Biology 2542 – Lab
LAB #11
(11th/19 Lab Sessions for Winter Quarter, 2008)
TOPICS TO BE COVERED:
»Introduction: Why enzymes are needed to digest your food?
»Description of the chemical composition of carbohydrates, lipids, and proteins
»Perform chemical tests
»Analyze results of chemical tests
DESIRED OUTCOMES:
After completing the activities described for this lab session, students should:
»Understand the basic chemical principles of nutrient macromolecule catabolism
»Be able to describe why enzymes are used to digest the food we eat
»Be able to summarize chemical composition of and digestion of proteins carbohydrates, and fats
»Be able to describe each chemical test performed on the substrates and analyze the results of
each experiment
MATERIALS NEEDED:
»Water Bath (heated to 37ºC) with thermometer
»Test tube racks at each Lab Bench
»Wax Marking pencils
»Bunsen burners
»Ring stands
»Test Tubes
»Safety glasses (or one’s own glasses)
»Four 250 ml beakers and four 50 ml beakers
»Template sets of 2 ml, 3 ml, 5 ml, and 10 ml test tube water “blanks” »Asbestos pads
»Hot-hand grippers
»Test-tube holders
»Specific items for Tests and Properties of Carbohydrates:
»5% fructose solution
»5% glucose solution
»10% glucose solution
»Thin starch paste
»Benedict’s solution
»Barfoed’s reagent
»1 M HCl
»1 M NaOH
»5% sucrose solution
»Seliwanoff’s reagent
»Lugol’s iodine (IKI)
»Specific items for Tests and Properties of Lipids:
»5% Pancreatin solution
»Blue litmus solution
»Sweet cream (fresh)
»5% NaHCO3
»Specific items for Tests and Properties of Proteins:
»Undiluted egg white
»1% albumin solution (egg white)
»1% CuSO4
»10% NaOH
»0.1% alcohol solution of glycine (or alanine)
»Buffer solution (pH8)
»5% Pancreatin solution
»Wooden stirring sticks
Activity #1: Introduction: Why are enzymes needed to digest your food?
Before nutrients can be converted to usable energy by cells, the large organic macromolecules in food
must be catabolized into monomers, the building blocks of macromolecules. These chemical reactions are controlled by enzymes produced by the digestive system. Enzymes are protein (organic) catalysts that lower activation energy, the energy required for a chemical reaction. Without enzymes, the body would have to heat up to
dangerous temperatures to provide the activation energy necessary to decompose ingested food. Enzymes have
Biology 251 – LAB #11 – continued
Page Two
a narrow range of physical conditions in which they operate at maximum efficiency. Temperature and pH are
two important factors of enzymatic reactions. For example, enzymes involved in protein digestion require quite
a different pH (around 2.2 – 2.3) than what is needed for carbohydrate digestion (around 8). The enzyme pepsin
is most active in the acidic conditions of the stomach. The next protein-digesting enzyme in the sequence,
trypsin, requires an alkaline environment.
An enzymatic reaction involves reactants, called the substrate, and results in one (or more) product(s).
The enzyme itself has an active site where the substrate binds. Only a substrate that is compatible with an enzyme’s active site is metabolized, and the enzyme is said to have specificity for compatible substrates. Upon
Completion of the chemical reaction, the product is released, and the enzyme, unaltered in the reaction, can bind
to another substrate molecule and repeat the reaction many times in succession.
Each enzyme has a narrow range of physical conditions in which it operates at maximum efficiency.
Temperature and pH are two important factors in enzymatic reactions. A good example of enzyme conditions
are those required in the protein digestion experiment in this exercise. The stomach produces an inactive enzyme, a proenzyme, called pepsinogen. When pepsinogen is mixed with stomach acid, the proenzyme is converted into its active protein-digesting enzyme form, pepsin, which begins to catabolize large protein molecules
in the food. When materials from the stomach move into the small intestine, the pancreas releases sodium
bicarbonate which buffers the stomach acid and raises the ambient pH to 7 – 8. Because pepsin is active only at
an acidic pH, it becomes less active in the small intestine while other enzymes take their respective turns metabolizing the substrate. Like all protein molecules, an enzyme’s function is related to its structural shape, much as
the shape of a key determines which lock it fits. Some enzyme experiments in this exercise will be incubated in
a warm-water bath set at body temperature, 37ºC. Too high a temperature denatures an enzyme, causing a
change in shape, and destroys the enzyme.
Your digestive system catabolizes the food you ingest with a complex sequence of enzymes. If an
enzyme is absent or secreted in an insufficient quantity, the substrate cannot be digested. Some individuals, for
example, do not produce the enzyme lactase which digests lactose, commonly called “milk sugar”. If a lactoseintolerant individual consumes dairy products, which are high in lactose, the sugar remains in the distestive tract
and is only very slowly digested by bacteria. This results in gas, intestinal cramps, and diarrhea.
Enzymes as a group are involved in both catabolic (decomposition) and anabolic (synthesis) reactions.
A specific enzyme, however, functions in only one type of reaction. Enzymes of the digestive system generally
cause catabolic reactions to metabolize your food into smaller molecules which can cross cell membranes and
supply raw materials for cellular respiration.
Digestion includes both mechanical digestion, mechanically breaking food into smaller pieces, and
chem.ical digestion, the enzymatic breakdown of large molecules into smaller ones that can be absorbed into the
bloodstream. Mechanical digestion begins in the mouth and continues in the stomach and small intestine. Chew
ing, or mastication, breaks down large pieces of food into smaller ones that are swallowed. In the stomach, peristaltic waves mechanically churn and mix the food with gastric juices to enhance digestion. Churning continues
in the small intestine by smooth muscle contractions called segmentations, or back and forth movements of portions of the small intestine, that mix the chyme with digestive enzymes prior to peristaltic contractions moving
the contents along. Mechanical digestion enhances the speed of chemical digestion by creating a greater surface
area for digestive enzymes to do their work.
Chemical digestion involves digestive enzymes breaking the bonds of the food macromolecules. The
food we eat contains macromolecules of carbohydrates, lipids, and proteins. (It contains the nucleic acids RNA
and DNA, too, however, we don’t usually think of these as “nutrient” molecules!) Because these molecules are
too large to be absorbed as is by the gastrointestinal tract, enzymes secreted by various parts of the digestive system catabolize (break down) large molecules into small ones that can be absorbed. Starch, a carbohydrate, is digested into disaccharides and monosaccharides by the action of specific enzymes called amylases. Lipids are
macromolecules that are catabolized to glycerol and fatty acids by the enzyme lipase. Proteases digest proteins
into peptides and amino acids. Peptidases digest peptides into amino acids. Monosaccharides, glycerol and
fatty acids, and amino acids are small enough to be absorbed across the wall of the gastrointestinal tract and are
used by cells for building new macromolecules or to provide energy (ATP).
Biology 251 – LAB #11 – continued
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Activity #2: Description of the chemical composition of Carbohydrates, Lipids, and Proteins
All carbohydrates contain the elements carbon, hydrogen, and oxygen, the last two of which are present
in the same ratio as in water (2:1). Carbohydrates may be classified as mono-, di-, or polysaccharides. The
mono- and disaccharides resemble each other considerably (are sweet to the taste, form a white, granular substance,
and are very soluble in water), but polysaccharides bear little resemblance to the other two classes.
Activity #3: Chemical Tests of Carbohydrates, Proteins, and Lipids
Each of the Lab Tables will be assigned one of the three sets of tests; however, you will be responsible
for knowing how ALL of the tests are done, what the substrate is, what the enzyme(s) are (where applicable),
whether the test is positive or negative for certain products.
Here are the three sets of tests:
I.
Carbohydrates:
1. Test for Starch and Starch Hydrolysis
This is a qualitative test for localizing starch molecules.
Procedure:
1) First, have one student collect “spit” into one of the small, 25 ml beakers. (This “volunteer”
will be doing this while the others in the group are proceeding with the lab protocol. )
2) Into each of two test tubes, place 5 ml of the prepared thin starch paste.
3) Using a grease pencil, number the tubes Tube 1 and Tube 2
4) To Tube 1, add one drop of Lugol’s iodine solution
5) Note any color change.
6) To Tube 2, add 4 – 5 drops of saliva (as collected from the “volunteer”)
7) Set Tube 2 in a 37ºC water bath for ½ hour or more.
8) Remove Tube 2 from water bath and test its contents by adding one drop of Lugol’s iodine.
9) Is there a color change when the iodine solution is added?
10) Explain the results.
2. Benedict’s Test
This is a qualitative test specific for localizing reducing sugars, which include all monosaccharides –
glucose, fructose, and galactose; and also some disaccharides - mannose, maltose, and lactose.
Procedure:
1) Place 5 ml of Benedict’s reagent into a test tube; label this Tube 1.
2) To Tube 1, add four to five drops of 5% glucose solution.
3) Place Tube 1 in a beaker water-bath over a Bunsen burner; boil for 2 minutes.
4) Remove Tube 1 and let cool slowly in a test tube rack.
5) While cooling, if there is any color change, or precipitate formation, this indicates a
positive reaction.
6) Make a note of any color change (what color is it?), or if there is any precipitate formation.
7) Place 5 ml of Benedict’s solution into another test tube; label this Tube 2.
8) To Tube 2, add four to five drops of 5% sucrose solution.
9) Place Tube 2 in a beaker water-bath over a Bunsen burner; boil for 2 minutes.
10) Remove Tube 2 and let cool slowly in a test tube rack.
11) While cooling, if there is any color change, or precipitate formation, this indicates a
positive reaction.
12) Make a note of any color change (again, what color?), or if there is any precipitate formation.
Biology 251 – LAB #11 – continued
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3. Barfoed’s Test
This is a qualitative test specific for localizing monosaccharides only.
Procedure:
1) Place 5 ml of Barfoed’s reagent in a test tube.
2) Add five to six drops of 1o% glucose solution.
3) Place in a beaker water-bath over a Bunsen burner; boil for 1 minute.
4) Remove tube from water bath and set aside in a test-tube rack for 15 – 20 minutes to cool.
5) While cooling, watch for any color change, or precipitate formation. If either or both of
these occur, it indicates a positive reaction.
6) Make a note of any color change/precipitate formation.
4. Seliwanoff’s Test
This is a qualitative test specific for distinguishing fructose from glucose and localizing fructose only.
Procedure:
1) Run this test in duplicate, using two tubes which you pre-label Tube 1 and Tube 2.
2) Into each of Tubes 1 and 2, place 5 ml of Seliwanoff’s reagent.
3) Into Tube 1, add five drops of 5% glucose solution.
4) Into Tube 2, add five drops of 5% fructose solution.
5) Place both Tubes 1 and 2 into a beaker water-bath over a Bunsen burner; boil for 1 minute.
6) Note any color change (a color change indicates a positive reaction.) What color is it?
5. Inversion of Sucrose
Since the disaccharide sucrose, is composed of one glucose molecule and one fructose molecule covalently bonded to each other via a dehydration synthesis reaction; then, via a hydrolysis reaction, sucrose
can be decomposed to its original two monosaccharide monomers. This test provides the means by
which such a hydrolysis reaction takes place.
Procedure:
1) Add 5 ml of 5% sucrose solution to each of two test tubes; label these Tube 1 and Tube 2.
2) To Tube 1, add 5 cc of Benedict’s reagent and bring to a boil in the beaker water bath.
3) Remove Tube 1 from heat immediately.
4) To Tube 2, add two drops of dilute (1M) HCl and boil in the beaker water bath for 1 minute.
5) Remove Tube 2 from heat and neutralize with an equal amount and strength of NaOH.
(Explain why this is important to do.)
6) Test the contents of Tube 2 with 5 cc of Benedict’s reagent.
7) Explain the different results for these two Tubes / tests.
II.
Lipids:
Fats are the esters (organic salts) formed by the union of a fatty acid and an alcohol. The three common
fats in our foods and in the body are oleic, palmitic, and stearic, which are formed, respectively, from oleic,
palmitic, and stearic acids united chemically with the alcohol glycerol (glycerin). One molecule of glycerol can
unite with three fatty acids via a dehydration synthesis reaction as follows:
1. Digestion of Emulsified Fat
This test cleverly uses a pH indicator substance and an indirect method of showing that lipid (fat) has
been chemically broken down.
Biology 251 – LAB #11 – continued
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Procedure:
1) Pour 10 ml of sweet cream into a clean test tube; label it Tube 1.
2) Add 10 drops of 1% blue litmus solution to the cream to impart a light blue color.
3) Mix well with a wooden stirring stick.
4) Dispense half of this mixture (about 5 ml) into another test tube and label it Tube 2.
5) Add 3 ml of 5% Pancreatin solution to Tube 2.
6) Add 3 ml of 0.5% NaHCO3 to Tube 1.
7) Place both tubes in the 37ºC water bath for one hour.
8) Remove both Tubes from the water bath. Do you observe any color change in either tube?
9) Explain the results.Use a schematic representation of the chemical steps that have occurred.
III. Proteins:
1. Biuret Reaction
The Biuret reaction is specific for compounds containing two or more peptide bonds.
Procedure:
1) To 3 ml of 1% albumin solution (egg white), add 3 ml of 10% NaOH and one drop of 1%
CuSO4.
2) Mix.
3) Add additional drops of CuSO4 until a violet color is obtained, indicating a positive reaction.
2. Ninhydrin Reaction
The Ninhydrin reaction is a test for alpha amino acids.
Procedure:
1) To 5 ml of 0.1% alcohol solution of glycine (or alanine), add 0.5 ml of 0.1% freshly prepared
Ninhydrin.
2) Heat in a water bath. NOTE color formation.
3) Repeat steps 1 + 2 using 3 cc of 1% albumin solution instead of an amino acid solution.
4) Heat to boiling the albumin to which Ninhydrin was added.
5) Allow to cool. (What is the color?_________________)
3. Heat Coagulation
This treatment demonstrates the irreversible denaturing effect on protein of exposure to high heat.
Procedure:
1) Place 5 ml of undiluted albumin (egg white) in a test tube and place in a beaker water-bath
over a Bunsen burner.
2) Boil.
3) Describe the change you observe.
4. Digestion of Protein
This treatment simulates one of the chemical digestion reactions in the body for protein.
Procedure:
1) Place 1 – 2 chunks of chopped cooked egg white from the Heat Coagulation exercise above
into each of two test tubes.
2) Using a grease pencil, number the tubes Tube 1 and Tube 2.
3) With a wooden stirring stick, break the egg-white chunk in each tube into somewhat
smaller pieces; but do not “mash” entirely)
4) To Tube 1, add 10 ml distilled water (buffered to pH 8).
5) To Tube 2, add 10 ml of 5% Pancreatin solution.
6) Place both tubes in the 37ºC water bath for 1 ½ - 2 hours.
7) Observe; then describe your results.
Biology 251 – LAB #11 – continued
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Activity #4: Analysis of the Results of Chemical Tests
These are all colorimetric tests – tests that change color to indicate some chemical change has taken place.
(They are not quantitative tests, but qualitative only.)