Aerobic co-oxidation of hemoglobin and aminoacetone, a putative source of methylglyoxal

Article abstract of DOI:10.1016/j.freeradbiomed.2021.02.023

Aminoacetone (1-aminopropan-2-one), a putative minor biological source of methylglyoxal, reacts like other α-aminoketones such as 6-aminolevulinic acid (first heme precursor) and 1,4-diaminobutanone (a microbicide) yielding electrophilic α-oxoaldehydes, ammonium ion and reactive oxygen species by metal- and hemeprotein-catalyzed aerobic oxidation. A plethora of recent reports implicates triose phosphate-generated methylglyoxal in protein crosslinking and DNA addition, leading to age-related disorders, including diabetes. Importantly, methylglyoxal-treated hemoglobin adds four water-exposed arginine residues, which may compromise its physiological role and potentially serve as biomarkers for diabetes. This paper reports on the co-oxidation of aminoacetone and oxyhemoglobin in normally aerated phosphate buffer, leading to structural changes in hemoglobin, which can be attributed to the addition of aminoacetone-produced methylglyoxal to the protein. Hydroxyl radical-promoted chemical damage to hemoglobin may also occur in parallel, which is suggested by EPR-spin trapping studies with 5,5-dimethyl-1-pyrroline-N-oxide and ethanol. Concomitantly, oxyhemoglobin is oxidized to methemoglobin, as indicated by characteristic CD spectral changes in the Soret and visible regions. Overall, these findings may contribute to elucidate the molecular mechanisms underlying human diseases associated with hemoglobin dysfunctions and with aminoacetone in metabolic alterations related to excess glycine and threonine.

Full text of DOI:10.1016/j.freeradbiomed.2021.02.023

Free Radical Biology and Medicine 166 (2021) 178–186
Contents lists available at ScienceDirect
Free Radical Biology and Medicine
journal homepage: www.elsevier.com/locate/freeradbiomed
Original article
Aerobic co-oxidation of hemoglobin and aminoacetone, a putative source
of methylglyoxal
a
,
b
a
,
c
,
d
e
e
Luiz D. Ramos , Mariana C. Mantovani
, Adriano Sartori , Fernando Dutra ,
a
a,c,*
Cassius V. Stevani , Etelvino J.H. Bechara
a
Departamento de Química Fundamental, Universidade de S a˜ o Paulo, S a˜ o Paulo, SP, Brazil
Centro Universit a´ rio Anhanguera, UniA, Santo Andr ´e , SP, Brazil
Instituto de Ci ˆe ncias Ambientais, Químicas e Farmac ˆe uticas, Universidade Federal de S a˜ o Paulo, Diadema, SP, Brazil
Instituto de Pesquisas Energ ´e ticas e Nucleares, Universidade de S a˜ o Paulo, S a˜ o Paulo, SP, Brazil
Centro de Ci ˆe ncias Exatas e Tecnologia, Universidade Cruzeiro Do Sul, S a˜ o Paulo, SP, Brazil
b
c
d
e
A R T I C L E I N F O
A B S T R A C T
Keywords:
Aminoacetone (1-aminopropan-2-one), a putative minor biological source of methylglyoxal, reacts like other
Aminoacetone
Methylglyoxal
Hemoglobin
α
-aminoketones such as 6-aminolevulinic acid (first heme precursor) and 1,4-diaminobutanone (a microbicide)
yielding electrophilic -oxoaldehydes, ammonium ion and reactive oxygen species by metal- and hemeprotein-
α
catalyzed aerobic oxidation. A plethora of recent reports implicates triose phosphate-generated methylglyoxal in
protein crosslinking and DNA addition, leading to age-related disorders, including diabetes. Importantly,
methylglyoxal-treated hemoglobin adds four water-exposed arginine residues, which may compromise its
physiological role and potentially serve as biomarkers for diabetes. This paper reports on the co-oxidation of
aminoacetone and oxyhemoglobin in normally aerated phosphate buffer, leading to structural changes in he-
moglobin, which can be attributed to the addition of aminoacetone-produced methylglyoxal to the protein.
Hydroxyl radical-promoted chemical damage to hemoglobin may also occur in parallel, which is suggested by
EPR-spin trapping studies with 5,5-dimethyl-1-pyrroline-N-oxide and ethanol. Concomitantly, oxyhemoglobin is
oxidized to methemoglobin, as indicated by characteristic CD spectral changes in the Soret and visible regions.
Overall, these findings may contribute to elucidate the molecular mechanisms underlying human diseases
associated with hemoglobin dysfunctions and with aminoacetone in metabolic alterations related to excess
glycine and threonine.
α
-aminoketones
Electron transfer
Superoxide radical
1
. Introduction
diabetes [6–13].
Methylglyoxal (MG), the simplest and most widely studied -oxoal-
α
Alpha hydroxy and
α
-aminoketones are carbohydrate and protein
dehyde due to its involvement in age-associated diseases, is generated
mostly by the spontaneous degradation of the triose phosphates of the
glycolytic pathway and subsequent action of methylglyoxal synthase
(MS) [14–20]. To a lesser extent, MG is reportedly a product of the cyt
catabolites prone to enolization and further chemical or enzymatic
oxidation yielding highly reactive
-oxoaldehyde electrophiles [1–5]. By
bearing a vicinal electron-withdrawing carbonyl group, -oxoaldehydes
α
α
and aldoses reportedly undergo condensation reactions with the amino
groups of basic amino acids such as lysine (Lys) and arginine (Arg) and
P450-catalyzed aerobic oxidation of acetone (a ketone body) and of
aminoacetone (AA), a substrate of a semicarbazide-sensitive amine ox-
idase (SSAO) [14]. Aminoacetone (AA) also is a putative threonine and
glycine catabolite produced during reactions catalyzed by the sequential
action of L-threonine 3-dehydrogenase (TDH) and 2-amino-3-ketobuty-
rate CoA ligase (KBL) (Fig. 1) [21]. In vitro, aminoacetone has been
′
′
of nucleobases 2 -deoxyadenosine (dAdo) and 2 -deoxyguanosine
(
dGuo), yielding Schiff’s base. This leads to protein adducts and aggre-
gation and the formation of ethene DNA adducts when accumulated in
the tissues of patients suffering from metabolic disorders such as
Abbreviations: AA, aminoacetone; MG, methylglyoxal; oxyHb, oxyhemoglobin; metHb, methemoglobin; ALA, 5-aminolevulinic acid; DAB, 1,4-diamino-2-
butanone.
*
Corresponding author. Departamento de Química Fundamental, Instituto de Química, Av. Prof. Lineu Prestes 748, 05508-000, S a˜ o Paulo, SP, Brazil.
E-mail address: ebechara@iq.usp.br (E.J.H. Bechara).
https://doi.org/10.1016/j.freeradbiomed.2021.02.023
Received 28 December 2020; Received in revised form 10 February 2021; Accepted 13 February 2021
Available online 23 February 2021
0
891-5849/© 2021 Elsevier Inc. All rights reserved.

L.D. Ramos et al.
Free Radical Biology and Medicine 166 (2021) 178–186
⦁
⦁
shown to undergo non-enzymatic oxidation catalyzed by adventitious
yields a resonant enoyl radical (AA ) [CH
3
–C(O ) = CHNH
2
↔
⦁
⦁-
2
iron ions propagated by superoxide ion radicals [17,21–24].
CH
3
–CO–C NH
2
] and the superoxide radical-anion (O
), which propa-
+
Threonine conversion to aminoacetone is initiated by NAD -depen-
gates the reaction by abstracting one electron from AA (eq. (1)) [5,37,
⦁
⦁-
2
dent oxidation to 2-amino-3-ketobutyrate catalyzed by threonine de-
hydrogenase [22]. Highly unstable, the latter catabolite undergoes
spontaneous decarboxylation that yields aminoacetone [27], which is a
substrate for a semicarbazide-sensitive amine oxidase, SSAO [3,4,22–24,
40]. Further electron transfer from AA to oxygen regenerates O
and
–
forms AAimino (eq. (2)) (CH
3
–C(O)–CH
–
NH), whose hydrolysis yields
+
⦁-
2
MG and NH
4
ions (eq. (3)). The reaction ends with O
dismutation to
⦁
⦁-
2
H
2
O
2
(eq. (4)) and annihilation of AA by O
radical, which also yields
2
7], whose products are highly reactive MG and H
2
O
2
(Fig. 1). Inter-
AAimino (eq. (5)).
estingly, threonine and glycine supplementation of cultures of Strepto-
myces sahachiroi, a soil–dwelling microorganism able to synthesize the
antitumor azinomycin A, yields an aminoacetone derivative [21]. Like
AA + O•ꢀ + H →AA + H
+
•
O
(1)
(2)
(3)
(4)
(5)
2
2
2
•
→AAim ino + O •2 ꢀ + H+
AA + O
2
AA, other -aminoketones are of biomedical interest, such as 5-aminole-
α
vulinic acid (ALA), a heme precursor known to accumulate in blood and
other tissues of patients of acquired and inborn porphyria, e.g., in lead
poisoning [28–32], intermittent acute porphyria [33,34], and heredi-
tary tyrosinemia Type 1 [35]. In addition, the 1,4-diamino-2-butanone
O →MG + NH+4
+
AAim ino + H
3
•ꢀ
+
2O
+ 2H →H
2
O
2
+ O
2
2
(
DAB), a putrescine analogue, acts as a large spectrum microbicide,
including against Trypanosoma cruzi [36–38].
The -aminoketones AA, ALA, and DAB have been shown to undergo
•
•ꢀ
+
AA + O2 + H →AAim ino + H
2
O
2
Accordingly, AA oxidation to MG is inhibited by the addition of CuZn
superoxide dismutase (SOD-1) and its rate of formation increases in
response to the addition of the xanthine/xanthine oxidase system, a
α
in vitro enolization at physiological pH in phosphate buffer, followed by
metal-catalyzed and superoxide-propagated aerobic oxidation to the
⦁
-
2
long-known source of O radical [1,4]. A remarkable set of potentially
corresponding
α
-oxoaldehydes, respectively, MG, 4,5-dioxovaleric acid
+
toxic reactive species are thus produced by the aerobic oxidation of AA:
(
DOVA), and 4-amino-2-oxobutanal, plus H
2
O
2
and the NH
4
ion (Fig. 2)
+
oxygen- and carbon-centered radicals, H
2
O
2
, NH
4
ion, and electrophilic
[
2,4,37]. Proteins (ferritin, ceruloplasmin, cytochrome c), supercoiled
MG. Hence, AA has been shown to trigger the radical-mediated release
plasmid DNA, isolated rat liver mitochondria, insulin-producing cell
of Fe2 ions from ferritin and copper ions in human plasma cerulo-
+
cultures and rats have been shown, in vitro or in vivo, to undergo
2
+
plasmin [41–43], inducing Ca -mediated membrane permeabilization
and oxidative damage to isolated rat liver mitochondria [42] and
causing oxidative imbalance and death to insulin-producing RINm5f
cells [44]. Moreover, AA has been shown to directly reduce
oxidative injury when challenged with
Similar to ALA (pK 8.1) and DAB (pKa1 7.5; pKa2 9.5), the AA (pK
.8) reaction is believed to be triggered by electron-transfer from
α-aminoketones [3,5,39].
a
a
6
–
tautomeric enol AA [CH
3
–C(OH)
–
CHNH ] to oxygen. This reaction
2
Fig. 1. Proposed pathways for enzymatic generation of methylglyoxal (MG) from triose phosphates [25], acetone [26], and aminoacetone (AA) [4]. Also reported is
in vitro metal-catalyzed generation of MG from aminoacetone, initiated by a superoxide anion radical source such as the xanthine/xanthine oxidase system [1] or by
2 2
an H O -provided oxo-iron AA complex [17].
1
79

L.D. Ramos et al.
Free Radical Biology and Medicine 166 (2021) 178–186
Fig. 2. A-aminoketones reported to undergo metal-catalyzed oxidation by molecular oxygen to the corresponding
α-oxoaldehydes and ammonium ion. Values of pKa
a
b
c
obtained from Ref. [4] [37], , and [40] .
ferricytochrome c to its ferrous form under anaerobiosis, ultimately
2. Materials and methods
+
yielding MG and NH
4
ion, and also H
2
O
2
when in the presence of oxygen
[
17]. However, contrary to the studies conducted by Oliveira et al. [45]
2.1. Reagents
and more recently by Mercado-Uribe and colleagues [46] on MG-treated
cytochrome c, no significant alterations in tertiary structure and protein
aggregates were found under AA treatment, although enhanced fluo-
rescence intensities were detected, attributable to the oxidation of Trp
and Tyr cyt c residues [44].
Reagents of the purest quality available and buffer salts were pur-
chased from Sigma-Aldrich (St. Louis, MO). The spin trap 5,5-dimethyl-
1-pyrroline-N-oxide (DMPO) was obtained from Dojindo Molecular
Technologies (Rockville, MD), and HPLC grade solvents were acquired
from Merck KGaA (Darmstadt, Germany).
Earlier findings indicate (i) that the physiological concentrations of
MG form hydroimidazolone adducts with solvent accessible Arg residues
of human hemoglobin (Hb) without heme release [47], and (ii) that
bovine oxyhemoglobin (oxyHb) increase the rate of ALA aerobic
2
.2. Synthesis of
α
-aminoacetone, AA
oxidation to an -oxoaldehyde (DOVA) chemically similar to MG while
α
Alpha-aminoacetone was prepared as described by Hepworth [48],
being oxidized to methemoglobin (metHb) [2,40]. Consequently, we
launched an investigation into oxyHb-induced AA oxidation to MG,
under the assumption that it may be relevant to help clarify a possible
role of AA in diabetes as a source of MG.
with slight modifications. Briefly, a stoichiometric mixture of glycine,
pyridine and acetic anhydride was kept under reflux and stirring for 6 h.
Excess reagents were removed by evaporation at reduced pressure
without heating, yielding a yellow oil. This oil was dissolved in 6 M HCl
and refluxed under Argon atmosphere for 6 h. The solution was
concentrated by evaporation at reduced pressure, yielding a brown oil
that was recrystallized from ethanol: ether (8:2), sealed in Eppendorf
◦
vials in a nitrogen glove box, and stored at ꢀ 20 C. Stock solutions of
1
2
00 mM AA were prepared in N -purged Milli-Q purified water
1
80

L.D. Ramos et al.
Free Radical Biology and Medicine 166 (2021) 178–186
immediately before use. Elemental analysis of C
3
H
8
ClNO (Calc./Found):
2.9. Statistical analysis
1
2
C(32.89/31.92); N(12.79/12.25); H(7.36/7.21). HNMR.in D O-d2,
2
00 MHz, δ/ppm): 2.08 (3H, s); 3.88 (2H, s).
The results obtained from at least three independent experiments
were analyzed using one-way ANOVA followed by Dunnett’s multiple
comparisons test (GraphPad Prism 7), with a significance of p < 0.05.
2
.3. Preparation of oxyHb and metHb
3
. Results
To obtain oxyHb, commercial Hb solution (0.05 mM) was prepared
by diluting Hb (Sigma-Aldrich) in Chelex-treated phosphate buffer (50
mM, pH 7.4). Sodium hydrosulfite (SHS) solution was added with SHS:
3
.1. Oxygen uptake studies
Hb = 1:1 (molar ration), respectively. NaHSO
3
-treated bovine blood
As previously reported, 5 mM AA slowly consumes molecular oxygen
hemoglobin was purified by gel filtration through Sephadex G-25 and
quantified spectrophotometrically as already reported [2]. The metHb
was prepared in the same way, except that Hb was treated with potas-
sium ferrocyanide.
in air-equilibrated Chelex-treated phosphate buffer (50 mM, pH 7.4 at
◦
⦁-
3
7 C) (Fig. 3d, Table 1), through a reaction propagated by O
2
radicals
⦁
and amplified by HO radicals formed from superoxide dismutation (eq.
(
7)) and Fenton-type reaction (eq. (8)). Despite pretreatment with
Chelex, residual contaminant transition metal ions must have catalyzed
the molecular oxygen-promoted AA oxidation, since autoxidation is
spin-forbidden [4,49].
2
.4. Oxygen uptake
A Hansatech oxygraph equipped with a Clark-type electrode was
Fe3 + O •2 ꢀ →Fe2+ + O
+
(6)
used to measure oxygen uptake by AA (1.0–15 mM) in air-equilibrated
buffer at 37 C, in both the absence and presence of micromolar
2
◦
+ SOD
amounts of oxyHb. Catalase (5
μ
M) and superoxide dismutase (SOD 1,
2O •2 ꢀ + 2H ̅→H
2
O
2
+ O
2
(7)
(8)
CuZnSOD) (50 U/mL) were used throughout this work to demonstrate
Fe2 + H
+
O
→Fe + HO + HOꢀ
3+
•
the involvement of H
reaction mechanism.
2
O
2
and superoxide species, respectively, in the
2
2
Accordingly, oxygen consumption is slowed down by the addition of
either SOD (Fig. 3e) or catalase (Fig. 3f), which are known superoxide
radical-anion and hydroxyl radical suppressors, respectively. Ten
micromolar oxyHb are shown to enhance by fivefold the rate of oxygen
uptake (Fig. 3a, Table 1), which is also inhibited in the presence of SOD
(Fig. 3b), and much more significantly by that of catalase (Fig. 3c).
Initiation of the reaction is here rationalized in terms of previous
attachment of AA, an electron donor, to the outer coordination sphere of
oxyHb heme Fe(II). This step is based on a similar mechanism long
proposed by Wallace and Caughey [50–52] to explain faster oxyHb
oxidation to metHb by nucleophiles, and more recently recalled to
describe the oxidation of AA catalyzed by cytochrome c [17]. Next,
concomitant one-electron transfer from coordinated AA and heme Fe(II)
2
.5. UV–vis spectrophotometry
A Varian Cary 50 Bio spectrophotometer was used to record the
temporal variations of the absorption spectra of oxyHb (7.8 M) in the
μ
presence and absence of AA (1–5 mM) in 50 mM phosphate buffer at
3
◦
7
C. Spectral changes of oxyHb in the range of 300–700 nm were
tracked at 2-min intervals for 20 min.
2
.6. EPR spin trapping
EPR spin-trapping studies were conducted by using a Bruker EMX
, a potential source of HO⦁
to molecular oxygen would yield H O
2 2
spectrometer with the AA/oxyHb reaction mixtures in the presence of
5 mM DMPO and either catalase, SOD or ethanol to verify the inter-
radical. Hydroxyl radical could then efficiently initiate the AA
superoxide-propagated chain reaction. This hypothesis is consistent
with the slower AA oxidation observed in the presence of oxyHb and
2
mediacy of oxyradicals. EPR spectra were tracked 3 min after the
addition of AA under operating conditions indicated in the figure
legends.
2
.7. CD analysis
The CD spectra of fivefold diluted 1.0–5.0 mM AA/50
μ
M oxyHb-
containing solutions incubated for 24 h were tracked in the range of
1
90–450 nm (Far-UV, Near-UV, and Soret regions) at room temperature
in a Jasco J-720 spectropolarimeter. Quartz cells with 0.10- and 0.50-cm
light paths were used for measurements in the far-UV and near-UV/Soret
regions, respectively. All the spectra were corrected by subtracting their
backgrounds. The spectra were acquired with 10 nm/min resolution,
applying an average of four scans per spectrum. Spectral deconvolution
was performed using CONTIN software.
2
.8. SDS-PAGE studies
◦
OxyHb (50
μ
M) was incubated for 2 h at 37 C with AA (0.0–5.0
◦
mM). The eightfold diluted samples were heated (100 C for 5 min) in
the presence of sample buffer (TRIS pH 6.8 (50 mM); glycerol (10%);
SDS (2%); Bromophenol blue (0.1%); 2-Mercaptoethanol (5%). The
prepared samples were loaded in 12% polyacrylamide SDS-PAGE gel
and separated at 90 V. The protein bands were stained with Coomassie
blue dye.
Fig. 3. Effects of SOD and catalase on oxygen consumption by the AA/oxyHb
system. The experiments were conducted with 5 mM AA and 10
μ
M oxyHb in
◦
phosphate buffer (50 mM, pH 7.4, 37 C). AA + oxyHb (a); AA + oxyHb + SOD
(50 U/mL) (b); AA + oxyHb + catalase (5
μM) (c); AA (d); AA + SOD (50 U/
mL) (e); AA + catalase (5 M) (f); oxyHb (g).
μ
1
81

L.D. Ramos et al.
Free Radical Biology and Medicine 166 (2021) 178–186
Table 1
superoxide dismutase and catalase to the AA/oxyHb-containing reaction
Relative rates of O
2
consumption by the AA/oxyHb system in normally aerated
mixture. The EPR spectra depicted in Fig. 5a–c (black lines) can be
◦
5
0 mM phosphate buffer, pH 7.4, at 37 C).
assigned to the DMPO-OH spin adduct (4 lines 1:2:2:1; a
= a
-hydroxyethyl
= 1.58 mT)
= 1.49
H
N
a
System
O
2
consumption (ΔA/min)
mT) [17,40], whereas the red lines describe a DMPO-
α
•
3
radical [ CH(OH)–CH ] adduct (6 lines; a
H
β = 2.28 mT; a
N
AA + oxyHb (a)
5.041 ± 0.009
2.132 ± 0.003
0.553 ± 0.004
1.000 ± 0.004
0.443 ± 0.008
0.724 ± 0.005
0.465 ± 0.008
in the presence of ethanol [53]. The EPR studies with the AA/oxyHb
system and the control runs with only AA [40] are depicted in Fig. S2. A
AA + oxyHb + SOD (b)
AA + oxyHb + catalase (c)
AA (d)
3
-min incubation time was adopted to obtain additional information
AA + SOD (e)
confirming the involvement of the reactive oxygen species in the initi-
ation step (Fig. 5), albeit reflecting an apparent absence of oxyHb effect
on the intensity of the EPR spectrum compared to that of the complete
system.
AA + catalase (f)
oxyHb (g)
a
Values were normalized for 5,0 mM AA.
As expected, the effect of catalase inhibition observed here (Fig. 5-
black-c) clearly predominates over that of SOD (Fig. 5-black-b), which is
consistent with the assumption that the reaction is initiated by
concomitant two-electron transfer from AA and oxyHb(FeII) to molec-
catalase (Fig. 3c vs d). Similar kinetic effects of the presence of oxyHb,
SOD and catalase on the aerobic oxidation of another -aminoketone,
α
namely ALA, have been reported previously [40]. Ferritin and cerulo-
plasmin have also previously been shown to amplify AA oxidation
2 2
ular oxygen, yielding H O and metHb [2,40]. These findings corrobo-
through the release of catalytic metal ions [4,41]. In both cases, the
rate the results of the oxymetric studies depicted in Fig. 3, which shows
that SOD and catalase decrease the rate of oxygen consumption by the
reaction.
intermediacy of reactive O⦁
-
and H
was corroborated by the use of
2
2
O
2
SOD and catalase.
EPR spin-trapping experiments in the presence of ethanol (Fig. 5-red)
•
were performed to demonstrate that the four-line signal [DMPO-OH]
3
.2. UV–visible spectrometric analysis
•
adduct does not result from spontaneous [DMPO-OOH] decomposition
(
t
1/2 = 27 s at pH 5.0 and 91 s at pH 9.0) [54]. Ethanol does not react
Previous studies have shown that superoxide radical-anion and
, produced in the adventitious metal-catalyzed oxidation of ALA
with superoxide radical-anion, but is able to donate a hydrogen atom to
H
2
O
2
•
•
HO , yielding a stable adduct with DMPO {[DMPO–CHOH–CH
3
] , aHβ
and amplified in response to the addition of Fe(III), drive the co-
oxidation of oxyHb to metHb [4,40]. Now we report that the addition
of AA to oxyHb-containing buffered phosphate solutions also promotes
bleaching, hypsochromic shift and heme iron oxidation of oxyHb as a
function of AA concentration and incubation time (eq. (9), Fig. 4 and
Fig. S1). This is indicated by the decay of oxyHb absorbance at 400, 545,
and 577 nm with concomitant increased absorption at 630 nm, and
isosbestic points at 350, 523, and 580 nm, which are characteristic of the
conversion of oxyHb to metHb [40].
=
2.28 mT; aN = 1.58 mT} [53]. A six-line spectrum can be assigned to
the DMPO-ethanol radical adduct (Fig. 5-red) and it is thus in accor-
dance with the data presented (Fig. 5-black). The use of SOD and cata-
lase only demonstrated a partial inhibitory effect, suggesting that the
•
[
DMPO-OH] radical adduct derives from the Fenton reaction
(
Fig. 5-black).
3
.3. SDS-PAGE experiments
→MG + m etHb + NH+ + H
O
(9)
SDS-PAGE studies were performed using an oxyHb-treated AA so-
AA + oxyHb + O
2
2
2
4
lution to determine if the system can cause structural and molecular
weight damage to the protein (Fig. 6). Albeit not yet identified, hemo-
globin alterations by AA-generated products were observed only in the
presence of 5 mM AA, which showed a small, but significant decrease in
the gel band densitometry. Indeed, numerous studies have reported
oxidative damage to proteins promoted by radicals and reactive alde-
Spin-trapping EPR experiments with DMPO and ethanol were carried
out to confirm the participation of superoxide radical-anion, H , and
2 2
O
hydroxyl radical intermediates, which were checked by adding
hydes, including
α
-oxoaldehydes [55,56]. It was previously reported
47] that upon treatment with MG (10
M–200 mM), hemoglobin (100
M) undergoes structural and chemical modifications. According to the
[
μ
μ
authors, hemoglobin undergoes condensation reactions with MG (Schiff
base formation) in solvent exposed arginine residues, namely Arg-92
and Arg 141 in the
yielding hydroimidazolone derivatives. Two other buried arginine res-
idues – Arg-31 in the chain and Arg-30 in the β chain – are not
α-chain and Arg-40 and Arg-104 in the β-chain,
α
modified. The authors suggested that Hb-MG adducts may be harnessed
as possible molecular biomarkers of diabetes and other diseases asso-
ciated with MG accumulation, similarly to non-enzymatically glycated
hemoglobin (HbA1c). Noteworthy is the observation of significant he-
moglobin alterations only at a higher AA concentration (5 mM), in ex-
periments using up to 200 mM MG [47].
3
.4. Circular dichroism studies
The CD spectra of oxyHb in the far-UV range (190–250 nm), near UV
Fig. 4. Spectrophotometric profile of AA and oxyHb co-oxidation. Temporal
(
250–350 nm) and the Soret band (390–450 nm), were obtained in the
variations of 7.8
μ
M hemoglobin spectrum in the presence of 5.0 mM AA in 50
◦
absence and presence of AA in order to verify any spatial structural
protein modifications. In response to AA treatment, the far-UV spectrum
presents a slight concentration-dependent decrease in the 200 nm peak
(Fig. 7A). Spectral deconvolution showed only minor changes in the
mM phosphate buffer, pH 7.4, at 37 C, at 2-min intervals for 20 min. 0 min.
(
(
(
black); 2 min (red); 4 min (green); 6 min (blue); 8 min (cyan); 10 min
magenta); 12 min (yellow); 14 min (dark yellow); 16 min (navy); 18 min
purple); 20 min (wine). (For interpretation of the references to colour in this
figure legend, the reader is referred to the Web version of this article.)
percentage of protein secondary structure (
α-helix = 98.9%; β - sheet =
1
82

L.D. Ramos et al.
Free Radical Biology and Medicine 166 (2021) 178–186
Fig. 5. EPR spin trapping studies of the AA/oxyHb system. Three minutes of incubation of 15 mM AA/150
μ
M oxyHb using 25 mM DMPO as spin trap in phosphate
◦
buffer (50 mM, pH 7.4, at 37 C) black lines, or with 30% ethanol (v/v) red lines. AA + oxyHb (a); AA + oxyHb + SOD (150 U/mL) (b); AA + oxyHb + catalase (5
μ
M) (c). The instrumental conditions were as follows: microwave power = 20 mW, modulation amplitude = 0.1 mT, modulation frequency = 100.00 KHz, time
6
constant = 163.83 ms, receiver gain = 1.1 × 10 . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of
this article.)
Fig. 6. Hemoglobin band optical densitometries. OxyHb (7.8
Hb control (1); Hb + AA (5.0 mM) (2), Hb + AA (2.0 mM) (3), Hb + AA (1.0 mM) (4), incubated Hb control (5). Statistical parameters: not significant p > 0.05, *p <
.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
μ
M) were incubated with increasing concentrations of AA and stained with Coomassie blue dye. Fresh
0
1
.1%; random structure = 0%). The tertiary structure of the protein
Fig. 1). These data are interpreted through different reaction mecha-
nisms of AA metal-catalyzed oxidation or oxyHb-mediated AA oxidation
by molecular oxygen, based on previous studies of several metabolites
(
Fig. 7B) proved to be more affected by higher AA concentrations,
possibly as a result of the attachment of AA-oxidized MG product to Arg
residues of the protein, which would decrease the interaction between
aromatics and apoprotein, placing them in a more symmetrical envi-
ronment [47,57]. The Soret band is also AA-concentration dependent,
which may be ascribed to the decreased interaction of the heme moiety
with aromatic residues of the protein [58].
belonging to the classes of
tone phosphate [1], glucose and of
AA [4], and DAB [36–38]. In the absence of iron or hemeproteins, these
compounds were shown to be oxidized to the corresponding -oxo-
α
-hydroxycarbonyls, such as dihydroxyace-
α
-aminocarbonyls such as ALA [40],
α
carbonyl products via superoxide radical intermediate, which explains
the stronger inhibitory effect of added SOD. The lower effect of catalase
⦁
-
4
. Discussion
2 2 2
is due to the greater generation of H O from O dismutation from the
⦁
Fenton reaction driven by adventitious metals. Once formed, HO radi-
cals can lead to electron abstraction from AA, thereby amplifying the AA
metal-catalyzed oxidation. This is corroborated by the EPR spin trapping
studies conducted with DMPO in the absence of oxyHb depicted in
Fig. S2-black-a. Conversely, in the presence of oxyHb, the effect of
catalase prevails over that of SOD, which has been interpreted as due to
previous coordination of AA to the heme iron, followed by pari passu
In this report, hemoglobin is shown to undergo co-oxidation with AA
•- •
to MG via H O , O , OH intermediates, with concomitant heme iron
2 2 2
oxidation and modification of the secondary and tertiary protein struc-
tures. Oxygen uptake measurements show that oxyHb accelerates AA
oxidation (fivefold in the experimental conditions of this study; Fig. 3),
2 2,
and is oxidized to metHb (Fig. 4). Unambiguous intermediacy of H O
and oxyradicals is confirmed by the inhibitory effects of the kinetics of
oxygen consumption exerted by the antioxidant enzymes SOD and
catalase. Both enzymes inhibit the reaction to some extent, but SOD is
more efficient in the absence of oxyHb (Fig. S2), whereas catalase
operates mainly in the presence of hemeprotein (Fig. 5) [2,4,5,40,53].
This is in accordance with the proposed mechanisms (equations and
2 2 2
one-transfer from both AA and Fe(II) to O yielding H O , a source of the
⦁
HO radical initiator (Fig. 1). The simultaneous operation of the two
mechanisms is supported by EPR spin-trapping studies in the presence of
ethanol (Fig. 5-red and S2), where both catalase and SOD exhibited a
•
quenching effect, albeit to different degrees, on the DMPO- CHOH–CH
3
adduct signal during the experiment carried out with the AA/oxyHb
1
83

L.D. Ramos et al.
Free Radical Biology and Medicine 166 (2021) 178–186
not identified in the SDS-PAGE experiment, as only the native protein
monomer band (approximately 14 kDa) was detected. The determina-
tion of oxyHb bands by optical densitometry revealed that only a high
AA concentration (5 mM) resulted in significantly reducing the intensity
of the protein band. This finding is similar to that reported by Wang and
Gao in their study of hemoglobin tagging with MG, thus suggesting that
SDS-PAGE loss of native oxyHb may be due to the chemical attachment
of MG product to water-exposed protein Arg residues [47] and Hb ag-
gregation [55,56].
The UV/visible absorption bands (Fig. 4 and S1) indicate that AA
promotes bleaching of the Soret band, causes a hypsochromic shift in the
Soret band, and diminishes the absorption intensities attributed to heme
iron (545 nm and 577 nm), culminating in the characteristic spectrum of
metHb. Additionally, the CD spectra recorded in the UV and visible re-
gions suggest as-yet unidentified secondary and tertiary structural al-
terations in hemoglobin.
5
. Conclusion
In summary, our work presents relevant data that elucidates the role
of aminoacetone as a possible contributing source of MG, whose putative
accumulation in vivo has been implicated in age-related diseases such as
diabetes [20] and in threonine disorders such as threoninemia and
cri-du-chat, a rare inborn disease [4]. Here, we propose that co-oxidation
of Hb and aminoacetone occurs by simultaneous one-electron transfer
from oxyHb Fe(II) and aminoacetone to molecular oxygen, yielding
2
metHb (FeIII) and H O2, as previously reported by Wallace et al. [51] for
electron donors-treated oxyHb and by our group [2,40]. This has now
been shown to be accompanied by structural alterations of the apopro-
tein promoted by AA-generated oxyradicals and electrophilic MG, both
species able to elicit adverse effects of AA if accumulated in vivo [14].
5
.1. Biomedical implications
OxyHb exposure to MG and other
α
-oxoaldehydes generated by
triose phosphates, and to a lesser extent to Thr or Gly/CoA, reportedly
leads to structural and oxidative modifications of protein that may
impair its biological functions [4,14,39]. Likewise, their determination
of HbA1c to evaluate the glycemic status of normal and diabetic in-
dividuals led Wang and Gao [47] to suggest that blood indices of Hb-MG
adduct may also help clarify pathological conditions linked to excess MG
in diabetes, other age-related diseases, and albeit rare, in threoninemia
[
5]. Moreover, it is important to underscore the biochemical signifi-
cance of potential oxidative and carbonyl stress triggered by iron- and
hemeprotein-catalyzed aerobic oxidation of -aminocarbonyl metabo-
lites such as ALA, AA and -amino-substituted aldoses and ketoses for
α
α
their ability to generate oxy-radicals, H
bonyls [4,37,40].
2
O
2
, and electrophilic -dicar-
α
Declaration of competing interest
The authors declare that they have no conflict of interests.
Acknowledgements and funding
The authors are grateful to the funding agencies that supported the
elaboration of this work. Funding: S a˜ o Paulo Research Foundation –
FAPESP (EJHB 2017/22501–2; LDR 2019/24515–6; CVS 2017/
Fig. 7. Effect of AA concentration on the CD spectra of oxyHb in the far-UV (A),
near UV (B), and Soret band region (C). Experimental conditions: oxyHb (15
μ
M) (black); oxyHb + AA (1.0 mM) (red); oxyHb + AA (2.0 mM) (green);
2
2501–2), by the Brazilian National Council for Scientific and Techno-
oxyHb + AA (5.0 mM) (blue). (For interpretation of the references to colour in
logical Development – CNPq (EJHB 306460/2016–5), and John Simon
Guggenheim Memorial Foundation (EJHB Fellowships). We are
indebted to Ohara Augusto and Iseli Nantes for EPR and CD assistance,
respectively.
this figure legend, the reader is referred to the Web version of this article.)
system. The lower efficiency of catalase in the absence of oxyHb (Fig. S2)
may also only be apparent because the enzyme-catalyzed decomposition
2 2
of H O partly replenishes the consumed molecular oxygen.
Fragmentation and oligomerization of oxyHb induced by AA were
1
84

L.D. Ramos et al.
Free Radical Biology and Medicine 166 (2021) 178–186
Appendix A. Supplementary data
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