COMPARISON OF HUMAN RED-CELL LYSIS BY HYPOCHLOROUS AND HYPOBROMOUS ACIDS - INSIGHTS INTO THE MECHANISM OF LYSIS

Human red blood cells are lysed by the neutrophil-derived oxidant hypo chlorous acid (HOCI), although the mechanism of lysis is unknown. Hypo bromous acid (HOBr), a similarly reactive oxidant, lysed red cells app rox. 10-fold faster than HOCI. Therefore we compared the effects of th ese oxidants on thiols, membrane lipids and proteins to determine whic h reactions are associated with lysis. There was no difference in the loss of reduced glutathione or membrane thiols with either oxidant, bu t HOBr reacted more readily with membrane lipids and proteins. Bromohy drin derivatives of phospholipids and cholesterol were seen at approx. one-tenth the level of oxidant than chlorohydrins were. However, thes e products were detected only with high concentrations of HOC1 or HOBr , which caused instant haemolysis. Membrane protein modification occur red at much lower doses of oxidant and was more closely correlated wit h lysis. SDS/PAGE analysis showed that band 3, the anion transport pro tein, was lost at the lowest dose of HOBr and at the higher concentrat ions of HOCI. Labelling the red cells with eosin 5-maleimide, a fluore scent label for band 3, suggested possible clustering of this protein in oxidant-exposed cells. There was also irreversible cross-linking of all the major membrane proteins; this reaction occurred more readily with HOBr. The results indicate that membrane protein modification is the reaction responsible for HOCl-mediated lysis. These effects, and p articularly cross-link formation, might result in clustering of band 3 and other membrane and cytoskeletal proteins to form haemolytic pores .