Krystal G, Pankratz H R, Farber N M, Smart J E
Blood. 1986 Jan;67(1):71-9.
Human urinary erythropoietin (Ep) has been purified using a simple five-step procedure to yield preparations with potencies of 80,000 U/mg in 25% yield. The five steps involve: (1) affinity chromatography on CM Affi-Gel Blue, (2) chromatofocusing, (3) wheat germ lectin (or hydroxylapatite) chromatography, (4) reverse-phase high-performance liquid chromatography (HPLC) using a phenyl column, and (5) preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Ep activity was determined at each stage using a highly sensitive and specific in vitro assay that measures [3H]-thymidine incorporation into erythroid cells from spleens of phenylhydrazine-treated mice. The step 5 material was also tested with the in vivo polycythemic mouse assay procedure and was found to have a similar potency to that obtained in the [3H]-thymidine in vitro assay. SDS-PAGE analysis of the step 5 material revealed a single 38.5-kd band that comigrated with Ep bioactivity. Homogeneity was confirmed by amino acid sequence analysis. Starting with urine containing approximately 13 U/mg of protein, the cumulative degrees of purification achieved with each step were: step 1,25-fold; step 2, 75-fold; step 3, 300-fold; step 4, 1,500-fold; and step 5, 5,000-fold. Corresponding overall recoveries after each step were: greater than 100%, 70%, 45%, 30%, and 25%. These recoveries could be obtained when as little as 5,000 U of starting urinary Ep were processed because of the introduction of Tween 20 and SDS into buffers used at various stages of the purification procedure. In addition, a rapid method for determining Ep purity which involves reverse-phase HPLC of trypsinized 125I-labeled Ep is presented. This allows the establishment of purity with far less material than is required for amino acid sequencing.
人尿促红细胞生成素（Ep）已通过一个简单的五步程序进行纯化，以25%的产率获得了效价为80,000 U/mg的制剂。这五个步骤包括：（1）在CM Affi-Gel Blue上进行亲和色谱；（2）色谱聚焦；（3）麦胚凝集素（或羟基磷灰石）色谱；（4）使用苯基柱的反相高效液相色谱（HPLC）；以及（5）制备性十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）。在每个阶段，使用一种高度灵敏且特异的体外测定法来测定Ep活性，该方法测量[³H]-胸苷掺入经苯肼处理的小鼠脾脏中的红系细胞。步骤5的材料也用体内红细胞增多症小鼠测定程序进行了测试，发现其效价与在[³H]-胸苷体外测定中获得的效价相似。步骤5材料的SDS-PAGE分析显示出一条与Ep生物活性共迁移的单一38.5-kd条带。通过氨基酸序列分析确认了其均一性。从含有约13 U/mg蛋白质的尿液开始，每个步骤实现的累积纯化倍数为：步骤1，25倍；步骤2，75倍；步骤3，300倍；步骤4，1500倍；步骤5，5000倍。每个步骤后的相应总回收率为：大于100%、70%、45%、30%和25%。由于在纯化程序的各个阶段所使用的缓冲液中引入了吐温20和SDS，因此当起始尿Ep低至5000 U时也能获得这些回收率。此外，还提出了一种测定Ep纯度的快速方法，该方法涉及对经胰蛋白酶消化的¹²⁵I标记的Ep进行反相HPLC。这使得用比氨基酸测序所需材料少得多的材料就能确定纯度。
