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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

Rotofor based separation of B. g. rhinoceros venom proteins.A. SDS-PAGE (10%) was run with 50 µg of B. g. rhinoceros venom and stained with Coomassie brilliant blue. Several proteins with different molecular weights are present in this venom. B. 2 mg of venom were mixed with non-reducing rotofor buffer containing ampholytes with pI 6–8 and separated under non-denaturing conditions. In total 10 fractions (indicated by the numbers at the top of the gel) were collected. 10 µl of each fraction were run in SDS-PAGE (10%) and stained with Coomassie brilliant blue. C. 20 µl of each rotofor fraction were used to measure serine protease activity using Arg-AMC fluorescent substrate. The data represents the mean ± S.D. (n = 3). The hydrolytic activity measured for fraction 5 was taken as 100%.
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pone-0009687-g001: Rotofor based separation of B. g. rhinoceros venom proteins.A. SDS-PAGE (10%) was run with 50 µg of B. g. rhinoceros venom and stained with Coomassie brilliant blue. Several proteins with different molecular weights are present in this venom. B. 2 mg of venom were mixed with non-reducing rotofor buffer containing ampholytes with pI 6–8 and separated under non-denaturing conditions. In total 10 fractions (indicated by the numbers at the top of the gel) were collected. 10 µl of each fraction were run in SDS-PAGE (10%) and stained with Coomassie brilliant blue. C. 20 µl of each rotofor fraction were used to measure serine protease activity using Arg-AMC fluorescent substrate. The data represents the mean ± S.D. (n = 3). The hydrolytic activity measured for fraction 5 was taken as 100%.

Mentions: Protein profile of venom- Fractionation of whole B. g. rhinoceros venom using reducing SDS-PAGE yielded 16 visible protein bands of varying staining intensities (figure 1A). The molecular masses of these proteins ranged between 10 kDa and 150 kDa. Eight proteins were visible between 25 kDa and 40 kDa, the molecular weight range where serine proteases have been identified.


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)

Rotofor based separation of B. g. rhinoceros venom proteins.A. SDS-PAGE (10%) was run with 50 µg of B. g. rhinoceros venom and stained with Coomassie brilliant blue. Several proteins with different molecular weights are present in this venom. B. 2 mg of venom were mixed with non-reducing rotofor buffer containing ampholytes with pI 6–8 and separated under non-denaturing conditions. In total 10 fractions (indicated by the numbers at the top of the gel) were collected. 10 µl of each fraction were run in SDS-PAGE (10%) and stained with Coomassie brilliant blue. C. 20 µl of each rotofor fraction were used to measure serine protease activity using Arg-AMC fluorescent substrate. The data represents the mean ± S.D. (n = 3). The hydrolytic activity measured for fraction 5 was taken as 100%.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0009687-g001: Rotofor based separation of B. g. rhinoceros venom proteins.A. SDS-PAGE (10%) was run with 50 µg of B. g. rhinoceros venom and stained with Coomassie brilliant blue. Several proteins with different molecular weights are present in this venom. B. 2 mg of venom were mixed with non-reducing rotofor buffer containing ampholytes with pI 6–8 and separated under non-denaturing conditions. In total 10 fractions (indicated by the numbers at the top of the gel) were collected. 10 µl of each fraction were run in SDS-PAGE (10%) and stained with Coomassie brilliant blue. C. 20 µl of each rotofor fraction were used to measure serine protease activity using Arg-AMC fluorescent substrate. The data represents the mean ± S.D. (n = 3). The hydrolytic activity measured for fraction 5 was taken as 100%.
Mentions: Protein profile of venom- Fractionation of whole B. g. rhinoceros venom using reducing SDS-PAGE yielded 16 visible protein bands of varying staining intensities (figure 1A). The molecular masses of these proteins ranged between 10 kDa and 150 kDa. Eight proteins were visible between 25 kDa and 40 kDa, the molecular weight range where serine proteases have been identified.

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