CHARACTERIZATION OF MAIN ENZYMATIC PROPERTIES OF ISOLATED BASIC PLA₂ OF THE Bothrops jararacussu and Crotalus durissus ssp USING CHROMOGENIC SUBSTRATE 4-(NITRO-3-OCTANOYLOXY) BENZOIC ACID

BONFIM, V.L.; TOYAMA, M.H.; PONCE-SOTO, L.A.; NOVELLO, J.C.; MARANGONI, S.

Department of Biochemistry, Institute of Biology/ State University of Campinas (UNICAMP), Campinas, SP, Brasil

Objectives: In this work, we showed the main enzymatic characteristics of several basic PLA₂ from the Bothropic and Crotalic venoms using chromogenic substrate laminate. Methods and Results: In this work we performed several enzymatic studies on the Bj-IV and Bj-V, F6 C. d. cascavella and F6 C. d. collilineatus using a 96 well micro plates system (SpectraMax 340 Molecular Devices). The comparison kinetics parameters between the PLA_2s were temperature, pH optima, substrate concentration, influence of ions, PLA₂ activity and inhibition of PLA₂ activity by crotapotins. Maximum enzyme activity temperature occurred in Bj-IV and Bj-V, F6 C. d. cascavella and F6 C. d. collilineatus  went around 35ºC–40ºC. The pH optima went around to the of7.5 at 8.3. All PLA₂ study here required Ca²⁺ (10mM) for full activity. However those showed enzymatic activity on minimum concentration of the 1mM Ca²⁺. The PLA₂ Bj-IV and Bj-V were diminished by cations Cu²⁺ and Zn^2+, and by Cu²⁺ and Mg²⁺ in the presence and absence of Ca²⁺, respectively. The crotapotins from rattlesnake venom significantly inhibited the enzymatic activity of PLA_2s as well as other PLA₂ sources including the bee and bovine pancreas_. Conclusion:All PLA₂ assayed in this experimental showed an allosteric behavior using chromogenic substrate 4-(nitro-3-octanoyloxy) benzoic acid under our experimental conditions. Both PLA₂ isoforms (Bj-IV and Bj-V) from B. jararacussu, F6 from C. d. collilineatus and F6 from C. d. cascavella, exhibition a lot of similarity of activity kinetic because they are Asp49 with characteristic physical-chemical similar. The inhibition found by crotapotin against several PLA₂ suggest a possible usage of this protein as anti-inflammatory drug.

CORRESPONDENCE TO:

Vera Lucia Bonfim, Rua Frei São Carlos 66, Campinas, SP, CEP: 13080-061, Brasil, E-mail: veralb@yahoo.com