Schlesinger D H, Frangione B, Walter R
Proc Natl Acad Sci U S A. 1972 Nov;69(11):3350-4. doi: 10.1073/pnas.69.11.3350.
The completed amino-acid sequence of bovine neurophysin-II, a major neurohypophyseal hormone-binding protein in the hypothalamo-neurohypophyseal complex of cows, set the stage for the localization of the disulfide bonds of this sulfur-rich molecule. Neurophysin-II was digested with subtilisin or a pepsin-trypsin mixture. The resulting peptides were subjected to first-dimensional electrophoresis at pH 6.5, oxidized with performic acid, and subjected to second-dimensional electrophoresis under identical conditions as the first-dimensional separation, but in a perpendicular direction. Cysteic acid peptides were eluted (several after additional electrophoretic purification at pH 3.5) for amino-acid composition and NH(2)- and COOH-terminal analyses. Our assignment of the seven disulfide bridges present in neurophysin-II is as follows: Cys(10)-Cys(93); Cys(13)-Cys(95); Cys(21)-Cys(27); Cys(28)-Cys(44); Cys(54)-Cys(61); Cys(67)-Cys(73); Cys(74)-Cys(79). The assignment of disulfide bridges associated with Cys(27) and Cys(28) is tentative as it is derived from evolutionary consideration. The high disulfide content reduces drastically the allowed number of biofunctional conformers of neurophysin-II. It is suggested that neurophysin-II possesses a globular topography with minimal alpha-helix structure.
牛神经垂体素-II是奶牛下丘脑-神经垂体复合体中一种主要的神经垂体激素结合蛋白，其完整的氨基酸序列为确定这种富含硫的分子中二硫键的位置奠定了基础。用枯草杆菌蛋白酶或胃蛋白酶-胰蛋白酶混合物消化神经垂体素-II。将所得肽段在pH 6.5条件下进行一维电泳，用过甲酸氧化，然后在与一维分离相同的条件下，但在垂直方向上进行二维电泳。洗脱半胱氨酸肽（在pH 3.5下进行额外的电泳纯化后有几种）用于氨基酸组成分析以及氨基和羧基末端分析。我们对神经垂体素-II中存在的七个二硫键的分配如下：半胱氨酸（10）-半胱氨酸（93）；半胱氨酸（13）-半胱氨酸（95）；半胱氨酸（21）-半胱氨酸（27）；半胱氨酸（28）-半胱氨酸（44）；半胱氨酸（54）-半胱氨酸（61）；半胱氨酸（67）-半胱氨酸（73）；半胱氨酸（74）-半胱氨酸（79）。与半胱氨酸（27）和半胱氨酸（28）相关的二硫键分配是暂定的，因为它是基于进化考虑得出的。高含量的二硫键极大地减少了神经垂体素-II可能的生物功能构象数量。有人提出神经垂体素-II具有最小α-螺旋结构的球状拓扑结构。
