Probing the binding behavior and conformational states of globular proteins in reversed phase high-performance liquid chromatography

Reversed-phase high-performance liquid chromatography (RP-HPLC) is a widely used technique for the separation of proteins under low pH aquo-organic so lvent gradient elution conditions, typically carried out at ambient tempera tures. These conditions can however induce conformational effects with prot eins as evident from changes in their biological or immunological activitie s, By monitoring the influence of temperature on the retention and band-bro adening characteristics of proteins, the role of conformational processes i n these lipophilic environments can be examined. These processes can then b e interpreted in terms of a two-state model involving a native (N) and a fu lly unfolded species (U) or more complex folding/unfolding models, In the p resent study, the gradient elution RP-HPLC behavior of sperm whale myoglobi n (SWMYO) and hen egg white lysozyme (HEWL) has been investigated at temper atures between 5 and 85 degrees C with n-octadecyl (C-18)- and n-butyl (C-4 )-silica reversed-phase sorbents, The interaction of these proteins with th ese reversed-phase sorbents has also been examined in terms of the contribu tions that the heme prosthetic group of SWMYO and the disulfide bonds in HE WL make to the stabilization of the native conformation of these proteins i n these hydrophobic environments, The observed inter-conversions of multipl e peak zones of SWMYO and HEWL in the presence of C-18 and C-4 ligands have been subsequently analyzed in terms of the unfolding processes that these proteins can undergo at low pH and at elevated temperatures. The ability of hydrocarbonaceous ligands to trap ensemblies of partially unfolded conform ational intermediates of proteins in these perturbing environments has been examined, Pseudo-first-order rate constants have been derived for these pr ocesses from analysis of the dependencies on time of the concentration of t he different protein species at specified temperatures. The relationship of these processes to the conformational transitions that these proteins can undergo via molten globule-like intermediates (i.e., compact denatured stat es with a significant amount of residual secondary structure) in solution h as also been examined. This study thus further documents an experimental st rategy to assess the folding/unfolding behavior of globular proteins in the presence of hydrophobic surfaces and aquo-organic solvents, whereby the sy stem parameters can potentially affect the preservation of native conformat ions, and thus the function, of the protein under these conditions.