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

Merchant, M.E.; Verret, B.; Elsey, R.M.

Comparative Biochemistry and Physiology. Part B Biochemistry and Molecular Biology 141(3): 289-293


ISSN/ISBN: 1096-4959
PMID: 15922642
DOI: 10.1016/j.cbpc.2005.03.006
Accession: 012525081

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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.