Real time partitioning of octadecyl rhodamine B into bead supported lipid bilayer membranes reveals quantitative differences in saturable binding sites in DOPC and 1:1:1 DOPC/SM/Cholesterol membranes

摘要

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 to were used examine real time transfer of octadecyl rhodamine B (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 is raised from 0 to 10 mol% in membranes. The data show a > 2-fold enhancement in the quenching level of the probes that are reconstituted in DSC relative to DOPC membranes at the highest concentration of R18. We have parameterized 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 were the critical aggregation concentration (CAC) of the probe is defined as 14nM. The mass action kinetics of association of R18 with DOPC and DSC lipobeads are shown to be are similar. However, the saturable capacity for accepting exogenous probes is found to be 37% higher in DOPC relative to 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 towards a nano-scale probing of membrane heterogeneity in living cells by analyzing differential local FRET among sites of unique receptor expression in living cells.