engleski

The EPR investigation of membrane fluidity upon external oxidative stimulus

Reactive oxygen species (ROS) appear in vivo and are involved in physiological signaling. However, the production of very high ROS levels can be triggered under various pathophysiological conditions causing the loss of structural and functional capacity of living cell. The free radical mediated membrane damage has been postulated in the oxidative stress and proposed as a key feature in different disorders. Concomitantly, antioxidative species/compounds has been suggested. In this contribution electron paramagnetic resonance (EPR) was applied to study the changes in human sperm fluidity upon oxidation induced by the iron/ascorbate treatment. Two different stearic acid nitroxides (NS) were used to label the plasma membrane: 5-NS, whose paramagnetic center resides near the lipid-water interface, and 13-NS, in which the nitroxide moiety localizes deep in the lipid bilayer (1). The spin labeling was performed after the membrane has been oxidised. The experimental EPR spectra reveal the increase in the maximal hyperfine splitting of 5-NS upon the oxidation. On the other hand, no difference in the hyperfine splitting of 13-NS between the contol and oxidised sample could be traced. Based on the previous study of the protective role of gangliosides in the oxidation process (2) the cells were preincubated with gangliosides (mixture of bovine brain gangliosides and individual ganglioside, GT1b). The results indicate that oxidation induced spectral changes of 5-NS are diminished in the presence of gangliosides. In order to understand the observed changes in the experimental EPR spectra the theoretical simulation was performed assuming a fast nitroxide rotation about the normal to the membrane surface. The comparison of the calculated and experimental data indicate that 5-NS nitroxide motional restrictions (two spectral components with different order parameter). Upon oxidation of the cell membrane the contribution of the spectral component with a smaller order parameter decreases accompanied with the increase of the rotational correlation time what is effectively detected as an increase of the apparent maximal hyperfine splitting. In the presence of gangliosides the oxidation induced changes of the spectral component assigned to the more fluid domain (smaler order parameter) proceed slower. (1) F.R. Ochsendorf, D.Rinne, J.Fuchs, P.Such, G. Zimmer, Andrologia 32, 169, (2000) (2) M. Kveder, G. Pifat, M. Gavella, V. Lipovac, Biophys. Chem. 104, 45-54, (2003)

EPR; membrane fluidity; ROS; gangliosides

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