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TEST FOR PROTEIN IDENTIFICATION
Activity 8
Biochemistry Laboratory
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TEST FOR PROTEIN IDENTIFICATION
Protein, highly complex substance that is present in
all living organisms.
Proteins are of great nutritional value and are directly
involved in the chemical processes essential for life.
Protein is built from building blocks called amino
acids which are linked by peptide bonds.
• Protein is found throughout the body—in muscle, bone,
skin, hair, and virtually every other body part or tissue.
• It makes up the enzymes that power many chemical
reactions and the hemoglobin that carries oxygen in your
blood.
• At least 10,000 different proteins make you what you are
and keep you that way.
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A variety of tests exists to determine whether a
substance or a solution contains proteins or protein-
breakdown products (e.g. proteoses, peptones,
polypeptides, and in some tests, amino acid).
These depend on the presence of certain functional
groups which react with a specific reagent to produce a
certain change in color, odor, and/or solubility.
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Common Tests for Proteins
1. Biuret’s Test – is a general test for proteins
comparable to Molisch’s test for carbohydrates.
Biuret’s reagent is accomplished by adding 0.1%
CuSO4 with NaOH. Only with peptide bonds.
2. Ninhydrin Test - is a chemical test which is used
to check whether a given analyte contains amines
or α-amino acids
3. Millon’s Test – this is a test for the presence of a
phenolic ring. Millon’s reagent is made by dissolving
mercury in nitric acid.
4. Xanthoproteic Acid Test – detects the presence of
benzene rings on which there are amino acids (e.g.
tryptophan) of hydroxyl groups (e.g. tyrosine) to
give colored aromatic nitro compounds
(xanthoproteic acid).
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5. Hopkin’s–Cole Test – the reagent used in this
test contains glyoxylic acid (HOOCCHO) which
condenses with indole derivatives, in the presence
of strong acids to form colored complexes.
6. Reduced Sulfur Test – is a test for sulfur-
containing acids cysteine and cyctine (except for
methionine where its sulfur is not reactive), which
reacts with lead acetate.
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Objectives:
• To detect the presence of amino acids and
proteins by performing several color tests.
• To explain the result of each color test in terms
of the presence of amino acids
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Materials:
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Procedure: BIURET’S TEST
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Procedure: MILLON’S TEST
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Procedure: XANTHOPROTEIC TEST
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Procedure:HOPKINS-COLE TEST
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Procedure:
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RESULTS (Based on Theory not on actual exp.)
Test Albumin Casein
Biuret Appearance of Purple Color Appearance of Purple Color
solution solution
Millon Pink to Brick red solution Pink to Brick red solution
Xanthoproteic Yellow solution and Yellow solution and Precipitate
Precipitate
Hopkins-Cole Formation of purple ring Formation of purple ring
Reduce Sulfur Formation of Black Formation of Black precipitate
precipitate
TEST FOR PROTEINS
Biuret Test
• The Biuret Test is often used to determine the presence
of peptide bonds in protein.
• This is a general test for identification of proteins. This is
positive for all compounds containing more than one
peptide bonds.
• The biuret test generates complex compounds in
which copper atom binds to nitrogen atoms of a
peptide chain by coordinate bond.
• The biuret test is used for quantitative
determination of high protein concentration in
serum and other biological fluids as well.
Determination of lower levels of proteins requires
more sensitive methods.
OBSERVATIONS INTERPRETATION
No change (solution remains blue ) Proteins are not present
The solution turns from blue to violet (deep Proteins are present
purple)
The solution turns from blue to pink Peptides are present (Peptides or peptones
are short chains of amino acid residues)
Ninhydrin Test
• ninhydrin (a chemical compound with the
formula C9H6O4; IUPAC name: 2,2-
dihydroxyindane-1,3-dione) is added to a test
solution of the analyte
• The development of a deep blue colour indicates
the presence of ammonia, primary/secondary
amines, or amino acids in the analyte.
Millon’s Test
• Millon’s test is specific test for identification of tyrosine. Tyrosine
or tyrosine containing protein when reacts with acidified mercuric
sulphate solutions gives yellow precipitate of mercury protein
complex.
• On addition of sodium nitrite solution and heating, the yellow
complex of mercury phenolate forms, which is red in colour.
Interpretation:
• Formation of pinkish red
color indicates the presence
of hydroxy phenyl (phenol)
group containing amino acid
tyrosine.
Xanthoproteic Test
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 from yellow to orange.
Hopkins-Cole Test
• The Hopkins-Cole reaction, also known as
the glyoxylic acid reaction, is a test used for
detecting the presence of tryptophan in proteins.
• Adamkiewicz–Hopkins’ test
• The compounds that have indole ring can
condense with aldehydes (more readily with
formic aldehyde) to form colourful condensation
products. Among protein amino acids, only
tryptophan undergoes this reaction.
• Interpretation
A purple ring appears between
the two layers if the test is
positive for tryptophan.
REDUCED SULFUR TEST
• Test for sulfur containing acids cysteine (except
methionine where its sulfur is not reactive)
which reacts to lead acetate
Sulfur-containing protein ----> NaOH----> S2- ----
Pb2+----> PbS
• A black deposit is formed with albumin
• while a slight black turbidity is obtained with
casein due to its lower content of sulfur.
• Gelatin gives negative result.
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Guide Questions
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Suppose you were given two test tubes, one containing an
amino acid and the other a polypeptide, how would you
distinguish one from the other using the color tests?
• Biuret’s Test- Test for the Protein due to the
presence of peptide bonds. If the sample contain
only an amino acid, it gives a negative result because
it lacks peptide bond.
• The Biuret reagent contains
• Hydrated Copper sulphate
• Potassium hydroxide solution
• Potassium sodium tartrate
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• The Biuret test is based on the ability
of Cu (II) ions to form a violet-
coloured chelate complex with
peptide bonds (-CONH- groups) in
alkaline conditions.
Lone electron pairs from
4 nitrogen atoms in the peptide
bond coordinate a copper (II) ion to
form the chelate complex.
• The chelate complex absorbs light
at 540 nm so appears violet. Hence a
color change from blue to violet
indicates that proteins are present.
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OBSERVATIONS INTERPRETATION
No change (solution remains blue ) Proteins are not present
The solution turns from blue to violet (deep Proteins are present
purple)
The solution turns from blue to pink Peptides are present (Peptides or peptones are
short chains of amino acid residues)
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Why is it important to know the level of
the protein creatinine in the blood?
• The kidneys maintain the blood creatinine in a
normal range. Creatinine has been found to be a
fairly reliable indicator of kidney function.
Elevated creatinine level signifies impaired
kidney function or kidney disease.
• As the kidneys become impaired for any reason,
the creatinine level in the blood will rise due to
poor clearance of creatinine by the kidneys.
• Abnormally high levels of creatinine thus warn
of possible malfunction or failure of the kidneys.
It is for this reason that standard blood tests
routinely check the amount of creatinine in the
blood.
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Do not be confuse between Creatinine
and Creatine.
CREATININE CREATINE
Creatinine is a compound formed Creatine is a compound formed
during the metabolism of creatine during the protein metabolism and is
and is excreted in urine. involved in the supply of energy for
muscular contraction
A waste produce by the metabolism Used as supplement to increase the
of creatine muscle mass
Produced in the skeletal muscles by Produced in liver, kidney, and
the breakdown of creatine phosphate pancreas and send it to skeletal
muscles.
Helps in diagnosing the function of Helps to supply energy to muscles.
the kidney.
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Why do nitric acid stain the human skin
yellow?
• Proteins and amino acids that contain phenyl rings
form a yellow colored compound when treated with
concentrated nitric acid.
• The yellow stains on the skin are caused due to the
reaction of nitric acid with protein keratin present in
the skin.
• This reaction is called xanthoproteic reaction.
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What happen in the red blood cells of a patient with
a sickle cell anemia? And what are the possible cause
of the abnormality of shape of the red blood cell?
• In sickle cell anemia, the red blood cells
become rigid and sticky and are shaped
like a sickles or crescent moons.
• These irregular shaped cells can get
stuck in small blood vessels, which can
slow or block blood flow and oxygen to
parts of the body.
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• Mutations in the HBB gene cause sickle cell disease.
• Hemoglobin consists of four protein subunits, typically, two
subunits called alpha-globin and two subunits called beta-globin.
• The HBB gene provides instructions for making beta-globin.
Various versions of beta-globin result from different mutations in
the HBB gene.
• One particular HBB gene mutation produces an abnormal version of
beta-globin known as hemoglobin S (HbS).
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