Hydrophobic Interaction Chromatography

Herbert P Jennissen, Universitätsklinikum, Universität Duisburg‐Essen, Essen, Germany

Published online: February 2013

DOI: 10.1002/9780470015902.a0002678.pub3

Hydrophobic interaction chromatography (HIC) involves the separation of protein molecules in their native and biologically active state owing to a differential interaction of these molecules with hydrophobic sites on the surface of a solid support. In the separation process, hydrophobic patches on the protein surface interact with hydrophobic molecules (e.g. alkyl residues) immobilised on the hydrophilic solid phase surface (e.g. agarose). In the paper, a historical overview is followed by a consideration of the biochemical and biophysical mechanisms involved in HIC and a discussion of the critical hydrophobicity method and other approaches to hydrophobic interaction chromatographic methods. A detailed protocol for the individual synthesis of gel libraries for specific needs has been published. The treatise is concluded with a discussion on the predictability of chromatographic results and an outlook into the future.

Key Concepts:

  • Positive and negative cooperativity manifests itself by binding isotherms of proteins not governed by the Langmuir equation, that is, a single binding constant but depends on site–site interactions leading to multiple binding constants.
  • Adsorption–desorption hysteresis manifests itself by an adsorption isotherm, which is not retraced by the desorption isotherm, due to conformational changes on the surface, dissipation of entropy and an increase in affinity.
  • Metastability of adsorption states occurs during adsorption–desorption hysteresis and indicates energetically that a local energy minimum exists (i.e. nonequilibrium state) in distinction to the global energy minimum, that is, true equilibrium.
  • Critical hydrophobicity is the threshold hydrophobicity under low salt concentrations (e.g. 50 mM Tris, 150 mM NaCl and pH 7.0) at which the adsorption of a protein can first be measured.
  • A quantised hydrophobicity gradient is a hydrophobicity gradient separated into a library of distinct gels of varying hydrophobicity for determining the critical hydrophobicity.
  • A homologous gel library consists of gels carrying covalently immobilised hydrocarbons (e.g. alkyl residues) comprising a homologous series.
  • The lattice-site binding function is a protein adsorption isotherm (often showing positive cooperativity) as a function of the surface concentration of immobilised hydrocarbons (e.g. alkyl residues).
  • The bulk ligand binding function is a protein adsorption isotherm on immobilised hydrocarbons (often showing negative cooperativity) as a function of the bulk protein concentration.
  • Predicting chromatographic behaviour from sorption isotherms is a major aim in understanding HIC.
  • Predicting chromatographic behaviour from protein structure is the ultimate aim in understanding and utilising HIC.
  • The critical hydrophobic patch area hypothesis is a method of predicting the chromatographic behaviour of proteins in HIC from protein structure, utilising the area of hydrophobic patches on the surface of proteins.

Keywords: agarose/Sepharose 4B; adsorption hysteresis; cooperativity; critical hydrophobicity; fibrinogen; hofmeister series; irreversibility; multivalent interactions; phosphorylase; salting-in; salting-out

Figure 1. The lattice-site concept and parameters of HIC. (a) Simplified scheme of a planar two-dimensional binding site lattice on a butyl-Sepharose 4B disregarding variations in the orientation of butyl-residues. The spacing between residues of c. 1.5–2.0 nm is in the range of a surface concentration of 30 mol/ml packed gel; the length of a butyl residue is c. 0.5 nm. Phosphorylase b (6.3×5.5×10.9 nm) will cover an area containing more than 10 residues (see Jennissen (1976b)). (b) Variation of the chain length parameter in an uncharged homologous series of alkyl agaroses. The surface concentration of the immobilised residues should be constant, no charges should be introduced and leakage should be low. The CDI method is optimal for coupling of alkyl amines (see Jennissen (2005)). (c) Variation of the surface concentration parameter of immobilised alkyl residues of a fixed chain length. The surface concentration shown here as a one-dimensional array of residues increases from (a) to (c). The number of alkyl residues capable of interacting with the protein (i.e. the valence) and thus the affinity of binding increases as the surface concentration of alkyl residues and concomitantly the hydrophobicity is enhanced.
Figure 2. Interrelationship between the chain length parameter and the surface concentration parameter for the binding of a single protein to various series of alkyl agaroses as shown in model curves. The sigmoidal curves shown here for an idealised system correspond to adsorption isotherms of the lattice-site binding function type (Jennissen, 1988, 2005). This form of the curves is due to the cooperative interaction of multiple alkyl residues with the protein (multivalence). For simplicity, the curves were calculated with the same increment, although there are indications that the increment can vary (Jennissen and Heilmeyer, 1975). An increase in the chain length of the immobilised alkyl residue shifts the curves from the right to the left. CH indicates the threshold region of critical hydrophobicity, which corresponds to the three different points of origin for the sigmoidal curves on the x-axis. n, number of carbon atoms (n>3).
Figure 3. Inverse salt dependence of the chromatography of purified phosphorylase b (phos. b) on Seph-C1 (30 mol mL–1 of packed gel). The equilibration buffer contained 10 mmol L–1 of sodium -glycerophosphate, 20 mmol L–1 of mercaptoethanol, 2 mmol L–1 of EDTA, 20% sucrose, 0.5 mol L–1 of phenylmethylsulfonyl fluoride and pH 7.0 (buffer A), to which either 1.1 mol L–1 of ammonium sulfate or NaCl was added. Phosphorylase b (6 mg/3 mL–1) was added to 20 mL Seph-C1 in a 2.0 cm i.d.×17 cm column at 5 °C. Fractions of 6.5 mL were collected. The gel was prepared by the CNBr procedure. (a) Application of enzyme to a column equilibrated with buffer without ammonium sulfate (NH4)2SO4 or NaCl. (1) Application of phosphorylase b in buffer A. (2) Elution with buffer A+1.0 mol L–1 of NaCl. (b) Application of enzyme to a column equilibrated with buffer of (NH4)2SO4. (1) Application of phosphorylase b in buffer A+1.1 mol L–1 of (NH4)2SO4. (2) Elution with buffer A. (3) Elution with buffer A+NaCl. For further details see the text and (Jennissen, 1976a).
Figure 4. Determination of the critical surface concentration (critical hydrophobicity) of Seph-C5 for the adsorption of purified fibrinogen. The uncharged pentyl-agaroses were synthesised by the CDI method. Purified human fibrinogen (1.0 mg) was applied in 1.0 mL to a column (0.9 cm i.d.× 12 cm) containing 2.0 mL of packed gel in 50 mmol L–1 of Tris HCl, 150 mmol L–1 of NaCl, 1.0 mmol L–1 of EGTA and pH 7.4. Fractions of 1.5 mL were collected at room temperature. The column was washed with 15 mL of buffer followed by elution either with 7.5 mol L–1 of urea or, at high hydrophobicity of the gel, with 1% sodium dodecyl sulfate for the determination of the amount of protein bound. 100% equals to 1.0 mg of fibrinogen adsorbed to 2.0 mL of packed gel of Seph-C5. The total amount of adsorbed fibrinogen, corrected for the amount adsorbed to unsubstituted control Sepharose 4B, is shown. For further details see the text and (Jennissen, 2005; Jennissen and Demiroglou, 2006).
Figure 5. One-step purification of fibrinogen from human blood plasma by HIC at the critical hydrophobicity point of Seph-C5. (a) 19 mL of fresh unclotted human blood plasma was applied (arrow 1) to 20 mL of packed Seph-C5 (13.6 mol mL–1 of packed gel in a column 1.4 cm i.d.×13 cm) equilibrated with 50 mmol L–1 of Tris HCl and 1.5 mol L–1 of NaCl (pH 7.4), at a flow rate of 70 mL h–1 and a fraction volume of 6.0 mL at room temperature. The nonadsorbed protein was washed out with 200 mL equilibration buffer. Elution (arrow 2) was facilitated by equilibration buffer containing a 10-fold lower salt concentration of 150 mmol L–1 NaCl. (b) The fractions 30–32 contain pure fibrinogen with a clottability of 93–100% with a total yield of 25%. For further details see the text, legend to Figure 4 and (Jennissen, 2005; Jennissen and Demiroglou, 2006).
Figure 6. Comparison of equilibrium adsorption/desorption isotherms of phosphorylase b with zonal chromatography on low-affinity butyl-Sephrose 4B (5 mol/ml packed gel). The experiments were performed in 10 mM Tris/maleate, 5 mM dithioerythritol, 1.1 M ammonium sulphate, 20% sucrose and pH 7.0 at 5°C. (a) The adsorption and desorption isotherms are described by the Freundlich equations (a= c1/n), a=1.4 (c0.82) (mg/ml packed gel) and a=1.2 (c0.39) (mg/ml packed gel), respectively (a: amount adsorbed, c: equilibrium concentration, : Freundlich constant and 1/n: Freundlich exponent). For further details on the isotherms, apparent binding constants and saturation and thermodynamic data, see Table 1 and references Jennissen and Botzet (1979) and Jennissen (1981). Data from (Jennissen and Botzet, 1979; Jennissen, 1981). (b) Isocratic chromatography of phosphorylase b: () control run with unsubstituted Sepharose 4B (sample: phosphorylase b, 0.39 mg/mL); (£) butyl Sepharose 4B, (sample: 0.39 mg/ml); () butyl Sepharose 4B, (sample: 3.6 mg/mL). Column: 0.62 cm I. D. x 16.5 cm bed height; flow rate: 5 mL/h; fraction volume: 2.9 ml; sample volume 0.2 mL. Vt=5.0 mL, Vo/Vt=0.4 and the dead volume Vd=3.2 mL. In the chromatogram no corrections for Vd were made. The protein yields were between 77% and 100%. Vm and Ve were determined according to the one-half peak width at one-third maximum height method (Crone, 1971) to V′m=8.41 mL, V′e1=17.98 mL and V′e2=17.98 mL (uncorrected for Vd). The peak width at one-third maximum height was () 5.5 mL, (£) 13.1 mL and () 18.9 mL. For further details and corrected elution volumes, see Table 1 and Jennissen (1981). Jennissen and Botzet (1979) and Jennissen (1981) © Elsevier.
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 Further Reading
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Related Sub-Topics:

Protein Structure | Biomolecular Interactions | Proteins: Structure, Function, Metabolism
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Article Title: Hydrophobic Interaction Chromatography
 
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Jennissen, Herbert P(Feb 2013) Hydrophobic Interaction Chromatography. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002678.pub3]