Thesis final - after defense-7 - Jacobs University

Chapter 3
3.1.3.3. Influence of the ligands chemistries and average protein properties
3.1.3.3.1. Average hydrophobicity (AH)
Average hydrophobicity is actually the average of the hydrophobicities of the overall residues
in the primary structure of a protein. Average hydrophobicity has been reported for its relation
with the protein structure (66). However, average hydrophobicity has been rarely investigated
in the context of HIC. There are different hydrophobicity scales proposed by several research
groups but there is still a debate on which one scale is the best (32). Average hydrophobicity
was calculated for the tabulated proteins on the scales proposed by Cowan-Whittaker,
Miyazawa-Jernigan and Tanford (Tables 5-7) (25, 92, 95). A statistically significant linear
correlation (r 2 = 0.82, p < 0.0001) was reported between the average hydrophobicity and
protein retention behavior (Figures 15 A-C). This confirmed that protein with high average
hydrophobicity will take longer time to elute during chromatography due to the high
hydrophobic interactions between proteins and adsorbents. In contrast, the protein with less
hydrophobic residues in its sequence will result in less hydrophobic interactions and
ultimately the protein will elute at high salt concentrations in the beginning of the
chromatography. It was reported before with model proteins, that in addition to a hydrophobic
surface area, it is important to have a higher overall number of hydrophobic residues in the
protein sequence for a strong binding on HIC adsorbents (62). The high abundance of
hydrophilic amino acids in the contact surface area was also reported to have an inverse
relationship with the protein retention (113). However, these results confirmed the role of
average hydrophobicity in HIC with the process proteomics approach. The hydrophobicity
value for the same protein on the scale of Miyazawa-Jernigan was differed with 1 and 2 digits
to the scales of Cowan-Whittaker and Tanford, respectively. The reason for this could be the
different direct and indirect approaches applied by researchers while determining the
hydrophobicity indices for individual amino acids (25, 92, 95). The hydrophobicity indices of
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