Anomalous fluorescence enhancement of Cy3 and Cy3.5 versus anomalous fluorescence loss of Cy5 and Cy7 upon covalent linking to IgG and noncovalent binding to avidin
Anomalous fluorescence enhancement of Cy3 and Cy3.5 versus anomalous fluorescence loss of Cy5 and Cy7 upon covalent linking to IgG and noncovalent binding to avidin
复制标题
DOI：
10.1021/bc000015m
复制
发表时间：
2000-09-01
影响因子：
4.7
通讯作者：
Knaus, HG
中科院分区：
文献类型：
--
作者：
Gruber, HJ;Hahn, CD;Knaus, HG
这项研究提供了一个关键的检查蛋白质标记与Cy 3，Cy 5，和其他Cy染料。测试了两种替代情况。(i)用Cy染料琥珀酰亚胺酯以各种荧光团/蛋白质比率共价标记抗体，并将标记抗体的荧光与游离Cy染料的荧光进行比较。(ii)用一个生物素和一个Cy 3（或Cy 5）残基衍生乙二胺，合成了荧光生物素衍生物。在所有配体/（链霉）抗生物素蛋白比率（0小于或等于n小于或等于4）下检查这些生物素-Cy染料缀合物的荧光性质。结果显示了Cy 3和Cy 5之间惊人的差异：Cy 3标记的抗体即使在高Cy 3/蛋白质比率下也能很好地发出荧光，并且同样适用于具有多达四种结合的生物素-Cy 3缀合物的（strept）亲和素。相比之下，具有6个共价结合的Cy 5标记的抗体（用推荐的程序获得）几乎是非荧光的，仅在2-3个Cy 5标记/IgG下获得一些中等的荧光。通过类比，与弱荧光生物素-Cy 5缀合物相比，即使在高配体/抗生物素蛋白比率下，生物素-Cy 3缀合物也强烈地发荧光。三种机制负责Cy 3和Cy 5之间的差异。(i)Cy 3与蛋白质表面的连接导致荧光异常增强（2-3倍），而Cy 5没有增强。(ii)IgG结合的Cy染料通过共振能量转移的相互猝灭对于Cy 5标记比对于Cy 3显著得多。(iii)在具有六个结合的Cy 5标记的IgG中，约三分之一的标记采用非荧光状态，其特征在于在600 nm处而不是在650 nm处具有大的紫外-可见吸收最大值。发现Cy3.5模拟Cy 3的性质，而Cy 7以及在某种程度上Cy5.5与Cy 5相似。总之，Cy染料系列分为两组：具有多个Cy 3或Cy3.5标记的抗体产生明亮的荧光，而在用Cy 5和Cy 7标记的抗体中发生广泛的淬灭。
This study provides a critical examination of protein labeling with Cy3, Cy5, and other Cy dyes. Two alternate situations were tested. (i) Antibodies were covalently labeled with Cy dye succinimidyl ester at various fluorophore/protein ratios and the fluorescence of the labeled antibodies was compared to that of free Cy dye. (ii) Fluorescent biotin derivatives were synthesized by derivatizing ethylenediamine with one biotin and one Cy3 (or Cy5) residue. The fluorescence properties of these biotin-Cy dye conjugates were examined at all ligand/(strept)avidin ratios (0 less than or equal to n less than or equal to 4). The results showed an astounding discrepancy between Cy3 and Cy5: Cy3-labeled antibodies fluoresced very well, even at high Cy3/protein ratios, and the same applied to (strept)avidin with up to four bound biotin-Cy3 conjugates. In contrast, antibodies with six covalently bound Cy5 labels (obtained with the recommended procedure) were almost nonfluorescent, only at 2-3 Cy5 labels/IgG some moderate fluorescence was obtained. By analogy, the biotin-Cy3 conjugate fluoresced intensely, even at high ligand/avidin ratio, in contrast to the weakly fluorescing biotin-Cy5 conjugate. Three mechanisms are responsible for the discrepancy between Cy3 and Cy5. (i) Attachment of Cy3 to a protein's surface causes an anomalous enhancement in fluorescence (by 2-3-fold) while no enhancement occurs with Cy5. (ii) Mutual quenching of IgG-bound Cy dyes by resonance energy transfer is much more pronounced for Cy5 labels than for Cy3. (iii) In IgG with six bound Cy5 labels, about one-third of the labels adopt a nonfluorescent state which is characterized by a large UV-vis absorption maximum at 600 nm instead of at 650 nm. Cy3.5 was found to mimick the properties of Cy3, while Cy7, and to some extent also Cy5.5, were similar to Cy5. In conclusion the Cy dye series is divided into two groups: Antibodies with multiple Cy3 or Cy3.5 labels yield bright fluorescence while extensive quenching occurs in antibodies labeled with Cy5 and Cy7.