الفهرس 
Snake Venom Phospholipase A2Only 14 pages are availabe for public view
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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.