Residual emission, decay time

Even in proteins containing a sin e tryptophan residue, multiexponential decay kinetics have been observed suggesting mobility of the protein structure during emission . However, recent time-resolved fluorescence studies of the tryptophan zwitterion and tryptophan peptides indicate that this fluorc hore does not decay... 

Temperature Dependence. The temperature dependence of the intensity at 475 n.m. at various times after irradiation out to 4 msec, are presented in Figure 7. At temperatures above 300°K. there is a decrease in the residual luminescence with increasing temperature. Below 300°K., however, the residual emission at a given time after irradiation has a maximum value at a particular temperature. This maximum occurs at lower temperatures as the observation time after irradiation is increased. All the decay curves, whether directly observed at constant temperature, or plotted at intermediate temperatures from the smoothed curves of Figure 7, are non-exponential in form and change gradually from the shape at high temperature to that at 93 °K. 

In this expression the t, values are the decay times of the individual residues and , values are the preexponential factors. The contribution of each residue to the emission is... 

To resolve the emission spectra of each residue similar data were collected at closely spaced wavelengths across the emission. The assumption was made that the decay time of each tryptophan residue was independent of emission wavelength, and constant across the emission. The measured decay law was used to calculate the individual spectra using... 

These spectra are shown in Fig. 15. The shorter-lived trp-314 (3.8 nsec) showed a shorter-wavelength emission, whereas the longer-lived trp-15 emitted at longer wavelengths. Hence, the emission spectrum and decay time of each tryptophan residue in LADH were resolved. 

Both melittin and S, Nuclease contain a single tryptophan residue. The data illustrated the point that the emission from such simple proteins can be multiexponential. Even though only a single residue is responsible for the emission, it was not possible to fit the data using a single decay time. This is shown by the failure of... 

Tune-resolved measurements are widely used in fluorescence spectroscopy, particularly for studies of biological macromolecules. This is because time-iesolved data fie-quently contain more information than is available frcnn the steady-state data. For instance, consider a protein which contains two tryptophan residues, each with a distinct 11 fetime. Because of spectral overlap of the absorption and emission, it is not usually possible to resolve the onission from the two residues. However, the tune-resolved data may reveal two decay times, which can beused to resolve the emission spectra and relative intensities of the two tryptophan residues. Then one can question how each of the tryptophan residues is affected by the interactions of the protein with its substrate or other macromolecules. Is one of the tryptophan residues close to the binding site Is a tryptophan residue in a distal domain affected by substrate binding to another domain Such questions can be answered if one measures the decay times associated with each tryptophan residue. 

Figure 17.12. Fluorescence decay of RNaseTi analyzed by using a single-exponenUal model. The points represent expeiimental data, and the solid curve represents die best single-decay-time fit RES and AC are the residuals and die auuKorrelation function, respectively, for die fit. Excitation was at 295 nra, and emission was monitored at 350 nm. The sample was in 0.05M acetate buffer, pH 5.5, ionic strength 0.5Af, 25 C. Redrawn from Ref. 50.

DAS have been determined for a number of proteins, with an emphasis on proteins which contain two tryptophan residues. In these cases one hopes that each tryptophan will display a single decay time, so that the I S represent the emission spectra of the individual residues. One example is provided by a study of yeast 3-phospho-glycerate kinase (3-FGK), which has two tryptophan residues. Rom a number of pH- and wavelengdi-dependent measurements, the 0.6-ns component in die decay was associated with one residue, and the 3.1- and 7.0-ns components were associated widi die second tryptophan residue. The wavelength-dependent intensity decays were... 

Lakowicz et al.(]7] VB) examined the intensity and anisotropy decays of the tyrosine fluorescence of oxytocin at pH 7 and 25 °C. They found that the fluorescence decay was best fit by a triple exponential having time constants of 80, 359, and 927 ps with respective amplitudes of 0.29, 0.27, and 0.43. It is difficult to compare these results with those of Ross et al,(68) because of the differences in pH (3 vs. 7) and temperature (5° vs. 25 °C). For example, whereas at pH 3 the amino terminus of oxytocin is fully protonated, at pH 7 it is partially ionized, and since the tyrosine is adjacent to the amino terminal residue, the state of ionization could affect the tyrosine emission. The anisotropy decay at 25 °C was well fit by a double exponential with rotational correlation times of 454 and 29 ps. Following the assumptions described previously for the anisotropy decay of enkephalin, the longer correlation time was ascribed to the overall rotational motion of oxytocin, and the shorter correlation time was ascribed to torsional motion of the tyrosine side chain. 

The statistics of processes such as radioactive decay and emission of light that produce a flux of particles or distributive polymerase enzymes that add residues at random to growing polymer chains obey the Poisson distribution (see Chapter 14). The number of particles measured per unit time or the number of residues added to a particular chain varies about the mean value x according to equation 6.41. 

A popular probe molecule which has b n employed for such studies is 1,6 diphenyl 1,3,5 hexatriene (DPH). This molecule has both absorption and emission along the long molecular axis and is thought to dissolve in the hydrocarbon interior. Time-resolved fluorescence depolarization studies with DPH probe molecules have been performed on the following bflayer syrtems dKdihydrosteraculoyl)pho halidyl choline dipalmitoyl phosphatidyl choline L-a-dimyristollecithin residues egg lecithin residues and mouse leukaemic L 1210cells In all reports the time-dependence of the emission anisotropy was found to decay non xponentially indkat-ing either... 

As a final example of the influence of solvent on dynamical processes in biopolymers, we examine the decay of the fluorescence emission anisotropy for Trp-62 and Trp-63 in lysozyme the model used is described in Chapt. XI.C).324 In this model the fluorescence anisotropy, r(t), as a function of time for a given residue is given by (see Eq.110)... 

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