Marek M, Kaiser K, Gruber H J
Institute of Biophysics, J. Kepler University, Linz, Austria.
Bioconjug Chem. 1997 Jul-Aug;8(4):560-6. doi: 10.1021/bc970088e.
Conventional biotin-fluorophore conjugates with approximately 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 PEG1900 derivative since quenching was approximately 50% in 4:1 complexes with avidin or streptavidin. In contrast, strong quenching and concomitant excimer fluorescence was seen with the biotin-PEG800-pyrene conjugate, providing for a new fluorescence assay to accurately measure avidin and streptavidin concentrations at > or = 40 and > or = 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 approximately 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.
带有约14个原子间隔基的传统生物素-荧光团共轭物与抗生物素蛋白或链霉抗生物素蛋白结合时会强烈猝灭，而如果典型的荧光团通过聚乙二醇（PEG）链与生物素相连，荧光对受体结合则不敏感（格鲁伯等人，见本期三篇论文中的第二篇）。在本研究中，以生物素-PEG-芘共轭物作为模型化合物，更深入地研究了PEG-PEG排斥与荧光团相互作用之间的拮抗作用。在PEG1900衍生物中，亲水性PEG链与疏水性芘标记之间的拮抗趋势大致平衡，因为在与抗生物素蛋白或链霉抗生物素蛋白形成的4:1复合物中猝灭约为50%。相比之下，生物素-PEG800-芘共轭物出现了强烈猝灭并伴随准分子荧光，从而提供了一种新的荧光测定法，可分别准确测量抗生物素蛋白浓度≥40 nM和链霉抗生物素蛋白浓度≥10 nM。根据芘荧光变化分析了缔合/解离动力学，并推导了解离常数。与抗生物素蛋白相比，链霉抗生物素蛋白的亲和力约高3倍，且几乎未见PEG链长度的影响。当使用生物素-PEG-四甲基罗丹明共轭物时，所有亲和力均增加了约3倍。荧光团变化对生物素结合的影响出乎意料地小；因此，动力学/热力学数据似乎总体上代表了生物素-PEG共轭物。
