Measuring Partition Coefficient between Liquid-Disordered (LD) and Liquid-Ordered Phases. Why are Phase Diagrams Important to Know?

Abstract

We study and exploit methods to measure the partition coefficient of fluorescent molecules between liquid-disordered (Ld) and liquid-ordered (Lo) phases in 3 or 4-components lipid mixtures. A trajectory is created of 60-70 different lipid composition samples along a thermodynamic tieline, and the fluorescence signal of a single probe is measured for each sample. To determinate the partition coefficient, the single dye fluorescence is fitted by a hyperbolic curve based on the fraction of Ld or Lo phase. Because self-quenching could disturb the fluorescence profile along the tieline, we develop a self-quenching correction and formulate an equation to account for this effect. Briefly, we first studied how the fluorescence self-quenching behaves for increased dye concentration. Then we applied this correction along the tieline, since the dye concentration and the self-quenching effects vary with the fraction of Ld and Lo phases. Without this correction for fluorescence self-quenching, significant partition coefficient errors can occur. We show experiments using different dye concentrations where our corrected equation conveys the same partition coefficient measurement. We tested dyes that favor Ld phase, TopFluorPC and C12:0DiI, and TopFluor Ceramide that favors the Lo phase. We also compare our results to partition coefficient measurements using giant unilamellar vesicles (GUVs). We call attention to how well-resolved phase diagrams can be crucial for any measurement of the partition coefficient.