Selective labeling of alpha-bungarotoxin with fluorescein isothiocyanate and its use for the study of toxin-acetylcholine receptor interactions.
The main product of the reaction of fluorescein isothiocyanate (FITC) and bungarotoxin (Bgt) under near stoichiometric conditions is a monofluorescein derivative preferentially labeled at Lys 26, a highly conserved residue known to be involved in the binding (McDaniel, C.S., Manshouri, T., and Atassi, M.Z. (1987) J. Prot. Chem. 6, 455-461; Garcia-Borron, J.C., Bieber, A.L., and Martinez-Carrion, M. (1987) Biochemistry 26, 4295-4303) of postsynaptic neurotoxins specific for the nicotinic acetylcholine receptor (AcChR). The fluorescently labeled toxin retains a high affinity for the AcChR, and an unaltered specificity. Binding of FITC-Bgt to AcChR results in a significant decrease in the fluorescence intensity of the probe. This AcChR-mediated quenching of FITC-Bgt fluorescence allows for a continuous monitoring of the binding process. The quenching of free and bound FITC-Bgt by charged and neutral quenchers shows few fluorophore accessibility changes as induced by the toxin-bound state. The results are consistent with a model in which the positively charged concave surface of the toxin interacts with a negatively charged complementary surface in the receptor molecule.
Garcia-Borron
J C
JC
Division of Molecular Biology and Biochemistry, School of Basic Life Sciences, University of Missouri, Kansas City 64110-2499.
Chinchetru
M A
MA
Martinez-Carrion
M
M
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002038:Bungarotoxins / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D004557:Electric Organ; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007700:Kinetics; D011485:Protein Binding; D011950:Receptors, Cholinergic / Q000032:analysis / Q000187:drug effects / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013861:Thiocyanates; D014101:Torpedo
D002038:Bungarotoxins; D005452:Fluoresceins; D005456:Fluorescent Dyes; D011950:Receptors, Cholinergic; D013861:Thiocyanates; D016650:Fluorescein-5-isothiocyanate