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Purification of M5, a fibrinolytic proteinase from Crotalus molossus molossus venom that attacks complement
1Department of Biological Sciences, University of Texas at El Paso 79968-0519, USA.
Abstract:
Crotalus molossus molossus (northern blacktailed rattlesnake) venom contains agents that affect blood coagulation. A fibrin(ogen)olytic proteinase, called M5, was isolated and purified from this venom by ion exchange chromatography in a two-step procedure. M5 consists of a single non-glycosylated polypeptide chain with a molecular weight of 25 kDa and an isoelectric point of 7.6. It hydrolyses the A alpha and B beta chains of fibrinogen and the alpha and beta chains of fibrin. It also exhibits caseinolytic activity, but has no effect on synthetic substrates cleaved by thrombin, plasmin, kallikrein, or trypsin. The proteolytic activity of the enzyme against fibrinogen, fibrin, and casein is inhibited by ethylenediaminetetraacetic acid (EDTA) and the loss of activity by EDTA treatment can be prevented by addition of Zn2+. This suggests that M5 is a zinc metalloproteinase. M5, at doses of 50 micrograms and higher, induces significant hemorrhage when injected subcutaneously into mice. In addition, it inactivates guinea-pig complement in a dose-dependent fashion and hydrolyses human C2, C3, and C4.
Insights
Northern blacktailed rattlesnake venom contains a proteinase, M5, that degrades fibrinogen and fibrin, impacting blood coagulation. This zinc metalloproteinase also causes hemorrhage and inactivates complement in mice.
Area of Science:
- Biochemistry
- Toxicology
- Molecular Biology
Background:
- Snake venoms are complex mixtures of biologically active molecules.
- Proteolytic enzymes in venom can affect hemostasis and immune functions.
- Crotalus molossus molossus venom's specific components affecting coagulation are not fully characterized.
Purpose of the Study:
- To isolate and characterize a fibrin(ogen)olytic proteinase from Crotalus molossus molossus venom.
- To investigate the enzymatic activity and biochemical properties of the isolated proteinase.
- To assess the in vivo effects of the proteinase, including hemorrhage and complement inactivation.
Main Methods:
- Ion exchange chromatography was used for protein isolation and purification.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) determined molecular weight.
- Enzyme activity assays were performed using fibrinogen, fibrin, and casein as substrates.
- Complement inactivation assays were conducted using guinea-pig complement.
- Hemorrhagic activity was assessed in mice.
Main Results:
- A single-chain proteinase, designated M5, was purified with a molecular weight of 25 kDa.
- M5 hydrolyzed both fibrinogen and fibrin A alpha/B beta and alpha/beta chains.
- The enzyme exhibited caseinolytic activity but not activity against synthetic substrates for thrombin, plasmin, kallikrein, or trypsin.
- M5 was identified as a zinc metalloproteinase, inhibited by EDTA and reactivated by Zn2+.
- Subcutaneous injection of M5 induced significant hemorrhage in mice.
- M5 dose-dependently inactivated guinea-pig complement and hydrolyzed human C2, C3, and C4.
Conclusions:
- M5 is a novel zinc metalloproteinase from Crotalus molossus molossus venom with potent fibrin(ogen)olytic and hemorrhagic activities.
- The enzyme's ability to degrade complement components suggests a role in venom's immune evasion strategies.
- M5 represents a significant toxic component of this rattlesnake's venom with implications for understanding venom-induced coagulopathy and immune modulation.