Techniques to separate

Amino acids and proteins

 Objectives
To discuss:
•Methods used for isolating and studying
proteins, principles, types and clinical
applications of chromatography
•Principles, types and clinical applications of
electrophoresis
 Learning Objectives

• Outline the methods used for isolating and studying

proteins.
• Explain the principles of chromatography.
• Distinguish the different types of chromatography
• Discuss the principles of electrophoresis
• Describe the techniques used to study primary structure
of proteins.
TECHNOQUES TO SEPARATE AMINO
ACIDS AND PROTEINS
T
 Protein purification process

• Detailed studies on function

• Determination of structure

• Industrial/pharmaceutical applications
 Protein purification process

A major portion of most biochemical investigations

involved the purification of the materials because these
substances must be relatively free of contaminations if
they are to be characterized.

• The material of interest may be unstable and exist in very

small amount. Typically, a substance that comprised
<0.1% of a tissue’s dry weight is brought to ~98% purity

• Mild conditions must be used during extraction &

isolation to avoid denaturation.
• Buffered solution must be used for extraction of proteins
from lysed cells.
General Structure of Aminoacids
 Steps involved in Protein Purification

I. Isolation 2. Separation 3. Identification

Isolation

1. Cell disruption (mechanical)- Homogenization

2. Ultra centrifugation

3. Filtration

4. Protein precipitation (concentration)

Homogenization
Sonication - process of applying
ultrasound energy (20–30 kHz) to
agitate particles in a sample. An
ultrasonic bath or an ultrasonic probe,
known as a sonicator is often used to
disrupt cell membranes and release
cellular contents.

Homogenization -
Blending of the tissues in a
Teflon homogenizer
process that involves
breaking apart cells -
releasing organelles and
cytoplasm.
Chemical method
The use of enzymatic methods to remove cell wall, is well-
established for preparing cells for disruption.

Enzymes commonly used include: lysozyme, lysostaphin,

zymolase, cellulase, mutanolysin, glycanase, proteases,
mannase etc.

Detergents disrupt the lipid barrier in surrounding cells,

Triton X (non-ionic detergent, SDS- ionic detergent)
2. ULTRA CENTRIFUGATION
 3. Filtration-Dialysis/Ultrafiltration
• Semi permeable membrane
allows passage of small
molecules but exclude the
passage of proteins.
• Very convenient to transfer a
protein to a lower salt (or
different pH) buffer.

Ultrafiltration (UF) is a variety of membrane filtration in which

hydrostatic pressure forces a liquid against a semi permeable
membrane. Suspended solids and solutes of high molecular weight are
retained, while water and low molecular weight solutes pass through
the membrane.
 4.PROTEIN PRECIPITATION

Protein precipitation can be promoted by agents such as

neutral salts, organic solvents, high molecular mass
polymers, or by appropriate pH.

•The addition of small quantities of neutral salts to a protein

solution often increases protein solubility; the 'salting in'
effect.

•Increasing salt concentrations above an optimal level leads

to destabilization of proteins in solution and eventually
promotes their precipitation. This is known as 'salting out'.
 Solubility of Proteins
 Solubility of proteins are strongly influenced
by pH and salt concentrations.

Solubility
 pH dependence of solubility

In general, solubility is least at pI (isoelectric pH

point) because of least electrostatic
interactions with solvent
pI
 Salt dependence of solubility

Salting in : phenomenon that solubility

increases as salt concentration increases.
Salting out : phenomenon that solubility
decreases as salt concentration increases.

The classic protein fractionation method Salting in

Salting out
called ammonium precipitation - based -on
Solubility
the salting out phenomenon.

Salt concentration
 PROTEIN SEPARATION
Common methods used in protein separation from cells
or tissues:
1. CHROMATOGRAPHY-Differential Partitioning of
solutes between mobile phase and stationary phase.
2. ELECTROPHORESIS- Electrical charge and size.

TYPES OF CHROMATOGRAPHY
 Gel filtration-size exclusion
 Molecular sieving- based on size and shape
 Affinity chromatography-based on specific ligand
binding
 Ion exchange chromatography-based on net charge
of protein at the working pH
 Mobile phase
solvent

Stationary
phase
 Column Chromatography

• Stationary bed is within a tube

• Protein mixture is loaded onto column.
• Because of different binding affinities for column matrix,
some proteins are retained longer on the column & so elute
out later.
• Binding properties obviously depend on what type of
stationary phase (column matrix) is used.
 Gel Filtration – Based on size and shape
• Stationary phase "beads" of
a polysaccharide material

• Mobile phase - solvent

• Large molecules can’t get

into the smaller pores in the
beads and emerge from the
column sooner.

• Smaller molecules and ions

enter the pores in the beads
and elute out later.
 Ion exchange - based on net charge of protein

• Stationary phase : Ion exchange resins have charged

groups (either positive or negative) covalently attached
to the stationary phase.

– Cation exchange chromatography -- column

packing beads have negatively (-) charged groups

– Anion exchange chromatography -- column packing

beads have positively (+) charged groups

• Proteins bind to the matrix by electrostatic interactions.

• Strength of these interactions depends on net charge on
the protein and buffer concentration.
 Cation and anion exchangers

Cation exchange resin

Anion exchange resin

 Affinity Chromatography- ligand binding
• STATIONARY PHASE: A
ligand (Antigen) specifically
recognized by the protein of
interest, covalently attached to
the column material.
•
• Only proteins that bind strongly
to the ligand will stick, while
other proteins pass through the
column.

• Finally protein of interest is

eluted with a buffer containing a
free ligand, which competes
with the column ligand to bind
to the protein.

• The protein of interest is then

eluted out of the column.
Electrophoresis - Gel electrophoresis is most commonly among
method used for macromolecule separation.

 In an electric field, a protein or other charged macromolecule will

move with a velocity that depends directly on the charge of the
macromolecule and inversely on its size and shape
 pH obviously important in determining net charge
 Gel electrophoresis is carried out in some supporting media,
usually polyacrylamide or agarose, with pores big enough to allow
passage of the macromolecule.
 Electric field is applied, and molecules move towards electrode
opposite to their net charge, but they’re slowed down ("friction")
by the gel larger /more elongated shaped molecules move slowly,
smaller/most compact molecules move faster. Proteins in the gel
are easily stained for detection purposes.
Gel electrophoresis
 Sodium Dodecyl Sulfate - PolyAcrylamide Gel
Electrophoresis

• SDS-PAGE is a variant of electrophoresis in which the

buffers contain SDS, a detergent that binds to proteins.

• SDS binds to the hydrophobic regions of proteins and

breaks the disulphide bonds, separates most of them into
their component subunits.

• SDS binding imparts a large negative charge to the

denatured, randomly coiled polypeptides.
• Protein mobility is INVERSELY proportional to the log of
the MASS of individual polypeptide chains.
 Isoelectric Focusing  
• Separation is based on pI.

• pH gradient is set up
using a mixture of
ampholytes (molecules
having both acidic and
basic ions) to give range
of pIs,

• Mixture of molecules
(proteins) is then applied.

• When electric field is

turned on, each protein
moves to its pI value
where its net charge is
 Two-dimensional Electrophoresis

• Isoelectric focusing in first dimension.

• SDS-PAGE in the 2nd dimension at 90o to the first dimension.
Protein Identification – Studying of the primary
structure of proteins and amino acids

Determination of amino acid composition.

Determination of the number of polypeptide chains in

the protein - protein sequencing
Determination of amino acid composition
Detection of proteins and amino acids
Determination of number of polypeptide chains
Protein Sequencing from the N-terminus
1. Edman Degradation
• One residue at a time from the amino terminus can be
chemically derivatized, removed and identified.
• Peptide (one residue shorter) is ready for next round
of derivatization, removal and identification of the
next N-terminal amino acid residue in the sequence.
• Essential steps:

• Coupling (labeling): of the α-amino group of a

• peptide by
the PITC (phenylisothiocyanate, Edman reagent)
• Cleavage ("release"): of the derivatized amino acid
• Conversion to phenylthiohydantoin (PTH) derivative
for identification by comparison with PTH standards
• Process is repeated.
• Whole procedure can also be automated.
Steps in Edman degradation

PITC

PTH aa
 2. Sanger’s reaction

• Reaction of 1-fluoro-2,4-dinitrobenzene (FDNB,

Sanger's reagent) with the N-terminal residue under
alkaline conditions to yield yellow dinitrophenyl
derivatives.
• Yellow color derivatized N-terminal residue is
released from the original peptide.
• α-amino derivatives (N-terminal residue) identified
by chromatographic analysis and comparison with
known standards.
 Clinical significance of studying protein structure

• To study diseases related to abnormal proteins

Mutant hemoglobins - Sickle cell hemoglobin (HbS) –

The change of a single amino acid alters the structure of
hemoglobin molecules to sickle shape.

Electrophoretic analysis of plasma proteins used in

diagnosis of liver diseases.
 References
1. Murray, Granner, Mayes and Rodwell: Harper’s
Biochemistry, 27th Edn
2. Lippincott’s Illustrated Reviews: Biochemistry 3rd
edition, Pamela C. Champe & Richard A. Harvey,
Lippincott Williams & Wilkins Publication.
3. DM Vasudevan & Sreekumari S; Textbook of
Biochemistry (For Medical Students), 4th edition.
Jaypee Bros.
4. TM Devlin; Textbook of Biochemistry 6th Edn.
5. N.V. Bhagavan: Medical Biochemistry 4th Edn