Thesis final - after defense-7 - Jacobs University

Chapter 3
such as D3-SP, B4-PS and B4-SP were eluted earlier during chromatography than E1-SP, C1-
PS and C1-SP, respectively although they have almost similar ASH. This could be due to the
higher flexibilities of the proteins which allow them to retain longer during chromatography.
The same behavior was also reported with model proteins where ribonuclease S has shown
higher retention on Sepharose-Butyl than other proteins, although they had almost identical
surface hydrophobicity. It was claimed that higher flexibility of ribonulcease S might be
favoring longer retention (63). The highly flexible proteins have revealed more unfolding and
most of the hydrophobic residues were exposed which has given tight binding and in result
most of them eluted at the end of the chromatography. Less flexible proteins were not able to
stay longer due to their compact nature and most of hydrophobic parts were in the inner core,
which resulted in less hydrophobic interactions and elution occurred at the beginning of the
chromatography. Although the flexibility has no decisive role in retention, however it can be
considered as the contributing parameter during HIC. The flexibility has given a correlation
with retention of proteins; however it has not exhibited any differences among the base
supports for their hydrophobic characters.
The average bulkiness was also calculated for the tabulated proteins using Zimmerman’s scale
(Tables 12-14). The average bulkiness of the proteins has revealed no significant relationship
(r 2 = 0.04, p = 0.38) with the chromatographic separation of proteins in HIC (Figure 28). The
bulkiness has been never reported for its contribution in HIC. In the previous report,
Zimmerman had investigated to correlate polarity and bulkiness of cytochrome c protein in
different organisms (96). However, no significant trend was observed between polarity and
bulkiness in his studies. This also confirmed that if bulkiness has no relation with the polarity
of the proteins, then it is supposed to have no relation with protein hydrophobicity as
evidenced here. The bulkiness has also exhibited no differences among the different base
supports.
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