Two-step affinity chromatography. Model systems and an example using biotin-avidin binding and a fluoridolyzable linker
Two-step affinity chromatography. Model systems and an example using biotin-avidin binding and a fluoridolyzable linker
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DOI：
10.1021/jo00024a029
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发表时间：
1991-11
影响因子：
3.6
通讯作者：
Wu Lin;T. Morton
中科院分区：
文献类型：
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作者：
Wu Lin;T. Morton
TweStep亲和层析是分离过程的总称，它包括以下两个连续步骤：生物分子与固体载体的特定结合，然后使用化学特定的裂解剂选择性洗脱，以切断附着在生物分子上的系链。以酶木瓜蛋白酶在其活性部位共价修饰的情况为例说明了该方法。生物素分子通过含有可氟解的CH20SiMe20CMez单元的亲水系链连接到半胱氨酸残基。然后，生物素化的木瓜蛋白酶与链霉亲和素分子(一种与生物素有四个相同结合位点的蛋白质)结合，然后这个复合体又与生物素分子结合，生物素分子与固体载体共价连接。在这第一步之后，共价修饰的木瓜蛋白酶通过氟化物催化的亲水系链中的氧硅键的水解而被选择性地洗脱。模型体系在水溶液中的均相动力学表明，氟化物催化的硅氧键的水解率没有饱和的证据。
Twestep affinity chromatography is a general designation for a separation procedure that involves the following pair of sequential step: specific binding of a biomolecule to a solid support then selective elution using a chemically specific cleaving agent to sever the tether that is attached to the biomolecule. This procedure is exemplified for the case of the enzyme papain covalently modified at its active site. A molecule of biotin is connected to a cysteine residue via a hydrophilic tether incorporating a fluoridolyzable -CH20SiMe20CMezunit. The biotinylated papain then binds a molecule of streptavidin (a protein that has four identical binding sites for biotin), and this complex in turn binds to a molecule of biotin that is covalently linked to a solid support. After this first step, the covalently modified papain is then selectively eluted by fluoride-catalyzed hydrolysis of an oxygensilicon bond in the hydrophilic tether. Homogeneous kinetics of model systems in aqueous solution show no evidence for saturation of the rate of fluoride-catalyzed hydrolysis of silicon-oxygen bonds.