Characterization of Fluorescent Dyes Frequently Used for Bioimaging: Photophysics and Photocatalytical Reactions with Proteins

Abstract

Derivatives of the rhodamine-based dye 5-TAMRA (5-carboxy-tetramethylrhodamine) and the indocarbocyanine-type Cy3B (cyclized derivative of the cyanine dye Cy3), both representing important fluorophores frequently used for the labeling of biomolecules (proteins, nucleic acids) and bioactive compounds, such as receptor ligands, were photophysically investigated in aqueous solution, i.e., in neat phosphate-buffered saline (PBS) and in PBS supplemented with 1 wt % bovine serum albumin (BSA). The dyes exhibit comparable absorption (λabs,max: 550–569 nm) and emission wavelengths (λem,max: 580–582 nm), and similar S1 lifetimes (2.27–2.75 ns), and their excited state deactivation proceeds mainly via the lowest excited singlet state (triplet quantum yield ca. 1%). However, the probes show marked differences with respect to their fluorescence quantum yield and photostability. While 5-TAMRA shows a lower quantum yield (37–39%) than the Cy3B derivative (ca. 57%), its photostability is considerably higher compared to Cy3B. Generally, the impact of the protein on the photophysics is low. However, on prolonged illumination, both fluorescent dyes undergo a photocatalytic reaction with tryptophan residues of BSA mediated by sensitized singlet oxygen resulting in a tryptophan photoproduct with an absorption maximum around 330 nm. The overall results of this work will assist in choosing the right dye for the labeling of bioactive compounds, and the study demonstrates that experiments performed with 5-TAMRA or Cy3B-labeled compounds in a biological environment may be influenced by photochemical modification of experimentally relevant proteins at aromatic amino acid residues.

Illuminating Neuropeptide Y Y4 Receptor Binding: Fluorescent Cyclic Peptides with Subnanomolar Binding Affinity as Novel Molecular Tools

“…Interestingly, comparison to the Cy3B derivative, which was obtained by preparation of an amide from succinimidyl ester 12 (Cy3B-SE, cf. Scheme ) and 2-aminoethanol, reveals that the peptide moiety in fluorescent ligand 16 mediates an increase in fluorescence quantum yield by ca. 10%.…”

“…As Cy3B exhibits a fluorescence lifetime (τ­(PBS) = 2.27 ns, τ­(PBS + 1% BSA) = 2.74 ns) compatible with FA measurements, the Cy3B-labeled fluorescent ligand 16 was investigated in FA-based Y 4 R binding assays. For these experiments, Y 4 R displaying budded baculovirus particles (BBVs) were used.…”

“…[ 3 H]­Propionyl-pNPY (molar activity: 1.39 TBq/mmol) was prepared according to a previously reported procedure with minor modifications that were described elsewhere . Compounds 8 and 9 , the fluorescent dyes 12 (Cy3B-SE), 14 (Py-1), and 22 (Py-5), as well as the fluorescent dummy ligands 23 and 24 were prepared according to reported procedures. Millipore water was consistently used for the preparation of stock solutions, buffers, and aqueous eluents for HPLC.…”

Design, Synthesis, and Characterization of New δ Opioid Receptor-Selective Fluorescent Probes and Applications in Single-Molecule Microscopy of Wild-Type Receptors

“…Moreover, the second generation of probes exhibited a higher affinity and selectivity for μOR, presumably due to the longer distance between the two parts of the molecule which should result in fewer interactions between the fluorophore and the binding site. , Based on the above, we set out to develop δOR probes that would be suitable for cutting-edge microscopy methods. Cyanine 5 (Cy5) and cyanine 3 (Cy3) are established fluorophores and are used in many state-of-the-art assays. − There have been recent reports indicating that the photophysics of cyanine dyes is more complex than previously thought, including lower photostability for dyes bearing an increased polymethine chain, photooxidation leading to sensitivity in oxygen, and FRET fluctuations due to cyanine acceptor blinking. − Nonetheless, the Cy5 and Cy3 dyes have been successfully used in many newly developed fluorescent probes for the ORs, while they can also be employed as a FRET pair in relevant studies. − ,,,,, …”