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BIOCHEMISTRY FOR MEDICAL LABORATORY SCIENCE LABORATORY Lesson 8: Qualitative Tests for Proteins / Amino Qualitative Tests Acids Biuret Test 1. Add 1mL of...

BIOCHEMISTRY FOR MEDICAL LABORATORY SCIENCE LABORATORY Lesson 8: Qualitative Tests for Proteins / Amino Qualitative Tests Acids Biuret Test 1. Add 1mL of 10% NaOH to 3mL of each of the Proteins protein suspensions (albumin, casein, and Proteins (Polypeptides) are biochemical gelatin - in 3 separate test tubes) and mix. compounds made up of amino acids 2. Add a drop of 0.01M CuSO4 to each and arranged in a linear chain mix. If no definite color develops, add Like other biomolecules, proteins are another drop or of the CuSO4 solution. important part of organisms and A purplish violet color means a positive test. participate almost all processes inside the Take note of the color that will be produced cells, thereby providing essential functions by the reaction. in our body They can be found in digestive enzymes, Ninhydrin Test antibodies, muscle contraction and 1. Add 5 drops of 0.1% Ninhydrin solution to movement, and hormones 2mL of each of the 3 protein suspensions 2. Heat in boiling water for 10 minutes. Amino Acids Observe and record the results. The monomer units of proteins A purplish violet color means a positive test. Are molecules containing an amino group, Take note of the color that will be produced a carboxyl group, and a side chain by the reaction. At a particular pH, amino acid have no overall charge Xanthoproteic Test This pH is known as the isoelectric point of 1. Slowly add 1mL of concentrated HNO3 to an amino acid 3mL of each of the protein suspensions. When amino acids have no overall charge, 2. Place in a water bath for about 30 seconds they known as zwitterions and place each test tube in a rack to cool Nonpolar amino acids are symmetrical and the solutions. contains hydrocarbons (C and H atoms) 3. Add slowly drop by drop, saturated NaOH to each test tube until the solutions are Materials, Reagents, and Equipment alkaline (or basic). Test tubes Take note of the color that will be produced Test tube rack by the reaction. Test tube holder 10mL graduated cylinder Millon’s Test 500mL beaker 1. Add 5 drops of fresh Millon’s reagent to Tripod 3mL of each protein suspension. Wire gauze 2. Carefully heat each mixture in boiling water Alcohol lamp for 5 minutes. Dropper 3. Cools the test tubes and notes the colors Serological pipette formed. Aspirator bulb If too much reagent is usd, the color may Litmus paper disappear in boiling. Test solutions: Take note of the color that will be produced ○ Glycine by the reaction. ○ Tryptophan ○ Tyrosine Sakaguchi Test ○ Glutamic acid 1. Add 1mL of 10% NaOH and 1mL of 0.02% ○ Gelatin (Biuret and Xanthoproteic alpha naphthol solution to 3mL of each of tests) the protein suspensions. ○ Casein (Biuret and Xanthoproteic 2. After 3 minutes, add 2-4mL of bromine tests) water to each. A strong red color can be ○ Albumin (Biuret and Xanthoproteic stabilized by adding urea to destroy the tests) excess hypobromite. ○ Distilled water Take note of the color that will be produced by the reaction. NIX 1 BIOCHEMISTRY FOR MEDICAL LABORATORY SCIENCE LABORATORY Hopkins-Cole Test Expected Reaction 1. Add 2mL of Hopkins-Cole reagent to 3mL For ammonia, primary/secondary amines, of each of the three protein suspensions. and amino acids, deep purple color is 2. Mix. incline each test tube and with a obtained dropper, add slowly about 1mL conc. For hydroxyproline and proline, a yellow H2SO4 down the side of the tube so that color is obtained two layers will form. Do not stir. For asparagine, brown color is obtained 3. Let stand for 1-2 minutes and note the color If no color change is observed, the analyte formed in between the layers. does not contain amino acids, amines, or ammonia Lead Acetate Test 1. Add 5mL of NaOH and a few crystals of Xanthoproteic Test Pb(Ac)2 (lead acetate II) to 3mL of each Principle protein suspension. A qualitative test to determine the amount 2. Heat in boiling water for 5-10 minutes with of protein in a solution using nitric acid occasional mixing of the contents of the The test is named after the yellow color tube. Describe the color change. substance produced after the reaction - Xanthoproteic acid Biuret Test Xantho is derived from the Greek word Principle xanthos meaning yellow Also known as Piotrowski’s test, it positively identifies the presence of Expected Reaction proteins and peptide bonds (not less than Positive - Formation of yellow to orange two peptides). color The reaction in this test involves the Negative - No color change complex formation of the proteins with Cu2+ ions in a strongly alkaline solution Millon’s Test Despite the name of the test, the reagent Principle used to perform the procedure does not Millon’s test is specific to phenol containing contains Biuret, a chemical derived from structures (Tyrosine) urea Its reagent is conc. HNO3, in which mercury It is named so because it also gives a is dissolved positive reaction to the peptide-like bonds As a result of the reaction, a red precipitate in the Biuret molecule or a red solution is considered a positive test Expected Reaction A yellow precipitate of Mercuric (II) oxide Positive - Purple colored solution (HgO) is not a positive reaction but usually Negative - Blue colored solution indicates that the solution is too alkaline The test was developed by a French Ninhydrin Test chemist, Auguste Nicolas Eugene Millon Principle A chemical test which is used to check Expected Reaction whether a given analyte contains amines or Positive - Brick red colored solution or red α-amino acids precipitate In this test, ninhydrin (a chemical Negative - No red color compound with the formula C9H6O4; IUPAC name: Sakaguchi Test 2,2-dihydroxyindane-1,3-dione) is added to Principle test solution of the analyte It is based on the principle of reaction The development of a deep blue color between 1-naphthol and the guanidinium indicates the presence of ammonia, groups on arginine, in the presence of an primary/secondary amines, or amino acids oxidizing agent in the analyte The exact mechanism of the reaction is not yet known; however, the reaction results in the formation of a red-colored complex due to the formation of an indole-like structure NIX 2 BIOCHEMISTRY FOR MEDICAL LABORATORY SCIENCE LABORATORY The Sakaguchi reagent consists of sodium hypobromite and 1-naphthol The sodium hypobromite acts as an oxidizing agent that facilitates the hydrogen bonding between two arginine molecules Expected Reaction Positive - Demonstrated by the formation of red color. This indicates the presence of arginine or guanidinium compounds. Negative - Demonstrated by the absence of red color. This indicates the absence of arginine or guanidinium compounds. Hopkins-Cole Test Principle A chemical test for the determination of the presence of heterocyclic side chain (indole group) of tryptophan in proteins The test is named after Frederick Gowland potawalapaakoreviewinunakoinom Hopkins and Sydney W. Cole for their work on tryptophan isolation Expected Reaction Positive - Purple colored ring at the interface Negative - No formation of purple colored ring Lead Acetate Test Principle Also known as Lead Sulfide Test, is a biochemical test for the detection of amino acids like cysteine and cystine It is a specific test for the detection of amino acids containing Sulfur; S-S (Sulfide) group in cysteine, and S-H (Thiol) group in cystine. Methionine does not give a positive result in this test Expected Reaction Positive - Represented by the formation of black precipitate at the bottom of the test tube. This indicates the presence of cysteine or cystine in the solution. Negative - Represented by the absence of black residue in the test tube. This indicates the absence of cysteine or cystine. NIX 3