Naturally-occurring tetrahydro-β-carboline alkaloids derived from tryptophan are oxidized to bioactive β-carboline alkaloids by heme peroxidases

Article abstract of DOI:10.1016/j.bbrc.2014.07.047

β-Carbolines are indole alkaloids that occur in plants, foods, and endogenously in mammals and humans, and which exhibit potent biological, psychopharmacological and toxicological activities. They form from naturally-occurring tetrahydro-β-carboline alkaloids arising from tryptophan by still unknown way and mechanism. Results in this research show that heme peroxidases catalyzed the oxidation of tetrahydro-β-carbolines (i.e. 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid and 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid) into aromatic β-carbolines (i.e. norharman and harman, respectively). This oxidation followed a typical catalytic cycle of peroxidases through redox intermediates I, II, and ferric enzyme. Both, plant peroxidases (horseradish peroxidase, HRP) and mammalian peroxidases (myeloperoxidase, MPO and lactoperoxidase, LPO) catalyzed the oxidation in an efficient manner as determined by kinetic parameters (V_MAX and K_M). Oxidation of tetrahydro-β-carbolines was inhibited by peroxidase inhibitors such as sodium azide, ascorbic acid, hydroxylamine and excess of H₂O ₂. The formation of aromatic β-carbolines by heme peroxidases can help to explain the presence and activity of these compounds in biological systems.

Full text of DOI:10.1016/j.bbrc.2014.07.047

Biochemical and Biophysical Research Communications 451 (2014) 42–47
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Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
Naturally-occurring tetrahydro-b-carboline alkaloids derived from
tryptophan are oxidized to bioactive b-carboline alkaloids by heme
peroxidases
⇑
Tomás Herraiz , Juan Galisteo
Instituto de Ciencia y Tecnología de Alimentos y Nutrición (ICTAN-CSIC), Spanish National Research Council (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 July 2014
Available online 15 July 2014
b-Carbolines are indole alkaloids that occur in plants, foods, and endogenously in mammals and humans,
and which exhibit potent biological, psychopharmacological and toxicological activities. They form from
naturally-occurring tetrahydro-b-carboline alkaloids arising from tryptophan by still unknown way and
mechanism. Results in this research show that heme peroxidases catalyzed the oxidation of tetrahydro-b-
carbolines (i.e. 1,2,3,4-tetrahydro-b-carboline-3-carboxylic acid and 1-methyl-1,2,3,4-tetrahydro-b-carb-
oline-3-carboxylic acid) into aromatic b-carbolines (i.e. norharman and harman, respectively). This
oxidation followed a typical catalytic cycle of peroxidases through redox intermediates I, II, and ferric
enzyme. Both, plant peroxidases (horseradish peroxidase, HRP) and mammalian peroxidases (myeloper-
oxidase, MPO and lactoperoxidase, LPO) catalyzed the oxidation in an efficient manner as determined by
kinetic parameters (VMAX and KM). Oxidation of tetrahydro-b-carbolines was inhibited by peroxidase
inhibitors such as sodium azide, ascorbic acid, hydroxylamine and excess of H2O2. The formation of aro-
matic b-carbolines by heme peroxidases can help to explain the presence and activity of these com-
pounds in biological systems.
Keywords:
Alkaloids
b-Carboline
Tetrahydro-b-carboline
Horseradish peroxidase
Myeloperoxidase
Lactoperoxidase
Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction
cytotoxic against microorganisms [1] and participate in inflamma-
tory pathologies [3,4]. Mammalian peroxidases have been found in
Heme-containing peroxidases use hydrogen peroxide (H2O2) as
electron acceptor to catalyze oxidative reactions in biosynthesis
and metabolism of biological molecules [1]. In that reaction, native
ferric enzyme reacts with one equivalent of H2O2 under a two elec-
tron process being oxidized to redox intermediate compound I (Fe
(IV) = O+ꢀ). Compound I is reduced back directly to native ferric
enzyme by a two electron process (e.g. the oxidation of halides
by mammalian peroxidases), or instead by alternative substrates
(AH) via intermediate compound II (PorFeIV = O) through two suc-
cessive one-electron reductions [2]. Compounds I and II oxidize
substrates (AH) to give radicals (AHꢀ) that evolve to stable prod-
ucts. Heme peroxidases are widely distributed in nature and
include plant peroxidases (e.g. horseradish peroxidase, HRP) and
mammalian peroxidases (e.g. myeloperoxidase, MPO; lactoperoxi-
dase, LPO, eosinophil peroxidase, EPO). Peroxidases play a protec-
tive role against oxidative damage and in response toward
wounding or stress in plants. In mammals, peroxidases catalyze
the oxidation of halides (e.g. ClÀ) to hypohalous acids being
brain where they can oxidize dopamine to dopaminochrome exert-
ing cytotoxicity [5,6] and are involved in the bioactivation of pro-
toxins and procarcinogens [7–9].
b-Carbolines are naturally-occurring bioactive alkaloids origi-
nally detected in plants and which also appear in mammalian flu-
ids and tissues, including the human brain [10–14]. In addition,
b-carbolines occur in foods and cigarette smoke, suggesting human
uptake and exposure to these compounds [10,15–17]. They exhibit
an array of biological, psychopharmacological and toxicological
activities, including antitumor, antimicrobial, antimalarial,
anti-inflammatory, vasorelaxant, antioxidant, neuroactive, psycho-
active or neurotoxic actions [10,11,18–20]. Biosynthetically, b-
carboline alkaloids arise from the amino acid tryptophan which
affords the nitrogen atoms and carbon skeleton. Thus, an indoleth-
ylamino acid or amine derivative reacts with an aldehyde or a-keto
acid under an enzymatic or chemical reaction (Pictet–Spengler) to
give an intermediate Schiff base that cyclizates to give tetrahydro-
b-carbolines [21] (Fig. 1). Subsequently, tetrahydro-b-carboline
molecules are oxidized to b-carbolines. However, an enzymatic
step able to accomplish the latter bioconversion is currently
unknown and lacking. This research reports the biotransformation
⇑
Corresponding author.
E-mail address: tomas.herraiz@csic.es (T. Herraiz).
http://dx.doi.org/10.1016/j.bbrc.2014.07.047
0006-291X/Ó 2014 Elsevier Inc. All rights reserved.

T. Herraiz, J. Galisteo / Biochemical and Biophysical Research Communications 451 (2014) 42–47
43
Incubations were at least in duplicate and control reactions were
carried out in absence of enzyme, H2O2 or tetrahydro-b-carbolines,
and in presence of catalase (0.05–0.2 mg prot./ml). Reactions were
also carried out in presence of 1 mM ascorbic acid, 1 mM sodium
azide, 1 mM hydroxylamine or excess H2O2 (2 mM) as inhibitors
of peroxidase and substrate (250 lM). On the other hand, cyto-
chrome P450 and catalase (heme proteins), and glutathione perox-
idase were incubated with tetrahydro-b-carbolines to assess
possible oxidation, as follows: (a) 0.2 ml phosphate buffer 50 mM,
pH 7.0, containing a mixture of cytochrome P450s (Supermix)
(20 pmol P450) and THCA and/or MTCA (250
NADPH (1 mM) and H2O2 (50–500 M); (b) 1 ml phosphate buffer
50 mM, pH 7, containing catalase (0.05–0.2 mg prot./ml), THCA or
MTCA (250 M) and H2O2 (100–500 M); (c) 1 ml phosphate buffer
50 mM (pH 7), containing glutathione peroxidase (9.6 g prot./ml),
H2O2 (50 M) and MTCA or THCA (500 M). The mixtures
were incubated (37 °C, 25 min), the reaction stopped with
10 % v/v HClO4/methanol (1:1), centrifuged and analyzed by
RP-HPLC-DAD.
lM), with or without
l
l
l
l
l
l
Fig. 1. Biosynthesis of naturally-occurring b-carboline alkaloids in biological
systems. The oxidation step from tetrahydro-b-carbolines to aromatic b-carboline
alkaloids can be accomplished by heme peroxidases occurring in plants and
mammals as suggested here.
2.3. Spectral measurements
of tetrahydro-b-carboline-3-carboxylic acids, which are naturally-
occurring alkaloids derived from tryptophan, into aromatic b-carb-
olines as catalyzed by heme peroxidases arising from both
mammalian and plant origin. This metabolic conversion contrib-
utes to rationalize the biological presence and activity of b-carbo-
lines (e.g. norharman and harman) found in biological fluids and
tissues and the possible implications are discussed.
UV–vis spectra of enzymatic incubations were obtained at room
T in a Beckman-Coulter DU 800 spectrophotometer. Concentration
of peroxidases (HRP, LPO, MPO) was determined by absorbance
using the extinction coefficients of the respective Soret bands
(403, 412, 430 nm) [22]: e403 (HRP) = 102 mMÀ1 cmÀ1
, e412
(LPO) = 112 mMÀ1 cmÀ1, and
e
430 (MPO) = 91 mMÀ1 cmÀ1. H2O2
= 39.4 MÀ1 cmÀ1).
concentration was calculated at 240 nm (
e
2. Materials and methods
2.4. RP-HPLC and HPLC-MS (electrospray ionization)
2.1. Chemicals and enzymes
The chromatographic analysis of incubation media was per-
formed by RP-HPLC coupled to diode array (DAD) and fluorescence
detectors and by HPLC-MS (electrospray) [9,16]. A 150 Â 3.9 mm,
Horseradish peroxidase (HRP) (type II) and bovine milk lacto-
peroxidase (LPO) were obtained from Sigma; human myeloperox-
idase (MPO) from polymorphonuclear leucocytes was obtained
from Calbiochem (Merck); human cytochrome P450s Supermix
expressing CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and
CYP3A4 enzymes was obtained from Gentest Co (BD) (Woburn,
MA, USA); catalase from bovine liver and glutathione peroxidase
from bovine erythrocytes were from Sigma. All enzymes were pre-
pared in phosphate buffer and used as supplied. Norharman (9H-
pyrido[3,4-b]indole), Harman (1-methyl-9H-pyrido[3,4-b]indole),
(1S,3S)-1-methyl-1,2,3,4-tetrahydro-b-carboline-3-carboxylic acid
(MTCA) and 1,2,3,4-tetrahydro-b-carboline-3-carboxylic acid
(THCA) were obtained from Sigma. Hydrogen peroxide was
obtained from a 30% solution (Scharlau Chemical) and diluted to
desired concentration.
4 lm, Nova-pak C18 column (Waters, Milford, MA) was used for
separation. Conditions were: 50 mM ammonium phosphate buffer
(pH 3) (buffer A) and 20% A in acetonitrile (buffer B). Gradient from
100% A to 32% B in 8 min and 90% B at 10 min; flow rate 1 ml/min;
the column temperature, 40 °C and injection volume 20 ml. Absor-
bance detection was 254 nm for b-carbolines and 280 nm for tetra-
hydro-b-carbolines, and concentration was determined from
calibration curves of area vs concentration of standards. Analysis
by HPLC-MS was carried out by using a Hewlett–Packard 1100
HPLC-mass spectrometer working under ESI positive ionization
mode. Chromatographic separation was done with
a
2.1 Â 150 mm Zorbax SB C18 column (Agilent technologies) using
0.5% formic acid (eluent A) and 0.5% formic acid in acetonitrile
(eluent B). The gradient was 0–80% B in 30 min. The flow rate
was 0.3 ml/min and the column temperature, 40 °C. The drying
gas temperature was 350 °C, flow of 11 l/min, nebulizer pressure
of 55 psi, capilar voltage of 4000 V and fragmentator of 100 V.
Acquisition was from 50 to 700 u.
2.2. Enzymatic metabolism
Fresh solutions of peroxidases were prepared to make: 0.32
lM
HRP, 0.178 M LPO or 0.0126 M MPO in 50 mM phosphate buffer,
l
l
pH 7 (0.5 ml final volume for MPO or 1 ml for HRP or LPO) contain-
ing in separate, 1,2,3,4-tetrahydro-b-carboline-3-carboxylic acid
(THCA) or 1-methyl-1,2,3,4-tetrahydro-b-carboline-3-carboxylic
acid (MTCA) in a range from 0 to 2 mM. The reaction was initiated
2.5. Kinetic studies
Peroxidase-catalyzed oxidations were studied as a function of
the concentration of substrates and the apparent KM and VMAX were
determined from non-linear regression fitting to Michaelis–Men-
ten curves (GraphPad prism). Peroxidases have not true VMAX and
kcat, and a pseudo-first order rate constant k4 corresponding to
the global oxidation leading to b-carbolines was calculated from KM-
by addition of H2O2 solution (5–500
the samples incubated at 37 °C for 40 min. Following addition of
HClO4 + methanol (1/1) (50 or 100 l, 10% v/v), the tubes were cen-
lM, final concentration), and
l
trifuged at 10,000 rpm, 10 min, and the reaction products were ana-
lyzed by HPLC and HPLC-MS. Selected concentrations of H2O2 used
for kinetic studies with tetrahydro-b-carbolines were as follows:
app
and kcat as kcat/KaMpp [23]. The reaction rate (v) was determined
100
25
l
M for HRP/THCA, 50
l
M for HRP/MTCA; 10
l
M for LPO/THCA;
as the amount of b-carboline formed as a function of time and con-
centration of enzyme.
l
M for LPO/MTCA, and 25
l
M for THCA/MPO and MTCA/MPO.

44
T. Herraiz, J. Galisteo / Biochemical and Biophysical Research Communications 451 (2014) 42–47
indicate that oxidation of tetrahydro-b-carbolines by HRP, LPO or
3. Results and discussion
MPO followed a typical catalytic cycle of peroxidases through com-
pounds I and II. Other heme enzymes such as catalase or cyto-
chrome P-450, and glutathione peroxidase (non-heme
peroxidase) were unable to accomplish this oxidation, suggesting
it is heme peroxidase-dependent.
Reaction rates were determined as a function of the concentra-
tion of substrates. They reached a maximum at relative low con-
centrations of H2O2 (6100 lM) with inhibition detected at higher
Incubation of tetrahydro-b-carboline-3-carboxylic acids (i.e.
THCA and MTCA) with heme peroxidases (HRP, LPO or MPO) and
H2O2 gave oxidation products that were identified as the corre-
sponding aromatic b-carbolines by HPLC-DAD (kmax at 248, 302
and 365–370 nm), fluorescence spectra (kmax for emission at
around 435–445 nm for exc. 300/254 nm) and co-elution with
authentic standards (Fig. 2). These results were confirmed by
HPLC-MS analysis (positive ESI). Thus, incubation of THCA with
peroxidases gave norharman (m/z 169, (M+H)+) whereas MTCA
gave harman (m/z 183 (M+H+)). Formation of b-carbolines
increased with incubation time and peroxidase concentration and
it was negligible in absence of enzyme, H2O2 or in presence of cat-
alase (0.2 mg/ml).
The time-course of peroxidase-catalyzed reaction was followed
by spectrophotometry (Fig. 3). Changes in the Soret bands were
monitored to follow the formation of peroxidase compounds I
and II. Following addition of H2O2 to HRP (403 nm), compound I
was formed as detected by a lower intensity Soret band (418 nm)
(Fig. 3A). The addition of MTCA (an electron donor) to H2O2-HRP
formed compound II (more intense Soret band at 420 nm) that
later disappeared to recover the native ferric enzyme (403 nm).
This process was accompanied by the presence of a new spectral
band that corresponded to formation of harman (1-methyl-b-carb-
oline) (350–365 nm). Similar patterns were observed for LPO and
MPO although compound I was not easily detected for these
enzymes [22]. Thus, following addition of MTCA to H2O2-LPO,
LPO-compound II was formed (Soret band red-shifted from 412
to 430 nm) and simultaneously, MTCA oxidized to harman (band
at 356 nm) (Fig. 3B). Then, LPO-compound II disappeared and
native enzyme recovered (Soret band at 412 nm). Similar spectral
features were obtained for MPO with native enzyme (430 nm) giv-
ing rise to compound II (456 nm) while MTCA oxidized to harman
(result not shown). Inserts in Fig. 3 illustrate spectral changes in
characteristic absorption maxima. Thus, after mixing LPO, H2O2
and MTCA, compound II formed (430 nm) and remained for several
minutes while native enzyme decreased (412 nm) and MTCA oxi-
dized to harman (356 nm). Formation of b-carboline decreased or
stopped when native enzyme (412 nm) recovered. These results
concentrations. In Fig. 4 are the curves corresponding to the forma-
tion of norharman by HRP, LPO and MPO as a function of the con-
centration of tetrahydro-b-carboline (similar curves obtained for
harman). These curves were used to calculate the apparent kinetic
parameters KM, VMAX and kcat (Table 1). A pseudo-first order rate
constant (k4) was calculated from KM and kcat that reflects the glo-
bal oxidation process of substrates by peroxidases [9,23]. The three
peroxidases catalyzed the oxidation of THCA and MTCA in an effi-
cient manner. The values of KaMpp were relatively similar for THCA
app
and MTCA and the three enzymes. V
and turnover (kcat) were
MAX
higher for the oxidation of THCA than MTCA, suggesting that THCA
was a better substrate. The k4 was also higher for the oxidation of
THCA than MTCA. Turnover (kcat) and k4 were higher for LPO and
MPO, suggesting that they were better catalysts of tetrahydro-b-
carboline oxidation than HRP. Finally, the formation of norharman
or harman by peroxidases was strongly inhibited (from 65% to
100%) in presence of peroxidase inhibitors such as sodium azide,
hydroxylamine, ascorbic acid and excess of H2O2, with hydroxyl-
amine and ascorbic acid being the best inhibitors (Fig. 4D).
The results described above have shown that heme peroxidases
efficiently catalyze the metabolic conversion of naturally-occurring
tetrahydro-b-carboline-3-carboxylic acids (THCA and MTCA) alka-
loids into aromatic b-carbolines. This conversion was higher for
mammalian peroxidases (MPO and LPO) than for plant peroxidase
(HRP). Tetrahydro-b-carbolines were good substrates and electron
donors of peroxidase-redox intermediates and their oxidation fol-
lowed a typical catalytic cycle of peroxidases through compounds
I, II and native enzyme. Peroxidases oxidize indoles by a one-elec-
tron oxidation to give nitrogen centered radicals (i.e. indolyl cation
or neutral radical) [1,22,24–27] that evolve to indole-ring cleavage
products. Indoles and tetrahydro-b-carbolines also form indolyl
mAU
A
200
100
min
10
0
2
4
6
8
mAU
200
B
100
0
0
2
4
6
8
10
min
Fig. 2. RP-HPLC chromatograms (absorbance at 254 nm) of incubation media containing tetrahydro-b-carbolines and heme peroxidases: (A) THCA (250
(0.32 M) and (B) MTCA (250 M) and LPO (0.178 M). Incubation and chromatographic conditions as indicated in Section 2.
lM) and HRP
l
l
l

T. Herraiz, J. Galisteo / Biochemical and Biophysical Research Communications 451 (2014) 42–47
45
0.3
A
403 nm
0.3
420 nm
356 nm
0.2
8
7
0.1
0.2
6
0.0
-10
0
10 20 30 40 50 60 70
Time (min)
5
4
1
2
0.1
3
9
0.0
300
325
350
375
400
425
450
λ (nm)
412 nm
430 nm
356 nm
0.15
0.2
0.1
0.0
B
0.10
0.05
0.00
7
6
-10
0
10 20 30 40 50 60 70
Time (min)
5
2
1
4
3
300
325
350
375
λ (nm)
400
425
450
Fig. 3. Time-course of peroxidase reaction followed by spectrophotometry. (A) MTCA (250
HRP + H2O2 + MTCA initial (3), and after 5 min (4), 10 min (5), 20 min (6), 30 min (7), 40 min (8); spectrum of harman (1-methyl-b-carboline) (9). (B) MTCA (250
oxidation with LPO (0.9 M): LPO (1), LPO + H2O2, 25 lM (2), LPO + H2O2 + MTCA initial (3), and after 5 min (4), 10 min (5), 20 min (6), and 30 min (7). Inserts illustrate
l
M) oxidation with HRP (1.28
l
M): HRP (1), HRP + H2O2, 50
lM
(2),
l
M)
l
spectral changes in Soret bands of HRP and LPO and formation of b-carbolines (356 nm).
radicals when acting as radical scavengers [28–30]. Therefore,
oxidation of tetrahydro-b-carbolines by heme peroxidases sug-
gests a stepwise oxidation in which peroxidases catalyze the initial
one-electron oxidation to a tetrahydro-b-carboline nitrogen radical
(e.g. indolyl radical) which is further oxidized to afford the aro-
matic b-carboline accompanied by decarboxylation [29,31,32].
Peroxidases use hydrogen peroxide (H2O2) that is a major reactive
oxygen species (ROS) involved in oxidative stress [7,8,33]. As THCA
and MTCA are substrates of peroxidases, they may exert antioxi-
dant actions because they are good electron donors of peroxidase
redox intermediates and consume H2O2 during enzymatic oxida-
tion. These compounds are also radical scavengers as reported else-
where [29,31].
Tetrahydro-b-carbolines and b-carbolines (i.e. pyridoindole
alkaloids) are widely distributed in plants and marine organisms.
They exert many biological activities including antitumor, antimi-
crobial, antimalarial, anti-inflammatory, neuroactive and psycho-
active actions [10,19]. Most of these activities often relay on the
aromatic b-carboline (pyrido[3,4-b]indole) ring-unit. Their biosyn-
thesis starts with indolethylamines or indolethylamino acids
that cyclizate to tetrahydro-b-carboline alkaloids (Fig. 1). Then,
tetrahydro-b-carbolines experiment an oxidation step to afford
b-carbolines. Results in this research highlight the basis for heme
peroxidases as key catalysts for this oxidation in nature. Indeed,
peroxidases abound in plants and fungi, take part in the metabo-
lism, extracellular defenses and stress [7] and are involved in key
steps within metabolic pathways of indole alkaloids [34,35] linked
to oxidative stress [36].
Mammalian peroxidases (e.g. MPO, EPO and LPO) abound in
neutrophils, eosinophils and secretory cells of human exocrine
glands. They take part in inflammatory processes and oxidize
halides to oxidants (e.g. hypochlorous acid) that help to kill micro-
organisms [37–39]. Peroxidases are involved in diseases and are
current targets for inhibitory drugs [4–6,25,26,40,41]. MPO occurs
at sites of neuroinflammation and increases in Alzheimer’s disease
[4,42] while its ablation or inhibition mitigates neurodegeneration
[40,43–45]. Oxidation of physiological donors and xenobiotics by
peroxidases often results in biological and toxic effects [3,7,8]. Tet-
rahydro-b-carboline-3-carboxylic acids and their b-carboline prod-
ucts occur in human biological tissues and fluids, and humans are
exposed to these compounds through the diet and environment
(e.g. smoking) [10,15,18,29]. Then, human peroxidases could per-
form the oxidation of tetrahydro-b-carboline-3-carboxylic acids
(THCA and MTCA) which are relatively abundant in biological

46
T. Herraiz, J. Galisteo / Biochemical and Biophysical Research Communications 451 (2014) 42–47
10
25
20
15
10
5
A
B
8
6
4
2
0
0
0
250
500
THCA ( M)
750
1000
0
250
500
THCA (
750
1000
μ
M)
μ
30
20
10
0
C
D
100
75
50
25
0
HRP
LPO
)
M
d
e
e
n
d
ci
i
m
m
a
c a
azi
i
m
u
(2
0
250
500
THCA (μM)
750
1000
O 2
H 2
orb
roxyl
Asc
Sodi
Hyd
Fig. 4. Formation of the b-carboline norharman (nmoles/min/nmol prot.) from THCA by (A) HRP (0.32
lM) and H2O2 (100 lM); (B) LPO (0.178 lM) and H2O2 (10 lM); and (C)
MPO (0.0126 M) and H2O2 (25 M); (D) inhibition (%) of oxidation of THCA (250 M) by HRP or LPO in presence of ascorbic acid (1 mM), sodium azide (1 mM),
l
l
l
hydroxylamine (1 mM), and excess of H2O2 (2 mM). Results are from duplicates.
Table 1
Apparent kinetic parameters for the oxidation of tetrahydro-b-carboline-3-carboxylic acids by heme peroxidases.
kcat (minÀ1
)
k4 (ml nmolÀ1 minÀ1
)
app
Substrate
V
(nmol b-carboline/min nmol prot.)
KaMpp
(
lM)
MAX
HRP
LPO
THCA
MTCA
11.1 1.1
9.6 0.5
335.0 81.3
393.5 63.2
11.1
9.6
0.033
0.024
THCA
MTCA
27.5 0.5
8.8 0.2
279.6 14.0
254.1 22.4
27.5
8.8
0.098
0.034
MPO
THCA
MTCA
29.7 1.9
11.9 0.7
397.3 58.8
243.2 40.6
29.7
11.9
0.075
0.049
k4 is calculated from kcat/KaMpp [23]; the b-carboline products are norharman from THCA (1,2,3,4-tetrahydro-b-carboline-3-carboxylic acid) and harman from MTCA (1-methyl-
1,2,3,4-tetrahydro-b-carboline-3-carboxylic acid). Incubations and amount of enzymes and H2O2 are described in Section 2.
systems resulting in an increase of aromatic b-carbolines (norhar-
man and harman) that exhibit singular biological actions. Aromatic
b-carbolines bind to brain receptors and inhibit monoamine oxi-
dase (MAO) exerting psychopharmacological and antidepressant
actions [11,16,46]. On the other hand, aromatic b-carbolines are
involved in toxicological processes [47–49]. They accumulate in
the substantia nigra [12,50] and could be bioactivated to neuro-
toxic b-carbolinium cations [9,18,49,51,52]. Indeed, a higher pres-
ence of aromatic b-carbolines and their b-carbolinium cations have
been found in parkinsonian patients [13,53].
Acknowledgments
The authors are grateful to Spanish government (MICINN) for
financial support (AGL2010-18448) and CSIC -Spain (200470E658).
References
[1] H.B. Dumford, Heme Peroxidases, Wiley-VCH, New York, 1999.
[2] J. Arnhold, P.G. Furtmüller, G. Regelsberger, C. Obinger, Redox properties of the
couple compound I/native enzyme of myeloperoxidase and eosinophil
peroxidase, Eur. J. Biochem. 268 (2001) 5142–5148.
In summary, this research have shown that mammalian (MPO
and LPO) and plant (HRP) heme peroxidases efficiently catalyze
the biotransformation of the naturally-occurring tetrahydro-b-
carboline-3-carboxylic acid alkaloids derived from tryptophan into
aromatic b-carboline alkaloids. This oxidation followed a typical
catalytic cycle of heme peroxidases. Tetrahydro-b-carbolines and
heme peroxidases abound in nature, including the human body.
Then, oxidation of tetrahydro-b-carbolines by heme peroxidases
can help to explain the formation and presence of b-carboline alka-
loids in biological systems. This oxidation might have implications
as b-carbolines (e.g. norharman and harman) exhibit potent biolog-
ical, pharmacological and toxicological actions.
[3] S.J. Klebanoff, Myeloperoxidase: friend and foe, J. Leukoc. Biol. 77 (2005) 598–
625.
[4] P.S. Green, A.J. Mendez, J.S. Jacob, J.R. Crowley, W. Growdon, B.T. Hyman, J.W.
Heinecke, Neuronal expression of myeloperoxidase is increased in Alzheimer’s
disease, J. Neurochem. 90 (2004) 724–733.
[5] J. Everse, P.W. Coates, Role of peroxidases in Parkinson disease: a hypothesis,
Free Radical Biol. Med. 38 (2005) 1296–1310.
[6] A. De Iuliis, A.P. Burlina, R. Boschetto, P. Zambenedetti, P. Arslan, L. Galzigna,
Increased dopamine peroxidation in postmortem Parkinsonian brain, Biochim.
Biophys. Acta Gen. Subj. 1573 (2002) 63–67.
[7] P.J. O’Brien, Peroxidases, Chem. Biol. Interact. 129 (2000) 113–139.
[8] S. Tafazoli, P.J. O’Brien, Peroxidases: a role in the metabolism and side effects of
drugs, Drug Discov. Today 10 (2005) 617–625.
[9] T. Herraiz, H. Guillén, J. Galisteo, N-methyltetrahydro-b-carboline analogs
of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxin are

T. Herraiz, J. Galisteo / Biochemical and Biophysical Research Communications 451 (2014) 42–47
47
oxidized to neurotoxic b-carbolinium cations by heme peroxidases, Biochem.
Biophys. Res. Commun. 356 (2007) 118–123.
[31] T. Herraiz, J. Galisteo, Tetrahydro-b-carboline alkaloids that occur in foods and
biological systems act as radical scavengers and antioxidants in the ABTS
assay, Free Radic. Res. 36 (2002) 923–928.
[32] J.M. Bobbitt, J.P. Willis, Electrochemistry of natural products. 7. Oxidative
decarboxylation of some tetahydro-b-carbolinecarboxylic acids, J. Org. Chem.
45 (1980) 1978–1984.
[33] B. Halliwell, J.M.C. Gutteridge, Free Radicals in Biology and Medicine, Oxford
University Press, 1999.
[34] S.E. O’Connor, J.J. Maresh, Chemistry and biology of monoterpene indole
alkaloid biosynthesis, Nat. Prod. Rep. 23 (2006) 532–547.
[35] S. Kumar, A. Dutta, A.K. Sinha, J. Sen, Cloning, characterization and localization of
a novel basic peroxidase gene from Catharanthus roseus, FEBS J. 274 (2007) 1290–
1303.
[36] H.N. Matsuura, M.R. Rau, A.G. Fett-Neto, Oxidative stress and production of
bioactive monoterpene indole alkaloids: biotechnological implications,
Biotechnol. Lett. 36 (2014) 191–200.
[10] T. Herraiz, b-Carbolines in foods, in: J. Gilbert, H.Z. Sßenyuva (Eds.), Bioactive
Compounds in Foods, Blackwell Publishing, 2008, pp. 199–223.
[11] E.S.J. Robinson, N.J. Anderson, J. Crosby, D.J. Nutt, A.L. Hudson, Endogenous b-
carbolines as clonidine-displacing substances, Ann. N. Y. Acad. Sci. 1009 (2003)
157–166.
[12] D. Fekkes, W.T. Bode, Occurrence and partition of the b-carboline norharman
in rat organs, Life Sci. 52 (1993) 2045–2054.
[13] K. Matsubara, T. Gonda, H. Sawada, T. Uezono, Y. Kobayashi, T. Kawamura, K.
Ohtaki, K. Kimura, A. Akaike, Endogenously occurring b-carboline induces
parkinsonism in nonprimate animals:
a possible causative protoxin in
idiopathic Parkinson’s disease, J. Neurochem. 70 (1998) 727–735.
[14] M.M. Airaksinen, I. Kari, b-Carbolines, psychoactive compounds in the
mammalian body. 1. Occurrence, origin and metabolism, Med. Biol. 59
(1981) 21–34.
[15] T. Herraiz, Relative exposure to b-carbolines norharman and harman from
foods and tobacco smoke, Food Addit. Contam. 21 (2004) 1041–1050.
[16] T. Herraiz, C. Chaparro, Human monoamine oxidase is inhibited by tobacco
smoke: b-carboline alkaloids act as potent and reversible inhibitors, Biochem.
Biophys. Res. Commun. 326 (2005) 378–386.
[37] H. Parker, A.M. Albrett, A.J. Kettle, C.C. Winterbourn, Myeloperoxidase associated
with neutrophil extracellular traps is active and mediates bacterial killing in the
presence of hydrogen peroxide, J. Leukoc. Biol. 91 (2012) 369–376.
[38] D. Roos, C.C. Winterbourn, Immunology – Lethal weapons, Science 296 (2002)
669–671.
[17] T. Herraiz, Occurrence of 1,2,3,4-tetrahydro-b-carboline-3-carboxylic acid and
1-methyl-1,2,3,4-tetrahydro-b-carboline-3-carboxylic acid in fruit juices,
purees, and jams, J. Agric. Food Chem. 46 (1998) 3484–3490.
[18] T. Herraiz, b- Carbolines as neurotoxins, in: L. Antkiewicz-Michaluk, H.
Rommelspacher (Eds.), Isoquinolines and b-carbolines as neurotoxins and
neuroprotectants. New vistas in Parkinson´ s disease theraphy, Current Topics
in Neurotoxicity, vol. 1, Springer, 2012, pp. 77–103.
[19] R.H. Cao, W.L. Peng, Z.H. Wang, A.L. Xu, b-Carboline alkaloids: biochemical and
pharmacological functions, Curr. Med. Chem. 14 (2007) 479–500.
[20] C.C. Shi, S.Y. Chen, G.J. Wang, J.F. Liao, C.F. Chen, Vasorelaxant effect of harman,
Eur. J. Pharmacol. 390 (2000) 319–325.
[39] J. Arnhold, J. Flemmig, Human myeloperoxidase in innate and acquired
immunity, Arch. Biochem. Biophys. 500 (2010) 92–106.
[40] D.K. Choi, S. Koppula, M. Choi, K. Suk, Recent developments in the inhibitors of
neuroinflammation and neurodegeneration: inflammatory oxidative enzymes
as a drug target, Expert Opin. Ther. Pat. 20 (2010) 1531–1546.
[41] J. Flemmig, J. Remmler, F. Rohring, J. Arnhold, (À)-Epicatechin regenerates the
chlorinating activity of myeloperoxidase in vitro and in neutrophil
granulocytes, J. Inorg. Biochem. 130 (2014) 84–91.
[42] D.L. Lefkowitz, S.S. Lefkowitz, Microglia and myeloperoxidase:
a deadly
partnership in neurodegenerative disease, Free Radic. Biol. Med. 45 (2008)
726–731.
[21] E.D. Cox, J.M. Cook, The Pictet-Spengler condensation – a new direction for an
old reaction, Chem. Rev. 95 (1995) 1797–1842.
[22] W. Jantschko, P.G. Furtmüller, M. Allegra, M.A. Livrea, C. Jakopitsch, G.
Regelsberger, C. Obinger, Redox intermediates of plant and mammalian
peroxidases: a comparative transient-kinetic study of their reactivity toward
indole derivatives, Arch. Biochem. Biophys. 398 (2002) 12–22.
[43] D.-K. Choi, S. Pennathur, C. Perier, K. Tieu, P. Teismann, D.-C. Wu, V. Jackson-
Lewis, M. Vila, J.-P. Vonsattel, J.W. Heinecke, S. Przedborski, Ablation of the
inflammatory enzyme myeloperoxidase mitigates features of Parkinson’s
disease in mice, J. Neurosci. 25 (2005) 6594–6600.
[44] P. Teismann, Myeloperoxidase in the neurodegenerative process of Parkinson’s
disease, Dtsch. Med. Wochenschr. 139 (2014) 99–102.
[23] T. Michon, M. Chenu, N. Kellershon, M. Desmadril, J. Gueguen, Horseradish
peroxidase oxidation of tyrosine-containing peptides and their subsequent
polymerization: a kinetic study, Biochemistry 36 (1997) 8504–8513.
[24] M. Allegra, P.G. Furtmüller, G. Regelsberger, M.L. Turco-Liveri, L. Tesoriere, M.
Perretti, R.A. Livrea, C. Obinger, Mechanism of reaction of melatonin with human
myeloperoxidase, Biochem. Biophys. Res. Commun. 282 (2001) 380–386.
[25] J. Soubhye, I. Aldib, B. Elfving, M. Gelbcke, P.G. Furtmüller, M. Podrecca, R.
Conotte, J.M. Colet, A. Rousseau, F. Reye, A. Sarakbi, M. Vanhaeyerbeek, J.M.
Kauffmann, C. Obinger, J. Neve, M. Prevost, K.Z. Boudjeltia, F. Dufrasne, P. Van
Antwerpen, Design, synthesis, and structure-activity relationship studies of
novel 3-alkylindole derivatives as selective and highly potent
myeloperoxidase inhibitors, J. Med. Chem. 56 (2013) 3943–3958.
[26] W. Jantschko, P.G. Furtmüller, M. Zederbauer, K. Neugschwandtner, I. Lehner, C.
Jakopitsch, J. Arnhold, C. Obinger, Exploitation of the unusual thermodynamic
properties of human myeloperoxidase in inhibitor design, Biochem. Pharmacol.
69 (2005) 1149–1157.
[27] H.R. Hallingback, R.R. Gabdoulline, R.C. Wade, Comparison of the binding and
reactivity of plant and mammalian peroxidases to indole derivatives by
computational docking, Biochemistry 45 (2006) 2940–2950.
[28] A.G. Turjanski, R.E. Rosenstein, D.A. Estrin, Reactions of melatonin and related
indoles with free radicals: a computational study, J. Med. Chem. 41 (1998)
3684–3689.
[29] T. Herraiz, J. Galisteo, Tetrahydro-b-carboline alkaloids occur in fruits and fruit
juices. Activity as antioxidants and radical scavengers, J. Agric. Food Chem. 51
(2003) 7156–7161.
[30] T. Herraiz, J. Galisteo, Endogenous and dietary indoles: a class of antioxidants
and radical scavengers in the ABTS assay, Free Radic. Res. 38 (2004) 323–331.
[45] N. Stefanova, B. Georgievska, H. Eriksson, W. Poewe, G.K. Wenning,
Myeloperoxidase inhibition ameliorates multiple system atrophy-like
degeneration in a transgenic mouse model, Neurotox. Res. 21 (2012) 393–404.
[46] D. Farzin, N. Mansouri, Antidepressant-like effect of harmane and other b-
carbolines in the mouse forced swim test, Eur. Neuropsychopharmacol. 16
(2006) 324–328.
[47] H. Rommelspacher, L.G. Schmidt, T. May, Plasma norharman (b-carboline) levels
are elevated in chronic alcoholics, Alcohol. Clin. Exp. Res. 15 (1991) 553–559.
[48] E.D. Louis, E. Rios, K.M. Pellegrino, W. Jiang, P. Factor-Litvak, W. Zheng, Higher
blood harmane (1-methyl-9H-pyrido-3,4-b-indole) concentrations correlate
with lower olfactory scores in essential tremor, Neurotoxicology 29 (2008)
460–465.
[49] M.A. Collins, Alkaloids, alcohol and Parkinson’s disease, Parkinsonism Relat.
Disord. 8 (2002) 417–422.
[50] A. Östergren, A. Annas, K. Skog, N.G. Lindquist, E.B. Brittebo, Long term
retention of neurotoxic b-carbolines in brain neuromelanin, J. Neural Transm.
111 (2004) 141–157.
[51] D.A. Gearhart, M.A. Collins, J.M. Lee, E.J. Neafsey, Increased b-carboline 9N-
methyltransferase activity in the frontal cortex in Parkinson’s disease,
Neurobiol. Dis. 7 (2000) 201–211.
[52] T. Herraiz, H. Guillén, J. Galisteo, Metabolite profile resulting from the
activation/inactivation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and
2-methyltetrahydro-b-carboline by oxidative enzymes, Biomed. Res. Int.
248608 (2013).
[53] W. Kuhn, T. Muller, H. Grosse, H. Rommelspacher, Elevated levels of harman
and norharman in cerebrospinal fluid of parkinsonian patients, J. Neural
Transm. 103 (1996) 1435–1440.