The aim of our investigation was to study the red blood cell (RBC) membrane effects of NaNO(2)-induced oxidative stress. Hyperpolarization of erythrocyte membranes and an increase in membrane rigidity have been shown as a result of RBC oxidation by sodium nitrite. These membrane changes preceded reduced glutathione depletion and were observed simultaneously with methemoglobin (metHb) formation. Changes of the glutathione pool (total and reduced glutathione, and mixed protein-glutathione disulfides) during nitrite-induced erythrocyte oxidation have been demonstrated. The rates of intracellular oxyhemoglobin and GSH oxidation highly increased as pH decreased in the range of 7.5-6.5. The activation energy of intracellular metHb formation obtained from the temperature dependence of the rate of HbO(2) oxidation in RBC was equal to 16.7+/-1.6 kJ/mol in comparison with 12.8+/-1.5 kJ/mol calculated for metHb formation in hemolysates. It was found that anion exchange protein (band 3 protein) of the erythrocyte membrane does not participate significantly in the transport of nitrite ions into the erythrocytes as band 3 inhibitors (DIDS, SITS) did not decrease the intracellular HbO(2) oxidation by extracellular nitrite.

No abstract

A method based on the measurement at room temperature of tryptophan phosphorescence (RTTP) gives the unique possibility to investigate the dynamic structure of membrane proteins without their isolation from cells. This method was used to study the influence of tert- butyl hydroperoxide (t -BHP) on Chinese hamster fibroblasts. The treatment of fibroblasts with t -BHP in a concentration range of 0. 5-2 m m for 60 min caused an increase of frequency and amplitude of membrane protein motions with lifetimes of hundreds miliseconds (a decrease of RTTP tau(2)). In parallel, cell viability was studied by trypan blue exclusion test and the content of thiobarbituric acid reactive substances was measured in cells. The dependences of the RTTP tau(2)and cell viability on t -BHP concentration were similar. Contrary to this, t -BHP did not induce the activation of lipid peroxidation processes in cells. This indicates that cell death is connected with the excessive increase of intramolecular dynamics of membrane proteins during t -BHP action.