PROTEINS

BIOCHEMISTRY CLUSTER
LABORATORY EXPERIMENT

Department ofDepartment
Medical Technology,
of MedicalBiochemistry
Technology, Biochemistry
Cluster – 2022-2023
Cluster – 2022-2023
 CONCEPT OVERVIEW
PROTEIN COLOR REACTIONS

Department of Medical Technology, Biochemistry Cluster – 2022-2023

QUALITATIVE ANALYSIS OF PROTEINS
Ninhydrin Test
Biuret Test
Xanthoproteic Test
Millon’s Test
Sakaguchi Test
Hopkins-Cole Test
Fohl’s Test
Reduces Sulfur Test
Pauly Test

From the experiment Other tests

Department of Medical Technology, Biochemistry Cluster – 2022-2023

NINHYDRIN TEST
• Test for amines or α-amino acids/free
amino acids
• When exposed to ninhydrin, the amino
acid undergoes oxidative deamination,
releasing CO2 and an aldehyde, while
simultaneously transferring the amino
group to ninhydrin.
• The amino-containing ninhydrin then
forms a complex with another
ninhydrin molecule to form
diketohydrin, otherwise called
Ruhemann’s complex, which is
responsible for the dark purple color

Department of Medical Technology, Biochemistry Cluster – 2022-2023

BIURET TEST

• Test for peptide bonds, minimum of two

peptide bonds/3 AAs
• All proteins should be positive in the
Biuret test. Free amino acids yield
negative result.

• Cu2+ ions form a violet-colored chelate

complex with peptide bonds in alkaline
conditions; the greater the peptide
bonds, the greater the color intensity

Department of Medical Technology, Biochemistry Cluster – 2022-2023

REDUCES SULFUR TEST
• This test is specific for sulfur-containing
amino acids – cysteine, cystine, and
methionine.

• On boiling the sulfur-containing amino

acids, the sulfur is split off from these
amino acids as metallic sulfides.

• By the addition of lead acetate, they are

oxidized to black-colored lead sulfides.

Department of Medical Technology, Biochemistry Cluster – 2022-2023

MILLON’S TEST
• Test for phenol groups/derivatives, tyrosine
• Not specific to proteins and will test
positive for other phenol derivatives.

• Hydroxybenzene radicals form a red-

colored complex with mercury.
• The only amino acid with a
hydroxybenzene (phenol) group is
tyrosine. Initially, the phenol group of
tyrosine is nitrated. Upon the
application of heat, the nitrated
tyrosine form as a complex with
mercury.

Department of Medical Technology, Biochemistry Cluster – 2022-2023

XANTHOPROTEIC TEST
• Test for aromatic amino acids, tyrosine and
tryptophan
• Phenylalanine yields a negative result
due to the stability of its side chain

• Aromatic compounds react with HNO3 in a

nitration reaction which results to a yellow
color.
• Upon the addition of a base, the color
further intensifies and turns almost
orange-yellow.

Department of Medical Technology, Biochemistry Cluster – 2022-2023

HOPKINS-COLE TEST
• Test for indole groups, tryptophan
• ‘glyoxylic acid reaction’

• In the presence of indole-containing

compounds like tryptophan, glyoxylic acid
undergoes a condensation reaction,
forming a purple-colored product.
• H2SO4 hydrolyzes the protein and
liberates amino acids, among which
would be tryptophan, hence the
formation of a purple color at the
interface

Department of Medical Technology, Biochemistry Cluster – 2022-2023

SAKAGUCHI TEST

• Test for guanidine, arginine

• Under alkaline conditions,

the guanidine group of
arginine is able to react with
napthol and NaOBr, forming
a red complex.

Department of Medical Technology, Biochemistry Cluster – 2022-2023

FOHL’S TEST
• Test for sulfur, cysteine and cystine (two cys
residues bound by a disulfide bond.
• Methionine does not yield a positive
result due to the inert nature of its sulfur
atom.
• ‘Reduced Sulfur Test’

• When heated in alkaline conditions, the sulfur

in sulfur-containing amino acids reacts with
lead, resulting to a black precipitate in the
form of lead sulfide (PbS).

Department of Medical Technology, Biochemistry Cluster – 2022-2023

PAULY TEST

• Test for imidazole, histidine, and

phenol/hyroxybenzyl groups, tyrosine

• A diazo reagent (contains an N≡N group)

is first prepared through sulfanilic acid
with NaNO2.
• In alkaline conditions, the diazo
reagent will form a red-colored
complex with the imidazole or phenol
groups present in proteins.

Department of Medical Technology, Biochemistry Cluster – 2022-2023

 QUALITATIVE TESTS POSITIVE RESULTS
NINHYDRIN BIURET MILLON’S XANTHOPROTEIC

Protein AA (+)
(+)

Department of Medical Technology, Biochemistry Cluster – 2022-2023

 QUALITATIVE TESTS POSITIVE RESULTS
HOPKINS-COLE SAKAGUCHI FOHL’S PAULY

Department of Medical Technology, Biochemistry Cluster – 2022-2023

 PROCEDURE
COLOR REACTIONS

Department of Medical Technology, Biochemistry Cluster – 2022-2023

COLOR REACTIONS
1. Biuret test – Mix 2mL of egg albumin solution with 1mL of dilute NaOH and 10 drops of dil.
CuSO4 solution in a test tube. Shake well. Note the change in color.
2. Millon’s test – To 2mL egg albumin, add 10 drops of Millon’s reagent. Shake well and heat
over water bath to boiling. Note the color change.
3. Hopkins-Cole test – To 5 drops of egg albumin solution, add 5 drops of glyoxylic acid.
Carefully run down 5 drop of conc. H2SO4 on the side of the test tube. Note the color
formed at the point of contact between the two liquids.
4. Reduces Sulfur test – To 2mL of egg albumin solution in a test tube, add 1mL of 20%
NaOH and 10 drops of 10% Lead acetate solution. Cover the test tube with a cork and boil
in a water bath for two minutes. Observe color change and explain.

Department of Medical Technology, Biochemistry Cluster – 2022-2023

 CONCEPT OVERVIEW
PROTEIN PRECIPITATION

Department of Medical Technology, Biochemistry Cluster – 2022-2023

PROTEIN PRECIPITATION BY HEAT
Reagents required: 1% acetic acid
Reaction: When the albumin solution is heated, the
white coagulum is obtained because
albumin is denatured by heat (Albumin is
a coagulable protein). After the addition
of 1 drop of 1% acetic acid, the coagulum
increases because the pH of the albumin
solution is shifted towards the isoelectric
point. At this pH solubility is minimum and
more protein is precipitated from its
solution.

Department of Medical Technology, Biochemistry Cluster – 2022-2023

PROTEIN PRECIPITATION BY HEAVY
METAL SALTS
Reagents Required: Lead acetate and ferric chloride
Reaction: Proteins are precipitated from their solution by
heavy metal ions. These metal ions
precipitate the protein from their solution. On
the alkaline side of isoelectric pH, Protein
dissociates as a protein anion which
combines with a positive metal ion (cation) to
form an insoluble precipitate of metal
proteinates such as the lead albuminate and
silver albuminate.

Department of Medical Technology, Biochemistry Cluster – 2022-2023

PROTEIN PRECIPITATION BY STRONG
MINERAL ACIDS
Reagents required: Concentrated Nitric acid
Reaction: When native protein solution is
treated with Concentrated HNO3, a
white precipitate ring is obtained
due to denaturation of protein.

Department of Medical Technology, Biochemistry Cluster – 2022-2023

PROTEIN PRECIPITATION BY
ALKALOIDAL REAGENTS
Reagents required: Tannic acid and picric acid
Reaction: These organic acids exist as negative ions
when organic acids are added to albumin
solution proteins are precipitated from their
solution because, on the acidic side of
isoelectric pH, proteins dissociate as the
cation (protein +ion ) which combine with
anions (protein - ion ) of organic acids to
form a salt of protein

Department of Medical Technology, Biochemistry Cluster – 2022-2023

PROTEIN PRECIPITATION BY ALCOHOL
Reagents Required: Absolute Alcohol
Reaction:
• organic solvent such as alcohol, acetone, and
chloroform when added to albumin solution,
decreases the dielectric constant of solvent &
displaces some of the water molecules
(dehydration) associated with protein, and
decreases the concentration of water.
• The mechanism of precipitation, in this case,
is dehydration, denaturation & removal of
charges.

Department of Medical Technology, Biochemistry Cluster – 2022-2023

 PROCEDURE
PRECIPITATION OF PROTEINS

Department of Medical Technology, Biochemistry Cluster – 2022-2023

PRECIPITATION OF PROTEIN
1. By heat – Heat 5 drops of egg albumin solution to boiling. Observe if coagulation
takes place. Add 1 drop of Acetic acid and note the effect.
2. By heavy metal salts – Prepare 2 clean test tubes and place 2mL of egg albumin to
each test tube. To the first test tube, add 2 drops of 5% Ferric Chloride and to the
second test tube, add 1 drop of 10% Lead Acetate. Shake and observe.
3. By strong mineral acids – To two clean test tubes, place 2mL egg albumin solution.
Using a pipette, add 1 drop of conc. Nitric acid to the first tube allowing the acid to slide
down the side of the test tube. To the second test tube, add similarly, 1 drop conc.
Sulfuric acid. Note the color change at the junction of the protein-acid layer.

Department of Medical Technology, Biochemistry Cluster – 2022-2023

PRECIPITATION OF PROTEIN
4. By alkaloidal reagents – To 2mL of egg albumin, add 2 drops of Tannic acid in a
test tube. Shake well. Repeat the test using 2 drops of Picric acid in place of Tannic acid.
Compare the results.

5. By alcohol – Place 2mL of egg albumin in a test tube. Add 1 or 2 drops of Acetic acid
and 2mL of 95% Ethyl alcohol. Shake and note the precipitate formed.

Department of Medical Technology, Biochemistry Cluster – 2022-2023

PROTEINS
BIOCHEMISTRY CLUSTER
LABORATORY EXPERIMENT

Department ofDepartment
Medical Technology,
of MedicalBiochemistry
Technology, Biochemistry
Cluster – 2022-2023
Cluster – 2022-2023