Systematic evaluation of avidin–biotin interaction by fluorescence spectrophotometry

The avidin–biotin interaction was evaluated systematically by fluorescence spectroscopy under different conditions of temperature, pressure, pH, metal ions, incubation time and initial avidin concentration. The binding constant was calculated according to the modified Stern–Volmer equation, which deduced the existence of static quenching mechanism. The data obtained revealed that avidin–biotin interaction exhibited temperature, pH, metal ions, incubation time and initial avidin concentration sensitivity. The binding constants decreased with increase in temperature, while the binding sites were independent of temperature. The values of thermodynamic parameter ΔH (−149.85 kJ mol−1) and ΔS (−284.26 J mol−1 K−1) suggested hydrogen bonds and van der Waals played a major role in the reaction. The binding constants between avidin and biotin increased firstly and then decreased gradually with the increase of pH values. Metal ions can also affected the binding constants between avidin and biotin. The association kinetics firstly acquired by the combination of the change in fluorescence per unit time and the modified Stern–Volmer equation indicated that the reaction time required to reach equilibrium was 2200 s, and the average reaction rate for the binding process was very high in the first 180 s. Reaction of the avidin in the first 180 s was more than 40% of the total avidin involved in the whole process.

The avidin–biotin interaction was investigated systematically using biotin-4-fluorescein by fluorescence spectrophotometry. The quenching mechanism, binding constants, thermodynamic parameters, the equilibrium time and kinetic parameters of binding were obtained.Figure optionsDownload as PowerPoint slideHighlights
► We evaluated systematically the avidin–biotin interaction by spectroscopic methods.
► The mechanism of fluorescence quenching was static quenching.
► Hydrogen bonds and van der Waals forces were the main forces.
► Temperature, pH and metal ion affected the avidin–biotin interaction.
► Equilibrium and kinetics of reaction between avidin and biotin were investigated.