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
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- 作者：Hermann J. Gruber ; Christoph D. Hahn ; Gerald Kada ; Christian K. Riener ; Gregory S. Harms ; Werner Ahrer ; Thomas G. Dax ; and Hans-Gü ; nther Knaus
- 刊名：Bioconjugate Chemistry
- 出版年：2000
- 出版时间：September 2000
- 年：2000
- 卷：11
- 期：5
- 页码：696 - 704
- 全文大小：155K
- 年卷期：v.11,no.5(September 2000)
- ISSN：1520-4812
文摘
This study provides a critical examination of protein labeling with Cy3, Cy5, and other Cy dyes. Twoalternate situations were tested. (i) Antibodies were covalently labeled with Cy dye succinimidyl esterat various fluorophore/protein ratios and the fluorescence of the labeled antibodies was compared tothat of free Cy dye. (ii) Fluorescent biotin derivatives were synthesized by derivatizing ethylenediaminewith one biotin and one Cy3 (or Cy5) residue. The fluorescence properties of these biotin-Cy dyeconjugates were examined at all ligand/(strept)avidin ratios (0 
n 4). The results showed anastounding discrepancy between Cy3 and Cy5: Cy3-labeled antibodies fluoresced very well, even athigh Cy3/protein ratios, and the same applied to (strept)avidin with up to four bound biotin-Cy3conjugates. In contrast, antibodies with six covalently bound Cy5 labels (obtained with therecommended procedure) were almost nonfluorescent, only at 2-3 Cy5 labels/IgG some moderatefluorescence was obtained. By analogy, the biotin-Cy3 conjugate fluoresced intensely, even at highligand/avidin ratio, in contrast to the weakly fluorescing biotin-Cy5 conjugate. Three mechanismsare responsible for the discrepancy between Cy3 and Cy5. (i) Attachment of Cy3 to a protein's surfacecauses an anomalous enhancement in fluorescence (by 2-3-fold) while no enhancement occurs withCy5. (ii) Mutual quenching of IgG-bound Cy dyes by resonance energy transfer is much morepronounced for Cy5 labels than for Cy3. (iii) In IgG with six bound Cy5 labels, about one-third of thelabels adopt a nonfluorescent state which is characterized by a large UV-vis absorption maximum at600 nm instead of at 650 nm. Cy3.5 was found to mimick the properties of Cy3, while Cy7, and tosome extent also Cy5.5, were similar to Cy5. In conclusion the Cy dye series is divided into twogroups: Antibodies with multiple Cy3 or Cy3.5 labels yield bright fluorescence while extensivequenching occurs in antibodies labeled with Cy5 and Cy7.
n 4). The results showed anastounding discrepancy between Cy3 and Cy5: Cy3-labeled antibodies fluoresced very well, even athigh Cy3/protein ratios, and the same applied to (strept)avidin with up to four bound biotin-Cy3conjugates. In contrast, antibodies with six covalently bound Cy5 labels (obtained with therecommended procedure) were almost nonfluorescent, only at 2-3 Cy5 labels/IgG some moderatefluorescence was obtained. By analogy, the biotin-Cy3 conjugate fluoresced intensely, even at highligand/avidin ratio, in contrast to the weakly fluorescing biotin-Cy5 conjugate. Three mechanismsare responsible for the discrepancy between Cy3 and Cy5. (i) Attachment of Cy3 to a protein's surfacecauses an anomalous enhancement in fluorescence (by 2-3-fold) while no enhancement occurs withCy5. (ii) Mutual quenching of IgG-bound Cy dyes by resonance energy transfer is much morepronounced for Cy5 labels than for Cy3. (iii) In IgG with six bound Cy5 labels, about one-third of thelabels adopt a nonfluorescent state which is characterized by a large UV-vis absorption maximum at600 nm instead of at 650 nm. Cy3.5 was found to mimick the properties of Cy3, while Cy7, and tosome extent also Cy5.5, were similar to Cy5. In conclusion the Cy dye series is divided into twogroups: Antibodies with multiple Cy3 or Cy3.5 labels yield bright fluorescence while extensivequenching occurs in antibodies labeled with Cy5 and Cy7.