Measurement of the binding of retinoic acid to β-lactoglobulin B by affinity capillary electrophoresis

✍ Scribed by Frédéric Lynen; Wim Van Thuyne; Frans Borremans; Gerd Vanhoenacker; Pat Sandra

• Book ID: 102440651
• Publisher: John Wiley and Sons
• Year: 2003
• Tongue: English
• Weight: 355 KB
• Volume: 26
• Category: Article
• ISSN: 1615-9306
• DOI: 10.1002/jssc.200390014

✦ Synopsis

Abstract

The quantification of the binding strength between retinoic acid and the primary binding site of β‐lactoglobulin B by dynamic equilibrium affinity capillary electrophoresis (DE‐ACE) is described. Although the peaks for retinoic acid were broad, a distinctive shift in migration time could be observed upon complexation allowing the construction of a binding curve and three linearised plots. By performing the corresponding linear and non‐linear regression analyses, an apparent dissociation constant varying between 1.4 and 2.2 μM was measured in a 100 mM Tris‐acetate buffer at pH 8.2 with 30 mM Na~2~SO~4~ and 1% EtOH as additives. The sample was prepared by adding a concentrated solution of retinoic acid in ethanol to the background electrolyte, such as to obtain a final 1% solution of ethanol and 5 μM retinoic acid in the buffer. It was shown that an MEKC‐based approach, attempted to improve the solubility of retinoic acid in the aqueous buffer, could not be used to perform a binding study.