Steady-state fluorescence polarization

W. van der Meer, R. B. van Hoeven, and W. J. van Blitterswijk, Steady-state fluorescence polarization data in membranes. Resolution into physical parameters by an extended Perrin equation for restricted rotation of fluorophores, Biochim. Biophys. Acta 854, 38-44 (1986). 

Steady-state fluorescence polarization studies have been carried out with a number of peptides, including model peptides, ACTH, glucagon, melittin, and thyrocalcitonin. This work has been reviewed 5 and will not be discussed in the present article. More recently, interesting information on the rotational behavior and structural flexibility of various peptides has been obtained from fluorescence anisotropy decay measurements. 

Table 1. Binding constants (K) for host-guest complexes of artificial receptors with 11, and microenvironmental polarity parameters ( ) and steady-state fluorescence polarization values (P) for the guest bound to hosts in aqueous solution at 30.0 °C ...

In order to evaluate microscopic viscosity around the guest incorporated into the hybrid assemblies, the steady-state fluorescence polarization measurements were performed for ANS and TNS (Table 3). The P value for ANS... 

Figure 11.3 Steady-state fluorescence polarization vs. lemperalure/viscosity ratio for Mble Zn. Data were obtained by thermal variation of temperature. Xex = 517 nm and X,-m = 600 nm.

The phosphorescence of trivalent cations (as analogues of Ca ) is also widely used in binding studies. The photobinding of phenothiazine derivatives has been studied for different types of biological membranes. The specificity of binding is low, although general, and can be used to identify and localize membrane proteins. The influence of Ca " and phase behaviour in synaptosomal lipids have been examined by the steady-state fluorescence polarization of A fluorescent probe of the tumour promoter phorbol... 

The steady-state fluorescence polarization measured for 9,10-DMA attached to PAA and PMA at degrees of neutralization (o) in the range... 

Figure 7. Steady-state fluorescence polarization for PMA and PAA with 9,10-DMA end-groups as a function of degree of neutralization (a) of the carboxylic acid groups.

Andrich, M.P., and Vanderkooi, J.M., Temperature dependence of 1,6-diphenyl-1,3,5-hexa-triene fluorescence in phospholipid artificial membranes, Biochemistry, 15, 1257, 1976. Blitterswijk, W.J.V., Hoeven, R.P.V., and Dermeer, B.W.V., Lipid structural order parameters (reciprocal of fluidity) in biomembranes derived from steady-state fluorescence polarization measurements, Biochem. Biophys. Acta, 644, 323, 1981. 

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.

Figure 8.9. Steady-state fluorescence polarization veisis temperatm / viscosity ratio for 2.6-TNS- ni acid glycoprotein cxmiplex. DSa were obtained by Ihennal variation of temperature. X u 320 nm and XcB, 430 nm. Source Albani, J. l992,Biopliys.Chem.44,129-137.

Figure 20.25. Steady-state fluorescence polarization of [Re(bGpXCX 03(4-COOHPy)i-HSA at various concentrations of IgO specific for HSA (anti-HSA ) or ncaispecific IgO 0. Revised and reprinted, with pemiNlon, frrai Ref. S3, Copyriglit C1998, American Chemical Sodety.

Absorption measurements were carried out using Hitachi 150-20 or Varian Cary Bio50 absorption spectrometers. Steady-state fluorescence measurements were recorded using Hitachi 4010,Varian Eclipse or Spex Fluorolog-2 spectrofluorimeters. Steady-state fluorescence polarization spectra were recorded using a Hitachi 4500 spectrofluorimeter equipped with a fluorescence polarization accessory, and using excitation and emission bandpass values of 3 nm. 

Fluorescence measurements. Steady-state fluorescence polarization measurements were performed with a SIi4 8000 spectrofluorimeter at 20 C. Vesicles were labelled with 1 yM DPH by incubation for 30 min in the dark, at room temperature. Data were expressed as plots of the anisotropy parameter [(ro/r - which provides a quantitative index of the environmental... 

Steady-State Fluorescence Polarization Spectroscopy as a Tool to Determine Microviscosity and Structural Order in Lipid Systems... 

Some practical applications of steady-state fluorescence polarization spectroscopy in food-related research are presented in this section. The nature of the... 

The viscosities of the fats and oils in the temperature range chosen can be determined by steady-state fluorescence polarization by measuring the anisotropy... 

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