Fig 1.
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.
Fig 2.
Homogeneity of the purified protein, daboxin P.
(A) 12.5% glycine SDS-PAGE profile of the purified protein after silver staining. Lane 1: PageRulerTM pre-stained protein marker (170–10 kDa). Lane 2: daboxin P after treatment with β-mercaptoethanol. (B): ESI-MS spectra. The spectra show a series of multiple charged ions corresponding to a homogenous peptide. Inset: Reconstructed mass of daboxin P (cps: counts per second, amu: atomic mass unit).
Fig 3.
Primary structure of daboxin P.
(A): Amino acid sequence was deciphered by Edman degradation sequencing and ESI-LC MS/MS. The peptide sequences obtained from N-terminal sequencing and chemical cleavage by BNPS-skatole and hydroxylamine hydrochloride are shown with two headed solid arrows whereas peptide sequences obtained after ESI-LC MS/MS of the tryptic digested fragments are indicated with two headed doted arrows. (B): Multiple sequence alignment of daboxin P with the PLA2 enzymes from different subspecies of Daboia russelii (24638087: D. r. russelii, 408407675: D. r. siamensis, 31615955: D. r. pulchella, 49259309: D. r. russelii, 31615954: D r.pulchella, 109157490: D. r. pulchella, 48425253: D. r. pulchella, 298351762: D. r. russelii, 81174981: D. r. russelii). The conserved cys residues are highlighted in grey and the amino acid substitutions in daboxin P are underlined. * indicates the His residue at the active site. The predicted anticoagulant region is highlighted with a solid black line.
Fig 4.
Far-UV circular dichroism (CD) spectra of daboxin P (0.4 mg/ml).
(A): in milli q water at 25°C, (B): at different pH (3.0, 7.4 & 12) at 25°C. (C) Melting curve of daboxin P (dissolved in milli q water) at 222 nm considering temperature as a function. The curve was plotted using sigmoidal curve fit and Tm value was determined by Boltzman equation using Origin (OriginLab).
Fig 5.
Phospholipase A2 activity of daboxin P.
(A) Progress curve of diheptanoyl thio-PC cleavage by daboxin P, bee venom PLA2 enzyme and histidine modified daboxin P* at 414 nm. (B) Michaelis-Menten’s curve for sPLA2 assay. (C) The Lineweaver-Burk plot of daboxin P for determination of Km and Vmax.
Fig 6.
Cytotoxic effect of crude Daboia r. russelii venom and daboxin P.
Microscopic images were photographed at 10X magnification under Inverted microscope (Axio Vert A1., Zeiss) after treatment with venom samples for 24 h (A): HEK-293 cells treated with 0.9% NaCl were considered as negative control (B): HEK-293 cells treated with crude Daboia r. russelii venom (5 μg/ml) (C): HEK-293 cells treated with daboxin P (5 μg/ml) (D): MCF-7 cells treated with 0.9% NaCl were considered as negative control (E): MCF-7 cells treated with crude Daboia r. russelii venom (5 μg/ml) (F): MCF-7 cells treated with daboxin P (5 μg/ml). Percentage cell viability (G): HEK-293; (H): MCF-7 after treatment with crude venom and daboxin P using MTT based colorimetric assay. Percentage cell viability was calculated by considering the cells without venom treatment as 100% viable. The results are mean ± SD of three independent experiments.
Fig 7.
Anticoagulant activities of daboxin P on platelet poor human plasma (PPP).
(A): Recalcification time, different concentrations of daboxin P (0.001, 0.01, 0.1) were pre-incubated with 150 μl of plasma at 37°C for 2 min. 100 μl of 50 mM CaCl2 was added to initiate clot formation. (B): Activated partial thromboplastin time, daboxin P (0.01, 0.1 & 1 μM) was pre-incubated with 50 μl of plasma and 50 μl APTT reagent (Liquecelin) for 3 min at 37°C. 50 μl of 25 mM CaCl2 was added to form clot. (C): Prothrombin time, different concentrations of daboxin P (0.01, 0.1 & 1 μM) were pre-incubated with 50 μl of plasma at 37°C for 2 min. 50 μl of PT reagent (Uniplastin) was added to initiate the clot formation. (D): Stypven time, daboxin P (0.01, 0.1 & 1 μM) was pre-incubated with 75 μl plasma for 3 min at 37°C. 75 μl RVV-X (10 ng/ml) was added and incubated for 2 min. 25 mM of CaCl2 was added to initiate clot formation. For all the experiments, the clot formation was monitored using Tulip Coastat-1 coagulo analyser and the time taken for clot formation in the presence of Tris-Cl buffer (20 mM, pH 7.4) was considered as normal clotting time (NCT). The results are mean ± SD of three independent experiments.
Fig 8.
Effect of daboxin P on the time to occlusion (TTO) in FeCl3 induced carotid artery thrombosis in mice.
C57BL/6 male mice anesthetized with ketamine (75 mg/kg) and medetomidine (1 mg/kg) (i.p) were injected (i.p.) with daboxin P (10 mg/kg) in tail vein. Saline treated mice were considered as negative control. Each data-point represents the time-to-occlusion (TTO) of a single mouse. Maximum experimental time was considered for 60 min after FeCl3 induction.
Fig 9.
Percentage residual amidolytic activity of various serine proteases and complexes pre-incubated with daboxin P.
(A): Residual activity of FXIIa, FXIa, FXa, FIXa and FVIIa; (B): Activity of extrinsic tenase complex (ETC) (C): intrinsic tenase complex (ITC), in the presence or absence of phospholipid and alkylated daboxin P (indicated by *). (D): Residual activity of prothrombinase complex. Daboxin P was either pre-incubated with FXa followed by addition of FVa (pre-complex) or after reconstitution of FXa-FVa complex (post-complex). The rate of hydrolysis of respective chromogenic substrates for all the assays was measured at 405 nm using Multiskan Go spectrophotometer. Activity of the serine protease/complex without daboxin P was considered as 100%. The results are mean ± SD of three independent experiments.
Fig 10.
Inhibition curve (IC50) of daboxin P for the extrinsic and intrinsic tenase complex.
Different concentrations of daboxin P (1x10-6, 1x10-5, 1x10-4, 1x10-3, 1x10-2, 1x10-1, 1, 3x10-6, 3x10-5, 3x10-4, 3x10-3, 3x10-2, 3x10-1 and 3 μM) were pre-incubated with reconstituted tenase complexes as described above. The IC50 was calculated by fitting the points by non-linear curve fit using Origin (OriginLab, Northampton, MA).
Fig 11.
Interaction of daboxin P with FX and FXa.
(A): Fluorescence emission spectra of daboxin P, FX, and the complex (daboxin P + FX). (B) Fluorescence emission spectra of daboxin P, FXa, and the complex (daboxin P + FXa). 1 μM of daboxin P (DP) was pre-incubated with either 0.05 μM of FX or 0.1 μM of FXa for different time intervals (10 min & 20 min) at room temperature. The emission spectra of the individual proteins and the complexes were measured from 200 to 500 nm with an excitation wavelength of 280 nm using quartz cuvette (1 cm path length). (C): Electrophoretic profile of the flow through and elute obtained after affinity column chromatography. Lane 1: PageRulerTM Plus pre-stained protein ladder (250–10 kDa), Lane 2: flow through (FX) Lane 3: FX after elution with 1.0 M NaCl, Lane 4: control (FX); Lane 5: blank; Lane 6: PageRulerTM Plus pre-stained protein ladder (250–10 kDa), Lane 7: flow through (FXa); Lane 8: FXa after elution with 1.0 M NaCl, Lane 9: control (FXa).
Fig 12.
3D ribbon model of the docked complex of FXa and daboxin P.
The interface surface residues involved in the interaction were predicted by PDBsum. The Ca+2 binding loop (Trp30, Gly31, Gly32); helix C (Asp48, Tyr51, Gly52); anticoagulant region (Asn58) and C-terminal region (Phe113) of daboxin P interact with the heavy chain of FXa (Thr132, Arg165, Lys169, Asn166, Leu170, Tyr225 and Arg125) are represented in scaled ball and stick. Inset: Diagram illustrating the interaction of the seven residues of chain B (heavy chain of FXa) with eight residues of chain C (daboxin P) as predicted by PDBsum server. Orange line denotes non-bonded contacts and blue line denote hydrogen bond.
Table 1.
Critical residues of daboxin P and FXa involved in interaction based on PDBsum analysis.
Fig 13.
Contact map of daboxin P-FXa complex generated using online server Contact Map Analysis.
(A): residue to residue contact of light chain of FXa and daboxin P (B): residue to residue contact of heavy chain of FXa and daboxin P. The residue to residue contact area of the interacting amino acid residues for the chains has been considered above 8 Å2 for the design of the contact map.