Quantitative high-performance affinity chromatography: evaluation of use for analyzing peptide and protein interactions.

To examine analytical high-performance affinity chromatography as a microscale method for characterizing macromolecular interactions, the chromatographic behavior was evaluated of Arg8-vasopressin on bovine neurophysin II covalently immobilized in its monomer form on several new high-flow and pressure-resisting affinity supports. Zonal elution of both tritiated and unlabeled peptide hormone and an extension of theoretical treatment of analytical affinity chromatography allowed determination of equilibrium dissociation constants of hormone binding to immobilized bovine neurophysin II. Microamounts of hormone, ranging from 0.05 to 15 micrograms, were eluted within 20-30 min, with a quantitative recovery of the amount injected. For zones containing more than 5 micrograms, continuous elution monitoring was possible by ultraviolet absorbance, providing greater speed and accuracy in data analysis. The values obtained for the equilibrium dissociation constants were in good agreement with those previously measured in solution. The above hormone-protein evaluation system has led to identification of several pressure-resistant affinity supports, including silica-, agarose- and glass-based matrices, which are appropriate for use with high-performance liquid chromatographic instrumentation for affinity chromatographic analysis of macromolecular interactions.