Real-Time Partitioning of Octadecyl Rhodamine B into Bead-Supported Lipid Bilayer Membranes Revealing Quantitative Differences in Saturable Binding Sites in DOPC and 1:1:1 DOPC/SM/Cholesterol Membranes

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Abstract
Quantitative analysis of the staining of cell membranes with the cationic amphimphile, octadecyl rhodamine B (R18), is confounded by probe aggregation and changes to the probes’ absorption cross section and emission quantum yield. In this paper, flow cytometry, quantum-dot-based fluorescence calibration beads, and FRET were used to examine real-time transfer of R18 from water to two limiting models of the cellular plasma membrane, namely, a single-component disordered membrane, dioleoyl-l-α-phosphatidylcholine (DOPC), and a ternary mixture of DOPC, cholesterol, and sphingomyelin (DSC) membranes, reconstituted on spherical and monodisperse glass beads (lipobeads). The quenching of R18 was analyzed as the probe concentration was raised from 0 to 10 mol % in membranes. The data show a > 2-fold enhancement in the quenching level of the probes that were reconstituted in DSC relative to DOPC membranes at the highest concentration of R18. We have parametrized the propagation of concentration-dependent quenching as a function of real-time binding of R18 to lipobeads. In this way, phenomenological kinetics of serum-albumin-mediated transfer of R18 from the aqueous phase to DOPC and DSC membranes could be evaluated under optimal conditions where the critical aggregation concentration (CAC) of the probe is defined as 14 nM. The mass action kinetics of association of R18 with DOPC and DSC lipobeads are shown to be similar. However, the saturable capacity for accepting exogenous probes is found to be 37% higher in DOPC relative to that for DSC membranes. The difference is comparable to the disparity in the average molecular areas of DOPC and DSC membranes. Finally, this analysis shows little difference in the spectral overlap integrals of the emission spectrum of a fluorescein derivative donor and the absorption spectrum of either monomeric or simulated spectrum of dimeric R18. This approach represents a first step toward a nanoscale probing of membrane heterogeneity in living cells by analyzing differential local FRET among sites of unique receptor expression in living cells.