Erythrocyte Membrane Fluidity Analysis

The erythrocyte membrane ordering parameter was estimated by using a steady—state fluorescent polarization technique. The suspension of erythrocytes (2 ml of 0.05% hematocrit in 0.9% NaCl) was labelled with a fluorescent probe (DPH or TMA-DPH, respectively) at a concentration of 1 μM (10 min, 37 °C). The fluorescence measurements were carried out at 37 °C using a Perkin-Elmer LS-55 (Perkin-Elmer, UK) spectrofluorometer equipped with a fluorescence polarization device. The readings were taken at intervals of 2 s. Changes in membrane fluidity after addition of tannins in the concentration range of 2–10 µM were determined based on polarization values of the samples (r).
The polarization values (r) were calculated by the fluorescence data manager program using the Jablonski equation: $$r=\frac{I_{\mathit{VV}}-G{I}_{\mathit{VH}}}{I_{\mathit{VV}}+2G{I}_{\mathit{VH}}}$$ where I_VV and I_VH are the vertical and horizontal fluorescence intensities, respectively to the vertical polarization of the excitation light beam. The factor G = I_HV/I_HH (grating correction factor) corrects the polarizing effects of the monochromator. The excitation wavelengths were 348 nm (DPH) and 340 nm (TMA-DPH) and the fluorescence emission was measured at 426 nm for DPH and 430 nm for TMA-DPH^{78 (link)}.
Based on the data obtained, the membrane ordering parameter was calculated using the equation⁴⁷ : $$S=\frac{\sqrt{\left[1-2\left(\frac{r}{r_0}\right)+5{\left(\frac{r}{r_0}\right)}^2\right]}-1+\frac{r}{r_0}}{2\left(\frac{r}{r_0}\right)}$$ where r₀ is the fluorescence anisotropy of DPH or TMA-DPH in the absence of any rotational motion of the probe. The theoretical value of r₀ of DPH and TMA-DPH is 0.4.