Observation of "hook effects" in the inhibition and dose-response curves of biotin assays based on the interaction of biotinylated glucose oxidase with (strept)avidin

Abstract

The inhibition of highly biotinylated enzymes by avidin and streptavidin has been used in the development of homogeneous assays for biotin and other analytes. Usually, this inhibition occurs in a similar fashion for both avidin and streptavidin. Specifically, the curves that relate the inhibition of the enzymatic activity with the concentration of avidin or streptavidin have a sigmoidal shape; I.e., the inhibition of the enzyme-biotin conjugates increases gradually with increasing amounts of avidin or streptavidin and arrives at a plateau at high binding protein concentrations. However, when these two biotin-specific binding proteins interact with biotinylated glucose oxidase a significant difference in their inhibitory action is observed. In particular, the inhibition curves have a sigmoidal shape for streptavidin, while those for avidin exhibit a maximum ("hook") at low avidin concentrations. This difference in the reactivity of the two proteins with biotinylated enzymes influences both the shape of the dose-response curve and the detection limits of homogeneous enzyme-linked competitive binding assays for biotin.

Avidin/PSS membrane microcapsules with biotin-binding activity

“…The catalytic activity of the immobilized enzyme was lower than that of free F-b-GOx, whose catalytic activity was 7.7 Â 10 À3 mole min À1 g À1 . The apparent kinetic parameters of b-GOx immobilized to avidin matrix are often different from those of free b-GOx [34,35]. The reduced activity of GOx may be ascribed in part to the effects of the inner and outer PAH/PSS layers of the microcapsule, by which diffusion of glucose and other substrates involved in the enzyme reaction was probably suppressed to some extent.…”

Determination of the Extent of Protein Biotinylation by Fluorescence Binding Assay

“…Biotinylation of proteins is generally achieved by an amidation reaction in which the free amino groups on lysine residues and the N terminus of proteins are reacted with an activated biotin derivative that is typically an N-hydroxysuccinimide (NHS) ester (Bayer and Wilchek, 1990). Sometimes, derivatives that introduce a spacer between the biotin and the protein surface are used to increase the accessibility of biotin toward (strept)avidin (Barbarakis et al, 1993a). Knowledge of the degree of biotinylation is of importance in the characterization of biotinylated proteins for use in optimized binding assays, bioreactors, and biosensors.…”

Interaction of Immobilized Avidin with an Aequorin–Biotin Conjugate: An Aequorin-Linked Assay for Biotin

“…Under certain circumbilization supports. The effect was more pronounced stances, however, biphasic curves in which the dosewhen low concentrations of aequorin-biotin conjuresponse curve has a maximum at low concentrations gate were used, and diminished at a high conjugate of ligand have been observed (14)(15)(16)(17)(18). These ''hook'' anism.…”