ANS fluorescence and displacement binding curves

8-anilino-1-naphthalenesulfonic acid (ANS) fluorescence binding curves and the binding-induced reduction in ANS fluorescence resulting from competition with tested ligands across a range of concentration facilitated the measurement of IC₅₀ and an estimate of K_d values using the binding models and procedures described previously⁷⁰. To measure the affinity of each ANS-PR10 interaction and obtain the corresponding K_d value, binding curves were generated by measuring the fluorescence of a series of assays using a fixed 10 µM concentration of ANS and increasing concentrations of purified recombinant MLP/PR10 proteins (0 to either 80 or 100 µM). ANS was dissolved in dimethylsufoxide at a concentration of 20 mM, stored at −20 °C until dilution and protected from exposure to light. ANS was diluted to 20 µM and individual MLP/PR10 proteins were diluted to various concentrations up to 200 µM in citrate-buffered saline, pH 5.5 (CBS, 10 mM citrate, 127 mM NaCl, 2.7 mM KCl). 40 µl of the ANS solution and 40 µl of each MLP/PR10 protein solution were combined to 80 µl final reaction volumes, which were incubated at 21 °C for 10 min to allow equilibration of binding. Triplicate 18-µl aliquots were withdrawn from each 80 µl reaction, transferred to optically clear PCR tubes (Diamed, Canada) and ANS fluorescence was measured following an additional 3-min incubation at 30 °C using a Qiagen Rotor-gene Q 6-plex with excitation and emission filters of 365 ± 20 nm and 460 ± 20 nm, respectively. An identical reaction series whereby protein was omitted from the mixtures was measured under identical conditions and trace fluorescence was subtracted from each MLP/PR10 protein-containing sample. Each data set was scaled so that the maximum observed fluorescence corresponded to 100% and individual data points were plotted, and a rectangular hyperbolic ligand binding function was fit, using least-squares analysis (Prism 5 software, Graphpad, USA). Experiments were repeated at least twice with independently prepared protein. For PR10-10, the experiment was repeated four times as described above, and twice more with substantially altered MLP/PR10 protein and ANS concentrations.

To measure the ability of major BIAs (i.e., thebaine, morphine, codeine, papaverine and noscapine) to compete with ANS for interaction with the MLP/PR10 proteins, ANS displacement assays were conducted with fixed equimolar concentrations of protein and ANS (10 µM), and a range of BIA ligand concentrations (0 to 3 mM, dependent on solubility). Displacement of ANS is associated with reduced fluorescence. The use of CBS buffer, pH 5.5, as the buffer was required to solubilize papaverine and noscapine. Briefly, 40 µl of 20 µM ANS and individual MLP/ PR10 protein solutions were combined in CBS buffer, pH 5.5, incubated at 21 °C for 5 min, mixed with 40 µl of separate 0 to 6 mM BIA solutions in CBS buffer, pH 5.5, and the 80 µl reactions were subsequently incubated for 10 min at 21 °C. Fluorescence was measured as described above, including subtraction of any fluorescence in a blank series whereby protein was omitted. Individual data points were plotted with fluorescence scaled to 100% and BIA concentrations expressed in their log₁₀ form, which allowed fitting of a sigmoidal dose-response model curve and determination of IC₅₀ values in Prism 5 software. Each experiment was repeated at least twice with independently prepared MLP/PR10 proteins and BIA ligand. Average IC₅₀ values were used, together with corresponding average PR10-ANS K_d values, to estimate MLP/PR10-BIA K_d value based on an approximate relationship described previously⁷¹.