D

i Different levels of structural hierarchy in proteins

f
f
e
r
e
n
t
l
e
v
e
l
o
f
Working with Proteins:

Protein Isolation, Purification and Identification

Protein source: Examples: Tissue samples, plant materials,

cultured mammalian or plant cells
yeast, bacteria

Solubiliozation of Proteins:

Homogenization of the source material:

•Cell rupture by hypotonic solution followed by homogenization
•Cell lysis by detergents followed by homogenization
•For yeast and bacteria rigorous grindings and enzymatic treatments
are used to break the cells.
Sub-cellular fractionation: To obtain and study proteins from various
cellular compartments, cells are ruptured by hypotonic shocks to keep
all membranes intact. A differential centrifugation protocol is used to
Separate fractions e.g. nuclear, mitochondrial, membranous and
fraction. See next figure.

Once proteins are extracted out from their cellular environments

They are unstable:

•May get denatured (loss of structure and activity)

•May get proteolysed by several proteases released from cell
rupture
•May get modified (oxidized)

To avoid this most of the time, all prification steps are carried out
At 0-4 oC and in presence of general protease inhibitors.
Assay of proteins:
•Total proteins and
• specific protein which is under investigation (by enzymatic activity or
specific probe)

Total protein amount can be assayed by

Biuret assay
Lowry Method
Both these methods are based on the binding of Cu2+ with the N of
peptide bond in alkaline condition producing tetra-dentate complex
This complex produces blue colour which can be measured (absorption
at 550 for biuret and 750 nm for lowry’s method). Increase in
absorption is proportional to the anount of proteion.

Coomassie Blue Dye binding assay (Bradford Method): This dye binds
to protein in acidic condition.
There should be some way of assessing the specific protein being
Purified during each step of purification.

For proteins with enzymatic activities such as peroxidase, phosphatase’

Protease, nucleases, polymerases etc, there are convenient assays

These assays use substrates which are converted to fluorogenic product

or product with different absorption maxima (colourimetric product).

Measurement of the amount of product in reaction mixture is

correlated with the amount of protein enzyme.

For proteins without any enzymatic activity, specific probes like

antibodies to them are used to quantify the amount of protein.

ELISA is one of the most common procedure used in diagnostic and

Research laboratories to quantify specific proteins or peptides.
ELISA: Enzyme linked immunosorbent assay
Radioimmume assy (RIA): It is very sensitive technique to estimate the
Amount of known protein/peptide in any solution.

Using a known concentration of radiolabelled peptide and

immobilized antibody, the amount of the unlabelled peptide in
unknownsolution is estimated. The unlabelled peptide in the sample
competes with labelled one.

Amount of radioactive peptide displaced (which can be measured easily

by unlabelled peptide (the unknown concentration) corresponds to the
amount of unlabelled peptide.

A standard curve using different known amount of unlabelled peptide

in this assay can be made. By putting the value of radioactive peptide
released by unknown solution on standard curve one can easily
calculate concentration of peptide in sample solution.
Strategy for purification of a protein:

Once the protein extract is prepared, in order to prify a single protein

from this extract different fractionation methodologies can be used.

•Salting in and salting out

•pH dependent precipitation

•Protein charge based methods: Ion exchange chromatography,

electrophoresis, and isoelectric focussing

•Protein size based methods: Dialysis, ultrafiltration, gel

Electrophoresis, gel filtration chromatography and ultrafiltration

•Polarity based methods: Reverse phase chromatography,

Hydrophobic interaction chromatography, adsorption
chromatography
Effect of salt concentration on the solubility of proteins

Ionic strength (I) of salt affects the solubility of proteins.

I = 1/2 c iZ i2

Ci is molar concentration of the ionic species

Zi is the charge of the ion

Salting in: As the salt concentration (ionic strength) increases, the

Additional ions more effectively shield the protein molecule’s
multiple charges increasing its solubility. Different proteins get
solubilized at different ionic strengths. (see figure 5-2 of the book)
Salting out: At higher ionic strength, the solubility of proteins decreases
This because, the availability of solvent molecules becomes limited as
Most of them are used up by salt ions.

Ammonium sulphate is the most preferred salt used in biochemistry

For salting out proteins. This is because it has very high solubility (close
To 4 M at zero degree Celsius). Refer to fig. 5-3 in the book.

Effect of pH on solubility: In solution of moderate salt solution,

the solubility proteins are minimum at pH around the isoelectric
Point (pI) of the protein. Some proteins are fractionated using this
chriterion