TOPIC3 Organic Compounds OBJECTIVES 1. Compare the major groups of biologically important or- ‘ganic compounds — carbohydrates, lipids, proveins, and Aue acids —with respect to their chemical composi- tion and function. 2 Distinguish among monosecchatides, disaccharides, and polysaccharides, giving examples ofeach, 5. Desorbe neutal fats and give the biclogial function of this type of molecule, INTRODUCTION Tre various molecules found in all life forms are called organic compounds because they ae produced boy organisms. Organic molecules are carbon based and range from small molecules to ones that are encr~ mousinsize. These large molecties are notas complex as they ‘first seem because they are composed of simpler, smaller molecules or monomers linked to- gether into long chains or polymers. ‘There are four major classes of biologically impor- tant organic compounds. Within the living cell, each of these four major classes has specific important functions. 1, Carbohydrates are energy sources and provide structural support asin the cell wall of plants, Car- bon, hydrogen, and oxygen are the elements found, in carbohydrates. Carbohydrates may be classified as monosaccharides, disaccharides, ot polysac- charides (Fig 3-1). Monosaccharides are simple ‘sugars such as fructose and glucose. Glucose, for example, is @ readily usable energy source, Two 6 4. Describe the fonetions and chemical stuctte of pro= 5. Describe the chemical stuctre of milotdes and nue lec acide and explain the importance ofthese corn pounds in living orgasms 6, Nae the specie laboratory test used to identify (a) reducing sogae,¢) starch, (neural a, protein, ara (¢) lei acd ‘monosaccharides bonded together form a disac- charide, A common disaccharide is sucrose table sugar), which consists of a glucose and fructose molecule. Three or more bonded monosaccharides form a polysaccharide. Starch, cellulose, and gly- ‘cogen are polysaccharides. Starch, produced by plants, and glycogen, produced by animals, are storage forms of energy. Cellulose isthe structaral component of plant cell walls 2. Lipids are a varied group of molecules most of ‘which are insoluble in water. Like carbohydrates, carbon, hydrogen, and oxygen are the principal elements of lipids although the oxygen content is, much reduced, Lipids are essential components of ‘membranes, 2 good means of storing energy and some are essential hormones. Neutral fats, phos- pholipids, steroids, carotenoids, and waxes are lipids, Fatty acids ate the simplest lipids, Neutral fats are the most abundant group of lips in the biological world, Neutral fats are composed of three fatty acid molecules bonded to a molecule of the alcohol glycerol. Neutral fats have at least‘urge the energy-sloring capacity per unit weight as carbohydrates do. Cholesterol is an important stepoid that is @ part of some hormones. Steroids differ from most other lipids by virtue of the struc- ‘turtofseroidsconsisting of carbon ringsinstead of chains, FIGURE “Thin section electron micrograph of Bacillus cereus showing polysaccharide produced by the cells, 50,000X. lor mi- ageaph by Dr. Raber Aten? 3, Proteins have numerous importantrolesin the iv- ing organism, Proteins form many structural fea- ‘sees such as hair, hooves, and tendons, The ele- ments i proteins are carbon, hydrogen, Oxygen, nitrogen, and, in some proteins sulfur. All proteins are polymers of amino acids covalently bonded in Jong chains that subsequently coil and fold into ‘comptex shapes that determine the function of the resulting protein molecule, The chemical bond that holds amino acids together is called a peptidle ond. The bond forms between the functional ‘groups of two arrino acids, A functional xoup is the portion of a molecule that takes part in @ chern- ical reaction, Specifically, a peptide bond occurs between the carboxyl group (COOH) of one Introduction 37 amino acid and the amino group (NH) of its neighboring amino acd, Many amino acide inked together form a polypeptide. Proteins are poly- peptides. A particularly important clase of proteing « polypeptides in biology are the enaymes that controt the many chemical reactions teat keep the cell alive 4, Nucleic acids are extremely large and complex iolecules that havea variety of important biologi- ‘al roles. The elements of nucleic acids are caroor, Inydrogen, oxygen, nitrogen, and phosphorus. One general type of nucleic acid (DNA, deoxyribonu- cleic acid) isthe genetic information (Fig, 3-2), the FIGURES ‘Thin section electron micrograph of Escherichia eli, '50,000%, showing condensed DNA, etn mireeaph by Dr Robert A fier)28 Topie3 Lab3.A genetic code of eels. A second type (RNA, eibonu- Gleic acd} is involved in the expression of the ge- neti code when amino acids are assembled into proteins. Nucleotides ae the monomersin the na cleic acid polymess. ATP (adenosine triphosphate) fs a nucleotide existing in nonpolymer form. ATP supplies energy forthe many chemical reactions of the cell In the laboratory exercises ofthis chapter, you ‘will gain some useful knowledge about the fost major biologically impostant molecules — carbohydrates lipid, proteins, and nucleic acids — and experience in performing laboratory tests 10 identify those types of molecules PRELAB QUESTIONS 1. What is (are) the function(s) of carbohydrates? 2. What classification of carbohydrates is celulose? 3, What are the major components of a neutral fat? 4, What smaller molecules or monomers bond to- gether to form a protein? 5. What type of organic compound is DNA? LAB 3.A TESTING FOR CARBOHYDRATES: REDUCING SUGARS MATERIALS REQUIRED 4 test tubes Benediet’s reagent test tube rack disilled water test tube holder glucose solution boiling water bath sucrose solution ‘wax pencil starch solution small ruler PROCEDURE ‘Some carbohydrates called reducing sugars have an aldehyde functional group as part oftheir molecular structure (Fig. 3-3) which makes them react with Benedict's reagent when heated. The reaction pro- duces a color change in the Benedict’ reagent from biue to green or orange-red, depending on how much reducing sagaris present. Therefore, this colar change Js a positive test for reducing suger. Reducing sugars are so named because they accept an oxygen atom from the Benedict's reagent causing the xeagent to become reduced. You will test three kinds of carbohy- rates with Benedict's reagent to determine which of them are reducing sugars (See Fig. 3-4). FIGURE. ‘Structural formule ofthe reducing sugar bose showing the aldehyde functional group. ‘Note: In all the chemical tests of this exercise, you ‘will alvays test a substance, such as distilled water, ‘that gives a negative result. This is known asa control and it serves as an unchanging standard for judging, positive reactions, 1. Use the ruler and wax pencil to mark Lem and cm from the bottom of 4 clean test tubes. Label the top end of the test tubes as J, 2,3, and 4 2. Fill each test tube up to the I em mark with one of the following; distilled water (tube 1), glucose so- lution (tube 2), sucrose solution (tube 3), and starch solution (tube 4). 43, Add Benedict's reagent up fo the 3.cm mark of all ‘bes. 4. Place ail four test tubes in a boiling water bath and ‘eat for 3 minutes. 5. Remove the test tubes from the hot waterbath with ‘the test tube holder and place them in the test tube rack, Record the color of each tube in Table 3-1‘Teatng for Carbotyarates:Relucing Supers 29 Giacose Sucrose Starch FIGURE 3-4 Proceduee for determining the presente of reducing suger.30 Topic3 Lab 3.8 ‘Table 3-1. Results of Benedict's Test fer Reducing Sugars Color of Tube Alter Boing Tube Number Tube Contents var + Benes reagent 2 glucose + Bones Feagent svcroe-+ Benedie's reagent toch} ener reagent POSTLAB QUESTIONS 1, Which carbohydrate solution contains reducing sugar? 2, What functional group present in this carbohy- rate molecule produces a positive reaction with Benedict's reagent? 3. Ifonion juice mixed with Benedict's ceagent givesa mustard yellow color, in what form is sugar stored in onions? Discard the contents of the test tubes and sinse them for use in the next exercise. Shake out excass water from the tubes, LAB 3.B TESTING FOR CARBOHYDRATES: STARCH MATERIALS REQUIRED 4 test tubes sucrose solution test mabe rack starch solution wax pencil fresh potato smell euler razor blade iodine solution clean side disiled water coverslip glucose colution PROCEDURE Todine reacts with starch reslting in « dark, blue- back color thats postive tes for starch, The reac- tion occurs because starch isa ceiled polysaccharide rade of repested glucose molecules (Fig. 3-8). The ceiled shape of the starch molecule binds with the iodine molecules and produces the color change (See Fig 3-6) 1, Use the rulerand wax pencil to mark 1 cm from the bottom of 4 clean test tubes. Label the top end of the test tubes as 1, 2, 3, and 4. 2, Fill each test tube up to the 1 cm mark with one of the following: distilled water (tube 1), glucase so- ution (pube 2), sucrose solution (tube 3), and starch solution (tube 4. 3. Add3 drops offodine solution to each test tube and swirl to mix, Record the color of each tube in Table 3-2. ‘Table 3-2 Results of Iodine Test for Starch Discard the contents of the test tubes and rinse them for use in the following exercises. Shake out ‘excess water from the tubes. Starch is stored in the seeds, roots, and tubers (lleshy underground stems) of plants as an energy reserve. Thestarch is converted to simple sugars and is used by the plant during times of growth or when low levels of photosynthesis may not meet its energy re- quirements. The potato tuber is an example of starch storage in plants, 1. Usearazorblade to cuta small extremely thin slice of fresh potato. The potato slice should be thin enough to be nearly transparent.‘Testing for Carbokydrates: Starch 31 HOH FIGURE 5-5 ‘Molecular structure of starch it a ‘consisting of repented glucose ‘molecales bonded. together fnto long chains. This Bgure Hoe represents only tiny segment cuon of a large starch molecule Draw the cells and any details you see in the box provided ( qh p Swirl each tube to ris FIGURE 3-6 Procedure for determining the presonce of starch,32 Topic Lab3.C POSTLAB QUESTIONS 1, Do you see a blue-black color in any of the carbo- hydrate solutions other than the starch? 2. Does the iodine test help you distinguish starch from sucrose? Why or why not? 3, Starch and cellulose are both polymers of glucose molecules, but starch gives a positive iodine test ‘and cellulose does not. What is @ plausible expla- nation for this difference? 4. Did you see small dark bodies vithin the potato cells? If you did not, review your slide and, if need bee, contact your instructor for assistance, 5, What molecule ig obviously contained in these dark bodies? 6. If youmix potato juice with the Benedict's reagent in the previous experiment, would a color change ‘occur? Why ox why not? Discard the potato slice. Clean your microscope slide and return it and the scope to their proper place LAB 3.C ‘TESTING FOR PROTEINS: BIURET TEST MATERIALS REQUIRED. 4 test tubes honey solution test tube rack com oil small ruler egg white solution ‘wax pencil 10% NaQH solution istilled water 1% CuSO, solution. PROCEDURE, Protein moleculesconsist of multiple amino acid mol- ecules linked together in long chains (Fig, 3-7). The amino acids are linked by peptide bonds that react, ‘with copper sulfate that is used in the biuret test pro- ducing 2 violet color, Therefore, the violet color indi- cates a positive biuret test {or protein (see Fig. 38). FIGURE 3-7 Inthe Biurat testa complex is formed between the copper tom and four ritragen ators, 1, Mark 4 test tubes at 2 emandd cm measured from the bottom of the test tube. Label the top end of the test tubes as 1, 2, 3, and 4. 2, Fill each test tube up to the 2 cm mark with one of the following: distilled water (tube 1}, honey solu- tion (tube 2), com oil (tube 3), and egg white solu- tion (tube 4). 3, Add 10% NaOH (sodium hydroxide) solution to ‘each test ube up to the 4 cm mar, CAUTION Sodium hydroxide can cause skin ‘burns. Thoroughly wash with soap and water if contact occurs with skin o clothing. 4, Add S drops of 1% CuSO, (copper sulfate) solution to each test tube and swirl to mix. Examine the tubes for the appearance of any violet color and record your results in Table 3-3,Testing for Nucleic Acids: Dische Diphenylamine Test 39 ‘Swf 620d tube tnx » ieuso, P}cuso, F>1e180, Ftous0, 4 4 & 4 6 0 = fan 2em Disilled Honey water solution o FIGURES-8 ‘Procedure for datermining the presence of proteins. Resuls of Bist Test for Protein otorot Tube Contents ‘tebe vwter-+NaOH F C280, 2 | oney + ns0H + C380, 3 | comail+neoe + Cu50, 4 | egguite +Ns0rt + C80, POSTLAB QUESTIONS 1. Which solution(s) contain(s) protein? 2. What type of chemical bond causes the biuret re- agent to react and to give the characteristic violet color? 3. Would you expect a solution containingan enzyme to yield a positive biuret test? Why or why not? 4. What color change would occur if iodine was added to the egg white? Discard the contents of the tubes, wash thoroughly ‘with detergent, and singe them well foruse in the next ‘exercises, Shake out excess water from the tubes LAB 3D TESTING FOR NUCLEIC ACIDS: DISCHE DIPHENYLAMINE TEST MATERIALS REQUIRED. Stest tubes test tbe holder test habe rack distilled water34 Topic3 Lab 3.0 boiling water bath ‘small ruler ‘wax penal DNA solution Dische diphenylamine. RNA solution reagent PROCEDURE ‘Nucleic acids, RNA and DNA, are large molecules composed of many nucleotides bonded together in Jong strands (Fig. 3-9). Each nucleotide contains a five-carbon sugar as part ofits structure, Specifically, DNA contains the sugar deoxyribose that reacts with the diphenylamine reagent when heated forming a blue cotor—a positive test for DNA (Fig. 3-10) 1. Mark3 test tubes at em and 4 em measured from the bottom of the test tube, Label the top end of the test tubes as J, 2, and 3. 2, Filleach test tube up to the 2 em mark with one of the following: distilled water (tube 1), DNA solu- tion (tube 2), and RNA solution (tube 3). 3. Add Dische diphenylamine reagent to each test tube up to the 4 cm mark, Dating DNA RNA water solution solution ® FIGURE 3-10 Procedure for determining the presence of DNA. Phosphate group Os endo aught FIGURE 3-9 Generatized diagram of a nucleic aid molecule componed of cautiple nucleotides. Each nucleotide consists of three paste fve-carbon sugae, a phosphate group, and a base. 5 15 Ey 2 itesCAUTION Dische diphenylamine reagent contains strong acids. Use great care when han- dling the reagent. If the reagent contacts skin or Gothing, Hush the area under running water for several minutes, 4. Bol the test tabes inthe hot water bath for 2 min- ‘utes or until ohe of the tubes tuins blue. Do not coves boll the test tubes —this causes the contents to boil out ofthe tubes. 5, After boiling, use the test tube holder to place the tubes into the test tube rack. Record the color of each tube in Table 3-4 ‘Table 3-4 Results of Dische Diphenylamine Test — Color of Tube Tube Number Tube Contents 1 water + diphenylanine reagent [DNA + aiphenylemine agent ANA + diphenylamine roagent POSTLAB QUESTIONS 1. Did the test fube containing DNA yield a positive ‘est for this nucleic aig? Alyce H-c~o-c— 1 H FIGURE 3-11 _ ‘Testing for Lipids Sudan I Test 38 2. Based on the chernical composition of DNA and RNA nucleotides, explain why the nucleic acid RNA did not yield 2 positive diphenylamine test. Discard the contents of the tubes in the container des- ‘ignated for diphenylamine reagent dispose! —DO NOT pour tiuids containing eiphenylamine down, the sink dain! Wash the test bes well for ase i the next exercise. Shake out excess water from the hibes. LAB 3.E TESTING FOR LIPIDS: SUDAN WM TEST MATERIALS REQUIRED ‘test tubes egg white solution test tube rack honey solution Sudan IN dye com oll wax pencil distilled water ‘small ruler PROCEDURE Lipids exe eonpolar compounds composed of a glye- ‘erol molecule bonded to three fatty acid molecules (Fig. 3-11), Because lipids are nonpolat they do-nct dissolve in water or other polar liquids. Lipids are soluble in nonpolar solvents such as ether. Liquid Sadan Il yes ade with ether asits solvent. There= fore, solubility in Sudan lll dye can be used 28 a posi~ Baty ain a ‘A lipid (at) molecule consisting of three fatty acids bonded to one glycerol molecule36 Topic3 Lab 3.8 Sean st! WSadan tc sudan ut jrreceecocoa fi? Disillat Honey water salen solution ® FIGURE 3-12 Procedure for determining the presence of lipids, tive test for lipids. Nonlipid substances will not dis- solvein Sudan Ill dyeand form twodistinctfayers ina teat tube (Rg. 2-12}, 1. Matk 4 test tubes at 2 cm measured from the bot~ tom of the tube, Label the top end of the test tubes as1,2,3,and4. 2, Fill each test tube up to the 2 cm mark with one of the following: distilled water (tube £), honey solu- ‘ion (tube 2), com oil (tube 3), and egg white solu- fion (tube 4). 3. Add ten drops of Sudan III dye to each test tube. Cc eg white Sedan mt q ‘Stench tube tc » present” (even mixing of liquid and Sudan IT dye) or “lipids absent” (layers of liquid and Sudan IIL dye form). ‘Table Results of Sudan Il Test for Lipids ‘Tube Number____Tube Contents water + Suomi aye a honey + Sudan 6 aye CAUTION Sudan Ill is a powerful stain Avoid contact with skin and clothing. ‘otn ol + Sudan OL aye ‘Swirl the tubes to mix, Examine the tubes for re- sults. Record your zesults in Table 3-5 as “lipids ap, white + Sea MeyePOSTLAB QUESTIONS 1, Which compounel(s) contain(s) lipids? 2. Which tube served as the control? 3, Which compound contains more calories, honey or corn oll? Why? Discard the contents of all tubes in the container des- ‘grated for Sudan fl disposal DO NOT pour Sudan TTI down the sink drain! Thoroughly brash your test tubes with detergent and water, particularly the tubes containing com oil. Shake out any excess water re- -mainingin the tubes, This will ensure good testresults for those students in the next lab period. FOR FURTHER READING “Alberts, Bet al, 1989. Moleculer Biology of the Cll, New ‘York Garland Publishing, ‘Atkins, PW, 1987, Molecufes, New York: Scientife Ameri: ean Library. Bettelheim, FA, and). March, 1990. luroduetiont General, ‘Organic and Biochemisny Psladalpbia: Saunders College Publishing, Bloomfield, I 1980, Chemistry end the Living Organism. ‘New York John Wiley and Sons. (Coleman, David Cand Brian Fry, eds. 1991, Carbo tsotope Techniques. San Diego: Acadersic Press. Testing for Lipids: Sudan HII Test 37 Creighton, T. &. 1990. Protein Function: A Practical ap preach. New York: IRL Press. Denyer, 8. and W. B. Hugo, eds, 1991, Mechenisms of ‘Acton of Cherce! Biocides: Their Study and Explotavion. Boston: Blackwell Scene Paalionions. Glick, David M1990. Glossary of Bechemisty ard Molecutar Biology. New York: Raven Press. ‘Hames, B.D, and D. Rickwood. 1990, Get Electrophoresis of Proven: A Practical Approach. New York: IRL Press at Oxford University Pres Haris, EL. V, and S, Angal, eds, 1990, Protein Purification “Applications: A Practical approach. New York: IRL Press ‘eth, H1,, and B. Market, eds. 1990, Element Concentration dusters in Ecosystems, New York: VCH Publishers. Moody, Pater C. E. 1990, Protein Engineering. New York: TRL Press at Oxford University Press. Moore, Geoffrey R. 1999. Cytociveme c: Evolutionary, Sie tel, and Physichemical Aspects. New York: Springer- ‘Verlag. Morimoto, Richard 1, sh, eds. 1990, Sess Proteins x Biology and Modine, Cold Spring Harbor, N.Y: Cole Spring Harbor Laboratory. Reddy, C. Channa, ot at, eds. 1990, Biological Oxidation ‘Systems, San Diego: Academie Pres, Revzin, Azo ed. 1990, The Bialagy of Nonspecific DNA- Protein Ieteretins. Boca Raton, Fl: CRC Press. Siddle, K. and T. Creighton, eds. 1999. Peptide Hormone ‘Acton: A Practice! Approack. New York: IRL Poess at OX ford University Pres, Soxensen, Elsa M, B. 199%. Metal Boisoning in Fish, Boca Raton, Fz CRC Press. ‘Stein, Stanley. 1990, The Fundamentals of Protein Biotecnol- gy. New York: M, Deke, Stein, Wilfeed D_ 1990, Chawvels, Carers, and Pumps: An Introduction to Mente Transport San Diego: Academic Press, ‘Stryer,L. Blockenastry. 1988. New York: Freeman,