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8/7/2019 Lect 5 b HIC
1/22
HIC
Topic 6Hydrophobic Interaction Chromatography
Principle of HIC
Advantages of using HIC
What are the factors affecting HIC
Example: Isolating linamarase with
HIC
Source of protein
Extraction
Separation
Purity &
characterization
8/7/2019 Lect 5 b HIC
2/22
HIC
Hydrophobic interaction chromatography
Principle
Separation of substances is based ontheir varying strength of interactionwith hydrophobic groups attached toan uncharged gel matrix
Hydrophobic groups on proteins aresufficiently exposed to bind to the
hydrophobic groups on the matrix.
How is this achieved?
Source of protein
Extraction
Separation
Purity &
characterization
8/7/2019 Lect 5 b HIC
3/22
HIC
Hydrophobic interaction chromatography
General concept
Salt-promoted adsorption Porath(1986)
L H Soluble protein
Hydrophobic groups on gel matrix and soluble
proteins are shielded by water molecules. To expose
these hydrophobic regions, water must be removed,
and this can be achieved by adding ammonium
sulfate
8/7/2019 Lect 5 b HIC
4/22
HIC
Hydrophobic interaction chromatography
General concept
L H Soluble protein
L
H Soluble protein
Exposed hydrophobic regions of proteins and
ligand group will interact, leading to binding of
protein to ligand
In medium containing
ammonium sulfate
8/7/2019 Lect 5 b HIC
5/22
HIC
Hydrophobic interaction chromatography
General concept
L H Soluble protein
L
H Soluble protein
To release the bound protein from the ligand,
dissociation can be achieved by eluting bound
protein with medium of low ionic strength
In medium of low ionic strengthor water
8/7/2019 Lect 5 b HIC
6/22
HIC
Steps in HIC
1. Bind protein to HIC media at high salt
(0.5 M ammonium sulfate)2. Wash column with buffer of similar
salt concentration
3. Elute column with decreasing saltconcentration
4. Gradient type: For example, 0.5 to 0
M5. Batch wash: 0.5M, 0.3M, 0M
8/7/2019 Lect 5 b HIC
7/22
HIC
What is the experimental set up like?
Column
Recorder
UV monitor
Buffer
Fraction
collector
8/7/2019 Lect 5 b HIC
8/22
HIC
Hydrophobic Interaction Chromatography
Principle of HIC
Advantages of using HIC
What are the factors affecting HIC
Example: Isolating linamarase with
HIC
Source of protein
Extraction
Separation
Purity &
characterization
8/7/2019 Lect 5 b HIC
9/22
HIC
Advantages of HIC
Large volume of sample can
be loaded
Samples with high ionic
strength can be used
Well suited to use before gel-filtration, ion-exchange and
affinity chromatography
Sample eluted with low salt
Purification steps that generate
large sample volume can becoupled with this method
Good for samples after
ammonium sulfate fractionation.
These techniques may require
pretreatment of samples (eg
reducing ionic strength)
Sample can be used in ion-
exchange chromatography step
8/7/2019 Lect 5 b HIC
10/22
HIC
Hydrophobic Interaction Chromatography
Principle of HIC
Advantages of using HIC
What are the factors affecting HIC
Example: Isolating linamarase with
HIC
Source of protein
Extraction
Separation
Purity &
characterization
8/7/2019 Lect 5 b HIC
11/22
HIC
Factors affecting HIC
Ligand type and degree of substitution
Type of base matrix
Type and concentration of salt
pHTemperature
Additives
8/7/2019 Lect 5 b HIC
12/22
HIC
Factors affecting HIC
Ligand type affects protein
absorption because interactionsmay not be strictly hydrophobic
Straight chain alkyl ligands show
pure hydrophobic character
whereas with aryl ligands both
aromatic and hydrophobic
interactions are possible.
Choice of ligand is empirical and
must be established by
experiments
8/7/2019 Lect 5 b HIC
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HIC
Factors affecting HIC
Degree of substitution
Binding capacity of protein
to HIC increases with
increased alkyl chainlength (A) and increased
degree of substitution of
immobilised ligand (B)
Caution: protein can bind
via multipoint attachment,
thus difficult to elute
8/7/2019 Lect 5 b HIC
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HIC
Factors affecting HIC
Type of base matrix
Important to take note that selectivity will not be exactly
the same even with the same type of ligand if the base
matrix is different
Two widely used supports are cross-linked agarose and
synthetic copolymer materials
May be necessary to modify adsorption and elution
conditions
Implication: when reading protocol, take notice
8/7/2019 Lect 5 b HIC
15/22
HIC
Factors affecting HIC
Type and concentration of salt
Salts that produce relativelyhigher salting out (eg Na, K orammonium sulfates) effectively
promote ligand-proteininteractions in HIC
Amounts of protein boundincreases almost linearly with
increase salt concentration Bound proteins are desorbed
by washing the HIC columnwith dilute buffer solutions(near neutral pH) or water.
8/7/2019 Lect 5 b HIC
16/22
HIC
Factors affecting HIC: pH
Effect of pH on HIC is not straight forward.
In general an increase in pH weakens hydrophobic interactions. Itcould be due to increased titration of charged groups leading to an
increase in hydrophilicity of the proteins
Decrease in pH leads to an apparently increase in hydrophobic
interaction
Implication: Important factor to consider for optimisation of HIC
interaction. It is observed that proteins which do not bind to HICadsorbent at neutral pH, bind at acidic pH.
8/7/2019 Lect 5 b HIC
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HIC
Factors affecting HIC: temperature
Visser and Strating (1975): that role of temperature is a
complex issue and differ from observation of Hierten. Binding of proteins to HIC adsorbents is entropy driven
(Hjerten, 1976), ie interaction increases with increase in
temperature Discrepancy in views could be due to differential effects
by temperature on the conformational state of differentproteins and solubility in solution
Practical terms: To be aware that procedure developedat room temperature may be different if used in the coldroom
8/7/2019 Lect 5 b HIC
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HIC
Factors affecting HIC: additives
Salts that cause salting-in will weaken
protein-ligand interactions
Alcohols and detergents (non-polar parts)
can compete with protein for HIC absorbentsites and may displace proteins
8/7/2019 Lect 5 b HIC
19/22
HIC
Hydrophobic Interaction Chromatography
Principle of HIC
Advantages of using HIC
What are the factors affecting HIC
Example: Isolating linamarase with
HIC
Source of protein
Extraction
Separation
Purity &
characterization
8/7/2019 Lect 5 b HIC
20/22
HIC
Linamarase preparation using HIC:
Peel (root)
3 ml Phenyl
Sepharose
Final enzyme preparation
(90 min)
Leaf
Novelty: instead of ammonium sulfate precipitating the
protein, use salt to precipitate undesired protein
2 g tissue in 10ml 100 mM Na
citrate pH 6; add
0.1 g PVP, then
homogenize and
followed by 12
ml 2M Ammsulfate
Bind filtrate to
column; was
with 6 ml 0,6M
Amm sulfate;
discard eluate;wash column
with 6 ml
water; collect
eluate
8/7/2019 Lect 5 b HIC
21/22
HIC
Crude enzyme extract
Salt precipitation
Gel filtration Ion exchange
Dialysis step
ChromatofocusingHydrophobic
Interaction
Chromatography
8/7/2019 Lect 5 b HIC
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HIC
Summary
Understand the principle of HIC interaction
Know the factors that can affect HIC interactions
Exploit this method for protein separation
See the value of using this procedure
Develop own procedures using HIC