Evaluation of the binding of Acanthamoeba profilin to pyrene-labeled actin by fluorescence enhancement

We have used a fluorescence assay to measure the binding of Acanthamoeba profilin to monomeric Acanthamoeba and rabbit skeletal muscle actin labeled on cysteine-374 with pyrene iodacetamide. The wavelengths of the pyrene excitation and emission maxima are constant at 346 and 386 nm, but the fluorescence is enchanced up to 50% by profilin. The higher fluorescence is largely due to higher absorbance in the presence of profilin. The fluorescence enhancement has a hyperbolic dependence on the concentration of profilin, suggesting a single class of binding sites. Linear Scatchard plots yield an estimate of the dissociation constant, K_d, of the complex of profilin with pyrenyl-actin. In low-ionic-strength buffers with 2 to 6 mm imidazole (pH 7.0) and 0.1 mm CaCl₂ the K_d is 9 μm for both muscle and Acanthamoeba actin. In 50 mm KCl the K_d for the complex with Acanthamoeba actin is 16 μm, while the K_d for the complex with muscle actin is greater than 50 μm.