title: 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.
authors.affiliation: Department of Chemistry, University of Kentucky, Lexington 40506-0055.
authors.forename: M S
authors.initials: MS
authors.lastname: Barbarakis
authors.forename: W G
authors.initials: WG
authors.lastname: Qaisi
authors.forename: S
authors.initials: S
authors.lastname: Daunert
authors.forename: L G
authors.initials: LG
authors.lastname: Bachas
mesh_terms: D001360:Avidin / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D001667:Binding, Competitive; D001710:Biotin / Q000032:analysis*; D002352:Carrier Proteins; D004305:Dose-Response Relationship, Drug; D005949:Glucose Oxidase / Q000378:metabolism*; D019809:Streptavidin
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D001360:Avidin; D001710:Biotin; D019809:Streptavidin; D005949:Glucose Oxidase