[20] Limulus Clotting Factor C: Lipopolysaccharide-Sensitive Serine Protease Zymogen

This chapter describes the lipopolysaccharide-sensitive serine protease zymogen. The chapter also mentions the purification and biochemical properties of limulus clotting factor C. The bacterial endotoxin lipopolysaccharide (LPS)-induced clotting phenomenon of the hemocyte lysate from horseshoe crab is thought to be a defense mechanism that serves to immobilize invading gram-negative bacteria. Because of this specific property and its extreme sensitivity to endotoxins, Limulus hemolymph has been applied in the past decade to estimate or quantify nanogram quantities of endotoxins in human body fluids and pharmaceutical products. The amidase activity of factor C after activation in the presence of LPS is measured using Boc-Val-Pro-Arg-p-nitroanilide (pNA) as the substrate. On sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) the purified zymogen gives a single band before reduction and two protein bands after reduction by 2-mercaptoethanol suggesting that zymogen factor C consists of two polypeptide chains connected by a disulfide linkage(s). The zymogen factor C preparation does not exhibit any amidase activity toward either Boc-Leu-Gly-ArgpNA or Boc-Val-Pro-Arg-pNA.