LAB 25: PROTEINS TE EMISTRY OF ASEIN AND ALBUMIN PURPOSE: To isolate asein (milk protein) the base for glue and paint. To denature albumin (egg protein). To perform color tests to identify the presence of proteins. SAFETY ONERNS: Always wear safety goggles. andle 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 AIDS & 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). N R O N R' O N R'' O O 2 2 O + N R O O O + N O + R' N O R'' O uman 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 (N - 2 ) and the acidic carboxyl group (-OO) 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 p less than 3, and yellow in a solution of p greater than 4. The indicator phenolphthalein used to be used as a laxative in Ex-Lax ; it is colorless in a solution of p less than 7.8 and pink in a solution having a p greater than 9.6. ISOELETRI 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 p, the compound will have a neutral charge (charge = 0); this p is the isoelectric point of the protein (or amino acid). Each protein has a characteristic isoelectric point. At this p, the molecules coagulate and are removed from solution. We will analyze asein, the major storage protein in milk to determine its isoelectric point. 106 Lab 25: Proteins (W13) 65
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 p, 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. PROEDURES: ATIONS: I. Denaturation of Proteins: 1. Prepare a fresh egg albumin solution by mixing the white from one egg 1 with 200 ml of deionized water. 2 2. Obtain six separate test tubes and label them A through F. 3. Place 2-3 mls 2 of egg albumin solution into each of the tubes. 4. For each indicated tube perform the following: A. eat: Using a test tube holder, heat the egg albumin solution A over a low flame. B. Acid: Add 2 mls of 10% NO 3 (Nitric acid) to tube B. 3. Base: Add 2 mls of 10% NaO (sodium hydroxide) to tube. 3 D. Alcohol: Add 4 mls of 95% Ethanol (EtO, 2 5 O) to tube D. 3 E. eavy Metal Ions: Add 10 drops of 1% AgNO 3 (Silver nitrate) to tube E. 3 F. Alkaloids/Tannins: Add 1 ml (20 drops) of 10% tannic acid solution to tube F. 3 NOTES: 1 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 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. 5. Describe any changes. Give a brief explanation for the results. 66 106 Lab 25: Proteins (W13)
II. asein Products: A. Isolation of asein from Milk: 1. Weigh a 150 ml beaker. Add about 50 ml of nonfat milk to the beaker and weigh again. alculate the mass of the nonfat milk sample. 2. Record the p of the nonfat milk. 4 3. Warm the sample on a steam bath until the temperature of the milk reaches about 40 o then remove from heat. 4. Add 5% acetic acid ( 3 O 2, vinegar) a little at a time 5 with gentle stirring until the casein (milk protein) becomes insoluble. When no further precipitation occurs, stop adding acid. 6 5. Determine and record the p at which the casein becomes insoluble in solution. This p is the isoelectric point of casein. 7 6. Separate the solid casein protein from the soluble whey protein by decanting off the liquid, keeping the solid casein behind in the beaker. 8 7. Wash the solid casein with two 10 ml portions of water. Stirring and then decanting off the liquid each time to rinse away any remaining whey protein or acid. Squeeze dry by pressing the protein between pieces of filter paper or squeezing through cheesecloth. 8. Weigh a watch glass. Transfer the protein to the watch glass and let the protein dry. Weigh. alculate the mass of milk protein. 9. alculate the percentage of casein in the nonfat milk. 9 B. asein Glue: 10. Mix a chunk of casein 10 (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 NaO 3, (sodium bicarbonate, baking soda) to neutralize any acid remaining from the vinegar and make the mixture alkaline. 11 Watch for bubbles of O 2 gas produced. Add a little more sodium bicarbonate until no more bubbles appear. 4 For best result use a p meter or indicator paper having approximately a 4.5 to 8.5 p range. 5 It may take from 4-6 mls. 6 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 p that the protein becomes least soluble. 8 Rather than decanting you could filter through cheesecloth or filter using a Buchner funnel apparatus. 9 (g s of solid casein / g s milk) x 100 = % 10 If the casein is too clumpy grind the water and casein together with a mortar and pestle to insure a good mix. 11 When dissolved in an alkaline solution casein becomes sticky so can be used as glue. 12 The glue you made is similar to non-toxic commercial white glue. You can make fancy glues of various colors by adding food coloring. 12. The substance in the beaker is glue. 12 Test the adhesive properties of your product with various materials. 106 Lab 25: Proteins (W13) 67
. asein Paint: Pigment Preparation: 1. Prepare paint pigments according to the following instructions using the 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. an 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. NOTES: 13 asein 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. Pigment Reactants Product White A. 0.3g al 2 B. 0.3g Na 2 O 3 ao 3 (blackboard chalk) Green A. 0.3g K 4 Fe(N) 6 o 2 Fe(N) 6 B. 0.2g ol 2 Brown A. 0.2g N 4 Fe(SO 4 ). 2 3 2 O Fe(O) 3 B. 0.2g Na 2 O 3 Blue (dark) A. 0.2g N 4 Fe(SO 4 ) 2 B. 0.2g K 4 Fe(N) 6 KFe 2 (N) 6 (Prussian Blue) Orange A. 0.2g N 4 Fe(SO 4 ) 2 Fe 2 (SiO 3 ) 4 + Fe(O) 3 B. 1.0mL sat Na 2 SiO 3 Blue (royal) A. 0.2g ol 2 osio 3 B. 1.0mL sat Na 2 SiO 3 Lavender A. 0.2g ol 2 oo 3 B. 0.2g Na 2 O 3 Red Add powdered Iron (III) oxide to Fe 2 O 3 casein Black Add powdered charcoal to casein Paint Preparation: 2. hop 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 68 106 Lab 25: Proteins (W13)
LAB 25: PROTEINS: PRE LAB EXERISES: NAME DATE 1. At the isoelectric point of a protein, A. the p of the solution will be 7 B. the number of positive charges on the protein will equal the number of negative charges.. the p of the solution will always be greater than 7. D. the p 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.. 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 %. 75 % D. 33 % E. 0.033 % 4. After casein is isolated from milk sodium bicarbonate (NaO 3 ) is added to neutralize any acid remaining from the vinegar. Write the balanced equation for the reaction accounting for the gas produced. 5. omplete and balance the equation for the double replacement reaction of obalt (II) chloride with Sodium carbonate to form Lavender paint pigment. 106 Lab 25: Proteins (W13) 69
70 106 Lab 25: Proteins (W13)
LAB 25: PROTEINS: REPORT: I. Denaturation of Proteins: Observations Protein Used: circle one asein Whey Albumin NAME PARTNER DATE Explanation/onclusion (If denaturation occurs explain how/why. Be specific) A. eat B. Acid. Base D. Alcohol E. eavy Metal Ion: Ag 1+ F. Tannic Acid Explanation/Analysis: Why are the results as they are? Explain any anomalies. 106 Lab 25: Proteins (W13) 71
II. asein A. Isolation from Milk: Mass of Milk used Initial p pi = Isoelectric point Observations Explanation: Why does adding acid cause a precipitate? Explain why the most precipitate occurs at the pi. Mass of casein produced Percentage of casein in nonfat milk (show calculation & circle answer) B. Glue: Observations Glue a sample here: Explanation/Analysis: Why are the results as they are? ow/why does casein act as glue? Explain any anomalies. 72 106 Lab 25: Proteins (W13)
. Paint: Paint pigments made: Observations Paint a picture here: Explanation/Analysis: Why are the results as they are? Why use casein in paint? Explain any anomalies. 106 Lab 25: Proteins (W13) 73
RELATED EXERISES: 1. Precipitation of a protein takes place when A. the p is equal to 7 so the protein is neither acidic nor basic. B. the p is equal to the isoelectric point (pi) because at this point the protein is negatively charged.. the p 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 p. 2. Paints containing Lead compounds are poisonous because A. Lead is a heavy metal that denatures life supporting protein enzymes by complexing with S side groups and thus changing their shape. B. Lead catalyzes the hydrolysis of proteins so that life supporting proteins are no longer available.. 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 (AgNO 3 ) 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.. Silver is expensive and thus hospitals can charge patients more money. 4. Isopropyl alcohol a good disinfectant because A. alcohol acts as a solvent to dissolve bacteria. B. alcohol denatures the enzyme proteins of bacteria by disrupting the hydrogen bonds.. alcohol hydrolyzes proteins so kills bacteria. D. 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. (int; think about the chemical make up of skin.) 6. Baby vomit looks and smells similar to urds and Whey. Explain why. 74 106 Lab 25: Proteins (W13)