Synergistically acting PLA₂: peptide hemorrhagic complex from Daboia russelii venom

Snake venoms are complex mixture of enzymatic and non-enzymatic proteins. Non-covalent protein-protein interaction leads to protein complexes, which bring about enhanced pharmacological injuries by their synergistic action. Here we report identification and characterization of a new Daboia russelii hemorrhagic complex I (DR-HC-I) containing phospholipase A₂ (PLA₂) and non-enzymatic peptide. DR-HC-I was isolated from the venom of D. russelii by CM-Shepadex-C25 and gel permeation chromatography. Individual components were purified and identified by RP-HPL chromatography, mass spectrometry and N-terminal amino acid sequencing. DR-HC-I complex was lethal to mice with the LD₅₀ dose of 0.7 mg/kg body weight with hemorrhagic and neurotoxic properties. DR-HC-I complex consists of non-hemorrhagic PLA₂ and neurotoxic non-enzymatic peptide. The non-enzymatic peptide quenched the intrinsic fluorescence of PLA₂ in a dose dependent manner, signifying the synergistic interaction between two proteins. PLA₂ and peptide toxin in a 5:2 M ratio induced skin hemorrhage in mice with MHD 20 μg. However, addition of ANS (1-Anilino-8-naphthalene sulfonate) to DR-HC-I complex inhibited skin hemorrhagic effect and also synergic interaction. But there was no impact on PLA₂ due to this synergistic interaction, and indirect hemolytic or plasma re-calcification activity. However, the synergistic interaction of PLA₂ and non-enzymatic peptide contributes to the enhanced venom-induced hemorrhage and toxicity of Daboia russellii venom.