Thesis final - after defense-7 - Jacobs University

Chapter 3
The polarity value for the same protein on Grantham’s scale was almost one digit higher than
Zimmerman’s scale due to the different approaches adopted by them. The r 2 values of the
polarity with the protein retention were 0.89 and 0.76 for the scales of Grantham and
Zimmerman, respectively. An inverse relationship between polarity and retention volume
proved that proteins with less polarity will have a more non polar surface area and will result
in stronger binding to HIC surface. In contrast, proteins with high polarity will have more
polar and less non polar surface area, resulting in less binding to hydrophobic adsorbents. In
other words, polarity has an inverse relationship with protein hydrophobicity. An inverse
relationship was also observed between polarity and flexibility; the proteins with high polarity
were less flexible and eluted at high salt concentrations. Although polarity has no decisive
role in retention but still it can be considered as a contributing parameter during HIC. The
average polarity has shown a correlation with the retention behavior of protein, but it has no
potential to differentiate among the ligands. The reason can be that this property was derived
from the primary structure of a protein instead of the exposed surface residues in the three
dimensional structure. HIC has more relevance with the surface residues of a protein and is
rarely affected by the total residues. Instead, RPC is a chromatographic method based on the
total hydrophobic residues of a protein. Due to this reason, the average polarity might have
the potential to differentiate the hydrophobic characters of the reverse phase chromatographic
adsorbents.
3.1.3.3.3. Average flexibility (AF)
The average flexibility was calculated for the tabulated proteins using the Bhaskaran and
Ponnuswamy’s scale (Tables 5-7) (98). The statistical analysis revealed a direct significant
correlation (r 2 = 0.80, p < 0.0001) of average flexibility with the retention behavior of the
proteins during chromatography (Figure 17). The direct relationship of flexibility with
protein retention could be related to the abundance of cystein residues in the primary structure
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