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Topic 6

Hydrophobic Interaction Chromatography

Source of protein
‰ Principle of HIC
‰ Advantages of using HIC
Extraction ‰ What are the factors affecting HIC
‰ Example: Isolating linamarase with
HIC
Separation

Purity &
characterization

HIC
Hydrophobic interaction chromatography
Source of protein Principle
‰ Separation of substances is based on
their varying strength of interaction
Extraction with hydrophobic groups attached to
an uncharged gel matrix

Separation ‰ Hydrophobic groups on proteins are


sufficiently exposed to bind to the
hydrophobic groups on the matrix.
Purity &
characterization
‰ How is this achieved?

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
HIC
Hydrophobic interaction chromatography
General concept

L H Soluble protein

In medium containing
ammonium sulfate
L
H Soluble protein

Exposed hydrophobic regions of proteins and


ligand group will interact, leading to binding of
protein to ligand
HIC
Hydrophobic interaction chromatography
General concept

L H Soluble protein

In medium of low ionic strength


or water
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
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 salt
concentration
4. Gradient type: For example, 0.5 to 0
M
5. Batch wash: 0.5M, 0.3M, 0M

HIC
What is the experimental set up like?

Fraction
Column
collector

Recorder

Buffer

UV monitor

HIC
Hydrophobic Interaction Chromatography

Source of protein
‰ Principle of HIC
; Advantages of using HIC
Extraction ‰ What are the factors affecting HIC
‰ Example: Isolating linamarase with
HIC
Separation

Purity &
characterization

HIC
Advantages of HIC

‰ Large volume of sample can ‰ Purification steps that generate


be loaded large sample volume can be
coupled with this method

‰ Samples with high ionic ‰ Good for samples after


strength can be used ammonium sulfate fractionation.

‰ Well suited to use before gel- ‰ These techniques may require


filtration, ion-exchange and pretreatment of samples (eg
affinity chromatography reducing ionic strength)

‰ Sample eluted with low salt ‰ Sample can be used in ion-


exchange chromatography step

HIC
Hydrophobic Interaction Chromatography

Source of protein
‰ Principle of HIC
‰ Advantages of using HIC
Extraction ; What are the factors affecting HIC
‰ Example: Isolating linamarase with
HIC
Separation

Purity &
characterization

HIC
Factors affecting HIC

Ligand type and degree of substitution


Type of base matrix
Type and concentration of salt
pH
Temperature
Additives

HIC
Factors affecting HIC

‰ Ligand type affects protein


absorption because interactions
may 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

HIC
Factors affecting HIC

‰ Degree of substitution

‰ Binding capacity of protein


to HIC increases with
increased alkyl chain
length (A) and increased
degree of substitution of
immobilised ligand (B)

‰ Caution: protein can bind


via multipoint attachment,
thus difficult to elute

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

HIC
Factors affecting HIC

‰ Type and concentration of salt

‰ Salts that produce relatively


higher “salting out” (eg Na, K or
ammonium sulfates) effectively
promote ligand-protein
interactions in HIC
‰ Amounts of protein bound
increases almost linearly with
increase salt concentration
‰ Bound proteins are desorbed
by washing the HIC column
with dilute buffer solutions
(near neutral pH) or water.

HIC
Factors affecting HIC: pH

‰ Effect of pH on HIC is not straight forward.


‰ In general an increase in pH weakens hydrophobic interactions. It
could 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 HIC
adsorbent at neutral pH, bind at acidic pH.

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 different
proteins and solubility in solution
‰ Practical terms: To be aware that procedure developed
at room temperature may be different if used in the cold
room

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 absorbent
sites and may displace proteins

HIC
Hydrophobic Interaction Chromatography

Source of protein
‰ Principle of HIC
‰ Advantages of using HIC
Extraction ‰ What are the factors affecting HIC
; Example: Isolating linamarase with
HIC
Separation

Purity &
characterization

HIC
Linamarase preparation using HIC:
Novelty: instead of ammonium sulfate precipitating the
protein, use salt to precipitate undesired protein

Leaf
Bind filtrate to
column; was 3 ml Phenyl
with 6 ml 0,6M Sepharose
Amm sulfate;
2 g tissue in 10 discard eluate;
ml 100 mM Na wash column
citrate pH 6; add with 6 ml
0.1 g PVP, then water; collect
homogenize and eluate
followed by 12
ml 2M Amm
sulfate
Peel (root) Final enzyme preparation
HIC
(90 min)
Crude enzyme extract

Salt precipitation

Dialysis step

Gel filtration Ion exchange

Hydrophobic
Chromatofocusing
Interaction
Chromatography

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

HIC