Lab Assignment #3: Macromolecules PDF

Preview

Summary

This is a lab assignment for our biology class. If one does not complete their lab assignments then he/she will fail the course. This lab assignment talks about macromolecules. We learned that a molecule is a very complex structure. These molecules are made up of a bunch of atoms and they make proti...

Description

BIOLOGICAL MOLECULES OBJECTIVES By the end of this exercise you should be able to: 1. Explain the importance of controls in biochemical testing. 2. Describe how to carry out a simple laboratory test for each of the following: monosaccharide, polysaccharide, protein, and lipid. 3. Define hydrolysis and give an example of the hydrolysis of carbohydrates. INTRODUCTION Living organisms contain four main classes of organic molecules: carbohydrates, proteins, lipids, and nucleic acids. Each class has characteristic monomers or subunits which are linked together by covalent bonds to form macromolecules. When two monomers are joined, a hydroxyl (-OH) is removed from one monomer and a hydrogen (-H) is removed from the other. This reaction is referred to as dehydration (removal of H2O) synthesis. CLASS

MONOMERS

MACROMOLECULES

Carbohydrates

Monosaccharides

Polysaccharides

Proteins

Amino Acids

Polypeptides (Proteins)

Lipids

Glycerol & Fatty Acids

Fats

Nucleic Acids

Nucleotides

DNA & RNA

In today’s lab, you will study carbohydrates (monosaccharides, disaccharides & polysaccharides), proteins and lipids. Most of the biochemical tests used in the following exercises rely on indicators, chemicals that change color when a specific substance is present. It is easier to determine if an appropriate color change has occurred if you have controls for comparison. Controls are samples that demonstrate the positive and negative results of each test: negative control - substance that does not react in the test; will show that there is no contamination positive control - substance known to give a reaction in the test; will show that the test worked correctly (no problem with reagents, instruments or technique). All valid experiments contain controls to help evaluate results and to verify that the experiment worked properly. Your first activity will be to establish a set of positive and negative controls for use in later experiments.

25

P Positive and d Negative Controls 1. Work in pairs. mbered tube s in a test tube Take 8 test tubes; number eac h tube 1 throough 8 and pplace the num rack. NOTE E: a separaate disposable plastic piipette (1 ml..) must be used u for eac h solution; contamination will ruin yourr experimen nts and pos sibly s those of o the stude nts in follow wing lab sections A. Ben nedict’s tes st for Red ucing u Suga a rs up with a do uuble-bondedd oxygen (C= =O) that reaccts Reduccing sugars contain a funnctional grou with th he copper ioons in the alk kaline Beneddict’s reagent to produc e a colored p recipitate (soolid). This iss an oxidatioon-reductionn reaction in which oxidaation of the sugar s occurs simultaneo usly with reduction of the Benedictt’s reagent.

Reducinng Sugar

Copp per ion (blu e solution)

Oxidizedd Sugar

Precipitate (gre e en to red)

When he e ated, the Benedict’s B reagent changes from m clear bluee to green, yellow, y oran nge, or red, depending d upon u the am mount of re educing suggar present.. All monossaccharidess and some disaccharides are reduccing sugarss. m of gluco ose solution to test tube #1. 1. Add 2 ml (dispossable pipettt es hold 1 ml) Add 2 ml of disti l ed water t o test tube # 2.

1 ml plastic Pasteur P pipe ette

2. Add one o pipette full (1 ml) o f Benedict’’s reagent to t test tube e #1 and #2 . Careffully swirl thhe tubes to mix the con ntents. 3. Careffully place the test tub bes in the boiling water bath for 5 minutes.

26

est tube. 4. Using the test tubbe holder, re emove the tuubes and obsserve the coll or in each te 5. What happened h to the color in tube #1 ? __________ _ ___________ ________ h to the color in tube # 2 ? _______________________________ 6. What happened 7. Descrii be a positivee Benedict’ss test. _________________________________

NOTE E: Save tu bes #1 and d #2 for us e in later experiment e ts

ol’s test fo r starch B. Lulgo Lugol’s test can b e used to disstinguish starch from monossaccharides, disaccharid es and otherr polysaccha rides. Starchh (amylose) is i a glucose polymer. A single moleccule of starch consists of 1,000 or more glucose uunits linked iin a long x that reactss with the ioddine in Lugool’s coiled chain (heli x) reagennt (I 2KI) to give g adistinc tive blue-blaack color. Otther glucosse polymers lack this preecise three-ddimensional ccoiling and will not react with iodine to produce the t dark coloor.

h (amylose) solution s to teest tube #3. 1. Add 2 ml of starch

Sta arch (amylose)

Add 2 ml of distillled water to test tube #4. 2. Add 2 drops of Luugol’s reagennt to test tu be b #3 and #44. Carefuully swirl thee tubes to miix the contennts. 3. What c olor is the liquid l in tubee #3 ? __________________________ _______ l in tubee #4 ? __________________________ _______ 4. What c olor is the liquid ____________________________ 5. Descrii be a positivee Lugol’s tesst. _______

NO OTE: Save ttubes #3 and #4 for usee in later experiments

27

6. Paper towels are made of 100% cellulose, a glucose polymer, derived mainly from wood, rags and certain grasses. Place on drop of Lugol’s iodine on a piece of paper towel.



Place a drop of Lugol’s iodine in this box.

Based on your results, what can you conclude about this paper ? ___________________________________________________________

C. Biuret test for protein Proteins are polymers of amino acids joined together by peptide bonds. The Biuret reagent reacts with the peptide bonds to produce a violet color. 1. Add 2 ml of albumin (protein in egg white) solution to tube # 5. Add 2 ml of distilled water to tube # 6. 2. Add 1 ml of Biuret reagent to tubes # 5 and # 6. Carefully swirl the tubes to mix the contents. 3. Allow at least 2 min. for the reaction to occur. 4. What is the color of the liquid in tube # 5 ? ___________________________ 5. What is the color of the liquid in tube # 6 ? ___________________________ 6. Describe a positive Biuret Test. ___________________________________

NOTE: Save tubes #5 and #6 for use in later experiments

D. Sudan IV test for lipids Lipids are a large class of organic compounds (fats, oils, cholesterol, wax etc.) which are nonpolar and therefore, insoluble in water. The Sudan IV test is based on solubility; “like dissolves like”. Sudan IV is a nonpolar dye that dissolves in nonpolar substances like fats and oils but not in water. If lipids are present, the red dye on the wooden stick will dissolve and produce floating red droplets or a floating red layer.

28

1. Add 2 ml of distilled water to tubes # 7 and # 8. 2. Add 1 ml of corn oil to tube # 7. 3. Wooden applicator sticks with Sudan IV dye at one end have already been prepared. Place the red end of a wooden stick into tube # 7 and one into tube # 8. 4. Vigorously stir the liquid with the stick. 5. What do you observe in tube # 7 ? ________________________ 6. What do you observe in tube # 8 ? ________________________ 7. Describe a positive Sudan IV test. _________________________________ NOTE: Save tubes #7 and #8 for use in later experiments

Record your observations on the following table:

Results of Testing Controls

TEST

SUBSTANCE DETECTED

NEGATIVE CONTROL

BENEDICT’S

LUGOL’S

BIURET

SUDAN IV

29

POSITIVE CONTROL

TESTING OF FOOD SAMPLES

1. Work in groups of 4; each student will analyze a different food sample and share his/her results with the rest of the group 2. Label 4 clean test tubes A, B, C, D and place them in your test tube rack. 3. Take 3 scoops of minced food and place into test tubes A and B. 4. Add 10 ml of distilled water; cover the top of test tubes A and B with your thumb and shake vigorously. 5. Transfer 2 ml of liquid from tube A into test tube C and perform the Benedict’s test. Don’t be too hasty in recording results; some samples require extra cooling time. Compare your result to your controls: tube # 1 and tube # 2. 6. Transfer another 2 ml of liquid from tube A into test tube D and perform the Biuret test. Allow at least 5 min. for samples to react. Compare your results to your controls: tube # 5 and Tube #6. 7. Place tube A with the minced food and remaining liquid into the water bath for 5 min. before adding 6 drops of Lugol’s solution directly to the tube. Compare your results to your controls: tube # 3 and tube # 4. Because starch is not water soluble, cell walls must be ruptured by boiling in order to get a strong reaction with the Lugol’s test. 8. Place the B tube in the boiling water bath for 5 min. Because fat is not water soluble, cell walls must be ruptured by boiling in order to get a reaction with the Sudan IV test. 9. Remove the B tube from the water bath and place the Sudan IV dye applicator stick directly into this tube. Stir vigorously. Some samples may take 5 min. to react. Compare your results to your controls: tube # 7 and tube # 8.

These directions are outlined in the Flow Chart on the next Page

30

Minced Food 3 scoops

3 scoops

Tube A

Tube B

add 10 ml DW

add 10 ml DW

Shake

remove 2 ml

remove 2 ml

Tube C

Tube D

Benedict’s test

Biuret test

Shake

Boil 5 min

Boil 5 min

Lugol’s test

Sudan IV test

Tube A

Refer to pages 26 to 29 for testing procedures; continue using the same controls.

10. Record your results and those of the other members of your group on the following chart:

Results of Food Testing (+ or -) FOODS TESTED

REDUCING SUGAR

STARCH

PROTEIN

FAT (LIPID)

CLEAN UP: Using soap and a test tube brush located by the sink, wash and rinse all tubes. Place the test tube rack on paper towels and invert the tubes in the rack to dry.

Interpretation of Results 1. Which results were unexpected ?

2. What factors might result in a false negative test (that is, the food contains a macromolecule but the test results are negative) ?

3. Why might a plant storage organ (such as a fruit or tuber) contain both starch and reducing sugar ?

31

Hydrolysis of Carbohydrates During digestion, enzymes break apart carbohydrates, proteins, lipids and nucleic acids in to their monomers by hydrolysis. Covalent bonds between the monomers are broken by the addition of water molecules. HYDROLYSIS MONOMER-MONOMER + H20  MONOMER-H + MONOMER-OH

In this experiment, sucrose (disaccharide) and starch (polysaccharide) will be hydrolyzed by heating with acid. The hydrolysis cannot be seen directly but it can be detected by carbohydrate analysis with Benedict’s reagent and Lugol’s reagent. Fill in the product(s) of hydrolysis: Sucrose + H20  _____________________ Starch + H20  _____________________

Work in pairs. 1. a) In a test tube rack, place 3 rows of 4 test tubes. Label the 1st row A1, A2, A3, A4; the 2nd row B1, B2, B3, B4; and the 3rd row C1, C2, C3, C4 b) add the following substances to the tubes as specified:

TUBE

DW

A1 B1 C1 A2 B2 C2 A3 B3 C3 A4 B4 C4

2 ml 2 ml 2 ml

GLUCOSE

SUCROSE

STARCH

2 ml 2 ml 2 ml 2 ml 2 ml 2 ml 2 ml 2 ml 2 ml

32

PART A: Carbohydrate Composition before Hydrolysis 1. Perform Benedict’s test on all A tubes. Record results in the Table on page 34. 2. Perform Lugol’s test on all B tubes. Record results in the Table (p. 33).

PART B: Hydrolysis 1. To all C Tubes add 2 ml distilled water; next add 2 ml N HCl to each tube

HCl is a strong acid. Handle it with caution. Do not lay the used pipette down on the bench.

TUBE

DW

N HCl

C1 C2 C3 C4

2 ml 2 ml 2 ml 2 ml

2 ml 2 ml 2 ml 2 ml

2. Gently swirl each tube to mix; heat in hot water bath 15 min. 3. Take 4 clean test tubes and label them D1, D2, D3, D4 4. Remove C Tubes from water bath. Transfer 2 ml from each C Tube to the corresponding D Tube; be sure to use a clean pipette for each transfer.

C Tubes from water bath C1 C2 C3 C4

transfer 2 ml     

33

new D Tubes D1 D2 D3 D4

PART C: Carbohydrate Analysis after Hydrolysis 1. Benedict’s Test of C TUBES Hydrolysis was performed by heating with acid. Benedict’s test can not be performed at this pH; the acid must be neutralized first. a) Add a 1 cm piece of litmus paper to each C Tube. What color is the paper ? ______________ b) Add NaOH (sodium hydroxide, a strong base) one dropperful at a time until the litmus paper turns blue and remains blue. NaOH is a strong base. Handle it with caution. Do not lay the used pipette down on the bench. What happens to the acid as you add the base ? __________________ c) Perform the Benedict’s test on the C Tubes; record your results in the Table below. 2. Perform the Lugol’s test on the D Tubes and record your results in the Table below.

Results of Hydrolysis Experiment (+ or -) BEFOREHYDROLYSIS TEST

DW (Tube1)

GLUCOSE (Tube 2)

SUCROSE (Tube 3)

STARCH (Tube 4)

BENEDICT’S (A Tubes) LUGOL’S (B Tubes)

AFTERHYDROLYSIS BENEDICT’S (C Tubes) LUGOL’S (D Tubes)

CLEAN UP: Using soap and a test tube brush located by the sink, wash and rinse all tubes. Place the test tube rack on paper towels and invert the tubes in the rack to dry. Return all materials to their proper location

34

Interpretation of Results 1. Explain the results you obtained using the Benedict’s test on the sucrose solution.

2. Explain the results you obtained using the Benedict’s test and Lugol’s test on starch.

Questions For Further Thought and Review

1. What monomers make up: carbohydrates ? proteins ? fats ? nucleic acids ?

2. During urinalysis, a lab technician observed that distilled water turned violet when he added Biuret reagent. Suggest an explanation and a course of action.

3. Why might a substance taste sweet yet give a negative reaction with the Benedict’s test ?

What procedure could you use to check your answer to the previous question ?

4. An unknown sample is tested with Biuret and Benedict’s reagents. The solution mixed with Biuret reagent is blue. The solution boiled with Benedict’s reagent is also blue. What does this tell you about the sample ?

5. Since potatoes have starch in them, why don’t they taste sweet after they are boiled ?

35

6. Describe how you would test an unknown solution for the presence of a) starch

b) lipid

c) reducing sugar

d) protein

7. Why must an acid solution be neutralized before the Benedict’s test is conducted ?

8. Describe the varied results of the Benedict’s test.

9. Will a solution of an amino acid, such as glycine, give a positive Biuret test ? Explain why or why not.

36...