1H-nuclear magnetic resonance study of the oxidation/reduction chemistry of penicillamine in intact human erythrocytes.

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.