Isolation, enzymatic characterization and antiedematogenic activity of the first reported rattlesnake hyaluronidase from Crotalus durissus terrificus venom
| Autor: | Márcio G. Perino, José Roberto Giglio, Karla de Castro Figueiredo Bordon, Eliane Candiani Arantes |
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| Rok vydání: | 2012 |
| Předmět: |
Male
Molecular Sequence Data Hyaluronoglucosaminidase Hyaluronidase Venom Sodium Chloride Biochemistry Substrate Specificity Diffusion Mice Sequence Analysis Protein Phospholipase A2 Crotalid Venoms medicine Animals Edema Magnesium Amino Acid Sequence Hyaluronic Acid Antiedematogenic activity chemistry.chemical_classification Chromatography Phospholipase A Sequence Homology Amino Acid Edman degradation biology Crotalus Fast protein liquid chromatography General Medicine Crotoxin Crotalus durissus terrificus Enzyme assay Kinetics Phospholipases A2 Enzyme chemistry Snake venom Biocatalysis biology.protein BIOQUÍMICA Calcium Electrophoresis Polyacrylamide Gel Kinetic studies medicine.drug |
| Zdroj: | Repositório Institucional da USP (Biblioteca Digital da Produção Intelectual) Universidade de São Paulo (USP) instacron:USP |
| ISSN: | 0300-9084 |
| DOI: | 10.1016/j.biochi.2012.08.014 |
| Popis: | A hyaluronidase (CdtHya1) from Crotalus durissus terrificus snake venom (CdtV) was isolated and showed to exhibit a high activity on hyaluronan cleavage. However, surveys on this enzyme are still limited. This study aimed at its isolation, functional/structural characterization and the evaluation of its effect on the spreading of crotoxin and phospholipase A(2) (PLA(2)). The enzyme was purified through cation exchange, gel filtration and hydrophobic chromatography. After that, it was submitted to a reverse-phase fast protein liquid chromatography (RP-FPLC) and Edman degradation sequencing, which showed the first N-terminal 44 amino acid residues whose sequence evidenced identity with other snake venom hyaluronidases. CdtHya1 is a monomeric glycoprotein of 64.5 kDa estimated by SDS-PAGE under reducing conditions. It exhibited maximum activity in the presence of 0.2 M NaCl, at 37 °C, pH 5.5 and a specificity to hyaluronan higher than that to chondroitin-4-sulphate, chondroitin-6-sulphate or dermatan. Divalent cations (Ca(2+) and Mg(2+)) and 1 M NaCl significantly reduced the enzyme activity. The specific activity of CdtHya1 was 5066 turbidity reducing units (TRU)/mg, against 145 TRU/mg for the soluble venom, representing a 34.9-fold purification. The pure enzyme increased the diffusion of crotoxin and PLA(2) through mice tissues. CdtHya1 (32 TRU/40 μL) potentiated crotoxin action, as evidenced by mice death, and it decreased the oedema caused by subplantar injections of buffer, crotoxin or PLA(2), thus evidencing the relevance of hyaluronidase in the crotalic envenoming. This work yielded a highly active antiedematogenic hyaluronidase from CdtV, the first one isolated from rattlesnake venoms. |
| Databáze: | OpenAIRE |
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