On the metabolism of the thrombin-like enzyme from the venom of bothrops atrox

Abstract

A clot promoting enzyme has been isolated from the venom of Bothrops atrox and found to possess a thrombin-like activity. On incubation of this enzyme with normal human serum or dog serum a slow inhibition of the enzymatic activity was demonstrated. For further studies of this phenomenon the enzyme was labelled with 125I-isotope. After labelling the enzyme had lost about 70% of its original activity but on immunoelectrophoresis and on polyacrylamide gel electrophoresis the mobility was the same as for native enzyme. By means of autoradiography and immunoelectrophoresis of serum samples incubated with the labelled enzyme it was demonstrated that the precipitin are for α 2-macroglobulin contained radioactivity. Purified human α 2-macroglobulin was shown to inhibit the clotting activity of the snake venom enzyme while its amidolytic activity was unaffected. Studies by means of gel filtration revealed that only 30–60% of the iodine labelled enzyme was capable of forming complex with α 2-macroglobulin. No dissociation of this complex could be demonstrated by gel filtration after incubation of isolated α 2-macroglobulin-enzyme complex for 24 hours at 37°. When the isolated complex was dialyzed against water and freeze-dried a dissociation could be demonstrated by gel filtration. When 125I-labelled enzyme was injected into two dogs 10–25% of the radioactivity was found to be associated with a plasma protein, most likely α 2-macroglobulin. A “half life” of the enzyme was estimated to 3–10 hours. The labelled enzyme was slowly excreted by the urine. The excreted enzyme had no enzymatic activity.