Thesis final - after defense-7 - Jacobs University

Chapter 3
of a protein forming disulfide bridges. The protein with many cystein residues / or disulfide
bridges will be less flexible towards conformational changes. In contrast, the protein with
high flexibility will be more flexible towards conformational changes and when it come in
contact of HIC surface, it will unfold or expose the internal surface in order to increase
hydrophobic interactions with the adsorbent, consequently resulting in longer retention during
chromatography (33, 62). The role of average flexibility in chromatographic separation has
been confirmed here for the first time with the process proteomics approach. The direct effect
of flexibility on the retention behavior has also revealed the direct relationship of flexibility
with proteins hydrophobicity. If a protein has high hydrophobic residues in its sequence, the
higher will be the flexibility and compressibility of that protein, which is in agreement of the
previous paper (119). The proteins eluted at high salt concentrations at the beginning of the
chromatography have less flexible nature than those proteins eluted at the end of the
chromatography. The less flexible proteins have compact structures and hydrophobic parts of
the proteins exist in the inner core and resulted in less hydrophobic interactions and earlier
elution during chromatography. In contrast, the highly flexible proteins will unfold during
chromatography and a hydrophobic part will come to the surface which will ensue high
hydrophobic interactions and resulted in later elution during chromatography. Some proteins
such as C4-TH and E4-TB were eluted earlier than D1-TH and F1-TB, respectively although
they have almost identical ASH. This could be due to the higher flexibilities of the latter
proteins resulting in longer retention during chromatography. The same behavior was also
observed with model proteins where ribonuclease S has shown higher retention on Butyl-
Sepharose than other proteins, although they have identical surface hydrophobicity. It was
claimed that the higher flexibility of ribonulcease S might be favoring longer retention (63).
63