Spector T, Averett D R
Anal Biochem. 1983 Oct 15;134(2):467-70. doi: 10.1016/0003-2697(83)90324-x.
Assays of ribonucleotide reductase in extracts of Detroit 98 (human) cells were found to be complicated by the rapid depletion of the substrate (CDP) by nucleoside diphosphate kinase. Assays of either 100,000g supernatants or ammonium sulfate-fractionated extracts resulted in the conversion of greater than 90% of the substrate to CTP within 2 min. It was therefore desirable to separate nucleoside diphosphate kinase from ribonucleotide reductase. Chromatography of the fractionated extract on an ATP-agarose column resulted in the delivery of nondissociated ribonucleotide reductase in the void volume and the retention of greater than 99.9% of the nucleoside diphosphate kinase. The kinase could be eluted by 2 mM ATP. The ribonucleotide reductase was recovered from this commercially available gel with an apparent yield of greater than 200%. It could be accurately assayed with only minimal extraneous depletion of substrate. Furthermore, it was stable to storage at -80 degrees C. Tris-HCl was found to inhibit the enzyme. When HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)-Na buffer was used in place of Tris-HCl, the rate of CDP reduction was increased by 2.5-fold. Since the above procedure selectively removes nucleoside diphosphate kinase from crude preparations of ribonucleotide reductase, it should have general applicability for purifying ribonucleotide reductase from other sources.
在底特律98（人）细胞提取物中进行核糖核苷酸还原酶的测定时，发现由于核苷二磷酸激酶会迅速消耗底物（CDP），使得测定变得复杂。对100,000g上清液或硫酸铵分级分离提取物进行测定时，在2分钟内超过90%的底物会转化为CTP。因此，需要将核苷二磷酸激酶与核糖核苷酸还原酶分离。将分级分离的提取物在ATP-琼脂糖柱上进行色谱分离，结果是未解离的核糖核苷酸还原酶在空体积中流出，而超过99.9%的核苷二磷酸激酶被保留。激酶可用2 mM ATP洗脱。从这种市售凝胶中回收的核糖核苷酸还原酶，其表观产率大于200%。它可以在底物仅有极少额外消耗的情况下进行准确测定。此外，它在-80℃储存时很稳定。发现Tris-HCl会抑制该酶。当使用HEPES（4-(2-羟乙基)-1-哌嗪乙磺酸）-Na缓冲液代替Tris-HCl时，CDP还原速率提高了2.5倍。由于上述方法能从核糖核苷酸还原酶的粗制品中选择性地去除核苷二磷酸激酶，它应该对从其他来源纯化核糖核苷酸还原酶具有普遍适用性。
