Copper amine oxidases are important enzymes, which contribute to the regulation of monoand

polyamine levels. Each monomer contains one Cu(II) ion and 2,4,5-trihydroxyphenylalanine

(TPQ) as cofactors. They catalyze the oxidative deamination of primary amines to aldehydes

with a ping-pong mechanism consisting of a transamination. The mechanism is followed by the

transfer of two electrons to molecular oxygen which is reduced to hydrogen peroxide. Inhibitors

are important tools in the study of catalytic properties of copper amine oxidases and they also

have a wide application in physiological research. In this study, purification of the chickpea

seedling amine oxidase, was done via salting out by ammonium sulfate and dialysis, followed by

DEAE-cellulose column chromatography. By using the Lineweaver - Burk plot, the Km and Vm

of the enzyme were found to be 3.3 mM and 0.95 mmol/min/mg, respectively. In this study, the

interaction of chickpea diamino oxidase with tetraethylene- pentamine was studied. Analysis of

kinetic data indicated that tetraethylenepentamine (with Ki=0.1 mM) inhibits the enzyme by

linear mixed inhibitory effect

اسدی, اسداله

امانی, مجتبی

طلایی, سونیا

English

ArUMS Digital Repository (سامانه اطلاعات زیست پزشکی و سلامت)

Inhibition of chickpea seedling copper amine oxidases by

tetraethylenepentamine

Molecular Biology Research Communications 2012;1(1):27-32 MBRC
* Address for correspondence: Deptarment of Biology, Faculty of Science, University of Mohaghegh Ardabili, Ardabil, Iran
E-mail: asady@uma.ac.ir
ISSN 2322-181X
Original Article Open Access
Inhibition of chickpea seedling copper amine oxidases by
tetraethylenepentamine
Sona Talaei1, Asadollah Asadi1,*, Mojtaba Amani2
1) Deptarment of Biology, Faculty of Science, University of Mohaghegh Ardabili, Ardabil, Iran.
2) Deptarment of  Science, Faculty of Medicine, University of Medical science, Ardabil, Iran.
A B S T R A C T
Copper amine oxidases are important enzymes, which contribute to the regulation of mono-
and polyamine levels. Each monomer contains one Cu(II) ion and 2,4,5-trihydroxyphenylalanine
(TPQ) as cofactors. They catalyze the oxidative deamination of primary amines to aldehydes
with a ping-pong mechanism consisting of a transamination. The mechanism is followed by the
transfer of two electrons to molecular oxygen which is reduced to hydrogen peroxide. Inhibitors
are important tools in the study of catalytic properties of copper amine oxidases and they also
have a wide application in physiological research. In this study, purification of the chickpea
seedling amine oxidase, was done via salting out by ammonium sulfate and dialysis, followed by
DEAE-cellulose column chromatography. By using the Lineweaver - Burk plot, the Km and Vm
of the enzyme were found to be 3.3 mM and 0.95 mmol/min/mg, respectively. In this study, the
interaction of chickpea diamino oxidase with tetraethylene- pentamine was studied. Analysis of
kinetic data indicated that tetraethylenepentamine (with Ki=0.1 mM) inhibits the enzyme by
linear mixed inhibitory effect.
Key words: Chickpea, Copper-containing amine oxidases, Tetraethylenepentamine, Linear
mixed.
INTRODUCTION
Amine oxidases (AOs) present a class of enzymes, which are divided into two main groups
based on the chemical nature of the attached cofactor, including the AOs containing flavin
adenine dinucleotide (FAD-AOs), and copper-containing amine oxidases (CuAOS), which have a
tightly bound copper ion in their molecule [1-9]. FAD-AOS and CuAOS are present in all kinds of
organisms such as bacteria, yeasts, mushrooms, plants, and animals. They catalyze the oxidative
deamination of mono- and polyamines (essential compounds for cell growth and proliferation) to
the corresponding aldehydes and hydrogen peroxide according to the following reaction [2, 5-
10]: RCH2NH2 + O2 + H2O → RCHO + NH3 + H2O2.
Asadi et al. /Mol Biol Res Commun 2012;1:27-32 MBRC
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Therefore, amine oxidases and products deriving from the oxidation of amines are essential in
physiological processes. Inhibitors play important roles in the study of catalytic properties of
AOs and can be divided into reversible and irreversible groups [11, 12]. The use of inhibitors
provides substantial information to understand the catalytic mechanism of plant copper amine
oxidases [3]. Mixed inhibition refers to a combination of competitive and uncompetitive
inhibition. In mixed inhibition, the inhibitor binds to the free enzyme or the enzyme-substrate
complex, which is a site different from the active site where the substrate binds [13, 14]. In the
present study, we report on the inhibitory effect of tetraethylenepentamine on the chickpea
seedling amine oxidase.
MATERIALS AND METHODS
Chemicals: DEAE-cellulose, horseradish peroxidase and putrescine dihydrochloride were
purchased from Sigma-Aldrich. Tetraethylenepentamine and guaiacol were purchased from
Merck.
Protein purification: The preparation of a sufficient amount of purified chickpea amine
oxidase was carried out in three steps: (a) the seeds’ germination, (b) ammonium sulfate
fractionation, (c) ion-exchange chromatography on diethylaminoethylcellulose. The last two steps
were carried out at 4 °C.
Germination: Chickpea (Cicer arietinum) seeds were washed with 0.5% KMnO4 solution to
keep mold and bacteria from growing. Chickpea seeds, layered on suitable wet cotton containing
trays, were watered with warm tap water (30 °C) for 2 min. This procedure was repeated every
30 min. The trays were kept at a temperature 28 °C.
Homogenate preparation and ammonium sulfate fractionation: After 3–6 days of
germination, the seedlings were collected. Upon excision of the cotyledons, the epicotyls and
roots were homogenized in a Waring blender, with an equal amount (v/w) of 0.2 M potassium
phosphate, pH 7.2, containing EDTA (1 mM) and 0.1mM phenylmethylsulfonyl fluoride as
protease inhibitor.
The homogenate was squeezed through a clean cotton cloth and the obtained crude extract
was treated with 35% saturated ammonium sulfate. After 1h incubation, the solution was
centrifuged at 13,500g for 30min and the pellet was discarded. The supernatant was again
precipitated with 65% saturated ammonium sulfate, and after 1h incubation, a new pellet was
collected by centrifugation (13,500g for 30 min).
The pellet was dissolved in a small volume of 10 mM potassium phosphate, pH 6.8, and
dialyzed overnight against the same buffer. The dialyzed solution was frozen in liquid nitrogen
and stored at -80 °C.
Diethylaminoethyl-cellulose chromatography: For further purification, the dialysed
solution was loaded to a diethylaminoethyl (DEAE) cellulose column (Ø = 1.5 cm, l = 50 cm)
equilibrated with 0.2 M potassium phosphate, pH 6.8, containing 0.1 mM EDTA. The column
was washed with the same buffer. The enzyme was eluted from the column, while most of the
impurities were retained by the column. All purification steps were carried out at 4 °C. SDS–
Asadi et al. /Mol Biol Res Commun 2012;1:27-32 MBRC
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polyacrylamide gel electrophoresis were performed according to the Laemmli method [15]. Gels
were stained for the protein with Coomassie brilliant blue G-250 and Silver Nitrate.
Enzyme assay: Chickpea amine oxidase activity was measured by the oxidation of guaiacol
in the presence of hydrogen peroxide and horseradish peroxidase. The reaction mixture contained
0.1 M potassium phosphate buffer, pH 7.0, 130 mM putrescine dihydrochloride as a substrate, 1
U/ml horseradish peroxidase and 5 mM guaiacol in a final volume of 1 ml. The increase in
absorbance at 470 nm was recorded using a Rayleigh UV–2100 Spectrophotometer.
Measurements of inhibitory kinetics: The inhibitory effect of tetraethylenepentamine on
chickpea amine oxidase was measured by the oxidation of guaiacol in the presence of hydrogen
peroxide and horseradish peroxidase. The reaction mixture contained 0.1 M potassium phosphate
buffer, pH 7.0, 1.6-51.2 mM putrescine dihydrochloride as a substrate, 1 U/ml horseradish
peroxidase, 5 mM guaiacol and 2.5 mM tetraethylenepentamine as inhibitor in a final volume of
1 ml. The decrease in absorbance at 470 nm was recorded using a UV–2100 Spectrophotometer.
RESULTS AND DISCUSSION
The purified enzyme, obtained according to the procedure, was characterized by gel
electrophoresis and spectroscopic techniques. Apparent molecular weights estimated, from SDS–
PAGE gels, to be about 75 kDa for each consisting monomer (Figure 1). The efficiency of
different amine oxidase purification steps is shown in Table 1.
3       2       1
b a
Figure 1: Polyacrylamide gel electrophoresis of purified chickpea seedling amine oxidase. Lane 1, molecular
weight standards; lane 2, chickpea seedling amine oxidase purified by ammonium sulfate; lane 3, chickpea seedling
amine oxidase purified by diethylaminoethylcellulose chromatography. b) The gel was stained for proteins with
Silver Nitrate, chickpea seedling amine oxidase purified by diethylaminoethylcellulose chromatography.
Table 1. Characterization of the steps of the amine oxidase purification process from chickpea seedlings
Fraction Protein (mg/ml) Activity (U) Specific activity (U/mg)
Crude enzyme 30 0.0364 0.001
(NH4)2SO4 fractionation 8 0.0582 0.007
DEAE-cellulose chromatography 1.05 0.0606 0.057
Asadi et al. /Mol Biol Res Commun 2012;1:27-32 MBRC
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We found that tetraethylenepentamine inhibited chickpea amine oxidase with the inhibition
constants Ki = 0.1 mM.
Several reversible and irreversible inhibitors have been described and used to investigate the
structure–function relationships of plant amine oxidases [16-18]. For instance, diamino- and
monoamino-ketonic compounds were synthesized and evaluated as inhibitors of pea seedling
amine oxidase [19]. Noncompetitive inhibition of plant amine oxidases has also been observed
with chelating agents such as cyanide and azide [20]. Moreover, diethylentriamine (Ki = 72 mM)
and triethylentriamine (Ki = 0.57 mM) have been found to be strong noncompetitive inhibitors of
pea seedling amine oxidase [21]. Pyridine carbaldoximes and alkyl pyridyl ketoximes also act as
strong noncompetitive inhibitors of pea seedling amine oxidase. Finally, competitive inhibition of
pea seedling amine oxidase by tetraethylenepentamine (Ki = 20 μM) and pentaethylenehexamine
(Ki = 148 μM) were reported by Vianello et al [22].
By analyzing the Lineweaver - Burk plot, the kinetic parameters Km and Vm of the purified
enzyme (by using guaiacol oxidation in the presence of hydrogen peroxide, peroxidase and
putrescine dihydrochloride as a substrate) were found to be 3.3 mM and 0.95 mmol/min/mg,
respectively (Figure 2).
Figure 2: Kinetic parameters of chickpea diamine oxidase
The results showed that the presence of tetraethylenepentamine increased the Km value by
62.85 mM and reduced the Vm value by 0.55 mmol/min/mg (Figure 3).
Figure 3: Kinetic parameters of chickpea diamine oxidase in the presence of tetraethylenepentamine
R² = 0.97
0
1
2
3
4
5
0 0.2 0.4 0.6 0.8
1/V
1/Km
0
10
20
30
40
50
0 0.1 0.2 0.3 0.4
1/V
1/Km
Asadi et al. /Mol Biol Res Commun 2012;1:27-32 MBRC
http://mbrc.shirazu.ac.ir 31
Therefore, the inhibitory effect of tetraethylenepentamine on chickpea amine oxidase is a
linear mixed inhibition and could hence be applied as an effective tool for further research on the
enzyme-bound copper function in chickpea amine oxidase, which aids to elucidate the structure-
function relationships and its detailed physiological roles. In a linear mixed inhibition, the
inhibitor can bind to the enzyme at the same time as the substrate; in other words, the binding of
the inhibitor affects the substrate's binding, and vice versa. Although this type of inhibition can
be reduced, it cannot overcome the increments in substrate concentration. In this case, increasing
the concentration of putrescine dihydrochloride can only reduce the inhibition in a linear mixed
manner. This type of inhibition generally results from an allosteric effect where the inhibitor
binds to a different site on an enzyme [13, 14]. In other words, the binding of
tetraethylenepentamine to this allosteric site changes the conformation (tertiary structure or three-
dimensional shape) of the amine oxidase so that the affinity of the putrescine dihydrochloride for
the active site is reduced.
Acknowledgments
This study was funded by the Iran National Foundation of Sciences (INFS) and the Research
Council of the University of Mohaghegh Ardabili.
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http://eprints.arums.ac.ir/3760/1/oxidases.pdf

Inhibition of chickpea seedling copper amine oxidases by tetraethylenepentamine

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