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Structural and functional characterization of basic PLA2 isolated from Crotalus durissus terrificus venom
D G Oliveira1, M H Toyama, J C Novello
1Departamento de Bioquimica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), SP, Brasil.
Journal of Protein Chemistry
|May 23, 2002
Summary
Fractionated Crotalus durissus terrificus venom yielded potent phospholipase A2 (PLA2) and crotapotins with antimicrobial activity. A specific PLA2 isoform (F17) also demonstrated significant anticoagulant effects, highlighting its multifaceted biological roles.
Area of Science:
- Biochemistry
- Toxicology
- Microbiology
Background:
- Crotalus durissus terrificus venom contains complex protein mixtures, including crotoxin, a neurotoxic complex.
- Understanding the specific activities of individual venom components is crucial for potential therapeutic applications.
Purpose of the Study:
- To isolate and characterize bioactive components from Crotalus durissus terrificus venom.
- To investigate the antimicrobial and anticoagulant properties of purified venom fractions.
Main Methods:
- Reverse-phase High-Performance Liquid Chromatography (HPLC) for venom fractionation.
- Antimicrobial assays against Xanthomonas axonopodis pv. passiflorae.
- Anticoagulant activity assays.
- Amino acid sequencing and molecular weight determination (Tricine SDS-PAGE, 2D electrophoresis, MALDI-TOFF).
Main Results:
- High-purity crotapotins (F5, F7) and phospholipases A2 (PLA2s) (F15, F16, F17) were obtained.
- PLA2s and crotapotins exhibited antimicrobial activity, unlike unseparated crotoxin.
- PLA2 isoform F17 showed significant anticoagulant activity, dependent on specific amino acid residues (Glu 53, Trp 61).
- F17 PLA2 displayed allosteric behavior and its amino acid sequence showed slight variations from known crotoxin subunits, with 60-90% homology to other crotalid PLA2s.
Conclusions:
- Purified PLA2s and crotapotins possess distinct antimicrobial activities.
- PLA2 isoform F17 exhibits potent anticoagulant properties, with enzymatic activity likely contributing to both bactericidal and anticoagulant effects.
- The specific amino acid sequence and structural features of F17 PLA2 are critical for its observed biological functions.