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    R Reactions of Proteins and Amino Acids Additional Notes

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    R Reactions of Proteins and Amino Acids Additional Notes

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    chickenducktales
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    Rp

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    r-Reactions-of-Proteins-and-Amino-Acids-Additional-Notes

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    R Reactions of Proteins and Amino Acids Additional Notes

    Uploaded by

    chickenducktales
    Rp

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    © All Rights Reserved
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    COLOR REACTIONS OF PROTEINS AND AMINO ACIDS

    Test Expected Color Reaction Inference


    Xanthoproteic Acid Reaction Yellow Aromatic amino acid
    Biuret Test Pink or Violet Peptide linkages
    Ninhydrin Reaction Blue (Ruhemann’s blue) Alpha amino group
    Millon’s Test Flesh to Red precipitate Phenol group in tyrosine
    Glyoxylic acid reaction Violet Indole nucleus in tryptophan
    Sakaguchi Reaction Red Guanidine group in arginine
    Nitroprusside Reaction Purple Sulfhydryl group in cysteine

    PROCEDURES

    A. XANTHOPROTEIC TEST
    1. Place 1 mL of egg albumin solution in a test tube.
    2. Add 0.5mL of concentrated nitric acid.
    3. Heat in a water bath for 5 minutes. Note the color of the precipitate formed.
    4. Cool and add sodium hydroxide solution in excess. Note the changes in the mixture.
    The yellow color produced in the Xanthoproteic test is due to the formation of aromatic into compounds
    and is therefore due to the presence of aromatic amino acid in the protein molecules. Phenylalanine,
    however, does not nitrate readily and proteins containing only this aromatic nucleus do not usually give a
    positive test.

    B. BIURET TEST
    1. Mix 1ml of egg white solution and 10drops of 10% NaOH in a test tube.
    2. Add 1 drop of 0.5% CuSO4 solution. Mix well and record your observation.
    Biuret test is a general test for proteins. A positive reaction is obtained with all native proteins and most of
    the derived proteins. A violet color is obtained with long chain proteins while a pink color is obtained with
    shorter chain. This is due to the varying amount of peptide linkage. This is not however a specific test for
    peptide linkage. Any compound containing carbonyl group joined together through a single nitrogen or
    carbon atom will give a positive test

    C. NINHYDRIN TEST
    1. Place 1mL egg albumin solution in a test tube.
    2. Add 0.5% mL solution of ninhydrin.
    3. Heat to boiling.
    4. Allow to cool and observe the color procedure.
    This is one of the most sensitive tests for detecting the presence of protein or –amino acids as low as 1 part
    of 100,000 part of water can be detected. A blue color is produced when a solution of protein is heated with
    dilute aqueous solution of ninhydrin (triketohydrindene hydrate). This is due to the split protein products

    Biochemistry for Medical Laboratory Science


    (alpha amino acids) that undergoes dehydrogenation resulting to the formation of ammonia and an
    aldehyde. The ammonia condenses with ninhydrin and its reduction products to form a blue colored
    compound.

    D. MILLON’S TEST
    1. Place 1 mL of egg albumin solution in a test tube.
    2. Add 1drop of Millon’s reagent.
    3. Mix and heat in the water bath. OBSERVE

    E. GLYOXYLIC ACID REACTION (HOPKINS-COLE)


    1. Mix 1mL of egg albumin solution and 1 mL of Hopkins-Cole reagent.
    2. Incline the tube and allow 1mL of pure concentrated sulfuric acid to flow on the side of the tube to
    form a layer of acid beneath the protein mixture (the sulfuric must be pure).
    3. Observe the color produced at the point of contact of the two liquids. If no color is produced, make a
    gentle shake to cause slight mixing

    F. SAKAGUCHI REACTION FOR ARGININE


    1. To 3mL of egg albumin solution, add 1mL of 10% NaOH.
    2. Add 6 drops of dilute alcohol a-naphthol solution.
    3. Mix well and add 10 drops of sodium hypobromite solution.
    4. Note the results.

    G. NITROPRUSSIDE REACTION FOR SULFHYDRYL GROUP


    1. To 1mL of egg solution, add 0.5mL of saturated solution, add 0.5mL saturated sodium carbonate solution
    and 5 drops of 2% sodium nitroprusside.
    2. If the test is negative, add 2 drops of 1% NaOH. Purple color indicates the presence of disulfide
    compounds.

    Biochemistry for Medical Laboratory Science

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