ISOLATION AND PARTIAL CHARACTERIZATION OF A FIBRIN(OGEN)OLYTIC METALLOPROTEASE AND A PLASMINOGEN ACTIVATING SERINE PROTEASE FROM THE VENOM OF Bothrops brazili

MODESTO, J.C.A.(1, VENTURA, J.S.(1), OLIVA, M.L.V.(2), CHUDZINSKI-TAVASSI, A.M.(1)

(1)Laboratório de Bioquímica e Biofísica, Instituto Butantan, São Paulo, SP, (2)Departamento de Bioquímica, Universidade Federal de São Paulo (UNIFESP), São Paulo, SP, Brasil.

Snake venoms contain a complex mixture of proteins that participate and interfere with the control and regulation of a number of important biological processes of their prey. Some of these proteases, inhibitors and toxins directly affect the haemostatic system. Systematic disturbances observed in victims of snake bites by Bothrops sp. are hemorrhage and blood incoagulability caused by a coagulopathy consumption. Metalo and serine proteases are responsible for procoagulant and fibrino(geno)lytic activities of Bothrops snake venoms. In Brazil, the Bothrops brazili snake is found in a few specific regions of the Amazon Forest and the proteins isolated from this venom have not been purified and characterized. We present here the isolation and partial characterization of two proteins presents in the Bothrops brazili venom, one is a fibrin(ogen)olytic enzyme and the second is a plasminogen activator. The enzymes were isolated using ion-exchange chromatography (DEAE Sephadex A-50), followed by two steps in the FPLC system: molecular exclusion (Superdex 200) and ion-exchange (Resource Q). The homogeniety was evaluated by SDS PAGE (10%). The fibrin(ogen)olytic activity was tested based on fibrinogen degradation in fibrin and fibrin-agarose plates. The plasminogen activator activity was measured indirectly by the formation of plasmin using the Solid Fibrin Assay (SOFIA) and S-2251 as substrate. The fibrin(ogen)olytic enzyme was characterized as a metalloprotease (approximate molecular mass 25 kDa) that degrades both the A and Bchains of fibrinogen  and fibrin by a path independent of plasminogen activation. Fibrinogen and fibrin products are different from those induced by plasmin. The fibrin(ogen)olytic enzyme did not hydrolyse quenched substrates based on the -chain of insulin. The plasminogen activator protein (approximate molecular mass 30 kDa) is a serine protease which requires fibrin fragments, so it can be classified as a t-PA like protein.

Supported by: FAPESP

CORRESPONDENCE TO:

Jeanne Claine de Albuquerque Modesto, Avenida Vital Brasil, 1500, Butantã, São Paulo, SP, CEP: 05503-900, Brasil, Email: clainealbuquerque@hotmail.com