Red blood cells from neonatal calves, but not from adult cows, rapidly hemolyze in buffered 300 mM solutions of a variety of nonelectrolytes and
amino acids. Of these compounds,
sucrose is chosen to elucidate the mechanism by which this preferential
hemolysis takes place. As in other mammalian red cells, both calf and cow cells are found to be impermeable to
sucrose and, in an isosmolar
sucrose solution, to undergo volume shrinkage caused by the net loss of
chloride ions with concomitant increase in intracellular pH. To test the potential role of intracellular pH change associated with
chloride loss in promoting
hemolysis, intracellular pH was altered by: (a) a direct addition of fixed
acid or base to
sucrose solution; (b) the removal of dissolved CO(2) from
sucrose solution; and (c) the addition of cells to isotonic NaHCO(3)
solution in the absence of
sucrose. In all cases, only calf and not cow cells underwent
hemolysis. Moreover, 4-acetamido-4'-isothiocyano-2,2'-stilbene disulfonic
acid, a potent
anion transport inhibitor, completely protected calf cells from
hemolysis and caused a nearly total inhibition of both
chloride loss and intracellular alkalinization. Furthermore, the hemolytic process is closely related to the integrity of a
membrane protein, the
band 3 protein, which can be cleaved to varying degrees by the combined treatment of
pronase and
lipase.
Hemolysis is progressively inhibited as the
band 3 protein undergoes proteolysis, until a total inhibition of
hemolysis takes place when almost all of the
band 3 protein is digested into smaller
protein components with a mol wt of 65,000 and 35,000 daltons. These results suggest that the intracellular alkalinization process leading to a structural instability of the membrane
band 3 protein is responsible for this calf cell
hemolysis.