Chemistry of Proteins (Part - IV) Properties of proteins .

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Colloid property
• Diameter: 1~100nm, in the range of colloid;
• Are heavier than water and sink
• Proteins shows colloidal properties.

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Colloidal Osmotic Pressure
• Colloidal protein molecules exert osmotic pressure.
• The osmotic pressure generated by plasma proteins is
often called the colloidal osmotic pressure or oncotic
pressure of plasma.
• The osmotic pressure of protein is proportional to its conc.,
but inversely proportional to its molecular weight.
• • In blood plasma, albumin contributes 75-80% of osmotic
pressure (although it represents no more than half the
plasma proteins), because its molecular weight is lower.
• Oncotic pressure exerted by protein is clinically important
in maintaining blood volume.

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Hydration of Proteins
• Proteins, when come in contact with
water, absorb water and swell up.
• Polar groups (COOH, NH2, OH) on the
surface form a hydration shell;
• Hydration shell and electric repulsion
make proteins stable in solution.
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Isoelectric PH (PI) of proteins
The PH at which protein exist as zwitter ion.
At isoelectic PH…
Maximum
Precipitation
Minimum
Migration under electrical field.
Solubility
Buffuring capacity
Viscosity

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Isoelectric PH (PI) of proteins
The isoelectric PH of some proteins are ……
Pepsin = 1.1
Casein = 4.6
Albumin = 4.7
Globulin = 6.4
Applications
This property of proteins is used in
Separation of protein from the mixture

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Precipitation reactions of proteins
PRECIPITATION BASED ON DENATURATION
Precipitation by Heat (Heat Coagulation Test)
Heller’s test
PRECIPITATION BASED ON DEHYDRATION
Precipitation by Alcohol
PRECIPITATION BY SALTS
Half Saturation Test
Full Saturation Test
PRECIPITATION BY HEAVY METALS
PRECIPITATION BY ALKALOIDAL REAGENT

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Precipitation of proteins
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isoelectric point
(hydrophilic)
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positively charged
(hydrophobic)
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Instable protein
(deposition)
acidbase
acid base
acidbase
dehydrationdehydrationdehydration
negatively charged
(hydrophobic)
positively charged
(hydrophilic)
negatively charged
(hydrophilic)

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Protein denaturation
The process in which a protein loses its native
conformation under the treatment of denaturants is
referred to as protein denaturation.
leading to disruption of the secondary, tertiary and
quaternary structure of proteins due to cleavage of the
non-covalent bonds, with loss of biological activity.
Primary structure of proteins, i.e. peptide bond is not
affected.

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Causes of denaturation:
1. Physical agents:
-heating above 70 ºC
- vigorous shaking and stirring
- repeated freezing and thawing
- ultraviolet rays, X-rays
- exposure to high pressure.
2. Chemical agents:
- Strong acids and bases (extreme pH)..
- Mercaptoethanol (destroys S-S bonds by
reduction).

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The denatured proteins tend to
- decrease in solubility;
- increase the viscosity;
- lose the biological activity;
- lose crystalizability;
- be susceptible to enzymatic digestion.

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Renaturation
• Once the denaturants are removed, the
denatured proteins tend to fold back to their
native conformations partially or fully.
• The renatured proteins can restore their
biological functions.

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PRECIPITATION PROTEINS BASED ON
DENATURATION
Heat Coagulation Test
Heat disrupts hydrogen bonds of secondary and tertiary protein
structure while the primary structure remains unaffected.
The protein increases in size due to denaturation and coagulation occurs.
A coagulum (solid clump) which is insoluble in water, dilute acids or alkali.
Importance: This is the simplest test to diagnose renal function at bedside.

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PRECIPITATION PROTEINS BASED ON
DEHYDRATION
Precipitation by Alcohol.
Organic solvents like ethanol and Acetone also precipitate
proteins by reducing the water concentration and reducing
the dielectric constant.

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PRECIPITATION PROTEINS BY
SALTS (Salting Out)
Protein molecules contain both
hydrophilic and hydrophobic amino acids.
In aqueous medium, hydrophobic amino
acids form protected areas while
hydrophilic amino acids form hydrogen
bonds with surrounding water
molecules (solvation layer).

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When proteins are present in salt solutions
(e.g. ammonium sulfate), some of the water
molecules in the solvation layer are attracted
by salt ions. When salt concentration
gradually increases, the number of water
molecules in the solvation layer gradually
decreases until protein molecules coagulate
forming a precipitate; this is known as
“salting out”.
As different proteins have different
compositions of amino acids, different
proteins precipitate at different
concentrations of salt solution.
The salt concentration used is described as
“Full Saturation” (Albumin) or “Half
Saturation” (Casein & Gelatin). Globulins are
also precipitated at Half saturation.

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Precipitation by Full saturation
Test
3ml of protein solution in a test tube and add
Ammonium sulphate crystals in a repeated
small quantities until it is saturated. This is
indicated by some crystals of salts remaining
settled in the bottom. Allow the tube stand
for 5 minutes. Filter it and perform the
Biuret test with the filtrate using 40%
NaOH.
Observation
White colour precipitate is formed.
On filtrate Biuret test is negative.
Inferance
Indicate the presence of the
protein.
Note: Filtrate contains ammonium sulphate ions which interferes in the Biuret test.
This is minimized by using 40% NaOH instead of 5% NaOH.

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PRECIPITATION OF PROTEINS BY
HEAVY METALS
Heavy metals (e.g. Hg2+, Pb2+, Cu2+) are high molecular
weight cations.
The positive charge of these cations counteracts the
negative charge of the carboxylate group in proteins
giving a precipitate.

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PRECIPITATION OF PROTEINS BY
ALKALOIDAL REAGENT
Alkaloidal reagents (e.g. Tricholoroacetic acid,
Esbach’s reagent, and sulphosalicyclic acid) are high
molecular weight anions.
The negative charge of these anions counteracts the
positive charge of the amino group in proteins giving a
precipitate.

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Importance of Precipitation
Reaction
Preparations of protein free filtrate for various
estimations as proteins for turbidity and interfere in
colorimetry.
To separate plasma proteins.
To diagnose renal function at bedside.

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