الفهرس 
General Introduction of Snake VenomsOnly 14 pages are availabe for public view
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Abstract
The present study includes purification and characterization of a 5’–nucleotidase enzyme from crude Naja nigricollis venom.
In this work, the 5’NT was purified by three 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 5’NT activity but the most enzyme activity and the most specific activity is in fraction IIa, so we choose fraction IIa for purification by ion exchange chromatography.
The second step: Ion exchange chromatography on DEAE-sephadex A-50, elution was performed by using a starting buffer of Ammonium acetate (0.05 M, pH 6.8) for 30 test tubes then a gradient of ammonium acetate was performed from (0.05 M, pH 6.8) to (0.5 M, pH 8).Fraction IIa was resolved into 4 fractions designated as Ib, IIb, IIIb, IVb. Fractions (Ib, IIb, IIIb) were eluted in the starting buffer, while Fraction IVb was eluted in the gradient buffer. Only fraction IIb shows 5’NT activity, so we choose it for further purification by gel filtration chromatography on sephadex G-50.
The third step: gel filtration chromatography on sephadex G-50, elution was performed using ammonium acetate buffer (0.02 M, pH 4.8). Fraction IIb was resolved into 2 fractions: Ic, IIc. Only fraction IIc shows 5’NT activity.
Disc SDS-PAGE of fraction IIc obtained from gel filtration chromatography on sephadex G-50 showed two bands, the first band was at 50 kDa and the second one was at 35 kDa, which indicates partially purification or might indicate that fraction IIc is a heterodimer where the two subunits are linked by non-covalent bonds which are disrupted under denaturing conditions of SDS PAGE.
Fraction IIc (5’NT) shows carbohydrate content 60 μg/mg protein, optimum pH 7, with maximum activity at 37˚C, increase in 5’NT activity with CaCl2 and MgCl2.While MnCl2 has no effect on 5’NT activity.
The activity of the fraction IIc inhibited by serine protease inhibitors e.g benzamidine, PMSF, but thiol protease inhibitors e.g iodoacetate had no effect.
EDTA showed inhibitory effect on the 5’NT enzyme while the activity was restored 100 % and 70 % by adding 10 mM CaCl2 with 5mM, 10 mM EDTA respectively, as EDTA is Ca2+ chelator.
Furthermore kinetic study was done for 5’nucleotidase enzyme purified from Naja nigricollis venom (fraction IIc), Km of 2.7 mM and a Vmax of 0.465 u/ml/min were calculated from the Lineweaver-Burk plot. That was done by using AMP which is the most commonly used substrate for 5’nucleotidase enzyme .