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Daboxin P, a Major Phospholipase A2 Enzyme from the Indian Daboia russelii russelii Venom Targets Factor X and Factor Xa for Its Anticoagulant Activity.

Sharma M, Iyer JK, Shih N, Majumder M, Mattaparthi VS, Mukhopadhyay R, Doley R - PLoS ONE (2016)

Bottom Line: It exhibits strong anticoagulant and phospholipase A2 activity but is devoid of any cytotoxic effect on the tested normal or cancerous cell lines.It is found to be stable at acidic (pH 3.0) and neutral pH (pH 7.0) and has a Tm value of 71.59 ± 0.46°C.Molecular docking analysis reveals the interaction of the Ca+2 binding loop; helix C; anticoagulant region and C-terminal region of daboxin P with the heavy chain of factor Xa.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biotechnology, Tezpur University, Tezpur-784028, Assam, India.

ABSTRACT
In the present study a major protein has been purified from the venom of Indian Daboia russelii russelii using gel filtration, ion exchange and Rp-HPLC techniques. The purified protein, named daboxin P accounts for ~24% of the total protein of the crude venom and has a molecular mass of 13.597 kDa. It exhibits strong anticoagulant and phospholipase A2 activity but is devoid of any cytotoxic effect on the tested normal or cancerous cell lines. Its primary structure was deduced by N-terminal sequencing and chemical cleavage using Edman degradation and tandem mass spectrometry. It is composed of 121 amino acids with 14 cysteine residues and catalytically active His48 -Asp49 pair. The secondary structure of daboxin P constitutes 42.73% of α-helix and 12.36% of β-sheet. It is found to be stable at acidic (pH 3.0) and neutral pH (pH 7.0) and has a Tm value of 71.59 ± 0.46°C. Daboxin P exhibits anticoagulant effect under in-vitro and in-vivo conditions. It does not inhibit the catalytic activity of the serine proteases but inhibits the activation of factor X to factor Xa by the tenase complexes both in the presence and absence of phospholipids. It also inhibits the tenase complexes when active site residue (His48) was alkylated suggesting its non-enzymatic mode of anticoagulant activity. Moreover, it also inhibits prothrombinase complex when pre-incubated with factor Xa prior to factor Va addition. Fluorescence emission spectroscopy and affinity chromatography suggest the probable interaction of daboxin P with factor X and factor Xa. Molecular docking analysis reveals the interaction of the Ca+2 binding loop; helix C; anticoagulant region and C-terminal region of daboxin P with the heavy chain of factor Xa. This is the first report of a phospholipase A2 enzyme from Indian viper venom which targets both factor X and factor Xa for its anticoagulant activity.

No MeSH data available.


Related in: MedlinePlus

Purification of the major protein from the crude venom of Indian Daboia r. russelii.(A): Size exclusion chromatography of crude D. r. russelii venom. 20 mg of crude venom was dissolved in 50 mM of Tris–Cl pH 7.4 and fractionated on Hiload 16/600 superdex 75 preparative grade column pre-equilibrated with the same buffer. Fractions were eluted at a flow rate of 1 ml/min and monitored at 215 & 280 nm. For each, 1 ml fractions were collected and peaks were pooled (P1 to P8). (B): Ion exchange chromatography profile of P6: The gel filtration peak, P6 was loaded onto CM FF 16/10, a weak cation exchanger column pre-equilibrated with 50 mM of Tris-Cl pH 7.4. Fractionation was carried out at a flow rate of 2.25 ml/min and eluted with a linear gradient of the same buffer containing 0.8 M NaCl and monitored at 215 nm. (C): Rp-HPLC profile of CM-II: Ion exchange fraction CM-II was loaded on Jupiter C18 column pre-equilibrated with buffer A (0.1% TFA). Fractionation was carried out at a flow rate of 0.8 ml/min with a linear gradient of buffer B (80% MeCN+0.1% TFA) and monitored at 215 nm. (D): Recalcification time of the fractions obtained from the chromatographic steps. Clotting time of plasma in presence of Tris-Cl buffer (20 mM, pH 7.4) was considered as normal clotting time (NCT). 1 μg of each fraction was incubated with plasma for 2 min followed by addition of 50 mM CaCl2 to initiate clot formation which was monitored using Tulip Coastat-1 coagulo analyser. (E): PLA2 activity of the chromatographic fractions using sPLA2 assay kit. 0.01 μg of each fraction was used for screening PLA2 activity using diheptanoylthio-phosphatidylcholine as the substrate. The amount of substrate hydrolyzed was quantified at 414 nm for 10 min at room temperature. * indicates the peak of interest in each purification step.
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pone.0153770.g001: Purification of the major protein from the crude venom of Indian Daboia r. russelii.(A): Size exclusion chromatography of crude D. r. russelii venom. 20 mg of crude venom was dissolved in 50 mM of Tris–Cl pH 7.4 and fractionated on Hiload 16/600 superdex 75 preparative grade column pre-equilibrated with the same buffer. Fractions were eluted at a flow rate of 1 ml/min and monitored at 215 & 280 nm. For each, 1 ml fractions were collected and peaks were pooled (P1 to P8). (B): Ion exchange chromatography profile of P6: The gel filtration peak, P6 was loaded onto CM FF 16/10, a weak cation exchanger column pre-equilibrated with 50 mM of Tris-Cl pH 7.4. Fractionation was carried out at a flow rate of 2.25 ml/min and eluted with a linear gradient of the same buffer containing 0.8 M NaCl and monitored at 215 nm. (C): Rp-HPLC profile of CM-II: Ion exchange fraction CM-II was loaded on Jupiter C18 column pre-equilibrated with buffer A (0.1% TFA). Fractionation was carried out at a flow rate of 0.8 ml/min with a linear gradient of buffer B (80% MeCN+0.1% TFA) and monitored at 215 nm. (D): Recalcification time of the fractions obtained from the chromatographic steps. Clotting time of plasma in presence of Tris-Cl buffer (20 mM, pH 7.4) was considered as normal clotting time (NCT). 1 μg of each fraction was incubated with plasma for 2 min followed by addition of 50 mM CaCl2 to initiate clot formation which was monitored using Tulip Coastat-1 coagulo analyser. (E): PLA2 activity of the chromatographic fractions using sPLA2 assay kit. 0.01 μg of each fraction was used for screening PLA2 activity using diheptanoylthio-phosphatidylcholine as the substrate. The amount of substrate hydrolyzed was quantified at 414 nm for 10 min at room temperature. * indicates the peak of interest in each purification step.

Mentions: Fractionation of crude venom of Daboia r. russelii on size exclusion chromatography has resolved it into 8 prominent peaks (P1 to P8) (Fig 1A). Each individual peaks were screened for PLA2 and anticoagulant activity. Peaks, P1 to P5 were found to be devoid of PLA2 activity but exhibited procoagulant effect on human plasma while peaks P6 to P8 showed PLA2 activity and anticoagulant effect on human plasma (Fig 1D & 1E). Peak P6 which exhibited highest anticoagulant (>600 s) and PLA2 activity (93.69 μmol/min) was subjected to cation exchange chromatography. It resolved P6 into one major (CM-II) and two minor peaks (CM-I & CM-III) (Fig 1B). The major peak CM-II which showed highest anticoagulant (>600 s) and PLA2 activity (77.82 μmol/min) (Fig 1D & 1E), was further fractionated using Rp-HPLC, which resolved into one minor (Rp-1) and one major (Rp-2) peaks (Fig 1C). The homogeneity of the major peak, Rp-2 with highest PLA2 activity and anticoagulant activity was assessed by SDS-PAGE under reduced state and named as daboxin P (Fig 2A). The molecular mass of the protein was determined by ESI-MS which showed the presence of 6 mass to charge (m/z) ratio peaks ranging from +8 to +13 charges corresponding to a deconvoluted molecular mass of 13597.62 ± 1.28 Da (Fig 2B).


Daboxin P, a Major Phospholipase A2 Enzyme from the Indian Daboia russelii russelii Venom Targets Factor X and Factor Xa for Its Anticoagulant Activity.

Sharma M, Iyer JK, Shih N, Majumder M, Mattaparthi VS, Mukhopadhyay R, Doley R - PLoS ONE (2016)

Purification of the major protein from the crude venom of Indian Daboia r. russelii.(A): Size exclusion chromatography of crude D. r. russelii venom. 20 mg of crude venom was dissolved in 50 mM of Tris–Cl pH 7.4 and fractionated on Hiload 16/600 superdex 75 preparative grade column pre-equilibrated with the same buffer. Fractions were eluted at a flow rate of 1 ml/min and monitored at 215 & 280 nm. For each, 1 ml fractions were collected and peaks were pooled (P1 to P8). (B): Ion exchange chromatography profile of P6: The gel filtration peak, P6 was loaded onto CM FF 16/10, a weak cation exchanger column pre-equilibrated with 50 mM of Tris-Cl pH 7.4. Fractionation was carried out at a flow rate of 2.25 ml/min and eluted with a linear gradient of the same buffer containing 0.8 M NaCl and monitored at 215 nm. (C): Rp-HPLC profile of CM-II: Ion exchange fraction CM-II was loaded on Jupiter C18 column pre-equilibrated with buffer A (0.1% TFA). Fractionation was carried out at a flow rate of 0.8 ml/min with a linear gradient of buffer B (80% MeCN+0.1% TFA) and monitored at 215 nm. (D): Recalcification time of the fractions obtained from the chromatographic steps. Clotting time of plasma in presence of Tris-Cl buffer (20 mM, pH 7.4) was considered as normal clotting time (NCT). 1 μg of each fraction was incubated with plasma for 2 min followed by addition of 50 mM CaCl2 to initiate clot formation which was monitored using Tulip Coastat-1 coagulo analyser. (E): PLA2 activity of the chromatographic fractions using sPLA2 assay kit. 0.01 μg of each fraction was used for screening PLA2 activity using diheptanoylthio-phosphatidylcholine as the substrate. The amount of substrate hydrolyzed was quantified at 414 nm for 10 min at room temperature. * indicates the peak of interest in each purification step.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835082&req=5

pone.0153770.g001: Purification of the major protein from the crude venom of Indian Daboia r. russelii.(A): Size exclusion chromatography of crude D. r. russelii venom. 20 mg of crude venom was dissolved in 50 mM of Tris–Cl pH 7.4 and fractionated on Hiload 16/600 superdex 75 preparative grade column pre-equilibrated with the same buffer. Fractions were eluted at a flow rate of 1 ml/min and monitored at 215 & 280 nm. For each, 1 ml fractions were collected and peaks were pooled (P1 to P8). (B): Ion exchange chromatography profile of P6: The gel filtration peak, P6 was loaded onto CM FF 16/10, a weak cation exchanger column pre-equilibrated with 50 mM of Tris-Cl pH 7.4. Fractionation was carried out at a flow rate of 2.25 ml/min and eluted with a linear gradient of the same buffer containing 0.8 M NaCl and monitored at 215 nm. (C): Rp-HPLC profile of CM-II: Ion exchange fraction CM-II was loaded on Jupiter C18 column pre-equilibrated with buffer A (0.1% TFA). Fractionation was carried out at a flow rate of 0.8 ml/min with a linear gradient of buffer B (80% MeCN+0.1% TFA) and monitored at 215 nm. (D): Recalcification time of the fractions obtained from the chromatographic steps. Clotting time of plasma in presence of Tris-Cl buffer (20 mM, pH 7.4) was considered as normal clotting time (NCT). 1 μg of each fraction was incubated with plasma for 2 min followed by addition of 50 mM CaCl2 to initiate clot formation which was monitored using Tulip Coastat-1 coagulo analyser. (E): PLA2 activity of the chromatographic fractions using sPLA2 assay kit. 0.01 μg of each fraction was used for screening PLA2 activity using diheptanoylthio-phosphatidylcholine as the substrate. The amount of substrate hydrolyzed was quantified at 414 nm for 10 min at room temperature. * indicates the peak of interest in each purification step.
Mentions: Fractionation of crude venom of Daboia r. russelii on size exclusion chromatography has resolved it into 8 prominent peaks (P1 to P8) (Fig 1A). Each individual peaks were screened for PLA2 and anticoagulant activity. Peaks, P1 to P5 were found to be devoid of PLA2 activity but exhibited procoagulant effect on human plasma while peaks P6 to P8 showed PLA2 activity and anticoagulant effect on human plasma (Fig 1D & 1E). Peak P6 which exhibited highest anticoagulant (>600 s) and PLA2 activity (93.69 μmol/min) was subjected to cation exchange chromatography. It resolved P6 into one major (CM-II) and two minor peaks (CM-I & CM-III) (Fig 1B). The major peak CM-II which showed highest anticoagulant (>600 s) and PLA2 activity (77.82 μmol/min) (Fig 1D & 1E), was further fractionated using Rp-HPLC, which resolved into one minor (Rp-1) and one major (Rp-2) peaks (Fig 1C). The homogeneity of the major peak, Rp-2 with highest PLA2 activity and anticoagulant activity was assessed by SDS-PAGE under reduced state and named as daboxin P (Fig 2A). The molecular mass of the protein was determined by ESI-MS which showed the presence of 6 mass to charge (m/z) ratio peaks ranging from +8 to +13 charges corresponding to a deconvoluted molecular mass of 13597.62 ± 1.28 Da (Fig 2B).

Bottom Line: It exhibits strong anticoagulant and phospholipase A2 activity but is devoid of any cytotoxic effect on the tested normal or cancerous cell lines.It is found to be stable at acidic (pH 3.0) and neutral pH (pH 7.0) and has a Tm value of 71.59 ± 0.46°C.Molecular docking analysis reveals the interaction of the Ca+2 binding loop; helix C; anticoagulant region and C-terminal region of daboxin P with the heavy chain of factor Xa.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biotechnology, Tezpur University, Tezpur-784028, Assam, India.

ABSTRACT
In the present study a major protein has been purified from the venom of Indian Daboia russelii russelii using gel filtration, ion exchange and Rp-HPLC techniques. The purified protein, named daboxin P accounts for ~24% of the total protein of the crude venom and has a molecular mass of 13.597 kDa. It exhibits strong anticoagulant and phospholipase A2 activity but is devoid of any cytotoxic effect on the tested normal or cancerous cell lines. Its primary structure was deduced by N-terminal sequencing and chemical cleavage using Edman degradation and tandem mass spectrometry. It is composed of 121 amino acids with 14 cysteine residues and catalytically active His48 -Asp49 pair. The secondary structure of daboxin P constitutes 42.73% of α-helix and 12.36% of β-sheet. It is found to be stable at acidic (pH 3.0) and neutral pH (pH 7.0) and has a Tm value of 71.59 ± 0.46°C. Daboxin P exhibits anticoagulant effect under in-vitro and in-vivo conditions. It does not inhibit the catalytic activity of the serine proteases but inhibits the activation of factor X to factor Xa by the tenase complexes both in the presence and absence of phospholipids. It also inhibits the tenase complexes when active site residue (His48) was alkylated suggesting its non-enzymatic mode of anticoagulant activity. Moreover, it also inhibits prothrombinase complex when pre-incubated with factor Xa prior to factor Va addition. Fluorescence emission spectroscopy and affinity chromatography suggest the probable interaction of daboxin P with factor X and factor Xa. Molecular docking analysis reveals the interaction of the Ca+2 binding loop; helix C; anticoagulant region and C-terminal region of daboxin P with the heavy chain of factor Xa. This is the first report of a phospholipase A2 enzyme from Indian viper venom which targets both factor X and factor Xa for its anticoagulant activity.

No MeSH data available.


Related in: MedlinePlus