Name: Phoebe Joyce Montajes

Date submitted: October 26, 2016

Group number: 2

Date performed: September 15, 2016

Exercise # 5
COLOR REACTIONS OF PROTEINS AND AMINO ACIDS

I.
II.

Objectives: To determine the presence of amino acids to a protein.

To detect the existence of amino acid to a protein.
Discussion:

Amino acids are building blocks of all proteins, and are linked in series by peptide bond (-CONH-) to form
the primary structure of a protein. Amino acids possess an amine group, a carboxylic acid group and a varying
side chain that differs between different amino acids. Amino acids respond to all typical chemical reactions
associated with compounds that contain carboxylic acid and amino groups, usually under conditions where the
zwitter ions form is present in only small quantities. All amino acid except glycine exhibit optical activity due to
the presence of an asymmetric - Carbon atom. Amino acids with L configuration are present in all naturally
occurring proteins, whereas those with D forms are found in antibiotics and in bacterial cell walls.
Gelatin is a protein which contains no tryptophan, and only small amounts of cysteine, methione, and
histidine so it will give a negative results in
Hopkins-Cole (Test is used to detect the presence of tryptophan)
Hopkins-Cole reagent (magnesium salt of oxalic acid) gives positive results with proteins containing the
essential amino acid Tryptophan indicating a high nutritive value specific test for detecting tryptophan The
indole moiety of tryptophan reacts with glyoxilic acid in the presence of concentrated sulphuric acid to give a
purple colored product. Glyoxilic acid is prepared from glacial acetic acid by being exposed to sunlight.
Ninhydrin test
Amino acids contain a free amino group and a free carboxylic acid group that react together with
ninhydrin to produce a colored product. When an amino group acid is attached to the first, or alpha, carbon, on
the amino acids carbon chain, the amino groups nitrogen atom is part of blue-purple product. Proteins also
contain free amino groups on the alpha carbon and can react with ninhydrin to produce to produce a blue-purple
product. Amino acids that have secondary amino group attachments also react with ninhydrin. However, when
the amino group is secondary, the condensation product is yellow.

Detection of amino acids reaction of amino acids with ninhydrin leads to their decarboxylation,
domination (formation of CO2 and ammonia) and formation of aldehyde which has one carbon atom less in its
structure.
The reduced ninydrin condenses with ammonia and non- reduced ninhydrin molecule which leads to the
formation of the violet-blue condensation product:
In the pH range of 4-8, all - amino acids react with ninhydrin (triketohydrindene hydrate), a powerful
oxidizing agent to give a purple colored product (diketohydrin) termed Rhuemanns purple. All primary amines
and ammonia react similarly but without the liberation of carbon dioxide. The imino acids proline and
hydroxyproline also react with ninhydrin, but they give a yellow colored complex instead of a purple one.
Besides amino acids, other complex structures such as peptides, peptones and proteins also react positively
when subjected to the ninhydrin reaction.
Xanthoproteic acid test
Aromatic amino acids, such as Phenyl alanine, tyrosine and tryptophan, respond to this test. In the
presence of concentrated nitric acid, it gives a color when heated with proteins containing tyrosine (yellow
color) or tryptophan (orange color); the color is due to nitration. Aromatic phenyl ring is nitrated to give yellow
colored nitro- derivatives. Alkaline pH, the color changes to orange due to the ionization of the phenolic group.
The Xanthoproteic test uses nitric acid to produce an intense yellow color in samples containing proteins. It
works by nitrating (adding a NO2 group to) the aromatic ring in certain amino acid groups (tyrosine,
tryptophan, and phenylalanine). Casein contains amino acids with these rings and would give a positive result.
Paulys Reaction
This test is specific for the detection of Tryptophan or Histidine. The reagent used for this test contains
sulphanilic acid dissolved in hydrochloric acid. Sulphanilic acid upon diazotization in the presence of sodium
nitrite and hydrochloric acid results in the formation a diazonium salt. The diazonium salt formed couples with
either tyrosine or histidine in alkaline medium to give a red coloured chromogen (azo dye).
Millon's test
Phenolic amino acids such as Tyrosine and its derivatives respond to this test. Compounds with a
hydroxybenzene radical react with Millons reagent to form a red colored complex. Millons reagent is a
solution of mercuric sulphate in sulphuric acid.
Sakaguchi test

Specific test for Arginine or protein containing Arginine. Proteins treated with hypobromite and
-naphthol develop an intensive red color in an alkaline medium. In this case, hypobromite oxidizes the
guanidine group of arginine, and the resulting compound condenses with -naphthol a colored solution.
Positive for the amino acid containing the guanidine group in Arginine. Guanidine group present in the amino
acid reacts with Naphthol and alkaline hypobromite to give red- colored complex. Under alkaline condition, naphthol (1-hydroxy naphthalene) reacts with a mono-substituted guanidine compound like arginine, which
upon treatment with hypobromite or hypochlorite, produces a characteristic red color.

Lead acetate test

Sulphur containing amino acids, such as cysteine, methionine and cystine.The protein solution upon
adding a few drops of lead acetate and heating in an alkaline medium becomes dark. The reaction takes place
also with free amino acids. By heating in an alkaline medium, they produce sulfur and sodium sulphide:

Bromine water test

The bromine in water reagent reacts with sites of unsaturation, even aromatic rings, through a complex
addition reaction. The hydroxyl group of the ring activates the ring toward reaction with the electrophilic
bromine. The advantage to using water as the solvent in this reaction is that the polarity significantly enhances
the stability of the reaction intermediate and increases the overall rate of the reaction. Additional catalysts are
not necessary. Aromatic compounds without the phenol functional group rarely react with bromine through this
mechanism. Bromine water also reacts in an electrophilic addition with alkenes and alkynes.
Biurets test
The Biuret Test is a general test to detect peptide bond or not. Biuret, H2NCONHCONH2, reacts with
copper (II) ions in a basic solution to form a deep violet complex. The peptide linkages in proteins resembles
those in biuret and also form deep violet complexes with basic copper (II) ions in solution. The general or biuret
complex formed between the protein linkages and the copper (II) ion of the biuret test is shown:

The Biuret reagent (copper sulphate in a strong base) reacts with peptide bonds in proteins to form a
violet complex known as the Biuret complex. Two peptide bonds are atleast required for the formation of this
complex, this is why amino acids give negative results with Biuret test. Casein is a water soluble protein, so it
should give a positive test result (light blue to deep purple) when reacted with Biuret reagent.
Albumin is an umbrella term for a type of protein which is water soluble.

http://vlab.amrita.edu/?sub=3&brch=63&sim=1094&cnt=1
https://www.ukessays.com/essays/biology/quantitative-tests-for-aminoacids-and-proteins-biology-essay.php
Results:
Hopkins-Cole Test:

Tryptophan
Gelatin
Peptone
Casein
Albumen

(+) Dark solution

(-) Clear solution
(-) Light violet solution
(-) Purple solution
(-) Light violet solution

Ninhydrin test

Glycine
Gelatin
Peptone
Casein
Albumen

(+) Solution turned dark

blue
(+) turned into light blue
(+) turned blue
(+) blue solution but blurry
(-) No color change

Xanthoproteic acid test

Tryptophan
Tyrosine
Gelatin
Peptone
Casein
Albumen
Paulys Reaction

(-) Brown solution

(+) Faint yellow
(-) Clear solution
(+) Yellow precipitate
(+) Yellow precipitate
(+) Yellow precipitate

Tyrosine
Gelatin
Peptone
Casein
Albumen

(+) Bright red solution

(-) Dark orange solution
(+) Red orange solution
(+) Bright red solution with
precipitate
(-) Orange solution

Millon's test
Tyrosine
Gelatin
Peptone
Casein
Albumen

Sakaguchi test

(+) Yellow solution

(-) Clear solution
(-) Light violet solution
(-) Light violet solution
(-) Light violet solution

Arginine

(+) Light brown solution

Gelatin

(+) Dark brown solution

Peptone

(+) Darker brown solution

Casein

(+) Brown solution

Albumen

(-) Dark brown solution

Cysteine
Hair Sample
Gelatin
Peptone
Casein
Albumen

(-) Clear solution

(+) Black solution
(-) Clear solution
(-) Clear solution
(-) Clear solution
(-) Clear solution

Bromine water test

Tryptophan
Gelatin
Peptone
Casein
Albumen
Biurets test

(+) Light violet solution

(-) Light yellow solution
(-) clear solution
(-) Light violet solution
(-) blurry solution

Lead acetate test

Glycine
Gelatin
Peptone
Casein
Albumen
Blank

(-) Light blue solution

(+) Light purple solution
(+) Light purple solution
(+) Light purple solution
(+) Light purple solution
(-) Turned blue