EXPERIMENT 6

Qualitative Analysis of Proteins and Amino Acids

Objectives
To identify the amino acid present in a protein sample based on its color reaction with specific reagents.

Qualitatively analyze amino acids and proteins

FLOW DIAGRAM

1. BIIURET TEST

2 ml pepton e +

1 ml NaOH +

+ 5 drops CuSO4 solution

Mix

Observe color

2.NINHYDRIN TEST
2 ml peptone + 1 ml Ninhydrin reagent

heat to boiling in water bath

describe color

3.XANTHOPROTEIC TEST
1 ml peptone + 5 drops conc.HNO3

mix

heat to water bath

heat for another 1 minute

cool under tap H 2O

add few drops conc. NH4OH

note the color

4.MILLONS TEST
1 ml peptone + 4 drops Millons reagent

observe the color of precipitate

boil in water bath for 10 minutes

add 4 drops NaNO2

cool in running water

5.HOPKINS COLE TEST

1ml peptone + 2 drops Hopkins Cole reagent

Mix

Add 1 ml conc. H2SO4 along the side of inclined test tube

Note the color of the junction between 2 layers

6.SAKAGUCHI TEST
3 ml peptone + + 2 drops NaOH solution + 4 drops naphthol solution + 10 drops bromine water

Observe the color

7.LEAD ACETATE TEST

1 ml peptone + 5 drops NaOH solution boil in water bath for 3 minutes

Cool
add 3 drops lead acetate with mixing

color of precipitate

Results and Data

Biuret Test green Ninhydrin Test Blue Xanthoproteic Test Light Yellow

Millons Test Light Brown

Hopkins Cole Test- Yellow Sakaguchi Test- Brown Lead Acetate Test- White

LEARNINGS and VALUES:

Values -teamwork -patience -discipline Proteins are complex, high molecular weight biomolecules composed of amino acids joined together by peptide bonds. The twenty amino acids normally found in proteins differ in their amino groups in the side chains. These respond to specific qualitative tests with certain chemical compounds chemical reagents and thus become the basis of their detection whether as free amino acids or in combined form as in peptides or proteins

A peptide or a peptone is a short chain of amino acid residues. A polypeptide is a long-chain of amino acid residues. A protein consists of one or more polypeptides which are folded in a globular or fibrous form, so that it performs a biological function. A peptone is a secondary derived protein form by hydrolysis of proteoses. It is not heat coagulable protein. Peptone contains traces of cysteine and cystine amino acid.

BIURET TEST
A biuret test is a chemical test used for detecting the presence of peptide bonds. It works by copper sulphate ions reacting with the peptide bonds of proteins to produce a pink to purple color in an alkaline solution. Comparison of colour is made from a standard solution whose protein concentration is known.

NINHYDRIN TEST
Ninhydrin test is the general test given by all the amino acids. One can identify the amino acids by performing this test. The amino group of amino acids reacts with the ninhydrin an oxidizing agent and the amino acid gets itself oxidative decarboxylated and delaminated to an aldehyde. It reacts with amino groups of protein and free amino acid to give a blue or bluish purple complex (Ruhemanns purple).

Xanthoproteic Test (Mulders test)

This test gives positive result with aromatic amino acids containing phenylg roups in their structures ( phenyl alanine, tyrosine & tryptophan).Peptone gives a yellow color on this test.

Xanthoprotein is a yellow acid substance formed by the action of hot nitric acid on albuminous or protein matter and is changed to a deep orange-yellow colour by the addition of ammonia.

Principle: This test is based on the ability of aromatic amino acids containing substituted phenyl groups to react with concentrated nitric acid to give dinitro derivates. These are further converted to quinoid structures by reacting with strong alkali.

Millons test
It is generally Known as MillonNasses Test. This test gives a positive result with tyrosine amino acid it gives a positive result both in free state as well as being present as constitutive of protein.

Peptone gives a pinkish red color due to the presence of tyrosine.

PRINCIPLE: Phenyl /radical or hydroxy benzene radical present in tyrosine reacts with millons reagent to yield a purple red nitro-hydroxy phenyl mercurial, on heating with mercuric sulfate and sodium nitrite. This reaction is also given by phenolic compound. In amino acids only tyrosine and its derestatives gives the positive result.

Glyoxylic acid test (Hopkin-cole test):

This test is given by those amino acids which have the indole groups. The indole groups present in the tryptophan molecules react with glyoxylic acid in presence of con. H2SO4 forms a condensation product which is reddish violet, the sulfuric acid acts as dehydrationg agent, eliminating a molecule of water. Tryptophan glyoxylic condensation product ( reddish violet). Theres a violet ring at the junction of two solutions due to the presence of tryptophan.

SAKAGUCHI TEST
PRINCIPLE : The amino acid containing guanidino group reacts with alpha- Naphthol and on oxidizing agent like bromine water to give red colour product. This test is positive for arginine. In a test for proteins, the guanido group is in the basic amino acid Arginine. The guanido (NH2 C = NH) group of free arginine or arginine residues in the protein reacts naphthol and sodium hypobromite(bromine water and NaOH) to give a bright red colored complex. Peptone gives bright red color due to the presnce of arginine.

LEAD ACETATE TEST

When proteins containing cysteine or cystine residues are boiled with strong alkali,organinc sulphur is converted to sodium sulphide. The sodium sulphide reacts with lead acetate to form lead sulphide precipitate (insoluble) which is black or brown in color. Peptone gives light black or brown color because it traces the presence of cysteine and cystine.

APPLICATION:
Amino Acid Disorders Screening Amino acid disorder screening checks for inherited disorders in amino acid metabolism. Tests are most commonly done on newborns. Two tests are available, one using a blood sample and the other a urine sample.

Urine Test
Protein in the urine is a warning sign. It may indicate kidney damage or disease or it may be a transient elevation due to an infection, medication, vigorous exercise, or emotional or physical stress. When kidney damage is present, the amount of protein present is generally associated with the severity of damage, and increasing amounts of protein over time indicate increasing damage and decreasing kidney function. Proteinuria is associated with many diseases and conditions

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