48] Competitive binding assays for biotin-binding proteins

Abstract

This chapter describes the competitive binding assays for biotin-binding proteins. Radioligand binding assays of egg white avidin and egg yolk biotin-binding protein (BBP) are complicated by the presence of indeterminate amounts of biotin in samples. In each case, the exceptionally tight binding of biotin and its slow rate of dissociation mask occupied binding sites from exchange with added radiolabeled biotin. Heat-accelerated exchange to isotopic equilibrium between free and bound biotin at several sample concentrations followed by separation and measurement of protein-bound radioactivity enables the quantitation of total biotin-binding sites and the proportion of those sites occupied by endogenous biotin. A hyperbolic equation that describes the binding of radiolabeled biotin as a function of sample volume (V) at equilibrium can be transformed into the linear equation. Linearly transformed equations such as the Lineweaver–Burk equation for enzyme assays, it is convenient to apply linear regression analysis to the data to obtain values for the slopes and intercepts. However, this is statistically inappropriate because of the unequal weighting of data in double-reciprocal plots.