This document discusses fluorescence quenching, which decreases fluorescence intensity. There are three types of quenching: collision/dynamic, static, and apparent. Collisional quenching involves diffusion of a quencher to collide with the fluorophore during its excited state lifetime. Static quenching involves formation of a non-fluorescent complex between fluorophore and quencher. Apparent quenching is not truly quenching but involves optical effects. The Stern-Volmer equation relates fluorescence intensity in the presence and absence of quencher. Deviations from linearity of this equation can indicate a combination of static and dynamic quenching. Fluorescence quenching can provide information about fluorophore accessibility and conformational

1. Fluorescence Quenching
Any process which decreases the fluorescence intensity of a sample
Collision/
Static Apparent
Dynamic
Quenching Quenching
Quenching
Collision returns fluorophore to G.S. Binding, Optical density,
without photon emission, Complex formed is non-fluorescent turbidity, etc not
Quencher must diffuse to fluorophore useful
during lifetime of excited state. Amines, chlorinated hydrocarbons 
excited-state charge-transfer complex.
Molecular oxygen  best Fluorescence from complex is quenched in
Paramagnetic, spin-orbit coupling, polar solvents.
intersystem crossing to long-lived, easily
quenched triplet state.
Iodine, Bromine (heavy atoms) F0/F = 1 + KS[Q]  Stern-Volmer Eqn.
intercept slope
F0/F = 1 + KDτ0[Q]
F0/F
Fluorescence lifetime
KD = Kqτ0 in the absence of KS or KD
quencher
1 --
Bimolecular quenching constant
[Q]

2. Deviation from Linearity
Linearity  all fluorophores are equally accessible to quenchers
In this case, quenching is either Static or Dynamic but not both.
How do we decide which mechanism is at play?
Static – Dynamic Bend towards x-axis  Quenching
τ0/τ = 1 -- τ0/τ = F0/F starts to saturate because few of
Slope falls wit T – rises with T
Absorption spectra changes – no change
the fluorophore molecules are
inaccessible (How many Trp
residues are on the surface of
protein?)
Bend towards y-axis  Combination of Static quenching and Dynamic quenching
(second order in [Q])
F0/F = (1 + KS[Q])(1 + KD[Q])
Kapp
F0/F = 1 + Kapp[Q]
KSKD
KS + KD --
Kapp = ((F0/F) – 1) / [Q] = KS + KD + KSKD [Q]
[Q]

3. Application of Quenching
The Bimolecular Quenching constant reflects:
1. Efficiency of Quenching
2. Diffusion Coefficient of Quencher
3. Accessibility of Fluorophore to Quencher
 localization, membrane permeability
Here, we can see a change in protein
conformation due to substrate binding
because the extent of quenching changes.