PROPERTIES OF SUBUNITS OF AVIDIN COUPLED TO SEPHAROSE
PROPERTIES OF SUBUNITS OF AVIDIN COUPLED TO SEPHAROSE
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DOI：
10.1042/bj1330687
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发表时间：
1973-01-01
影响因子：
4.1
通讯作者：
TOMS, EJ
中科院分区：
文献类型：
--
作者：
GREEN, NM;TOMS, EJ
亲和素偶联到经溴化氰活化的Sepharose 4 B上，保留了90%以上的生物素结合能力。当使用低浓度的CNBr时，通过用氯化胍（6 m）洗涤，可以从Sepharose上去除约75%的蛋白质。剩余的25%，共价结合的亚基，对生物素的能力几乎没有减弱，但亲和力降低。在氯化胍中加入亲和素亚基，然后稀释或透析，恢复了原始的生物素结合能力和亲和力。三类结合位点存在于亚基的制备中。大约25%是弱的（K= 5X 10 − 8 m），大约三分之一在几分钟内交换了它们的生物素（K = 10− 10 m），其余的与天然四聚体无法区分。最后命名的交换其结合的生物素在pH 5和在pH 2以相似的速率，他们没有失去他们的生物素在6 m-胍氯化物，他们是抵抗胰蛋白酶消化的情况下，生物素。当偶联到Sepharose上的亚基在37°C孵育时，这些稳定位点的比例可增加到65%。这种增加被逆转的胍氯化物，这表明它是由一个温度依赖性的协会共价连接的亚基。这又意味着琼脂糖凝胶的琼脂糖基质的温度依赖性迁移率。对琼脂糖凝胶珠内亚基空间分布的分析得出结论，亚基的缔合意味着它们可以移动超过20 nm（几百nm）的距离。这种流动性和随后形成的四聚体大大减少时，亲和素亚基偶联到琼脂糖，已交联与二乙烯基砜。
Avidin that had been coupled to Sepharose 4B activated with CNBr retained over 90% of its biotin-binding capacity. When low concentrations of CNBr were used about 75% of the protein could be removed from the Sepharose by washing with guanidinium chloride (6m). The remaining 25%, the covalently bound subunits, had an almost undiminished capacity for biotin but a decreased affinity. Addition of avidin subunits in guanidinium chloride to the coupled subunits followed by dilution or dialysis restored the original biotin-binding capacity and affinity. Three classes of binding sites were present in preparations of the subunits. About 25% were weak (K=5X10−8m), about one third exchanged their biotin in a few minutes (K∼10−10m) and the remainder were indistinguishable from the native tetramer. The last-named exchanged their bound biotin at a similar rate at pH5 and at pH2, they did not lose their biotin in 6m-guanidinium chloride and they were resistant to tryptic digestion in the absence of biotin. The proportion of these stable sites could be increased to 65% when the subunits coupled to Sepharose were incubated at 37°C. This increase was reversed by guanidinium chloride, which suggested that it was caused by a temperature-dependent association of covalently linked subunits. This in turn implies a temperature-dependent mobility of the agarose matrix of the Sepharose. Analysis of the spatial distribution of subunits within the Sepharose beads led to the conclusion that the association of subunits implied that they could move through distances greater than 20nm (several hundred Å). This mobility and consequent formation of tetramer was greatly decreased when avidin subunits were coupled to Sepharose that had been cross-linked with divinyl sulphone.