Stability of gels formed following coagulation of Limulus amebocyte lysate: lack of covalent crosslinking of coagulin.
Incubation of lysates prepared from amebocytes of the horseshoe crab (Limulus polyphemus) with bacterial endotoxin results in coagulation and formation of a solid gel. Although Limulus gels remained solid indefinitely, if undisturbed, they were easily disrupted by mechanical agitation. Chemical solubility studies of gelled lysates demonstrated rapid solubilization of gels in monochloroacetic acid, a property of clots that have not been covalently stabilized; but in contrast demonstrated resistance to solubilization by urea, a property of stabilized clots. Analysis of solubilized proteins by polyacrylamide gel electrophoresis in SDS demonstrated coagulin, the designation for the activated form of coagulogen (the clottable protein) that forms a gel, only in samples derived from clotted lysate that had been previously incubated with monochloroacetic acid, but not in samples following incubation with urea, confirming the results of the chemical solubility studies. Enzymatic assays for transpeptidase (Factor XIII-like) activity in either native or gelled Limulus lysates were negative. Furthermore, analysis for covalently crosslinked peptides in gelled coagulin confirmed the absence of intermolecular gamma-glutamyl-epsilon-lysyl bonds. Therefore, the stable gels formed following coagulation of Limulus lysate by bacterial endotoxin are not covalently crosslinked.
Department of Laboratory Medicine, University of California School of Medicine, San Francisco.
R I
RI
Roth
J C
JC
Chen
J
J
Levin
D000818:Animals; D001780:Blood Coagulation Tests; D055598:Chemical Phenomena; D002621:Chemistry; D004731:Endotoxins / Q000494:pharmacology; D005782:Gels; D006737:Horseshoe Crabs / Q000502:physiology*; D009391:Nephelometry and Turbidimetry; D010458:Peptidyl Transferases / Q000378:metabolism; D012995:Solubility; D013925:Thromboplastin
D004731:Endotoxins; D005782:Gels; C044101:endotoxin, Escherichia coli; D013925:Thromboplastin; D010458:Peptidyl Transferases