PURIFICATION AND PARTIAL CHARACTERIZATION OF A NOVEL PHOSPHOLIPASE A₂ FROM Micrurus dumerilii carinicauda CORAL SNAKE VENOM

Belo, C.A.D.(1), Leite, G.B.(1), Toyama, M.H.(2), Marangoni, S.(2), Fontana, M.D.(1), Hyslop, S.(1), Rodrigues-Simioni, L.(1)

(1)Departamento de Farmacologia, FCM, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, (2)Departamento de Bioquímica, Instituto de Biologia, UNICAMP, Campinas, SP, Brasil.

Objective: The aim of this work was to purify and partially characterize the PLA₂ from Micrurus dumerilii carinicauda venom and to examine its neuromuscular activity. Material and Results: Venom (10 mg samples) was fractionated by reverse-phase HPLC on a Sephasil Peptide C18 5 mm St 4.6/250 column (Pharmacia Biotech) equilibrated with 0.1% trifluoroacetic acid (TFA). Proteins were eluted (1 ml/min) with a linear gradient (0-65%) of acetonitrile (66% in 0.1% TFA).  The elution profile was monitored at 214 nm and 280 nm and fractions were collected manually and lyophilized. PLA₂ activity was assayed colorimetrically. Sequencing was done with an Applied Biosystems model Procise f Protein Sequencer and the phenylthiohydantoin amino acid derivatives were identified in a PTH amino acid analyzer. Fractionation resulted in 22 peaks. Peak 16 contained most PLA₂ activity and consisted of a single protein (confirmed by RP-HPLC). N-terminal sequencing showed the first 36 residues to be NKIDFLNMIQCTTPGREPLVAFTNYGCYCGKGGSGT which shared high homology with PLA₂ enzymes from Micropechis ikaheka venom (group IB PLA₂). In mouse phenic nerve-diaphragm preparations, peak 16 (5 mg/ml) produced some facilitation (15±3%, n=3) with slight neuromuscular blockade after 120 min. In contrast, peak 9 (5 mg/ml) produced rapid blockade (100% in 40 min, n=4), similar to the venom (100% in 80 min, n=3), but contained no PLA₂ activity. Conclusion: The PLA₂ from M. d. carinicauda venom contains Lysat residue 2 and causes slight facilitation, but has only weak neuromuscular activity compared to the principal neurotoxic peak and the whole venom.

Financial support: FAPESP

CORRESPONDENCE TO:

Cháriston André Dal Belo, Rua João Jacob Rohwedder, 19, Sumaré, SP, CEP: 13.170-310, Brasil, Email: charistondb@yahoo.com.br