Endo T, Inagaki F, Hayashi K, Miyazawa T
Eur J Biochem. 1981 Nov;120(1):117-24. doi: 10.1111/j.1432-1033.1981.tb05677.x.
The 270-MHz proton NMR spectra were analyzed of the long neurotoxins alpha-bungarotoxin from Bungarus multicinctus and Toxin B from Naja naja. The aromatic proton resonances were completely assigned to individual nuclei for alpha-bungarotoxin and in part for toxin B. The pH dependences of proton chemical shifts were analyzed by the nonlinear least-square method, for obtaining pKa values and protonation shifts. The pKa values of Tyr-25, an invariant residue of neurotoxins, are 12.1 for alpha-bungarotoxin and 11.3 for toxin B, suggesting the presence of a strong hydrogen bond involving Tyr-25 in alpha-bungarotoxin. The Trp-29 residues of both toxins show a common titration shift due to the carboxylate group of Asp-31 and a similar structural arrangement of functionally invariant pair of Trp-29 and Asp-31 is implied. From the temperature dependences of the chemical shifts of His-68 and a methyl group of alpha-bungarotoxin, the local structure around His-68 near the tail part is shown to be more flexible than the other part. The six main-chain amide protons of alpha-bungarotoxin exchange most slowly with solvent deuterons and are found by interproton nuclear Overhauser effects to be in the beta-sheet near the aromatic ring of Tyr-25 residue. Hydrogen leads to deuterium exchange rates in 2H2O solution at 37 degrees C were measured of slowly exchanging amide protons of alpha-bungarotoxin, toxin B, and two short neurotoxins, namely cobrotoxin and erabutoxin b. The two long neurotoxins have amide protons with relatively long half-times spanning as long as 10-100 h, but the two short neurotoxins do not have amide protons with half-times longer than 3 h. The distributions of the half-times of amide proton exchange indicate the structural rigidity of neurotoxins in the order, alpha-bungarotoxin greater than toxin B greater than cobrotoxin approximately erabutoxin b, in agreement with the order of neurotoxicity as reported previously by Chicheportiche et al. and by Lee and Chen.
对多环眼镜蛇的长链神经毒素α-银环蛇毒素和眼镜蛇的毒素B进行了270兆赫质子核磁共振谱分析。已将α-银环蛇毒素的芳香质子共振完全归属到各个核上，对毒素B则部分进行了归属。采用非线性最小二乘法分析了质子化学位移的pH依赖性，以获得pKa值和质子化位移。神经毒素的不变残基Tyr-25的pKa值，α-银环蛇毒素为12.1，毒素B为11.3，这表明α-银环蛇毒素中存在涉及Tyr-25的强氢键。两种毒素的Trp-29残基由于Asp-31的羧基显示出共同的滴定位移，这意味着Trp-29和Asp-31这一功能不变对具有相似的结构排列。根据His-68和α-银环蛇毒素一个甲基的化学位移的温度依赖性，显示尾部附近His-68周围的局部结构比其他部分更灵活。α-银环蛇毒素的六个主链酰胺质子与溶剂氘核交换最慢，通过质子间核Overhauser效应发现它们位于Tyr-25残基芳香环附近的β-折叠中。测定了α-银环蛇毒素、毒素B以及两种短链神经毒素（即眼镜蛇毒素和海蛇毒素b）在37℃的2H2O溶液中酰胺质子与氢发生氘交换的速率。两种长链神经毒素的酰胺质子具有相对较长的半衰期，长达10 - 100小时，但两种短链神经毒素的酰胺质子半衰期不超过3小时。酰胺质子交换半衰期的分布表明神经毒素的结构刚性顺序为：α-银环蛇毒素＞毒素B＞眼镜蛇毒素≈海蛇毒素b，这与Chicheportiche等人以及Lee和Chen之前报道的神经毒性顺序一致。
