Alkylation of Histidine Residues of Bothrops moojeni Venom and its Myotoxins I and II Attenuates their Toxicity without any Harmfull Effect on their Antigenicity against other Venoms and Toxins

Soares, A.M.(1,2), Sestito, W.P.(2), Andrião-Escarso, S.H.(2), MARCUSSI, S.(1), STABELI, R.G.(2), Cunha O.A.B.(2), Vieira C.A.(2), GIGLIO, J.R.(2)

(1)Departamento de Biotecnologia, UNAERP, (2)Departamento de Bioquímica e Imunologia, FMRP, USP, Ribeirão Preto, SP, Brazil

Myotoxin-I (MjTX-I) and Myotoxin-II (MjTX-II) were purified to homogeneity from the venom of Bothrops moojeni by ion-exchange chromatography on CM-Sepharose. M_r of MjTX-I or II, estimated by SDS-PAGE, and that of MjTX-II by mass-spectrometry, were 13,500 and 13,698, respectively. After alkylation of B. moojeni crude venom (MjCV) and MjTX-II by p-bromophenacyl bromide (BPB), increases in the i.p. LD₅₀ from 6.0 to 15.7 mg/kg  and from 8.0 to 45.0 mg/kg (mice) were observed, respectively. In addition, doses of 5 LD₅₀ of alkylated MjTX-I did not cause a single obit in mice. Also no myonecrosis was detected for the alkylated toxins, although both of them still induced edema. Antibodies to native and modified crude venom or myotoxins cross-reacted with 12 purified class II myotoxic phospholipases A₂ found in snake venoms of the genus Bothrops. Myotoxic PLA₂s from class I  and class III  were not recognized by the above antibodies. These results suggest that the overall antigenic structure is conserved among class II myotoxic PLA₂s, despite differences in their corresponding amino acid sequences. Anti-MjTX-I-BPB and anti-MjTX-II-BPB rabbit serum, obtained against the modified myotoxins, were more efficient than those obtained against the native myotoxins. In neutralization experiments, pre-incubation of crude venom or isolated myotoxins with antibodies to the native or modified toxins inhibited their PLA₂ and myotoxic activities. Therefore, alkylation of His-48 by BPB strongly reduces the local tissue damage induced by B. moojeni venom or isolated myotoxins while keeping and even increasing antigenicity, thus leading to an enhanced and more efficient  antiophidian serum production procedure for practical purposes.

Financial supported by: FAPESP and CNPq.

CORRESPONDENCE TO:

A. M. Soares, Departamento de Biotecnologia, UNAERP, Ribeirão Preto, 14040-000, SP, Brazil, Email: andreimar@unaerp.br