Thesis final - after defense-7 - Jacobs University

Chapter 3
calculated. The ASP parameter can be used for the prediction of protein retention time in HIC.
The average polarity has revealed no differences among the base supports for their
hydrophobicity.
3.2.3.3.3. Average surface hydrophobicity (ASH)
There is no universally accepted method to be considered for the measurement of protein
hydrophobicity; however it can be estimated through several hydrophobicity scales proposed
by different co-workers (25, 92, 95). The hydrophobicity scale proposed by Cowan-Whittaker
(92) was used to calculate ASH. This scale was based on a direct method and was derived
from the amino acid retention time in RP-HPLC. The approach of J.C. Salgado et al (32, 92)
was used for calculating ASH utilizing the bioinformatics tools. This property was
demonstrated by averaging the hydrophobic potentials of the surface amino acid residues
present in the three dimensional structure of a protein. All the amino acids on the surface of a
protein have contributed in the ASH; however the eight non polar hydrophobic amino acids
such as alanine, leucine, isoleucine, tryptophan, methionine, phenylalanine and proline have
the major contributions. The ASH values have been calculated for the tabulated proteins
(Tables 12-14). The statistical analysis revealed a significant direct correlation (r 2 = 0.91, p <
0.002) between ASH and protein retention behavior on the respective adsorbents (Figure 26).
The proteins were classified into three groups depending on the ASH and the corresponding
salt concentrations on which the proteins were eluted. The proteins eluted at high salt
concentrations in the beginning of the chromatography have less ASH values and were
considered as less hydrophobic than the proteins eluted at the middle and / or at the end of the
chromatography. Those proteins which have many hydrophobic residues on their surfaces
were retained longer and eluted at the end of the chromatography. The data obtained for the
proteins eluted at low salt concentrations such as 0.6 M, 0.2 M and 0 M have high importance
for industrial applications, where the use of low salts is the preferred choice in HIC (24). ASH
93