Fig. 2. Equilibrium titration of AChBP with α-bungarotoxin and epibatidine.

AChBP at 300 nm was titrated in a 4 × 4-mm cuvette with incremental quantities of the peptide antagonist and the alkaloid agonist. Since quenching by α-bungarotoxin is only 15-20% of the unliganded receptor fluorescence, a receptor-gallamine complex was formed by addition of 2 μm gallamine to enchance fluorescence prior to the α-bungarotoxin addition. The contribution of the fluorescence from the added α-bungarotoxin was subtracted from the titration curve. The single tryptophan in α-bungarotoxin has less than 2% of the emission intensity at 335 nm of the tryptophans in the receptor-gallamine complex, so only a small correction is necessary. Fluorescence was recorded in a SPEX Fluoromax2 spectrofluorometer at 25 °C. Protein content was determined by quantitative amino acid analysis with a subunit molecular weight of 26,551. Fluorescence excitation was at 280 nm; emission maxima were measured over the range of 337–343 nm.