A simple quantitative assay of 125I-labeled α-bungarotoxin binding to soluble and membrane-bound acetylcholine receptor protein

Abstract

This method involves the irreversible formation of a complex between 125I-labeled α-bungarotoxin and the acetylcholine receptor in either its membrane-bound or purified state. The separation of the labeled toxin-receptor complex from unreacted toxin is accomplished by chromatography on carboxymethylcellulose cation-exchange resin. The method described was developed to satisfy the following experimental requirements that could not be dealt with in their entirety by employing any of the published methods: (i) the complete recovery of reacted and unreacted species in relatively small volumes; (ii) an efficient and precise isolation of the specific and irreversible 125I-labeled α-bungarotoxin-receptor complex when the complexation reactions demand a large excess of unlabeled α-bungarotoxin for quenching (a 20-fold molar excess of unlabeled over labeled toxin); (iii) this isolation of the toxin-receptor complex allows one to determine the protein concentrations in the samples, a necessity in experiments covering a wide range of receptor concentrations; (iv) a consistent low blank for binding site concentrations ranging over two or three orders of magnitude; (v) simplicity and rapidity.