Enzymatic and structural characterization of new PLA2 isoform isolated from white venom of Crotalus durissus ruruima.

This work reports the structural and enzymatic characterization of a new sPLA2 from the white venom of Crotalus durissus ruruima, nominated PLA2A. The homogeneity of the PLA2A fraction and its molecular mass were initially evaluated by SDS-PAGE and confirmed by MALDI-TOF spectrometry, indicating a molecular mass of 14,299.34Da. Structural investigation, through circular dichroism spectroscopy, revealed that PLA2A has a high content of alpha helix and beta-turn structures, 45.7% and 35.6% respectively. Its amino acid sequence, determined by Edman degradation and "de novo amino acid sequencing", exhibited high identity to PLA2 Cdt F15 from Crotalus durissus terrificus. The enzymatic investigation, conducted using the synthetic substrate 4-nitro-3-(octanoyloxy)-benzoic acid, determined its V(max) (7.56nmoles/min) and K(M) (2.76mM). Moreover, PLA2A showed an allosteric behavior and its enzymatic activity was dependent on Ca(2+). Intrinsic fluorescence measurements suggested that Ca(2+) induced a significant increase of PLA2A fluorescence, whereas its replacement for Mg(2+), Mn(2+), Sn(2+) and Cd(2+) apparently induced no structural modifications. The optimal pH and temperature for the enzymatic activity of PLA2A were 8.4 and 40 degrees C, respectively, and the minimal concentration of p-BPB and crotapotin that significantly inhibited such activity was 0.75mM and 0.4muM, respectively. In addition, PLA2A showed a significant antibacterial effect that was not strictly dependent on the enzymatic activity of such sPLA2.

Molecular modeling and inhibition of phospholipase A2 by polyhydroxy phenolic compounds.

Phospholipases A(2) are enzymes responsible for the hydrolysis of membrane phospholipids that release arachidonic acid, which serves as substrate for pro-inflammatory mediators, such as prostaglandins and leucotriens. The design of specific inhibitors for PLA(2) might help in the development of new anti-inflammatory drugs. Polyhydroxy phenolic compounds, such as flavonoids, vitamin E, rosmarinic acid and aristolochic acid, are able to inhibit PLA(2) from different sources. Herein, we have studied the kinetic behavior and the capacity of inhibiting edema formation induced by PLA(2) of five different polyhydroxy phenolic compounds (two phenolic derivatives and three acetophenone hydroxylated derivatives) extracted from the venom of Crotalus adamanteus. The results showed that compounds 1,3-dihydroxy benzene, 1,3,5-trihydroxy benzene and 2,4,6-trihydroxy acetophenone were the most efficient in the inhibition of the enzymatic activity and edema induction by PLA(2). It was also verified that the number of hydroxyls in each molecule is not a limiting factor for the inhibition capacity of these compounds. Molecular modeling studies indicated that the most active compounds are linked to the amino acid Asp 49 and that they destabilize the coordination of the calcium atom, which is essential to the catalytic activity. The study of potential surfaces showed that there are conditions in which the potential values must be adequate for enzyme complex formation with polyhydroxy phenolic compounds. When the potential over the hydroxyl surfaces is very high, formation of stable complexes does not occur and the enzyme does not act intensely. These results might be helpful in the design of a drug that specifically inhibits PLA(2).

Synthesis and evaluation of nitrostyrene derivative compounds, new snake venom phospholipase A2 inhibitors.

Several nitrostyrene derivatives were synthesized and their inhibitive activities on phospholipase A(2) (PLA(2)) from Bothrops jararacussu venom were evaluated. Some compounds were very efficient as inhibition agents against edema-inducing, enzymatic and myotoxic activities. Data revealed that the size of the substitute and substitution position in the nitrostyrene moiety had important influence on the inhibition capacities. The enzymatic kinetic studies show that the nitrostyrene derivatives compounds inhibit PLA(2) in a non-competitive manner. The electronic, molecular and topologic parameters were calculated using ab initio quantum calculations (density functional theory-DFT) and analyzed by chemometric methods (principal component analysis (PCA) and hierarchical cluster analysis (HCA)) in order to build models able to establish relationships between the electronic features and the structure-activity presented by the target compound. Compounds with the nitro group in the ortho, meta and para position (compounds 2-4) on the aromatic ring were more efficient in the inhibition of PLA(2) activity in all tests. These results indicate that the influence of the nitro group in the aromatic ring is, in fact, important. In addition, quantum chemistry calculations show that compounds with a higher capacity of inhibiting PLA(2) present lower values of highest occupied molecular orbital (HOMO) energy and polarizability, suggesting the formation of a charge-transferring complex between the nitrostyrene compounds and PLA(2).