Effects of chemical modifications of crotoxin B, the phospholipase A(2) subunit of crotoxin from Crotalus durissus terrificus snake venom, on its enzymatic and pharmacological activities

Crotoxin B, the basic Asp49-PLA(2) subunit from crotoxin, the main componen t of Crotalus durissus terrificus venom, displays myotoxic, edema-inducing, bactericidal (upon Escherichia coli), liposomal-disrupting and anticoagula nt activities. Chemical modifications of His (with 4-bromophenacyl bromide, BPB), Tyr (with 2-nitrobenzenesulphonyl fluoride, NBSF), Trp (with o-nitro phenylsulphenyl chloride, NPSC) and Lys (with acetic anhydride) residues of this protein, in addition to cleavage with cyanogen bromide (CNBr) and inh ibition with ethylenediaminetetraacetic acid (EDTA), were carried out in or der to study their effects on enzymatic and pharmacological activities. Let hality was reduced after modification of His or Lys residues, as well as af ter cleavage with CNBr, while enzymatic activity was completely abolished a fter modification of His or incubation with EDTA. Modification of Lys or Ty r, or cleavage with CNBr, partially reduced enzymatic activity. Anticoagula nt activity was modified similarly to enzymatic activity, evidencing the de pendency of this pharmacological effect on catalytic activity. Myotoxicity was reduced after modification of His or Lys, as well as after cleavage wit h CNBr, whereas EDTA reduced this effect to a lesser extent. Bactericidal e ffect was significantly reduced only after modification of Lys and after cl eavage with CNBr. Edema-inducing activity was partially inhibited after tre atment with EDTA and strongly reduced after acetylation of Lys residues and cleavage with CNBr, being only partially reduced after His alkylation. On the other hand, liposome disrupting activity was only partially reduced aft er modification of His and Tyr or after cleavage with CNBr. Modification of Trp residue partially reduced lethality and myotoxicity but did not affect enzymatic or anticoagulant activities. These data indicate that enzymatic activity is relevant for some pharmacological effects induced by crotoxin B (mainly lethal, myotoxic and anticoagulant activities), and also evidence that this subunit of crotoxin displays regions different from them active c atalytic site which are involved in some of the toxic and pharmacological e ffects induced by this phospholipase A(2). (C) 2001 Elsevier Science Ltd. A ll rights reserved.