从牛小肠中亲和层析纯化碱性磷酸酶。
Affinity-chromatography purification of alkaline phosphatase from calf intestine.
作者信息
Brenna O, Perrella M, Pace M, Pietta P G
出版信息
Biochem J. 1975 Nov;151(2):291-6. doi: 10.1042/bj1510291.
A crude preparation of alkaline phosphatase (EC 3.1.3.1) from calf intestinal mucosa was purified by affinity chromatography on Sepharose-bound derivatives of arsanilic acid, which was found to be a competitive inhibitor of the enzyme. Three biospecific adsorbents were prepared for the chromatography, and the best results were obtained with a tyraminyl-Sepharose derivative coupled with the diazonium salt derived from 4-(p-aminophenylazo)phenylarsonic acid. Alkaline phosphatase was the only enzyme retained by the affinity column in the absence of Pi. The enzyme eluted by phosphate buffer had a specific activity of about 1200 units per mg of protein at pH 10.0, with 5.5mM-p-nitrophenyl phosphate as the substrate.
从小牛肠黏膜中提取的碱性磷酸酶（EC 3.1.3.1）粗制品，通过在与对氨基苯胂酸结合的琼脂糖衍生物上进行亲和层析进行纯化，发现对氨基苯胂酸是该酶的竞争性抑制剂。制备了三种生物特异性吸附剂用于层析，其中由酪胺基琼脂糖衍生物与源自4-（对氨基苯偶氮）苯胂酸的重氮盐偶联得到的吸附剂效果最佳。在没有无机磷酸盐（Pi）的情况下，碱性磷酸酶是亲和柱唯一保留的酶。用磷酸盐缓冲液洗脱的酶，以5.5mM对硝基苯磷酸为底物，在pH 10.0时，每毫克蛋白质的比活性约为1200单位。
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