Ligand Receptor Dynamics at Streptavidin-Coated Particle Surfaces:  A Flow Cytometric and Spectrofluorimetric Study

Abstract

We have the studied the binding of 5- ((N-(5-(N-(6-(biotinoyl)amino)hexanoyl)amino)pentyl)thioureidyl)fluorescein (fluorescein biotin) to 6.2 µm 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, the extent of fluorescein quenching upon binding to the bead, and the association and dissociation kinetics. We estimate the site number to be ≈1 million per bead. The binding of the fluorescein biotin ligand occurs in steps where the insertion of the biotin moiety into one receptor pocket is followed immediately by the capture of the fluorescein moiety by a neighboring binding pocket; fluorescence quenching is a consequence of this secondary binding. At high surface coverage, the dominant mechanism of quenching appears to be via the formation of nonfluorescent nearest-neighbor aggregates. 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 ≈(1.3 ( 0.3) × 10 7 M -1 s -1 . The rate of binding of a second biotin may be reduced due to steric interference. The early time dissociative behavior is in sharp contrast to the typical stability associated with this system. The dissociation 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.

The development of quantum dot calibration beads and quantitative multicolor bioassays in flow cytometry and microscopy

“…Quantum ™ FITC beads are based on the properties of a NIST-calibrated fluorescein solution. Thus in order to be used accurately, the end user must have to account for the inevitable changes in quantum yields and spectral mismatches that occur when fluorescein is functionalized into a fluorescent tag (21,26).…”

“…The laser output is fixed at 488 nm. In general bead samples from the centrifugation assays were analyzed as previously described (26).…”

“…It is also instructive to note that the quantum yields of some fluorophores (e.g. fluorescein biotin) (37) or octadecyl rhodamine B (38)) depends on surface density due to self quenching at high surface occupancies (37). Thus, unless the characteristics of surface coverage-dependent changes in fluorophore quantum yield are known, calibration beads are generally most useful at low surface densities (9,10,37).…”

Rapid-mix flow cytometry measurements of subsecond regulation of G protein-coupled receptor ternary complex dynamics by guanine nucleotides

“…The results, interpreted according to a ternary complex model [15], were consistent with the idea that the dissociation of the α and βγ subunits in the presence of GTPγS was slow compared to the typical kinetics of intracellular signals such as intracellular Ca 2+ elevation and therefore could not likely account, kinetically, for cell activation. While fluorescent formyl peptides and FPR-GFP made it possible to study ternary complex dynamics in real-time, sample mixing and delivery times in flow cytometry limited kinetic resolution [16]. To minimize the dead time, online mixing schemes have been developed to achieve subsecond resolution [17].…”

Small‐volume rapid‐mix device for subsecond kinetic analysis in flow cytometry

“…Because flow cytometers can discriminate between free and cell-or particle-bound fluorophores, homogeneous and real-time measurements of ligand-receptor interactions on cells or beads can be made. Typical kinetic flow cytometer measurements require the removal of the sample tube from the cytometer, manual reagent mixing, and resumption of data collection, resulting in a dead time of 5 to 10 s or longer (4,5). To minimize the dead time, several online mixing schemes have been developed.…”