A comparative investigation of snake venom neurotoxins and their triplet-state tryptophan-disulfide interactions using phosphorescence and optically detected magnetic resonance.

We have investigated the luminescence and optically detected magnetic resonance (ODMR) of the highly homologous snake venom neurotoxins alpha-bungarotoxin (BgTX), alpha-cobratoxin (CbTX), and cobrotoxin (CoTX) in frozen aqueous glasses. The phosphorescence intensity and lifetime of the single invariant tryptophan, Trp29, are found to be diminished in BgTX and CbTx relative to CoTX both at 77 K and at 4.2 K. Selective reduction of the Cys30-Cys34 disulfide proximal to Trp29 in BgTX and CbTX, that is absent in CoTX, results in the enhancement of the phosphorescence to fluorescence intensity ratio of Trp29 and identifies this disulfide as the source of the triplet-state quenching. Variations of the phosphorescence parameters are observed for differently frozen BgTX and CbTX samples. We argue that this observation is consistent with conformational flexibility in the region of Trp29. For BgTX and CbTX, changing the wavelength of excitation from 285 to 300 nm results in a small bathochromic phosphorescence shift of 0.4 nm, an average decrease in the lifetime, and a change in the polarity of the normally positive D-E ODMR signal. From the small excitation-dependent emission shift, we infer that Trp29 is in a relatively hydrophobic environment. The excitation-dependent changes in lifetime and ODMR signal parameters arise from subtle heterogeneity in the disposition of Trp29 with respect to Cys30-Cys34. We discuss the mechanism of disulfide-induced quenching of the Trp29 triplet state in BgTX and CbTX and argue that it most probably is due to electron transfer.