POLYMER-COATED REVERSED-PHASE PACKINGS WITH CONTROLLED HYDROPHOBIC PROPERTIES .1. EFFECT ON THE SELECTIVITY OF PROTEIN SEPARATIONS

A novel approach to analytical and preparative protein separations by reversed-phase chromatography is described. Porous and non-porous silica supports were coated with polymethacrylate-based polymers or copolymers to produce tailored stationary phases of varying hydrophobicity. Through the application of a model protein mixture of lysozyme, cytochrome c and myoglobin, it was demonstrated that selective unfolding of proteins can be achieved by varying the hydrophobicity of the polymer coat permitting manipulation of the chromatographic pattern of analytical protein separations. Thus, proteins may be maintained in their native, folded state or may be partially or completely unfolded, depending on the choice of packing and/or run conditions. In addition, on the non-porous packings, such manipulation of protein elution profiles is achievable at run times of <5 min. A potential preparative role for the polymer-coated packings was demonstrated through their application to the reversed-phase chromatography of a three-protein complex, rabbit skeletal troponin. Through packing and or temperature manipulation, it was demonstrated that even such a multi-protein complex, stabilized only by non-covalent interactions. may be maintained during purification by reversed-phase chromatography.