Thesis final - after defense-7 - Jacobs University

Chapter 3
the amino acids in a protein had a contribution in the retention behavior and due to this reason,
the correlation of average hydrophobicity with retention seem sensible. The average
hydrophobicity has been also reported for its significance in reverse phase chromatography
(RPC). The RPC is based on the overall hydrophobicity derived from the primary structure
instead of only surface residues of a protein (126). This revealed that average hydrophobicity
and average surface hydrophobicity have decisive roles in HIC and RPC, respectively. The
analysis of the two methods of protein hydrophobicity confirmed that ASH was more relevant
to HIC than average hydrophobcity in terms of chromatographic separations. The trend lines
(Figure 24 A-C) revealed that although average hydrophobicity has a role in HIC, but it has
no ability to differentiate among the hydrophobic characters of the three base supports.
3.2.3.3.2. Average polarity (AP)
The average polarity was for the first time investigated for its correlation with protein
retention behavior in HIC as a function of the cell proteome. The average polarity was
calculated for the proteins using the scales proposed by Grantham and Zimmerman (Tables
12-14) (96, 97). A statistically significant (r 2 = 0.82, p < 0.0001) inverse relationship was
observed between polarity and retention behavior of the proteins on different base supports.
The proteins eluted at high salt concentrations were highly polar than the proteins eluted at
low salt concentrations. In other words, the proteins eluted at the beginning of the
chromatography were more polar than those proteins eluted at the end of the chromatography.
The average polarity values of the Grantham’s scale were higher in early eluted proteins (>
0.49) than late eluted proteins (< 0.41). The same trend was also observed on Zimmerman’s
scale, where early eluted proteins had high average polarity (> 0.35) than late eluted proteins
(< 0.24). The reason can be that proteins with high polar residues retained for a shorter space
of time than those with low polar residues. In a previous study, it has been mentioned that
membrane proteins had low polarity below 40% while soluble proteins had higher polarity
89