Conformational effects on reversed-phase chromatography of proteins with particle beam LC/FT-IR spectrometry and free solution capillary electrophoresis

In this study, reversed-phase HPLC (RPC), particle beam FT-IR spectrometry, and capillary electrophoresis (CE) were employed to ascertain the dynamic conformational structure empirically of various forms of bovine ribonuclease A (RNase A) to investigate the interdependence of protein conformation and RPC separation, RNase A was analyzed in four confotmational states: native, partially denatured, completely unfolded, and completely unfolded and disulfide-reduced (denatured), Each form was analyzed individually and produced similar multizoned RPC profiles composed of several low-response peaks before the primary peak. Particle beam FT-IR spectrometry results from the RPC fractions of the partially denatured and unfolded forms were identical to one another and exhibited a loss of ordered structure. The infrared spectra of the RNase A that was introduced into the HPLC in the denatured and reduced form showed an excess of beta-sheet. content, particularly the primary peak, We believe this to be a non-native form of RNase A. CE migration times of RNase A samples that were reduced in 2-mercaptoethanol and unfolded in increasing concentrations of urea increased with the degree of unfolding. Mobilities of RNase A RPC fractions were compared to those of the mea unfolded samples, The migration time of thee primary RPC fraction of the denatured form showed an intermediate migration rate between that of the native, spherical geometry and denatured, open geometry forms. It is unclear whether this beta-structure formed during column propagation or upon elution.