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العنوان
Purification and characterization Of
L-Amino acid oxidase from Naja
Nigricollis Venom\
المؤلف
Labeb, Mariam Ezzat.
هيئة الاعداد
باحث / Mariam Ezzat Labeb
مشرف / Mohamed Farid El Asmer
مشرف / Hanaa El Tayeb Nasser
مناقش / Ayman Ragaa Basheer
تاريخ النشر
2014.
عدد الصفحات
163P. :
اللغة
الإنجليزية
الدرجة
ماجستير
التخصص
Organic Chemistry
تاريخ الإجازة
1/1/2014
مكان الإجازة
جامعة عين شمس - كلية الطب - Basic Science (Biochemistry)
الفهرس
Only 14 pages are availabe for public view

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from 163

Abstract

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.
Summary
‐ 117 ‐
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
Summary
‐ 118 ‐
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.