BIO462

CHAPTER 3:
PROTEINS & PROTEINS PURIFICATION
TECHNIQUES
 LEARNING OUTCOMES

 Describe different levels of protein.

 Understand methodology of protein analysis
 Distinguish between different types of column
chromatography
 Understand the electrophoresis
PROTEIN STRUCTURE
 LEVEL OF PROTEIN STRUCTURE

 Primary structure 

 Secondary structure 

 Tertiary structure 

 Quaternary structure 
Four levels of
protein structure
 PRIMARY STRUCTURE OF PROTEINS

 The primary structure refers to the sequence of the different amino

acids of the peptide or protein that held together by peptide bonds.
 The two ends of the polypeptide chain are referred to as the
carboxyl terminus (C-terminus) and the amino terminus (N-
terminus)
 SECONDARY STRUCTURE

 Secondary structures are defined by patterns of hydrogen

bonds between the carboxyl groups and the amino groups of
different amino acids in the polypeptide backbone.
 These hydrogen bonds can twist and stabilize the amino
chain so that it is woven together into :
 A series of sheet

 A series of spiral

 A single chain of amino acids can be woven into pleated

sheets in one area, and alpha helixes in a different area.
 α-helix

 This structure resembles a coiled spring and is secured by

hydrogen bonding in the polypeptide chain.
 The helix is stabilized by hydrogen bonds between the N-
terminal of one amino acid and the C=O group on the 4th
amino acid away from it.
β-pleated sheet

 Composed of two or more straight chains that are

hydrogen bonded side by side.
 If the amino terminus are on the same end of each
chain, the sheet is termed parallel, and if the chains run
in the opposite direction, the sheet is termed
antiparallel.
 TERTIARY STRUCTURE
 Determined by a variety of
bonding interaction between
the side chain on the amino
acids
 Bonding interaction may be
stronger than the hydrogen
bonds between amide group
holding the helical structure
 As a result, bonding
interaction between side
chain may cause a number of
folds, bends, and loops in the
protein chain
 QUATERNARY STRUCTURE

 Clustering of several individual peptide or protein chains

into a final specific shape
 A variety of bonding interaction including hydrogen
bonding, salt bridge, and disulphide bonds hold the
various chains into a particular geometry
 There are two categories of proteins with quaternary
structure
 Fibrous

 Globular
Fibrous Proteins

 Fibrous proteins: contain polypeptide chains organized

approximately parallel along a single axis. They
 consist of long fibers or large sheets
 tend to be mechanically strong
 play important structural roles in nature
Globular Proteins

 Globular proteins: proteins which are folded to a more or

less spherical shape
 they tend to be soluble in water and salt solutions
 most of their polar side chains are on the outside and interact
with the aqueous environment by hydrogen bonding and ion-
dipole interactions
 most of their nonpolar side chains are buried inside
 DENATURATION OF PROTEIN

 The tertiary structure of protein is result of many intra-molecular

interaction that can be disrupted by change of the environment,
causing the protein to become denatured
 Enzyme lose all catalytic activity when denatured
 Denaturation can be brought about by heat, pH, detergent and
urea
DETERMINATION PROTEIN STRUCTURE
PROTEIN STRUCTURES
 PROTEIN FOLDING
 Protein must fold to function
 Some diseases are cause by misfolding
 Eg: mad cow disease
 caused by the misfolding of a protein known as PrP
 affects cow’s nervous system – lose control
 Domains
 Domains have been described as:
 Geometrically separate regions of protein structure
 Structure with a recognized function
 Sub-sequences that can fold independently into a stable
structure
PROTEIN STRUCTURE DETERMINATION

 High resolution structure determination

 X-ray crystallography ~1A
 Nuclear magnetic resonance (NMR) ~ 1-2.5A

 Low resolution structure determination

 Cryo-EM (electron microscopy) ~10-15A
 X-RAY CRYSTALLOGRAPHY

 More accurate
 Extremely pure protein sample is needed
 Protein sample must form crystal
 Many protein are not amenable to crystallization at
all
NUCLEAR MAGNETIC RESONANCE (NMR)

 Used in advanced medical imaging techniques, such

as in magnetic resonance imaging
 Fairly accurate
 No need for crystal
 Limited to small and soluble protein only
Determining the Primary Structure of a Protein

 Step 1: to establish which amino acids are present and in

what proportions  amino acid analyzer
 Step 2: identities of the N-terminal and C-terminal are
determined
 Step 3 and 4: protein is cleaved into smaller fragments
and the amino acid sequence is determined  Edman
degradation
Edman Degradation

The amino-terminal residue is

labeled and cleaved from the
peptide without disrupting
the peptide bonds between
other amino acid residues
 PROTEIN SIZE

 In general, proteins contain > 40 residues

 Minimum needed to fold into tertiary structure
 Usually 100-1000 residues
 Percent of each AA varies
 Proteins separated based on differences in size and
composition
 Proteins must be pure to analyze, determine
structure/function
PROTEIN PURIFICATION
 PROTEIN PURIFICATION

 Many different proteins exist in a single cell

 A detailed study of the properties of any one protein
requires a homogeneous sample consisting of only
one kind of molecule
 Separation techniques focus on size, charge and
polarity – the source differences between molecules
Overview of protein analysis

 Homogenization.
sample preparation
 Differential centrifugation.

 Protein determination
Salting out
analysis
Chromatography
Electrophoresis
X-ray crystallography
 HOMOGENIZATION

 Before the real purification steps can begin, the protein must
be releases from the cells and subcellular organelles.
 Homogenizer = a thick-walled test tube and tight-fitting
plunger.
 After the cells are homogenized, they are subjected to
differential centrifugation.
Differential centrifugation
SEPARATING PROTEINS:
CHROMATOGRAPHY TECHNIQUE

1. Ion exchange chromatography

2. Gel filtration chromatography (size exclusion)
3. Affinity chromatography
 CHROMATOGRAPHY

 Mobile phase
 a solvent that flows through
the supporting medium
 Stationary phase
 a layer/coating (porous solid
matrix) on the supporting
medium that interacts with the
analytes
 Components of mixture pass
through the column at
different rates based on
properties
 CHROMATOGRAPHY

 The expansion of the protein

band in the mobile phase is
caused by separating of
protein with different
properties and diffusion
spreading
ION EXCHANGE CHROMATOGRAPHY

 Separate based on net charge

of proteins
 Stationary phase = chemically
modified to include charged
groups
 Affected by pH
 Anion exchangers
 positively charge resin
 Cation exchangers
 negatively charge resin
GEL/SIZE-EXCLUSION CHROMATOGRAPHY

 Size/molecular exclusion
chromatography
 Stationary phase = column
matrix is a cross-linked
polymer /gels with pores of
particular size
 Molecules separate based on
size
 Small molecules caught in

pores
 Large molecules pass

through
AFFINITY CHROMATOGRAPHY

 Separate protein by their

binding specificities
 Stationary phase = column
matrix is covalently linked to
some compound called
ligand
 Protein retained on the
column are those that bind
specifically to a ligand, while
other proteins pass through
column
 ELECTROPHORESIS

 Separation of protein based on migration of ions in an electric

field
 The positively charged proteins move towards the negative
electrode (cathode) – attract cation
 The negatively charged proteins move toward the positive
electrode (anode) – attract anion
 A protein at its pI (neutral) will not migrate in either direction
 Variety of supports (gel, paper, starch, solutions)
SDS-PAGE

 Sodium dodecyl sulfate polyacrylamide gel

electrophoresis, is a technique widely used in
biochemistry, forensics, genetics and molecular biology
 To separate proteins according to their electrophoretic
mobility (a function of length of polypeptide chain or
molecular weight).
SDS-PAGE

 Following electrophoresis, the gel may be stained (most

commonly with Coomassie Brilliant Blue R-250 or silver stain),
allowing visualization of the separated proteins

 It is common to run molecular weight size markers of known

molecular weight in a separate lane in the gel, in order to
calibrate the gel and determine the weight of unknown
proteins by comparing the distance traveled relative to the
marker
ISOELECTRIC FOCUSING

 Is another variation of gel electrophoresis

 Different proteins have different isoelectric points
(pH at which a particular molecule carries no net
electrical charge)
 At the pI, the number of +ve charges exactly balances
the number of –ve charges
 In isoelectric focusing, a gel is prepared with a pH
gradient that parallels the electric field gradient
Each protein
remains at the
position on the gel
corresponding to
its pI
 ENZYME PURIFICATION

 Extraction of a single enzyme from cell/ tissues may

contain more than 1000 different proteins and lots of
other biomolecules
 There is no recipe that is sure to work for the purification
of all enzymes and each may have steps that are specific to
the properties of the enzyme
 There are features that tend to be common in methods of
recovery of proteins – enzyme are polymers of amino acids
with many common features
WHY PURIFY ENZYME?
REQUIREMENT OF ENZYME PURIFICATION
LOCATION OF ENZYMES

 Intracellular enzyme : cytoplasmic, organelle

 Extracellular enzyme : released by the cell
 Cell-wall bound : attach to a cell membrane
EXTRACTION TECHNIQUE

 Extraction must be carried out as soon as possible

after sample are obtained
 Otherwise, freeze / cool samples
 Cooling will reduce protease activity and general
tissues breakdown
ISOLATION OF ENZYMES

1. Centrifuge
 Low speed (<3000rpm)
 Moderate speed (<20,000 rpm)
 Very high speed (>20,000 rpm)
2. Filtration
- Cotton wool
- Membrane (>1µm)
SEPARATE CELL DEBRIS

 Centrifugation
 Supernatant
 Pellet

 Filter
PRECIPITATION / SALTING OUT

 Most common method for purification after

extraction and isolation

 3 general methods
 Salting out of protein – ammonium precipitation
 Addition of organic solvent – ethanol, acetone

 Additional of polymers with high molecular weights –

polyethylene glycol
SALTING OUT OF PROTEIN

 A method of separating protein based on the

principle that protein are less soluble at high salt
concentration
 The salt concentration needed for the protein to
precipitate out of the solution differs from protein
to protein
 There are hydrophobic amino acids and
hydrophilic amino acids in protein molecules
 SALTING OUT OF PROTEIN

 After protein folding in the aqueous solution – hydrophilic

amino acids interact with the molecules of H20 and allow
protein to form hydrogen bond with surrounding water
molecule
 When the salt concentration is increased, some of the water
molecules are attracted by the salt ion – decrease the
number of water molecule available to interact with the
charged part of the protein
 Protein molecules coagulates by forming hydrophobic
interaction with each other
 Known as salting out
DIALYSIS

 Carried out after salting out to remove residual  used to

desalt and concentrate protein sample
 Removed ammonium sulphate and other small molecular
weight compounds such as sugar or organic acids
 Concept:
 Separate of big and small molecule
through permeable membrane
in buffer / distilled water
END OF THIS CHAPTER