Porphyrins as fluorescent probes for monitoring phase transitions of lipid domains in biological membranes. Factors influencing the microenvironment of haematoporphyrin and protoporphyrin in liposomes

Abstract The distribution properties of haematoporphyrin (HP) and protoporphyrin (PP) (concentration range 0.5–15 μM) in small unilamellar vesicles (SUVs) of dimiristoylphosphatidylcholine (DMPC), sometimes mixed with cholesterol (Chol) or cardiolipin (Card), were compared with those already reported for HP and PP in SUVs of dipalmitoylphosphatidylcholine (DPPC). In the latter system the study was extended to vesicles modified by addition of high concentrations of Chol (up to 55 mol% of total lipids) or Card (up to 37% w/w). These lipids are determinants for most biological membranes. The studies were performed by following the temperature dependence of the porphyrin fluorescence polarization and the quenching of porphyrin fluorescence by methyl viologen. In all lipid systems HP interacts with very polar, solvent-accessible regions of the inner monolayer. In DMPC liposomes HP is partitioned at the phospholipid headgroup-water interfaces over the whole concentration range. In contrast, discrimination of different areas (lipid-headgroup and headgroup-water interfaces) in the polar regions of the inner monolayer of DPPC liposomes is possible by modulating the dye concentration. HP shows no tendency to diffuse into pure lipid domains: a significant loading of the outer monolayer is observed only after an increase in the fluidity of the system by addition of Card or by increasing the temperature. A limit situation is obtained in DPPC liposomes enriched with high concentrations of Chol: in spite of the very low affinity of HP for Chol-rich domains of lipid membranes, porphyrin molecules do not move to the external monolayer but instead accumulate in mixed DPPC-Chol regions at the water-lipid inner interface. In all lipid systems PP is preferentially distributed in the most buried, hydrophobic regions of the lipid bilayer. A shift of the porphyrin molecules towards more quencher-accessible lipid domains is possible only in mixed DMPC-Chol or DMPC-Card liposomes brought to the fluid state with high temperatures.