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 SKIM/ SKIMMED MILK
▪ is an oil-in-water emulsion, ▪ is made when all
containing its 3.9% fat the cream (also
dispersed as micronsized called milkfat) is removed
globules 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
presence of ammonia
(No presence of Nitrogen
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
Detection of Red - Red

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:
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

Deep Red color w/ a-

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

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
HYDROLYSIS OF INTACT
PROTEINS
ANTONET TE LARRAZABAL
HYDROLYSIS
❖ chemical reaction in which water is used to break down bonds of a particular substance
❖reaction with water/ breaking using water
PROTEIN HYDROLYSIS

generally only used

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

HYDROLYSIS reduced by the addition of phenol

to
the HCl.
PROTEIN HYDROLYSIS

▪ is rarely used except for the

determination of glutamine
and asparagine, which are
ENZYMATIC converted to aspartic and
glutamic acids together with
HYDROLYSIS ammonia by acid hydrolysis..
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 ACI DS
- BY SA LTS OF HEAV Y M E TALS
- BY A LCOHOL
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
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