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Excitation E0 Molecular energy levels Excitation radiation is normally scanned into the spectrophotometer through the u.v. visible region (>200 nm preferable) and the emission spectrum (at lower wavelength) recorded Emission
Detector
Spectral output
Imposed (Extrinsic) fluorescence may be introduced when a protein is modified with a fluorescent reagent known as a fluorophore (fluor) this is useful as long as the fluor may be introduced in a unique location adequately responds to the D-R interaction does not affect the binding affinity of the drug with the receptor
CO2H
O O
N
CO2H
Fluorescent Fluoroscein
C S
IF
EX
.
EM max
IF
IF = 2.303QIocl
concentration
X R
Incident excitation photons Emission photon Q = no.of photons emitted no. of photons absorbed changes in the environment / binding of the fluor changes Q
X R
Incident excitation photons Emission photon Fluor in a polar environment shows a shift to lower wavelength with an increase in Q as environment becomes less polar Temperature effect in polar environments Q decreases with increasing temperature non polar environment very little change
I = 1 + K D [Q ] IF
Fluoresence intensity in the presence of quencher Quenching constant
o F
A linear plot (intensity ratio vs. [Q] ) is generally indicative of a single class of fluor. If two fluor populations are present with differing accessibilities the plots deviate towards the abscissa.
C IF IF P= C IF + IF
Intensities found with the polarising filter parallel to the analyser, or perpendicular, vary. These are a function of the ability of the fluor to rotate (see arrow) at the molecular level within the time course of the experiment.
Detector
Spectral output