Biotin-pyrene conjugates with poly(ethylene glycol) spacers are convenient fluorescent probes for avidin and streptavidin
Biotin-pyrene conjugates with poly(ethylene glycol) spacers are convenient fluorescent probes for avidin and streptavidin
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DOI：
10.1021/bc970088e
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发表时间：
1997-07-01
影响因子：
4.7
通讯作者：
Gruber, HJ
中科院分区：
文献类型：
--
作者：
Marek, M;Kaiser, K;Gruber, HJ
当与亲和素或链霉亲和素结合时，具有类似14个原子间隔的常规生物素-荧光团结合物被强烈猝灭，而如果典型的荧光团通过聚乙二醇链与生物素连接，则荧光变得对受体结合不敏感(Gruber等人，见本期三篇论文中的第二篇)。在本研究中，以生物素-聚乙二醇戊二烯偶合物为模型化合物，对聚乙二醇-聚乙二醇键斥力和荧光团相互作用之间的拮抗作用进行了更深入的研究。在与亲和素或链霉亲和素形成的4：1络合物中，亲水的聚乙二醇链与疏水的芘标记物之间的拮抗倾向在聚乙二醇(1900)衍生物中基本平衡，因为猝灭的程度类似于50%。相反，生物素-聚乙二醇(800)-芘结合物显示出强烈的猝灭和伴随的准分子荧光，这为准确测量亲和素和链霉亲和素浓度提供了一种新的荧光分析方法，分别大于或等于40和大于或等于10 nM。根据荧光变化分析了缔合/解离动力学，并推导出解离常数。链霉亲和素与亲和素的亲和力约为亲和素的3倍，而聚乙二醇链的长度对其影响很小。当使用生物素-聚乙二醇-四甲基罗丹明偶联物时，所有亲和力都增加了大约3倍。观察到的荧光团变化对生物素结合的影响出人意料地小；因此，动力学/热力学数据似乎总体上具有生物素-聚乙二醇偶联物的代表性。
Conventional biotin-fluorophore conjugates with similar to 14 atom spacers are strongly quenched when bound to avidin or streptavidin, whereas fluorescence becomes insensitive to receptor binding if typical fluorophores are linked to biotin via poly(ethylene glycol) (PEG) chains (Gruber et al., see the second of three papers in this issue). in the present study the antagonism between PEG-PEG repulsion and fluorophore interaction was examined more closely, using biotin-PEG-pyrene conjugates as model compounds. The antagonistic tendencies between hydrophilic PEG chains and hydrophobic pyrene labels were about balanced in the PEG(1900) derivative since quenching was similar to 50% in 4:1 complexes with avidin or streptavidin. In contrast, strong quenching and concomitant excimer fluorescence was seen with the biotin-PEG(800)-pyrene conjugate, providing for a new fluorescence assay to accurately measure avidin and streptavidin concentrations at greater than or equal to 40 and greater than or equal to 10 nM, respectively. Association/ dissociation kinetics were analyzed from pyrene fluorescence changes, and dissociation constants were deduced. About 3-fold affinities were observed for streptavidin as compared to avidin, and little influence of PEG chain length was seen. All affinities were increased by a factor of similar to 3 when biotin-PEG-tetramethylrhodamine conjugates were used. The observed effect of fluorophore variation upon biotin binding is unexpectedly small; thus, the kinetic/thermodynamic data appear to be representative for biotin-PEG conjugates in general.