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

Rafael Sutti,Stephen Hyslop,Thomaz Augusto Rocha-E-Silva,Mariana Tamascia

doi:10.1186/1678-9199-20-2

Abstract

BackgroundVenom 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.ResultsActive 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.ConclusionThe 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.

Highlights

Venom hyaluronidase (Hyase) contributes to the diffusion of venom from the inoculation site
In the present work we describe the purification and characterization of the hyaluronidase present in its venom
Hyase purification was initiated through a size exclusion chromatography, which provided three active fractions, pooled for the second stage (Figure 2A)

Summary

Introduction

Venom hyaluronidase (Hyase) contributes to the diffusion of venom from the inoculation site. Venom obtained by electrical stimulation of adult male and female V. dubius was initially fractionated by gel filtration on a Superdex® 75 column. Spider venoms comprise complex mixtures of components of low molecular weight, peptides and proteins [1]. 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 cylindrata/murina, Loxosceles recluse, Loxosceles rufescens, Loxosceles deserta, Loxosceles gaucho, Loxosceles laeta, Loxosceles recluse, and Loxosceles intermedia [1,2,3,4,5,6]. The. 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. Tarantula bites to humans are relatively rare [12,13,14], these spider venoms represent a rich source of bioactive molecules for scientific interest, basic research and possible therapeutic applications [15,16,17,18]

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