Search

The oxidation/reduction chemistry of penicillamine in human erythrocytes was characterized directly in intact erythrocytes by 1H-NMR spectroscopy. Spectra were measured by the Carr-Purcell-Meiboom-Gill pulse sequence to selectively eliminate interfering resonances from hemoglobin and membrane protons and from the intracellular water. Glucose-free penicillamine-containing erythrocytes were subjected to oxidative stress by titration with t-butyl hydroperoxide. The t-butyl hydroperoxide rapidly crosses the erythrocyte membrane and reacts with glutathione (GSH) and penicillamine (PSH), with the PSH being oxidized to penicillamine-glutathione mixed disulfide (PSSG) as indicated by the appearance of characteristic resonances in the high resolution 1H-NMR spectrum. Extracellular PSH is oxidized to penicillamine disulfide (PSSP) when t-butyl hydroperoxide is added to the cells and thereafter in amounts dependent on the oxygenation state of the sample. Following addition of glucose, both the oxidized glutathione and the PSSG are rapidly reduced at comparable rates. The results of additional experiments using erythrocyte lysate and of kinetic experiments on solutions containing PSSG and/or GSH, NADPH and glutathione reductase suggest that the predominant mechanism for reduction of PSSG is by a thiol-disulfide exchange reaction with GSH to form PSH and GSSG, which in turn undergoes enzyme-catalyzed reduction by NADPH.

The properties of integral membrane proteins obtained by dilute alkali extraction of red cell ghosts were examined. A variety of conditions promoted the disulfide-mediated aggregation of integral membrane proteins, principally band 3. Procedural modifications which minimized aggregation were the use of EDTA and S-alkylation. Integral membrane proteins were solubilized under non-denaturing conditions using Brij 36T, a lauryl polyoxyethylene ether with an NMR-determined average chain length of 8.2 (oxyethylene) units. Detergent gel filtration revealed a chromatographic shoulder due to aggregated band 3 when membrane proteins were not alkylated. Analyses of the column profile also revealed a discrete peak for sialoglycoproteins and two phosphate peaks, an early one due to phospholipid and a later one not identified, but probably due to phosphoinositide.

The behavior of glucose-6-phosphate dehydrogenase (G6PD)-deficient red cell membrane proteins upon treatment with diamide, the thiol-oxidizing agent (Kosower, N.S. et al. (1969) Biochem. Biophys. Res. Commun. 37, 593-596), was studied with the aid of monobromobimane, a fluorescent labeling agent (Kosower, N.S., Kosower, E.M., Newton, G.L. and Ranney, H.M. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 3382-3386) convenient for following membrane thiol group status. In diamide-treated G6PD-deficient red cells (and in glucose deprived normal cells), glutathione (GSH) is oxidized to glutathione disulfide (GSSG). When cellular GSH is absent, membrane protein thiols are oxidized with the formation of intrachain and interchain disulfides. Differences in sensitivity to oxidation are found among membrane thiols. In diamide-treated normal red cells, GSH is regenerated in the presence of glucose and membrane disulfides reduced. In G6PD-deficient cells, GSSG is not reduced, and the oxidative damage (disulfide formation) in the membrane not repaired. Reduction of membrane disulfides does occur after the addition of GSH to these membranes. A direct link between the thiol status of the cell membrane and cellular GSH is thereby established. GSH serves as a reductant of membrane protein disulfides, in addition to averting membrane thiol oxidation.

Human erythrocytes were exposed to oxidative stress by iodate and periodate. Oxidation causes a time- and concentration-dependent increase in membrane permeability for hydrophilic molecules and ions. The induced leak discriminates nonelectrolytes on the basis of molecular size and exhibits a very low activation energy (Ea = 1-4 kcal.mol-1). These results are reconcilable with the formation of aqueous pores. The pore size was approximated to be between 0.45 and 0.6 nm. This increase in permeability is reversible upon treatment with dithioerythritol. Blocking of membrane thiol groups with N-ethylmaleimide protects the membranes against leak formation. The oxidation causes dithioerythritol-reversible modification of membrane proteins as indicated by the gel electrophoretic behavior. These modifications can also be suppressed by blocking the membrane thiol groups with N-ethylmaleimide. About half of the membrane methionine is oxidized to acid hydrolysis-stable derivatives. A fast saturating increase in diene conjugation was observed in whole cells but not in isolated membranes, with only minor degradation of fatty acid chains. The oxidation of cell membrane lipids as well as oxidation of cell surface carbohydrates are not involved in leak formation. Taken together with earlier data (Deuticke, B., Poser, B., Lütkemeier, P. and Haest, C.W.M. (1983) Biochim. Biophys. Acta 731, 196-210), these findings indicate that formation of disulfide bonds by different oxidative mechanisms results in leaks with similar properties.