Interaction of Immobilized Avidin with an Aequorin–Biotin Conjugate: An Aequorin-Linked Assay for Biotin
| Autor: | Sylvia Daunert, Agatha Feltus, Sridhar Ramanathan |
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| Rok vydání: | 1997 |
| Předmět: |
Models
Molecular inorganic chemicals Protein Conformation Biophysics Aequorin Biotin Binding Competitive Sensitivity and Specificity Biochemistry Magnetics chemistry.chemical_compound Bioluminescence Biotinylation Molecular Biology Binding Sites Chromatography biology Ligand binding assay Cooperative binding Cell Biology Avidin Recombinant Proteins chemistry Luminescent Measurements embryonic structures biology.protein Conjugate |
| Zdroj: | Analytical Biochemistry. 254:62-68 |
| ISSN: | 0003-2697 |
| DOI: | 10.1006/abio.1997.2400 |
| Popis: | 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). |
| Databáze: | OpenAIRE |
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