Thesis final - after defense-7 - Jacobs University

Chapter 3
published papers with model proteins (32, 62). This revealed that a protein with less ASH
value retained for a shorter space of time than the protein with high ASH value. This kind of
behavior was also reported by several research groups that binding affinity increased with the
exposed hydrophobic residues of a protein. These results confirmed that hydrophobicity of the
adsorbents and proteins have the combine contribution to increase the entropy of the system
and resulted in hydrophobic interactions (118). The correlation coefficients (r 2 ) values of ASH
with the respective salt concentrations were 0.95, 0.94 and 0.80 for Toyopearl-Hexyl-, -Butyl
and -Ether, respectively. The r 2 values in the case of Toyopearl-Ether- was very less than -
Butyl and -Hexyl. The reason can be the elution of the highly hydrophobic proteins at the end
of the chromatography in the case of Toyopearl-Ether which exerted an effect on the overall
trend line. Some proteins were reported to have high or similar ASH values than others but
retained less i.e. C3-TH and E4-TB were eluted earlier than D3-TH and F1-TB, respectively.
This revealed that higher the molecular mass of the protein, the longer it will stay on the
adsorbent. The behavior based on protein molecular mass was not reported for Toyopearl-
Ether due to its less hydrophobic nature which resulted in less ability to entrap larger proteins.
Although ASH has been reported in the prediction of retention time of model proteins,
however this parameter has been never studied in HIC as a function of the cell proteome (26,
87). The salt and hydrophobicity ranges were defined for the first time in this work and can be
used for the separation of target proteins employing HIC. ASH has revealed a direct
relationship with the retention of proteins; however it has a limited ability to differentiate
among different ligands.
3.1.3.4. Comparative studies of the hydrophobic ligands based on average protein
properties
There are several reports where various ligands have been compared for their hydrophobicity
utilizing the retention time of model proteins (22, 33, 61, 62, 99). However, the
hydrophobicity of the ligands has been never explored with a cell proteome. The
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