Conformational Effects of Reversed-Phase HPLC on Ribonuclease Α and α-Chymotrypsin by Particle Beam LC/FT-IR Spectrometry

Abstract

The reversed-phase liquid chromatographic behavior of ribonuclease A (Rnase) and alpha-chymotrypsin (alpha-Chy) have been examined by particle beam LC/FT-IR spectrometry. Interaction of many proteins with a non-polar stationary phase is known to induce reversible/irreversible denaturation of the protein. Reverse-phase HPLC of such proteins often produces a series of chromatographic peaks representing different solution conformations. The chromatographic behaviors of Rnase and alpha-Chy were examined through selective sampling of the major peaks observed in the chromatographic profiles of each protein by particle beam LC/FT-IR spectrometry. These conformations were produced and separated in this study by the use of a C-4 column with an acetonitrile gradient. These bands were examined post-column with a diode array detector at 220 nm, followed by isolation of the different solution conformers by the particle beam technique. The aerosol-based particle beam apparatus has previously been shown to be successful in the isolation of intermediate conformations of proteins. ?he resulting protein deposits are examined off-line by FT-IR microscopy. Additionally, Rnase was examined in various concentrations of acetonitrile to assess the degree to which the organic modifier affects the secondary structural content of the protein. Differences in secondary structure elements were evident among the chromatographic peaks of both Rnase and alpha-Chy. Secondary structure differences were also evident in Rnase in different concentrations of acetonitrile.