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

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.

Pharmacological Characterization of the Edema Caused by Vitalius dubius (Theraphosidae, Mygalomorphae) Spider Venom in Rats

“…We have previously shown, using ELISA and immunoblotting, that V. dubius venom also cross-reacts with this antiarachnid antivenom produced by the Instituto Butantan (Rocha-e-Silva et al, 2009b). Immunoblotting showed that only venom components .10 kDa reacted with the antivenom (Rocha-e-Silva et al, 2009b), including hyaluronidase (45 kDa) (Sutti et al, 2014). Studies with T. serrulatus scorpion venom have shown that hyaluronidase has an important role in the action of this venom (Pessini et al, 2001;Horta et al, 2014), and we have also observed the V. dubius enzyme to be edematogenic.…”

“…We have previously reported on the venom gland structure (Rocha-e-Silva et al, 2009a) and general venom composition (Rocha-e-Silva et al, 2009b) of V. dubius. We have also isolated a nicotinic receptor antagonist (Rocha-e-Silva et al, 2013) and hyaluronidase (Sutti et al, 2014) from this venom. In this work, we have extended our investigation of V. dubius venom by examining its ability to increase vascular permeability in rat dorsal skin and hind paw.…”

Virocidal activity of Egyptian scorpion venoms against hepatitis C virus

“…It has been reported that the proteolytic activities of those enzymes were almost completely abolished by treatment with a matrix metalloproteinase inhibitor, 1, 10-phenanthroline [31,32,47]. Also, those enzymatic activities are known to be inactivated by heating at 60°C for 20 min [33,34,48]. We S. maurus palmatus venom (30 μg/ml) was treated with a metalloproteinase inhibitor (1, 10-phenan-throline; 5 mM) at 4°C or 60°C for 20 min.…”

“…Crude venom (30 μg/ml) was heated at 60°C for 20 min or treated with a metalloprotease inhibitor (1,10-phenanthroline; 5 mM) [31][32][33][34] at 4°C or 60°C for 20 min. The treated venom or untreated control was mixed with HCV for 2 hr at 37°C.…”

Purification of hyaluronidase as an anticancer agent inhibiting CD44

“…Chromatography technology plays a major role in downstream protein processing as the most fundamental step in biopharmaceutical purification. Animal venom Hyal is structurally comparable with that from the sperm acrosome membrane (El Zahar, Magdy, El‐Kosasy, & Bartlett, , Sutti, Tamascia, & Hyslop, ). Hyal produced is limited if produced by the animal, but unlimited if generated by microorganisms.…”