Effects of Ligand Heterogeneity in the Characterization of Affinity Columns by Frontal Analysis

Abstract

This study examined how the binding capacities and equilibrium constants measured by frontal analysis are affected by ligand heterogeneity in affinity columns. Equations derived for n- and two-site systems gave good agreement with results obtained for the binding of L-thyroxine to a column containing human serum albumin (HSA) and for the binding of (R)-warfarin to coupled columns containing HSA or pigeon serum albumin. The same equations were used to examine how different degrees of ligand heterogeneity affected the apparent binding capacities or equilibrium constants measured using the linear range of double-reciprocal frontal analysis plots. A large proportion of two-site systems gave good estimates (i.e., less than 10-20% error) for the true total column capacity and for the association constant of the highest affinity ligand in the column. A smaller, but still appreciable, fraction of all three- and four-site cases also produced good estimates of these values. The results of this work are not limited to protein-based affinity columns but should be applicable to any type of stationary phase that has well-defined binding regions and relatively fast, reversible interactions with solutes.

Measuring Binding Constants of His-Tagged Proteins Using Affinity Chromatography and Ni-NTA-Immobilized Enzymes

“…If the resulting plot is nonlinear (Fig. 5), then multi-site binding is indicated (see Note 14) and the resulting plot takes the following form [16]: …”

“…When multi-site binding occurs, an estimate of the binding constant can be determined by looking at the linear range obtained from the lowest range of concentrations. This is possible since at very low concentrations, the highest affinity sites are bound first and it is only at high concentrations that secondary binding sites bind the analyte [16].…”

“…Since we are using a very uniform and gentle method of attachment of the protein to the chromatographic support, we can assume that the distribution of the two binding sites is uniform and α 1 is equal to 0.5 and the error associated with the determination of K A1 is less than 10 % based on the study by Tweed et al [16].…”

“…While the physiological substrate for APX is ascorbate, it has also been observed to oxidize other nonphysiological substrates such as 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), guaiacol, and pyrogallol [15]. Using frontal analysis affinity chromatography, we were able to identify the presence of multi-site binding in our enzyme-substrate system and estimate the binding constant of the system using the method described by Tweed et al [16]. Other methods of determining binding constants include ultracentrifugation [17], NMR [18], and affinity capillary electrophoresis [19].…”

Affinity Chromatography as a Tool for Quantification of Interactions Between Drug Molecules and Their Protein Targets

“…A similar approach, but using a combination of both nonlinear and linear fits, can be used for more complex systems that involve multisite interactions (Jacobson et al, 1993;Tweed et al, 1997, as cited in Hage & Chen, 2006). .…”