Protein Tests Lab 4 PDF

Chemical composition of cells  Tests for protein 1. Test the peptide bond The Biuret Test is often used to determine the presence of peptide bonds in protein. As a student you will most likely will be testing for the presence of protein in foods. As an alternat...

Chemical composition of cells  Tests for protein 1. Test the peptide bond The Biuret Test is often used to determine the presence of peptide bonds in protein. As a student you will most likely will be testing for the presence of protein in foods. As an alternative to the Biuret Reagent, either of the following solutions may be used to yield comparable results. Sodium Hydroxide and Copper Sulfate Solutions Protein Peptide Negative  Tests for protein 2. Test the free amino acids Ninhydrin test Ninhydrin Test Definition  Ninhydrin test is a chemical test performed to detect the presence of ammonia, primary/secondary amines, or free amino acids.  This test involves the addition of ninhydrin reagent to the test sample that results in the formation of deep blue color, often termed as Ruhemann’s purple, in the presence of an amino group. Objectives of Ninhydrin Test  To detect the presence of amines and amino groups in the test solution.  To quantify the amino acids present in the sample.  To distinguish carbohydrates from amino acids. Principle of Ninhydrin Test  This assay is based on the fact that two molecules of ninhydrin (2, 2- dihydroxyindane-1, 3-dione) react with a free alpha-amino acid to produce a deep purple or blue color known as Ruhemann’s purple. ‫ارجواني‬  In this reaction, ninhydrin acts as an oxidizing agent and causes the deamination and decarboxylation of the amino acids at an elevated temperature.  This reaction is then followed by condensation between the reduced ninhydrin molecules, released ammonia, and the second molecule of ninhydrin.  By the end of the reaction, a diketohydrin complex is formed which has a deep purple color.  In amino acids like proline and hydroxyproline, this test yields an iminium salt, which is yellow-orange in color. ‫ملح اإلمينيوم‬  Similarly, proteins with a free amind group like asparagine, react with the ninhydrin reagent to form a brown colored product.  The intensity of the formed complex is proportional to the concentration of amino acids in the solution.  The color intensity, in turn, depends on the type of amino acid present. Reaction Reagents Procedure of Ninhydrin Test :  Ninhydrin reagent 1. Take 1 ml of standard protein solution in  Standard solution (1% protein solution) one test tube and 1 ml of the test sample in  Sample solution (amino acids solutions) another dry test tube. Materials require 2. Add a few drops of ninhydrin reagent to  Test tubes both the test tubes.  Test tube stand 3. Place the test tubes in the water bath for 5  Pipette minutes and then allow cooling to room Equipment temperature.  Water bath /(beaker with heated water) 4. Observe the formation of color and note down the result.  Tests for protein 3. Test the Aromatic amino acids Xanthoproteic Test: Phenyl rings containing an activating group can be nitrated producing a yellow product. Xanthoproteic Test Objective: This test is used for aromatic amino acids which give positive result from other amino acids. Such tyrosine , and tryptophan react with Xanthoproteic test, phenyl alanine does not respond with this test. Principle: Xanthoproteic test is used to detect amino acids containing an aromatic nucleus (tyrosine, tryptophan and phenylalanine) in a protein solution which gives yellow color nitro derivatives on heating with conc. HNO3. The aromatic benzene ring undergoes nitration to give yellow colored product. Phenylalanine gives negative or weakly positive reaction though this amino acid contains aromatic nucleus because it is difficult to nitrate under normal condition. On adding alkali to these nitro derivative salts, the color change for yellow to orange. Reagents: Procedures: 1. Take 1ml of albumin solution in dry 1-test solution: 1 % tyrosine, 1 % test tube. tryptophan, 1 % phenylalanine, 5 % egg 2. Add 1ml of conc. HNO3 in all test white (albumin). tubes and mix well. 3. Cool the solution under tap water, 2-Nitric acid Con. becomes yellow. 3-NaOH 40%. 4. Now add 2ml of 40 % NaOH to all test tubes, becomes orange.