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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 PMSF on rhinocerase activity.10 U of thrombin or 100 µg of native and deglycosylated rhinocerase were mixed with different concentrations of PMSF and incubated at 37°C for 30 minutes prior to the addition of Arg-AMC to a final concentration of 50 nM. The amount of Arg-AMC released was measured after 15 minutes of incubation using a spectrofluorometer. Each bar shows the mean ± S.D. (n = 3). The hydrolytic activity measured for rhinocerase without any PMSF was taken as 100%.
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pone-0009687-g007: Effect of PMSF on rhinocerase activity.10 U of thrombin or 100 µg of native and deglycosylated rhinocerase were mixed with different concentrations of PMSF and incubated at 37°C for 30 minutes prior to the addition of Arg-AMC to a final concentration of 50 nM. The amount of Arg-AMC released was measured after 15 minutes of incubation using a spectrofluorometer. Each bar shows the mean ± S.D. (n = 3). The hydrolytic activity measured for rhinocerase without any PMSF was taken as 100%.

Mentions: 1.2 mM PMSF is sufficient to inhibit the activity of 10 units of thrombin. 100 µg of rhinocerase activity were inhibited by 2.3 mM PMSF (figure 7). Comparison of thrombin and rhinocerase activity shows that the activity of 100 µg of rhinocerase is equivalent to only 4.05 units of thrombin, and thus we conclude that rhinocerase is less sensitive to PMSF than thrombin. Similarly, the deglycosylated rhinocerase was inhibited by the same concentration of PMSF as native rhinocerase indicating that the glycosylated moieties do not affect the binding of PMSF. A benzamidine affinity column was used to assess the sensitivity of rhinocerase to benzamidine. Rhinocerase was unable to bind the column indicating that benzamidine is not an inhibitor for rhinocerase (data not shown).


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 PMSF on rhinocerase activity.10 U of thrombin or 100 µg of native and deglycosylated rhinocerase were mixed with different concentrations of PMSF and incubated at 37°C for 30 minutes prior to the addition of Arg-AMC to a final concentration of 50 nM. The amount of Arg-AMC released was measured after 15 minutes of incubation using a spectrofluorometer. Each bar shows the mean ± S.D. (n = 3). The hydrolytic activity measured for rhinocerase without any PMSF was taken as 100%.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0009687-g007: Effect of PMSF on rhinocerase activity.10 U of thrombin or 100 µg of native and deglycosylated rhinocerase were mixed with different concentrations of PMSF and incubated at 37°C for 30 minutes prior to the addition of Arg-AMC to a final concentration of 50 nM. The amount of Arg-AMC released was measured after 15 minutes of incubation using a spectrofluorometer. Each bar shows the mean ± S.D. (n = 3). The hydrolytic activity measured for rhinocerase without any PMSF was taken as 100%.
Mentions: 1.2 mM PMSF is sufficient to inhibit the activity of 10 units of thrombin. 100 µg of rhinocerase activity were inhibited by 2.3 mM PMSF (figure 7). Comparison of thrombin and rhinocerase activity shows that the activity of 100 µg of rhinocerase is equivalent to only 4.05 units of thrombin, and thus we conclude that rhinocerase is less sensitive to PMSF than thrombin. Similarly, the deglycosylated rhinocerase was inhibited by the same concentration of PMSF as native rhinocerase indicating that the glycosylated moieties do not affect the binding of PMSF. A benzamidine affinity column was used to assess the sensitivity of rhinocerase to benzamidine. Rhinocerase was unable to bind the column indicating that benzamidine is not an inhibitor for rhinocerase (data not shown).

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