Conformation of two homologous neurotoxins. Fluorescence and circular dichroism studies.

Two homologous short neurotoxins isolated from snake venoms (Laticauda semifasciata erabutoxin b and Naja nigricollis toxin alpha) have been studied by means of fluorescence spectroscopy and in aqueous solution at various pH values. In parallel experiments, the stability of toxin conformations was analyzed on the basis of ultraviolet circular dichroism. Total luminescence spectra (77 K) were recorded for both toxins in neutral and alkaline solutions. The data obtained indicate that, at neutral pH, the fluorescence emission is only due to the single and invariant tryptophan (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. Also, 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 congruent to 4) and Lys-27 (pK = 9.6) for both toxins and Lys-26 (pK congruent to 9.6) for toxin alpha. No tyrosinate emission can be observed at neutral pH and 77 K. Excitation spectra of toxin alpha revealed that 50% of the light absorbed by Tyr-25 in water is transferred to Trp-29. From the energy transfer measurements, the distance separating these two aromatic chromophores in the native toxin was estimated to be 13 A. A similar experiment was made for toxin alpha dissolved in trifluorethanol. The data indicate that the distance separating the two aromatic side chains does not depend greatly on the nature of the solvent.