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The present study includes purification and characterization of a L-Amino acid oxidase enzyme from crude Naja nigricollis venom.
In this work, the LAAO 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 5.8,). Six fractions were obtained: Ia, IIa, IIIa, IVa, Va, and VIa. All fractions show LAAO activity but more enzyme activity in fraction Ia and VIa and the highest specific activity in fraction VIa then, in fraction Ia, but we choose fraction Ia for further purification by cation exchange chromatography due to:
1- Its molecular weight near from reported M.W of LAAO.
2- The total protein in fraction Ia =1.5 mg, but in fraction VIa =0.5 mg (total protein in fraction Ia more than in fraction VIa) and can use it in extra purification steps.
3- Molecular weight of protein in fraction Ia is more than molecular weight of protein in fraction VIa, so we can apply ultrafilteration to fraction Ia without losing the protein because the ultrafilteration membrane pores can pass proteins of MW less than 10.000 kDa.
The second step: Cation exchange chromatography on CM-sephadex C-25, elution was performed using Na acetate buffer (0.05 M, pH 5.8) for 20 test tubes then NaCl gradient (from 0.1 M to 1 M) by gradient mixer. Fraction Ia was resolved into 9 fractions designated as Ib, IIb, IIIb, IVb, Vb, VIb, VIIb, VIIIb and IXb. Only fraction VIIb shows LAAO activity.
All fractions obtained from these purification steps were preserved in glycerol 50% to prevent ice crystals formation which cause loss of enzyme activity.
Disc SDS-Page of fraction VIIb obtained from cation exchange chromatography showed single thick band which indicate either partially purified or due to differential glycosylation produces isoenzymes of close molecular weight. Its approximate molecular weight was 63 kDa.
Fraction VIIb (LAAO) shows carbohydrate content 400 μg/mg protein, optimum pH 7, with thermal stability up to 60 ˚C but maximum activity at 25˚C, increase in LAAO activity with CaCl2 and MnCl2. While MgCl2 has no effect on LAAO activity.
The activity of the fraction inhibited by serine protease inhibitors e.g benzamidine, PMSF, but thiol protease inhibitors e.g iodoacetate had no effect. Glutathione (reducing agent) has inhibitory effect on LAAO while β -mercaptoethanol (reducing agent) has no inhibitory effect on LAAO enzyme.
EDTA showed inhibitory effect on the LAAO 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 LAAO purified from Naja nigricollis venom (fraction VIIb), Km of 0.003 mM and a Vmax of 0.008 u/ml were calculated from the Lineweaver-Burk plot. That was done by using L-phenylalanine which is the most commonly used substrate for LAAO.