Spector T
Prep Biochem. 1985;15(3):183-8. doi: 10.1080/10826068508062271.
The use of an ATP-agarose column to purify ribonucleotide reductase from human D-98 cells was recently reported. The column selectively retains greater than 99.9% of the contaminating nucleoside diphosphate (NDP) kinase from crude preparations of ribonucleotide reductase. It was presently found, however, that extending the length of the column caused the ribonucleotide reductase to dissociate into subunits. One subunit appeared in the low ionic strength buffer wash while the other required 0.5 M KCl for elution. The enzyme could also be recovered intact (non-dissociated) by equilibrating the enzyme preparation and the column with 0.5 M KCl prior to chromatography. Either method greatly improved the overall yield and the specific activity of the ribonucleotide reductase because it prevented the binding and subsequent loss of any of the subunits. In addition, the use of a larger column permitted the gel-filtration properties of the ATP-agarose to separate the bulk of the residual (not bound) NDP kinase from the ribonucleotide reductase.
最近有报道称使用ATP-琼脂糖柱从人D-98细胞中纯化核糖核苷酸还原酶。该柱可从核糖核苷酸还原酶的粗制品中选择性保留超过99.9%的污染性核苷二磷酸（NDP）激酶。然而，目前发现延长柱的长度会导致核糖核苷酸还原酶解离成亚基。一个亚基出现在低离子强度缓冲液洗脱液中，而另一个亚基需要用0.5 M KCl洗脱。在层析之前，通过用0.5 M KCl平衡酶制品和柱子，也可以完整地回收（不解离的）酶。这两种方法都极大地提高了核糖核苷酸还原酶的总体产量和比活性，因为它防止了任何亚基的结合及随后的损失。此外，使用更大的柱子可利用ATP-琼脂糖的凝胶过滤特性将大部分残留的（未结合的）NDP激酶与核糖核苷酸还原酶分离。
