Measurement of Monovalent and Polyvalent Carbohydrate-Lectin Binding by Back-Scattering Interferometry
Measurement of Monovalent and Polyvalent Carbohydrate-Lectin Binding by Back-Scattering Interferometry
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DOI：
10.1021/ac900569c
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发表时间：
2009-06-15
影响因子：
7.4
通讯作者：
Bornhop, Darryl J.
中科院分区：
文献类型：
--
作者：
Kussrow, Amanda;Kaltgrad, Eiton;Bornhop, Darryl J.
碳水化合物-蛋白质结合对生物化学的许多领域都很重要。在这里，背散射干涉法（BSI）已被证明是一种方便和灵敏的方法，用于获得定量信息的强度和选择性的这种相互作用。玻璃微流控通道的表面用抗生物素蛋白外亲和素共价修饰，随后通过孵育和洗涤将生物素化的凝集素附着于其上。未修饰的碳水化合物与所得的抗生物素蛋白固定的凝集素的结合通过BSL监测。在几分钟内产生的剂量-响应曲线在多个洗涤/测量循环中高度可重复，提供的吸附系数显示甘露糖与伴刀豆球蛋白A（conA）结合的亲和力比葡萄糖高3.7倍，与文献值一致。观察到半乳糖选择性地结合凝集素BS-1，并具有相似的亲和力。多价糖包被的病毒颗粒的亲和性固定化刀豆蛋白A是远远高于单价聚糖，增加60-200倍，每个聚糖排列在豇豆花叶病毒或噬菌体Q β的外表面时。糖官能化的PAMAM树枝状聚合物显示出尺寸依赖性吸附，这与表面上凝集素的预期密度一致。BSI的灵敏度匹配或超过表面等离子体共振和石英晶体微天平技术，是敏感的结合事件的数量，而不是质量的变化。BSI的操作简单性和通用性，沿着与接近天然的条件下，目标结合蛋白被固定，使BSI的凝集素和其他蛋白质的结合功能的定量表征的一个有吸引力的方法。
Carbohydrate-protein binding is important to many areas of biochemistry. Here, backscattering interferometry (BSI) has been shown to be a convenient and sensitive method for obtaining quantitative information about the strengths and selectivities of such interactions. The surfaces of glass microfluidic channels were covalently modified with extravidin, to which biotinylated lectins were subsequently attached by incubation and washing. The binding of unmodified carbohydrates to the resulting avidin-immobilized lectins was monitored by BSL Dose-response curves that were generated within several minutes and were highly reproducible in multiple wash/measure cycles provided adsorption coefficients that showed mannose to bind to concanavalin A (conA) with 3.7 times greater affinity than glucose consistent with literature values. Galactose was observed to bind selectively and with similar affinity to the lectin BS-1. The avidities of polyvalent sugar-coated virus particles for immobilized conA were much higher than monovalent glycans, with increases of 60-200 fold per glycan when arrayed on the exterior surface of cowpea mosaic virus or bacteriophage Q beta. Sugar-functionalized PAMAM dendrimers showed size-dependent adsorption, which was consistent with the expected density of lectins on the surface. The sensitivity of BSI matches or exceeds that of surface plasmon resonance and quartz crystal microbalance techniques, and is sensitive to the number of binding events, rather than changes in mass. The operational simplicity and generality of BSI, along with the near-native conditions under which the target binding proteins are immobilized, make BSI an attractive method for the quantitative characterization of the binding functions of lectins and other proteins.