The high affinity (KD ~ 10−15 M) of biotin for avidin and streptavidin is the essential component in a multitude of bioassays with many experiments using biotin modifications to invoke coupling. Equilibration times suggested for these assays assume that the association rate constant (kon) is approximately diffusion limited (109 M-1s-1) but recent single molecule and surface binding studies indicate that they are slower than expected (105 to 107 M-1s-1). In this study, we asked whether these reactions in solution are diffusion controlled, which reaction model and thermodynamic cycle describes the complex formation, and if there are any functional differences between avidin and streptavidin. We have studied the biotin association by two stopped-flow methodologies using labeled and unlabeled probes: I) fluorescent probes attached to biotin and biocytin; and II) unlabeled biotin and HABA, 2-(4’-hydroxyazo-benzene)-benzoic acid. Both native avidin and streptavidin are homo-tetrameric and the association data show no cooperativity between the binding sites. The kon values of streptavidin are faster than avidin but slower than expected for a diffusion limited reaction in both complexes. Moreover, the Arrhenius plots of the kon values revealed strong temperature dependence with large activation energies (6–15 kcal/mol) that do not correspond to a diffusion limited process (3–4 kcal/mol). Accordingly, we propose a simple reaction model with a single transition state for non-immobilized reactants whose forward thermodynamic parameters complete the thermodynamic cycle, in agreement with previously reported studies. Our new understanding and description of the kinetics, thermodynamics, and spectroscopic parameters for these complexes will help to improve purification efficiencies, molecule detection, and drug screening assays or find new applications.
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Agricultural and Biological Sciences(all)