Lect 5 B HIC

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
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
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
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
Source of protein
Principle of HIC
; Advantages of using HIC
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
Source of protein
Principle of HIC
Extraction ; What are the factors affecting HIC
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
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
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
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
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
Source of protein
Principle of 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

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Topic 6

Hydrophobic Interaction Chromatography

Separation  Hydrophobic groups on proteins are

Hydrophobic groups on gel matrix and soluble

Exposed hydrophobic regions of proteins and

In medium of low ionic strength

To release the bound protein from the ligand,

1. Bind protein to HIC media at high salt

 Large volume of sample can  Purification steps that generate

 Samples with high ionic  Good for samples after

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

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

Ligand type and degree of substitution

 Ligand type affects protein

 Straight chain alkyl ligands show

 Binding capacity of protein

 Caution: protein can bind

 Type of base matrix

 Type and concentration of salt

 Salts that produce relatively

 Effect of pH on HIC is not straight forward.

 Implication: Important factor to consider for optimisation of HIC

 Visser and Strating (1975): that role of temperature is a

Salts that cause “salting-in” will weaken

Gel filtration Ion exchange

 Understand the principle of HIC interaction

Anda mungkin juga menyukai

Separation Hydrophobic groups on proteins are

Large volume of sample can Purification steps that generate

Samples with high ionic Good for samples after

Well suited to use before gel- These techniques may require

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

Ligand type affects protein

Straight chain alkyl ligands show

Binding capacity of protein

Caution: protein can bind

Type of base matrix

Type and concentration of salt

Salts that produce relatively

Effect of pH on HIC is not straight forward.

Implication: Important factor to consider for optimisation of HIC

Visser and Strating (1975): that role of temperature is a

Salts that cause “salting-in” will weaken

Understand the principle of HIC interaction