Perrin plot

Figure 1. Perrin plots of the two fluorophores (open circles and solid squares) in hydrophobic humic strong (a) and weak (b) adds. (Continued on next page.)...

Figure 3.40 Example of the Perrin plot of static quenching. Luminescence of a metal complex [Ru (bpy)32+] in rigid glycerol in the presence of increasing concentrations of methylviologen (quenching by electron transfer)...

Figure 9 Perrin plots for Na+-H (O) and Na+-BS (O) The surface areas for the calculation of the surface concentration are respectively 775 and 110 m2/g. The black dots are the points of Na+-H at small loadings recalculated with a surface area of 125 nr/g.

Figure 10s Perrin plot of Ca2+-L. The surface concentration is calculated with a surface area of 819...

The Perrin plot enables us to obtain information concerning fluorophore motion. When the fluorophore is tightly bound to the protein, its motion will correspond to that of the protein. In this case, r will be equal to the protein rotational correlation time p, and A0 obtained experimentally from the Perrin plot will be equal to that measured at —45°C (Figure 11.3). 

When fluorophore exhibits local motions, extrapolated value of A, A(o ), is lower than the A0 value obtained at —45°C. Information obtained from the Perrin plot slope depends on the fluorophore fluorescence lifetime and on the relative amplitudes of the fluorophore and protein motions. 

Energy transfer is a source of depolarization. For example, a high energy-transfer efficiency in hemoproteins can be evidenced by plotting Perrin plot at different temperatures. 

The main fluorescence lifetime (r=54 ps) and its important fractional intensity (fi = 92%) indicate that an important energy transfer occurs between Trp residues and heme. In an attempt to measure rotational correlation time of the protein, we have measured anisotropy of cytochrome b2 core Trp residues at different temperatures. Results are described with the classical Perrin plot (1 /A as a function of T/p) (Figure 11.4). 

The data yield a rotational correlation time equal to 38 ps instead of 5.9 ns calculated theoretically for the cytochrome b2 core, with an extrapolated value A(o) of 0.208, lower than that (0.265) usually found for Trp residues at Xex = 300 nm at —45°C. The fact that the extrapolated anisotropy is lower than the limiting anisotropy means that the system is depolarized as a result of global and local motions within the protein. In this case, the value of the apparent rotational correlation time ( a) calculated from the Perrin plot is lower than the global rotational time of the protein ( bp). However, the fact that 4>a is 1000 times lower than 4>p indicates that a third process different than the global and local rotations is... 

Plot the Perrin plot. What type of plot do you obtain and what information do you obtain ... 

The Perrin plot as a function of temperature (Figure 13.3) reveals the presence or absence of local motions of the fluorophore on the macro molecule. The absence of any residual motions yields a Perrin plot with a slope equal to that of the protein and an extrapolated polarization equal to the limiting one. 

Figure 13.3 Perrin plot of the fluorescein-LCA complex obtained at different temperatures. kex = 495 nm...

Figure 13.4 Perrin plot of the LCA-fluorescein complex at 20°C and at different sucrose concentrations. The slope of the high values gives a p of 18 ns and the other slope yields a <1>r of 72 ps.

Let us plot the Perrin plot 1/P vs. T/r] and calculate the rotational correlation time c is equal to that of the protein and P(0) to the limiting polarization, then the fluorophore is bound tightly to the protein. Otherwise, the fluorophore displays free and independent motions. 

FIGURE 2, Perrin plot of an Iq/I against concentration of cyclooctadiene (COD) for thermally oxidized poly(butadiene) in 1-1 tetrahydrofuran diethyl ether solution at 77K. Iq is intensity of phosphorescence from an ap-unsaturated carbonyl in absence of COD ... 

Figure 5.7. Steady-state fluorescence polarization versus temperature over viscosity ratio for Trp residues of human aj -acid glycoprotein prepared by acetonic precipitation. Data were obtained by thermal variations in the range 7-35" C. Xex = 300 nm. Xem = 330 nm. Protein concentration is equal to 10 pM. The rotational correlation time determined from the Perrin plot is equal to 13 ns at 20°C is in the same range as that (17 ns) expected for the protein at the same temperature, indicates the presence of residual motions. Also, the extrapolated anisotropy (0.264) is equal to that measured at -35 C (0.267). Source Albani, J. R. 1998, Spectrochimica Acta, Part A. 54, 173-183.

When the fluorophore lifetime is equal to or lower than the rotational correlation time of the protein, the extrapolated anisotropy will be lower than the limiting one and the rotational correlation time obtained from the slope of the Perrin plot will correspond to an apparent rotational correlation time segmental motion of the fluorophore (Fig. 5.8). 

When the fluotescence lifetime is much higher dian the totaticuial emulation time of the protein, the Perrin plot representation should enable us to obtain the value Smee the fluoropbore displays local motions, the extr xdated anisotropy will be lower than the limiling or intrinsic one (Fig. S 9.)... 

Fig. 5.10. Perrin plot for tiyptopliaii fluorescence of apo a-cliain (0.4 mg/ml) in 40 mM phosphate, pl-l S.3, 2S°C. The viscosity is vai ied by the addition of sucrose (0) or glycerol ( ). The polarization (P) was measured as described in the text. The excitation and emission wavelengths were 285 and 345 nm, respectively. The units of T / t) are degrees cemipoise". Source Oton J. Franchi D, Steiner R.F., Martinez, C. F. and Bucc, E. 984. Arch. Biocliem. Biophys 228.SI9-S24.

The experiment was carried out in the absence (0) or presence ( ) of 18 mM acetal phosphate (AcP). A the effect of ChcY concentration on the fluorescence anisotropy of fluorescein-labeled CheZ2l4FC (0.2 pM). B Perrin plot of tlic Huorescence anisotropy of fluorescein-labded ClieZ2l4FC. The medium viscosity was increased by sucrose up to 4.6 ePoise. In this viscosity range the change in fluorescence anisotropy reflects mainly the rotation of the whole CheZ-fluorescein conjugate and to a much lesser extent the free rotation of the probe (fluorescein). The latter rotation IS not expected to be affected by the presence of Che Y and AcP. Therefore, to a first approximation, slopes of these lines, can be taken as the inveise of... 

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

In general, when anisotropy varies as a function of quencher concentration, the Perrin plot can be written as... 

Energy transfer such as that observed between two or different chromophores, for examples between two tryptophan residues and / or from tryptophan to heme, is a source of depolarization. Energy transfer in hemoproteins between tryptophans and heme is commonly observed. The high energy transfer efficiency in hemoproteins can be put into evidence by plotting the Perrin plot by varying the polarization or the anisotropy as a function of the temperature. 

Steady-state fluorescence anisotropy of Trp residues in both preparations of ai-acid glycoprotein was performed at different temperatures. The Perrin plots (Fig. 5.7 and 5.8) reveal that Trp residues of ai-acid glycoproteiii s display free motions while those of a 1-acid glycoprotein follow the global rotation of the protein. 

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