Analytical high-performance affinity chromatography: evaluation by studies of neurophysin self-association and neurophysin-peptide hormone interaction using glass matrixes

Abstract

Bovine neurophysin II (BNP II) was covalently immobilized on both nonporous and porous (200-nm pore diameter) glass beads and incorporated in a high-performance liquid chromatograph to evaluate analytical high-performance affinity chromatography as a microscale method for characterizing biomolecular interactions. By extension of the theoretical treatment of analytical affinity chromatography, both the self-association of neurophysin and its binding of the peptide hormone vasopressin were characterized by using a single chromatographic column containing immobilized neurophysin predominantly in the monomer form. Both [3H] [Arg8]vasopressin (AVP) and 125I-BNP II were rapidly eluted (less than 25 min). The relatively symmetrical elution peaks obtained allowed calculation of both equilibrium dissociation constants and kinetic dissociation rate constants. The dissociation constant measured chromatographically for the AVP-immobilized neurophysin complex, KM/L = 11 microM with porous glass beads and 75 microM with nonporous glass (NPG) beads, was in reasonable agreement with those previously obtained by curve fitting of Scatchard plots (16-20 microM) and from binding to [BNP II]Sepharose (50 microM). The values obtained are larger than that for dissociation of AVP from BNP II dimer, by a factor consistent with the intended nature of immobilized BNP II as monomers. Chromatography of BNP II on the [BNP II]NPG gave a dimer dissociation constant of 166 microM, a value in excellent agreement with that derived from equilibrium sedimentation studies (172 microM). In contrast to the agreement of chromatographic equilibrium binding constants with those measured in solution, the dissociation rate, k-3, determined from the variance of the affinity chromatographic elution profile with nonporous beads, was several orders of magnitude smaller than the solution counterpart.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetic studies of biological interactions by affinity chromatography

“…These experiments utilized BNP II that was immobilized on either non-porous or porous glass beads. However, it was found that the rate constants determined by this approach were underestimated by orders of magnitude compared to solution phase values, especially when using porous supports [49, 74]. This difference may have been due to differences in the stagnant mobile phase mass transfer contributions for the retained and non-retained species or the use of relatively low flow rates and fraction collection for these experiments.…”

Measurement of Drug−Protein Dissociation Rates by High-Performance Affinity Chromatography and Peak Profiling

“…11,53,58 However, the plate height term H sm is also known to depend on retention and, therefore, could be quite different in size for a retained versus non-retained species. If this is the case, the H sm would be expected to also make up part of the difference noted in the values of H R and H M .…”

Analysis of stereoselective drug interactions with serum proteins by high-performance affinity chromatography: A historical perspective

“…The earliest approach that was developed for this purpose is one based on band-broadening measurements [39,147–149]. This approach was suggested as early as 1975 [150] and was used with low-performance affinity columns to study the self-association of neurophysin II and its binding to a neuropeptide [151]. This method was then explored for use with HPAC in the mid-1980s for the study of lectin-sugar interactions [39,152,153] and was adapted for use in the study of drug or solute interactions with HSA by the mid-1990s [93,154].…”