Fe(III)-resorcylate as a spectrophotometric probe for phospholipid-cation interactions

A simple spectrophotometric microplate assay that allows quantification of the interaction between phospholipids and metal ions or other small cationic compounds has been developed. The assay is based on the competition of the phospholipids for the Fe(3+) ion in the purple-colored Fe(III)-γ-resorcylate complex and for other cations. To compare the binding affinities of several cation-phospholipid interactions, K0.5 values were derived from binding curves constructed by determination of the absorbance of the Fe(III)-γ-resorcylate at 490 nm as a function of the cation concentration. The assay was used to analyze the binding of lanthanide ions, calcium ions, and amines (hydrochlorides of ethanolamine, spermidine, and hexyltrimethylammonium chloride) to small unilamellar vesicles (SUVs) and mixed micelles containing anionic lipids such as phosphatidic acid and phosphatidyl-p-nitrophenol. The method was evaluated by fluorescence measurements with Eu(3+) ions as tracer. Lanthanide ions such as La(3+) and Ce(3+) ions showed K0.5 values smaller by one to two orders of magnitude compared with Ca(2+) ions. In the presence of increasing amounts of detergents such as Triton X-100, the method also reflected transitions from SUVs to micelles. The binding capacity for metal ions was higher for phospholipid-containing micelles than for the corresponding SUVs.