Fluorophores in Enzymes

Intrinsic –  Phe, Tyr, Trp

Extrinsic – noncovalent and covalently attached probes (examples ANS, EDANS, IANBD)


Power of the method comes from the sensitivity of many fluorophores to the local environment

For example -  Trp emission dependence on environment   - change in ANS fluorescence


Both intrinsic and extrinsic fluorophores and be used to monitor folding, denaturation, substrate binding, and conformational changes - for example see

example: methods and uses of thiol reactive probes


- Any decrease in fluorescence emission is called quenching

Quenching can occur by various methods

• collisional quenching

• when emission intensity is excited stated is deactivated by collision with another molecule
• I-, Cs+, acrylamide
• quenching is  dependent on
• the concentration of the chemical quencher [Q]
• accessibility of the fluorophore
• used to
• monitor the location and environment of a fluorophore in enzymes (ie location of a probe)
• follow protein folding and conformational changes

• Static quenching

• when a another molecule forms a nonfluorescent complex with the fluorophore

• FRET - florescence resonance energy transfer

• occurs when the emission spectrum of the fluorophore (donor) overlaps with the absorption spectrum of a quencher molecule (acceptor)
• note: it is not direct absorption of emitted light by the donor
• no intermediate photon
• it is a direct coupling of the donor and acceptor energy states
• quenching is  dependent on
• distance between the acceptor and donor
• extent of spectral overlap
• used to
• measure distances between a donor and acceptor (spectroscopic ruler)
• interaction between a ligand and protein
• interaction between two proteins (acceptor attached to one, donor to the other)
• conformational changes (in which distance between the pairs change)
• peptidase activity (as in our  FRET peptides )