Haemolysis of human and sheep red blood cells in glycerol media: the effect of pH and the role of band 3

Haemolysis of red blood cells (RBC) in glycerol media may be measured spect rophotometrically. The haemolytic process in a rapid phase obeys a first or der rate law. The rate constant expresses the rate of haemolysis. To gain a better understanding of the mechanism of haemolysis in glycerol media, the effects of pH and band 3 inhibitors on the rare of haemolysis in human and sheep RBC were observed. Over the pH range used (pH 5.8-10.0), the rate of haemolysis decreased with increase in pH in sheep RBC. By contrast, the ra te of haemolysis increased from pH 5.8 to 6.4 and decreased above pH 6.4 in human RBC. The different effects of pH on the rate of haemolysis are due t o inhibition of glycerol permeability by H+ in human RBC but not in sheep R BC. This is supported by the different effects of temperature and Cu2+ on t he rate of haemolysis in human and sheep RBC. We did not observe complete i nhibition of haemolysis by the classical band 3 inhibitor, 4,4'-diisothiocy anatostilbene-2,2'-disulfonic acid (DIDS). Another band 3 inhibitor 4,4'-di nitrostilbene-2,2'-disulfonic acid (DNDS) showed only weak inhibition. Phen ylgloxal (PG), another band 3 inhibitor, had no effect whatsoever on the ra te of haemolysis. These results indicate that the anion pathway of band 3 i s not the preferred route of transport of glycerol in mammalian RBC. (C) 20 00 Elsevier Science Inc. All rights reserved.