Oury T D, Crapo J D, Valnickova Z, Enghild J J
Department of Pathology, Duke University Medical Center, Durham, NC 27710, USA.
Biochem J. 1996 Jul 1;317 ( Pt 1)(Pt 1):51-7. doi: 10.1042/bj3170051.
Studies examining the biochemical characteristics and pharmacological properties of extracellular superoxide dismutase (EC SOD) have been severely limited because of difficulties in purifying the enzyme. Recently EC SOD was found to exist in high concentrations in the arteries of most mammals examined and it is the predominant form of SOD activity in many arteries. We now describe a three-step, high-yield protocol for the purification of EC SOD from human aorta. In the first step, the high affinity of EC SOD for heparin is utilized to obtain a fraction in which EC SOD constitutes roughly 13% of the total protein compared with only 0.3% of that of the starting material. In addition, over 80% of the original EC SOD activity present in the aortic homogenate was retained after the first step of purification. EC SOD was further purified using a combination of cation- and anion-exchange chromatography. The overall yield of EC SOD from this purification procedure was 46%, with over 4 mg of EC SOD obtained from 230 g of aorta. Purified EC SOD was found to exist predominantly as a homotetramer composed of two disulphide-linked dimers. However, EC SOD was also found to form larger multimers when analysed by native PAGE. It was shown by urea denaturation that the formation of multimers increased the thermodynamic stability of the protein. Limited proteolysis of EC SOD suggested that there is one interchain disulphide bond covalently linking two subunits. This disulphide bond involves cysteine-219 and appears to link the heparin-binding domains of the two subunits.
由于纯化细胞外超氧化物歧化酶（EC SOD）存在困难，对其生化特性和药理性质的研究受到了严重限制。最近发现，在大多数被检测的哺乳动物动脉中，EC SOD以高浓度存在，并且是许多动脉中SOD活性的主要形式。我们现在描述一种从人主动脉中纯化EC SOD的三步高产方案。第一步，利用EC SOD对肝素的高亲和力获得一个组分，其中EC SOD占总蛋白的约13%，而起始材料中仅占0.3%。此外，在纯化的第一步之后，主动脉匀浆中超过80%的原始EC SOD活性得以保留。使用阳离子和阴离子交换色谱相结合的方法进一步纯化EC SOD。该纯化过程中EC SOD的总产率为46%，从230克主动脉中获得了超过4毫克的EC SOD。发现纯化的EC SOD主要以同四聚体形式存在，该同四聚体由两个通过二硫键连接的二聚体组成。然而，通过非变性聚丙烯酰胺凝胶电泳分析发现，EC SOD也会形成更大的多聚体。尿素变性实验表明，多聚体的形成增加了蛋白质的热稳定性。对EC SOD的有限蛋白酶解表明，存在一个链间二硫键共价连接两个亚基。这个二硫键涉及半胱氨酸-219，似乎连接了两个亚基的肝素结合结构域。
