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LAB 25: PROTEINS THE CHEMISTRY OF CASEIN AND ALBUMIN PURPOSE: To isolate Casein (milk protein) the base for glue and paint. To denature albumin (egg protein). To perform color tests to identify the presence of proteins. SAFETY CONCERNS: Always wear safety goggles. Handle the concentrated acids (hydrochloric, nitric, and sulfuric) with care. Avoid skin contact. Wash your hands after use. Ninhydrin may be irritating to skin and mucous membranes. Most organic compounds, such as ethanol are flammable. AMINO ACIDS & PROTEINS: Proteins, essential components of the living cell, are high molecular weight compounds which, upon hydrolysis, produce amino acids. In a protein the amino acids are linked together by series of peptide bonds. A peptide bond is an amide linkage; it is the functional group which can be hydrolyzed (split apart by water). H H O H H O H H O H N C C N C C N C C OH H R R' 2H2O H R H+ H H O H H O H N C C OH R'' + H N C C OH H H O + H R' H N C C OH H R'' Humans cannot synthesize ten of the twenty common amino acids found in protein; these essential amino acids must be supplied in the diet. The presence of both the basic amino group (NH2-) and the acidic carboxyl group (-COOH) accounts for the fact that amino acids (or proteins) in water solution can act as either acids or bases. Substances that can act as either acids or bases are amphoteric. In this experiment you will use indicators (phenolphthalein and methyl orange) to illustrate the amphoteric properties of casein, the principle protein found in cow's milk. Methyl orange appears red in a solution having a pH less than 3, and yellow in a ® solution of pH greater than 4. The indicator phenolphthalein used to be used as a laxative in Ex-Lax ; it is colorless in a solution of pH less than 7.8 and pink in a solution having a pH greater than 9.6. ISOELECTRIC POINT: Amino acids (and proteins) contain basic amino groups; when an amino group accepts a proton in an acid-base reaction, the compound becomes positively charged. When the carboxylic acid group of an amino acid (or protein) donates a proton, the substance becomes negatively charged. At a specific pH, the compound will have a neutral charge (charge = 0); this pH is the isoelectric point of the protein (or amino acid). Each protein has a characteristic isoelectric point. At this pH, the molecules coagulate and are removed from solution. We will analyze Casein, the major storage protein in milk to determine its isoelectric point. CH106 Lab 25: Proteins (W16) 57 DENATURATION OF PROTEINS: The primary structure of a protein refers to the exact order in which the amino acids are linked together by means of peptide bonds. The three-dimensional configuration of segments of the protein chain is called its secondary structure; common secondary structures are the alpha-helix and the pleated-sheet. The secondary structure is formed when amino acids hydrogen bond to other amino acids farther along the polypeptide chain. The tertiary structure is the three-dimensional shape of the entire polypeptide chain. Globular proteins, for example, are very tightly folded into a compact spherical form. This folding results from interactions between the R side chains of amino acids, and may involve hydrogen bonding as well as disulfide bondings, salt bridges, and hydrophobic interactions. Only proteins containing more than one chain, such as hemoglobin, have a quaternary structure; the manner in which the several peptide chains fit together describes a protein's quaternary structure. If the secondary, tertiary, or quaternary structures of a protein are disrupted, the protein loses is biological activity and is said to be denatured. Denaturation may result in coagulation with the protein being precipitated from solution. The factors that may cause the denaturation of proteins are pH, heat, certain organic solvents, heavy metal ions, alkaloidal reagents, reducing agents, and non-ionizing radiation. In this experiment you will study the denaturation of albumin, the protein found in egg white. PROCEDURES: NOTES: ACTIONS: 1 I. Denaturation of Proteins: 1. Prepare a fresh egg albumin solution by mixing the white from one egg1 with 200 mL of deionized water.2 2. Obtain six separate test tubes and label them A through F. 3. Place 2-3 mLs2 of egg albumin solution into each of the tubes. This makes enough albumin protein supply for 2 to 3 teams of students. 2 If needed, filter through cheesecloth into a beaker to remove any solid impurities. 2 4. For each indicated tube perform the following: A. Heat: Using a test tube holder, heat the egg albumin solution A over a low flame. Acid: Add 2 mLs of 10% HNO3 (Nitric acid) to tube B.3 Base: Add 2 mLs of 10% NaOH (sodium hydroxide) to tube C.3 Alcohol: Add 4 mLs of 95% Ethanol (EtOH, C2H5OH) to tube D.3 Heavy Metal Ions: Add 10 drops of 1% AgNO3 (Silver nitrate) to tube E.3 F. Alkaloids/Tannins: Add 1 mL (20 drops) of 10% tannic acid solution to tube F.3 B. C. D. E. 5. Describe any changes. Give a brief explanation for the results. 58 CH106 Lab 25: Proteins (W16) The measurement does not need to be exact. You could measure one and eyeball the others to match. 3 It may be necessary to stopper the tube and shake to mix after each addition. II. Casein Products: A. Isolation of Casein from Milk: 1. Weigh a 150 mL beaker. Add about 50 mL of nonfat milk to the beaker and weigh again. Calculate the mass of the nonfat milk sample. 4 For best result use a pH meter or indicator paper having approximately a 4.5 to 8.5 pH range. 5 It may take from 4-6 mLs. 6 2. Record the pH of the nonfat milk. 4 3. Warm the sample on a steam bath until the temperature of the milk reaches about 40oC then remove from heat. 4. Add 5% acetic acid (CH3CO2H, vinegar) a little at a time5 with gentle stirring until the casein (milk protein) becomes insoluble. When no further precipitation occurs, stop adding acid. 6 5. Determine and record the pH at which the casein becomes insoluble in solution. This pH is the isoelectric point of casein. 7 You’ve made curds and whey by artificially souring the milk. When milk sours naturally, the souring and curdling are caused by lactic acid formed by the action of bacteria. 7 At the isoelectric point (pI) the number of positive charges on a protein equals the number of negative charges giving it a net charge of zero. It is at this pH that the protein becomes least soluble. 8 6. Separate the solid casein protein from the soluble whey protein by vacuum filtration through a Buchner funnel.8 7. Wash the solid casein in the Buchner funnel with two 10 mL portions of water with stirring to rinse away any remaining whey protein. 9 Press excess water from the casein with a spatula in the Buchner funnel and continue the vacuum to further dry the protein. 8. Weigh a watch glass. Transfer the protein to the watch glass and let the protein air dry. Weigh. Calculate the mass of milk protein. As an alternative to vacuum filtration through a Buchner funnel you could filter through cheesecloth or simply decant off the liquid leaving the solid casein behind in the beaker. 9 If not using vacuum filtration then stir in the wash water and decant off the liquid or filter through cheesecloth again. Squeeze dry by pressing the protein between pieces of filter paper or squeezing through the cheesecloth. 10 9. Calculate the percentage of casein in the nonfat milk. 10 (g’s of solid casein / g’s milk) x 100 = % 11 B. Casein Glue: 10. Mix a chunk of casein11 (about the size of a nickel) with several drops of water in a small beaker to make a paste. 11. Stir in a pea sized amount of NaHCO3, (sodium bicarbonate, baking soda) to neutralize any acid remaining from the vinegar and make the mixture alkaline.12 Watch for bubbles of CO2 gas produced. Add a little more sodium bicarbonate until no more bubbles appear. 12. The substance in the beaker is glue.13 Test the adhesive properties of your product with various materials. CH106 Lab 25: Proteins (W16) If the casein is too clumpy grind the water and casein together with a mortar and pestle to insure a good mix. 12 When dissolved in an alkaline solution casein becomes sticky so can be used as glue. 13 The glue you made is similar to non-toxic commercial white glue. You can make “fancy” glues of various colors by adding food coloring. 59 C. Casein Paint: Pigment Preparation: NOTES: 1. Prepare paint pigments according to the following instructions using the 13 chart of reagents below to produce the desired colors: Place REAGENT A _______ in a small test tube. Add about 3 mLs of warm water. Shake the tube gently until the reagent dissolves. Can add a couple more mLs of warm water if needed to dissolve. Add REAGENT B _______ to the tube. Stopper the tube and shake it thoroughly. Filter the solution. Discard the liquid filtrate and save the precipitated pigment on the filter paper. Pigment Reactants Product White A. 0.3g CaCl2 B. 0.3g Na2CO3 A. 0.3g K4Fe(CN)6 B. 0.2g CoCl2 A. 0.2g NH4Fe(SO4)2.3H2O B. 0.2g Na2CO3 A. 0.2g NH4Fe(SO4)2 B. 0.2g K4Fe(CN)6 A. 0.2g NH4Fe(SO4)2 B. 1.0mL sat Na2SiO3 A. 0.2g CoCl2 B. 1.0mL sat Na2SiO3 A. 0.2g CoCl2 B. 0.2g Na2CO3 Add powdered Iron (III) oxide to casein Add powdered charcoal to casein CaCO3 (blackboard chalk) Co2Fe(CN)6 Green (olive) Brown Blue (dark) Orange Blue (royal) Lavender Red Black Fe(OH) 3 KFe2(CN) 6 (Prussian Blue) Fe2(SiO3) 4 + Fe(OH)3 CoSiO3 CoCO3 Fe2O3 C Paint Preparation: 2. Chop the dried solid casein prepared in Part IA into small pieces to dry. Then grind it into a fine powder with a mortar and pestle or a kitchen blender. 3. Place a small amount of ground casein (enough to cover a penny) in an evaporating dish. Add a few drops of water and mix to make a thick paste. 4. Mix the casein and the prepared color pigment of choice with a wooden splint or spatula until the desired color is obtained adding more casein or pigment as needed. 5. Paint a picture.13 60 CH106 Lab 25: Proteins (W16) Casein paints are water-based paints that are inexpensive. Most water-based paints have casein glue in them to help them stick to the walls and ceilings. LAB 25: PROTEINS: NAME_____________ PRE LAB EXERCISES: DATE______________ 1.___ At the isoelectric point of a protein, A. the pH of the solution will be 7 B. the number of positive charges on the protein will equal the number of negative charges. C. the pH of the solution will always be greater than 7. D. the pH of the solution will always be less than 7. 2.___ At the isoelectric point of a protein, A. the protein will be hydrolyzed into amino acids. B. the protein will be most soluble in water. C. the protein will be least soluble in water. D. the protein will oxidize to form disulfide bonds. 3.___ If 1.5 g’s of casein were isolated from acidification of 50. g’s of nonfat milk what is the percent yield of casein in the milk sample? (Show your work) A. 3.0 % B. 1.5 % C. 75 % D. 33 % E. 0.033 % 4. After casein is isolated from milk sodium bicarbonate (NaHCO3) is added to neutralize any acid remaining from the vinegar. Write the balanced equation for the reaction accounting for the gas produced. Show Structures. 5. Complete and balance the equation for the double replacement reaction of Cobalt (II) chloride with Sodium carbonate to form Lavender paint pigment. CH106 Lab 25: Proteins (W16) 61 62 CH106 Lab 25: Proteins (W16) LAB 25: PROTEINS: NAME___________________ REPORT: PARTNER_________DATE___ I. Denaturation of Proteins: Protein Used: circle one Casein Whey Albumin Observations Explanation/Conclusion (If denaturation occurs explain how/why. Be specific. If denaturation is not visible then indicate.) A. Heat B. Acid C. Base D. Alcohol E. Heavy Metal Ion: Ag1+ F. Tannic Acid Summary/Explanation/Analysis: Why are the results as they are? CH106 Lab 25: Proteins (W16) Explain any anomalies. 63 II. Casein A. Isolation from Milk: Mass of Milk used Initial pH pI = Isoelectric point Explanation: Explain why the most precipitate occurs at the pI. g Observations Why does adding acid cause a precipitate? Mass of casein actually produced (= your actual yield) g Expected mass of casein Your experimental percentage of casein in nonfat produced (= theoretical yield). milk (show calculation & circle answer) (If milk is 3.5% protein and that protein is 82% casein and 18% whey, then what is the theoretical yield of casein from 50 g milk?) ( ( g’s casein produced) x 100 = g’s milk used ) % casein (show calculation & circle answer) Percent yield (show your calculations) ( ( g actual yield ) x 100 = g Theoretical yield) Explanation/Analysis/Conclusion: Why are the results as they are? % yield Explain any anomalies. B. Glue: Observations Glue a sample here: Explanation/Analysis/Conclusion: Why are the results as they are? How/why does casein act as glue? Explain any anomalies. 64 CH106 Lab 25: Proteins (W16) C. Paint: Paint color made: _____________________________ Chemical Equation for reaction of pigment made: Observations Paint a picture here: Explanation/Analysis: Why are the results as they are? Why use casein in paint? Explain any anomalies. CH106 Lab 25: Proteins (W16) 65 LAB 25: PROTEINS NAME____________ RELATED EXERCISES: DATE _____________ 1.___ Precipitation of a protein takes place when A. the pH is equal to 7 so the protein is neither acidic nor basic. B. the pH is equal to the isoelectric point (pI) because at this point the protein is negatively charged. C. the pH is equal to the isoelectric point (pI) because at this point the protein has no net charge. D. a solution of protein is hydrolyzed regardless of the pH. 2.___ Paints containing Lead compounds are poisonous because A. Lead is a heavy metal that denatures life supporting protein enzymes by complexing with SH side groups and thus changing their shape. B. Lead catalyzes the hydrolysis of proteins so that life supporting proteins are no longer available. C. Lead causes disulfide bridges (S-S) to form in globular proteins and so changes the protein’s shape. D. Lead is a heavy metal that causes persons who ingest or breathe them to weigh more. E. More than one of these. 3.___ Drops of a dilute solution of Silver nitrate (AgNO3) are applied to the eyes of newborn infants because: A. Silver is a heavy metal that will denature the baby’s eyes and cause them to open. B. Silver is a heavy metal that will denature the enzyme proteins of bacteria that may cause gonorrheal conjunctivitis. C. Silver is expensive and thus hospitals can charge patients more money. 4.___ Isopropyl alcohol a good disinfectant because A. B. C. D. alcohol acts as a solvent to dissolve bacteria. alcohol denatures the enzyme proteins of bacteria by disrupting the hydrogen bonds. alcohol hydrolyzes proteins so kills bacteria. More than one of these. 5. Tannic acid and picric acid are useful in commercial products for the treatment of burns. Explain why that may be. (Hint; think about the chemical make up of skin.) 6. Baby vomit looks and smells similar to Curds and Whey. Explain why. 66 CH106 Lab 25: Proteins (W16)