CHARACTERIZATION OF AMEBOCYTE COAGULOGEN FROM THE HORSESHOE CRAB (LIMULUS-POLYPHEMUS)

The hemolymph of the horseshoe crab (Limulus polyphemus) contains a single type of cell, the amebocyte. When appropriately stimulated, these cells release a soluble protein (coagulogen) that can be converted to a gel (coagulin) by the action of bacterial endotoxins. The present studies concern characterization of this protein. Upon electrophoresis under acidic conditions (unreduced) or in dodecyl sulfate-containing gels (reduced or unreduced), purified coagulogen migrated as a single band and occupied the same position as did the corresponding coagulogen band from the starting material. Dodecyl sulfate gel electrophoretic analyses of unreduced samples indicated an apparent molecular weight ranging from 21,000 to 30,000 (mean 23,500 ± 2,500) whereas reduced samples had an apparent molecular weight ranging from 11,000 to 17,000 (mean 12,400 ± 1,500). Gel sieving chromatography of coagulogen in phosphate buffer, pH 7, yielded an apparent molecular weight value of 19,000 ± 1,000. The same type of analysis in solutions containing 8 M urea yielded a value of 11,500 ± 1,000. These findings, plus NH2-terminal sequence analysis indicating a single sequence Gly-Asp-Pro-Asn-Val-Pro-, are consistent with a molecular structure comprised of two identical chains joined by non-covalent forces. Endotoxin-mediated proteolytic conversion of coagulogen to coagulin, monitored by dodecyl sulfate gel electrophoresis, was characterized by cleavage at a minimum of two sites on each chain. © 1979.