Determination of soluble nAChR-binding activity of alpha-neurotoxins by an innovative precipitation with DEAE-Sephacel.
A simple and convenient method for determining the binding activity of alpha-neurotoxins toward soluble nicotinic acetylcholine receptor (snAChR) by precipitation with DEAE-Sephacel was established. The determination was carried out by incubation of 125I-neurotoxin with snAChR, followed by precipitation with DEAE-Sephacel. The DEAE-Sephacel particles bind negatively charged snAChR with high affinity and simultaneously precipitate the 125I-neurotoxin bound to the receptors. After centrifugation, the free 125I-neurotoxin in the supernatant was counted, and the amounts of neurotoxins bound to snAChR could be determined. Two alpha-neurotoxins, cobrotoxin and alpha-bungarotoxin, were employed to verify the feasibility of this determination. The different binding characteristics of cobrotoxin and alpha-bungarotoxin to snAChR could be distinguished. This method required only small quantities of DEAE-Sephacel (7 mg), snAChR (0.54 micrograms), and 125I-neurotoxin (90 fmol) for each reaction, and minimized the handling of isotopic materials as compared with the conventional methods. This method is reliable, reproducible, and superior to current methods for the determination of the snAChR-binding activity for alpha-neurotoxins.
D001667:Binding, Competitive; D002038:Bungarotoxins / Q000378:metabolism; D003039:Cobra Neurotoxin Proteins / Q000378:metabolism; D003636:DEAE-Cellulose / Q000031:analogs & derivatives / Q000378:metabolism; D009498:Neurotoxins / Q000378:metabolism*; D011485:Protein Binding; D011863:Radioimmunoassay; D011978:Receptors, Nicotinic / Q000378:metabolism*
D002038:Bungarotoxins; D003039:Cobra Neurotoxin Proteins; D009498:Neurotoxins; D011978:Receptors, Nicotinic; D003636:DEAE-Cellulose