CHARACTERIZATION OF CATIONIC BINDING-SITES OF NEUROTOXINS FROM VENOM OF THE SCORPION (CENTRUROIDES-SCULPTURATUS EWING) USING LANTHANIDES AS BINDING PROBES

Binding sites for cations were probed in the structures of protein neurotoxins from Centruroides sculpturatus by enhancement of terbium(III) fluorescence, detected by emission at 552 nm, when aromatic side-chains of the toxins were activated at 286 nm. Gadolinium, Gd(III), was used as a cation binding probe by observing its effects on nuclear magnetic resonance (NMR) spectra. Toxins CsE-v2 and v3, when bound to Tb(III), enhance luminescence of Tb(III) 20-fold whereas CsE-v1 enhances Tb(III) luminescence about 15-fold. Toxins CsE-I and V have no effect on the luminescence of Tb(III) implying that these latter two toxins have structures incompatible with efficient energy transfer from activated aromatic side-chains. Enhancement of fluorescence is pH dependent and is competitively inhibited by alkaline earth divalent cations and by other lanthanide(III) ions. Neodymium, Nd(III), with an ionic radius of 0.995 angstrom is the most efficient of the lanthanide ions and the divalent cations in displacement of Tb(III) from the toxins. Relaxation enhancements of aromatic CH resonances by Gd(III) are apparent with tyrosines 4, 42, 38, 14-40 peak and tryptophan 47. Results from pH vs fluorescence studies suggest that carboxyl groups are involved in binding of Tb(III). Association constants (K(a)) of the Tb(III)-CsE-v2 and v3 complexes are respectively 2.5 x 10(3) and 2.4 x 10(3) M-1 determined by fluorescence enhancement and 2.4 x 10(3) and 2.3 x 10(3) M-1 by equilibrium dialysis. Similarly K(a) values for toxins CsE I and V are respectively 1.9 x 10(3) and 1.8 x 10(3) M-1 determined by equilibrium dialysis. Experimental evidence suggests that at least two Tb(III)s are bound per toxin molecule. The results from these studies are discussed in relation to the tertiary structure of toxin CsE-v3.