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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

Purification of rhinocerase from rotofor separated venom fractions.A. Superdex 75 gel filtration chromatogram obtained during the elution of proteins from rotofor separated venom fractions 3 and 4. The peaks were numbered at the particular fractions which were used for analysis by SDS-PAGE and for analysis of the serine protease activity. B. 100 µl of the selected fractions shown in figure A were used to measure serine protease activity using Arg-AMC fluorescent substrate. Each bar shows the mean ± S.D. (n = 3). The hydrolytic activity measured for P2 was taken as 100%. C. 100 µl of the selected fractions indicated in figure A were analysed by SDS-PAGE (10%) and silver stained.
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pone-0009687-g002: Purification of rhinocerase from rotofor separated venom fractions.A. Superdex 75 gel filtration chromatogram obtained during the elution of proteins from rotofor separated venom fractions 3 and 4. The peaks were numbered at the particular fractions which were used for analysis by SDS-PAGE and for analysis of the serine protease activity. B. 100 µl of the selected fractions shown in figure A were used to measure serine protease activity using Arg-AMC fluorescent substrate. Each bar shows the mean ± S.D. (n = 3). The hydrolytic activity measured for P2 was taken as 100%. C. 100 µl of the selected fractions indicated in figure A were analysed by SDS-PAGE (10%) and silver stained.

Mentions: Purification of serine protease - To purify B. g. rhinoceros venom serine proteases size exclusion, ion-exchange and affinity column chromatography were tried either alone or in combination with limited success. We therefore used liquid phase isoelectric focusing which separates proteins based on their isoelectric points. Using an ampholyte range of 6–8 pI (under non-denaturing and non-reducing conditions), venom proteins were well separated (figure 1B) and high serine protease activity observed, which coincided with the presence of two proteins with approximate molecular masses of 36 kDa and 70 kDa in fractions 3 and 4 (figure 1C). These fractions were pooled and loaded onto a Superdex 75 gel filtration column. Five clearly resolved protein peaks (P1, P2, P3, P4 and P5) were identified (figure 2A). Serine protease activity was measured for single fractions with high absorbance in fractions P1.1-P1.3 and P2-P5, and was found to be restricted to peak P2 (figure 2B). Proteins present in these fractions were separated by reducing SDS-PAGE and visualised by silver staining. Peak P2 was found to contain a single protein with an approximate molecular mass of 36 kDa (figure 2C). Peaks P4 and P5 shown on the figure 2B do not show any proteins in the SDS-PAGE (figure 2C) indicating the elution of ampholytes, thiourea and urea present in the rotofor buffer. Consistent with the toxicological literature, we named this new purified venom serine protease ‘rhinocerase’. Based on the Bradford assay, 5 mg of whole B. g. rhinoceros venom yielded 1.12 mg of rhinocerase, thus this is a major active component which constitutes approximately 25% of the total protein in this venom.


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)

Purification of rhinocerase from rotofor separated venom fractions.A. Superdex 75 gel filtration chromatogram obtained during the elution of proteins from rotofor separated venom fractions 3 and 4. The peaks were numbered at the particular fractions which were used for analysis by SDS-PAGE and for analysis of the serine protease activity. B. 100 µl of the selected fractions shown in figure A were used to measure serine protease activity using Arg-AMC fluorescent substrate. Each bar shows the mean ± S.D. (n = 3). The hydrolytic activity measured for P2 was taken as 100%. C. 100 µl of the selected fractions indicated in figure A were analysed by SDS-PAGE (10%) and silver stained.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2837349&req=5

pone-0009687-g002: Purification of rhinocerase from rotofor separated venom fractions.A. Superdex 75 gel filtration chromatogram obtained during the elution of proteins from rotofor separated venom fractions 3 and 4. The peaks were numbered at the particular fractions which were used for analysis by SDS-PAGE and for analysis of the serine protease activity. B. 100 µl of the selected fractions shown in figure A were used to measure serine protease activity using Arg-AMC fluorescent substrate. Each bar shows the mean ± S.D. (n = 3). The hydrolytic activity measured for P2 was taken as 100%. C. 100 µl of the selected fractions indicated in figure A were analysed by SDS-PAGE (10%) and silver stained.
Mentions: Purification of serine protease - To purify B. g. rhinoceros venom serine proteases size exclusion, ion-exchange and affinity column chromatography were tried either alone or in combination with limited success. We therefore used liquid phase isoelectric focusing which separates proteins based on their isoelectric points. Using an ampholyte range of 6–8 pI (under non-denaturing and non-reducing conditions), venom proteins were well separated (figure 1B) and high serine protease activity observed, which coincided with the presence of two proteins with approximate molecular masses of 36 kDa and 70 kDa in fractions 3 and 4 (figure 1C). These fractions were pooled and loaded onto a Superdex 75 gel filtration column. Five clearly resolved protein peaks (P1, P2, P3, P4 and P5) were identified (figure 2A). Serine protease activity was measured for single fractions with high absorbance in fractions P1.1-P1.3 and P2-P5, and was found to be restricted to peak P2 (figure 2B). Proteins present in these fractions were separated by reducing SDS-PAGE and visualised by silver staining. Peak P2 was found to contain a single protein with an approximate molecular mass of 36 kDa (figure 2C). Peaks P4 and P5 shown on the figure 2B do not show any proteins in the SDS-PAGE (figure 2C) indicating the elution of ampholytes, thiourea and urea present in the rotofor buffer. Consistent with the toxicological literature, we named this new purified venom serine protease ‘rhinocerase’. Based on the Bradford assay, 5 mg of whole B. g. rhinoceros venom yielded 1.12 mg of rhinocerase, thus this is a major active component which constitutes approximately 25% of the total protein in this venom.

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