Determination of thermodynamic parameters for the interaction of a lipid-binding peptide and insulin with a reversed-phase column.

The effect of temperature was investigated on the reversed-phase chromatography of a synthetic lipid associating peptide (LAP), with the following sequence (LESFLKSWLSALEQALKA) and on insulin. The LAP was chromatographed on a muBondapak-alkylphenyl column with 1% aq. triethylammonium phosphate, pH 3.2-2-propanol (80:20) as the isocratic mobile phase. The insulin was separated on a Resolve-C18 column with a mobile phase that contained 0.1 M sodium phosphate, pH 2.0-acetonitrile (71.5:28.5). With the LAP the Van't Hoff plot (In k' vs. 1000/T) was linear and the value of enthalpy for association of the peptide with the reversed-phase column was large and negative. The phase ratio was estimated for the column used in the separation and then derivation of the corresponding entropic term demonstrated that the association was enthalpy and not entropy drive. By contrast the corresponding Van't Hoff plot derived or the insulin study was non-linear and with a positive slope. Further study indicated that the formation of the insulin-reversed phase complex could be attributed to a favorable entropy change. It is probable that the non-classical thermodynamics observed during the insulin chromatography could be related to conformational changes in the insulin structure during the chromatographic process.