【摘要】 本文报道制备顺丁烯二酰亚胺氮氧自由基纯品的方法。本方法为Hamilton-McConnell法的改进。在温和条件下,该化合物以共价键形式自旋标记到血清白蛋白的硫氢基和氨基等活性基团上。为利用自旋标记顺磁共振技术研究蛋白质分子的构象,提供了必要前提。经顺丁烯二酰亚胺氮氧自由基自旋标记的牛和羊血清白蛋白,由于标记位点和标记物所处的周围环境不同,给出了两类ESR波谱:即强固定化作用型和弱固定化作用型。用0.5 mMHgCl₂预处理牛血清白蛋白,阻断其硫氢基以后再自旋标记,仅给出弱固定化型波谱。从而进一步证明强固定化作用型波谱主要来自标记在蛋白质深层中的硫氢基;弱固定化作用型波谱则来自标记在靠近蛋白质表层的非硫氢基团上。通过对羊血清白蛋白自旋标记以后,再用8M尿素破坏其氢键的试验,发现原样品中强固定化型的谱线消失,仅剩下三条弱固定化型谱线。表明自旋标记物对蛋白质构象变化的反应是十分灵敏的。文中,对两类ESR波谱用自旋哈密尔顿理论进行了描述。更多还原

【Abstract】 The nitroxide-maleimide free radical, which we prepared by chemioal synthesis, tinder mild conditions,can be specially used to spin label such active groups of proteins as the snlfhydryl group of serum albumin as well as the amino group through covalent binding. It provides a favorable approach to the investigation of biological molecules in connection with electron spin resonance speotrosoopy.When the nitroxide-maleimide was used to spin label bovine and sheep’s serum albumin, two kinds of ESR spectra, i. e. "strongly immobilized" and "weakly immobilized", were obtained. This represents the difference between two types of labeling site and between their environments. Moreover, evidenoe from the SH group-blocking experiment with HgCl₂ shows that the strongly immobilized signal presumably arises from the labels of—SH groups located in the depth of the protein molecule, while spin labels of active amino groups on the surface layer give out the weakly immobilized spectrum.By virtue of studying conformational changes of serum albumin from native to unfolding states, it has been indicated that the ESR spectra are very sensitive to those changes of a protein carrying a spin label, with which the characteristics of spectra are consistent.更多还原