Mean fluorescence lifetime

We find that the fluorescence yield of freshly prepared covalent (+)-anti-BaPDE-DNA adducts in oxygen-free solutions is 66+2 lower than the yield of the tetraol 7,8,9,10-tetrahydroxytetrahydro-benzo(a)pyrene (BaPT) in the absence of DNA. Since the fluorescence lifetime of BaPT under these conditions is 200ns, the mean fluorescence lifetime of the adducts (see reference T7) can be estimated to have a lower limit of 3ns, which is close to the mean value of 0.52x1.6 + 0.42x4.0 = 2.7 ns estimated from the two short fluorescence components of Undeman et al (10). 

The radiative lifetime rr is l/kT. It is the real emission lifetime of a photon that should be measured independently of the other processes that deactivate the molecule. However, since these processes occur in parallel to the radiative process, it appears impossible to eliminate them during radiative lifetime measurements. Therefore, we will measure a time characteristic of all deexcitation processes. This time is called the fluorescence lifetime and is lower than the radiative lifetime. A fluorophore can have one or several fluorescence lifetimes in this case, we can determine the fractional contribution of each lifetime and calculate the mean fluorescence lifetime r0 or (r) ... 

The cytochrome b2 core from the yeast Hansenula anomala has a molecular mass of 14 kDa, and its sequence shows the presence of two tryptophan residues. Their fluorescence intensity decay can be adequately described by a sum of three exponentials. Lifetimes obtained from the fitting are equal to 0.054,0.529, and 2.042 ns, with fractional intensities equal to 0.922, 0.068, and 0.010. The mean fluorescence lifetime, r0, is 0.0473 ns. 

The mean fluorescence lifetime is the second order mean ... 

Table 2.5 yields the different values of the mean fluorescence lifetime and of intensity of fluorescein in presence of increased concentrations of KI. Lifetimes were measured with both frequency domain and Time correlated single photon counting methods. Figure 2.20 displays the normalized values at different Kl concentrations. 

Wc notice that for the 6 applied frequencies, tp < tm Thus, the dcca) is heterogeneous and at least two fluorescence lifetimes exist. In fact, the dsta yield two fluorescence lifetimes 2.320 and 0.333 ns Nith fiactional uitensities of 0.9 and O.I. respectively. The mean fluorescence lifetime is equal to 2.213 ns Actually, the possibility of measuring fluorescence lifetimes in the multi frequency method allows in principle to obtain much more accurate results Oian with small numbers of frequencies. However, below one nanosecond, the higher modulation frequencies necessary to make accurate measurements require an expensive laser-based excitation source. 

Table 2.7. Position of the emission peak (in nm), fluorescence lifetimes (t,) with their fractional intensities f, (in %) and the mean fluorescence lifetime (To) of wild-type NSCP and of three double protein mutants, in the apo form and in presence of calcium and magnesium ions.

The >imax of tryptophan 57 in the Ca state is very blue-shifted (317 nm). In presence of Mg " and in the apoform, the maximum shifts to 324 and 330 nm, respectively. Although this red shift indicates a modification in the microenvironment of the Trp residue, the positions of the maximum reveal that Trp 57 is buried in the hydrophobic core of the protein. We observe also that the fractional intensities of the fluorescence lifetimes are not the same with the state of ligation. The mean fluorescence lifetime is drastically modified when the protein goes from the apo to the ligated forms. 

The fluorescence quantum yield of the protein can be estimated from the ratio Tq / tr, where Xo is the mean fluorescence lifetime equal to 1.8 ns and tr is the radiative lifetime of tyrosine equal to 2.7 ns (Lux et al. 1977 Wu et al. 1994). Of = 0.067. The fact that steady-state and lifetime measurements yield the same value of Of indicates that the three tyrosines of 029 SSB contribute to its intrinsic fluorescence. Therefore, ground-state deexcitation of the tyrosine fluorescence by the surrounding environment is weak. 

Upon complex foimaflon with a drable stranded DNA, the intensity of Tip residiies of R.2 R) decreases by about 50%. This intenaQr decrease is accompained by a shift of the flucwescence emis on ma mum from 336 to 332 tun (Fig. 4.6) and by a decrease in the mean fluorescence lifetime from 1.46 to 0.71 ns. These results indicate that Tip residues environment is not the same in presence c DNA. I lowevcr, tins does not mean necessaiify that the envirenment of all the six tiypb ihans has been modified. 

The mean fluorescence lifetime is used to calculate the rotational correlation time from the Perrin plot (quenching resolved emission anisotropy experiment). 

The mean fluorescence lifetime of the Trp residues in the apoform of HiPlP decreases compared to the holo form. This decrease is the result of the modification in the fractional contribution (f) of the two longest lifetimes to the total emission (Table 7.2). 

The fluorescence intensity decay of the two tryptophan of cytochrome bi core extracted from the yeast Hansenula anomala, can be described as the sum of three fluorescence lifetimes xi = 0.054 ns, %2 0.529 ns and T3 = 2.042 ns with fractional intensities f equal to 0.922, 0.068 and 0.010, respectively. The mean fluorescence lifetime Tq is equal to 0.04728 ns. One can notice also the presence of a very short fluorescence lifetime (54 ps) characteristic of the efficient energy transfer from the tryptophans to the heme. 

The results obtained and the conclusions drawn from the data on the Trp residues are confirmed by the fluorescence lifetimes measurements obtained on calcofluor. In fact, the mean fluorescence lifetime of calcofluor bound to the carbohydrate residues of sialylated ai-acid glycoprotein decreases from 4.8 ns to 3.9 ns in the asialylated one. The efficiency of quenching is equal to 0.1875, a value close to that (0.148) found for the Trp residues. Therefore, in presence of sialic acids, the spatial conformation of the carbohydrate residues is different from that observed in their absence. Asialylated ai-acid glycoprotein possesses the carbohydrates structure and backbone closer to the protein matrix than the sialylated form, inducing by that a decrease in the fluorescence lifetime of calcofluor as the result of the molecular interaction between the carbohydrate residues and the protein matrix. 

We measured also tlie fluorescence lifetime of protoporphyrin IX at 20°C in different solvents. The results shown in Table 9.5 indicate clearly that the fluorescence intensity decays with two lifetimes. The major component (94 % of the total fluorescence) corresponds to 17 ns. Water decreases the contribution of the larger component and increases the heterogeneity of the emission. The mean fluorescence lifetime decreases in presence of water indicating that aggregation is occurring between the porphyrin molecules. Fluorescence quenching via energy transfer and/or local dynamics induces a decrease in the mean fluorescence lifetime... 

Table 9.5. Values of the fluorescence lifetimes (ns), the corresponding fractional intensities of protoporphyrin IX measured in different solvents and the mean fluorescence lifetime Tq.

Fig. 7 Typical dependence of the mean rotation correlation time Tc and the mean fluorescence lifetime Tp of dansyl-labeled PMA on pH...

The mean fluorescence lifetime may also be determined by continuous intensity measurements, if the exciting light intensity is modulated at a high frequency. Fluorescence is excited by light modulated sinusoidally at a known frequency (ajln Hz). The emission is a forced response to the excitation, and is therefore modulated at the same frequency, but with a phase shift, due to the time-lag between absorption and emission. The intensities of the two beams are monitored by photomultipliers. The difference in phase (0) between the two intensities is determined electronically. The lifetime r is given by cox = tan<. The modulation frequency must be made comparable to the decay rate, e.g., around 30 MHz for a mean lifetime of 30 ns. Such frequencies can be achieved by using a hydrogen lamp actuated by a suitably modulated current source. Commercial equipment is available. The method has been applied to quinine sulphate, fluorescein, and acridine, for example, with a precision of 1-2%. It is especially useful for very short (sub-nanosecond) lifetimes. 

Fig. 6 The comparison of the pH dependence of mean rotation correlation times from different samples in solutions with the same ionic strength (collapsed on one master curve ) with the pH dependence of the mean fluorescence lifetime Tp for the sample, the components rpi and rp2 of which are depicted in the Fig. 5. Springer, Self Organized Nanostmctures of Amphiphilic Block Copolymers I, 241, 2011, 187-249, figure 7, [105]. Copyright 2011. With kind permission from Springer Science and Business Media...

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