Nitration of tyrosine in three cobra neurotoxins

Abstract

The curarumimetic snake venom toxins always have a tyrosine residue in the same position in their sequences. This invariant tyrosine in three neurotoxins has been nitrated with tetranitromethane. The toxins were: (1) siamensis 3 of the structural type 71-5 (no. of amino acid residues and disulfides) and with one tyrosine, the main neurotoxin of the venom of Naja naja siamensis, (2) siamensis 7C (62-4 and two tyrosines), a minor neurotoxic component of the same venom, and (3) toxin α (61-4 and one tyrosine), the main neurotoxin of Naja nigricollis. The first toxin is a so-called long neurotoxin (a toxin with five disulfides and ca. 70 amino acid residues) and the other two short toxins (four and ca. 60). The 3-nitrotyrosyl derivatives of siamensis 3 and toxin α were isolated by gel filtration on Sephadex G-50 and ion-exchange chromatography on Bio-Rex 70. Both derivatives had i.v. ld 100-doses of 150 μg per kg mouse, corresponding to 67% of the initial toxicity. This high residual activity indicates that the invariant tyrosine does not have an essential role neither as a stabilizer of the active conformation nor as a functional group. The nitration of toxin 7C involved the modification of both tyrosine residues and was accompanied by great structural changes and loss of activity. The inactivation does not imply that the invariant tyrosine should be functionally essential in this toxin, but it is rather a consequence of the serious conformational changes accompanying the modification. The p K app of the invariant tyrosine is 10·5 in siamensis 3 and 11·6 in toxin α and probably about the same in toxin 7C. This indicates that this tyrosine in the long neurotoxin has an exposed position, whereas it is in the hydrophobic interior of the two short toxins, or that its ionization is greatly affected by neighbouring groups. In the discussion it is pointed out that the very strong binding between a neurotoxin and its target, the acetylcholine receptor is probably a result of the interaction between several groups in the toxin and the receptor. The majority of amino acid residues with reactive groups in the side chains have been modified with retention of a significant fraction of the initial activity. It seems likely that at least one of these groups (amino, carboxyl, tyrosinyl, tryptophanyl) should be in contact with the receptor in the toxin-receptor complex. If so, that should imply that it is possible to chemically modify such an interacting group; the interaction from the remaining groups should still be sufficient to bind the toxin to its target. There exists a great variety of cholinergica, which have only one obvious feature in common, a strongly positively charged group, which is suggested to be a recognition site, the function of which is to make it possible for the molecule to recognize the target. An invariant arginine residue is assumed to provide the recognition site in the neurotoxins.