Hayakawa K, De Felice C, Watanabe T, Tanaka T
Metabolism Research Laboratory, National Children's Medical Research Center, Tokyo, Japan.
J Chromatogr. 1993 Jul 2;616(2):327-32. doi: 10.1016/0378-4347(93)80403-q.
A reproducible, high-yield high-performance gel-permeation chromatographic method for proteins was developed and applied to the final step of purification of human serum biotinidase. One diol-type silica gel column, Tosoh TSK-gel 3000 SW (300 mm x 7.9 mm I.D.; average pore size 30 nm), and two Jasco Bio-Fine GFC SI 150-K columns (300 mm x 7.9 mm I.D.; average pore size 15 nm) were connected in series. A 0.1 M sodium phosphate buffer (pH 6.0) solution containing 0.3 M sodium chloride, glycerol (2.5%, v/v) and the non-ionic detergent Nonidet P-40 (NP-40, 0.15%, v/v) was utilized as an eluent. Recovery of the protein bovine serum albumin from the separating columns was measured by a spectrophotometric method and found to be 77.0 +/- 3.74% (mean +/- S.D.). The recovery of the total activity of human serum biotinidase was 72.6 +/- 13.0%. Since biotinidase activity was not recovered from the column in the absence of NP-40, the introduction of this non-ionic detergent to the mobile phase was shown to be essential for the final purification step of human serum biotinidase.
开发了一种可重现、高产率、高性能的蛋白质凝胶渗透色谱方法，并将其应用于人血清生物素酶纯化的最后一步。一根二醇型硅胶柱（东曹TSK-gel 3000 SW，300 mm×7.9 mm内径；平均孔径30 nm）和两根Jasco Bio-Fine GFC SI 150-K柱（300 mm×7.9 mm内径；平均孔径15 nm）串联连接。使用含有0.3 M氯化钠、甘油（2.5%，v/v）和非离子去污剂Nonidet P-40（NP-40，0.15%，v/v）的0.1 M磷酸钠缓冲液（pH 6.0）溶液作为洗脱剂。通过分光光度法测量分离柱中蛋白质牛血清白蛋白的回收率，发现为77.0±3.74%（平均值±标准差）。人血清生物素酶总活性的回收率为72.6±13.0%。由于在没有NP-40的情况下生物素酶活性不能从柱中回收，因此在流动相中引入这种非离子去污剂对于人血清生物素酶的最终纯化步骤至关重要。
