THE ISOLATION AND CHARACTERIZATION OF THREE BASIC PROTEINASES IN THE VENOM OF Bothrops moojeni

THESIS: S. M. de T. Serrano submitted this dissertation for her Master of Sciences degree in Molecular Biology publicly examined at the Escola Paulista School of Medicine – UNIFESP – São Paulo, SP, Brazil in 1991.

Advisor: Prof. Dr. Claudio A. M. Sampaio

ABSTRACT. Three basic proteolytic enzymes were isolated from the venom of Bothrops moojeni. They were isolated by gel filtration on Sephadex G-100, ion-exchange on DEAE-Sephacel followed by SP-Sephadex C-50, and gel filtration on Superose 12. Moojeni proteinase B (MPB) is a metalloproteinase active on casein. Its specific activity is similar to the crude venom and it is inhibited by EDTA and DTT. Moojeni serine proteinase 1 (MSP 1) and moojeni serine proteinase 2 (MSP 2) show esterolytic activity on N-alpha-tosyl-L-arginine methyl ester (TAME), respectively 6 and 33 times higher than that of the crude venom. Both enzymes are inhibited by phenylmethylsulfonyl fluoride (PMSF). The three proteinases do not show any of the other activities present in the crude venom, but still exhibit traces of coagulant activity.

The three proteinases are glycoproteins and show single broad protein bands on acidic polyacrylamide gel electrophoresis (pH 4.3). Isoelectrophocusing also shows broad bands which only allow the detection of pIs higher than 7.5. Under reducing conditions, the proteinases show bands on SDS-polyacrylamide gel electrophoresis corresponding to molecular weights of 65,000 and 55,000 for MPB, 34,000 and 32,500 for MSP 1, 38,000, 23,000 and 12,000 for MSP 2. MSP 1 and MSP 2 undergo autolysis with partial loss of enzymatic activity, suggesting that originally both contained only the polypeptide chains of molecular masses higher than 30,000.

According to kinetic parameters determined for MSP 1 and MSP 2 on several chromogenic peptides, the most susceptible for the amidolytic activity of both enzymes are the substrates for glandular kallikrein (D-Val-Leu-Arg-pNA) and thrombin (D-Phe-Pipecolyl-Arg-pNA).

MSP 1, unlike MSP 2, shows the property of aggregating platelets. MSP 1 not only induces platelet aggregation on platelet rich plasma (PRP) at a concentration of 10 M but also enhances platelet aggregation induced by ADP. The aggregating activity of MSP 1 is eliminated by PMSF and not by hirudin. The aggregating potency of MSP 1 on washed platelet suspensions is similar to that of thrombin.

Differences in hydrolytic specificity between the three proteinases are detected with the proteins fibrinogen, fibrin, type I collagen, and fibronectin. The fibrinogen molecule is degraded by the three enzymes, MPB being the most active. Alpha chain is easily degraded; beta and gamma chains are only degraded after prolonged incubation. Nevertheless, the action of the three proteinases was similar on fibrin. Only alpha chain and alpha-polymer are degraded. The proteinases digest type I collagen in a different manner. While collagen is easily and completely digested by MSP 1 giving products with Mr ranging from 80,000 to 10,000, MSP 2 causes limited degradation with products of Mr 100,000 to 80,000. Type I collagen is more resistant to the action of MPB. MSP 1 and MSP 2 cause similar limited degration on fibronectin with the removal of a peptide of Mr 10,000. In contrast, MPB rapidly hydrolyzes fibronectin, yielding products ranging from 100,000 to 20,000.

CORRESPONDENCE TO:

S. M. de T. SERRANO - Laboratório de Bioquímica e Biofísica, Instituto Butantan, Av. Vital Brasil, 1500, CEP: 05503-900, São Paulo, SP, Brasil.

Fone/Fax: [phone], E-mail: [e-mail]

Publication Dates

• Publication in this collection
  17 Sept 1999
• Date of issue
  1999