Fluorescent tetramethyl rhodamine derivatives of α-bungarotoxin: Preparation, separation, and characterization

α-Bungarotoxin is fluorescently labeled with tetramethyl rhodamine isothiocyanate and then fractionated on Sephadex G-25 and CM-Sephadex C-50 columns. The elution profile of the CM-Sephadex C-50 columns exhibits four distinct fluorescent peaks and a peak of unlabeled toxin. All four fluorescent peaks can fluorescently stain mouse diaphragm motor end plates. The most slowly eluting peak, Peak IV, has the highest quantum efficiency. Peak IV, which is identified as monolabeled tetramethyl rhodamine α-bungarotoxin, binds irreversibly to acetylcholine receptors on electroplax fragments and labels the fragments more intensely than Peaks I–III, which are identified as mixtures of multiply labeled tetramethyl rhodamine α-bungarotoxin.