Correlation of calcitonin structure with chromatographic retention in high-performance liquid chromatography

Chromatographic selectivity and position specificity were examined by using deletion and substitution analogues of calcitonin (CT), a 32-amino acid polypeptide. The biological activity of CT has been shown to be related to structural features, including a regular spacing of hydrophobic and hydrophilic residues in positions 8-22. The effect of structure on retention behavior in hydrophobic interaction chromatography (HIC) and reversed-phase chromatography (RPC) of 19 CT analogues was examined. No simple correlation was found between chromatographic retention and amino acid composition. Deletion of one leucine residue reduced retention in both chromatographic systems, but the magnitude of the change depended upon the site at which the deletion occurred. For example, deletion analogues des(Leu16)-, des-(Leu12)-, des(Leu10)-, des(Leu9)-, des(Leu4)-, and salmon calcitonin had retention times of 5.22, 8.44, 11.64, 13.53, 15.45, and 20.28 min, respectively, in HIC in contrast to RPC retentions of 15.19, 15.84, 28.53, 27.57, 25.92 and 34.79 min, respectively. Serine deletion was also shown to be position-specific. Non-amphiphilic analogues were eluted before amphiphilic ones. Circular dichroic spectral studies showed that the CT analogues possessed little secondary structure in the HIC solvents in contrast to alpha-helix formation in RPC solvents. The HIC data provided indirect evidence of secondary structure, induced in the amphiphilic CT analogues at the HIC solvent-chromatographic surface interface. Solute-solvent interactions contributed to differences observed between the selectivity of RPC and HIC.