RESUMO
A stationary phase for high-pressure liquid chromatography has been prepared by derivatizing microparticulate silica gel with functionality mimicking the side chain of isoleucine. The chromatographic retentions of a series of hydrophobic and amphiphilic amino acid analytes on this stationary phase (Ile MSP) using an aqueous mobile phase were measured as a function of temperature from 273 K to 323 K. Observed temperature dependencies are consistent with a constant change in heat capacity, DeltaC degrees P, upon binding of the analyte to the stationary phase. The curvatures of plots of retention data versus temperature (related to the magnitude of DeltaC degrees P) are distinctly different for retention of aromatic and aliphatic analytes, with retention of aliphatic analytes Val, Ile, and Leu exhibiting the characteristic signature of the hydrophobic effect, i.e., a large negative DeltaC degrees P upon desolvation from water and a maximum of retention around room temperature. Retention of aromatic analytes (Trp, Phe, and Tyr) involves smaller heat capacity changes and pronounced negative enthalpies of interaction with the stationary phase. Estimates of DeltaC degrees P for the interactions of analyte side chains with the Ile side chain were obtained by fitting the temperature dependence of retention to an expression derived from thermodynamic considerations and chromatographic theory. Similar estimates were made for interactions with the Phe side chain, using previously published data for a phenylalanine mimic stationary phase (Phe MSP) (. Protein Sci. 1:786-795). As with the Ile MSP, the retentions of aliphatic analytes show temperature dependencies markedly different from those of aromatic analytes. Data from both phases indicate that a realistic differentiation can be made between the interactions of various types of amino acid side chains tested (i.e., aliphatic/aliphatic, aliphatic/aromatic, and aromatic/aromatic) by comparison of the corresponding thermodynamic functions for pairwise interactions. The retention of leucine on the Phe MSP and that of phenylalanine on the Ile MSP showed similar DeltaC degrees P values, suggesting that the aromatic-aliphatic interaction is reasonably independent of the residue attached to the stationary phase. This result is consistent with a one-to-one interaction and suggests a simple way to estimate the column-dependent phase factor, making it possible to compare entropies and free energies of interaction obtained using different MSPs. The possibilities for using MSP-derived interaction potentials in folding simulations are discussed.
