Binding isotherms for soluble immobilized affinity ligands from spectral titration
- 5 December 1992
- journal article
- research article
- Published by Wiley in Biotechnology & Bioengineering
- Vol. 40 (10), 1263-1270
- https://doi.org/10.1002/bit.260401016
Abstract
The method of spectral titration has been applied to binding equilibria between proteins and soluble immobilized ligands and evaluated using the interaction between Cibacron blue–dextran conjugates and lysozyme. The method is both simple and rapid and provides a convenient screening technique for characterization of soluble adsorbents designed for use in aqueous two‐phase affinity extraction or as liquid‐phase models for affinity chromatography systems. The results indicate that regardless of ligand density a constant 28% of the total coupled dye is available for high‐affinity protein binding at saturation. The dissociation constant for the dye–protein interaction, however, decreases with dye loading. The potential for kinetic investigations has been demonstrated using a stopped‐flow apparatus. The results indicate that a simple rate equation is inadequate to describe the data for lysozyme binding to dye–dextran conjugates. A modified model, which better describes the data, was developed by including a second rate limiting process, the transition from stacked to unstacked dye ligands on the dextran backbone. This effect could have practical significance for protein binding kinetics in affinity chromatography, especially in high‐performance liquid affinity chromatography applications where mass transfer is rapid. © 1992 John Wiley & Sons, Inc.Keywords
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