Green N M, Toms E J
Biochem J. 1973 Aug;133(4):687-700. doi: 10.1042/bj1330687.
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 (6 m). 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(-8)m), about one third exchanged their biotin in a few minutes (K approximately 10(-10)m) 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 6 m-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 degrees 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 A). 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.
与经溴化氰活化的琼脂糖4B偶联的抗生物素蛋白保留了其90%以上的生物素结合能力。当使用低浓度的溴化氰时，约75%的蛋白质可通过用6mol/L的氯化胍洗涤从琼脂糖中去除。其余25%，即共价结合的亚基，对生物素的结合能力几乎未减，但亲和力降低。将氯化胍中的抗生物素蛋白亚基添加到偶联的亚基中，然后进行稀释或透析，可恢复其原始的生物素结合能力和亲和力。亚基制剂中存在三类结合位点。约25%为弱结合位点（K = 5×10⁻⁸mol/L），约三分之一在几分钟内就能交换其生物素（K约为10⁻¹⁰mol/L），其余的与天然四聚体难以区分。最后提到的这类结合位点在pH5和pH2时以相似的速率交换其结合的生物素，它们在6mol/L的氯化胍中不会失去生物素，并且在没有生物素的情况下对胰蛋白酶消化具有抗性。当与琼脂糖偶联的亚基在37℃下孵育时，这些稳定位点的比例可增加到65%。这种增加可被氯化胍逆转，这表明它是由共价连接的亚基的温度依赖性缔合引起的。这反过来意味着琼脂糖的琼脂糖基质具有温度依赖性流动性。对琼脂糖珠内亚基的空间分布分析得出结论，亚基的缔合意味着它们能够移动的距离大于20nm（几百埃）。当抗生物素蛋白亚基与用二乙烯砜交联的琼脂糖偶联时，这种流动性以及随之形成的四聚体大大降低。
