7/3/2016

PROTEINS
 Denaturation
 Purification and Characterization
Techniques

Denaturation
the loss of the structural order (2°, 3°, 4°,
or a combination of these) that gives a
protein its biological activity; that is, the
loss of biological activity

1
 7/3/2016

Denaturation of a Protein

Denaturation
Denaturation can be brought about by
◦ heat
◦ large changes in pH, which alter charges on
side chains, e.g., -COO- to -COOH or –NH4+
to –NH3
◦ detergents such as sodium dodecyl sulfate
(SDS) which disrupt hydrophobic interactions
◦ urea or guanidine, which disrupt hydrogen
bonding
◦ mercaptoethanol, which reduces disulfide
bonds

2
 7/3/2016

Protein Purification and

Characterization Techniques

Isolation of Proteins from Cells

Many different proteins exists within one cell
• Many steps needed to extract protein of
interest, and separate from many
contaminants
• Before purification begins, protein must be
released from cell by homogenization

3
 7/3/2016

How We Get Proteins Out of Cells?

Salting Out
• After proteins solubilized, they can be purified
based on solubility (usually dependent on overall
charge, ionic strength, polarity)
• Ammonium sulfate (NH4SO4) commonly used to
“salt out”
• Takes away water by interacting with it, makes
protein less soluble because hydrophobic
interactions among proteins increases
• Different aliquots taken as function of salt
concentration to get closer to desired protein
sample of interest (30, 40, 50, 75% increments)
• One fraction has protein of interest

4
 7/3/2016

Differential Centrifugation

• Sample is spun, after lysis, to separate

unbroken cells, nuclei, other organelles
and particles not soluble in buffer used

• Different speeds of spin allow for

particle separation

5
 7/3/2016

Column Chromatography
• Basis of Chromatography
◦ Different compounds distribute themselves to a
varying extent between different phases
• Interact/distribute themselves
• In different phases
• 2 phases:
• Stationary: samples interacts with this phase
• Mobile: Flows over the stationary phase and
carries along with it the sample to be separated

6
 7/3/2016

Ion Exchange

• Interaction based on overall

charge (less specific than
affinity)

• Cation exchange

• Anion exchange

Ion exchange chromatography

Three chromatographic methods used in

protein purification.
(a) Ion-exchange chromatography exploits
differences in the sign and magnitude of the net
electric charges of proteins at a given pH. The
column matrix is a synthetic polymer containing
bound charged groups; those with bound anionic
groups are called cation exchangers, and those
with bound cationic groups are called anion
exchangers. Ion-exchange chromatography
on a cation exchanger is shown here. The
affinity of each protein for the charged groups on
the column is affected by the pH (which
determines the ionization state of the molecule)
and the concentration of competing free salt ions
in the surrounding solution. Separation can be
optimized by gradually changing the pH and/or
salt concentration of the mobile phase so as to
create a pH or salt gradient.

7
 7/3/2016

Size-Exclusion/Gel-Filtration
• Separates molecules based on size.
• Stationary phase composed of cross-linked
gel particles.
• Extent of cross-linking can be controlled to
determine pore size
• Smaller molecules enter the pores and are
delayed in elution time. Larger molecules do
not enter and elute from column before
smaller ones.

Size Exclusion/Gel-filtration
Three chromatographic methods used in protein
purification.
(b) Size-exclusion chromatography,
also called gel filtration, separates proteins according
to size. The column matrix is a cross-linked polymer
with pores of selected size. Larger proteins migrate
faster than smaller ones, because they are too
large to enter the pores in the beads and hence take a
more direct route through the column. The smaller
proteins enter the pores and are slowed by their more
labyrinthine path through the column.

8
 7/3/2016

Affinity Chromatography

• Uses specific binding properties of

molecules/proteins
• Stationary phase has a polymer that can be
covalently linked to a compound called a ligand
that specifically binds to protein

Affinity Chromatography

Three chromatographic methods used in protein

purification.
(c) Affinity chromatography separates proteins
by their binding specificities.
The proteins retained on the column are those that
bind specifically to a ligand cross-linked to the
beads. (In biochemistry, the term “ligand” is used to
refer to a group or molecule that binds to a
macromolecule such as a protein.) After proteins
that do not bind to the ligand are washed through
the column, the bound protein of particular interest is
eluted (washed out of the column) by a solution
containing free ligand.

9
 7/3/2016

Electrophoresis
• Electrophoresis - charged particles migrate in
electric field toward opposite charge
• Proteins have different mobility:
◦ Charge
◦ Size
◦ Shape
• Agarose used as matrix for nucleic acids
• Polyacrylamide used mostly for proteins

Electrophoresis

10
 7/3/2016

Isoelectric Focusing

• Isoelectric focusing - based on differing

isoelectric pts. (pI) of proteins
• Gel is prepared with pH gradient that parallels
electric-field. What does this do?
• Charge on the protein changes as it migrates.
• When it gets to pI, has no charge and stops

11
7/3/2016

12
 7/3/2016

Primary Structure Determination

How is 1˚ structure determined?

1) Determine which amino acids are present
(amino acid analysis)
1) Determine the N- and C- termini of the sequence
(amino acid sequencing)
2) Determine the sequence of smaller peptide
fragments (most proteins > 100 a.a)
4) Some type of cleavage into smaller units necessary

Primary Structure Determination

13
 7/3/2016

Protein Cleavage
Protein cleaved at specific sites by:
1) Enzymes - Trypsin, Chymotrypsin
2) Chemical reagents - Cyanogen bromide

Enzymes:
Trypsin - cleaves @ C-terminal of (+) charged side
chains
Chymotrypsin - cleaves @ C-terminal of aromatics

Peptide Digestion

14
 7/3/2016

Cleavage by CnBr
Cleaves @ C-terminal of INTERNAL methionines

Determining Protein Sequence

After cleavage, mixture of peptide fragments produced.
• Can be separated by HPLC or other chromatographic
techniques
• Use different cleavage reagents to help in 1˚
determination

15
 7/3/2016

Peptide Sequencing
• Can be accomplished by Edman Degradation

• Relatively short sequences (30-40 amino

acids) can be determined quickly

• So efficient, today N-/C-terminal residues

usually not done by enzymatic/chemical
cleavage

Edman Degradation

16
 7/3/2016

Example 1
Consider the heptapeptide:

Val-Leu-Lys-Phe-Ala-Glu-Ala

What would you expect to get if you treated the

peptide with chymotrypsin?

What would you expect to get if you treated the

peptide with trypsin?

Example 1

What would you expect to get if you treated the

peptide with chymotrypsin?

Val-Leu-Lys-Phe and Ala-Glu-Ala

What would you expect to get if you treated the

peptide with trypsin?

Val-Leu-Lys and Phe-Ala-Glu-Ala

17
 7/3/2016

Example 2
A peptide containing (Ala,Gly,Pro,Ser) gives
three dipeptides upon partial hydrolysis. After
sequencing, the dipeptides we get are:

Ser-Ala Ala-Gly Pro-Ser

What is the sequence of the original

peptide?

Example 2
Pro – Ser

Ser – Ala

Ala - Gly

Sequence: Pro - Ser – Ala - Gly

18
 7/3/2016

Problem 1
A peptide containing (Ala,Arg,Cys,Leu,Val)
gives four dipeptides upon partial hydrolysis.
After sequencing, the dipeptides we get are:

Cys-Arg Ala-Cys Leu-Ala Arg-Val

What is the sequence of the peptide?

19