Fig 1.
Purification of asparaginase from E. cloacae.
Elution profiles of asparaginase (a) Cation exchange chromatography of 60–90% ammonium sulphate precipitated fraction on DEAE-cellulose column (2×15 cm2, Sigma). The column was pre-equilibrated with 0.05 M Tris-HCl buffer (pH 9.6) and absorbed protein was eluted with a linear gradient of KCl (0–200 mM) prepared in 0.05 M Tris-HCl buffer (pH 8.6). (b) Gel filtration of fractions (5,6,7 and 8) from DEAE-cellulose column on a Sephadex G-100 column (2×20 cm2, Sigma), which was pre-equilibrated with 0.05 M Tris-HCl buffer (pH 8.6) and eluted with the same buffer at a flow rate of 0.2 ml min-1. (c) Assessment of homogeneity and molecular weight analysis of purified asparaginase on Native-PAGE, Lane 1- Protein marker; Lane 2 and 3- Sephadex G-100 purified asparaginase. (d) SDS-PAGE, Lane 1- Protein marker; Lane 2- Cell free crude asparaginase; Lane 3- Ammonium sulphate precipitated asparaginase; Lane 4-DEAE-cellulose purified asparaginase and Lane5- Sephadex G-100 purified asparaginase. (e) Two-dimensional electrophoretic resolution of purified asparaginase. Purified enzyme (30 μg) after resolved by IEF and SDS-PAGE gel was visualized by coomassie brilliant blue staining. Molecular weight was calculated with standard molecular weight markers.
Table 1.
Summary of various steps involved in purification of asparaginase from E. cloacae.
Fig 2.
Biochemical properties of asparaginase purified from E. cloacae.
Optimum pH (a) and pH stability of asparaginase (b) were determined by assessing the enzyme activity in the pH range of 4.5–10.5 with various pH buffers as follows: pH 4.5–7.5, 50 mM Potassium phosphate buffer; pH 8.0–10.5, 50 mM Tris-HCl buffer. Optimum temperature on the activity (c) and the thermostability of asparaginase (d) were determined at various temperatures (20–70°C). Effect of various metal ions (e) and modulators (f) on the asparaginase activity. Asparaginase was mixed with the corresponding metal salts (NaCl (50 mM), (KCl (150 mM), (CaCl2 (150 mM), (MgCl2 (40 mM), (ZnCl2 (100 mM), (MnCl2 (100 mM), (FeCl3 (100 mM), (CdCl2 (10 mM), (NiCl2 (10 mM), (HgCl2 (100 mM) or modulators EDTA (5 mM), DDT (5 mM), iodoacetamide (5 mM), SDS (2 mM), 2-mercaptoethanol (0.5 mM), glutathione (0.5 mM), L-cysteine (25 mM), thiourea (1 mM), and human serum (10%) in 50 mM phosphate buffer (pH 7.5) for 30 min at 37°C and enzyme activity was measured by Nesslerization reaction. No addition was used as control. Each value represents the mean ±SD for three determinations.
Table 2.
Substrate specificity of purified asparaginase from E. cloacae.
Fig 3.
The Lineweaver-Burk plot of E. cloacae asparaginase for calculating kinetic parameters.
The concentrations of the substrate L-asparagine were between 0.2–24 mM. The Km, Vmax and Kcat of purified asparaginase were 1.58×10−3 M, 2.22 IU μg-1, and 5.3×104 S-1, respectively. All data were average value of triplicate measurement.
Fig 4.
Physiological properties of asparaginase purified from E. cloacae.
In vitro (a) serum and (b) trypsin half life. Asparaginase was incubated with serum and trypsin, separately and relative activity was measured on the basis of the highest activity of purified asparaginase as 100%. Each value represents the mean ±SD for three determinations.
Fig 5.
Dose-dependent effect of asparaginases on viability of human cancer cells.
(a-d) MTT assay showing cytotoxic effect on HL-60, MOLT-4, MDA-MB-231, and T47D cells proliferation following treatment with purified asparaginase. Approximately, 1.5x104 cells ml-1 were cultured at 37°C, separately. Purified E. cloacae asparaginase and reference asparaginase preparation (E. coli asparaginase) procured from Sigma chemical Co. St. Louis were added at a concentration of 2, 5, 10, and 15 IU ml-1. After 48 h of treatments cells viability was determined by the MTT assay. Each value represents the mean ±SD for three determinations.
Fig 6.
Asparaginase induced morphological changes on HL-60 cells.
Cells were treated with the indicated concentrations of E. cloacae asparaginase and observed for morphological changes under microscope (1×81, Olympus). Photographs were taken by using DP-12 camera. The procedure is discussed in Materials and Methods.
Fig 7.
Alteration in nuclear morphology by treatment with purified E. cloacae asparaginase.
HL-60 cells were treated with different concentrations of E. cloacae asparaginase, collected after centrifugation at 1600 rpm, washed once with PBS, and then stained with DAPI for 10 min. The procedure is discussed in Materials and Methods.
Fig 8.
Effect of asparaginase purified from E. cloacae on cell cycle progression.
HL-60 cells (1×106) were seeded in 12-well plates and treated with different concentrations of purified E. cloacae asparaginase for 24 h. After treatment, cells were collected at 1600 rpm, washed once with PBS and fixed in 70% ethanol overnight. Cells were then washed once with PBS and stained with 100 μg of propidium iodide for 30 min. Modfit software was used to differentiate between phases and to determine the amount of apoptotic population. Histograms showed that G0/G1 phase arrest increases in dose dependent manner.
Fig 9.
Agarose gel electrophoresis of genomic DNA extracted from HL-60 cells following the treatment of asparaginase.
HL-60 cells treated with different concentrations of E. cloacae asparaginase for 24 h. Genomic DNA was isolated and electrophoresed on 1.8% agarose gel as described in Material and Method.
Fig 10.
(a) Morphological observation of asparaginase treated HL-60 cells after AO/EB double staining. HL-60 cells treated with indicated concentrations of E. cloacae asparaginase for 24 h, stained with AO/EB solution and immediately visualized under a fluorescence microscope. Live cells showed green color nuclei and apoptotic cells showed orange color nuclei. Photographs were taken by using DP-12 camera. (b) Percentages of apoptosis were calculated during each concentration of asparaginase treatment. Values are means ±SD of three independent experiments.
Fig 11.
Asparaginase induced concentration dependent MMP loss in HL-60 cells.
HL-60 cells (0.5×106) were treated for 24 h with the indicated concentrations of purified E. cloacae asparaginase, washed once with PBS, and stained with Rhodamine-123. MMP was measured as discussed in Materials and Methods.
Fig 12.
Toxicological evaluation of asparaginase purified from E. cloacae.
(a) Non-toxic effects of purified asparaginase on noncancerous human epithelial cell line FR-2. Approximately, 1.5x104 cells ml-1 were cultured at 37°C and treated with 2, 5, 10, and 15 IU ml-1 concentrations of purified asparaginasefor 48 h and cell viability was determined by the MTT assay. (b(i) Crude asparaginase formed clear translucent zone on blood agar plate due to lysis of erythrocytes. (b(ii) Purified asparaginase had no hemolytic effect. (c) Quantitative measure of the haemolytic activity. A: negative control (without asparaginase); B: positive control (Phosphate buffer); C: crude asparaginase; D: 15 IU ml-1; E: 7.5 IU ml-1; F: 3.75 IU ml-1 concentrations of asparaginase.