OXIDATION OF NEUTROPHIL GLUTATHIONE AND PROTEIN THIOLS BY MYELOPEROXIDASE-DERIVED HYPOCHLOROUS ACID

Neutrophils, when stimulated, generate reactive oxygen species includi ng myeloperoxidase-derived HOCl. There is an associated decrease in re duced glutathione (GSH) concentration. We have shown that neutrophil G SH levels decrease on exposure to reagent HOCl, whereas the equivalent concentration of H2O2 had no effect. GSH loss occurred without cell l ysis, was not reversible, and was accompanied by the loss of an equiva lent proportion of the total protein thiols. No glutathione disulphide was formed. Studies with S-35-labelled cells indicated that much of t he GSH lost was accounted for by mixed disulphides with protein and a product that co-migrated on HPLC with a novel compound formed in the r eaction of HOCl and pure GSH. The properties of this compound are cons istent with an intramolecular sulphonamide. Neutrophils stimulated wit h PMA lost 30-40 % of their GSH and a similar proportion of protein th iols. Little glutathione disulphide was formed and the products were t he same as seen with HOCl-treated cells. From these results and studie s with inhibitors and scavengers, we conclude that HOCl was responsibl e for the GSH loss. Propargylglycine and buthionine sulphoximine, inhi bitors of glutathione synthesis, enhanced GSH loss, but their effects were due to the production of long-lived chloramines that oxidized GSH with greater efficiency than HOCl, rather than to the inhibition of G SH synthesis. The lack of thiol selectivity by HOCl and irreversibilit y of oxidation means that GSH will provide limited antioxidant protect ion for thiol enzymes in stimulated neutrophils.