Proton nuclear magnetic resonance study of cobrotoxin

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Abstract
Cobrotoxin (Naga naga atra), which binds tightly to the acetylcholine receptors, contains no phenylalanines and only 2 His, 2 Tyr and 1 Try that result in well-resolved aromatic proton resonances in D2O at 360 MHz. His-32, Tyr-25 and the Trp are essential for toxicity and may interact with the acetylcholine receptor. Two titratable resonances (p[partial]Ka = 5.1) at .delta. = 9.0 and 7.5 ppm at pH 2.5 and at 7.7 and 7.1 ppm at pH 9.5 to the C-2 and C-4 ring protons, respectively, of His-4 are assigned. Two other titratable resonances (pKa = 5.7) at .delta. = 8.8 and 6.9 ppm at pH 2.5 and at 7.8 and 6.7 ppm at pH 9.5 are assigned to the C-2 and C-4 ring protons of His-32, respectively. The differences in .delta. values of the 2 his reflect chemically different microenvironments while their low pKa values could arise from nearby positive charges. A methyl resonance gradually shifts upfield to .delta. .apprx. 0.4 ppm as His-4 is deprotonated and is tentatively assigned to the methyl group of Thr-14 or Thr-15 which, from published X-ray studies of neurotoxins, are located in the vicinity of His-4. The aromatic resonances of the invariant Try and individual Tyr and the methyl resonance of 1 of the 2 Ile in the molecule are identified. Several broad nontitrating resonances of labile protons which disappear at pH > 9 may arise from amide groups of the .beta. sheet in cobrotoxin.