Shanbhag V P, Stigbrand T, Jensen P E
Department of Biochemistry, Umeå University, Sweden.
Arch Biochem Biophys. 1996 Sep 1;333(1):35-41. doi: 10.1006/abbi.1996.0361.
Sodium thiocyanate dissociates native human alpha 2-macroglobulin into half-molecules consisting of two disulfide-bonded subunits, when the salt concentration is equal to or exceeds 1.2 M. Incubation with 1.6 M sodium thiocyanate for 1 h at 22 degrees C dissociates about 90% of alpha 2-macroglobulin into half-molecules. The half-molecules remain stable when the concentration of sodium thiocyanate is reduced to 0.2 M or zero, demonstrating that reassociation does not occur under these conditions. The internal thiol esters of the half-molecules are intact because they can be exposed by treatment with methylamine or trypsin. The noncovalent interaction between the disulfide-bonded dimers is stronger in the "closed-trap" than in the "open-trap" conformation of alpha 2-macroglobulin. The cleavage in the bait region by trypsin makes alpha 2-macroglobulin completely stable toward dissociation, and alpha 2-macroglobulin remains in a tetrameric state in 2.2 M sodium thiocyanate even when trypsin is not covalently bound to it. The increase in fluorescence with time indicates that conformational changes occur as a consequence of dissociation.
当盐浓度等于或超过1.2M时，硫氰酸钠可将天然人α2-巨球蛋白解离为两个由二硫键连接的亚基组成的半分子。在22℃下用1.6M硫氰酸钠孵育1小时，约90%的α2-巨球蛋白会解离为半分子。当硫氰酸钠浓度降至0.2M或零时，半分子保持稳定，这表明在这些条件下不会发生重新缔合。半分子的内部硫酯键是完整的，因为它们可以通过用甲胺或胰蛋白酶处理而暴露出来。在α2-巨球蛋白的“封闭陷阱”构象中，二硫键连接的二聚体之间的非共价相互作用比“开放陷阱”构象中更强。胰蛋白酶在诱饵区域的切割使α2-巨球蛋白对解离完全稳定，即使胰蛋白酶未与其共价结合，α2-巨球蛋白在2.2M硫氰酸钠中仍保持四聚体状态。荧光随时间的增加表明构象变化是解离的结果。
