6 Characterization of Casein and Bovine Serum Albumin

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1 6 Characterization of Casein and Bovine Serum Albumin (BSA) Objectives: A) To separate a mixture of casein and bovine serum albumin B) to characterize these proteins based on their solubilities as a function of salt concentration. Introduction: Structurally, proteins are the most complex biomacromolecules. This structural complexity is due to the composition and sequence of the amino acids that make up proteins. The composition and sequence of amino acids is different for every protein. As such, the unique chemical and physical characteristics of a protein can be used to isolate it from other cellular components using basic chemical techniques. Once a protein has been isolated, one can initiate characterization studies, information such as ph- and heat-stability, that could prove helpful in ascertaining the protein s structure and/or function without knowing its amino acid sequence. Many different methods have been devised to separate proteins from other proteins or biomolecules. Several of these separation methods are based on the solubility of proteins under various conditions. In this week s experiment, two classes of proteins will be investigated. Casein is a heterogeneous mixture of proteins found in milk. Casein is a nutritionally-adequate protein. This means that it has all the essential amino acids required for normal growth and development. Casein is a phosphoprotein produced in mammary tissue, and is 3-5% of bovine and human milk. It is isolated from milk by removing the cream (lipid) and acidifying the resulting skim milk, which causes the casein to precipitate. Serum Albumin (we will use bovine serum albumin BSA) is the most abundant protein in the serum. It carries out several functions; among them are transport of fatty acids, keeping fluid from leaking out of the circulatory system, and a limited role in ph maintenance. Albumin is synthesized in the liver and normal human albumin levels are about 3.5 to 5.5 % (w/v). Low levels of albumin are indicative of either liver or kidney disease. Liver disease would inhibit the synthesis of albumin and kidney disease could cause the albumin to be excreted in the urine. Because of the high concentration of albumin in the blood it is fairly easy to purify and is often used to help stabilize dilute enzyme solutions in the lab, or to prevent non-specific binding of anti-bodies; proteins that have a tendency to bind to other proteins will bind to BSA instead of binding nonspecifically to the antibody.

2 The major part of the experiment today will be to answer this question: Can a mixture of Casein and BSA be separated using ammonium sulfate fractionation? Ammonium sulfate fractionation is often used as the first purification step in protein isolation. Different proteins are soluble to varying degrees in ammonium sulfate; some proteins may precipitate at 30% saturation while others may remain soluble at 80% saturation. This experiment entails examination of the solubility of this week s proteins as a function of salt concentration. Heat and ph can also be used to precipitate proteins. Tests that manipulate a protein s solubility usually involve protein denaturation. When a protein is denatured, the noncovalent interactions between the amino acid side chains and/or prosthetic groups are disrupted. Some denaturation methods are reversible; others are not. A method that irreversibly denatures the protein is generally not used if one wants to study the function of that protein. When a protein solution is heated, the proteins may be denatured, lose their solubility, and precipitate from solution. Since all proteins will not precipitate at the same temperature, this heat treatment can be useful in the separation of proteins. Many proteins are soluble in dilute salt solutions (salting-in effect) but insoluble in concentrated salt solutions (salting-out effect). One procedure for separating proteins involves the use of ammonium sulfate to achieve solutions of different salt concentrations to salt out some specific protein or proteins while others remain in solution. Proteins differ in solubility at a given ph. When the ph of a solution is brought to the isoelectric point (pi) of a protein in the solution, that protein has minimum solubility in the medium and may precipitate from solution, thus becoming separated from other dissolved proteins. This method, isoelectric precipitation, is a third means of isolating proteins based on solubility. Average Protein Stability Profiles Solubility Solubility Solubility pi ph Temperature [Salt]

3 Procedures: This week we are going to try something a little different. Instead of giving you precise instructions we will give you a mixture of BSA and Casein and let you try to work out the conditions that will best separate them. Percent saturation Grams ammonium sulfate to add to 1 ml of solution Using the table above you can calculate how much solid ammonium sulfate to add to get the desired % saturation. IMPORTANT NOTE In this context % is not meant to be grams / 100 ml. There is a difference between w/v % and % saturation. By % saturation we mean the % of the total solute that the solution can hold. You will need to select 4 or 5 concentrations, make your solution that concentration, set the tube on ice for 15 minutes, centrifuge, re-suspend the pellet in buffer (50 mm Tris ph 9.5). Carefully label the tubes, they will be kept frozen until the next lab period. How can we tell if the proteins have been purified? We need a mechanism to assay for the protein of interest. If we were trying to separate enzymes we would test for enzymatic activity. However, neither of these proteins has enzyme activity. We do know that these proteins have different sizes; casein

4 has a molecular weight of about 20,000 and BSA has a molecular weight of about 55,000. Running an SDS-PAGE gel will separate on the basis of size. Therefore we can run such a gel and determine if we have achieved purification of our mixture, and determine which protein precipitated in which ammonium sulfate fraction. Record procedures in your notebook: Since you have no data at this point a report will NOT be required until you run the gel. Take the time to study for the midterm exam.

5 Characterization of Casein and BSA STUDY GUIDE 1. Casein is a phosphoprotein. How are the phosphate groups linked to the protein? 2. What is a nutritionally adequate protein? 3. The albumin proteins of egg white are insoluble in half-saturated ammonium sulfate solution, while the globulins of egg white require fully saturated ammonium sulfate for precipitation. Both albumins and globulins dissolve in dilute NaCl solution. Suggest how the albumins and globulins of egg white might be separated. 4. What is the salting-in effect? The salting-out effect? 5. Protein denaturation does NOT include disruption of bonding/interactions. a) hydrogen b) hydrophobic c) covalent d) ionic 6. What happens to the solubility of a protein dissolved in an aqueous medium as it undergoes denaturation? 7. What is the net charge on a protein when the ph of the medium is below the isoelectric point of the protein? When the ph is above the pi? 8. A solution of proteins is treated with solid ammonium sulfate. The precipitate that forms is separated by centrifugation. The supernatant gives a brownish blue color when tested with Bradford reagent. What is the proper conclusion of this result? a) Ammonium sulfate precipitated all the protein from solution. b) Only a portion of the protein was precipitated from solution. c) Ammonium sulfate denatured all the protein in solution. d) Ammonium sulfate is present in the supernatant. e) There is something in the supernatant that interferes with the Bradford reagent is present in the supernatant. 9. Concentrated ammonium sulfate solution causes a) protein solubility to increase by salting-in the protein. b) protein denaturation by complexing with negatively charged amino acid side chains of the protein. c) protein precipitation by destroying hydrogen bonding in the protein molecule. d) protein precipitation by removing water molecules from around the protein. 10. From the data gathered in this experiment, describe how a solution containing both casein and globulin can be treated to achieve separation and isolation of these proteins.

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