用于结晶研究的豌豆幼苗胺氧化酶的纯化与特性分析
Purification and characterization of pea seedling amine oxidase for crystallization studies.
作者信息
McGuirl M A, McCahon C D, McKeown K A, Dooley D M
机构信息
Department of Chemistry and Biochemistry, Montana State University, Bozeman 59717.
出版信息
Plant Physiol. 1994 Nov;106(3):1205-11. doi: 10.1104/pp.106.3.1205.
Abstract
Pea (Pisum sativum L.) seedling amine oxidase (EC 1.4.3.6) is the first amine oxidase to be crystallized that diffracts to atomic resolution (2.5 A). Extensive modifications of a published purification procedure were necessary to obtain protein that would give diffraction-quality crystals. Here we report the improved purification and also use this high-purity protein to reexamine some fundamental characteristics of pea seedling amine oxidase. The extinction coefficient at 280 nm (epsilon 1%(280)) and the molecular mass of the protein are investigated by a variety of techniques, yielding epsilon 1%(280) = 20 cm-1 and a mass 150 +/- 6 kD. In addition, the stoichiometry of the metal and organic cofactors, Cu(II) and 6-hydroxy dopa (Topa) quinone, respectively, is examined. The ratio of Cu(II):Topa:protein monomer is found to be 1:1:1.
摘要
豌豆（Pisum sativum L.）幼苗胺氧化酶（EC 1.4.3.6）是首个结晶后能衍射至原子分辨率（2.5埃）的胺氧化酶。为获得能产生具有衍射质量晶体的蛋白质，有必要对已发表的纯化程序进行大量改进。在此，我们报告了改进后的纯化方法，并使用这种高纯度蛋白质重新审视豌豆幼苗胺氧化酶的一些基本特性。通过多种技术研究了该蛋白质在280纳米处的消光系数（ε1%（280））和分子量，得出ε1%（280） = 20厘米-1，质量为150 ± 6千道尔顿。此外，还研究了金属和有机辅因子（分别为Cu（II）和6-羟基多巴（Topa）醌）的化学计量比。发现Cu（II）:Topa:蛋白质单体的比例为1:1:1。
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