COLOR REACTIONS OF INTACT
PROTEIN AND HYDROLYZATE
GAERLAN - HERRERA - MENDOZA - ROQUE - SIMEON
INTRODUCTION
• Proteins are polymers of amino acids that are linked by
peptide bonds, which can be broken during hydrolysis.
• The protein used in this experiment is casein, the main
protein in milk. Casein is a phosphoprotein and it is
soluble in dilute acid and alkali.
BIURET TEST
• Often used to determine the presence of peptide bonds
in protein.
• Named after the substance biuret (H2NCONHCONH2).
• Biuret reagent contains hydrated copper sulfate,
potassium hydroxide solution, and potassium sodium
tartrate.
NINHYDRIN TEST
• Used to determine if there are amino acids in a
given sample.
• Ninhydrin (triketohydrindene hydrate in ethanol) is
an oxidating agent which leads to the oxidative
deamination of alpha-amino groups.
XANTHOPROTEIC TEST
• Reagents: conc. HNO3 & conc. NaOH
• General test for aromatic amino acids.
• Aromatic groups in the amino acids will be nitrated
by HNO3 via electrophilic aromatic substitution. The
nitro derivate show an intensely yellow color.
HOPKIN-COLE’S TEST
• The Hopkins-Cole test is specific for tryptophan
• The indole ring reacts with glyoxylic acid in the
presence of a strong acid to form a violet cyclic
product.
OBJECTIVE
• This experiment aims to characterize the intact
protein and acid and base hydrolyzate using
various chemical tests.
METHODOLOGY
Intact Casein
Cut into small pieces
Place in a mortar
Add 10 mL distilled water and grind
Fine protein suspension
BIURET TEST
Intact Casein Acid or Base Hydrolyzate
Add 1 drop of 2.5 M NaOH to 3 drops
of protein suspension or hydrolyzate
Add a drop or more of 0.01M CuSO₄
Note the colors produced.
NINHYDRIN TEST
Intact Casein Acid or Base Hydrolyzate
Add 1 mL of water was to 10 drops of protein
suspension. For acid/base hydrolyzate, use 1mL
without dilution with water.
Add 0.5mL of 0.1% Ninhydrin solution
Heat in a boiling water bath for 2-3 min.
Note the colors produced
XANTHOPROTEIC TEST
Intact Casein Acid or Base Hydrolyzate
Add 1 mL of water to 10 drops of protein
suspension. For acid/base hydrolyzate, use
1mL without dilution with water.
Add slowly 3 drops of conc. HNO₃. Mix
Note the colors produced.
Heat in a boiling water bath for 1 min.
Cool the solution with flowing water and add
slowly conc. NaOH drop by drop.
Continue addition of NaOH until the solution
is alkaline (test with litmus paper).
Note the colors produced.
HOPKIN-COLE’S TEST
Intact Casein Acid or Base Hydrolyzate
Add 1mL of Hopkins-Cole reagent to 2 drops of
protein suspension or hydrolyzate. Mix well.
Incline the tube and add slowly 1 mL conc.
H₂SO₄ down the side of the tube until two layers
form. Do not disturb the two layers.
Note the colors formed at the interphase.
RESULTS
INTACT PROTEIN
TEST POSITIVE RESULT RESULTS
Biuret Test Violet solution Positive
Ninhydrin Test Violet solution Negative
Xanthoproteic Test Yellow – Orange precipitate Positive
Hopkins-Cole Test Violet Ring in solution Positive
ACID HYDROLYZATE
TEST RESULTS
Biuret Test Light Blue (-)
Ninhydrin Test Dark Purple (+)
Xanthoproteic Test Yellow – Orange (+)
Hopkins-Cole Test Colorless Ring (-)
BASE HYDROLYZATE
TEST RESULTS
Biuret Test Colorless or light blue solution
(negative)
Ninhydrin Test Wine Red Solution (positive)
Xanthoproteic Test Yellow – orange solution (positive)
Hopkins-Cole Test Violet ring Present (positive)
BIURET TEST
• Purpose: Presence of peptide linkages
• Reagent: 2.5M NaOH, 0.01M CuSO4
• Positive Result: Violet solution
• Basis: Chelation of Cu (II) ions with peptide bonds
NINHYDRIN TEST
• Purpose: Presence of free amino acids
• Reagent: 0.1% Ninhydrin solution
• Positive Result: Deep blue solution
• Basis: The free NH2 group reacts with the ninhydrin
solution
XANTHOPROTEIC TEST
• Purpose: Presence of aromatic amino acids
• Reagent: Concentrated HNO3, NaOH
• Positive Result: Orange solution
• Basis: Nitration of aromatic rings via electrophilic
aromatic substitution
HOPKINS-COLE TEST
• Purpose: Presence of indole group
• Reagent: Hopkins-Cole reagent, concentrated
H2SO4
• Positive Result: Presence of violet ring
• Basis: Condensation of the glyoxylic acid with the
tryptophan
Acid Base
Test Intact Protein
Hydrolyzate Hydrolyzate
Biuret Test + - -
Ninhydrin Test - + +
Xanthoproteic
+ + +
Test
Hopkins-Cole
+ - +
Test
CONCLUSION
• The only positive for the Biuret test is the intact protein. It
can also be concluded that it contains an aromatic
amino acid tryptophan because it showed positive results
in the Xanthoproteic and Hopkins-Cole test.
• The acid and base hydrolyzates are both positive in the
Ninhydrin test which indicates that they contain free
amino acids or group. These are aromatic amino acids
since they both showed a positive result in the
Xanthoproteic test. The only difference is that the base
hydrolyzate contains tryptophan since it is positive for
Hopkins-Cole test while the acid hydrolyzate contains
either phenylalanine or tyrosine.
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