Separation and Characterization

Techniques for Proteins and

Amino Acids
I. Protein Isolation
Selection
 Sources
of Starting Material
 animal or plant tissues
 microorganisms
• Criteria for choosing a sample
– ease of obtaining sufficient quantity of tissue
– amount of biomolecule in the tissue
– any properties peculiar to the biomolecule of
choice
Methods of Solubilization
Separation Techniques
based on the characteristics of
biomolecules.

• Solubility
• Molecular size, weight, density
• Affinity
• Charge
Solubility
 Change in pH
Isoelectric precipitation
A procedure in which the pH of the protein mixture is adjusted to
the pI of the protein to be isolated to selectively minimize its
solubility.
Isoelectric precipitation
 Change in ionic strength
Salting in
Solubility of a protein at low ionic strength generally
increases with the salt concentration.
Salting out
Decrease in solubility of proteins and other
substances in aqueous solution at high ionic
strength. It is a result of the competition
between added salt ions and other dissolved
solutes for molecular solvation.
Based on Molecular Size
Centrifugation
Process of subjecting a suspension of sample at greatly increased
gravitational field (centrifugal force) by rapidly rotating a receptacle
containing the sample which will lead to sedimentation of particles.

Application:
Differential Centrifugation
For separation of crude mixtures of cellular components
Dialysis
 Itis the movement
of molecules by
diffusion from high
concentration to
low concentration.

A process that separates molecules by the

use of a semi-permeable membrane.
Ultrafiltration
 When macromolecular solution is forced
under pressure thru a semi-permeable
bag/disc.
Gel Filtration Chromatography
 Column is packed
with porous beads
 Small molecules enter
the beads and are
retarded, while, large
molecules cannot
enter and so they
migrate faster
Based on Affinity
Chromatography
 Separation of molecules in a mixture depends
on the affinity to either mobile or stationary
phase.
 Types of Chromatography based on the
polarity of each phase:
 Normal phase chromatography
 Reverse phase chromatography
Affinity Chromatography
A procedure based on the
ability of proteins to
interact with specific
molecules.
Based on Charge
 Electrophoresis
 It is the separation of charged particles in
an electric field thru a support medium.
Isoelectric focusing
 Involves electrophoresis
of protein mixtures thru
stable pH gradient
medium.
 Protein will migrate to the
region where pH = IpH.
Gel Electrophoresis

Types of Gel:
1. Agarose
2. Polyacrylamide
SDS-PAGE
 SDS: mask the intrinsic
charge of protein due
to large negative
charge it imparts on
it.
 Separates protein in
the order of their
MWs.
Ion Exchange
Chromatography
 Similarto affinity
chromatography
 Interaction is based
on net charge
 Column is packed
with resin that have
ligand (either positive
or negative in
charge)
 Anion
Exchanger

Cation
Exchanger
Determination of 10 Structure of Protein
A. Qualitative and Quantitative Analysis of
Amino Acids
1. Hydrolyze peptide with 6N HCl at 110 C for 24 hours
2. Separate mixture by an amino acid analyzer
B. Determination of Amino Acid Sequence
1.Methods for identification of N-terminal amino acid
residue
a. Sanger’s Reagent (DNFB)
b. Dabsyl chloride and Dansyl chloride
c. Edman Degradation
 d. Aminopeptidase

Gly – Arg – Phe – Ile – Lys – Met – Leu

2. Methods of Identification of C-terminal amino

acid residue
a. Carboxypeptidase

Gly – Arg – Phe – Ile – Lys – Met – Leu

b. Hydrazinolysis
3. Cleavage of Protein into Peptides
a. Chemical Method
Cyanogen bromide – cleaves at carboxyl side of Met
b. Enzymatic Method
Trypsin - cleaves on carboxyl side of Arg and Lys, but
not when Pro is present
Chymotrypsin - cleaves on carboxyl side of aromatic
amino acid, but not when Pro is present
Determine the peptide sequence.
1. After the reaction of chymotrypsin
N-E-S-R-V-I-W
T-L-M-I
M-V-S-T-K-L-F
2. After the reaction of trypsin
M-V-S-T-K
V-I-W-T-L-M-I
L-F-N-E-S-R
Determine the sequence of a peptide consisting
of 12 amino acids on the basis of the following
data.
1. Amino acid composition: A,M,S,P,F,R, K,I,
V,D,W,T
2. N-terminal analysis: A
3. Carboxypeptidase digestion: V
4. Trypsin digestion: 3 peptides (A,M,S,P,F,R) (K,I)
(V,D,W,T)
5. Chymotrypsin digestion: decapeptide
(A,M,S,P,F.R,K,I,W,T) and a dipeprtide (V and
D)
6. N-terminal analysis of (F-P-R) peptide: F
7. Cyanogen bromide treatment: tripeptide of
A,M,S, and a peptide with 9 amino acids