Conformation of two homologous neurotoxins. Fluorescence and circular dichroism studies

Abstract

Two homologous short neurotoxins isolated from snake venoms (Laticauda semifasciata erabutoxin b and Naja nigricollis toxin .alpha.) were studied by fluorescence spectroscopy and in aqueous solution at various pH values. In parallel experiments, the stability of toxin conformations was analyzed on the basis of UV circular dichroism. Total luminescence spectra (77.degree. K) were recorded for both toxins in neutral and alkaline solutions. A neutral pH, the fluoresence emission is only due to the single and invariant Trp (29). From a comparative study with erabutoxin a, which differs from erabutoxin b by a single residue, it is unambiguously shown that the protonation of His-26 of erabutoxin b is responsible for a decrease of Trp-29 fluorescence. On the basis of available X-ray data it is proposed that the protonation or deprotonation of the following titrable groups is responsible for an alteration of Trp-29 fluorescence. These are Asp-31 (pk .simeq. 4) and Lys-27 (pK = 9.6) for both toxins and Lys-26 (pK .simeq. 9.6) for toxin .alpha.. No tyrosinate emission can be observed at neutral pH and 77.degree. K. Excitation spectra of toxin .alpha. revealed that 50% of the light absorbed by Try-25 in water is transferred to Trp-29. From the energy transfer measurements, the distance separating these 2 aromatic chromophores in the native toxin was estimated to be 13 .ANG.. A similar experiment was made for toxin .alpha. dissolved in trifluoroethanol. The distance separating the 2 aromatic side chains does not depend greatly on the nature of the solvent.