5/30/2011 Protein Hydrophobicity

Protein Hydrophobicity
Dr Andrea Mahn
University of Santiago of Chile

Last updated on 23 December 2009

Hydrophobic Interactions

Hydrophobic interactions are the most important non-covalent forces that are responsible for different
phenomena such as structure stabilization of proteins (Privalov and Gill, 1988), binding of enzymes to
substrates (Erikkson, 1998), and folding of proteins (Dill, 1990). This kind of interaction appears when non-
polar compounds are put into water, and it is an entropy-driven process. The protein separation technique
based on hydrophobic interactions, Hydrophobic Interaction Chromatography (HIC) is an important
technique which exploits the reversible interaction between the hydrophobic patches on a protein’s surface
and the hydrophobic ligands of a chromatographic medium at moderately high concentrations of an
antichaotropic salt. The HIC process consists in injecting a protein sample in the column under conditions of
high ionic strength. Elution is normally achieved by reducing the ionic strength in the mobile phase. A protein
coming in contact with the hydrophobic ligands in the column suffers a spatial reorientation, and the
hydrophobic ligands interact with the hydrophobic zones on the protein surface to reversibly bind the protein
to the column (Mahn and Asenjo, 2005). HIC has been used for purifying a variety of biomolecules, such as
membrane proteins (McNair and Kenny, 1979), nuclear proteins (Comings et al., 1979), receptors (Kuehn et
al., 1980), recombinant proteins (Lienqueo et al., 2003) and serum proteins (Hrkal and Reimkova, 1982).

Modeling and predicting protein separation based on hydrophobicity

The main physicochemical property that determines protein retention in Hydrophobic Interaction
Chromatography is hydrophobicity, which can be estimated using different approaches. Tanford (1962) used
“average hydrophobicity”; Fisher (1964) used “polarity ratio”, and Erikkson used “net hydrophobicity” to
estimate this property. Based on the fact that retention in HIC occurs due to a surface adsorption
phenomenon, the use of "average surface hydrophobicity" (f surface) has been suggested. This parameter
can be estimated from knowledge of the three dimensional structure of a protein, by taking into account the
hydrophobic contribution of the amino acid residues on the protein’s surface. A methodology was proposed
to predict a protein’s retention time in HIC, starting from f surface and using quadratic models. The model
coefficients depend on the chromatographic conditions used in the HIC process (Lienqueo, Mahn et al.,
2002). This methodology has been validated for monomeric and dimeric proteins, and it has been used
successfully for predicting protein separation behaviour of rhSOD from a S. cerevisiae cell extract (Lienqueo
et al., 2003).

Hydrophobicity is heterogeneously distributed on protein surface, and thus, surface hydrophobicity

distribution has been considered in other predictive models (Mahn et al., 2004). A method to estimate
hydrophobicity distribution by identifying the zones on a protein surface that most probably would interact
with HIC supports was developed (Mahn et al., 2005). A parameter called "local hydrophobicity" was defined
as the average surface hydrophobicity of the interaction zone, and it was obtained by means of the three
dimensional structure of the protein and through molecular simulations. Quadratic models based on this
parameter were developed for predicting retention times of different ribonucleases in HIC (Mahn et al., 2005).
This method was extended for predicting retention time in HIC of several different proteins, with a reasonable
success (Lienqueo, Mahn et al., 2005).

In summary, the separation behaviour of proteins in HIC is mainly determined by surface hydrophobicity,
which can be calculated from the three dimensional structure of the proteins using simple matehmatical
models.

The parameters of the predictive models depend on the chromatographic conditions, and therefore, simple
mathematical models have been proposed to predict a protein’s retention time in HIC under certain
conditions, based on retention time of that protein under different conditions (Mahn et al., 2007a). These
models were validated, so they can be used to choose better operating conditions in a given purification
process.

Despite surface hydrophobicity is the main protein property affecting retention time in HIC, the system
characteristics have also a very important effect. The main factors are type of salt, initial ionic strength and
matrix properties. Additionally, the flow rate, the pH of the mobile phase and the temperature of the system
can also affect protein retention in HIC (Hjerten et al., 1986).

Recently, it was found that type of salt, initial ionic strength and substitution degree of the resin were the only
statistically significant variables of a HIC system (Mahn et al., 2007b), making it possible to predict protein

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5/30/2011 Protein Hydrophobicity
retention time in HIC by using them in functional models. The model obtained by Mahn et al. (2007b) was
optimized in order to maximize the difference between the retention time of two proteins in a binary mixture,
thus obtaining the optimal operating conditions to achieve the separation of two proteins.

Further reading
Lienqueo, M.E., Mahn, A.V., Asenjo, J.A. (2002). Mathematical Correlations for Predicting Protein Retention
Time in Hydrophobic Interaction Chromatography. J. Chromatogr. A 978: 71 - 79
Mahn A and Asenjo JA (2005). Prediction of protein retention in Hydrophobic Interaction Chromatography,
Biotechnol Adv 23 (5): 359 – 368 (Link »)
Mahn A, Lienqueo M. E, Asenjo JA. (2004). Effect of Surface Hydrophobicity Distribution on Retention of
Ribonuleases in Hydrophobic Interaction Chromatography. J Chromatogr A, 1043: 47 - 55.
Mahn A, Lienqueo M E, Salgado J C. (2009). Methods of calculating protein hydrophobicity and their
application in developing correlations to predict hydrophobic interaction chromatography retention, J
Chromatogr A 1216: (2009): 1838 - 1844
Mahn A, Lienqueo ME, Asenjo JA (2007). A simple method for the estimation of protein retention in
hydrophobic interaction chromatography under different operation conditions. The Open Biotechnology
Journal (1): 9 – 13 (Link »)
Mahn A, Lienqueo ME,. Asenjo JA (2007). Optimal operation conditions for protein separation in hydrophobic
interaction chromatography. J Chromatogr B 849: 236 – 242 (Link »)
Mahn A, Zapata-Torres G, Asenjo JA. (2005) A Theory of Protein - Resin Interaction in Hydrophobic
Interaction Chromatography. J Chromatogr A, 1066: 81 - 88

Selected links
ExPaSy Proteomics Server

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1. Increased protein hydrophobicity in response to aging and Alzheimer disease.
May 2010
Increased levels of misfolded and damaged proteins occur in response to brain aging and Alzheimer
disease ( AD), which presumably increase the amount of aggregation- prone proteins via elevations in
hydrophobicity. The proteasome is an intracellular . ..
[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=...]

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Membrane protein behavior should be predictable from first principles of physical chemistry. One such first
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[http://www.sciencedirect.com/science?_ob=GatewayUR...]

3. Increased protein hydrophobicity in response to aging and Alzheimer disease

May 2010
Abstract Increased levels of misfolded and damaged proteins occur in response to brain aging and
Alzheimer disease ( AD), which presumably increase the amount of aggregation- prone proteins via
elevations in hydrophobicity. The proteasome is an . ..
[http://www.sciencedirect.com/science?_ob=GatewayUR...]

4. A new non-hydrophobic cell wall protein (CWP10) of Metarhizium anisopliae enhances conidial

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hydrophobicity when expressed in Beauveria bassiana.
Jan 2010
A cell wall protein, CWP10, resolved from the conidial formic acid extract of a Metarhizium anisopliae isolate,
was characterized as a new 9.9-kDa protein with a 32-aa signal peptide with a central hydrophobic region (
ca. 10 residues) at its N-terminus. ...
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5. Heating and reduction affect the reaction with tannins of wine protein fractions differing in hydrophobicity
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Abstract During the storage, bottled white wines can manifest haziness due to the insolubilisation of the
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6. Methods of calculating protein hydrophobicity and their application in developing correlations to predict
hydrophobic interaction chromatography retention.
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Different methodologies to estimate the hydrophobicity of a protein are reviewed, which have been related to
the chromatographic behavior of proteins . ..
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hydrophobic interaction chromatography retention
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been related to the chromatographic ...
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adhesion - adsorbed - adsorbed protein - adsorption - aggregates - aggregation - albumins - algorithms -
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