PROTEINS

OUTLINE
▪ Sources of Proteins
▪ Isolation and Isoelectric precipitation of proteins
▪ Elementary composition of proteins
▪ Qualitative color reaction
▪Protein hydrolysis
▪Denaturation
PROTEINS
▪ a naturally occurring,
unbranched polymer in
which the monomer units
are amino acids.
▪ More specifically a protein is
a peptide in which at least
40 amino acids residues are
present.
PROTEINS SOURCES
Wheat
Milk
Flour

Minced
Beans
Beef

Egg
 MILK
▪ the most
nutritionally
complete food
found in nature.
 MILK
▪All kinds of milk, human or animal,
contain vitamins (principally
thiamine – VitB1, riboflavin –
VitB2, pantothenic acid – VitB5,
and vitamins A, B12, D), minerals
(calcium, potassium, sodium,
phosphorus, and trace metals),
proteins (mostly casein),
carbohydrates (principally lactose),
and lipids (fats).
COW’S MILK VS HUMAN MILK
WHOLE MILK vs SKIM/ SKIMMED
MILK
WHOLE MILK
▪ is an oil-in-water emulsion,
containing its 3.9% fat
dispersed as micronsized
globules
SKIM/ SKIMMED MILK
▪ is made when all
the cream (also
called milkfat) is removed
from whole milk.
▪It tends to contain around
0.1% fat
Casein
▪ main protein in milk
▪ is a phosphoprotein which has phosphate groups
attached to the hydroxyl groups of some of the amino
acids side-chains
▪Casein exists in milk as a calcium salt, calcium
caseinate

Calcium caseinate
Casein
▪ a mixture of three similar proteins, alpha, beta and kappa
caseins which form a micelle.
▪Alpha- and beta-casein are both insoluble in water and are
solubilized by the micelle surrounding them.
▪The kappa-casein which has a hydrophilic portion is responsible
for solubilizing the other two caseins by promoting the formation
of and stabilizing the micelles.
▪These micelles are responsible for the white opaque appearance
of milk.
Calcium caseinate
▪ has an isoelectric point of pH 4.6.
▪This means it is insoluble in solutions with a pH less than
4.6.
▪The pH of milk is 6.6, therefore, casein has a negative charge
at this pH and is solubilized as a salt
ISOELECTRIC POINT
▪ Is the pH at which an amino acid solution has no net charge because
an equal number of positive and negative charges are present.

▪That pH value is known as the isoelectric point (IEP) of the protein

and is generally the pH at which the protein is least soluble.
▪At this point, almost al amino acid molecules in a solution are
present at their zwitterion form.
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.
▪The prolamin and glutelin
from wheat (gliadin,
which is alcohol-soluble,
and glutenin, which is
only soluble in dilute
acids or alkalis) constitute
about 80% of the protein
contained in wheat fruit.
GLUTEN
▪(from Latin gluten, "glue")
▪is a protein composite found in wheat and related
grains, including barley and rye.
▪Gluten gives elasticity to dough, helping it rise and
keep its shape and often gives the final product a
chewy texture.
Washings remained clear after iodine
test, indicating removal of all starch.
▪ 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.
Elementary
composition of proteins
 Casein in test
tube

Incline and heat

on low flame

Burnt black
Detection of Moisture in the
color/ charring
test tube side
Carbon, of casein
Hydrogen and
Oxygen Presence of
Presence of
hydrogen and
carbon
oxygen
 1g soda lime in
mortar + 0.5g
casein

Mix , transfer to test

tube, heat

Red Litmus test

Red-Blue
Detection of Red-red
(No presence of Nitrogen
presence of ammonia
Nitrogen or ammonia )
(NH3)gas & urine like odor
(presence of Nitrogen )
 Casein in crucible + 2g solid sodium
fusion mixture (Na2CO3 + KNO3) 2:1

heat
Colorless mixture
(black to white) Cool , dissolve in a warm water

Filtration

Residue (discard) Filtrate sol’n (from fusion method)

Acidify w/ dilute HCl

Red-blue Red - Red

Detection of
Sulfur Heat to boiling, add 0.1N BaCl2 sol’n

White ppt.
(Barium Sulfate BaSO4)
 Heat to melt 2g fusion mixture in crucible +
0.5g casein

Heat ‘till fusion mixture clear

Cool, dissolve in water

Neutralize w/ conc. HNO3 until acidic

Litmus paper test

Red-Red Blue – Red

Filtration

Detection of Residue (discard) Filtrate

Phosphorus
Add of (NH4)2MoO4 Ammonium molybdate
Yellow ppt. (NH4)3PO4.12MoO3
ammonium phosphomolybdate Heat to nearly boiling

Presence of phosphorous
Qualitative Color
Reaction
Qualitative Color Reaction

Biuret Ninhydrin Xanthoproteic Millon’s

Lead acetate or
Hopkins-cole Sakaguchi Lieberman
lead sulphide

Sodium Sullivan/ sulliven

Molisch Follins reaction
Nitroprusside cysteine rxn

Pauly diazo test

 Write the Qualitative Color Reaction
test that matches with the description:
BIURET NINHYDRIN XANTHOPROTEIC MILLONS HOPKINS-COLE
Yellow w/ HNO3 & to Reddish violet ring at the
Flesh to red or brick red
Rose-pink to violet then Deep blue color to violet or orange w/ NaOH * - junction w/ Magnesium
complex with mercurous
purple – due to peptide purple - presence of free nitration of benzene ring of salts of oxalic acids –
nitrate in HNO3 , AA with
linkages amino group * aromatic amino acids presence of indole group/
phenol group, Tyrosine
(Y,W,F) tryptophan

SAKAGUCHI LEAD ACETATE LIEBERMAN MOLISCH SODIUM

NITROPRUSSIDE
Deep Red color w/
Black precipitate –
a-naphthol & sodium Violet ring – glycoprotein
methionine, cysteine, Violet color – tryptophan Red color – cysteine
hypochlorite or bromite – (ex. gamma-globulin)
cystine
arginine

FOLLINS REACTION SULLIVAN PAULY DIAZO

Deep red color in alkaline Red color w/ strong
Red color in alkaline
sol’n w/ sodium 1,2 reducing agent sodium
solution treated with
napthoquinonw-4sulfonate hydrosulfite – cysteine and
diazotized sulfanilic acid –
– as quantitative cystine quatitative
tyrosine & histidine
estimation of AA estimation
Qualitative Color Reaction

Biuret Ninhydrin Xanthoproteic Millon’s Hopkins-cole

Lead acetate
Sodium
Sakaguchi or lead Lieberman Molisch
Nitroprusside
sulphide

Sullivan/
Follins Pauly diazo
sulliven
reaction test
cysteine rxn
BIURET TEST (Piotrowski's test)
▪ is a chemical test used for detecting the
presence of peptide bonds/ linkages.
▪ In the presence of peptides,
a copper(II) ion forms violet-colored coor
dination complexes in
an alkaline solution

Positive color result:

▪ Rose – pink to violet to purple
NINHYDRIN TEST
▪ The ninhydrin test is used to detect
the presence of alpha amino acids
and proteins that contain free
amino groups, (-NH2).

▪ Except Proline and

hydroxyproline
 NINHYDRIN TEST
Ninhydrin spray is most
commonly used at
crime scenes to detect
latent fin1gerprints on
porous surfaces such as
paper

Positive color result:

▪ deep purple-blue
color
HYDROLYSIS OF INTACT
PROTEINS
ANTONETTE LARRAZABAL
 HYDROLYSIS
❖ chemical reaction in which water is used to break down bonds of a particular substance
❖reaction with water/ breaking using water
 RACEMATION
❖Process that occurs when a compound undergoes a reaction in which the transformation
produces an equal mixture of both possible enantiomers

ENANTIOMER-a.k.a optical isomer

- one in two molecule that are mirror image of each other
-non-superimposable
PROTEIN HYDROLYSIS

generally only used

ALKALINE in the determination
of tryptophan, since
HYDROLYSI other amino
S acids are degraded.
PROTEIN HYDROLYSIS
▪ cystine, cysteine, and
methionine undergo
variable degradation
ACID through oxidation
during acid hydrolysis
HYDROLYSI ▪ Tyrosine losses can also
occur due to oxidation, but
S this may be reduced by the
addition of phenol to
the HCl.
 Glutamine
Asparagine

PROTEIN HYDROLYSIS
▪ is rarely used except for
the determination of
ENZYMATIC glutamine
asparagine, which are
and

HYDROLYSI converted to aspartic and

glutamic acids together
S with ammonia by acid
hydrolysis..
ACID HYDROLYSIS
-Water molecule would give away proton (water acts as BASE)
-Tryptophan is destroyed
-Serine and Threonine are decomposed
ALKALINE HYDROLYSIS
-random breaking of nearly 40% of all the peptide bonds in protein
-also occurs in small intestines after we eat, hydrolysed by digestive enzymes
-function efficiently in slightly alkaline pH (8-8.5)
-Water acts as ACID

BARIUM HYDROXIDE- being easily removed after hydrolysis

-high reaction
- strong base
ENZYMATIC HYDROLYSIS
- a process in which enzymes enhance bond cleavage in molecules with the addition of the
elements of water
-determination of glutamine and aspargine which are converted to aspartic acid an glutamic acid

ENZYMES
Lipase- fats
Amylase- carbohydrates
Proteinases- proteins
ADVANTAGES & DISADVANTAGES OF
ENZYMATIC HYDROLYSIS
ADVANTAGES: DISADVANTAGES

▪Mild process ▪Low reaction rate

▪Requires less energy ▪High yield of sugar monomers
▪Produce less by-product ▪Usually incomplete
DENATURATION
DENATURATION
▪is the partial or complete disorganization of a protein’s
characteristic three-dimensional shape as a result of
disruption of its secondary, tertiary, and quaternary
structural interactions
▪Causes inactivation of protein activity
▪Causes loss of water solubility or renders the protein
insoluble at isoelectric point
DENATURATION
▪the process of altering the native/low free energy
conformation of a protein
▪Peptide bonds remain intact and the protein usually retains
its original primary structure
▪Principle: Denatured protein can return to its native state
and resume its specific biological activity.
COAGULATION vs FLOCCULATION
FLOCCULATION COAGULATION

– reversible clumping together of the – matted mass of heated

dispersed chains of denatured proteins
when the forces of mutual repulsion is flocculated protein (like in
at minimum. egg albumin)
◦ Agglomeration of destabilized particle into a
large size particle known as flocs which can
effectively be removed by sedimentation or
floatation.
DENATURING AGENTS and Mode of
action
PRECIPITATION:
PRECIPITANTS
- BY ACIDS
- BY SALTS OF HEAVY METALS
- BY ALCOHOL
Organic acids
▪ trichloracetic acid
▪ phosphomolybdic acid
▪ phosphotungstic
▪ Picric acid
▪ Tannic acids
Organic acids: TANNIC ACID
▪ Topically, tannic acid is used for cold sores and fever blisters, diaper rash and
prickly heat, poison ivy, ingrown toenails, sore throat, inflamed tonsils, spongy
or receding gums, acute dermatitis, and as a styptic.
▪Tannic acid is used orally and topically for bleeding, chronic diarrhea, dysentery,
bloody urine, painful joints, persistent coughs, and cancer.
▪Vaginally, tannic acid is used as a douche for leukorrhea.
▪In foods and beverages, tannic acid is used as a flavoring agent.
▪In manufacturing, tannic acid is used in hemorrhoidal ointments and
suppositories, for tanning hides and manufacturing ink, and to kill dust mites on
furniture.
TANNIC ACID
▪ Tannic acid has astringent effects
▪It dehydrates tissue, internally reducing secretions, and externally
forming a protective layer of harder, constricted cells.
▪Tannins show some evidence of antiviral, antimicrobial, CNS
depressant, and cariostatic effects
▪. Some evidence suggests that tannins might cause cancer, but other
evidence shows tannins might prevent it
▪. Regular consumption of herbs with high tannin concentrations
correlates with increased incidence of esophageal or nasal cancer .
Inorganic acids : HNO3/ Nitric acids
▪ used in detecting presence of proteins in
the urine through Heller’s test
▪Results: white ppt. turning yellow
Salts of heavy metals
▪ reaction: proteins from an alkaline solution + salts of heavy
metals = precipitation
▪Ex. lead nitrate, silver nitrate, mercuric chloride
▪ metallic salts – used as antiseptic and germicides through
precipitation.
▪Ex. AgNO3 – used for cauterization of wounds and ulcers.
▪ Egg whites and milk – antidote for metallic poisoning
Factors affecting protein solubility
1. Effects of neutral salts (NaCl, MgSO4,
(NH4)2SO4)
▪ salting in: increase solubility w/ low concentration
▪ salting out: aka antisolvent crystallization,
precipitation as a result of high concentration of
neutral salts
Factors affecting protein solubility
2. Effect of pH
- solubility is minimum at isoelectric point
and increases with increasing acidity or
alkilinity.
Factors affecting protein solubility
3. Effect of organic solvents
- organic solvents (miscible with water),
ex. methanol, ethanol and acetone.
- decrease dielectric constant
- increase electrostatic forces between
charged particles in solution.
Factors affecting protein solubility
4. Effects of Alcohol
▪ dilute solution: alcohol reduces the solubility of proteins
due to an increase in the electrical forces between charged
particles in solution.
▪ High alcohol concentration: protein molecules are
dehydrated. More precipitation.
▪95 % alcohol – less germicidal
▪ 70 % alcohol – with greater germicidal effect
END