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Protein Technologies – Bioseparation Products

Hydrophobic Interaction Chromatography

PHENYL AGAROSE
BUTYL AGAROSE
HEXYL AGAROSE
OCTYL AGAROSE
DECYL AGAROSE

Hydrophobic Interaction Chromatography (HIC) is used to separate proteins on the basis of relative hydrophobicity. Interactions between hydrophobic groups in water are promoted by the presence of water structuring salts, and at high ionic strengths, hydrophobic residues on the surface of a protein associate strongly with other hydrophobic species (the "salting-out" effect). At very high ionic strengths protein precipitation is observed. However, at intermedi­ate ionic strengths, proteins may be adsorbed from solution onto hydrophobic surfaces. This adsorption is reversible, and elution is achieved by simply lowering the ionic strength. Consequently, HIC is particularly useful for the purification from high ionic strength bio­logical extracts since binding is performed in the presence of salt, and elution in the absence of salt. The technique may be applied to the purification of most soluble proteins.

The strength of adsorption is determined by a number of factors including the inherent hydrophobicity of the protein, the hydropho­bicity of the adsorbent, temperature, and the concentration and type of electrolyte used to promote salting-out. Water structuring salts such as ammonium sulphate are normally used for promoting protein adsorption. The ability of salts to promote hydrophobic interactions may be determined from the "Hofmeister series" described below. Since proteins have very different hydrophobicites, and there is a limit to the amount of salt that can be added, it follows that a series of adsorbents with increasing hydrophobicity are required. This is achieved by increasing the size of the immobilized alkyl/aryl group. Relatively hydrophobic proteins require mildly hydrophobic ligands such as butyl groups (otherwise elution problems are encountered), whereas very hydrophilic proteins require relatively hydrophobic lig­ands such as decyl groups (otherwise excessive salt concentrations may be required for binding).

ProMetic BioSciences offers a comprehensive range of HIC media comprising five lig­ands of increasing hydrophobicity immobilized onto two forms of cross-linked agarose beads. Determination of the most suitable adsorbent for a given separation is facilitated by use of the PIKSI^TM-H Kit which is specifically designed for rapid screening of all ProMetic HIC adsorbents.

Properties

Pressure-Flow diagram for the cross-linked agaroses

Highest quality cross-linked agarose beads with very low non-specific binding properties are used to produce the ProMetic range of HIC adsorbents. This range comprises butyl, hexyl, octyl, decyl and phenyl groups immobilized on 6% cross-­linked agarose gels by a neutral and highly stable epoxide link­age. The 6% cross-linked gels typically exhibit higher flow rates and greater protein binding capacities for the majority of proteins. All HIC adsorbents are produced to stringent quality control standards and protein binding capacities are accurately defined to ensure batch-to-batch consistency and reproducible separations. The adsorbents are compatible with most biological buffers and commonly encountered buffer additives including chaotropes, detergents, chelating agents, metal ions and thiols.

Specifications

Protein Binding Capacities: mg BSA bound per ml of moist gel.

Parallel chromatograms of BSA on HIC media showing increasing retention with increased hydrophobicity of immobilized ligand.

Gels packed in identical columns (1.0cm. dia x 2 cm) and re-equilibrated in loading buffer. Bovine serum albumin (BSA) applied as a 5mg/ml solution in 50mM phosphate, pH 6.8 containing 1M ammonium sulphate. BSA eluted with linear gradient as indicated to 50mM phosphate, pH 6.8 containing no ammonium sulphate. Peaks detected spectrophoto­metrically at 280nm.

Purification of serum albumin from human plasma by hydrophobic interaction chromatography on Octyl Agarose 6XL

Column: 1 cm dia. x 5.5cm (bed volume: 4.3ml); flow rate: 0.5ml/min. Column equilibrated in 0.6M ammonium sulphate/50mM sodium phosphate, pH 6.0. Sample: 1:6 diluted human plasma in equilibration buffer (final ammonium sulphate concentration 0.6M): sample volume: 1ml. Step gradient: 0.6 -> 0M ammonium sulphate in 50mM sodium phosphate, pH 6.0. Regeneration: 1M NaOH (as indicated {R}. Eluted peaks: (1) Unbound contaminants: (2) human serum albumin (single bond by SOS-PAGE): (3) Residual bound contaminants.

Fractionation of Saccharomyces cerevisiae protein extract by hydrophobic interaction chromatography on Hexyl Agarose 4XL

Column: 1 cm dia. x 2.5cm (bed volume: 2.0ml); flow rate: 0.5ml/min. Column equilibrated in 1.5M ammonium sulphate/50mM sodium phosphate, pH 6.8. Sample: yeast enzyme concentrate {Sigma Type II} in equilibration buffer (2.5mg total protein/ml); sample volume: 2ml. Elution gradient: 1.5 -> 0M ammonium sulphate in 50mM sodium phosphate, pH 6.8. Peaks unassigned.

Fractionation of crude extract of Clostridium histolyticum showing separation of collagenase and clostripain activities by hydrophobic interaction chromatography on Hexyl Agarose 4XL

Column: 1.0cm dia. X 2.55cm (bed volume: 2ml); flow rate: 0.5ml/min. Column equilibrated in 1.5M ammonium sulphate/50mM Tris-HCl, pH 7.5. Sample: crude collagenase type l (Sigma) in 1.5M ammonium sulphate/50mM sodium phosphate, pH 6.8 (2.5mg/ml); sample volume: 2ml. Elution gradient: 1.5M -> 0M ammonium sulphate/50mM sodium phosphate, pH 6.8 (2.5mg/ml);sample volume 2ml. Elution gradient: 1.5M -> 0M ammonium sulphate/50mM Tris-HCI, pH 7.5. Total protein determined by absorbance at 280nm. Clostripain (∆) and collagenase (□) activities determined using the specific colorimetric substrates BAEE (N-a-benzoyl-L-arginine ethyl ester) and FAGPA (fury{ace-toyl}-Leu-GLY-Pro-Ala) respectively. Activities recovered: collagenase (102%); clostripain (105%).

Ordering information

Please quote both prefix and suffix codes when placing order (eg to order 500ml. Decyl Agarose 6XL, quote the code 0445-00500).

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