Role of divalent metal ions in serum complement activity of the American alligator (Alligator mississippiensis)

Treatment of alligator serum with different concentrations of EDTA resulted in a concentration-dependent inhibition of serum-mediated sheep red blood cell (SRBC) hemolysis. This inhibition of serum-dependent hemolysis was observed for other chelators of divalent metal ions, such as phosphate and citrate. Treatment of alligator serum with 5 mM EDTA completely inhibited SRBC hemolysis, which could be totally restored by the addition of 5 mM Ca(2+) or Mg(2+), but not Cu(2+) or Ba(2+). These data indicate a specific need for Ca(2+) and/or Mg(2+) in the serum-mediated hemolysis of SRBCs. Kinetic analyses revealed that the addition of 30 mM EDTA 1 min after incubation of SRBCs with serum resulted in only 30% inhibition of hemolytic activity. However, addition of EDTA as early as 3 min post-incubation resulted in complete SRBC hemolysis. Pretreatment of serum with EDTA inhibited the hemolytic activity, but the activity could be restored in a time-dependent manner by the addition of Ca(2+)or Mg(2+). These data indicate that, as in human serum, the need for divalent metal ions occurs early in the alligator serum complement cascade.