BIOCHEMISTRY I-II (BIO 202-203) LABORATORY MANUAL

by Assoc.Prof. Dr. Seyhun YURDUGL and Res.Assist.Gnce AHN

(Second edition)

Biology Department Abant Izzet Baysal University Bolu, TURKEY

2010

Emergency Phone Numbers/ Acil Telefonlar

110 Fire/tfaiye 112 Ambulance/Salk Acil Servis 118 Unknown Dials/Bilinmeyen numaralar 153 Municipal Office/Zabta 154 Traffic police/Alo Trafik 155 Police/Polis 156 Gendarme/Jandarma 187 Natural gas / Doalgaz arza AIBU District Security and Ambulance-AB ii Gvenlik ve Ambulans: 03742541933 Poison Information Center/Zehir Danma Merkezi Cemal Grsel Street/ Caddesi No: 18 Shhiye/ANKARA Phone/Tel: 08003147900-4 lines -4hat(Toll Free-cretsiz) Phone/Tel: 03124337001(cretli-with toll) Hospitals in Bolu I.Baysal Faculty of Medicine/Tp Fakltesi 03742534656 Bolu I.Baysal State Hospital/Devlet Hastanesi 03742154450 Krolu State Hospital/Devlet Hastanesi 03742704575 Hospitals in the Environ Provinces/evre Hastaneler Dzce Tp Fakltesi(Dzce Medical Faculty) 0380 542 13 90 Ankara Atatrk State Hospital/Devlet Hastanesi Bilkent/Ankara 0312 291 25 25 Ministry of Health/Ankara Hospital-Salk Bakanl Ankara Hastanesi ,Cebeci-Ankara, 0312 363 33 30 Hacettepe University Hospital-Hacettepe niversitesi 0312 310 35 45 Bayndr Hospital(private)- zel Bayndr Hastanesi-Stz 0312 287 90 00 Ankara Gven Hospital(Gven Hastanesi):Kavakldere, 0312 457 25 25 stanbul Marmara University Hospital(Marmara niversitesi Hastanesi), 0216 327 10 10 Anadolu Salk Merkezi(Anatolian Health center-Associated with Johns Hopkins center in the US)Gebze 0262 44 44 276 Toyotasa Hastanesi-Toyotasa Hospital Adapazar 0264-2293160 / 61

SELECTED GLASSWARES AND BASIC INSTRUMENTS IN THE LABORATORY

Erlenmayer flask

Beaker

Bunsen burner

Loop

Incubator

Autoclave

Vortex for test tube mixing

UV-VISIBLE spectrophotometer

Glass rod

Pasteur pipettes

GENERAL RULES FOR SAFETY

The biochemistry laboratory is a place of adventure and discovery. Some of the most important events in scientific history have happened in laboratories. The antibiotic powers of penicillin were discovered in a laboratory. The plastics used today for clothing and other products were first made in a laboratory. The list is endless. One of the first things any scientist learns is that working in the laboratory can be an exciting experience. However, the laboratory can also be quite dangerous if proper safety rules are not followed at all times. In order to prepare yourself for a safe year in the laboratory, read over the following safety rules. Then read them a second time. Make sure you understand each rule. If you do not, ask your teaching staff to explain any rules you are unsure of. You may even want to suggest further rules in the section labeled "Other Safety Rules" mentioned in Section I. A. Dress Code
1. Many materials in the laboratory can cause eye injury. To protect yourself

2. 3. 4.

5.

from possible injury, always wear safety goggles or glasses whenever you are working with chemicals, burners, or any substance that might get into your eyes. Laboratory aprons or coats should be worn everytime in the laboratory and keep them buttoned. Tie back long hair in order to keep it away from any chemicals, bunsen burners, and candles, or other laboratory equipment. Any article of clothing or jewelery that can hang down and touch chemicals and flames should be removed or tied back before working in the laboratory. Sleeves should be rolled up. Sandals and open toe shoes will not protect the feet from any spill.

B. General Safety Rules

1. Read all directions for an experiment several times. Follow the directions

exactly as they are written. If you are in doubt about any part of the experiment, ask your teaching staff for assistance.
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2. Never perform activities that are not authorized by your teaching staff. Always 3. 4.

5. 6. 7.

obtain permission before "experimenting" on your own. Never handle any equipment unless you have specific permission. Take extreme care not to spill any material in the laboratory. If spills occur, ask your teaching staff immediately about the proper clean-up procedure. Never simply pour chemicals or other substances into the sink or trash container. Never eat or drink in the laboratory. Wash your hands before and after each experiment. There should be no loud talking or horseplay in the laboratory. When performing a lab, make sure the work area has been cleared of purses, books , jackets, etc.

8. DO NOT SIT ON THE LABORATORY BENCH ZONE. 9. Know the location and use of all safety equipment (goggles, aprons, eyewash,

fire blanket, fire extinguishers, etc.) 10. Read your assignment before coming to class and be aware of all safety precautions. Follow the directions. C. Heating and Fire Safety
1. Never heat any chemical that you are not instructed to heat. A chemical that 2. 3. 4.

5. 6. 7.

is harmless when cool can be dangerous when heated. Always maintain a clean work area and keep all materials away from flames. Never leave a flame unattended. Never reach across a flame. Make sure you know how to light a Bunsen burner. (Your teaching staff will demonstrate the proper procedure for lighting a burner.) If the flame leaps out of a burner towards you, turn the gas off immediately. Do not touch the burner. It may be hot. And never leave a lighted burner unattended! Always point a test tube that is being heated away from you and others. Chemicals can splash or boil out of a heated test tube. Never heat a liquid in a closed container. The expanding gases produced may blow the container apart, injuring you or others. Never pick up any container that has been heated without first holding the back of your hand near it. If you can feel the heat on the back of your hand, the container may be too hot to handle. Always use a clamp or tongs when handling hot containers. Hot glassware looks the same as cool glassware.

D. Using Chemicals Safely

1. Never mix chemicals for the "fun of it." You might produce a dangerous,

possibly explosive substance. No unauthorized experiments should be performed. 2. Never touch, taste, or smell any chemical that you do not know for a fact is harmless. Many chemicals are poisonous. If you are instructed to note the
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fumes in an experiment, always gently wave your hand over the opening of a container and direct the fumes toward your nose. Do not inhale the fumes directly from the container. 3. Use only those chemicals needed in the activity. Keep all lids closed when a chemical is not being used. Notify your teaching staff when chemicals are spilled. 4. Dispose of all chemicals as instructed by your teaching staff. 5. Be extra careful when working with acids or bases. Pour such chemicals over the sink, not over your work bench. 6. When diluting an acid, always pour the acid into water. Never pour water into the acid. 7. Rinse any acids off your skin or clothing with water. Immediately notify your teaching staff of any acid spill. 8. Never use mouth-pipetting. 9. Be sure you use the correct chemical. Read the label twice. 10. Do not return any excess back to the reagent bottle. 11. Do not contaminate the chemical supply. 12. Keep combustible materials away from open flames (alcohol, carbon disulfide, and acetone are combustible). 13. Do NOT use the same spatula to remove chemicals from two different containers. Each container should have a different spatula. 14. When you remove the stopper from a bottle, do NOT lay it down on the desk, but place the stopper between your two fingers and hold the bottle so the label is in the palm of your hand so drips won't ruin the label, etc. Both the bottle and the stopper will be held in one hand. Be sure and rinse any drips that might have gotten on the outside of the bottle. 15. Be careful not to interchange stoppers from two different containers. 16. Replace all stoppers and caps on the bottle as soon as you finish using it. 17. Mercury spills must be informed to the teaching staff in the laboratory and cleaned up immediately. Use the new mercury sponge clean up kits put out by various companies.

E. Using Glassware Safely

1. Glass tubing should never be forced into a rubber stopper. A turning motion

and lubricant will be helpful when inserting glass tubing into rubber stoppers or rubber tubing. Your teaching staff will demonstrate the proper way to insert glass tubing. 2. When heating glassware, use a wire or ceramic screen to protect glassware from the flame of a Bunsen burner. 3. If you are instructed to cut glass tubing, always fire polish the ends immediately to remove sharp edges.

4. Never use broken or chipped glassware. If glassware breaks, notify your

teacher and dispose of the glassware in the proper trash container. 5. Never eat or drink from laboratory glassware. Always thoroughly clean glassware before putting it away.

F. Using Sharp Instruments

1. Handle scalpels or razor blades with extreme care. Never cut any material

towards you: always cut away from you. 2. Notify your teaching staff immediately if you are cut in the laboratory. 3. Properly mount, dissecting specimens to the dissecting pan before making a cut. G. Electrical Equipment Rules
1. Batteries should never be intentionally shorted. Severe burns can be caused

2.

3. 4. 5. 6.

7.

by the heat generated in a bare copper wire placed directly across the battery terminals. If a mercury type dry cell is shorted, an explosion can result. Never deliberately shock yourself or another person. Susceptibility to shock and possible resulting injury is unpredictable because of the many physical and physiological variables. Turn off all power when setting up circuits or repairing electrical equipment. Never use such metal articles as metal rulers, metal pencils or pens, nor wear rings, metal watchbands, bracelets, etc. when doing electrical work. When disconnecting a piece of electrical equipment, pull the plug and not the wire. Use caution in handling electrical equipment which has been in use and has been disconnected. The equipment may still be hot enough to produce a serious burn. Never connect, disconnect, or operate a piece of electrical equipment with wet hands or while standing on a wet floor.

H. End-of-Experiment Rules
1. When an experiment is completed, always clean up your work area and

return all equipment to its proper place. 2. Wash your hands after every experiment. 3. Make sure all candles and burners are turned off before leaving the laboratory. Check that the gas line leading to the burner is off as well. I. Other Safety Rules

1. Do not use hair spray or hair mousse during or even before coming to

laboratory class. These are highly flammable and might cause automatic ignition when in close proximity to a heat source. 2. Synthetic fingernails are also highly flammable and should not be worn in the lab.

Experiment I: How to prepare solutions in the laboratory: Normality Concept The Concentration, Molarity and

The concentration refers to the relative amount of a substance in a given volume of solution; but in some cases alternative expressions are used. Different ways of expressing concentration are listed below, which are mostly used in biochemical calculations. 1. Percent solution: Per cent concentration refers to a number of parts in 100 parts of solution Weight percent (% w/w) = weight of solute weight of solution x 100

%5 (w/w) NaCl solution is the one containing 5 g NaCl in 100 g of solution. Volume percent (% v/v) = volume of solute volume of solution x 100

%5 (v/v) NaCl solution of ethanol in water is the one containing 5 mL NaCl in 100 ml of solution.

Weight-volume percent (% w/v) =

weight of solute volume of solution x 100

%5 (w/v) NaCl solution is the one containing 5 gr NaCl in 100 mL of solution.

Example: How many grams of NaCl are necessary to prepare 500 mL of a 10 % (w/v) solution?

2. Concentration expression for very dilute solutions

Miligram percent (%mg) is the number of miligram present in 100 mL of solution. mg % = mg of solute 100 mL solution

Parts per million (ppm) means the number of miligrams of solute per liter of solution. ppm (liquids) = mg of solute = mg of solute = mg of solute 103g 103mL 1 L solution

for example: 1 L of solution that contains 5 ppm CdCl2 contains 5 mg of CdCl2. Example : How can you prepare 250 mL 100 ppm solution ?

Molarity The term molarity is defined as the number of moles of solute per liter of solution. M= n (number of moles) V number of moles = mass in grams molecular weight Dilute solutions are often expressed in terms of milimolarity and micromolarity. 1 mmole = 10-3 moles 1 mole = 10-6 moles 1 nmole = 10-9 moles 1 pmole = 10-12 moles Example : How many moles of NaCl are present in 150 mL of a 1.5 M solution? (M.W. NaCl : 58.5 )

How many grams of solid NaOH are required to prepare 500 mL of 0.04 M solution ? Express the concentration of this solution in terms of % (w/v) (M.W. of NaOH : 40 )

Normality Normality is defined as the number of equivalent weights per liter of solution. N= equivalent weight / liter Equivalent weight = molecular weight valance

Valance : the number of replaceable H+ or OH- per molecule (for acids and bases) OR the number of electrons lost or gained per molecule (for oxidizing and reducing agent) Example : What is the normality of 1 L of an aqueous solution of 98 gr of H2SO4 ? (m.w. H2SO4=98)

To prepare 6N NaCl ? (M.W. NaCl: 58.5)

Changing concentration It is often necessary to change the concentrations of one solution to that of a more dilute solution. In the process of dilution it is clear that, the total amount of solute remains unchanged, only its concentration is decreased. Hence it follows that; V1 x C1 = V2 x C2

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V1 = volume of initial solution C1 = concentration of initial solution V2 = volume of desired (final diluted solution) C2 = concentration of desired (final diluted solution)

Example : You have 5M NaOH. You need 100 mL of 1M solution. How much 5M would be required ?

Can you prepare 50 mL of a 2 N solution from a 5 N stock solution?

Can you dilute 7 mL of a 5 % solution to a 3 % solution ?

LITERATURE CITED 1. Segel, I.H. Biochemical Calculations, Wiley Publishing Co. New York, 1976.

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EXPERIMENT II SAPONIFICATION(SOAP MAKING) Fatty acids are straight-chain monocarboxylic acids. The most common fatty acids range in size from 10-20 carbons and most often have an even number of carbon atoms including the carboxyl group carbon. The carbon-carbon bonds in saturated fatty acids are all single bonds, while unsaturated fatty acids have one or more carbon-carbon double bonds in their chains. One example of a saturated fatty acid is palmitic acid, CH3-(CH2)14-CO2H. Fatty acids are seldom found as free molecules in nature but are most often a part of a larger molecule called a triglyceride. Triglycerides consist of a three-membered carbon chain (glycerol backbone) with a fatty acid bonded to each of the three carbon atoms in the glycerol backbone. Soap is produced by the saponification (hydrolysis) of a triglyceride (fat or oil) (Figure 1). The triglyceride is reacted with a strong base such as sodium or potassium hydroxide to produce glycerol and fatty acid salts. The salt of the fatty acid is called a soap. The bond between the fatty acid and the glycerol backbone is referred to as an ester linkage. In the saponification process the ester linkage is broken to form glycerol and soap.

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Figure 1. Saponification of a triglyceride.

Saponification value (or "saponification number", also referred to as "sap" in short) represents the number of milligrams of potassium hydroxide or sodium hydroxide required to saponify 1g of fat under the conditions specified. It is a measure of the average molecular weight (or chain length) of all the fatty acids present. As most of the mass of a fat/triester is in the 3 fatty acids, it allows for comparison of the average fatty acid chain length. If there are more moles of potassium hydroxide (KOH) used for 1 gram of fat then there are more moles of the fat. Therefore the chain lengths are smaller, due to the equation: The chemical compound potassium hydroxide (KOH), sometimes known as caustic potash, potassa, potash lye, and potassium hydrate, is a metallic base. Saponification of a lipid with potassium hydroxide. Fats consist of a wide group of compounds that are generally soluble in organic solvents and largely insoluble in water. The molecular mass of a substance (less accurately called molecular weight and abbreviated as MW) is the mass of one molecule of that substance, relative to the unified atomic mass unit u (equal to 1/12 the mass of one atom of carbon-12). In chemistry, especially biochemistry, a fatty acid is a carboxylic acid often with a long unbranched aliphatic tail (chain), which is either saturated or unsaturated.

Number of moles = mass of oil/relative atomic mass.

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The relative atomic mass would be smaller with smaller fatty acid chains, meaning more moles. The higher the saponification value, the smaller the chain lengths.

MATERIALS AND THE METHOD Caution: Oil will be hot, and may splatter or catch fire. Wear goggles at all times.

Preparation of the hot water bath 1. Assemble a hot water bath by filling an 800 mL beaker approximately 3/4 full with water and begin heating the water with a bunsen burner. 2. Add three or four boiling chips to the water in the hot water bath to prevent the water from boiling over. Preparing the reaction mixture 3. In a 150 mL beaker add the following ingredients. A. 15 mL of oil (or 10 g of solid shortening) B. 20 mL of 20% NaOH

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C. 10 mL of ethanol (Methanol can be used but since it is extremely toxic, ethanol is recommended. D. 3 boiling chips (These will help prevent the mixture from boiling over while it is being heating.) 4. Note the total volume (level) in the 150 mL beaker and how many layers the ingredients initially form. 5. Begin heating the reaction mixture by clamping the beaker and contents in the hot water bath. Heat the mixture for about 25 minutes after the water comes to a slow boil. The 150 mL beaker should be clamped so that the reaction mixture is below the level of the water in the water bath. Maintain the water level in the water bath by adding water as needed. 6. Using a stirring rod, stir the reaction mixture frequently so that it does not boil over. 7. Maintain the total volume of the reaction mixture by adding small quantities of 1:1 (volume/volume) ethanol-deionized water. 8. After the initial 25 minute heating there should be no separation of layers in the beaker. Testing the reaction mixture 9. Test the reaction mixture to determine if the saponification process is complete by carefully placing a few drops of the reaction mixture in a 6-inch test tube. Add 10 mL of cold water. If fat droplets form, add 5 mL of the 20% NaOH and 5 mL of ethanol to the beaker and continue to heat for an additional 10 minutes, or until no fat droplets form upon testing. Isolating the soap- CAUTION!!!!: Remember that the beaker and clamp are hot!

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10. When the saponification process is complete, turn the bunsen burner off, add 25 mL of deionized water to the beaker and place the beaker on the bench top to cool for about 5-6 minutes. Then place the soap reaction beaker into an ice bath and cool for about 10 minutes. 11. At this point, measure out about 50 mL of saturated NaCl solution and cool it also for about 5-6 minutes in the ice bath. 12. After the cooling time is complete for the soap reaction mixture, decant any liquid from the beaker. (Be careful not to pour off the soap.) 13. Next add, add the 50 mL cold, saturated NaCl solution to the soap beaker and stir thoroughly with a glass rod. This process separates the soap from the glycerol and excess base and is called "salting out." 14. Collect the solid soap, using a Buchner funnel. (Note: Decant as much liquid off before adding the solid soap to the Buchner funnel.) 15. While the air is being drawn over the soap in the funnel, wash the soap with two 20 mL volumes of ice cold deionized water. Continue to draw air over the soap for another 3 minutes. Soap disposal 16. Place the soap in the designated container. 17. Thoroughly rinse all of the glassware with water before storing. LITERATURE CITED 1. Segel, I.H. Biochemical Calculations, Wiley Publishing Co. New York, 1976. 2. Perry and Bassow, Chemistry, Laboratory Manual, 1985.

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Experiment III The Gelatinization and Retrogradation of Starch: Objectives: In this experiment, youll be able: -To realize the process of sol and gel formation in starch-water dispersions. -To predict how sugars, acids, lipids, and other ingredients affect the quality characteristics of starch-containing products. -To predict how processing, production, preparation, and storage of starch sols and gels affect the quality characteristics of the final product. To realize processing and modification of starches and relate this to dispersion characteristics. To be aware of the molecular and granule structure of various native starches to starch paste and gel characteristics. INTRODUCTION Starch is found in almost every typical meal. For a truly useful nutrient it is critical that there be some understanding of what starch is and how preparation processes will influence it. Starch is a polysaccharide (meaning "many sugars") made up of glucose units linked together to form long chains. The number of glucose molecules joined in a single starch molecule varies from five hundred to several hundred thousand, depending on the type

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of starch. Starch is the storage form of energy for plants, just as glycogen is the storage form of energy for animals. The plant directs the starch molecules to the amyloplasts, where they are deposited to form granules. Thus, both in plants and in the extracted concentrate, starch exists as granules varying in diameter from 2 to 130 microns. The size and shape of the granule is characteristic of the plant from which it came and serves as a way of identifying the source of a particular starch. The structure of the granule of grain is crystalline with the starch molecules orienting in such a way as to form radially oriented crystals. This crystalline arrangement is what gives rise to the phenomenon of birefringence. When a beam of polarized light is directed through a starch granule, the granule is divided by dark lines into four wedge-shaped sections. This cross-hatching or cross is characteristic of spherocrystalline structures.

There are two types of starch molecules: amylose and amylopectin. Amylose averages 20 to 30 percent of the total amount of starch in most native starches. There are some starches, such as waxy cornstarch, which contain only amylopectin. Others may only contain amylose. Glucose residues united by a 1,4 linkage form the linear chain molecule of amylose. Amylose is the linear fraction and amylopectin is the branched fraction.

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Amylose molecules contribute to gel formation. This is because the linear chains can orient parallel to each other, moving close enough together to bond. Probably due to the ease with which they can slip past each other in the cooked paste, they do not contribute significantly to viscosity. The branched amylopectin molecules give viscosity to the cooked paste. This is partially due to the role it serves in maintaining the swollen granule. Their side chains and bulky shape keep them from orienting closely enough to bond together, so they do not usually contribute to gel formation. Different plants have different relative amounts of amylose and amylopectin. These different proportions of the two types of starch within the starch grains of the plant give each starch its characteristic properties in cooking and gel formation. Starch in its processed, commercial form is composed of starch grains or granules with most of the moisture removed. It is insoluble in water. When put in cold water, the grains may absorb a small amount of the liquid. Up to 60 to 70C, the swelling is reversible, called as the retrogradation of starch. The degree of reversibility is dependent upon the particular starch. With higher temperatures in irreversible swelling called gelatinization begins. Starch begins to gelatinize between 60 and 70 C, the exact temperature dependent is the specific starch. For example, different starches exhibit different granular densities, which affect the ease with which these granules can absorb water. Since loss of birefringence occurs at the time of initial rapid gelatinization (swelling of the granule), loss of birefringence is a good indicator of the initial gelatinization temperature of a given starch. The largest granules,

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which are usually less compact, begin to swell first. Once optimum gelatinization of the grains has occurred, unnecessary agitation may fragment the swollen starch grains and cause thinning of the paste. The gelatinization range refers to the temperature range over which all the granules are fully swollen. This range is different for different starches. However, one can often observe this gelatinization because it is usually evidence by increased translucency and increased viscosity. This is due to water being absorbed away from the liquid phase into the starch granule. Raw starch that has not had moisture added does not undergo gelatinization. By definition, gelatinization is a phenomena which takes place in the presence of heat and moisture. The dry raw starch, if heated, would undergo dextrinization. This certainly would affect the starch paste viscosity and starch gel strength. decreased and gel strength decreased. If a "limited amount" of moisture is added to the raw starch you may get partial gelatinization. This condition exists in baked products. If a typical starch paste is allowed to stand undisturbed, inter-molecular bonds begin to form, causing the formation of a semirigid structure or gel. This gel is a structure of amylose, molecules bonded to one another and, slightly, to the branches of amylopectin molecules within the swollen granule. This The paste viscosity would be

phenomenon is sometimes called retrogradation. The conditions of gelation is critical to the ultimate rigidity of the final product. Gelation of starch occurs due to its 3-D structure. With heat removal, retrogradation of mixture to a paste-like mass of gel is formed.

PROCEDURE (Materials and method)

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Materials: Starch, Beaker, Glassware petri plates, Glass baget, Spoon a) Weigh 10 grams of starch, mix it with 100 mL distilled water and heat it until swelling at 100 C b) Weigh 10 grams of starch, mix it with 100 mL distilled water and heat it until swelling at 50-60 C. c) Take a piece of starch solution and put it onto the glassware petri plates d) Do not forget to look at the starch crystals under microscope.

LITERATURE CITED 1.Muhrbeck, P. and Eliasson, AC. 1987. Influence of pH and ionic strength on the viscoelastic properties of starch gels- a comparison of potato and cassava starches. Carbohydrate Polymers 7: 291-300 2. URL 1: http://food.oregonstate.edu/learn/starch.html(Cited 04.12.2008) 3. Johnson, J.M., E.A. Davis, and J. Gordon. 1990. Interactions of starch and sugar water measured by electron spin resonance and differential scanning calorimetry. Cereal Chemistry 67(3): 286-291. 4. Bean, M.M. and W.T. Yamazaki. 1978. Wheat starch gelatinization in sugar solutions. I. Sucrose: Microscopy and viscosity effects. Cereal Chemistry 55(6): 936-944. Experiment IV

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Determination of Vitamin C by Iodine Titration

Vitamin C - Ascorbic Acid

Vitamin C (ascorbic acid) is an antioxidant that is essential for human nutrition. Vitamin C deficiency can lead to a disease called scurvy, which is characterized by abnormalities in the bones and teeth. Many fruits and vegetables contain vitamin C, but cooking destroys the vitamin, so raw citrus fruits and their juices are the main source of ascorbic acid for most people.

One way to determine the amount of vitamin C in food is to use a redox titration. The redox reaction is better than an acid-base titration since there are additional acids in a juice, but few of them interfere with the oxidation of ascorbic acid by iodine.

The chemistry works like this: Iodine reacts with vitamin C. When this happens, both the iodine and vitamin C turn into different chemicals. If a sample contains vitamin C, iodine added to the sample will react with it and no longer be iodine. At the same time, the vitamin C that reacts with the iodine also is no longer vitamin C. When the amount of iodine added to the sample is greater than the amount of vitamin C that was there, all the vitamin C will be destroyed and there will be some iodine left over. So to find out how much vitamin C is in a sample, you can add small amounts of iodine, until the iodine you add no longer disappears as

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soon as it is added. The starch plays another role. Starch and iodine can pair up to make a dark blue material. In this combination, the iodine can still react with vitamin C. When that happens, the starch-iodine pair does not exist any more (because the iodine is gone), so the dark blue color disappears as well. So if you add the starch-iodine solution to the sample with vitamin C, the blue color of the sample will disappear until you have added enough iodine to react with all of the vitamin C. When that happens, the blue color will persist. It is much easier to see the blue color of the starch-iodine solution than to see the faint yellow color of a weak iodine solution. So the starch acts as an "indicator" for iodine. It makes it easy to see if any iodine is in the sample.

Iodine is relatively insoluble, but this can be improved by complexing the iodine with iodide to form triiodide:

I2 + I- <--> I3-

Triiodide oxidizes vitamin C to form dehydroascorbic acid:

C6H8O6 + I3- + H2O --> C6H6O6 + 3I- + 2H+

As long as vitamin C is present in the solution, the triiodide is converted to the iodide ion very quickly. However, when all the vitamin C is oxidized, iodine and triiodide will be present, which react with starch to form a blue-black complex. The blue-black color is the endpoint of the titration.

This titration procedure is appropriate for testing the amount of vitamin C in vitamin C tablets, juices, and fresh, frozen, or packaged fruits and vegetables. The titration can be

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performed using just iodine solution and not iodate, but the iodate solution is more stable and gives a more accurate result.

Purpose

The goal of this laboratory exercise is to determine the amount of vitamin C in samples, such as fruit juice.

Procedure

Preparing Solutions

1% Starch Indicator Solution

1. Add 0.50 g soluble starch to one-liter near-boiling distilled water. 2. Mix well and allow to cool before use.

Iodine Solution (Lugol solution can also work for this) 1. Dissolve 5.00 g potassium iodide (KI) and 0.268 g potassium iodate (KIO3) in 200 mL of distilled water. 2. Add 30 mL of 3 M sulfuric acid. 3. Pour this solution into a 500 mL graduated cylinder and dilute it to a final volume of 500 mL with distilled water. 4. Mix the solution. 5. Transfer the solution to a 600 mL beaker. Label the beaker as your iodine solution.

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Vitamin C Standard Solution

1. Dissolve 0.250 g vitamin C (ascorbic acid) in 100 mL distilled water. 2. Dilute to 250 ml with distilled water in a volumetric flask. Label the flask as your vitamin C standard solution.

Standardizing Solutions

1. Add 25.00 ml of vitamin C standard solution to a 125 mL Erlenmayer flask. 2. Add 10 drops of 1% starch solution. 3. Rinse your buret with a small volume of the iodine solution and then fill it. Record the initial volume. 4. Titrate the solution until the endpoint is reached. This will be when you see the first sign of blue color that persists after 20 seconds of swirling the solution. 5. Record the final volume of iodine solution. The volume that was required is the starting volume minus the final volume. 6. Repeat the titration at least twice more for the sample. The results should agree within 0.1 mL. Experiment V Benedict's Test for Reducing Sugars

The Benedict's test allows us to detect the presence of reducing sugars (sugars with a free aldehyde or ketone group). All monosaccharides are reducing sugars; they all have a free reactive carbonyl group. Some disaccharides like maltose have exposed carbonyl groups and are also reducing sugars (less reactive than monosaccharides). Other disaccharides such

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as sucrose are non-reducing sugars and will not react with Benedict's solution. Starches are also non-reducing sugars. Note that the -1,4 linkage will prevent the left-hand ring from opening up. In order to open up, the red C-O bond must break, which requires that the hydrogen that was on the green oxygen goes back to the red oxygen, but it's not there anymore. Let's use maltose as a specific example. The left-hand part of the molecule is stuck in the ring position. The right-hand ring, on the other hand, still can open because the hydrogen on the #1 OH can and will move back to the oxygen in the ring, thus, opening the ring and forming the double bond of the aldehyde group. C5 \ / C1 C4 / \ / \ C2 O C3 O

So, one ring can continue to open and this disaccharide can continue to act as a reducing sugar. However, one ring is stuck in the closed position because the hydrogen that originally came from the #5 OH and moved to the #1 OH when the ring closed was lost in the dehydration reaction and is not available to move back and open the ring. Note that only half of the glucose rings in maltose can open and close and form the double bond that allows for the reducing reaction. Consequently, maltose and other similar disaccharides will only reduce half as quickly and half as much as an equal weight of or other similar monosaccharides. That's something to keep in mind when you do your experiment for this laboratory. SUCROSE: When these two OH's react by an enzyme-catalyzed dehydration reaction, the resulting product is sucrose.

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+ H2O

The glycosidic bond for the sucrose is sometimes referred to as an - -1-2 bond, because there's an alpha-OH from the glucose bonding to a beta-OH from the sucrose, and we're going from the #1 carbon on the glucose to the #2 carbon on the fructose. There are a few other ways of indicating this designation, (-1)( -2) is probably the most descriptive, but they all try to say the same kind of thing, that we're dealing with the #1-OH in the alpha position bonded to the #2-OH in the beta position.

Non-reducing sugar Sucrose is an unusual disaccharide in that it is a nonreducing sugar. This is because both of the hydrogen atoms removed in the dehydration reaction came from OH groups that were formed during ring closure. Consequently, neither of the rings is able to open. The copper sulfate (CuSO4) present in Benedict's solution reacts with electrons from the aldehyde or ketone group of the reducing sugar to form cuprous oxide (Cu2O), a redbrown precipitate.

27

CuSO4

Cu++ + SO4-Cu+

2 Cu++ + Reducing Sugar (electron donor) Cu+ Cu2O (precipitate)

The final color of the solution depends on how much of this precipitate was formed, and therefore the color gives an indication of how much reducing sugar was present. Rating Scale: Increasing amounts of reducing sugar

blue (-)

green (+)

orange (++) (+++)

red

reddish brown (++++)

To Test For Sugar: 1) Add 2 ml of unknown/control solution. 2) Add 1.5 ml of Benedicts solution (WEAR GLOVES + GOGGLES!!). 3) Mix gently. Make sure tube is labelled on the top as the label will soak in water! 4) Place test tube in a boiling water bath. 5) Record the color development in 3-5 min using the rating scale. LITERATURE CITED 1.http://apbio.savithasastry.com/Units/Unit%201/labs/chp5_macromolecule_lab.doc(Cited: 06.01.2009) Experiment VI

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Starch Test For Polysaccharides Amylose in polysaccharides is responsible for the formation of a deep blue color in the presence of iodine. The iodine molecule slips inside of the amylose coil. To Test For Starch: 1) Add 1 ml of unknown/control in a test tube. 2) Add 1 drop of Iodine solution 3) Record color development against a white paper placed behind the test tube. Brown (-) Blue-Black (++++)

LITERATURE CITED 1.http://apbio.savithasastry.com/Units/Unit%201/labs/chp5_macromolecule_lab.doc(Cited: 06.01.2009)

Experiment VII Biurets Test for protein determination: The Biuret Reagent is made of sodium hydroxide and copper sulfate. The blue reagent turns violet in the presence of proteins, and changes to pink when combined with short-chain

29

polypeptides. In this test for proteins there is a reaction between the copper ions and the amino groups in the peptide bonds. To test for proteins: 1) Add 2 ml of unknown/control solution to a test tube. 2) Add 4 drops of Biurets Reagent (WEAR GLOVES AND GOGGLES!!). 3) Mix gently. 4) Record color development against a white background. Blue (-) Slight Purple/Pink (+) Violet (++++)

REFERENCES 1.http://apbio.savithasastry.com/Units/Unit%201/labs/chp5_macromolecule_lab.doc(Cited: 06.01.2009)

Experiment VIII Brown paper test for fats: Lipids make a translucent spot on a brown paper bag. 1) Rub the unknown/control on a brown paper bag mark the spot and label. 2) Dry thoroughly a) at room temperature for 30 min. b) at 120 C for 30 min.

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3) Hold against light. 4) Record appearance of translucent spot. No spot (-) REFERENCES 1.http://apbio.savithasastry.com/Units/Unit%201/labs/chp5_macromolecule_lab.doc(Cited: 06.01.2009) Experiment IX DNA Spooling only performed with the fruit strawberry or kiwi! The liquid detergent causes the cell membrane to break down and dissolves the lipids and proteins of the cell by disrupting the bonds that hold the cell membrane together. The detergent causes lipids and proteins to precipitate out of the solution. This is filtered out in the cheese cloth. Next, NaCl enables nucleic acids to precipitate out of an alcohol solution because it shields the negative phosphate end of DNA, causing the DNA strands to come closer together and coalesce. Translucent Spot (++++)

Procedure: 1. Add 2 grams of table salt (NaCl) and 10 mL of cleaning detergent (any brand) into a 100-mL measuring cylinder containing 90 mL distilled water. contents completely. This is the cell-lysis buffer. Swell to mix the

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2. Cut your fruit into small slices (about 10 mm thick) and smush it in a zipped bag for 2 min. Add 2 mL of the cell lysis buffer and mush it again for 1 min. Some fruits may need blending. [Warning: too much blending would shear the DNA molecules.] 3. Filter the slurry from the liquid through a cheese cloth (sitting in a filter funnel) into a 100-mL beaker. Each group should collect about 3 mL. [This filtering separates the cell wall material and protein (remains in the cheese cloth) from DNA, which is now in solution.] 4. Add equal volume (3 mL) of ice-cold ethanol slowly onto the surface of the fruit extract carefully. [The ethanol must be ice cold - kept in the freezer overnight beforehand.] 5. Immerse a bamboo skewer with a bent tip at the interface of the two layers (alcohol and fruit layer), stir slowly and continuously in a small circle (clockwise direction) to collect the DNA thread at the tip of the Pasteur pipette. [DNA doesn't dissolve in ethanol - it comes out of the lower layer into the upper layer. DNA has the appearance of white mucus.] LITERATURE CITED 1.http://apbio.savithasastry.com/Units/Unit%201/labs/chp5_macromolecule_lab.doc (Cited: 06.01.2009)

Experiment X Thin-layer chromatography

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Thin layer chromatography, which is typically abbreviated as TLC, is a type of liquid chromatography that can separate chemical compounds of differing structure based on the rate at which they move through a support under defined conditions. TLC is useful in detecting chemicals of security concern, including chemical weapons, explosives, stabilizing chemicals for rocket propellants, and illicit drugs. For example, the Forensic Service Center of Lawrence Livermore National Laboratory has designed a computerized and portable TLC machine that can be taken to the field, and which has the ability to analyze 20 samples at a time. Analysis can be completed within 30 minutes. TLC as it is still practiced today was introduced by Justus Kirchner in 1951. From its beginning, the technique was an inexpensive, reliable, fast, and easy to perform means of distinguishing different compounds from each other. The method was qualitativeit showed the presence of a compound but not how much of the compound was present. In the late 1960s, TLC was refined so that it could reliably measure the amounts of compounds. In other words, the technique became quantitative. Further refinement reduced the thickness of the support material and increased the amount of the separating material that could be packed into the support. In High Performance TLC (HPTLC) the resolution of chemically similar

compounds is better than with conventional TLC, and less sample is required. HPTLC requires specialized analysis equipment, and so is still not as popular or widespread as conventional TLC. In TLC a solution of the sample is added to a layer of support material (i.e., grains of silica or alumina) that has been spread out and dried on a sheet of material such as glass. The support is known as the plate. The sample is added as a spot at one end of the plate. The plate is then put into a sealed chamber that contains a shallow pool of chemicals (the solvent), which is just enough to wet the bottom of the plate. As the solvent moves up through the plate support layer by capillary action, the sample is dragged along. The different chemical

33

constituents of the sample do not move at the same speed, however, and will become physically separated from one another. The positions of the various sample constituents and their chemical identities are determined by physical methods (i.e., ultraviolet light) or by the addition of other chemical sprays that react with the sample constituents. i. Adsorbent: The most common adsorbent used is Silica gel (silicic acid combined with a small amount of gypsum as a binding agent). Many other material have also proven useful for specific purposes; for example, alumina for the separation of steroids and water soluble vitamins and Kieselgur for separation of sugars. ii. Solvents: Although it is frequently possible to select a good solvent system by the empirical methods, some time and effort may be saved by consideration of some principles of the chromatographic separation. Of particular importance in TLC are the elutropic series of solvents. This is a series of solvents arranged in the order of their eluting power; that is having increasing ability to remove compounds from an adsorbent. A knowledge of the relative adsorbent characteristics of the class of compounds to be separated is also important. For example, saturated hydrocarbons are adsorbed poorly while unsaturated hydrocarbons are adsorbed according to the increase in number of their double bonds.

iii. Development: The apparatus used for development must have certain features:

34

a)be capable of retaining any of a multitude of mixtures of original solvents. b)be air tight, to insure maintenance of a solvent saturated atmosphere.

c) be relatively easy to handle. iv. Visualization: Compounds separated can be visualized by: a)natural colour. b)spray reagents; yields coloured derivatives on reaction with the separated spots. c) iodine vapour. d) fluorescence and UV absorption. e) autoradiography.

EXPERIMENTAL PROTOCOL Handle the plates carefully. exposing to I2 vapour. 1. Using the template provided, mark the plates with a sharp pencil, as shown below(in the figure). 2. Line the chamber with chromatography paper. Prepare 202 mL of solvent system (Hexane: Ether: Acetic acid 60:40:1) in a 500 mL Erlenmayer flask. Mix and pour ~150 mL into the chamber. Cover and let the chamber saturate while loading the plates. 3. With a 10 L capillary pipette, spot 1-2 L of chlorophyll standard onto the TLC plate, as shown. Make sure the spot remains smaller than 4 mm in diameter. Move on to the other standards. After the spots have dried, repeat loading each standards until you have loaded NO FINGER PRINTS. These will be shown after

35

approx. 10 L each. Also, load 10 L of your lipid extract on one spot, and then the remainder of the extract as a line (i.e., a series of spots). 4. Let dry the spots. Make sure that the loading area is above the solvent. Place the plates in the chamber to develop. 5. Immediately close the cover and let run for approximately 30 min, until the solvent front has reached the upper line. 6. Remove the plate and leave to dry in the rack in the fume hood. Discard the solvent in the waste container provided, remove the chromatography paper and leave in the chamber. Leave the chamber in the fume hood to dry. 7. Now place the plate in the iodine tank in the fume hood. You will see the lipids as yellow spots after about 5 min or so. 8. Mark the edges of the spots with a pencil. Make a tracing on onion skin paper for a record.

9. Scrap off lipid fractions as shown, place in weighing paper, fold and roll to grind the clumps. 10. Meanwhile prepare columns to elute the lipids from silica gel. To do this insert a small amount of glass wool into a pasteur pipette, label the pipette and leave on the stand provided. 11. Carefully transfer the silica gel having different lipids into appropriate columns. Keep 7 mL glass vials beneath the columns. 12. Drip 1 mL of chloroform into each column except for the chlorophyll columns. To the latter, add >1 mL of 100 % methanol. 36

13. Shake the vials well. Place them in the fume hood evaporating set-up and evaporate off the solvent under a stream of N2(Ask the instructor before using the N2 tank). 14. Store the dry lipids under N2. 15. Label and leave the separated lipid in the -20 C freezer until transmethylation (day 2).

Results You should have obtained a good separation of the lipid classes, similar to the plate shown below. However, differences in the solvent composition will lead to altered mobility of the different components. A common problem occurs when the TLC tank is not

completely sealed, and the solvents can slowly evaporate. Since evaporation rate depends on the boiling point, solvents like hexane will evaporate faster. As a result, the mobile phase becomes more polar. Nevertheless, a positive identification of your spots should always be possible since you ran standards on the same plate as well.

LITERATURE CITED: URL 1: http://www.espionageinfo.com/Te-Uk/ThinLayer-Chromatography.html(Cited 06.01.2009) 37

URL

2: http://www.sfu.ca/bisc/bisc-429/TLC.html(Cited 06.01.2009)

EXPERIMENT XI THE ELECTROPHORESIS Electrophoresis on polyacrylamide gels in the presence of detergent sodium dodecyl sulfate[CH3 (CH2)10 CH2 OSO3 Na], abbreviated SDS is a rapid and often employed technique for the determination of the molecular weights of polypeptide chains. In addition, the electrophoresis is a common laboratory technique used for separating DNA fragments. DNA samples are placed in a special gel and subjected to an electric field. Because DNA is negatively-charged, it moves toward the positive electrode. The DNA fragments that are shortest will travel farthest, while the longest fragments will remain closest to the origin. Using the same basic principles, electrophoresis can also be used to separate RNA and proteins. First, follow our step-by-step instructions to build a gel electrophoresis chamber using inexpensive materials from local hardware and electronics stores. Then follow the below procedure to simulate DNA electrophoresis using food colors from your kitchen pantry.

Materials you will need: Electrophoresis chamber, gel form and comb, Power supply that produces 50-150 volts.Agar Agar granules, Salt solution, Commercial food colors, Filter paper circles (cut out with hole punch), Tweezers, Masking tape, Water.

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MELTING THE AGAR Add 1 gram agar to 100 milliliters of water in a glass container(a small canning jar). Cover the container with plastic wrap. Pierce a small hole in the plastic for ventilation. Heat the solution in the microwave oven on high power until it comes to a boil. Watch the solution closely; agar foams up and boils over easily. Remove the container (protect your hand with a pot holder or folded paper towel) and gently swirl it to re-suspend any settled agar. Continue this process until the agar dissolves completely. Cool the agar until you can comfortably touch the flask. POURING THE GEL Place tape across the ends of the gel form and place the comb in the form. Pour cooled agar into the form. The agar should come at least half way up the comb teeth. Immediately rinse and fill the agar flask with hot water to dissolve any remaining agar. When the agar has solidified, carefully remove the comb. Remove the tape from the ends of the gel form. LOADING THE SAMPLES Make a written record of which sample you will load in each well of the gel. You may find it helpful to load samples in every other well. Place the gel form on a black or dark surface to help you see the wells in the agar. Fold filter paper circle in half and hold sideways using tweezers. Dip filter paper into full-strength food color to saturate. Gently ease the filter paper into the well. Be careful not to puncture the bottoms of the wells as you load each sample. Repeat for remaining colors. Setting up the Gel Chamber

39

Place the gel in the electrophoresis chamber. Make sure that the wells are closest to the negative (black) electrode. To prepare the buffer, add a pinch of salt to one liter of tap water, deionized water, or distilled water (the water source that works for you may depend on your local water quality) and swirl to dissolve. Fill each half of the chamber, adding solution until it is close to the top of the gel. Gently flood the gel from the end opposite the wells to minimize sample diffusion. Place the lid on the chamber and connect the electrode leads to the power supply. Connect the black lead to the negative terminal and the red lead to the positive terminal. Running and Analyzing the Gel Turn on the power supply and adjust the voltage to 50-100 volts.

Run the gel for 5-10 minutes. Once the dyes have moved through the gel, turn off the power supply, disconnect the electrode leads, and remove the chamber lid. Remove the gel from the electrophoresis chamber and analyze your results. Did some colors move further than others? Did some colors separate into two?

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REFERENCES: 1. Arn, E. (1985) Experimental Biochemistry Laboratory Manual, Middle East Technical University, Department of Biological Sciences, Ankara, TURKEY. 2. URL 1: http://learn.genetics.utah.edu/content/labs/gel/electrophoresis/ (Cited:

06.01.2009) Experiment XII: The effect of glucose concentration on the growth of bakers yeast under aerobic and anaerobic conditions

In this experiment you will be able to observe the effect of glucose concentration on yeast growth. Different glucose concentrations (1% and 10%) were added to yeast extract broth and the growth will be monitored under: a) aerobic conditions b) anaerobic conditions(in anaerobic jar)

At the end you are expected to compare the ethanol production and the pH.

Experiment XIII: The effect of starch and aspartame concentration on the growth of bakers yeast under aerobic and anaerobic conditions

In this experiment you will be able to observe the effect of starch and aspartame concentration on yeast growth. Different glucose and fructose concentrations (1% and 10%) were added to yeast extract broth and the growth will be monitored under:

41

a)aerobic conditions b)anaerobic conditions(in anaerobic jar) At the end you are expected to compare the ethanol production and the pH.

Experiment XIV Spectrophotometry Spectrophotometry is an instrument used for measuring the transmission or reflection of light by comparing various wavelengths of the light. Also it is regarded as an analytic technique by which chemical substances are identified by sorting gaseous ions by mass using electric and magnetic fields. Different types of spectrophotometry is available like visible (360-700 nm wavelength), UV-visible spectrophotometry, atomic absorption

spectrophotometry, mass spectrophotometry, Fourier transform infrared spectrophotometry etc. For example, a mass spectrometer uses electrical means to detect the sorted ions, while a mass spectrograph uses photographic or other nonelectrical means; either device is a mass spectroscope. The process is widely used to measure masses and relative abundances of different isotopes, to analyze products of a separation by liquid or gas chromatography, to test vacuum integrity in high-vacuum equipment, and to measure the geological age of minerals. In analytical chemistry, atomic absorption spectroscopy is a technique for determining the concentration of a particular metal element in a sample. Atomic absorption spectroscopy can be used to analyze the concentration of over 62 different metals in a solution. Mass spectrometry begins by ionizing the molecules in the target sampleremoving one or more electrons to give them a positive charge. Molecules must be charged so they can be accelerated. The principle is the same as that used in a television or fluorescent light bulb: Charged particles are accelerated by being pulled toward something of the opposite charge. 42

In the mass spectrometer, the speed the molecules attain during acceleration is proportional to their mass (actually, their mass-charge ratio). By determining the speed of the molecules, researchers can calculate their mass. Turbid samples have large backgrounds which make obtaining accurate absorbance readings very difficult. The more turbid the sample, the more incorrect the answer obtained by an ordinary spectrophotometer will be. And, one cannot know that the results are incorrect with the ordinary spectrophotometer without further quantitative measurements. For many current applications, a spectrometer is increasingly becoming the measurement device of choice. Unlike other methods which give "single point" measurements for each calibration and unknown sample (i.e., pH, or single element Atomic Absorption), the spectrum of a sample contains many data points. Every response value in a spectrum has some relation to the properties or constituent(s) that make up the measured sample. Using a spectrum of a sample that has many data points has some distinct advantages over single point measurement techniques. One of the most important factors is that there are many more measurements per sample (spectral data points) to use in generating the calibration equations. As anyone who has performed quantitative analysis knows, the more measurements per sample, the more accurate the results. The problem for the analyst is to discover what those relationships are, and use a calibration model that reflects them accurately. One advantage of using spectroscopy as a measurement technique is that the Beer-Lambert Law (also know as Beer's Law) defines a simple linear relationship between the spectrum and the composition of a sample. This law, which should be familiar to all spectroscopists, forms the basis of nearly all other chemometric methods for spectroscopic data. Simply stated, the law claims that when a sample is placed in the beam of a spectrometer, there is a direct and linear relationship between the amount (concentration) of its constituent(s) and the amount of energy it absorbs. In mathematical terms:

43

Where ,

is the samples Absorbance value at specific wavelength (or frequency) ,

is the absorptivity coefficient of the material (constituent) at that wavelength, b or ( l ) is

the pathlength through the sample(in cuvette) and C is the concentration. The absorptivity coefficient for every material is different, but for a given compound at a selected wavelength, this value is a constant. The only problem that remains is to discover the "real" value of that constant.

In this experiment you will be able to learn about the basics of an ordinary spectrophotometer. The sample is the sour cherry juice; and you are responsible to meet with the spectrophotometry by means of the original juice and its diluted form(50 mL sour cherry juice will be diluted to 1:1; 1:5; 1:20 and 1:100) and it will be monitored through the spectrophotometer by recording its absorbance.

LITERATURE CITED 1. http://www.olisweb.com/products/upgrades/dw2.php(Cited 05.08.2009) 2. http://www.yourdictionary.com/spectrophotometer (Cited 05.08.2009)

Experiment XV Protein determination by Lowry method The Lowry protein assay method for protein concentration determination is one of the most venerable and widely-used protein assays. The Lowry method was first described in 1951 by Lowry et al. (Lowry et al., 1951). 44

Hydrolysis is probably the most accurate method of determining protein concentration followed by amino acid analysis. Most other methods are sensitive to the amino acid

composition of the protein, and absolute concentrations cannot be obtained. The Lowry procedure is sensitive, and is moderately constant from protein to protein. The Lowry protein estimation has been so widely used that it is a completely acceptable alternative to a rigorous absolute determination in almost all circumstances in which protein mixtures or crude extracts are involved. The method is based on both the Biuret reaction, in which the peptide bonds of proteins react with copper under alkaline conditions to produce Cu+, which reacts with the Folin reagent, and the FolinCiocalteau reaction, which is poorly understood but in essence phosphomolybdotungstate is reduced to heteropolymolybdenum blue by the coppercatalyzed oxidation of aromatic amino acids. The reactions result in a strong blue color, which depends partly on the tyrosine and tryptophan content. The method is sensitive down to about 0.01 mg of protein/mL, and is best used on solutions with concentrations in the range 0.011.0 mg/mL of protein. Equipment and Reagents: Spectrophotometer Solution A Solution B Solution C Bovine Serum Albumin(BSA) Procedure Modified Lowry Method 45

Solution A: Weigh out 2 g Na-K-tartrate and 100 g Na2CO3 and dissolve in 500 mL 1N NaOH and complete with distilled water to 1 liter. Solution B: Weigh out 2 g Na-K-tartrate and 1 g CuSO4.5H2O, dissolve in 90 mL distilled water and add 10 mL 1N NaOH. Solution C: Mix Folin Ciocalteu reagent with distilled water(1:15) Dilute the protein sample to 1 mL with distilled water and add 0.9 mL solution A. Allow the test tubes in a water bath at 50 C for 10 minutes. Cool to room temperature and add 0.1 mL solution B and leave at room temperature at least 10 min. Add 3 mL solution C rapidly to ensure mixing within 1 second, heat the tubes again at 50 C for 10 minutes and cool to room temperature. Read the absorbance at 650 nm. Prepare a standard curve using bovine serum albumin(BSA) as a stock solution. LITERATURE CITED URL 1: http://www.molecularstation.com/protein/lowry-protein-assay/ (Cited 07.01.2009) Experiment XVI Heat Transfer

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In this experiment you will be able to observe the heat conduction and heat convection, which are the milestones of the thermodynamic principles. Procedure Heat up 250 mL of water at the cooking pan for a)1 min b) 3 min c) 5 min d) 10 min and record the temperature 1) at the inner portion of the lid b) at the inner portion of the bottom. Experiment XVII The detection of proteins in the urine Proteins are normally not present in the urine. The normal concentration of proteins in urine is around 0-10 mg/dL. Sometimes proteins may be present in the urine due to defects of the metabolism. The kidney does two important jobs in the body. It filters out the waste products in the blood so it can be released in the form of urine. It also reabsorbs those materials the body still needs which got past the original filtering system. However, there are certain things in the blood which do not get past the filtering system of the kidney simply because they are too big. The red and white blood cells are a good example. These cells are entirely too large to pass through the tiny "holes" of the filter. This is good thing because we would otherwise need a blood transfusion every time we went to the bathroom. Proteins are large molecules which help make up our muscles, important parts of our immune system, and many other portions of our bodies. Most proteins are also too large to pass through the filtering system of the kidney. And since they are not supposed to pass into the kidney, there is no mechanism for proteins to be reabsorbed if they make it in there. Therefore, if protein is detected in the urine, it means there is something going on with the filter (called the glomerulus) that is allowing the proteins to pass. Proteins give pink color with the strong acids like nitric acid. The aim of this experiment is to detect the proteins in the urine.

47

Materials : fresh urine, test tube, pipette tips, strong acid, gloves. Method: 1. Put 5 mL of fresh urine into a test tube. 2. Take 2 mL from the nitric acid by a pipette. 3. Dip the pipette containing nitric acid into the test tube containing the nitric acid. Discard the pipette containing nitric acid into the tube. 4. Do not mix the tube. 5. Remove the pipette tip from the tube. 6. After a minute, a pink ring was formed just in the middle of the test tube. 7. This means that the urine contains protein.

Experiment 18 The dialysis of the proteins:

Introduction: The protein sample, in solution, is sealed in the semi-permeable membrane bag, manufactured with varying pore sizes which is then placed in a large volume of buffer, normally 200 500 times the volume of the sample. Due to the gradient between the sample and the buffer, the molecules in the sample attempt to flow from the dialysis bag where only those smaller than the pore size actually leave the sample. The power of dialysis lies in its ability to cleanse a sample of a number of contaminants at once, including small proteins, and small contaminating molecules such as salt.

48

The key to a successful separation based on dialysis is the selection of an optimal pore size. Membrane pore sizes are typically measured in Daltons such that pores measuring 1,000 Daltons will retain molecules a molecular mass of 1,000 Daltons. Various dialysis methods are commonly employed for the crystallization of proteins. Typical procedures include the use of dialysis bags and the dialysis buttons. The general principle involved is that the protein solution is gradually brought to a point of supersaturation by imposing a gradient of ionic strength or organic solvent concentration across the wall of the dialysis membrane. However, in some cases, the imposition of this gradient across the dialysis membrane can result in the formation of a large number of crystal nucleation sites, thereby giving rise to a reduction in the maximum size of the crystals which can be obtained. Materials: 1. Intestine. 2. A piece of comfort string. 3. The protein source, mentioned by your laboratory research assistant.

Method: 1. Put adequate amount of distilled water into a beaker according to the size of the intestine that you have used. 2. Pour a definite(known) amount of milk into the intestine and tighten it closely by the comfort string. 3. Watch the motion of the molecules outside the membrane of the intestine by turbidity. 4. Compare the untreated milk with the dialyzed milk according to its protein concentration by Biuret method.

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LITERATURE CITED: 1.http://en.wikibooks.org/wiki/Proteomics/Protein_Sample_Preparation/Contaminant_Elimin ation

Experiment 19: The ammonium sulfate precipitation of the proteins: Introduction:

Ammonium sulfate precipitation is a method of protein purification by altering solubility of protein. It is a specific case of a more general technique known as salting out. Ammonium sulfate is commonly used as its solubility is so high that salt solutions with high ionic strength are allowed.

The solubility of proteins varies according to the ionic strength of the solution, and hence according to the salt concentration. Two distinct effects are observed: at low salt concentrations, the solubility of the protein increases with increasing salt concentration (i.e. increasing ionic strength), an effect termed salting in. As the salt concentration (ionic

strength) is increased further, the solubility of the protein begins to decrease. At sufficiently high ionic strength, the protein will be almost completely precipitated from the solution (salting out). Since proteins differ markedly in their solubilities at high ionic strength, salting-out is a very useful procedure to assist in the purification of a given protein. The commonly used

50

salt is ammonium sulfate, as it is very water soluble, has no adverse effects upon enzyme activity. It is generally used as a saturated aqueous solution which is diluted to the required concentration, expressed as a percentage concentration of the saturated solution (a 100% solution). In the preliminary test, the ammonium sulfate concentration is increased stepwise, and the precipitated protein is recovered at each stage. This is usually done by adding solid ammonium sulfate, but calculating how much ammonium sulfate to add to a solution at one concentration to achieve a desired higher concentration is tricky, since addition of ammonium sulfate significantly increases the volume of the solution. The amount to add can be

determined either from published nomograms or by using an online calculator. Each protein precipitate is dissolved individually in fresh buffer and assayed for total protein content and amount of desired protein. The aim is to find the ammonium sulfate concentration which will precipitate the maximum proportion of undesired protein, whilst leaving most of the desired protein still in solution.

The precipitated protein is then removed by centrifugation and then the ammonium sulfate concentration is increased to a value that will precipitate most of the protein of interest whilst leaving the maximum amount of protein contaminants still in solution. The

precipitated protein of interest is recovered by centrifugation and dissolved in fresh buffer for the next stage of purification.

This technique is useful to quickly remove large amounts of contaminant proteins, as a first step in many purification schemes. It is also often employed during the later stages of purification to concentrate protein from dilute solution following the procedures such as gel filtration.

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Material: 1. The protein source, mentioned by your laboratory research assistant. Method: 1.Take aliquots of the extract, ideally 20-50 mL place in beakers and pre-chill to 4 C. 2.Calculate the amount of ammonium sulphate required to give 20, 30, 40, 50, 60, 70 and 80% saturation from the equation given in the ammonium sulfate precipitation table. Weigh out the required amounts of ammonium sulphate and ensure all lumps are removed(use a pestle and mortar).

3.Slowly add the ammonium sulphate to each aliquot whilst stirring(use a magnetic follower and stirrer). Leave each aliquot stirring for one hour at 4 C.

4.Centrifuge each aliquot at 3000 g for 40 minutes.

5.Remove the supernatants and drain the pellets.

Dissolve the pellets in buffer(e.g.

phosphate-buffered saline(PBS) or 50 mM Tris-HCl, pH=8.). Use the same volume for each pellet, approximately twice the volume of the largest pellet.

6.If undissolved material remains, centrifuge at 3000 g for 15 minutes.

7. Assay the supernatants for total protein using by Biuret(Dialyse the supernatants if ammonium sulfate interferes with the assay).

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8.Plot the concentration of total protein and the protein of interest against % saturation of ammonium sulphate. For an ammonium sulphate cut choose the maximum % saturation which does not precipitate the protein of interest, and the minimum % saturation that precipitates all the protein of interest.

The process will be repeated in the coming week with the denatured proteins by pH or temperature. LITERATURE CITED: 1.http://www.absoluteastronomy.com/topics/Ammonium_sulfate_precipitation(cited:05.05.20 09) 2. Harris, E.L.V. and Angal, S. Protein purification methods:A practical approach. IRL Press Inc. Oxford, 1993.

EXPERIMENT 20 PROTEIN DENATURATION IN EGGS

Protein molecules carry out many important tasks in living systems. Most important, the majority of proteins are quite specific about which task they perform. Protein structure is what dictates this specificity, and the three-dimensional (tertiary) structure is particularly important. When this specific three-dimensional structure is disrupted, the protein loses its functionality and is said to have undergone denaturation. The interactions, such as hydrogen bonding, that dictate the tertiary structure of proteins are not as strong as covalent chemical bonds. Because these interactions are rather weak, they can be disrupted with relatively modest stresses.

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If a solution containing a protein is heated, it will reach a temperature at which properties such as viscosity or the absorption of ultraviolet (UV) light will change abruptly. This temperature is called the melting temperature of the protein (because the measurement is analogous to that made for the melting of a solid). The melting temperature varies for different proteins, but temperatures above 41C (105.8F) will break the interactions in many proteins and denature them. This temperature is not that much higher than normal body temperature (37C or 98.6F), so this fact demonstrates how dangerous a high fever can be. A familiar example of heat-caused denaturation are the changes observed in the albumin protein of egg whites when they are cooked. When an egg is first cracked open, the "whites" are translucent and runny (they flow like a liquid), but upon heating they harden and turn white. The change in viscosity and color is an indication that the proteins have been denatured. Factors other than heat can also denature proteins. Changes in pH affect the chemistry of amino acid residues and can lead to denaturation. Hydrogen bonding often involves these side changes. Protonation of the amino acid residues (when an acidic proton H + attaches to a lone pair of electrons on a nitrogen) changes whether or not they participate in hydrogen bonding, so a change in the pH can denature a protein.

An egg white before the denaturation of the albumin protein causes the transucent substance to change in color and viscosity.

The heat-caused denaturation in albumin protein in egg whites causes the once translucent, runny substance into one that is white and firm.

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Changes in salt concentration may also denature proteins, but these effects depend on several factors including the identity of the salt. Some salts, such as ammonium sulfate, tend to stabilize protein structures and increase the melting temperature. Others, such as calcium chloride, destabilize proteins and lower the melting temperature and are called chaotropic. Salts in this category can also be used in the laboratory to help purify proteins that are being studied, by lowering their solubility and causing them to "salt out." Experimental: Materials Eggs Cooking pan Tap water Lowry solution

Procedure: Boil some tap water and put some eggs into the water very after boiling. Observe the eggs with respect to an egg which is not boiled, served as control. Calculate the protein content by using Lowry solution with respect to control. LITERATURE CITED http://www.chemistryexplained.com/Co-Di/Denaturation.html (Cited: 17.02.2011) EXPERIMENT 21 Determination of water holding capacity Water holding capacity was determined according to a method by Gould and others (1989). An amount of 3 g of the flour sample (dried) was weighed into a centrifugal tube, added with 30 mL distilled water and mixed using the vortex mixer (Vortex V1 Plus, Boeco, Germany) for 30 s. The sample was allowed to hydrate for 2 h at room temperature. This was followed by centrifugation using a bench top centrifuge (Kubota 5100 Bench Top Centrifuge, Fujioka, Japan) at 2800 rpm for 10 min. The supernatant was discarded and the

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hydrated

sample

was

weighed.

Results

were

expressed

as

LITERATURE CITED Gould JM, Jasberg BK, Cote GL. 1989. Structure-function relationships of alkaline-peroxide treated lignocellulose from wheat straw. Cereal Chem 66:2137.

EXPERIMENT 22 Determination of oil holding capacity Oil holding capacity was determined according to the method of Caprez and others (1986). A sample of 3 g flour (dried) was weighed into a centrifugal tube, added with 30 mL of corn oil, and mixed using a vortex mixer (Vortex V1 Plus, Boeco, Hamburg, Germany) for 30 s. The sample was allowed to stand for 1 h at room temperature. This was followed by centrifugation in a bench top centrifuge (Kubota 5100 Bench Top Centrifuge) at 2800 rpm for 10 min. The supernatant was discarded and the pellet was weighed. Results were expressed as:

LITERATURE CITED Caprez A, Arrigoni E, Amado R, Neukom H. 1986. Influence of different types of thermal treatment on chemical composition and physical properties of wheat bran. J Cereal Sci 4:233 9. 56

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