Purification and characterization of a hyaluronidase from venom of the spider Vitalius dubius (Araneae, Theraphosidae)

Journal of Venomous Animals and Toxins including Tropical Diseases, Feb 2019

Rafael Sutti, Mariana Leite Tamascia, Stephen Hyslop, Thomaz Augusto Alves Rocha-e-Silva

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Purification and characterization of a hyaluronidase from venom of the spider Vitalius dubius (Araneae, Theraphosidae)

RESEARCH Purification and characterization of a hyaluronidase from venom of the spider Vitalius dubius (Araneae, Theraphosidae) Rafael Sutti 1   Mariana Leite Tamascia 2   Stephen Hyslop 2   Thomaz Augusto Alves Rocha-e-Silva 1   *  1Department of Physiological Sciences, Santa Casa de Sao Paulo Medical School, Rua Cesário Motta Jr., 61, Vila Buarque, CEP 01.221-020 Sao Paulo, SP, Brasil. 2Department of Pharmacology, Schoolof MedicalSciences, Federal University of Campinas (UNICAMP), Campinas Sao Paulostate, Brazil. ABSTRACT Background Venom hyaluronidase (Hyase) contributes to the diffusion of venom from the inoculation site. In this work, we purified and characterized Hyase from the venom of Vitalius dubius (Araneae, Theraphosidae), a large theraphosid found in southeastern Brazil. Venom obtained by electrical stimulation of adult male and female V. dubius was initially fractionated by gel filtration on a Superdex® 75 column. Active fractions were pooled and applied to a heparin-sepharose affinity column. The proteins were eluted with a linear NaCl gradient. Results Active fractions were pooled and assessed for purity by SDS-PAGE and RP-HPLC. The physicochemical tests included optimum pH, heat stability, presence of isoforms, neutralization by flavonoids and assessment of commercial antivenoms. Hyase was purified and presented a specific activity of 148 turbidity-reducing units (TRU)/mg (venom: 36 TRU/mg; purification factor of ~4). Hyase displayed a molecular mass of 43 kDa by SDS-PAGE. Zymography in hyaluronic-acid-containing gels indicated an absence of enzyme isoforms. The optimum pH was 4-5, with highest activity at 37°C. Hyase was stable up to 60°C; but its activity was lost at higher temperatures and maintained after several freeze-thaw cycles. The NaCl concentration (up to 1 M) did not influence activity. Hyase had greater action towards hyaluronic acid compared to chondroitin sulfate, and was completely neutralized by polyvalent antiarachnid sera, but not by caterpillar, scorpion or snakes antivenoms. Conclusion The neutralization by arachnid but not scorpion antivenom indicates that this enzyme shares antigenic epitopes with similar enzymes in other spider venoms. The biochemical properties of this Hyase are comparable to others described. Key words: Hyaluronidase; Venom; Purification; Vitalius dubius Background Spider venoms comprise complex mixtures of components of low molecular weight, peptides and proteins [ 1 ]. Among the enzymatic activities, collagenase and hyaluronidase (Hyase) are often found and were formerly attributed to matrix degradation [ 1]. Hyase activity is found in several spider species, such as Cupiennius salei, Lycosa godeffroy, Lampona cylin-drata/murina, Loxosceles recluse, Loxosceles rufescens, Loxosceles deserta, Loxosceles gaucho, Loxosceles laeta, Loxosceles recluse, and Loxosceles intermedia [ 1 - 6 ]. The first report of hyaluronidasic activity was in venoms of the Brazilian spidersLycosa raptorial and Phoneutria nigriventer [ 7 , 8]. The first Hyase to be purified from a Theraphosidae spider was the one from the tarantula Dugesiella hentzi (Girard), which was characterized and identified as the major venom component [ 9]. The clinical relevance of Hyases is not confined to a toxin-spreading factor, since it acts as an allergen in a manner similar to hymenoptera venoms. Hyase, phospho-lipase A2 and melitin were identified as the tree major causes of allergic reactions involved in bee stings, and phospholipase A1 and antigen 5 are the major allergens in wasp venoms [ 10 , 11 ]. Although tarantula bites to humans are relatively rare [ 12 - 14 ], these spider venoms represent a rich source of bioactive molecules for scientific interest, basic research and possible therapeutic applications [ 15 - 18 ]. The tarantula Vitalius dubius (Mello-Leitão, 1923) is characterized as nonaggressive and is found in the very populous area of southeastern Brazil [19 ]. Recent studies have shown thatV. dubius venom has a complex biochemical and pharmacological composition, with different biological activities [ 20 , 21 ]. In the present work we describe the purification and characterization of the hy-aluronidase present in its venom. Methods Reagents Refined chemicals were purchased from Sigma Chemical Co. (USA) whereas heparin was purchased from Labora-tório Cristália (Brazil). Molecular mass markers for SDS-PAGE were acquired from BioRad (USA). The resins for column chromatography were purchased from GE Healthcare Life Sciences (Sweden). The HPLC column was a Jupiter C18 (4.6 mm χ 250 mm χ 12 μm) acquired from Phenomenex (USA). Spiders and venom extraction Specimens of V. dubius and Phoneutria nigriventer were provided by the Centers for Zoonosis Control for the cities in the region of Campinas, Sao Paulo state. The spiders were identified and kept in captivity, where (...truncated)


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Rafael Sutti, Mariana Leite Tamascia, Stephen Hyslop, Thomaz Augusto Alves Rocha-e-Silva. Purification and characterization of a hyaluronidase from venom of the spider Vitalius dubius (Araneae, Theraphosidae), Journal of Venomous Animals and Toxins including Tropical Diseases, pp. 1-7, 20, DOI: 10.1186/1678-9199-20-2