Interaction of immobilized avidin with an aequorin-biotin conjugate: an aequorin-linked assay for biotin.
Biotinylated recombinant aequorin was used in the development of a heterogeneous bioluminescence binding assay for biotin. This assay is based on a competition between a biotinylated aequorin conjugate and biotin for the binding sites of avidin immobilized on solid particles. Dose-response curves were obtained that relate solid-phase aequorin activity to the concentration of biotin. Under certain experimental conditions these curves were biphasic; i.e., as the biotin concentration increased, the solid-phase aequorin activity first increased reaching a maximum and then decreased at higher biotin concentrations. This "hook" effect was observed with four different types of immobilization supports. The effect was more pronounced when low concentrations of aequorin-biotin conjugate were used, and diminished at a high conjugate concentration. This behavior indicates a possible positive cooperativity in the interaction between the immobilized avidin and biotin. Scatchard plot analysis was also consistent with a positive cooperativity mechanism. By using the ascending portion of the dose-response curve, the detection limit of the assay for biotin was 1 x 10(-15) M (100 zmol of biotin in the sample).
Feltus
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Division of Medicinal Chemistry and Pharmaceutics, University of Kentucky, Lexington, Kentucky 40506-0055, USA.
Ramanathan
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Daunert
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D000331:Aequorin / Q000378:metabolism*; D001360:Avidin / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D001710:Biotin / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D019921:Biotinylation; D008163:Luminescent Measurements; D008280:Magnetics; D008958:Models, Molecular; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D012680:Sensitivity and Specificity
D011994:Recombinant Proteins; D001360:Avidin; D000331:Aequorin; D001710:Biotin