The effect of oxidative stress on the membrane dipole potential of human red blood cells.

The membrane dipole potential (ψ(d)) is an important biophysical determinant of membrane function and a sensitive indicator of lipid organisation. In this study we have used the environmentally sensitive probe di-8-anepps to explore the effects of oxidative stress on the membrane dipole potential of human erythrocytes. Cells suspended in 0.15mM phosphate buffered saline containing 0.1mg/ml albumin maintained a mean value for ψ(d) of 270 (±20) mV over the course of 1hour. In the presence of 0.4mM cumene hydroperoxide there was an increase in ψ(d) of 14 (±7)%, accompanied by a decrease in cell diameter of ~14 (±2)%. Exposure of the cells to 0.4mM hydrogen peroxide caused ψ(d) to decrease by 13 (±8)% at the centre of the cell and 8 (±5)% at the edge whilst the diameter remained constant. In both cases the changes were equivalent to a change in transmembrane electric field of a magnitude of ~10MVm(-1), sufficient to influence membrane function. Raman microspectrometry supported the conclusion that cumene exerts its effect primarily on membrane lipids whilst hydrogen peroxide causes the formation of spectrin-haemoglobin complexes which stiffen the membrane.