Isolation and characterization of fibrinogenase from western diamondback rattlesnake venom and its comparison to the thrombin-like enzyme, crotalase

Abstract

1. 1. A new type of fibrinogenase was isolated from the venom of the western diamondback rattlesnake ( Crotalus atrox). Unlike thrombin, the newly isolated fibrinogenase did not cause formation of a fibrin clot. Various properties of the fibrinogenase we isolated were compared with crotalase isolated from the venom of C. adamanteus. It was found that fibrinogenase has considerable similarity to crotalase isolated by Markland and Damus in 1971. Crotalase is a thrombin-like enzyme and produces a fibrin clot from fibrinogen. 2. 2. The Aα chain of fibrinogen was first split and the Bβ chain was cleaved later. The fact that no fibrin clot forms indicates that the cleavage sites in Aα and Bβ chains of fibrinogen must be different from thrombin sites. 3. 3. The fibrinogenase also released bradykinin by interacting with plasma proteins. It hydrolyzed TAME ( p- toluenesulfonyl- l-arginine methyl ester), BAEE ( N- benzoyl- l-arginine ethyl ester). TLME ( N-tosyllysine methyl ester) but not BAA ( N-benzoylarginine amide), TAA ( N-tosylarginineamide) or ATEE ( N-acetyltyrosine ethyl ester). 4. 4. The enzyme is an acidic protein with pI of 4.6 and a mol. wt of 31,000. It consists of 272 total amino acid residues, 21% of which are acidic amino acids. Fibrinogenase is a specific form of protease. A newly liberated amino group after hydrolysis of dimethyl-casein can be detected by the reagent trinitrobenzenesulfonic acid (TNBS). 5. 5. Fibrinogenase differs from trypsin as the soybean trypsin inhibitor does not inhibit the enzyme's action. Diisopropylfluorophosphate (DFP) inhibits the enzyme, suggesting that the serine hydroxyl group is important for enzyme activity. 6. 6. The enzyme is not lethal at 15 μg/g in mice and has no hemorrhagic activity, yet capillary permeability increases as shown by use of Evans blue dye.