Aird S D, Steadman B L, Middaugh C R, Kaiser I I
Department of Molecular Biology, University Station, University of Wyoming, Laramie 82071.
Biochim Biophys Acta. 1989 Aug 31;997(3):211-8. doi: 10.1016/0167-4838(89)90189-1.
Structures of four related neurotoxins and their purified subunits from the venoms of Crotalus durissus terrificus, C. vegrandis, C. s. scutulatus and C. viridis concolor were examined by circular dichroism (CD), deconvolution Fourier-transform infrared (FTIR) and fluorescence spectroscopy. CD spectra suggest that in general, the isolated subunits were decreased slightly in alpha-helix, while they were increased in beta-sheet structure, relative to intact toxins. These results were consistent with FTIR results. Fluorescence quenching (50-80%) was also observed in three of the four intact toxins as compared to spectra predicted by summation of free acidic and basic subunit spectra. It was tempting to conclude from these results that major conformational changes occur in individual subunits upon formation of the dimeric toxins. Intact crotoxin, however, when exposed to urea, yields spectra (CD, FTIR and fluorescence) that are virtually identical to control intact crotoxin. These findings suggest that the enhanced fluorescence exhibited by the isolated subunits, as well as the secondary structural changes in alpha-helix and beta-sheet, are artifacts resulting from irreversible structural changes that occur during subunit isolation by urea ion-exchange chromatography. In spite of these structural changes, LD50 values of intact crotoxin reassembled from isolated subunits are unaltered from those of native crotoxin.
利用圆二色性（CD）、去卷积傅里叶变换红外光谱（FTIR）和荧光光谱对来自剧毒矛头蝮、韦氏矛头蝮、中介亚种盾鳞棘背响尾蛇和墨西哥高原菱斑响尾蛇毒液中的四种相关神经毒素及其纯化亚基的结构进行了研究。CD光谱表明，总体而言，相对于完整毒素，分离出的亚基α-螺旋略有减少，而β-折叠结构有所增加。这些结果与FTIR结果一致。与通过游离酸性和碱性亚基光谱叠加预测的光谱相比，在四种完整毒素中的三种中还观察到了荧光猝灭（50 - 80%）。从这些结果很容易得出结论，在二聚体毒素形成时，单个亚基会发生主要的构象变化。然而，完整的响尾蛇毒素在暴露于尿素时，产生的光谱（CD、FTIR和荧光）与对照完整响尾蛇毒素几乎相同。这些发现表明，分离出的亚基所表现出的增强荧光以及α-螺旋和β-折叠中的二级结构变化，是尿素离子交换色谱法分离亚基过程中发生的不可逆结构变化导致的假象。尽管有这些结构变化，但从分离出的亚基重新组装的完整响尾蛇毒素的半数致死剂量（LD50）与天然响尾蛇毒素的LD50没有改变。
