Reactions of protein precipitation.
LABORATORY WORK 1
THE ANALYSIS OF PROTEINS AND AMINO ACIDS
Goals and Objectives: to study the methods of the extraction and analysis of substances from the biological objects, the structure and properties of amino acids and proteins; to master chromatographic method, to explain the possibility of using this method in medical practice.
The student must:
- Know: safety in the laboratory of biological chemistry; basic methods for the isolation and analysis of substances; the classification, structure, and properties of amino acids and proteins;
- Be able to: carry out the chromatographic analysis of the amino acids mixture, to make calculations of the retardation factor; to perform qualitative determination of amino acids and proteins;
- Master: the technique of laboratory work in the laboratory of biological chemistry; the method of thin layer chromatography; the method of collecting and analyzing experimental data.
Proteins can be called the basic molecules of life. Proteins are high molecular nitrogen-containing organic compounds found in all living cells. The building blocks of proteins are the twenty naturally occurring amino acids. Amino acid residues are joined by peptide bonds. Proteins differ only in the number, the nature, and the sequential order of their constituent amino acids. These amino acids are liberated when proteins are hydrolyzed.
The solubility of proteins in an aqueous solution containing salts depends on two opposing effects: on the one hand related to electrostatic interactions ("salting in"), and on the other hydrophobic interactions (salting out).
A protein is denatured when its specific three-dimensional conformation is changed by breaking some bonds without breaking its primary structure. Denaturing agents are physical or chemical agents. The denaturation may be reversible or irreversible. It causes a total or partial loss of biological activity. This is an important property of proteins.
Characteristic physical and chemical properties of proteins are high viscosity of solutions, low ability to diffusion, swelling capacity, optical activity, and mobility in an electric field, low osmotic pressure and high oncotic pressure. Protein molecules cannot penetrate through the semi permeable membrane.
Simple proteins are made of amino acid residues. When hydrolyzed, simple proteins form only amino acids. Simple proteins include albumins and globulins, protamins, histones, prolamins and glutelins.
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Conjugative proteins are made of two components – polypeptide chain and non-protein component. According to the nature of non-protein component there are classified as nucleoproteins, glycoproteins, lipoproteins, phosphoproteins, chromoproteins, metalloproteins.
Reagents and Equipment: 0.04M solutions of amino acids; mixture of butanol, acetic acid and water (15: 3: 7); 0.5% solution of ninhydrin in acetone; chromatographic plates; capillaries; chromatogram chamber with a cover; glass sticks; electric stove; spray-camera; spray tube; test tubes; water bath; pipettes; measuring cylinders.
Qualitative analysis of amino acid mixtures by thing layer chromatography method.
Prepare chromatographic plate. Draw the straight line by the pencil at 1.5-2 cm from the border of chromatographic plate. Than draw 4 points on this line. At points 1, 2, 3 apply amino acids solutions with capillary, and to point 4 - the solution of amino acid mixture. Dry the chromatogram over a warm electric stove.
Put the chromatogram into a chromatogram chamber, at the bottom of which a mixture of butanol, acetic acid and water (15:3:7) is poured so that the solvent does not reach the starting line. Close the chamber with a cover glass and leave the chromatogram for some time.
The aqueous component of the solvent system binds to the plate porous layer and forms a stationary phase. The organic component that migrates along the plate is the mobile phase. When the migration of the solvent is upwards, it is referred to as ascending chromatography. As the solvent flows, it takes along with the unknown substances. The rate of the migration of molecules depends on their relative solubility in the stationary phase (aqueous) and mobile phase (organic).
When the solvent traverses the path from the bottom to up remove the chromatogram from the chamber, mark the front line of the solvent and dry the plate. Apply 0.5% solution of ninhydrin in acetone on the dried chromatogram in the spray chamber with the help of the pulverize. When the acetone evaporates, the chromatogram is placed over a warm electric stove for some min. The positions of amino acids on the chromatogram are identified as purple spots.
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Calculate Rf (retardation factor) by the formula:
Rf = Хi/Х0,
where Х0 is the distance travelled by solvent (from the starting line to the front line of the solvent), Хi is the distance travelled by substance (from the starting line to the middle of corresponding spot).
The Rf value of each substance, characteristic of a given solvent system and paper, often helps for the identification of unknown. Comparing the values Rf of known amino acids and amino acids of the identifying mixture do the conclusion about the nature of amino acids in the mixture.
1. Draw the chromatogram.
2. Calculate Rf. Compare values.
Conclusion:
Reactions of protein precipitation.
Reagents and equipment: protein solution, conc. Nitric acid, conc. Nydrochloric acid, conc. Sulfuric acid, 20% solution of sulfosalicylic acid, 5% solution of trichloroacetic acid, 5% copper sulfate solution, 5% lead acetate solution, test tubes, pipettes, tube holder, spirit lampe.
Heat precipitation.
Pour 0.5 ml of protein solution in the test tube and heat.
Observations:
Conclusion:
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