HPLC-UV assays for evaluation of inhibitors of mono and diamine oxidases using novel phenyltetrazolylalkanamine substrates

Article abstract of DOI:10.1016/j.ab.2018.03.007

Recently, we have described an HPLC-UV assay for the evaluation of inhibitors of plasma amine oxidase (PAO) using 6-(5-phenyl-2H-tetrazol-2-yl)hexan-1-amine (4) as a new type of substrate. Now we studied, whether this compound or homologues of it can also

Full text of DOI:10.1016/j.ab.2018.03.007

AnalyticalBiochemistry549(2018)29–38
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Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio
HPLC-UV assays for evaluation of inhibitors of mono and diamine oxidases
using novel phenyltetrazolylalkanamine substrates
Kira Mergemeier, Matthias Lehr∗
Institute of Pharmaceutical and Medicinal Chemistry, University of Münster, Corrensstrasse 48, 48149 Münster, Germany
A R T I C L E I N F O
A B S T R A C T
Keywords:
Diamine oxidase
Recently, we have described an HPLC-UV assay for the evaluation of inhibitors of plasma amine oxidase (PAO)
using 6-(5-phenyl-2H-tetrazol-2-yl)hexan-1-amine (4) as a new type of substrate. Now we studied, whether this
compound or homologues of it can also function as substrate for related amine oxidases, namely diamine oxidase
(DAO), monoamine oxidase A (MAO A) and monoamine oxidase B (MAO B). Among these substances, 4 was
converted by DAO with the highest rate. The best substrate for MAO A and B was 4-(5-phenyl-2H-tetrazol-2-yl)
butan-1-amine (2). To validate the new assays, the inhibition values of known enzyme inhibitors were de-
termined and the data were compared with those obtained with the substrate benzylamine, which is often used
Monoamine oxidase A and B
Plasma amine oxidase
Phenyltetrazolylalkanamine
Aliphatic aldehyde derivative
TRIS
in amine oxidase assays. For the DAO inhibitor 2-(4-phenylphenyl)acetohydrazide an about 10fold lower IC50
-
value against DAO was obtained when benzylamine was applied instead of 4, indicating that 4 binds to the
enzyme with higher affinity than benzylamine. The IC50-values of clorgiline and selegiline against MAO A and B,
respectively, also decreased (two- and 30fold) replacing 2 by benzylamine. The discrepancies largely dis-
appeared, when the enzymes were pre-incubated with the inhibitors for 15 min. This can be explained with the
covalent inhibition mechanism of the inhibitors.
Introduction
Since an increased transmigration of leukocytes through the vascular
endothelium occurs during inflammatory responses, inhibitors of PAO
Amine oxidases are enzymes, which catalyze the oxidative de-
gradation of primary amines to aldehydes with the concomitant for-
mation of ammonia and hydrogen peroxide [1,2]. They can be cate-
gorized into two subclasses, the copper-containing amine oxidases [3]
and the flavin-adenine-dinucleotide (FAD)-containing amine oxidases
[4]. The copper-containing amine oxidase family consists of the dia-
mine oxidase (DAO, EC 1.4.3.22), the retina amine oxidase, the plasma
amine oxidase (PAO, EC 1.4.3.21) and the lysyl oxidase. A deficiency of
the histamine degrading DAO can result in an intolerance against his-
tamine ingested with food [5]. For this reason, DAO is used as food
supplement with the intention to decrease the histamine levels in the
gastrointestinal tract. Plasma amine oxidase (PAO), also known as
semicarbazide-sensitive amine oxidase (SSAO), copper containing
amine oxidase 3 (AOC3), or vascular adhesion protein-1 (VAP-1), is
localized to the extracellular surface of certain cells such as vascular
cells and adipocytes. Besides it exists as a soluble enzyme in blood
plasma, generated from proteolytic cleavage of the vascular PAO. The
enzyme is involved in physiological as well as pathophysiological
processes [6,7]. In particular, it acts as a vascular adhesion protein that
regulates leukocyte extravasation from the blood into tissues [8,9].
could be useful in the therapy of inflammation-related diseases [10].
The mitochondrial monoamine oxidases A and B (MAO A and MAO B,
EC 1.4.3.4) and the cytosolic polyamine oxidase belong to the group of
amine oxidases containing FAD. MAO A and MAO B play important
roles in the metabolism of neurotransmitters such as dopamine, nora-
drenaline and serotonin. Inhibitors of MAO A are marketed drugs
against depression, MAO B inhibitors are therapeutically used for the
treatment of Parkinson's disease [11].
In literature, various methods have been reported for the determi-
nation of the activity of amine oxidases and for screening of inhibitors
of these enzymes. PAO assays mostly employ the non-physiological
substrate benzylamine. The enzyme product benzaldehyde is quantified
by UV-spectrophotometry [12–18] or by HPLC and UV-detection after
derivatization with dinitrophenylhydrazine [19]. Alternatively, the
conversion of 14C-benzylamine to 14C-benzaldehyde is determined
radiometrically [20–22]. Moreover, peroxidase-coupled colorimetric
[23–30], fluorometric [31–34] or luminometric [35] assays are applied,
which measure the amount of H2O2 produced during benzylamine
oxidation. Besides, a (naphthalene-2-yl)methylamine substrate was
used in a fluorometric non-coupled assay [36].
∗
Corresponding author.
E-mail address: lehrm@uni-muenster.de (M. Lehr).
https://doi.org/10.1016/j.ab.2018.03.007
Received 28 December 2017; Received in revised form 5 March 2018; Accepted 12 March 2018
Availableonline15March2018
0003-2697/©2018ElsevierInc.Allrightsreserved.

K. Mergemeier, M. Lehr
AnalyticalBiochemistry549(2018)29–38
Diamine oxidase (DAO) is stored in epithelial cells and secreted into
the blood stream upon stimulation. Preferred substrates of this enzyme
are histamine, 1-methylhistamine, various diamines such as cadaverine
(pentane-1,5-diamine) and putrescine (butane-1,4-diamine), and the
polyamine spermidine. Despite benzylamine was reported to be only a
poor substrate for DAO, it also can be applied for determination of DAO
activity [37–40]. The most widely used DAO substrate is putrescine.
This diamine is converted to γ-aminobutyraldehyde, which undergoes a
spontaneous and rapid internal cyclization to Δ1-pyrroline. Some
methods detect this compound by colorimetry, GC or HPLC after its
derivatization with ninhydrin, o-aminobenzaldehyde, or o-amino-
benzaldehyde/CrO3 [41–45]. Alternatively, enzyme activity is mon-
itored by measuring the decrease of putrescine or histamine by ion
mobility spectrometry [46] or fluorometry after derivatization [47],
respectively. In the same way as described for PAO above, in several
DAO assays the formation of H2O2 formed during the oxidation of an
amine substrate is determined by using coupled reactions of peroxidase
and a dye leading to coloured [26,48–51] or luminescence [35,40]
products. Furthermore, the aromatic aldehydes produced by the en-
zyme from p-nitrobenzylamine or p-dimethylaminomethylbenzylamine
are quantified directly by UV/Vis-spectrophotometry [52,53]. Besides,
a variety of other methods has been reported, such as radiometric as-
says, which use 14C-putrescine or 14C-cadaverine as substrates [54,55],
or NADH-coupled spectrophotometric methods, which detect the al-
dehyde or the ammonia released by DAO from amine substrates
[56,57].
A large number of assays are described for the determination of
MAO A and/or MAO B activity using kynuramine as substrate. The
aldehyde formed by enzymatic oxidation of this compound undergoes a
complete cyclization to 4-hydroxyquinoline, which can be detected by
UV/Vis-spectrophotometry, fluorometry or mass spectrometry directly
or after LC separation [58–67]. A variety of different amine substrates
such as tyramine are used in MAO assays, in which the produced H2O2
is detected by generation of a chromogenic or fluorogenic compound in
a peroxidase-coupled reaction [25,26,34,68–71]. Tyramine is also used
as substrate in assays, in which the aldehyde produced by MAO is de-
rivatized with semicarbazide and dinitrophenylhydrazine before de-
tection by UV/Vis-spectrophotometry [72,73]. Some established test
systems for MAO A and MAO B inhibitor screening or for comparative
determination of activity use different substrates for both enzymes, e.g.
Fig. 1. Conversion of 6-(5-phenyl-2H-tetrazol-2-yl)hexan-1-amine (4) to 6-(5-phenyl-2H-
tetrazol-2-yl)hexanal (11) by PAO and derivatization of the aliphatic aldehyde product to
an 1,3-oxazolidine by TRIS.
derivatization with tris(hydroxymethyl)aminomethane (TRIS) leading
to the formation of an oxazolidine was carried out before analysis
(Fig. 1). In the present work, we describe HPLC-UV assays for the de-
termination of the activity of the amine oxidases DAO, MAO A and
MAO B using novel ω-(5-phenyl-2H-tetrazol-2-yl)alkan-1-amine sub-
strates. The procedures are suitable for screening inhibitors of these
important enzymes, and for testing the specificity of agents found to be
active towards PAO.
Material and methods
Reagents
Phosphate buffered saline tablets (one tablet dissolved in 200 mL of
deionized water yields 0.01 M phosphate buffer, 0.0027 M potassium
chloride and 0.137 M sodium chloride, pH 7.4, at 25 °C), glycerol, su-
crose, EDTA-Na2, dimethyl sulfoxide, benzylamine, berenil, clorgiline,
selegiline (Sigma-Aldrich, Steinheim, Germany); potassium dihydrogen
phosphate, dibasic potassium phosphate, tris(hydroxymethyl)amino-
methane (TRIS) base, benzaldehyde, ammonium acetate LC/MS-grade,
Triton X-100 (VWR, Darmstadt, Germany); acetonitrile HPLC-grade,
Brij35 (Fischer Scientific, Loughborough, UK); bovine plasma amine
oxidase (PAO) purified from bovine plasma (lyophilized powder, ac-
tivity: 32 Tabor units/mg protein) (Abnova, Taipei, Taiwan delivered
via Biozol, Eching, Germany); diamine oxidase (DAO) from porcine
kidney (powder, 0.11 units/mg protein, Cat. No. D7876), monoamine
oxidase A (MAO A) (human recombinant, expressed in baculovirus in-
fected BTI insect cells, 2.5 mg or 0.45 units/0.5 mL buffer solution, Cat.
No. M7317), monoamine oxidase B (MAO B) (human recombinant,
expressed in baculovirus infected BTI insect cells, 2.5 mg or 0.19 units/
0.5 mL buffer solution, Cat. No. M7441) (Sigma-Aldrich, Steinheim,
Germany); ω-(5-phenyl-2H-tetrazol-2-yl) alkanamines 2–6, the corre-
sponding aldehydes 9–13 and the PAO inhibitor 2-(4-phenylphenyl)
acetohydrazide were synthesized as published recently [97]; for the
synthesis of 3-(5-phenyl-2H-tetrazol-2-yl)propan-1-amine (1) and 3-(5-
phenyl-2H-tetrazol-2-yl)propanal (8) see Supplementary data.
serotonin for MAO
A
and benzylamine, 3-methoxy-4-hydro-
xybenzylamine or 2-phenylethylamine for MAO B. The generated al-
dehyde products are measured directly by UV-spectrophotometry, by
HPLC with UV, MS or electrochemical detection, respectively, by per-
oxidase-coupled reactions, or after derivatization with dini-
trophenylhydrazine by colorimetry [74–79]. Although benzylamine is
described to be only a poor substrate for MAO A [64], it is also used for
the determination of MAO A activity [58,80]. Furthermore, a luciferase-
coupled MAO assay [81], methods, in which the aldehyde produced by
MAO is oxidized to the corresponding carboxylic acid, which is quan-
tified by HPLC or capillary electrophoresis [82–85], and several test
systems applying radiolabelled MAO substrates [86–89] have been
published. Moreover, the neurotoxic illicit drug 1-methyl-4-phenyl-
1,2,3,6-tetrahydropyridine (MPTP) is applied as substrate in a MAO B
assay [90]. In contrast to the water soluble PAO and DAO, MAO A and B
are obligate membrane-associated enzymes embedded in the mi-
tochondrial membranes. Thus, for solubilization and stabilization of
these enzymes, often detergents are added to the reaction mixtures in
MAO A and B assays [58,59,91–96].
Determination of the conversion rates of different amine substrates by PAO,
DAO, MAO A and MAO B
Preparation of enzyme dilutions
PAO: The purchased enzyme (600 Tabor units [18], 19 mg) was
dissolved in 1 mL phosphate buffered saline (pH 7.4); an aliquot of this
solution was diluted 1:100 with phosphate buffered saline; final con-
centration: 0.19 mg/mL.
DAO: An aliquot of the purchased enzyme (27.5 units, 250 mg) was
dissolved in potassium phosphate buffer (0.1 M, pH 7.2); final con-
centration: 10 mg/mL.
MAO A: An aliquot of the purchased enzyme solution (0.45 units or
2.5 mg/0.5 mL) (40 μL) was diluted with potassium phosphate buffer
(0.1 M, pH 7.4, containing 0.25 M sucrose, 0.1 mM EDTA-Na2 and 5%
(v/v) glycerol) (104 μL); final concentration: 1.4 mg/mL.
MAO B: An aliquot of the purchased enzyme solution (0.19 units or
Recently, we have published a method for the detection of PAO
inhibitors, which monitors the conversion of the new substrate 6-(5-
phenyl-2H-tetrazol-2-yl)hexan-1-amine (4) to the enzyme product 6-(5-
phenyl-2H-tetrazol-2-yl)hexanal (11) by HPLC with UV-detection [97].
Since this aldehyde product only eluted with poor peak shape due to
hydrate formation in the aqueous mobile phase,
a pre-column
30

K. Mergemeier, M. Lehr
AnalyticalBiochemistry549(2018)29–38
2.5 mg/0.5 mL) (50 μL) was diluted with potassium phosphate buffer
(0.1 M, pH 7.4, containing 0.25 M sucrose, 0.1 mM EDTA-Na2 and 5%
(v/v) glycerol) (75 μL); final concentration: 2.0 mg/mL.
Determination of the reaction progress curves of the conversion of 6-(5-
phenyl-2H-tetrazol-2-yl)hexan-1-amine (4) by DAO and of 4-(5-phenyl-
2H-tetrazol-2-yl)butan-1-amine (2) by MAO A and MAO B
Incubation procedures
Incubation procedures
The reactions were performed as described above using 6-(5-phenyl-
2H-tetrazol-2-yl)hexan-1-amine (4) as substrate for DAO and 4-(5-
phenyl-2H-tetrazol-2-yl)butan-1-amine (2) as substrate for MAO A and
MAO B, making the following modifications: Enzyme preparations of
DAO, MAO A and MAO B prepared as described above were further
diluted by mixing one part of the appropriate enzyme preparation with
three parts of potassium phosphate buffer (0.1 M, pH 7.2) in case of
DAO or potassium phosphate buffer (0.1 M, pH 7.4) containing 0.25 M
sucrose, 0.1 mM EDTA-Na2 and 5% (v/v) glycerol in case of MAO A and
MAO B. The incubation time was variable (5–90 min). After termination
of the enzyme reactions by addition of acetonitrile (100 μL), aliquots of
the samples (175 μL) were diluted with aqueous TRIS buffer (100 mM,
pH 8.5 at 20 °C) (175 μL) and allowed to stand at room temperature for
30 min.
PAO: A solution of 3-(5-phenyl-2H-tetrazol-2-yl)propan-1-amine (1)
in DMSO (5 mM) (5 μL) was treated with an aliquot of the PAO dilution
(95 μL) and incubated at 37 °C for 60 min. The substrate concentration
in the final reaction mixture was 250 μM. The enzyme reaction was
terminated by the addition of acetonitrile (100 μL). The samples were
cooled in an ice bath for 10 min and centrifuged at 12 000 x g and 10 °C
for 5 min. Aliquots of the supernatants (175 μL) were diluted with water
(175 μL). The obtained mixtures were subjected to HPLC analysis. The
conversion rates of the substrates 2–7 by PAO have already been de-
termined using the same procedure [97].
DAO: To an aliquot of the DAO dilution (95 μL) pre-warmed at 37 °C
for 15 min was added a solution of the appropriate substrate (com-
pounds 1–7) in DMSO (5 mM) (5 μL). The mixtures were incubated at
37 °C for 60 min, and further treated as described above in the experi-
ment with PAO.
MAO A: To a solution of 0.2% (m/v) Brij35 in phosphate buffered
saline (pH 7.4) (90 μL) was added the MAO A dilution (5 μL). After
warming at 37 °C for 15 min, a solution of the appropriate substrate
(compounds 1–7) in DMSO (5 mM) (5 μL) was added. The mixtures
were incubated at 37 °C for 60 min, and further treated as described
above in the experiment with PAO.
MAO B: To a solution of 0.2% (v/v) Triton X-100 in phosphate
buffered saline (pH 7.4) (90 μL) was added the MAO B dilution (5 μL).
After warming at 37 °C for 15 min, a solution of the appropriate sub-
strate (compounds 1–7) in DMSO (5 mM) (5 μL) was added. The mix-
tures were incubated at 37 °C for 60 min, and further treated as de-
scribed above in the experiment with PAO.
HPLC analysis
The equipment described above was used. The injection volume was
50 μL. The temperature of the autosampler was set to 10 °C, the tem-
perature of the column oven was 20 °C. HPLC analysis was run iso-
cratically using acetonitrile/aqueous TRIS buffer (50 mM, pH 8.5 at
20 °C) (35:65, v/v in case of DAO and 30:70, v/v in case of MAO A and
MAO B) as eluent at a flow rate of 0.4 mL/min and a detection wave-
length of 238 nm.
Validation of the HPLC-UV method for measuring the amine oxidase
products 6-(5-phenyl-2H-tetrazol-2-yl)hexanal (11) and 4-(5-phenyl-2H-
tetrazol-2-yl)butanal (9)
References were prepared by mixing DMSO solutions (5 mM) of the
corresponding aldehydes 8–14 (5 μL) with phosphate buffered saline
(95 μL) in case of PAO, potassium phosphate buffer (95 μL) in case of
DAO, 0.2% (m/v) Brij35 in phosphate buffered saline (95 μL) in case of
MAO A, and 0.2% (v/v) Triton X-100 in phosphate buffered saline
(95 μL) in case of MAO B, respectively. The solutions were stored at
room temperature for 60 min. The aldehyde concentration in the final
mixtures was 250 μM. After addition of acetonitrile (100 μL), aliquots of
the obtained mixtures (175 μL) were diluted with water (175 μL). The
resulting dilutions were subjected to HPLC analysis.
Linearity
Linearity was evaluated using enzyme product spiked matrix sam-
ples at six concentration levels. These samples were prepared in du-
plicate as following:
DAO: To an aliquot of the DAO dilution (95 μL) pre-warmed at 37 °C
for 15 min was added a solution of 6-(5-phenyl-2H-tetrazol-2-yl)hex-
anal (11) in DMSO (0.030, 0.075, 0.15, 0.225, 0.30 und 0.45 mM, re-
spectively) (5 μL). The concentrations of 11 in the obtained solutions
(100 μL) ranged from 1.5 μM to 22.5 μM. The mixtures (n = 2 for each
concentration level) were incubated at 37 °C for 60 min. Acetonitrile
(100 μL) was added and the samples were cooled in an ice bath for
10 min, and centrifuged at 12 000 x g and 10 °C for 5 min. An aliquot of
each supernatant (175 μL) was mixed with aqueous TRIS buffer
(100 mM, pH 8.5 at 20 °C) (175 μL), allowed to stand at room tem-
perature for 30 min, and analyzed by HPLC as described above in the
reaction progress experiments.
HPLC-analysis
The HPLC-UV system consisted of a Thermo Scientific Dionex
UltiMate 3000 system including an autosampler and a variable wave-
length detector (Thermo Fisher Scientific, Germering, Germany).
Separation was achieved on a Luna 3u C8(2) analytical column (3 mm
inside diameter
x 150 mm, particle size 3 μm) (Phenomenex,
Aschaffenburg, Germany). The injection volume was 50 μL. The tem-
perature of the autosampler was set to 10 °C, the temperature of the
column oven was 20 °C. Gradient elution was used with solvent A
(acetonitrile/aqueous ammonium acetate buffer (10 mM, pH 6), 10:90,
v/v) and solvent B (acetonitrile/aqueous ammonium acetate buffer
(10 mM, pH 6), 90:10, v/v): 0–1 min: isocratic run at 20% B, 1–20 min:
linear gradient to 95% B, 20–23 min: isocratic run at 95% B, 23–25 min:
linear gradient to 20% B, 25–35 min isocratic run at 20% B. The flow
rate was 0.4 mL/min. For detection of the tetrazolyl-substituted alde-
hydes the effluents were monitored at 254 nm (experiments with PAO)
or 238 nm (experiments with DAO, MAO A and MAO B); benzaldehyde
was detected at 254 nm (experiments with PAO) or 250 nm (experi-
ments with DAO, MAO A and MAO B). The relative amount of substrate
cleaved by the amine oxidase was calculated by comparing the peak
areas of the aldehydes liberated by the enzyme with the peak areas of
the aldehydes of the reference solutions.
MAO A: To a solution of 0.2% (m/v) Brij35 in phosphate buffered
saline (90 μL) was added a MAO A dilution as used in the reaction
progress experiments (5 μL). After warming at 37 °C for 15 min, a so-
lution of 4-(5-phenyl-2H-tetrazol-2-yl)butanal (9) in DMSO (0.020,
0.050, 0.10, 0.15, 0.20 und 0.30 mM) (5 μL) was added. The con-
centrations of 9 in the obtained solutions (100 μL) ranged from 1.0 μM
to 15 μM. The mixtures were incubated at 37 °C for 15 min, and further
treated as described above in the experiments with DAO.
MAO B: To a solution of 0.2% (v/v) Triton X-100 in phosphate
buffered saline (90 μL) was added a MAO B dilution as used in the re-
action progress experiments (5 μL). After warming at 37 °C for 15 min, a
solution of 4-(5-phenyl-2H-tetrazol-2-yl)butanal (9) in DMSO (0.020,
0.050, 0.10, 0.15, 0.20 und 0.30 mM) (5 μL) was added. The con-
centrations of 9 in the obtained solutions (100 μL) ranged from 1.0 μM
to 15 μM. The mixtures were incubated at 37 °C for 60 min, and further
treated as described above in the experiments with DAO.
31

K. Mergemeier, M. Lehr
AnalyticalBiochemistry549(2018)29–38
Recovery
and absence of the test compounds corrected by the blank value. From
these data the IC50-values were calculated via Probit-log concentration
graphs.
The experiments using benzylamine (7) as substrate were carried
out in the same way with the modifications that aliquots (175 μL) of the
supernatants obtained after addition of acetonitrile and centrifugation
were diluted with water (175 μL) and acetonitrile/aqueous ammonium
acetate solution (10 mM, pH 6) (45:55, v/v) was used as eluent at a
detection wavelength of 250 nm.
MAO A: A solution of the inhibitor clorgiline (concentration vari-
able) (2.5 μL) was treated with a solution of 0.2% (m/v) Brij35 in
phosphate buffered saline (pH 7.4) (90 μL) followed by a solution of the
substrate 2 (10 mM) in DMSO (2.5 μL). The mixture was allowed to
stand at 37 °C for 15 min. Then a solution of MAO A as used in the
reaction progress experiments was added (5 μL) and incubation was
carried out at 37 °C for 15 min. After addition of acetonitrile (100 μL)
the samples were further treated and analyzed as described above in the
reaction progress experiments. Controls (n = 3) and a blank were pre-
pared analogously.
Recovery was determined for three different concentration levels of
enzyme product-spiked matrix samples prepared in the following way:
DAO: Mixtures containing the aldehyde product 11 in a final con-
centration of 1.5 μM, 7.5 μM and 15 μM (n = 5 for each concentration
level) were prepared and analyzed by HPLC as described above in the
linearity experiments. The recovery was calculated with the aid of a
matrix-free standard curve obtained as following: a solution of 11 in
DMSO (0.030, 0.075, 0.15, 0.225, 0.30 und 0.45 mM, respectively)
(5 μL) was added to phosphate buffered saline (95 μL). The mixtures
(n = 2 for each concentration level) were allowed to stand at room
temperature for 60 min. Then acetonitrile (100 μL) was added and the
samples were further treated and analyzed as described above. The
obtained data were also used to calculate precision.
MAO A: Mixtures containing the aldehyde product 9 in a final
concentration of 1.0 μM, 5.0 μM and 10 μM (n = 5 for each con-
centration level) were prepared and analyzed by HPLC as described
above in the linearity experiments. The recovery was calculated with
the aid of a matrix-free standard curve obtained as following: a solution
of 9 in DMSO (0.020, 0.05, 0.10, 0.15, 0.20 und 0.30 mM, respectively)
(5 μL) was added to phosphate buffered saline containing 0.2% Brij35
(m/v) (95 μL). The mixtures (n = 2 for each concentration level) were
allowed to stand at room temperature for 15 min. Then acetonitrile
(100 μL) was added and the samples were further treated and analyzed
as described above. The obtained data were also used to calculate
precision.
MAO B: Mixtures containing the aldehyde product 9 in a final
concentration of 1.0 μM, 5.0 μM and 10 μM (n = 5 for each con-
centration level) were prepared and analyzed by HPLC as described
above in the linearity experiments. The recovery was calculated with
the aid of a matrix-free standard curve obtained as following: A solution
9 in DMSO (0.020, 0.050, 0.10, 0.15, 0.20 und 0.30 mM, respectively)
(5 μL) was added to phosphate buffered saline containing 0.2% Triton
X-100 (v/v) (95 μL). The mixtures (n = 2 for each concentration level)
were allowed to stand at room temperature for 60 min. Then acetoni-
trile (100 μL) was added, and the samples were further treated and
analyzed as described above. The obtained data were also used to cal-
culate precision.
The experiments using benzylamine (7) as substrate were carried
out in the same way with the modifications described below in the pre-
incubation experiments with MAO A.
MAO B: A solution of the inhibitor selegiline (concentration vari-
able) (2.5 μL) was treated with a solution of 0.2% (v/v) Triton X-100 in
phosphate buffered saline (pH 7.4) (90 μL) followed by a solution of the
substrate 2 (10 mM) in DMSO (2.5 μL). The mixture was allowed to
stand at 37 °C for 15 min. Then a solution of MAO B as used in the
reaction progress experiments was added (5 μL) and incubation was
carried out at 37 °C for 60 min. After addition of acetonitrile (100 μL)
the samples were further treated and analyzed as described above in the
reaction progress experiments. Controls (n = 3) and a blank were pre-
pared analogously.
The experiments using benzylamine (7) as substrate were carried
out in the same way with the modifications described below in the pre-
incubation experiments with MAO B.
Experiments with pre-incubation of the enzyme with the inhibitor
PAO: To a solution of the inhibitor 2-(4-phenylphenyl)acetohy-
drazide (concentration variable) (2.5 μL) was added a solution of PAO
prepared as described above (95 μL). The mixture was pre-incubated at
37 °C for 15 min. Then the incubation was started at 37 °C by addition
of a solution of the substrate 4 (10 mM) in DMSO (2.5 μL). After 45 min
the enzyme reaction was terminated by the addition of acetonitrile
(100 μL). The samples were further treated and analyzed as described
above in the DAO inhibition experiments done without pre-incubation.
Controls (n = 3) and a blank were prepared analogously.
The experiments using benzylamine (7) as substrate were carried
out in the same way with the modifications that aliquots (175 μL) of the
supernatants obtained after addition of acetonitrile and centrifugation
were diluted with water (175 μL) and acetonitrile/aqueous ammonium
acetate solution (10 mM, pH 6) (45:55, v/v) was used as eluent at a
detection wavelength of 250 nm.
DAO: The experiments were carried out as described above for the
corresponding experiments with PAO with the modification that in-
stead of the PAO solution a solution of DAO as used in the reaction
progress experiments was added and that the incubation time was
60 min.
MAO A: A solution of the inhibitor clorgiline (concentration vari-
able) (2.5 μL) was treated with a solution of 0.2% (m/v) Brij35 in
phosphate buffered saline (pH 7.4) (90 μL) followed by a solution of
MAO A as used in the reaction progress experiments (5 μL). The mixture
was pre-incubated at 37 °C for 15 min. Then the incubation was started
at 37 °C by addition of a solution of the substrate 2 (10 mM) in DMSO
(2.5 μL). After 15 min the enzyme reaction was terminated by the ad-
dition of acetonitrile (100 μL). The samples were further treated and
analyzed as described above in the reaction progress experiments done
The variabilities of the assays were investigated by replicate de-
termination of the IC50- values of appropriate enzyme inhibitors at
different days (n = 3) according to the general procedures for inhibitor
screening described below.
General procedures for inhibitor evaluation
Experiments without pre-incubation of the enzyme with the inhibitor
PAO: The procedure for evaluation of the inhibitor 2-(4-phenyl-
phenyl)acetohydrazide using 4 and 7 as substrate was described pre-
viously [97].
DAO: A solution of the substrate (4) (10 mM) in DMSO (2.5 μL) was
treated with a DMSO solution of the inhibitor 2-(4-phenylphenyl)
acetohydrazide (concentration variable) (2.5 μL) followed by a DAO
solution as used in the reaction progress experiments (95 μL). After
incubation at 37 °C for 60 min, the enzyme reaction was terminated by
the addition of acetonitrile (100 μL). The samples were cooled in an ice
bath for 10 min, and centrifuged at 12 000 x g and 10 °C for 5 min. An
aliquot of each supernatant (175 μL) was mixed with aqueous TRIS
buffer (100 mM, pH 8.5 at 20 °C) (175 μL) and allowed to stand at room
temperature for 30 min. HPLC analysis was done as described above in
the reaction progress experiments. Controls with DMSO (2.5 μL) instead
of the DMSO solution of the inhibitor were prepared in the same
manner in parallel (n = 3). Furthermore, a blank incubation, in which
the enzyme solution was replaced by the buffer used for dissolving the
enzyme, was carried out. The relative inhibition values were de-
termined at different inhibitor concentrations and calculated as the
ratio of the areas of the aldehyde product 11 peaks formed in presence
32

K. Mergemeier, M. Lehr
AnalyticalBiochemistry549(2018)29–38
without pre-incubation. Controls (n = 3) and a blank were prepared
analogously.
The experiments using benzylamine (7) as substrate were carried
out in the same way with the modifications that aliquots (175 μL) of the
supernatants obtained after addition of acetonitrile and centrifugation
were diluted with water (175 μL) and acetonitrile/aqueous ammonium
acetate solution (10 mM, pH 6) (30:70, v/v) was used as eluent at a
detection wavelength of 250 nm.
MAO B: A solution of the inhibitor selegiline (concentration vari-
able) (2.5 μL) was treated with a solution of 0.2% (v/v) Triton X-100 in
phosphate buffered saline (pH 7.4) (90 μL) followed by a solution of
MAO B as used in the reaction progress experiments (5 μL). The mixture
was pre-incubated at 37 °C for 15 min. Then the incubation was started
at 37 °C by addition of a solution of the substrate 2 (10 mM) in DMSO
(2.5 μL). After 60 min the enzyme reaction was terminated by the ad-
dition of acetonitrile (100 μL). The samples were further treated and
analyzed as described above in the reaction progress experiments done
without pre-incubation. Controls (n = 3) and a blank were prepared
analogously.
The experiments using benzylamine (7) as substrate were carried
out in the same way with the modifications that aliquots (175 μL) of the
supernatants obtained after addition of acetonitrile and centrifugation
were diluted with water (175 μL) and acetonitrile/aqueous ammonium
acetate solution (10 mM, pH 6) (30:70, v/v) was used as eluent at a
detection wavelength of 250 nm.
Fig. 2. Structures of the amines, tested as substrates for amine oxidases, and the corre-
sponding aldehyde products.
B, and, therefore, can be used for the development of inhibitor assays
for these enzymes. First, the conversion rates of the homologue ω-(5-
phenyl-2H-tetrazol-2-yl)alkane-1-amines 1–6 and of benzylamine (7)
(Fig. 2) by DAO, MAO A and MAO B were determined. For stabilization
of the membrane-associated MAO A and MAO B, a detergent should be
added to the incubation solutions of these two enzymes. In preliminary
experiments, it was found that in presence of 0.2% Triton X-100 the
amine 1 was converted by MAO B but not by MAO A. In literature it has
been described that Triton X-100 can cause inhibition of MAO enzymes
at higher concentrations with MAO A being more susceptible to in-
hibition than MAO B [98,99], and that Brij35 can be used as stabilizer
in MAO A assays [93–95]. Thus, we investigated, whether the conver-
sion of 1 by MAO A is inhibited by the latter detergent. In a con-
centration range of 0.02%–0.2% the transformation of 1 by MAO A was
not effected by Brij35. Therefore, 0.2% Brij35 was added in the in-
cubations with MAO A and 0.2% Triton X-100 in case of MAO B.
For the evaluation of the usefulness of the amines 1–7 as substrates
for DAO, MAO A and B, these substances were incubated with the en-
zymes at a concentration of 250 μM for 60 min. The reactions were
stopped with acetonitrile. After centrifugation and dilution with water,
the aldehyde concentrations released were measured with reversed
phase HPLC and UV-detection at 238 nm (substrates 1–6) and 250 nm
(substrate 7), respectively, using an RP8 column and an acetonitrile/
ammonium acetate buffer (pH 6) gradient. At the applied enzyme
concentrations, all three enzymes transformed the investigated amines
to aldehydes more or less extensively (Table 1). The best phenylte-
trazolylalkanamine substrate of DAO was the hexan-1-amine 4. Inter-
estingly, this substance was also converted by PAO to a high degree
[97]. All compounds with shorter alkane chain (1–3) were oxidized less
Procedure for measuring Km-values
The Km-values of DAO for 4 and 7 and of MAO A and B for 2 and 7
were determined by measuring the reaction rates at different con-
centrations of the substrates.
DAO: Solutions with different concentrations of the substrates 4 and
7, respectively, in DMSO (5 μL) were incubated with the DAO enzyme
solution (10 mg/mL) (95 µL) at 37 °C for 5 min. The final concentrations
of the substrates in the assay medium (final volume 100 μL) ranged
from 10 to 133 μM. The enzyme reactions were terminated by the ad-
dition of acetonitrile (100 μL). The samples were further treated and
analyzed as described above in the DAO inhibition experiments done
without pre-incubation. The samples were run in duplicate. For each
concentration level corresponding blank solutions without enzyme
were analyzed analogously. References were prepared in triplicate by
mixing DMSO solutions (0.5 mM) of the corresponding aldehydes 11
and 14 (5 μL) with buffer (95 μL) and acetonitrile (100 μL).
MAO A and B: Solutions with different concentrations of the sub-
strates 2 and 7, respectively, in DMSO (5 μL) were diluted with 0.2%
(m/v) Brij35 in phosphate buffered saline (90 μL) in case of MAO A and
with 0.2% (v/v) Triton X-100 in phosphate buffered saline (90 μL) in
case of MAO B. Then solutions of MAO A and B (2.5 mg/0.5 mL), re-
spectively, were added (5 μL). The final concentrations of the substrates
in the assay medium (final volume 100 μL) ranged from 20 to 200 μM in
case of 2 and MAO A, from 10 to 133 μM in case of 2 and MAO B, from
200 to 2000 μM in case of 7 and MAO A and from 100 to 1000 μM in
case of 7 and MAO B. Incubation was carried out at 37 °C for 5 min. The
enzyme reactions were terminated by the addition of acetonitrile
(100 μL). The samples were further treated and analyzed as described
above in the MAO A and B inhibition experiments done without pre-
incubation. The samples were run in duplicate. For each concentration
level corresponding blank solutions without enzyme were analyzed
analogously. References were prepared in triplicate by mixing DMSO
solutions (0.5 mM) of the corresponding aldehydes 9 and 14 (5 μL) with
buffer (95 μL) and acetonitrile (100 μL). The Km-values were calculated
with the aid of Lineweaver-Burk plots.
Table 1
Conversion rates of different amines by PAO, DAO, MAO A and MAO B.
Substrate
Conversion of substrate [%]a
PAO
12
55
45
57
30
25
20
DAO
4
MAO A
20 0.6
1.2 21
MAO B
3-(5-Phenyl-2H-tetrazol-2-yl)
propan-1-amine (1)
4-(5-Phenyl-2H-tetrazol-2-yl)
butan-1-amine (2)
5-(5-Phenyl-2H-tetrazol-2-yl)
pentan-1-amine (3)
6-(5-Phenyl-2H-tetrazol-2-yl)
hexan-1-amine (4)
7-(5-Phenyl-2H-tetrazol-2-yl)
heptan-1-amine (5)
8-(5-Phenyl-2H-tetrazol-2-yl)
octan-1-amine (6)
0.8
0.1
7
0.9
2.6b 11
1.3b
0.4 17
0.6
1.4
7
0.2
8
6
8
2
7
0.2
14
9
1.4b 21
1.1b 13
1.2b 18
2.1b 29
0.3
1.8
2.0
1.4
0.2
0.5
0.1
0.3
0.5
0.1
5
n.d.
54
Benzylamine (7)
1.7
a
The substrate (250 μM) was incubated with the appropriate enzyme in a final volume
of 100 μL for 1 h. The amount of substrate converted was determined by HPLC with UV-
detection using analogous solutions of the corresponding enzyme products (aldehydes);
Results and discussion
values are means
standard deviations, n = 3; n.d.: not determined, because the en-
At the beginning of this study it was investigated, whether phe-
nyltetrazolylalkanamines are also substrates of DAO, MAO A and MAO
zyme product co-eluted with the substrate during the HPLC analysis.
b
Value already published in Ref. [97].
33

K. Mergemeier, M. Lehr
AnalyticalBiochemistry549(2018)29–38
Fig. 3. Reversed phase chromatograms of a solution of the substrate
4-(5-phenyl-2H-tetrazol-2-yl)butan-1-amine (2) incubated with MAO
A at 37 °C for 1 h. The enzyme reaction was terminated by addition
of acetonitrile followed by centrifugation. (A) Chromatogram ob-
tained in the experiments measuring the conversion rates. The
sample was diluted with water and elution was carried out with
acetonitrile/aqueous ammonium acetate buffer (10 mM, pH 6) ap-
plying gradient elution (26–86% acetonitrile). (B) Chromatogram
obtained in the reaction progress experiments. The sample was di-
luted with aqueous TRIS buffer (50 mM, pH 8.5 at 20 °C) and elution
was carried out isocratically with acetonitrile/aqueous TRIS buffer
(50 mM, pH 8.5 at 20 °C) (30:70, v/v). In both cases a Luna 3 μm
C8(2) column, 3 mm I.D. x 150 mm) was applied as stationary phase.
The injection volume was 50 μL each, the flow rate 0.4 mL/min and
the detection wavelength 238 nm. The enzyme concentration used in
case of chromatogram A was fourfold higher than in case of chro-
matogram B. 2: Amine substrate; 9: aldehyde product in equilibrium
with its hydrate; 15: condensation product of the liberated aldehyde
9 with TRIS. For representative chromatograms obtained during
inhibitor testing see Supplementary data.
efficiently by DAO, while in case of PAO the butan-1-amine 2 and the
pentan-1-amine 3 were reacted by the enzyme to a similar extent as the
hexan-1-amine 4. A second difference between PAO and DAO lies in the
conversion rate of benzylamine. While nearly all phenyltetrazolylalk-
anamines are better substrates for PAO than benzylamine, the latter
compound was the best DAO substrate of all compounds tested.
MAO A and MAO B differ considerably in their substrate preferences
from PAO and DAO (Table 1). In case of MAO A as well as MAO B the
phenyltetrazolylalkanamine substrate with the highest turnover rate
was the butan-1-amine 2. A comparable good substrate for MAO A was
the propan-1-amine 1 and for MAO B the pentan-1-amine 3. Benzyla-
mine was oxidized by MAO A to a significantly lower extent than 2,
while its transformation by MAO B was much more pronounced than
that of all tested phenyltetrazolylalkanamines. These results are in
agreement with the published data that benzylamine is only a poor
substrate for MAO A but a good substrate for MAO B.
the different substrates by the four amine oxidases, the HPLC analysis of
the phenyltetrazolylaldehyde products was carried out using gradient
elution with aqueous acetonitrile. As mentioned above, under these
conditions the aldehyde peaks showed a pronounced fronting (Fig. 3A).
This impaired peak form was caused by an aldehyde hydrate formation,
which could be proved by 1H-NMR spectroscopy. Thus, adding D2O to a
D6-acetone solution of a phenyltetrazolylaldehyde resulted in a de-
crease of the signal of the aldehyde proton at about 9.9 ppm, while a
new signal for this proton became visible at about 5.4 ppm. The area
ratio of these two signals was about 2:1 (see 1H-NMR-spectrum in the
Supplementary data). As published recently, the aldehydes can be forced
to elute as a single sharp peak when the sample is kept in TRIS buffer at
pH 8.5 due to the conversion of the aldehyde functionality with TRIS to
a non-hydratable 1,3-oxazolidine (Fig. 1) [97]. Therefore, the HPLC
analysis was carried out under these conditions in the following ex-
periments (determination of the reaction progress curves and validation
procedures). For illustration, Fig. 3B shows a HPLC chromatogram of
In the experiments for the determination of the conversion rates of
34

K. Mergemeier, M. Lehr
AnalyticalBiochemistry549(2018)29–38
the substrate 2 and its oxidation product 9 derivatized with TRIS to the
1,3-oxazolidine 15.
The conversion rates measured for the different phenylte-
trazolylalkanamines prompted us to use the hexane-1-amine 4 as sub-
strate for the DAO inhibition assay and the butan-1-amine 2 as substrate
for the corresponding MAO A and MAO B test systems. To determine
adequate incubation times, first the reaction progress curves for the
oxidation of 4 by DAO and of 2 by the two MAO enzymes were de-
termined (Fig. 4). The soluble DAO exhibited a nearly linear increase of
product formation during the observed time span (0–90 min). About 6%
(15 μM) of the initial amount of substrate (250 μM) was cleaved after
60 min. On the basis of this kinetic experiment, for the evaluation of
DAO inhibitors an incubation time of 60 min was chosen. The reaction
progress curves of the MAO enzymes differed significantly from that of
DAO. Here, a roughly linear increase of product formation only could
be measured at the beginning of the enzyme reaction. As a result of the
shape of the reaction progress curves (Fig. 4), for the evaluation of MAO
A and MAO B inhibitors incubation times of 15 min and 60 min, re-
spectively, were chosen.
The HPLC-UV methods were validated by measuring linearity and
recovery of the appropriate enzyme products and by determining the
IC50-values of inhibitors of DAO, MAO A and MAO B. Linearity was
evaluated using enzyme product spiked matrix samples (6-(5-phenyl-
2H-tetrazol-2-yl)hexanal (4) for DAO and 4-(5-phenyl-2H-tetrazol-2-yl)
butanal (2) for MAO A and MAO B) at six concentration levels. The
concentration of 4 in the DAO-containing solutions (final volume
100 μL) ranged from 1.5 μM to 22.5 μM, the concentration of 2 in the
solutions with MAO
A and MAO B (final volume 100 μL) was
1.0–15 μM. The mixtures were incubated at 37 °C for 60 min (DAO),
15 min (MAO A) and 60 min (MAO B), respectively, and then treated
with acetonitrile, cooled in ice, centrifuged and diluted with TRIS
buffer. HPLC analysis was performed with TRIS containing solvents.
The linearity parameters are shown in Tables 2 and 3.
Recovery was determined for three different concentration levels of
enzyme product-spiked matrix samples (1.5 μM, 7.5 μM and 15 μM in
case of 4, and 1.0 μM, 5.0 μM and 10 μM in case of 2), reflecting about
90%, 50% and 0% inhibition of product formation by an inhibitor.
These samples (n = 5 for each concentration level) were prepared by
mixing a DMSO solution of the aldehyde product 4 (DAO) or 2 (MAO A
and MAO B) with the appropriate enzyme dilutions. The mixtures were
incubated at 37 °C for 60 min (DAO), 15 min (MAO A) and 60 min
(MAO B), respectively, and further treated and analyzed as described in
the linearity experiments. The recoveries were calculated with the aid
of standard curves obtained in the same manner as described above in
the linearity experiments with the modifications that the enzymes
(matrix) were omitted and that the samples were allowed to stand at
room temperature for 60 min (DAO), 15 min (MAO A) and 60 min
(MAO B), respectively, prior addition of acetonitrile. The obtained data
were also used to calculate precision. The results obtained are listed in
Tables 2 and 3.
The stability of the analytes was determined by re-injection of the
samples of the linearity and recovery experiments after storing at 10 °C
for 12 h in the autosampler and at room temperature for an additional
12 h. After this period, no significant decrease of the reaction products
of the aldehydes of 4 and 2 with TRIS could be detected.
In addition, the assays were validated by measuring enzyme in-
hibition by certain inhibitors (Fig. 5). First, we tested berenil, which
was reported to be a DAO inhibitor, for inhibition of this enzyme. In
these experiments, a DMSO solution of the inhibitor or DMSO alone in
case of the controls was treated with a DMSO solution of the appro-
priate substrate followed by the enzyme solution and incubated for the
chosen time period. Enzyme reactions were stopped by addition of
acetonitrile, and the samples were further treated as described above.
Surprisingly, berenil was poorly active in our setting. At a concentration
of 100 μM the enzyme was only inhibited by 36%. On the other hand,
the known PAO inhibitor 2-(4-phenylphenyl)acetohydrazide proved to
Fig. 4. Reaction progress curves of the conversion of 6-(5-phenyl-2H-tetrazol-2-yl)hexan-
1-amine (4) by DAO (1) and of 4-(5-phenyl-2H-tetrazol-2-yl)butan-1-amine (2) by MAO A
(2) and MAO B (3). The final concentration of the substrate was 250 μM, the final volume
of the assay mixture was 100 μL. The experiments with MAO A and MAO B were carried
out in presence of the detergent Brij35 (0.2%, m/v) and Triton X-100 (0.2%, v/v), re-
spectively. Values are the means of duplicates.
35

K. Mergemeier, M. Lehr
AnalyticalBiochemistry549(2018)29–38
Table 2
Table 4
Linearity and recovery parameters for the DAO product 4 evaluated with appropriate
matrix-spiked samples.
Inhibitory potency of 2-(4-phenylphenyl)acetohydrazide against plasma amine oxidase
(PAO) and diamine oxidase (DAO) in dependence of the substrate and the assay condi-
tions.
Enzyme Correlation coefficient R
Recovery (%)b of 4 at a concentration of
of the standard curve of
Conditions and Substrate
PAO
DAO
4a
1.5 μM
7.5 μM
15 μM
IC50 (μM)a
DAO
0.9987
81.4
3.0
95.3
3.6
91.1
4.6
A) Measurement without pre-incubation of enzyme and inhibitor
0.03b
1.2b
0.17
1.5
0.01
0.1
a
Benzylamine (7)
6-(5-Phenyl-2H-tetrazol-2-yl)hexan-1-amine (4)
0.12
11
Concentration range: 1.5 μM–22.5 μM; linear equation of the curve of product 4:
y = 0.402 x + 0.062 (peak area y in mAU and concentration x in μM).
b
B) Measurement with pre-incubation of enzyme and inhibitor:
Mean
standard deviation, n = 3.
Benzylamine (7)
6-(5-Phenyl-2H-tetrazol-2-yl)hexan-1-amine (4)
0.11
0.29
0.003
0.02
0.11
0.24
0.01
0.02
Table 3
a
Mean
standard deviation, n = 3.
Linearity and recovery parameters for the MAO product 2 evaluated with appropriate
matrix-spiked samples.
b
Value already published in Ref. [97].
Enzyme Correlation coefficient R
Recovery (%)b of 2 at a concentration of
of the standard curve of
Table 5
2a
1.0 μM
5.0 μM
10 μM
Inhibitory potency of clorgiline against MAO A and selegiline against MAO B, respec-
tively, in dependence of the substrate and the assay conditions.
MAO A
MAO B
0.9998
0.9991
102.2
98.8
4.3 97.9
3.1 102.9
1.7
0.5 100.2
97.8
0.8
2.5
Conditions and Substrate
MAO A (clorgiline)
IC50 (nM)a
MAO B (selegiline)
a
Concentration range: 1.0 μM–15 μM; linear equation of the curve of product 2: assay
of MAO A: y = 0.441 x + 0.069; assay of MAO B: y = 0.420 x + 0.14 (peak area y in
mAU and concentration x in μM).
A) Measurement without pre-incubation of enzyme and inhibitor
b
Mean
standard deviation, n = 3.
Benzylamine (7)
29
68
2
9
38
2
4-(5-Phenyl-2H-tetrazol-2-yl)butan-1-
amine (2)
1100
150
B) Measurement with pre-incubation of enzyme and inhibitor:
Benzylamine (7)
4-(5-Phenyl-2H-tetrazol-2-yl)butan-1-
amine (2)
4.7
5.7
0.6
0.4
13
18
1
1
a
Mean
standard deviation, n = 3.
Table 6
m-values for the oxidation of the substrates benzylamine, 6-(5-phenyl-2H-tetrazol-2-yl)
K
hexan-1-amine (4) and 4-(5-phenyl-2H-tetrazol-2-yl)butan-1-amine (2) by different
amine oxidases.
Substrate
PAO
DAO
MAO A
MAO B
K
m-value (μM)
Fig. 5. Structures of the inhibitors investigated.
Benzylamine
4
2
62a
12a
–
32
16
–
378
–
82
280
–
14
be a good inhibitor of DAO, exhibiting an IC50 of 1.5 μM against this
enzyme (Table 4). Since we have found that the IC50 of 2-(4-phenyl-
phenyl)acetohydrazide against PAO is about 100fold lower when the
standard substrate benzylamine was used instead of tetra-
zolylhexanamine 4 [97], we also determined the IC50 of 2-(4-phenyl-
phenyl)acetohydrazide against DAO with benzylamine. Under these
conditions, an about tenfold decreased IC50 was measured (0.17 μM)
(Table 4). Thus, the extent of inhibition of DAO by the applied inhibitor
also strongly depends on the kind of substrate used. Similar results were
obtained with MAO A and MAO B. The IC50 of the inhibition of MAO A
by the selective inhibitor clorgiline decreased twofold when the tetra-
zolylbutanamine substrate 2 was replaced by benzylamine. Inhibition
a
Values already published in Ref. [97].
inhibitors under such conditions using the phenyltetrazolylalkanamines
and benzylamine as substrates. Applying a pre-incubation time of
15 min, all IC50-values declined significantly. Furthermore, the pro-
nounced deviation of the inhibition values observed in case of PAO,
DAO and MAO B in dependence of the substrate used mostly dis-
appeared (Tables 4 and 5). Obviously, the new phenyltetrazolylalk-
anamine substrates displace the covalent inhibitor more effectively
from binding to the active site of the enzyme than the standard ben-
zylamine substrate. As a result of this, the time for the formation of
covalent bonds between co-substrate and inhibitor is reduced by phe-
nyltetrazolylalkanamines more strongly than by benzylamine leading to
a lower enzyme inhibition. When enzyme and covalent inhibitor are
pre-incubated before the enzyme reaction is started, the effect of the
binding affinity of the substrate on inhibition is less pronounced, since
the inactivation reaction already takes place in absence of the substrate
to a high extent.
of MAO B by selegiline even resulted in a 30fold reduction of the IC50
value in the same experimental setting (Table 5).
-
2-(4-Phenylphenyl)acetohydrazide, clorgiline and selegiline are
known to inhibit DAO, MAO A and MAO B, respectively, by forming
covalent bonds with the co-substrate of the enzymes, which is topa-
quinone in case of DAO and FAD in case of MAO A and MAO B. In many
published MAO A and MAO B inhibition assays the inhibitor is pre-
incubated with the enzyme before the enzyme reaction is started by
addition of the substrate. This procedure provides the inhibitor more
time for an irreversible covalent modification of the enzyme, leading to
lower IC50-values. For this reason, we also tested the amine oxidase
These assumptions are confirmed by the Km-values measured for the
enzymes with the different substrates (Table 6). For all four amine
oxidases the Km-value of the appropriate tetrazolylalkanamine substrate
36

K. Mergemeier, M. Lehr
AnalyticalBiochemistry549(2018)29–38
was significantly lower than that of benzylamine. This reflects that the
tetrazolylalkanamines bind to the enzymes with higher affinity than
benzylamine.
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In conclusion, we have developed chromatographic assays suitable
for the evaluation of inhibitors of the amine oxidases DAO, MAO A and
MAO B applying novel phenyltetrazolylalkanamine substrates. Before
HPLC-UV-detection, the aliphatic aldehyde released by the enzyme was
treated with TRIS under mild conditions to give an 1,3-oxazolidine
derivative. For measuring DAO activity, the hexan-1-amine 4 was best
suited. MAO A and MAO B showed the highest conversion rates with
the butan-2-amine 2. The IC50-values of known inhibitors of the en-
zymes determined corresponded with published data. Although HPLC-
UV assays are more time-consuming than microtiter-plate assays, they
are advantageous when a high selectivity and specificity is needed in a
study. Furthermore, the investigations have shown that benzylamine,
which is often used in PAO, DAO, MAO A and MAO B assays, is only a
weak binding substrate of these enzymes. This can result in an over-
estimation of the potency of inhibitors, especially when they a tested
under competitive conditions. In combination with the method for the
determination of the activity of PAO, which we have described recently
[97], the new assays can be used for the assessment of inhibitor activity
and selectivity against the four most prominent amine oxidases present
in the organism.
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.
doi.org/10.1016/j.ab.2018.03.007.
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