n. Figure 6. Molecular structure of chitin (C6H12O6)n,
The repeating unit, glucose, is between brackets. showing two of the repeating N-acetyl-glucosamine
units in the bracket
.

The break down of carbohydrates by acid hydrolysis

With the help of heat and acid, carbohydrates can be broken down into their monomers.

Material

• washed tubes / colored caps • Benedict’s solution

• test tube holders tongues • 1, 6 % iodine solution
• test tube racks • dry blocks at 100°C
• 1% glucose solution • Sulfuric acid solution (H2SO4) - handled
• 1% fructose solution by TAs only
• 1% sucrose solution • Water
• 1% maltose solution • Sodium hydroxide solution (NaOH)
• 1% lactose solution • Glucose-testing strips
• 1% starch solution • Litmus paper strips, pH standard
• 1% cellulose solution solutions pH 4, 7 and 10 and
chart
• transfer pipets
• graduated pipets
• syringes

4
Methods

Each student will label test tubes from 1 to 4 and perform the treatment indicated in table 1
(iodine or Benedict’s). Label all tubes with the number before filling them with the solutions.
Make sure you do not cross-contaminate the reagents, when measuring and adding them.

1) Measure about 1 mL of one of glucose or your assigned carbohydrate in tubes #1 and #2.
2) Have your TA add the sulfuric acid
3) Add the water and incubate at 100 C for 15 minutes.
4) Remove from dry block and add 1 mL of Benedict's solution and cap the tube.
5) Place the tube in a dry block at 100°C
6) Watch it for about 5 minutes.

A reducing sugar causes a colored precipitate to form which changes in color from green
(presence of reducing sugar) to yellow, orange and finally to brick red (high presence of reducing
sugar).

Observe & record your results to be shared with the other student pairs. Keep all your reaction
mixtures, in order to show them to the other students. Those are qualitative, not quantitative
experiments.

Detect the presence of glucose with the testing strips

You can also test each one of your reaction mixtures with a glucose-testing strip.
Record your results in table 1.

Iodine test for starch

7) Measure about 1 ml of glucose and your assigned carbohydrate in tubes #3 and #4.
8) Add a drop or two of iodine solution.
9) Note any color change.

A blue-black or purplish color shows the presence of starch. Keep your reaction mixtures in order
to show them to the other students. Record your results in table 1.

Also test a cotton ball by placing 1-2 drops of iodine directly on it.
The break down of carbohydrates by enzymes

Enzyme specificity: Lactase and Amylase

Lactase hydrolyzes its disaccharide substrate, lactose (Fig. 7), into glucose and galactose
monomers.
C12H22O11 + H2O → C6H12O6 + C6H12O6 + heat
Lactose intolerance (hypolactasia) is caused by the lack of lactase, and results in the inability
to digest lactose. It can be managed by avoiding/minimizing dairy products, re-habituation to
dairy products, or lactase supplementation. The “lactose-free” dairy products are not actually
lactose- free, they are rather added with the enzyme lactase, so the lactose present is broken
down.

Figure7. Chemical structures of glucose, and the disaccharides lactose, maltose, and sucrose.

Amylase hydrolyzes starch (Fig. 4), into glucose monomers.

(C6H10O6-)n + n (H2O) → n (C6H12O6) + 1/2 O2n + heat

Objective
Demonstrate the specificity of enzymes
Again here, one partner tests glucose and the other partner, tests second carbohydrate, as
assigned by TA.

Materials
• washed test tubes /colored caps • amylase solution
• test tube racks • lactase solution
• 1% glucose solution • glucose-testing strips
• 1,6% iodine solution
• 1% fructose solution
• cotton balls
• 1% sucrose solution • dry blocks at 37C
• 1% maltose solution • transfer pipets
• 1% lactose solution • graduated pipets
• 1% starch solution • syringes
• 1% cellulose solution

6
Methods

Each student needs 2 tubes, one for amylase and the other for lactase. Each partner tests the
same carbohydrate as before (glucose or your assigned, other carbohydrate).
Please refer to Table 2.

1. Label each tube with the enzyme used, glucose or your assigned, second carbohydrate.
2. Using a transfer pipette, add 1 mL of glucose or your assigned carbohydrate to both tubes
3. Add 5 mL of amylase to one tube and incubate at 37 C for 15 minutes.
4. Add 5 mL to lactase to the other tube and let sit at room temperature for a few
minutes.

After the incubation time, use the glucose testing strips first and only after, add 1-2 drops of the
iodine solution to determine the presence of glucose and starch, respectively.

Catalase Activity: TA demonstration only

Please refer to Table 3.
Catalase is responsible for the breakdown of hydrogen peroxide to water and oxygen, the latter
of which will manifest as bubbles when it saturates the water. Although hydrogen peroxide is
naturally formed in living organisms, it is very harmful and has to be broken down by enzymes
such as catalase.
2H2O2 →2H20 + O2
Objectives
• Demonstrate the presence and activity of an enzyme in live tissue
• Observe enzyme specificity in living tissue
• Consider how boiling (canned pear) affects enzyme activity

Materials
• washed test tubes / colored caps • knife and cutting board
• test tube rack • water
• cubes of fresh pear (+/- 1 cm)
• 3% hydrogen peroxide (H 2O2)
• cubes of canned pear (+/- 1 cm)
• transfer pipets
• graduated pipets
• syringes

Methods
1. Obtain tubes from the central supply bench and label them as A, B, and C.
2. Add 5 mL of H2O2 to tubes A and B, and 5 mL of water to tube C (control).
3. Using a clean knife, cut two 1cm cubes of fresh pear and one 1 cm cube of canned pear.
4. Add the fresh pear cubes to tubes A and C, and the canned pear cube to tube B.
5. Observe the presence or absence of bubbles (oxygen gas released). It is important to
carefully monitor the tubes for the presence of bubbles. The pears should be added only when
one is ready to observe the results right away.
CLEAN UP NOTES

1. For all the Biol 112 labs, nothing is to be discarded in the large MIMM hazardous
waste cardboard box or hazardous liquid waste containers. The BIOL and MIMM
labs handle different chemicals and manage their respective hazardous wastes
separately as they could be incompatible, possibly creating reaction mixtures.
2. Only use the solid and liquid hazardous waste containers labeled “BIOL 111-
112” for our hazardous wastes, as instructed by your TA.

3. The nitril gloves go to our small orange bag(s), not the regular garbage or the
large MIMM box.

4. Glucose strips, litmus paper, cotton balls and fresh/canned pear cubes can be
thrown into the regular garbage

5. The Benedict’s test and iodine reaction mixture solutions should be poured
into the specified waste “BIOL 111-112” liquid waste container.

6. Rinse the test tubes thoroughly with warm water and place them neatly in the
“dirty” test tube metal basket by the sink, up- side down, so they are ready to
be placed in a dishwashing machine. No mountain of tubes in the basket
please.

7. The used colored plastic test tube caps should be thoroughly rinsed and placed
back in the original bag (along with the unused ones) for use by students on
the next lab, so they must be clean. No need to dry them.

8. Test tube racks should be returned neatly to their original location.

9. Used graduated pipettes are to be placed in the assigned container for proper
disposal as “sharps”. They should never be discarded in the regular garbage, as
they are “sharps” and could injure someone picking -up the garbage bag for
subsequent disposal.

8
Table 1. Acid Hydrolyses of Carbohydrates
The TA does the reactions with the remaining carbohydrates, if the student number is insufficient to cover them all.

(+/-)

Cellulose (+/-)
Maltose (+/-)

Sucrose (+/-)
Glucose (+/-)

Lactose (+/-)

Starch (+/-)
Tube
No Solution Add Treatment

Fructose
15 min at 100°C,
then
1 1 ml of 1% carbohydrate X 1.5 mL H2O Benedict’s test
(another 5 min. at 100C)

5 drops H2SO4 15 min at 100°C,

*after add 18 drops of NaOH
( byTA)
after do
2 1 ml of 1% carbohydrate X
Benedict's test
Let stand 10 min, (another 5 min. at 100C)
then add 1.5 mL H2O

15 min at 100°C,
3 1 ml of 1% carbohydrate X 1.5 mL H2O then
iodine test

5 drops H2SO4 15 min at 100°C,

( byTA) *after add 18 drops of NaOH
4 1 ml of 1% carbohydrate X
after do
Let stand 10 min, iodine test
then add 1.5 mL H2O

*You can check the pH of the reaction mixture is close to neutral (pH 7), before and after the addition of NaOH, with litmus paper strips.
Table 2. Enzyme Activity and Specificity

The TA does the reactions with any remaining carbohydrates, if the student number is insufficient to cover them all.

Amylase Amylase Lactase Lactase

glucose strip iodine glucose-strip iodine
(+/-) (+/-) (+/-) (+/-)

glucose
fructose
sucrose
maltose
lactose
starch
cellulose
cotton

Table 3. TA demo for Catalase Activity

A B B
Tissue Fresh pear Canned pear Fresh pear
Solution H2O2 H2O2 H2O
Bubbles (+ / - )