A fluorescence study of A23187 interaction with phospholipid vesicles

Abstract

The fluorescence of the ionophore A23187 has been monitored in suspensions of egg yolk phosphatidylcholine (EYPC) and dipalmitoyl phosphatidylcholine (DPPC) vesicles. Both the protonated form of A23187 and the Ca 2+ complex exhibit fluorescence enhancement when extracted into a hydrophobic environment. Measurements of fluorescence intensity versus lipid concentration were thus used to establish lower limits to the lipid/ water partition coefficients. Values obtained in this way were ≳ 50 ml water/mg phosphatidylcholine. Quenching of A23187 fluorescence by the spin labels 5NMS (methyl ester of 5-nitroxyl stearate), 12NMS, 16NMS, and TEMPO stearamide in EYPC and DPPC vesicles was also investigated. In EYPC all the labels yielded fairly linear Stern-Volmer plots, with TEMPO stearamide quenching about half as strong as the other probes. Quenching in DPPC was generally much stronger than in EYPC, but 12 NMS and 16NMS gave hyperbolic Stern-Volmer plots, apparently due to clustering of the labels. In all the cases the protonated form of A23187 was quenched approximately twice as efficiently as the Ca 2+ complex, possibly due to a longer fluorescence lifetime for the former. Calculations based on measured spectral properties were performed which indicate that the Förster transfer mechanism extends the nitroxides' quenching range to ∼- 10 Å.