The fluorescence result (eg: binding constant) at two different excitation wavelength 295 and 280 nm should be same or may be different like at exc wavelength 280 nm Kb is in the order of ~104 where at 295nm its ~103 .How to explain?
Hello Samima,
The major source of fluorescence in this wavelength region originates from tryptophan. Its emission is unusually complex arising from two excited states, the 1La and the 1Lb state. The 1La state is very sensitive to the local environment and can have a significant solvent induced stokes shift. This stokes shift would affect emission intensity differently across this wavelength region. Might I suggest, that the binding site is near enough to the tryptophanyl residue that binding affects the organization of solvent molecules or polar residues about the tryptophanyl residue. Please consult the text:
"Principles of Fluorescence Spectroscopy", by Lakowicz
Hi Samima, fluorescence is about the least reliable technique to monitor protein-protein binding constants.
There are so many things that need to be considered.
I strongly suggest that you consider other techniques to measure protein binding constants.
Hi Dr.
Fluorescence is a member of the ubiquitous luminescence family of processes in which susceptible molecules emit light from electronically excited states created by either a physical (for example, absorption of light), mechanical (friction), or chemical mechanism. Generation of luminescence through excitation of a molecule by ultraviolet or visible light photons is a phenomenon termed photoluminescence, which is formally divided into two categories,fluorescence and phosphorescence, depending upon the electronic configuration of the excited state and the emission pathway. Fluorescence is the property of some atoms and molecules to absorb light at a particular wavelength and to subsequently emit light of longer wavelength after
a brief interval, termed the fluorescence lifetime. The process of phosphorescence occurs in a manner similar to fluorescence, but with a much longer excited state lifetime.
At 280 nm tryrosine has a very high quantum yield and proteins generally do have more tyrosine than tryptophan. Check how many Trps and Tyrs your protein have and if possible look into the crystal structure to identify their locations. As protein do generally have more tyrs, at 280 tyrosine fluorescence contamination would be more. You should try to excite a single fluorophore while studying the binding constants. At 295 tyrosine barely emits.
Also look into the residual fluorescence. At 280 excitation what is the maximum quenchable fluorescence (i.e. Delta F_max). If the residual fluorescence is more, that means some of the fluorophores are not accessible and may be far away form the binding site. Go with the result where the residual fluorescence is less.
Finally, the values you mentioned are not called binding constants. If P+Q=PQ, Equilibrium constant is defined as Keq=[PQ]/[P][Q] with the dimension of M-1, whereas binding/dissociation constant is defined as Kd=[P][Q]/[PQ] with the dimension of M. In such binding studies equilibrium constants are also called affinity constant and written as Ka which is generally in the range of 103--105 M-1.
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