PROTEINS

P R E P A R E D B Y : C R I S T O P H E R P. Y T I N G , R P H
What are Proteins?
Polymers of Amino Acids
formed via Peptide bonds
Responsible for metabolic
capabilities & morphology of
organisms
AMINO ACID
BUILDING BLOCKS FOR PROTEINS
ISOLATION AND CHARACTERIZATION OF
PROTEINS
1. ISOLATION OF GLUTEN
2. ISOLATION OF BEAN PROTEIN
3. ISOLATION OF MYOGLOBIN FROM MUSCLE
4. ISOLATION OF ALBUMIN FROM EGG WHITE
Gluten
• Gluten is a protein naturally found in
some grains including wheat, barley, and
rye which acts as a binder.
• Gluten gives elasticity to dough, helping
it rise and keep its shape and often gives
the final product a chewy texture
Isolation of Gluten
 Washings remained clear after iodine
test, indicating removal of all starc
The principle involved in the isolation of gluten is difference in
solubility. The starch is partially soluble in water while gluten is
insoluble in water.
Thus, gluten can be separated from starch. Iodine solution is used to
test the complete removal of starch, which involves the formation of
blue-iodo starch complex. Refrigeration keeps the protein from
degrading.
CRUDE GLUTEN
is a mixture of two proteins, glutenin and gliadin.
It is also the composite of a prolamin and glutelin, which exist,
conjoined with starch, in the endosperm of various grass-related
grains.
BEAN PROTEIN
Soaking of Beans in Water
◦ Soaking allows water to be
distributed among starch and protein
fractions within the legume. As
soaking proceeds, water penetrates
the seed coat, travelling through the
cotyledons and towards the centre of
the bean. Such water absorption
causes the bean to become soft and
uniform in texture (Gowen et
al. 2007).
Predominant Protein found in Beans:
◦ globulin (50–70%) and albumin (10%).
(Ganesan et al, 2017)
Precipitation of Bean by an Acid
◦ Adding acids to the solution, lowers the pH
and leads to positively charging the protein,
due to the proton capture by amino groups.
In an aqueous solution the hydration sphere
surrounding a protein is disrupted; occurred
imbalance in structure leads to precipitation.
MYOGLOBIN
Myoglobin is a heme protein that's
found in your striated muscles
which supplies oxygen to the cells
in your muscles (myocytes).
Is a richly pigmented protein. The
more myoglobin there is in the
cells, the redder, or darker, the
meat.
ALBUMIN
Class of water-soluble proteins found in
egg white as well as milk and blood
serum.
Albumin is a protein made by your liver.
Albumin enters your bloodstream and
helps keep fluid from leaking out of your
blood vessels into other tissues. It is also
carries hormones, vitamins, and enzymes
throughout your body.
GENERAL TEST
BIURET TEST
•Test for the Presence of Peptide
Bonds/Linkage
•Biuret Reagent is composed of
Hydrated Copper Sulfate, Sodium
Hydroxide, and Rochelle salt
(sodium-potassium tartrate).
 PRINCIPLE: In the presence of peptides,
a copper(II) ion forms violet-
colored coordination complexes in
an alkaline solution
 Positive color result:
Rose – pink to violet to purple
NINHYDRIN TEST
Test for the presence of Alpha Amino Side Chain and proteins that
contain free amino groups, (-NH2).
 Except Proline and Hydroxyproline
When exposed to ninhydrin, the amino
acid undergoes oxidative deamination,
resulting in the liberation of CO2, NH3,
and an aldehyde along with
hydrindantin (which is a reduced form of
ninhydrin).
Positive color result:
Deep purple-blue color
PROLINE AND HYDROXYPROLINE
SPECIFIC TEST
 XANTHOPROTEIC TEST
TEST FOR AMINO ACIDS WITH AN AROMATIC NUCLEUS/ AROMATIC AMINO ACIDS

TYROSINE

TRYPTOPHAN
PHENYLALANINE
 Sample is treated with a hot,
concentrated nitric acid it reacts with
aromatic amino acids such as
phenylalanine, tyrosine and
tryptophan and forms a yellow
colored product known as Xantho
protein.
Positive Result : Dark yellow or
Orange-colored solution
MILLON’S TEST
TEST USED FOR DETECTION OF THE
AMINO ACID TYROSINE
PRESENCE OF PHENOLIC RING
Millon's reagent consists of Mercuric
nitrate and Mercurous nitrate dissolved
in nitric acid and distilled water.
TYROSINE
Principle: Nitrification of the
phenol group in tyrosine, which
then forms complexes with heavy
metals like Mercury.
Positive Result: Red or Pink
colored precipitate.
HOPSKIN-COKE TEST (GLYOXYALIC ACID)
TEST USED FOR THE DETECTION
OF AMINO ACID TRYPTOPHAN
PRESENCE OF INDOLE RING

TRYPTOPHAN
PRINCIPLE: Layering of concentrated
sulfuric acid over a mixture of
tryptophan-containing proteins with the
Hopkins's Cole reagent results in the
formation of a violet ring at the
interface.
POSITIVE RESULT: Purple colored Ring at
the junction of two Layers
SAKAGUCHI TEST
TEST USED FOR DETECTION OF
AMINO ACID ARGININE
PRESENCE OF GUANIDINE GROUP

ARGININE
Sakaguchi Reagent consists of : α –
napththol and Sodium Hypobromite
Principle: Arginine reacts with α –
napththol and an oxidizing agent such as
bromine water or sodium
hypochlorite/sodium hypobromite to give a
red colored product.
Positive Result: Formation of Red Color
LEAD ACETATE TEST
TEST USED FOR DETECTION OF
CYSTEINE AND CYSTINE
PRESENCE OF SULFUR
PRINCIPLE: Degradation of the S-H or S-S
group in amino acids under strongly alkaline
conditions. Amino acids like cysteine and
cystine release sulfur in the presence of
strong alkaline conditions at a high
temperature. The sulfur then combines with
the alkali (NaOH) to form Na2 The Na2S thus
formed reacts with lead acetate to form lead
sulfide, which results in a black residue.
POSITIVE RESULT: Black Precipitate