Elsevier

Toxicon

Volume 29, Issue 6, 1991, Pages 645-662
Toxicon

Characterization of cationic binding sites of neurotoxins from venom of the scorpion (Centruroides sculpturatus Ewing) using lanthanides as binding probes

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Abstract

R. M. David, N. R. Krishna and D. D. Watt. Characterization of cationic binding sites of neurotoxins from venom of the scorpion (Centruroides sculpturatus Ewing) using lanthanides as binding probes. Toxicon29, 645–662, 1991.—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 Å 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 (Ka) of the Tb(III)-CsE-v2 and v3 complexes are respectively 2.5 × 103 and 2.4 × 103M−1 determined by fluoresence enhancement and 2.4 × 103 and 2.3 × 103M−1 by equilibrium dialysis. Similarly Ka values for toxins CsE I and V are respectively 1.9 × 103 and 1.8 × 103M−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.

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      However, in the pharmacological group of MIBTXs, two toxins isolated from scorpions Tityus bahiensis and T. serrulatus have high percentages of identity with Ct-IT1: Tb2-II (59.09%) and Ts1 (58.46%), respectively (Pimenta et al., 2001; Sampaio et al., 1991). Ct-IT toxins also share some identity with Ts2 and CsE-V, isolated from the scorpions T. serrulatus and C. sculpturatus, respectively (David et al., 1991; Sampaio et al., 1991), despite these last toxins belong to α-NaScTxs. CsE-V, an Old World-like neurotoxin isolated from the venom of this New World scorpion, is an insect-specific neurotoxin with high specificity for insect sodium channels (Jablonsky et al., 2001).

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