Carroll R T, Muller J, Grimm J, Dunham W R, Sands R H, Funk M O
Department of Chemistry, University of Toledo, Ohio 43606.
Lipids. 1993 Mar;28(3):241-4. doi: 10.1007/BF02536646.
An efficient three-step purification technique has been developed for the reticulocyte 15-lipoxygenase from rabbit. Ammonium sulfate fractionated reticulocyte lysate was purified by size exclusion chromatography and preparative scale isoelectric focusing. The entire procedure was complete in less than eight hours and was carried out in batches which typically yielded 10 mg of purified enzyme. The identity and purity of the enzyme were evaluated by N-terminal sequencing, sodium dodecylsulfate polyacrylamide gel electrophoresis and specific activity determinations. The enzyme contained approximately one g-atom iron per mole of protein. The iron was present in an electron paramagnetic spectroscopy (EPR) silent, presumably high spin iron(II), form in the isolated enzyme. Treatment with one equivalent of 13-hydroperoxy-9(Z),11(E)-octadecadienoic acid resulted in the appearance of an EPR signal around g6.
已开发出一种高效的三步纯化技术，用于从兔网织红细胞中纯化15-脂氧合酶。经硫酸铵分级分离的网织红细胞裂解物通过尺寸排阻色谱法和制备规模的等电聚焦进行纯化。整个过程在不到八小时内完成，并且以分批方式进行，通常可获得10毫克纯化酶。通过N端测序、十二烷基硫酸钠聚丙烯酰胺凝胶电泳和比活性测定来评估酶的身份和纯度。该酶每摩尔蛋白质约含1克原子铁。在分离出的酶中，铁以电子顺磁共振光谱（EPR）沉默的形式存在，推测为高自旋铁（II）。用一当量的13-氢过氧-9（Z），11（E）-十八碳二烯酸处理后，在g6附近出现了EPR信号。
