Exercise 2: Color Reactions of

Intact Protein and Hydrolyzate

(Dumaguit, 2020)
 Intact Protein
Individual AA are linked
together by peptide bond
Hydrolyzate
Product of hydrolysis; peptide
bonds undergone cleavage
Spot Plate
• Biuret test
• Sakaguchi Test
Hydrolyzate
• Ninhydrin Test
• Xanthroproteic Test
• Hopkin’s Cole Test
(Dumaguit, 2020)
 Biuret Test (BT)
Presence of Intact Protein (Peptide Bonds)
Intact Protein Acid Hydrolysis Base Hydrolysis
+ - -
Reagents:
1) 0.01M CuSO4 : 5H2O (Chelating agent)
2) 2.5 M NaOH (raise the pH of medium to make
it alkaline)
Complexation Reaction
 Cu+2 & amide N atoms
 Based on the ability of Cupric ions to form
POSITIVE (+) NEGATIVE (-)
a violet-colored chelate complex with peptide
Violet Color bonds (-CONH- groups) in alkaline conditions. Blue Color
(Color intensity based  Chelate complex absorbs light at 540nm so it Has no peptide bonds
on peptide bond length) appears violet
Has 2 or more peptide
bonds

(Dumaguit, 2020)
 Ninhydrin Test (NT)
Presence of free amino acids (alpha Amino group)
Intact Protein Acid Hydrolysis Base Hydrolysis
- + +
Reagents:
1) 0.1% Ninhydrin = triketohydrindene hydrate in ethanol
(Strong Oxidizing Agent)
Oxidative Deamination & Condensation; Reduction of Ninhydrin
 Amino groups react with ninhydrin to form aldehyde, hydrindantin,
ammonia and carbon dioxide. One molecule of hydrindantin,
ninhydrin and ammonia complex forms Ruhemman’s compex
 When the analyte contains Imino-acids like proline, a yellow
coloured complex is formed.
 During oxidative deamination, an amino acid is converted into the
POSITIVE (+) corresponding keto acid by the removal of the amine functional NEGATIVE (-)
group as ammonia and the amine functional group is replaced by
Violet or Deep Blue the ketone group Yellow color
Color
(Enzyme+Ninhydrin)
(Supernatant from
enzyme + Ninhydrin)
Has no peptide bonds
Has 2 or more peptide
bonds
(Dumaguit, 2020)
 Xanthoproetic Test (XT)
Presence of Aromatic AA (F, Y, W)
Intact Protein Acid Hydrolysis Base Hydrolysis
+ + +
Reagents:
1) conc. HNO3 (nitration to react with the ring; used
to produce salt)
2) conc. NaOH (neutralize excess acids)
Nitration of Aromatic rings via Electrophilic Aromatic
Substitution
 White precipitate forms upon adding nitric acid, the
benzene ring of AAs undergo nitration with hot, strong
POSITIVE (+) HNO3 ,producing a yellow color in an acidic medium then NEGATIVE (-)
Yellow Precipitate to deepens to an orange color upon treatment with NaOH(to
neutralize)
No Color Change
Orange Solution  Presence of electron donating substituents enhances Has no aromatic amino
Has aromatic amino reaction rate
acids
acids

(Dumaguit, 2020)
 Hopkin’s Cole Test (HC)
Presence of Indole Group (W)
Intact Protein Acid Hydrolysis Base Hydrolysis
+ - +
Reagents:
1) Glyoxilic Acid (Mg + Oxalic Acid)
2) conc. H2SO4 (dehydrating agent)
Acid catalyzed-Condensation & Complexation
 Reduction of Oxalic Acid to Glyoxilic Acid
 The indole ring reacts with glyoxylic acid in the
POSITIVE (+) presence of sulfuric acid to form a violet cyclic NEGATIVE (-)
product at the junction of 2 layers of liquid
Violet Interphase with albumin and casein.
No Color Change
Contains tryptophan Has no tryptophan

(Dumaguit, 2020)
 Sakaguchi Test (ST)
Presence of Guanido group (R)
Intact Protein Acid Hydrolysis Base Hydrolysis
+ + -
Reagents:
1) α-napthol (condensation)
2) NaOH (catalyst)
3) NaOBr (oxidizing agent)
Base catalyzed-Condensation & Complexation
 Guanido group present in the amino acid
POSITIVE (+) reacts with α-napthol and NaOBr to give red- NEGATIVE (-)
colored complex
Red coloration which  Urea is used to stabilize color and destroy Yellow color
easily disappear excess OBr- anion Has no arginine
Contains arginine

(Dumaguit, 2020)