ABSTRACT
The influence of the As compounds on the physical properties of IUM was examined by fluorescence spectroscopy using DPH and laurdan (Molecular Probe, Eugene, OR, USA) fluorescent probes. DPH is widely used as a probe for the hydrophobic regions of the membrane phospholipid bilayer because of its favorable spectral properties. Their steady-state fluorescence anisotropy measurements were used to investigate the structural properties of IUM as it provides a measure of the rotational diffusion of the fluorophor, restricted within a certain region such as a cone due to the lipid acyl chain packing order. Laurdan, an amphiphilic probe, has high sensitivity of its excitation and emission spectra to the physical state of membranes. With the fluorescent moiety within a shallow position in the
bilayer, laurdan provides information about the polarity and/or molecular dynamics at the phospholipid glycerol backbone level. Quantification of the laurdan fluorescence spectral shift was effected by means of the generalized polarization (GP) concept (Parasassi and Gratton 1995). Erythrocytes were separated from heparinized venous blood samples obtained from normal casual donors by centrifugation and washing procedures. IUM were prepared by lysis, according to Dodge et al. (1963). DPH and laurdan were incorporated into IUM by addition of 2 µL mL−1 aliquots of 0.5 mM solutions of the probe in dimethylformamide and ethanol, respectively, in order to obtain final analytical concentrations of 1 µM, and incubated them at 37°C for 45 min. Fluorescence spectra and anisotropy measurements were performed in a phase shift and modulation K2 steady-state and time resolved spectrofluorometer (ISS Inc., Champaign, IL, USA) interfaced to computer. Software from ISS was used for both data collection and analysis. IUM measurements were made at 37°C using 10 mm path-length square quartz cuvettes. Sample temperature was controlled by an external bath circulator (ColeParmer, Chicago, IL, USA) and monitored before and after each measurement by means of an Omega digital thermometer (Omega Engineering Inc., Stanford, CT, USA). Anisotropy measurements were made in the L configuration using Glan Thompson prism polarizers (I.S.S.) in both exciting and emitting beams. Emission was measured through a WG-420 Schott high-pass filter (Schott WG-420, Mainz, Germany) with negligible fluorescence. DPH fluorescence anisotropy (r) was calculated according to the definition: r = (I|| − I⊥) / (I|| + 2I⊥), where I|| and I⊥ are the corresponding parallel and perpendicular emission fluorescence intensities with respect to the vertically polarized excitation light (Lakowicz 1999). Laurdan fluorescence spectral shifts were quantitatively evaluated using the GP concept (see above) which is defined by the expression GP = (Ib − Ir) / (Ib + Ir), where Ib and Ir are the emission intensities at the blue and red edges of the emission spectrum, respectively. These intensities have been measured at the emission wavelengths of 440 and 490 nm, which correspond to the emission maxima of laurdan in both gel and liquid crystalline phases, respectively (Parasassi et al. 1990). Each one of the As compounds was incorporated in IUM suspensions by addition of adequate (10 mM) aliquots of the corresponding solution in order to obtain the different concentrations used in this work. Samples thus prepared were then incubated at 37°C, for ca. 15 min and measured at 37°C because that is the normal temperature at which erythrocytes circulate in humans. Blank subtraction was performed in all measurements using unlabeled samples without probes. Data presented in the corresponding figures represent mean values and standard error of ten measurements in two independent samples. Unpaired Student’s t-test was used for statistical calculations.
