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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

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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

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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

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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

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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

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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

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HIC

What is the experimental set up like?

Column

Recorder

UV monitor

Buffer

Fraction

collector

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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

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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

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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

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HIC

Factors affecting HIC

Ligand type and degree of substitution

Type of base matrix

Type and concentration of salt

pHTemperature

Additives

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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

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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

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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

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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.

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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.

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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

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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

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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

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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

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HIC

Crude enzyme extract

Salt precipitation

Gel filtration Ion exchange

Dialysis step

ChromatofocusingHydrophobic

Interaction

Chromatography

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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