Fluorescence Intensity FI

Fluorescence intensity assays detect an increase or a decrease in the strength of a fluorescence emission. Based on the origin of this change in fluorescence, the two main classes are fluorogenic assays and fluorescence quench assays. 

An example of this technology is the HIV protease assay shown in Fig. 9, which was published by Wang and co-workers [67]. The peptide substrate is labeled at the amino terminus with EDANS (5-((2 -aminoethyl)amino)naphthalene-l-sulfonic acid) as a donor fluorophore and at the carboxyl terminus with DABCYL (4-((4 -(di-methylamino)phenyl)azo)benzoic acid) as the acceptor chromophore. In the intact peptide, fluorescence resonance energy transfer (FRET) from EDANS to DABCYL results in quenching of the EDANS fluorescence. On cleavage of the peptide by HIV protease, the fluorescence of EDANS is restored. 

The quench assay described by Peppard et al. [68] to determine the activity of matrix metalloproteases (MMP) is based on a generic substrate PEGEAARK that is 

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Fluorescence titration measurements are based on the proportion of fluorescence intensity to fluorophore concentration (concentration of fluorescent species in solution this is often a fluorescent guest, G). For a 1 1 complex with host, H, with stability constant Ku = [HG]/[H] [G] the fluorescence intensity Fis given by ... 

Most biochemical assays used to determine the potency of inhibitors against (1) aminopeptidases and (2) endopeptidases with minor contributions to the substrate binding efficiency by the S site are based on the measurement of fluorescence intensity (FI). The FI readout principle is shown in Figure 2.2. The dynamic range of the FI readout basically scales with the fluorescence quantum yield, that is, the efficiency of fluorescence emission of the dye label. For the FI readout, peptide substrates with fluorogenic groups such as acetylmethoxycoumarin (AMC), 7-amino-4-trifluoromethyl... 

Fluorescence-based detection methods are the most commonly used readouts for HTS as these readouts are sensitive, usually homogeneous and can be readily miniaturised, even down to the single molecule level.7,8 Fluorescent signals can be detected by methods such as fluorescence intensity (FI), fluorescence polarisation (FP) or anisotropy (FA), fluorescence resonance energy transfer (FRET), time-resolved fluorescence resonance energy transfer (TR-FRET) and fluorescence intensity life time (FLIM). Confocal single molecule techniques such as fluorescence correlation spectroscopy (FCS) and one- or two-dimensional fluorescence intensity distribution analysis (ID FID A, 2D FIDA) have been reported but are not commonly used. 

The following sections give an overview and some examples of the fluorescence detection methods currently used in HTS fluorescence intensity (FI) fluorescence polarization (FP) or fluorescence anisotropy (FA) fluorescence resonance energy transfer (FRET) Hfetime-based measurements (TRF and FLT) fluorescence corre-... 

Figure 1 Relationships of S with interfacial tension and emulsifying activity of proteins. I, bovine serum albumin 2, /3-lactoglobulin 3. trypsin 4, ovalbumin 5, conalbuntin 6, lysozyme 7, K-casein 8, 9, I0, II, and 12, denatured ovalbumin by heating at 85°C for l, 2, 3, 4, and 5 min respectively 13, 14, 15, 16. 17, and 18. denatured lysozyme by heating at 85"C for l, 2, 3, 4, 5, and 6 min respectively 19, 20, 21, 22, and 23, ovalbumin bound with 0.2, 0.3, 1.7, 5.7, and 7.9 mol of sodium dodecyl sulfate/mol of protein respectively 24, 25, 26, 27, and 28, ovalbumin bound with 0.3, 0.9, 3.1,4.8, and 8.2 mol of linoleate/mol of protein respectively. Interfacial tension measured at corn oil/0.20c protein interface with a Fisher Surface Tensiontat Model 21. Emulsifying activity index calculated from the absorbance at 500 nm of the supernatant after centrifuging blended mixtures of 2 ml of corn oil and 6 ml of 0.5% protein in 0.01 M phosphate buffer, pH 7.4 S initial slope of fluorescence intensity (FI) vs. percent protein plot. 10 /al of 3.6 mM m-parinaric acid solution was added to 2 ml of 0.002 to 0.1% protein in 0.01 M phosphate buffer, pH 7.4, containing 0.002% SDS. FI was measured at 420 nm by exciting at 325 nm. (From Ref. 2. Reprinted by permission.)...

Fig. 2.1 Fluorescence intensity /fi(/l) as a function of incident laser intensity for linear and nonlinear absorption...

Instead of measuring the fluorescence intensity /fi(A.l), the excitation spectrum can also be monitored via resonant two-photon ionization (RTPI). This is illustrated by Fig. 4.7, which shows a RTPI spectrum of a band head of the Cs2 molecule. 

If the first laser beam is interrupted at the time to for a time interval At that is short compared to the transit time T = z2 — Z )/v (this can be realized by a Pock-els cell or a fast mechanical chopper), a pulse of molecules in level /) can pass the pump region without being depleted. Because of their velocity distribution, the different molecules reach zi at different times t = to- -T. The time-resolved detection of the fluorescence intensity /fi(0 induced by the second, noninterrupted laser beam yields the distribution n T) = n(Az/v), which can be converted by a Fourier transformation into the velocity distribution n(v). Figure 4.13 shows as an example the velocity distribution of Na atoms and Na2 molecules in a sodium beam in the intermediate range between effusive and supersonic conditions. If the molecules Na2 had been formed in the reservoir before the expansion, one would expect the relation Up(Na) = V2up(Na2) because the mass m(Na2) = 2m(Na). The result of Fig. 4.13 proves that the Na2 molecules have a larger most probable velocity Up. This implies that most of the dimers are formed during the adiabatic expansion [410]. 

Fig. 6.99 (a) Potential diagram of Nal with the pump transition at ki and the tunable probe pulse at k2(R). (b) Fluorescence intensity /fi(AO as a function of the delay time At between pump and probe pulses (curve a) with k2 tuned to the atomic Na transition and (curve b) k2 tuned to k2(R) with R < Rc [S19]... 

This implies that the fluorescence intensity /fi(i) at times t < Tc, where aU excited atoms oscillate still in phase, is N times larger than in the incoherent case (Dicke... 

If the first laser beam is interrupted at the time to for a time interval Ar that is short compared to the transit time T = z2 z )/v (this can be realized by a Pockels cell or a fast mechanical chopper), a pulse of molecules in level /) can pass the pump region without being depleted. Because of their velocity distribution, the different molecules reach Z2 at different times t to + T. The time-resolved detection of the fluorescence intensity /fi(0 induced by the second, noninterrupted laser beam yields the distribution... 

The fluorescence intensity /fi( 2) induced by the probe laser with intensity h = /2o(l +cos2 t/20 on a transition starting from the lower level /) of the pump transition is... 

Fig. 14.30. Fluorescence intensity /fi(Ao ) of five Mg" " ions as a function of laser detuning for a disordered ion cloud and for an ion crystal between the arrows) [14.88]...

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