Preferential hemolysis of postnatal calf red cells induced by internal alkalinization.

Abstract

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 impermeable to sucrose and, in an isosmolar sucrose solution, undergo volume shrinkage caused by the net loss of Cl- with concomitant increase in intracellular pH. To test the potential role of intracellular pH change associated with Cl- loss in promoting hemolysis, intracellular pH was altered by: a direct addition of fixed acid or base to sucrose solution; the removal of dissolved CO2 from sucrose solution; and the addition of cells to isotonic NaHCO3 solution in the absence of sucrose. In all cases, only calf and not cow cells underwent hemolysis. Moreover, 4-acetamido-4''-isothio-cyano-2,2''-stilbene disulfonic acid, a potent anion transport inhibitor, completely protected calf cells from hemolysis and caused a nearly total inhibition of both Cl- loss and intracellular alkalinization. The hemolytic process is apparently 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 MW of 65,000 and 35,000 daltons. The intracellular alkalinization process leading to a structural instability of the membrane band 3 protein may be responsible for this calf cell hemolysis.