| Popis: |
The on-rate constants (kon) of biotin (B) binding to avidin (AV) and streptavidin (SAV) are believed to be diffusion limited (109 M−1s−1). In this study; we asked whether these reactions were actual diffusion controlled, what association model and thermodynamic cycle describe the process, and what are the functional differences between AV and SAV. We have studied the B association by two stopped flow methodologies that used: I) fluorescent probes attached to B; and II) unlabeled B and HABA [2-(4’-hydroxyazobenzene)-benzoic acid]. The reactions were carried out at several temperatures, pH's and under pseudo first-order conditions with: oregon green biocytin (BcO), biotin-4-fluorescein (BFl), biotin-DNA duplex, and unlabeled B. We obtained the spectroscopy properties of the bound dye-biotin complexes to have an insight of the chemical environment surrounding B. The association data showed not cooperativity between the 1st and the 4th binding sites of AV. The kon values of SAV were faster than AV's, but in both cases were slower than those expected for a diffusion limited reaction. Furthermore, the Arrhenius plots revealed strong temperature dependence with large activation energies (6-15 kcal/mol) that did not correspond either to a diffusion limited process (3-4 kcal/mol). The outcomes indicated that AV binding sites were deeper and less accessibility than SAV. In addition, we are reporting, for the first time, a second order displacement rate constant of a bound SAV complex when challenged with free B; results that are relevant for the purification technology base on these proteins. Finally, we propose a simple reaction model with a single transition state whose forward energetic parameters complete the thermodynamic cycle in excellent agreement with previous studies. |
