Improved Characterization of the Solution Kinetics and Thermodynamics of Biotin, Biocytin and HABA Binding to Avidin and Streptavidin

Abstract

The high affinity (K D ~ 10 -15 M) of biotin to 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 (k on ) is approximately diffusion limited (10 9 M -1 s -1 ) but recent single molecule and surface binding studies indicate they are slower than expected (10 5 to 10 7 M -1 s -1 ). In this study, we asked whether these reactions in solution are diffusion controlled, what reaction model and thermodynamic cycle described the complex formation, and the 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'-hydroxyazobenzene)-benzoic acid. Native avidin and streptavidin are homotetrameric and the association data show no cooperativity between the binding sites. The k on 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 k on 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). The data suggest that the avidin binding sites are deeper and less accessible than those of streptavidin. 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.3