Immobilization of α1-acid glycoprotein for chromatographic studies of drug–protein binding II. correction for errors in association constant measurements

Abstract

A new method for the immobilization of α 1 -acid glycoprotein (AGP) in HPLC columns was recently described for applications such as drug binding studies. Part of this earlier work used self-competition zonal elution studies to measure association equilibrium constants between immobilized AGP and R-or S-propranolol. It was later found that analysis of these data by a common equation derived for linear elution conditions gave erroneous values for experiments actually conducted under nonlinear conditions. This report discusses the nature of this error and uses frontal analysis to estimate the true binding strength between R-and S-propranolol and HPLC columns containing immobilized AGP.A recent paper examined the binding of R-and S-propranolol to a new type of α 1 -acid glycoprotein (AGP) column (1). Part of this earlier work used self-competition zonal elution studies for association equilibrium constant measurements, an approach which has since been noted to contain some errors that are not uncommon in the literature but that do need to be corrected to better estimate the true binding strength of R-and S-propranolol with AGP.The column used in Ref.(1) was prepared through the controlled and mild oxidation of AGP, followed by the immobilization of this protein to hydrazide-activated silica. Part of this paper evaluated binding of this AGP column to R-and S-propranolol by using a self-competition zonal elution experiment (i.e., the "perturbation method", "step and pulse method" or "system peak" method) (2-4). In this approach, a small sample of R-or S-propranolol was injected as a pulse onto the AGP column while a known, fixed concentration of the same compound was applied in the mobile phase as a competing agent. The resulting shift in the retention factor (k') for the observed peak was measured as a function of the mobile phase concentration of the analyte [A]. Plots of 1/k' versus [A] appeared to give a linear response for mobile phases containing 0 to 5 µM propranolol.In these experiments (1), association equilibrium constants were obtained by fitting the data to an equation from the literature that pertains to an analyte at infinite dilution, or "linear elution conditions" (see Eq. 1 in Ref. 1 and equivalent equations in Refs. 5 and 6). However, for an analyte at a finite concentration, this same equation can introduce errors when obtaining *Author for correspondence: Phone 402-472-2744; FAX 402-472-9402; Email, dhage@unlserve.unl.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author...

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“…This difference indicated that the mixed-mode model gave a better description of this data set. The same type of model has been found in prior work to be the best description for interactions by disopyramide and a number of other drugs (e.g., propranolol) with immobilized or soluble AGP [33–35]. …”

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“…The first one was often processed in solution, including nuclear magnetic resonance [2], ultraviolet and visible spectra [3], circular dichroism [4], fluorescence spectroscopy [5] and isothermal titration calorimetry [6]. The second one included surface plasma resonance (SPR) [7], enzyme linked immunosorbent assay [8] and frontal affinity chromatography (FAC) [9,10].…”

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