ARTS AND SCIENCES DEPARTMENT

Proteins
Experiment 2A
MC-2 LAB
OBJECTIVES of the Experiment

❏ To perform qualitative test for different types of proteins

❏ To identify proteins based on the different tests performed
❏ Relate the test results to the chemical structure of each protein or
amino acid
PROTEINS

❏ Protein is the most abundant substance in the cell next to water, comprising
15% of its over-all mass.
❏ Protein is composed of amino acids as its building block.
❏ It is linked together with peptide bonds with a positive charged nitrogen-
containing group at one end and a negatively charged carboxyl-group.
Samples in the Experiment
Aspartame – a dipeptide, a sweetener that has no sugar
component
Samples in the Experiment

Glutathione
– a tripeptide
– an antioxidant that prevents
damage to cellular components
caused by free radicals.
Samples in the Experiment
Egg Albumen
Composition of Albumen (Powrie 1973)
Proteins Lipids Carbohydrates Ash Water
9.7-10.6% 0.03% 0.4-0.9% 0.5-0.6% 87.9-89.4%

MAJOR PROTEINS IN ALBUMEN OF TOTAL PROTEINS (Powrie 1973)

● Ovalbumin 54%
● Conalbumin 13%
● Ovomucoid 11%
● Lysozyme 3.5%
● Globulins (G2, G3) 8.0% (?)
● Ovomucin 1.5%
● Other protein components include, flavoprotein (.8%), ovoglycoprotein (.5%), ovomacroglobulin (.5%),
ovoinhibitor (.l%) and avidin (.05%).
Samples in the
Experiment
Amino Acid
Content of the
major albumen
proteins
Samples in the Experiment
Casein
– Caseins and whey proteins are the main group of milk
proteins found in different ratios in various milk species.
– Human milk has casein to whey protein ratio of 40:60 while
cow, sheep, goat and buffalo milk has casein to whey protein of
80:20
Samples in the Experiment
Amino Acid Profile in Milk

Chemical Composition, Nitrogen Fractions and Amino Acids Profile of Milk from Different Animal Species
Saima Rafiq, Nuzhat Huma, Imran Pasha, Aysha Sameen, Omer Mukhtar,1 and Muhammad Issa Khan*
Samples in the Experiment
Amino Acid Profile in Milk

Chemical Composition, Nitrogen Fractions and Amino Acids Profile of Milk from Different Animal Species
Saima Rafiq, Nuzhat Huma, Imran Pasha, Aysha Sameen, Omer Mukhtar,1 and Muhammad Issa Khan*
Biuret Test
■ General test for proteins,
detecting peptide bonds
■ Reagents – KOH, hydrated
CuSO4, Potassium sodium
tartarate
■ Positive result – Violet solution
Chemical Reaction

■ The reaction is a formation of complex between copper and oxygen and

between copper and nitrogen
Experimental Results

Results showed purple color egg

albumin and very light purple for
milk
Ninhydrin Test
■ General test for proteins except
proline (yellow)

■ Test for the –NH2 group in free

amino acid
■ Reagent – Ninhydrin
■ Positive Result - a deep blue or
purple color solution
Chemical Reaction

■ Ninhydrin oxidizes the amino group in the amino acid and proceeds the
chemical reaction forming a complex called Ruhemann’s complex which give
the deep blue color.
Experimental Results

All samples showed a deep blue

coloration
Hopkins-Cole Test
■ Test for indole group,
tryptophan
■ Reagent: Hopkin’s Cole reagent
and conc. H2SO4

■ Positive Result: purple ring at

the interface of two liquids

Credits:
www.biocheminfo.com
Chemical Reaction

■ The indole group of tryptophan reacts with glyoxylic acid in the presence of
concentrated H2S04 to give a purple color.
Experimental Results

Only egg albumin showed distinct

purple color in the junction
between reagents
Xanthoproteic Test
■ Test for activated aromatic rings,
Tyrosine and tryptophan

■ Reagents: Conc. Nitric Acid, ammonium

hydroxide

■ Positive result: Yellow or dark orange

solution or precipitate

Tyrosine
Tryptophan

Credits image:
Phenylalanine https://en.wikipedia.org/wiki/X
anthoproteic_reaction
Chemical Reaction

■ When the sample is treated with a concentrated nitric acid it undergoes nitration
with the aromatic amino acids such as tyrosine and tryptophan and forms a
yellow colored

■ Phenylalanine is not an activated benzene ring so it will not react

Experimental Results

Only the egg and milk samples exhibit

yellow coloration and formation of
principates
Sakaguchi Test
■ Test for guanidine group, arginine
■ Reagent:  α-Naphthol and a drop of
sodium hypobromite
■ Positive Result: red-colored
complex solution

Credits:
Guanidine group Arginine www.biocheminfo.com
Chemical Reaction

■ Bromine water of sodium hypochlorite oxidizes the guanidinium group in

arginine to give a red complex
Experimental Results

All samples showed negative

results
Experimental Results

SAMPLE BIURET NINHYDRIN HOPKINS-COLE XANTOPROTEIC SAKAGUCHI

TEST TEST TEST TEST TEST

Aspartame Negative Positive Negative Negative Negative

Glutathione Negative Positive Negative Negative Negative

Milk Positive Positive Negative Positive Negative

Egg Albumin Positive Positive Positive Positive Negative

Theoretical Results

SAMPLE BIURET NINHYDRIN HOPKINS-COLE XANTOPROTEIC SAKAGUCHI

TEST TEST TEST TEST TEST

Aspartame Negative Positive Negative Negative Negative

Glutathione Negative Positive Negative Negative Negative

Milk Positive Positive Positive Positive Positive

Egg Albumin Positive Positive Positive Positive Positive