Ligand receptor dynamics at streptavidin-coated particle surfaces: A flow cytometric and spectrofluorimetric study

We have the studied the binding of 5-((N-(5-(N-(6-(biotinoyl)amino)hexanoyl fluorescein (fluorescein biotin) to 6.2 mu m diameter, streptavidin-coated polystyrene beads using a combination of fluorimetric and flow cytometric methods. We have determined the average number of binding sites per bead, t he extent of fluorescein quenching upon binding to the bead, and the associ ation and dissociation kinetics. We estimate the site number to be approxim ate to 1 million per bead. The binding of the fluorescein biotin ligand occ urs in steps where the insertion of the biotin moiety into one receptor poc ket is followed immediately by the capture of the fluorescein moiety by a n eighboring binding pocket; fluorescence quenching is a consequence of this secondary binding. At high surface coverage, the dominant mechanism of quen ching appears to be via the formation of nonfluorescent nearest-neighbor ag gregates. At early times, the binding process is characterized by biphasic association and dissociation kinetics which are remarkably dependent on the initial concentration of the ligand. The rate constant for binding to the first receptor pocket of a streptavidin molecule is approximate to(1.3 +/- 0.3) x 10(7) M-1 s(-1). The rate of binding of a second biotin may be reduc ed due to steric interference. The early time dissociative behavior is in s harp contrast to the typical stability associated with this system. The dis sociation rate constant is as high as 0.05 s(-1) shortly after binding, but decreases by 3 orders of magnitude after 3 h of binding. Potential sources for the time dependence of the dissociation rate constant are discussed.