Purification and Characterization of Phospholipase A2 Enzyme from Naja nigricollis Snake Venom
NESMA MOHAMED FAWZY MOSTAFA;
Abstract
The present study includes purification and characterization of a phospholipase A2 enzyme from crude Naja nigricollis venom.
In this work, the PLA2 was purified by two steps of fractionation.
The first step: gel filtration chromatography on sephadex G-75, using ammonium acetate buffer (0.02 M, pH 4.8). Six fractions were obtained: Ia, IIa, IIIa, IVa, Va, and VIa. All fractions show PLA2 activity but the highest enzyme activity and the specific activity in fraction Va, so we choose fraction Va for purification by ion exchange chromatography.
The second step: Ion exchange chromatography on DEAE-Sephadex A-50, elution was performed in a stepwise manner using 50 ml of each of the following solutions: 1. 0.05 M ammonium acetate buffer, pH 8.
2. 0.15 M ammonium acetate buffer, pH 7.
3. 0.3 M ammonium acetate buffer, pH 6.
4. 0.5 M ammonium acetate buffer, pH 5.
Fraction Va was resolved into 5 fractions designated as Ib, IIb, IIIb, IVb, Vb. Only fraction Ib shows PLA2 activity.
Disc SDS-Page of fraction Ib obtained from Ion exchange chromatography showed single band. Its approximate molecular weight was 25 kDa.
Fraction Ib (PLA2) shows carbohydrate content 100 μg/mg protein, optimum pH 7, with thermal stability up to 60˚C but maximum activity at 40˚C, increase in PLA2 activity with CaCl2 and MgCl2. While MnCl2 has no effect on PLA2 activity.
The activity of the fraction inhibited by phenol (5%), formaldehyde (0.6%) and thiol protease inhibitors e.g., iodoacetate, but phenol (1%) had no effect on PLA2 enzyme activity.
EDTA showed inhibitory effect on the PLA2 enzyme while the activity was restored 100 % and 80 % by adding 10 mM CaCl2 with 5mM, 10 mM EDTA respectively, as EDTA is Ca2+ chelator.
Furthermore kinetic study was done for PLA2 purified from Naja nigricollis venom (fraction Ib), Km of 0.13 mM and a Vmax of 14.2 moles/min were calculated from the Lineweaver-Burk plot. That was done by using Leicithin which is the most commonly used substrate for PLA2.
In this work, the PLA2 was purified by two steps of fractionation.
The first step: gel filtration chromatography on sephadex G-75, using ammonium acetate buffer (0.02 M, pH 4.8). Six fractions were obtained: Ia, IIa, IIIa, IVa, Va, and VIa. All fractions show PLA2 activity but the highest enzyme activity and the specific activity in fraction Va, so we choose fraction Va for purification by ion exchange chromatography.
The second step: Ion exchange chromatography on DEAE-Sephadex A-50, elution was performed in a stepwise manner using 50 ml of each of the following solutions: 1. 0.05 M ammonium acetate buffer, pH 8.
2. 0.15 M ammonium acetate buffer, pH 7.
3. 0.3 M ammonium acetate buffer, pH 6.
4. 0.5 M ammonium acetate buffer, pH 5.
Fraction Va was resolved into 5 fractions designated as Ib, IIb, IIIb, IVb, Vb. Only fraction Ib shows PLA2 activity.
Disc SDS-Page of fraction Ib obtained from Ion exchange chromatography showed single band. Its approximate molecular weight was 25 kDa.
Fraction Ib (PLA2) shows carbohydrate content 100 μg/mg protein, optimum pH 7, with thermal stability up to 60˚C but maximum activity at 40˚C, increase in PLA2 activity with CaCl2 and MgCl2. While MnCl2 has no effect on PLA2 activity.
The activity of the fraction inhibited by phenol (5%), formaldehyde (0.6%) and thiol protease inhibitors e.g., iodoacetate, but phenol (1%) had no effect on PLA2 enzyme activity.
EDTA showed inhibitory effect on the PLA2 enzyme while the activity was restored 100 % and 80 % by adding 10 mM CaCl2 with 5mM, 10 mM EDTA respectively, as EDTA is Ca2+ chelator.
Furthermore kinetic study was done for PLA2 purified from Naja nigricollis venom (fraction Ib), Km of 0.13 mM and a Vmax of 14.2 moles/min were calculated from the Lineweaver-Burk plot. That was done by using Leicithin which is the most commonly used substrate for PLA2.
Other data
| Title | Purification and Characterization of Phospholipase A2 Enzyme from Naja nigricollis Snake Venom | Other Titles | تنقية ودراسة خواص انزيم الفوسفوليبازأ2 من سم ثعبان الناجا نيجريكوليس | Authors | NESMA MOHAMED FAWZY MOSTAFA | Issue Date | 2015 |
Attached Files
| File | Size | Format | |
|---|---|---|---|
| G12116.pdf | 306.29 kB | Adobe PDF | View/Open |
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