Zymographic assay of plant diamine oxidase on entrapped peroxidase polyacrylamide gel electrophoresis. A study of stability to proteolysis.

Abstract

A zymographic assay of diamine oxidase (DAO, histaminase, EC 1.4.3.6), based on a coupled peroxidase reaction, and its behavior at proteolysis in simulated gastric and intestinal conditions, are described. The DAO activity from a vegetal extract of Lathyrus sativus seedlings was directly determined on sodium dodecyl sulfate polyacrylamide electrophoretic gels containing entrapped horseradish peroxidase, with putrescine as substrate of histaminase and ortho-phenylenediamine as co-substrate of peroxidase. The accumulation of azo-aniline, as peroxidase-catalyzed oxidation product, led to well-defined yellow-brown bands on gels, with intensities corresponding to the enzymatic activity of DAO. After image analysis of gels, a linear dependency of DAO content (Coomassie-stained protein bands) and of its enzymatic activity (zymographic bands) with the concentration of the vegetal extract was obtained. In simulated gastric conditions (pH 1.2, 37 degrees C), the DAO from the vegetal extract lost its enzymatic activity before 15 min of incubation, either in the presence or absence of pepsin. The protein pattern (Coomassie-stained) revealed that the DAO content from the vegetal extract was kept almost constant in the simulated intestinal fluid (containing pancreatin or not), with a slight diminution in the presence of pancreatic proteases. After 10 h of incubation at 37 degrees C, the DAO enzymatic activity from the vegetal extract was 44.7% in media without pancreatin and 13.6% in the presence of pancreatin, whereas the purified DAO retained only 4.65% of its initial enzymatic activity in the presence of pancreatin.

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ORIGINAL PAPER
Zymographic assay of plant diamine oxidase on entrapped
peroxidase polyacrylamide gel electrophoresis. A study
of stability to proteolysis
Carmen Calinescu & Rodolfo Federico &
Bruno Mondovi & Mircea Alexandru Mateescu
Received: 4 August 2009 /Revised: 6 November 2009 /Accepted: 8 November 2009 /Published online: 29 November 2009
# The Author(s) 2009. This article is published with open access at Springerlink.com
Abstract A zymographic assay of diamine oxidase (DAO,
histaminase, EC 1.4.3.6), based on a coupled peroxidase
reaction, and its behavior at proteolysis in simulated gastric
and intestinal conditions, are described. The DAO activity
from a vegetal extract of Lathyrus sativus seedlings was
directly determined on sodium dodecyl sulfate polyacryl-
amide electrophoretic gels containing entrapped horseradish
peroxidase, with putrescine as substrate of histaminase and
ortho-phenylenediamine as co-substrate of peroxidase. The
accumulation of azo-aniline, as peroxidase-catalyzed oxi-
dation product, led to well-defined yellow-brown bands on
gels, with intensities corresponding to the enzymatic
activity of DAO. After image analysis of gels, a linear
dependency of DAO content (Coomassie-stained protein
bands) and of its enzymatic activity (zymographic bands)
with the concentration of the vegetal extract was obtained.
In simulated gastric conditions (pH 1.2, 37 °C), the DAO
from the vegetal extract lost its enzymatic activity before
15 min of incubation, either in the presence or absence of
pepsin. The protein pattern (Coomassie-stained) revealed
that the DAO content from the vegetal extract was kept
almost constant in the simulated intestinal fluid (containing
pancreatin or not), with a slight diminution in the presence
of pancreatic proteases. After 10 h of incubation at 37 °C,
the DAO enzymatic activity from the vegetal extract was
44.7% in media without pancreatin and 13.6% in the
presence of pancreatin, whereas the purified DAO retained
only 4.65% of its initial enzymatic activity in the presence
of pancreatin.
Keywords Diamineoxidase.Zymographicassay.
Entrappedperoxidase polyacrylamidegel.Proteolytic
stability.Simulatedgastro-intestinal conditions
Introduction
Plant diamine oxidases (DAOs), also referred as histami-
nases [1], are homodimeric copper amine oxidases (EC
1.4.3.6), each subunit containing a single copper ion and
2,4,5-trihydroxyphenylalanine quinone/TPQ, a cofactor
derived from the post-translational oxidation of a tyrosine
residue [2]. They present a high specificity for primary
diamines, able to oxidate biogenic amines to corresponding
aldehyde, ammonia (NH3), and hydrogen peroxide (H2O2).
Current DAO assays in solution measure the amine oxidase
activity by spectrophotometrical methods, monitoring
directly the absorbance of formed aldehydes [3] or by
subsequent condensation of different compounds [4]. Other
methods are based on radiometric assays, with [1,4-14C]
putrescine as substrate [5], on oxymetric or polarographic
methods measuring the rate of oxygen consumption in the
presence of substrate [6] or on fluorimetric determinations
[7], where homovanillic acid is converted into a highly
fluorescent compound by the released H2O2in the presence
of peroxidase. All these methods are not giving information
C. Calinescu:M. A. Mateescu (*)
Department of Chemistry and Centre BioMed,
Université du Québec à Montréal,
CP 8888, Succ. A,
Montréal, QC H3C 3P8, Canada
e-mail: mateescu.m-alexandru@uqam.ca
R. Federico
Department of Biology, 3rd University of Rome,
00146 Rome, Italy
B. Mondovi
Department of Biochemical Sciences “Rossi-Fanelli”,
University of Rome “La Sapienza”,
00185 Rome, Italy
Anal Bioanal Chem (2010) 396:1281–1290
DOI 10.1007/s00216-009-3306-7

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on the loss of molecular integrity of DAO (i.e., to acidic or
proteolytic hydrolysis). Thus, supplemental information can
be obtained using polyacrylamide gel electrophoresis (PAGE)
by monitoring the protein pattern (staining gels with
Coomassie Blue) and the enzymatic activity (zymography).
There are several major advantages of polyacrylamide
(PAA) gels, such as: the high homogeneity of the gels with
the density which can be easily modified to allow the best
enzymes separation. The gels can be stained with Coomassie
Brilliant Blue for protein profile and, keeping the same
running conditions, a zymographic pattern can be directly
visualized in some specific conditions and the enzyme
activity can be quantified by densitometry. The zymographic
PAGE is easy to run and the results are highly reproducible.
As the H2O2is the product of almost all oxidases, the gel
areas occupied by DAO after its electrophoretic separation
can be visualized via a coupled peroxidase reaction.
Peroxidase as second enzyme is widely used to detect
oxidase-producing H2O2in presence of oxidizable dyes as
its second substrate. Frequently used as donor substrate of
peroxidase are: tetramethyl benzidine [8], 3,3′-diaminoben-
zidine [9], ortho-dianisidine [10], and guaiacol [11]. The
chromogenic guaiacol method is one of the most commonly
used on gels. However, this method has the disadvantage
that the bands are stable only for a short period, requiring
stabilization by Coomassie Brilliant Blue [11]. Similarly,
the 3,3′-diaminobenzidine may also be used as a peroxidase
chromogenic substrate, but developed bands have to be
intensified by other treatments [9].
Some previous reports concern amineoxidases staining
on PAA gels. Thus, Houen and Leonardsen [12] developed
a specific staining method for DAO activity in the presence
of peroxidase. The diaminobenzidine inhibited DAO and
gave rise to unspecific staining, the most suitable among
the substrates used being 4-Cl-1-naphtol. Lee et al. [13]
used a peroxidase-coupled reaction in the presence of 3-
amino-9-ethylcarbazole to detect plasma amineoxidase
activities after native polyacrylamide gel electrophoresis.
In the mentioned studies, after the electrophoresis, the gels
were kept in the presence of peroxidase solution to detect
amineoxidase enzymatic activities. This present study
proposes a method to detect the DAO enzymatic activity
from Lathyrus sativus seedlings using sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
with the peroxidase immobilized in the PAA gel. This
method allowed the study of DAO stability to proteolysis in
simulated gastro-intestinal conditions. To our knowledge,
this is the first zymographic assay reported for vegetal
histaminase in the presence of SDS using the second
enzyme (peroxydase) immobilized in the PAA gel. In the
presented method, we proposed the ortho-phenylenedi-
amine (OPDA) as donor substrate of peroxidase, with the
formation of a stable product, azo-aniline, easily monitored
on gels. The redox dyes, once oxidized, change in color and
some of them also in solubility (from soluble when reduced
to insoluble when oxidized). In our case, in the presence of
H2O2and peroxidase, OPDA changed from colorless to a
yellow-brown compound stable and easy to detect on the
gel. The two coupled enzymatic reactions to monitor the
enzymatic activity of DAO on the SDS polyacrylamide gel
containing entrapped peroxidase are:
Putrescine þ 2O2þ 2H2O ? ? ? ? !
DAOAldehyde þ 2NH3þ 2H2O2
ð1Þ
2H2O2þ 2OPDA ? ? ? ? ? ? ? ? ? !
PeroxidaseAzo ? aniline þ 4H2O
ð2Þ
In our approach, the peroxidase is entrapped into the
SDS polyacrylamide gels during polymerization and the
DAO samples are deposited onto the gels for electropho-
resis. After the electrophoretic run, gels are placed in
solutions of putrescine (DAO substrate) and OPDA
(peroxidase substrate). Both substrates diffuse into the gel
and yellow-brown bands are developed in situ,
corresponding to enzymatic activity of DAO.
A histaminase (DAO) of vegetal origin, more efficient
than that of animal origin, was proposed for the general
treatment of histamine-related pathologic conditions, such
as allergic and septic shock, allergic asthma, anaphylaxis,
allergic rhinitis and conjunctivitis, urticaria and atopic
dermatitis, in which the histamine is the principal chemical
mediator [14]. Plant histaminase can be obtained from
different vegetal sources and can be used as a crude extract
or as a purified enzyme. As previously shown with other
copper oxidases (such as ceruloplasmin and serum bovine
amine oxidase), which presented antioxidant, cardiomodu-
latory, and cardioprotective effects [15], vegetal DAO have
beneficial effects in cardiac anaphylactic response [16] and
in myocardial ischemia and reperfusion injury [17]. Plant
histaminase has also some beneficial effects in asthma-like
reaction [18], or in splanchnic artery occlusion/reperfusion
injury [19]. As histamine and reactive oxygen species are
involved in the pathophysiology of inflammatory bowel
disease, hog kidney DAO had been intraperitoneally
administered on experimental ulcerative colitis in rats
[20], and the DAO treatment positively modified the
inflammatory reaction. Thus, it is also expected that
exogenous DAO could protect against oxidative damage
[17–19], as reactive species also play an important role in
inflammatory diseases. In this context, the vegetal DAO
stability to proteolysis in the presence of digestive enzymes
has to be known. A proteolysis study on a vegetal DAO
purified from pea (Pisum sativum) seedlings was previously
reported [21] in the presence of 0.01% pepsin (pH 2) and of
1282 C. Calinescu et al.

Page 3

0.1% trypsin (pH 7.2). To our knowledge, there are no
studies on vegetal histaminase in more acidic conditions
(pH 1.2) and over longer periods in simulated intestinal
fluid, in the presence of pancreatin (containing trypsin and
other proteases, together with other various digestive
enzymes such as amylase, lipase, ribonuclease) [22]. In
this context, the aim of this work was also to study in vitro
the behavior of DAO from L. sativus seedlings extract to
proteolytic action of digestive enzymes, in simulated gastric
and intestinal conditions, using the zymographic assay
described above.
Experimental
Materials
1,4-Diaminobutane dihydrochloride (putrescine), ortho-
phenylenediamine dihydrochloride, peroxidase type I (from
horseradish, 96 purpurogallin units/mg solid), Bradford
Reagent, pepsin (from porcine gastric mucosa, 882 units/
mg protein), and pancreatin (from porcine pancreas) were
purchased from Sigma-Aldrich Chemical Company (St.
Louis, MO, USA). Acrylamide, N,N′-methylene-bis-
acrylamide and protein molecular weight standards (Broad
Range) were from Bio-Rad Laboratory (Richmond, VA,
USA).
Preparation of vegetal extract from L. sativus seedlings
and purification of DAO
The vegetal extract and the purified DAO from grass pea
L. sativus seedlings were prepared as previously described
[23], with minor modifications. Briefly, 500 g of freshly
collected shoots of etiolated L. sativus seedlings were
homogenized in a Waring blender with 1 L of 30 mM
NaH2PO4 (final pH 4.4), and then filtered. In these
conditions, the DAO remains ionically linked to the
insoluble fraction. The solid residue, mainly constituted
by cell walls and vascular fibers, was washed with the same
buffer and the enzyme was finally eluted from the solid
residue with 500 mL of 0.1 M sodium phosphate buffer
(pH 7) and, then, centrifuged. The supernatant containing
the DAO was lyophilized. The purification of DAO was
done as previously described [23], onto a DE52-cellulose
and a HiTrap SP Sepharose column.
Determination of protein concentration and of enzymatic
activity of DAO preparations from L. sativus seedlings
Different concentrations of vegetal extract (1, 5, 10, 15, 25,
40 mg/mL) or of purified DAO (3 mg/mL) were kept for
2 h at 4 °C in phosphate buffer solution (PBS, pH 7.4)
under agitation and then, filtered and rapidly frozen. Protein
concentrations of the vegetal extract and of the purified
DAO were determined by the method of Bradford [24],
using bovine serum albumin as standard. The enzymatic
activity of DAO was assayed spectrophotometrically with
the same two coupled reactions as for zymography assay, in
the presence of putrescine (30 mM) as substrate for DAO
and of peroxidase as a second enzyme reaction, where
OPDA is enzymatically oxidized into a colored compound
(azo-aniline) by the released H2O2. The incubation mixture
contained 640 µL of PBS (10 mM sodium phosphate
buffer, pH 7.4), 10 µL of peroxidase solution (0.1 mg/mL),
50 µL of OPDA solution (30 mM), 200 µL of putrescine
solution (30 mM) and 100 µL of DAO samples of unknown
concentrations. The mixtures containing PBS, peroxidase,
OPDA, and putrescine were incubated for 5 min at 37 °C,
and then, the DAO samples were added. The enzymatic
reactions were incubated at 37 °C for 10 min, when 100 µL
of HCl (4 M) were added and the final absorbance was read
at 484 nm using a Beckman DU®-6 spectrophotometer. The
standard curve was prepared with serial concentrations of
H2O2from 0 to 68 µM.
One enzymatic unit (EU) of DAO is defined as the
amount of enzyme catalyzing the oxidation of 1.0 µmole of
putrescine per 10 min at pH 7.4, at 37 °C.
Proteolysis of DAO
DAO was incubated in simulated gastro-intestinal condi-
tions, with and without pepsin in simulated gastric fluid
(SGF) or with and without pancreatin in simulated
intestinal fluid (SIF) [22]. Samples of 40 mg of vegetal
extract powder were each incubated for 0, 5, 10, 15, 30, 60
and 120 min in 875 µL SGF (pH 1.2), containing or not
pepsin (0.32% powder with 882 units/mg protein). In
parallel, the same amounts of vegetal extract (40 mg) were
each incubated for 0, 1, 2, 4, 6, 8, and 10 h in 1 mL SIF
(pH 6.8), containing or not pancreatin. The SIF containing
pancreatin only was also incubated (as control) for the same
periods of time as above. The incubations were done at
37 °C and 50 rpm using an incubator shaker (series 25D,
New Brunswick Scientific Co., New Jersey, USA).
After each indicated SGF period, every SGF sample was
neutralized with 125 µL of 6.66% sodium bicarbonate
solution and maintained under agitation for 2 h at 4 °C.
Then, the neutralized SGF samples (final concentration,
40 mg/mL) were filtered and frozen. The SIF samples were
only filtered and rapidly frozen. Purified DAO (3 mg/mL)
was incubated only for 10 h in SIF containing pancreatin
(pH 6.8, 37 °C and 50 rpm), then filtered and rapidly
frozen.
The DAO standards were represented by the vegetal
extract (40 mg/mL) or by the purified DAO (3 mg/mL) in
Zymographic assay of plant diamine oxidase 1283

Page 4

PBS solution (pH 7.4). Both standards were kept for 2 h
under agitation at 4 °C and then filtered and frozen.
All the samples of histaminase were run in SDS-PAGE
under non-reducing conditions.
Peroxidase entrapment in polyacrylamide gels
For the enzymatic detection of DAO on gels via the
peroxidase-coupled reaction, the peroxidase was entrapped
in the PAA gels. Thus, during the 8% PAA resolving gels
preparation, 1 mL of horseradish peroxidase (1 mg/mL)
was added to the gel solutions prior to polymerization (final
volume of 5 mL). Stacking gels contained no peroxidase.
To verify the homogenous distribution of peroxidase in the
polymerized gel, an 8% PAA resolving gel containing
peroxidase (19.2 purpurogallin units) was electrophoreti-
cally tested for 1 h (room temperature, 120 V) with no
samples loaded on it. Then, the gel was immersed in a
staining solution containing equal volumes of H2O2
(30 mM) and OPDA (30 mM) and kept under weak
agitation for 1 h. Another 8% PAA resolving gel, contain-
ing no entrapped peroxidase and no samples loaded on it
was treated in the same conditions (control).
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis
The DAO protein content and the enzymatic activity of the
vegetal samples incubated in PBS or in simulated gastric
and intestinal conditions were evaluated by SDS-PAGE,
using the electrophoresis system Mini-Protean II (Bio-Rad).
Each sample was run in two PAA gels: one with entrapped
peroxidase for zymography and another peroxidase-free for
protein pattern.
Thus, the samples of vegetal extract at different concen-
trations in PBS (1, 5, 10, 15, 25, 40 mg/mL), the
neutralized SGF samples (40 mg vegetal extract/mL in
SGF, with or without pepsin), the SIF samples (40 mg
vegetal extract/mL in SIF, with or without pancreatin) and
the samples of purified DAO (3 mg/mL in SIF with
pancreatin), prepared as described above, were defrosted
and treated (1:1, v/v) with SDS electrophoresis sample
buffer containing 0.12 M Tris–HCl (pH 6.8), 4% SDS
(138 mM), 20% glycerol, 0.004% bromophenol blue and
no beta-mercaptoethanol. The mixtures were not heated.
Then, 30 µL of each treated sample were loaded and
resolved by SDS-PAGE for 1 h and 15 min (room
temperature, 120 V) on 8% PAA peroxidase-free gels for
Coomassie Blue staining of proteins or on 8% PAA gels
containing entrapped peroxidase (19.2 purpurogallin
units) for zymographic revelation of DAO enzymatic
activity. The electrophoresis buffer used in electrophoresis
runs contained 0.025 M Tris–Base, 0.192 M glycine, and
0.1% SDS.
The molecular weight protein standards were diluted in
SDS reducing sample buffer (with beta-mercaptoethanol),
heated for 5 min at 95 °C, then cooled and loaded 10 µL/
well to the PAA gel, as indicated in specifications from
Bio-Rad.
Coomassie Blue protein staining
After electrophoresis, the 8% PAA gels were incubated for
30 min (mild agitation) in a fixation solution containing
methanol:acetic acid:water (50/10/40, v/v/v) followed by
1 h of staining with 0.5% Coomassie Brilliant Blue G-250
in a methanol:acetic acid:water (40/10/50, v/v/v) solution.
Detection of DAO enzymatic activity on polyacrylamide
gels
After electrophoresis, the PAA gels containing entrapped
peroxidase and electrophoretically separated DAO samples,
were rinsed with distilled water and placed in a solution of
putrescine (30 mM) for a few minutes. Then, the incubation
medium was completed with an equal volume of OPDA
solution (17 mM). The DAO enzymatic activity was
detected on PAA gels (zymographic bands) in function of
newly produced substrate (H2O2) and in saturating concen-
tration of chromogenic substrate (OPDA). Each substrate
solution was prepared in PBS (pH 7.4) and kept at 4 °C
before utilization. The gels immersed in the two mentioned
solutions were incubated at 37 °C (1 h) under dark
conditions and weak agitation. The position of DAO
enzymatic activity bands on gels (zymograms) was deter-
mined by comparison of the electrophoretic patterns
obtained from Coomassie staining, using DAO in PBS
solution (pH 7.4) as standard. The densitometry image
analysis of gel bands was carried out using the Quantity
One program (Bio-Rad). The DAO enzymatic activity
measured by densitometry of bands was correlated with
the DAO initial enzymatic activity (DAO in PBS, pH 7.4),
spectrophotometrically determined. The DAO percentages
were reported to standard, considering the standard (DAO
in PBS, pH 7.4) as 100%.
Influence of SDS on the enzymatic activity of DAO
The estimation of the DAO enzymatic activities monitored
on the zymographic gels (EU/electrophoretic load of 30 µL,
in the presence and the absence of SDS) and the
dependence of DAO specific enzymatic activity on the
time of incubation with SDS was studied spectrophotomet-
rically at 25 °C, in the same conditions as for electropho-
resis. Thus, samples of vegetal extract in PBS (1, 5, 10, 15,
25, 40 mg/mL) were treated (1:1, v/v) with an electropho-
resis buffer containing 0.12 M Tris–HCl (pH 6.8), 20%
1284C. Calinescu et al.

Page 5

glycerol, in the presence or the absence of 4% SDS
(138 mM), for the determination of the DAO enzymatic
activities as EU/electrophoretic load of 30 µL. The
dependence of DAO specific enzymatic activity on the
time of incubation was studied after 0, 30, 75, and 135 min
at 25 °C, on a sample of 40 mg vegetal extract/mL, in the
same electrophoresis buffer and in the presence or in the
absence of SDS. The DAO enzymatic activities were
determined by the same spectrophotometrical method
described above, using the two coupled reactions in
solution.
Results and discussion
The protein content of L. sativus vegetal extract, deter-
mined by Bradford assay [24], was in a linear dependency
(R2=0.9978) with the vegetal extract concentrations (1, 5,
10, 15, 25, 40 mg/mL), indicating a good homogeneity of
the extract powder (data not shown). The same concen-
trations of vegetal extract as mentioned above (with a total
protein content of 15±1.4 µg/mL, 132±8 µg/mL, 302±
13 µg/mL, 435±17 µg/mL, 719±33 µg/mL and, respec-
tively, 1,091±27 µg/mL) were then used on electrophoretic
gels to follow the DAO protein content (Coomassie
coloration) and the DAO enzymatic activity (zymography).
The same migration patterns of molecular weight standards
and of vegetal extract were obtained for either gels
containing or not entrapped peroxidase after Coomassie
coloration (data not shown). Thus, throughout the whole
study, we used gels containing entrapped peroxidase only for
the enzymatic activity evaluation of DAO (zymography).
To our knowledge, this is the first zymographic assay for
plant histaminase with the second enzyme (peroxidase)

y = 335.42x
R2 = 0.9819
0
2000
4000
6000
8000
10000
12000
14000
16000
DAO content
(intensity)
y = 98.364x
R2 = 0.9434
0
1000
2000
3000
4000
5000
0102030 4050
Concentration of vegetal extract (mg/mL)
010
Concentration of vegetal extract (mg/mL)
20 304050
0
Concentration of vegetal extract (mg/mL)
10 2030 40 50
DAO enzymatic activity
(intensity)
y = 0.0047x
R2 = 0.9937
y = 0.0225x
R2 = 0.9996
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
DAO enzymatic activity
(EU/electrophoretic load)
DAO with SDS
DAO without SDS
200 –
116 –
97 –
66 –
45 –
31 –
kDa Std 1 5 10 15 25 40 mg/mL
a)
b)
c)
1 5 10 15 25 40 mg/mL
d)
e)
Fig. 1 Electrophoretic pattern
of vegetal extract from
L. sativus seedlings. Coomassie
staining: a SDS-PAGE (8%
PAA resolving gel without
peroxidase) of different concen-
trations of DAO vegetal extract
(1, 5, 10, 15, 25, 40 mg powder/
mL), treated (1:1, v/v) with non-
reducing SDS loading buffer
(30 µL/well). The gels were
stained with Coomassie Brilliant
Blue G-250. b Image analysis of
DAO bands intensity with the
Quantity One program. n=3.
Zymographic DAO enzymatic
activities: c different concentra-
tions of DAO vegetal extract
(1, 5, 10, 15, 25, 40 mg powder/
mL), treated (1:1, v/v) with non-
reducing SDS loading buffer
(30 µL/well). Oxidase activity
was directly monitored on 8%
PAA gels (containing entrapped
peroxidase) after the electro-
phoretic run, in the presence
of putrescine and ortho-
phenylenediamine. d Image
analysis of DAO bands intensity
(enzymatic activity) with the
Quantity One program. e Enzy-
matic activities of DAO on gel
(EU/electrophoretic load of
30 µL), spectrophotometrically
determined, in function of the
vegetal extract concentrations.
Protein content of vegetal ex-
tract: 15±1.4 µg/mL (1 mg/mL),
132±8 µg/mL (5 mg/mL), 302±
13 µg/mL (10 mg/mL),
435±17µg/mL(15mg/mL),719±
33 µg/mL (25 mg/mL), 1,091±
27 µg/mL (40 mg/mL). n=3
Zymographic assay of plant diamine oxidase 1285