Analysis of the biotin-binding protein actinavidin using affinity capillary electrophoresis.

Abstract

Affinity capillary electrophoresis (ACE) was applied to study the bioaffinity of ligand-receptor interaction between the microbial biotin-binding protein actinavidin and biotin. The ACE method is based on short time incubation of a mixture of actinavidin and increasing concentrations of biotinylated oligonucleotide (bio-ON), which was found to be an effective affinity ligand. Separation of intermediate loading forms of actinavidin from unbound ligand in the presence of micellar phase and by capillary zone electrophoresis enabled the quantitation of free bio-ON, permitting the evaluation of the biotin-binding capacity of actinavidin in absence and presence of sodium dodecyl sulfate (SDS). Although in the latter case actinavidin lost a part of its binding capacity (not more than 12%), it was still possible to develop an indirect, noncompetitive assay for the determination of actinavidin in culture liquid, utilizing the combination of micellar electrokinetic capillary chromatography (MEKC) and ACE. Due to the affinity interaction, actinavidin in the sample decreases the amount of bio-ON added, enabling quantitation of the protein. SDS, which is required in this assay to prevent protein adsorption to the capillary wall, greatly enhances the reproducibility and peak shape. Actinavidin levels determined are in agreement with those obtained by commonly used solid-phase analysis. The limit of detection was about 500 ng/mL. Thus the proposed method was found to be well suited for the evaluation of actinavidin affinity and monitoring of its levels in cultivation process.