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

This study provides a critical examination of protein labeling with Cy3, Cy 5, and other Cy dyes. Two alternate situations were tested. (i) Antibodies were covalently labeled with Cy dye succinimidyl ester at various fluoropho re/protein ratios and the fluorescence of the labeled antibodies was compar ed to that of free Cy dye. (ii) Fluorescent biotin derivatives were synthes ized by derivatizing ethylenediamine with one biotin and one Cy3 (or Cy5) r esidue. The fluorescence properties of these biotin-Cy dye conjugates were examined at all ligand/(strept)avidin ratios (0 less than or equal to n les s 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 bou nd biotin-Cy3 conjugates. In contrast, antibodies with six covalently bound Cy5 labels (obtained with the recommended procedure) were almost nonfluore scent, only at 2-3 Cy5 labels/IgG some moderate fluorescence was obtained. By analogy, the biotin-Cy3 conjugate fluoresced intensely, even at high lig and/avidin ratio, in contrast to the weakly fluorescing biotin-Cy5 conjugat e. Three mechanisms are responsible for the discrepancy between Cy3 and Cy5 . (i) Attachment of Cy3 to a protein's surface causes an anomalous enhancem ent 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 muc h 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 whic h is characterized by a large UV-vis absorption maximum at 600 nm instead o f at 650 nm. Cy3.5 was found to mimick the properties of Cy3, while Cy7, an d 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 la bels yield bright fluorescence while extensive quenching occurs in antibodi es labeled with Cy5 and Cy7.