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Purification and functional characterisation of rhinocerase, a novel serine protease from the venom of Bitis gabonica rhinoceros.

Vaiyapuri S, Harrison RA, Bicknell AB, Gibbins JM, Hutchinson G - PLoS ONE (2010)

Bottom Line: Like many viper venom serine proteases, this enzyme is glycosylated; the estimated molecular mass of the native enzyme is approximately 36 kDa, which reduces to 31 kDa after deglycosylation.Other viper venom serine proteases have been shown to exert distinct biological effects, and our preliminary functional characterization of rhinocerase suggest it to be multifunctional.The activities of the enzyme are consistent with the known in vivo effects of Bitis gabonica envenoming, including bleeding disorders, clotting disorders and hypotension.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Reading, Reading, United Kingdom.

ABSTRACT

Background: Serine proteases are a major component of viper venoms and are thought to disrupt several distinct elements of the blood coagulation system of envenomed victims. A detailed understanding of the functions of these enzymes is important both for acquiring a fuller understanding of the pathology of envenoming and because these venom proteins have shown potential in treating blood coagulation disorders.

Methodology/principal findings: In this study a novel, highly abundant serine protease, which we have named rhinocerase, has been isolated and characterised from the venom of Bitis gabonica rhinoceros using liquid phase isoelectric focusing and gel filtration. Like many viper venom serine proteases, this enzyme is glycosylated; the estimated molecular mass of the native enzyme is approximately 36 kDa, which reduces to 31 kDa after deglycosylation. The partial amino acid sequence shows similarity to other viper venom serine proteases, but is clearly distinct from the sequence of the only other sequenced serine protease from Bitis gabonica. Other viper venom serine proteases have been shown to exert distinct biological effects, and our preliminary functional characterization of rhinocerase suggest it to be multifunctional. It is capable of degrading alpha and beta chains of fibrinogen, dissolving plasma clots and of hydrolysing a kallikrein substrate.

Conclusions/significance: A novel multifunctional viper venom serine protease has been isolated and characterised. The activities of the enzyme are consistent with the known in vivo effects of Bitis gabonica envenoming, including bleeding disorders, clotting disorders and hypotension. This study will form the basis for future research to understand the mechanisms of serine protease action, and examine the potential for rhinocerase to be used clinically to reduce the risk of human haemostatic disorders such as heart attacks and strokes.

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Related in: MedlinePlus

Effect of rhinocerase on platelets.100 µg of rhinocerase in a 50 µl volume was added to 450 µl of washed human platelets or platelet rich plasma in a siliconised glass cuvette in an optical platelet aggregometer with continuous stirring (1200 g) at 37°C. As positive controls, 5 µg/ml concentration of CRP and 1 U/ml concentration of thrombin were used at same conditions. The traces show nil effect of rhinocerase and aggregation effects of CRP and thrombin on human washed platelets (A) and platelet rich plasma (B).
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pone-0009687-g006: Effect of rhinocerase on platelets.100 µg of rhinocerase in a 50 µl volume was added to 450 µl of washed human platelets or platelet rich plasma in a siliconised glass cuvette in an optical platelet aggregometer with continuous stirring (1200 g) at 37°C. As positive controls, 5 µg/ml concentration of CRP and 1 U/ml concentration of thrombin were used at same conditions. The traces show nil effect of rhinocerase and aggregation effects of CRP and thrombin on human washed platelets (A) and platelet rich plasma (B).

Mentions: Rhinocerase was found to be incapable of stimulating platelet aggregation both in the washed platelets and PRP. This assay was repeated three times using blood platelets or PRP from three different donors but no aggregation was observed, although positive controls (thrombin and collagen related peptide) did aggregate platelets (figure 6).


Purification and functional characterisation of rhinocerase, a novel serine protease from the venom of Bitis gabonica rhinoceros.

Vaiyapuri S, Harrison RA, Bicknell AB, Gibbins JM, Hutchinson G - PLoS ONE (2010)

Effect of rhinocerase on platelets.100 µg of rhinocerase in a 50 µl volume was added to 450 µl of washed human platelets or platelet rich plasma in a siliconised glass cuvette in an optical platelet aggregometer with continuous stirring (1200 g) at 37°C. As positive controls, 5 µg/ml concentration of CRP and 1 U/ml concentration of thrombin were used at same conditions. The traces show nil effect of rhinocerase and aggregation effects of CRP and thrombin on human washed platelets (A) and platelet rich plasma (B).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2837349&req=5

pone-0009687-g006: Effect of rhinocerase on platelets.100 µg of rhinocerase in a 50 µl volume was added to 450 µl of washed human platelets or platelet rich plasma in a siliconised glass cuvette in an optical platelet aggregometer with continuous stirring (1200 g) at 37°C. As positive controls, 5 µg/ml concentration of CRP and 1 U/ml concentration of thrombin were used at same conditions. The traces show nil effect of rhinocerase and aggregation effects of CRP and thrombin on human washed platelets (A) and platelet rich plasma (B).
Mentions: Rhinocerase was found to be incapable of stimulating platelet aggregation both in the washed platelets and PRP. This assay was repeated three times using blood platelets or PRP from three different donors but no aggregation was observed, although positive controls (thrombin and collagen related peptide) did aggregate platelets (figure 6).

Bottom Line: Like many viper venom serine proteases, this enzyme is glycosylated; the estimated molecular mass of the native enzyme is approximately 36 kDa, which reduces to 31 kDa after deglycosylation.Other viper venom serine proteases have been shown to exert distinct biological effects, and our preliminary functional characterization of rhinocerase suggest it to be multifunctional.The activities of the enzyme are consistent with the known in vivo effects of Bitis gabonica envenoming, including bleeding disorders, clotting disorders and hypotension.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Reading, Reading, United Kingdom.

ABSTRACT

Background: Serine proteases are a major component of viper venoms and are thought to disrupt several distinct elements of the blood coagulation system of envenomed victims. A detailed understanding of the functions of these enzymes is important both for acquiring a fuller understanding of the pathology of envenoming and because these venom proteins have shown potential in treating blood coagulation disorders.

Methodology/principal findings: In this study a novel, highly abundant serine protease, which we have named rhinocerase, has been isolated and characterised from the venom of Bitis gabonica rhinoceros using liquid phase isoelectric focusing and gel filtration. Like many viper venom serine proteases, this enzyme is glycosylated; the estimated molecular mass of the native enzyme is approximately 36 kDa, which reduces to 31 kDa after deglycosylation. The partial amino acid sequence shows similarity to other viper venom serine proteases, but is clearly distinct from the sequence of the only other sequenced serine protease from Bitis gabonica. Other viper venom serine proteases have been shown to exert distinct biological effects, and our preliminary functional characterization of rhinocerase suggest it to be multifunctional. It is capable of degrading alpha and beta chains of fibrinogen, dissolving plasma clots and of hydrolysing a kallikrein substrate.

Conclusions/significance: A novel multifunctional viper venom serine protease has been isolated and characterised. The activities of the enzyme are consistent with the known in vivo effects of Bitis gabonica envenoming, including bleeding disorders, clotting disorders and hypotension. This study will form the basis for future research to understand the mechanisms of serine protease action, and examine the potential for rhinocerase to be used clinically to reduce the risk of human haemostatic disorders such as heart attacks and strokes.

Show MeSH
Related in: MedlinePlus