Fluorescent labeling reagents are used to attach a fluorescent group to peptides, proteins and other biomolecules. Therefore, fluorescently labeled products can be accurately detected in very low concentrations, making them very useful for both in vitro and in vivo applications. Fluorescent labeled substrates are very useful in studying substrate receptor binding, identifying unknown receptors or identifying receptor substrates. A significant application of fluorescence-labeled compounds is fluorescence microscopy to study the location of receptors in living cells.
Fluorescent groups, or fluorophores, absorb light at a certain wavelength and reemit light at a longer wavelength. Furthermore, a fluorescent molecule absorbs a photon of light putting an electron into a higher energy level. Some of the energy is dispersed through collisions and molecular vibrations, then the molecule emits energy as a photon of light at a longer wavelength.
Fluorescent compounds are characterized by two spectra, the absorbance or excitation spectrum and the emission spectrum. The difference between the maximum of the excitation spectrum and the maximum of the emission spectrum is the Stokes shift. Generally, if the Stokes shift is large, the fluorescence can be more accurately measured than if the Stokes shift is very small.
Many fluorescent tags are commercially available. Some common ones are the fluorescene-based tags (FAM, FITC), TAMRA, Cy dyes (Cy3, Cy5), and the Alexa dyes. In addition, new fluorescent dyes are being developed to expand the color range of available dyes and to improve absorbance and emission characteristics. Table of Common Fluorescent Tags
When preparing a labelled peptide, it is crucial that the label does not interfere with the binding of the peptide to its receptor. Often a linker is utilized to provide space between the fluorescent tag and the peptide so that the tag does not sterically interfere with the peptide. Fluorescent labels are often attached to the N-terminal through amino hexanoic acid or beta-alanine. The C-terminal of a peptide can be linked to common fluorophores through linkers such as ethylene diamine. As this is often difficult to achieve through solid phase synthesis methods, it is utilized as often.
A different method of incorporating a fluorescent label at the C-terminal is to add a lysine residue with the fluorescent group attached to the side chain. This method is fully compatible with solid phase peptide synthesis. The lysine side chain also acts as a self-contained linker to provide space between the peptide and the label. This method is only useful if the addition of the lysine residue does not significantly affect the binding of the peptide.
FRET, which is an abbreviation of fluorescence resonance energy transfer or Förester resonance energy transfer, occurs when the energy from an excited fluorophore is transferred by non-radiative interactions to a nearby energy-absorbing group, resulting in quenching of the fluorophore. If the energy-absorbing group is moved farther away, the fluorescence of the fluorophore returns.
FRET is effective only at distances up to tens of nanometers, and is very useful for studying protein conformation changes and substrate binding to receptors. It also affords a highly sensitive means of studying the kinetics of proteases.
There must be significant overlap of the emission spectrum of the fluorophore, or donating group and the absorption spectrum of the energy-absorbing or acceptor group, for efficient energy transfer to occur. Additionally, the acceptor group should have little or no fluorescence, hence the acceptor group is often called a “quencher.”