Quantitative affinity high-performance liquid chromatography of neuroendocrine polypeptides using porous and non-porous glass derivatives

Abstract

Analytical affinity HPLC was developed to isolate and characterize neuroendocrine peptide/protein components. Bovine neurophysin II (NP-II) was covalently immobilized on succinamidopropyl derivatives of both controlled-pore glass (CPG) and non-porous glass (NPG). These derivatives were packed into 25 × 0.46 cm I.D. stainless-steel columns and incorporated into a high-performance liquid chromatograph. Interaction of [ 3H]Arg 8-vasopressin ([ 3H]AVP) with NP-II was examined by chromatography of AVP on both CPG and NPG affinity matrices. Zonal elution profiles of [ 3H]AVP on NPG matrix showed, as predicted theoretically, a linear dependence of retardation on the concentration of hormone injected. The data permit calculation of the equilibrium dissociation constant for the NP-II/AVP interaction. Elution characteristics also were measured by frontal analysis of large-zone chromatography experiments, the results of which were in good agreement with the zonal elution analysis. Affinity resulting from dimerization also was studied by chromatography of [ 1 2 5I]NP-II on the NPG matrix. In this case, concentration dependence of retardation was non-linear, again as predicted theoretically. Off-rate kinetic constants for dissociation of the mobile interactant from the stationary phase also were obtained. The studies illustrate the utility of analytical affinity HPLC on non-porous beads for measuring relative affinities for various soluble ligands with small amounts of material. Chromatography on the CPG column proved useful for purification of microscale amounts of [ 3H]AVP.