Effect of amino acids on the interaction between cobalamin(II) and dehydroascorbic acid

Article abstract of DOI:10.1134/S0036024416030080

The kinetics of the reaction between one-electron-reduced cobalamin (cobalamin(II), Cb(II)) and the two-electron-oxidized form of vitamin C (dehydroascorbic acid, DHA) with amino acids in an acidic medium is studied by conventional UV-Vis spectroscopy. It is shown that the oxidation of Cbl(II) by dehydroascorbic acid proceeds only in the presence of sulfur-containing amino acids (cysteine, acetylcysteine). A proposed reaction mechanism includes the step of amino acid coordination on the Co(II)-center through the sulfur atom, along with that of the interaction between this complex and DHA molecules, which results in the formation of ascorbyl radical and the corresponding Co(III) thiolate complex.

Full text of DOI:10.1134/S0036024416030080

ISSN 0036-0244, Russian Journal of Physical Chemistry A, 2016, Vol. 90, No. 3, pp. 596–600. © Pleiades Publishing, Ltd., 2016.
Original Russian Text © I.A. Dereven’kov, Thu Thuy Bui Thi, D.S. Salnikov, S.V. Makarov, 2016, published in Zhurnal Fizicheskoi Khimii, 2016, Vol. 90, No. 3, pp. 390–394.
STRUCTURE OF MATTER
AND QUANTUM CHEMISTRY
Effect of Amino Acids on the Interaction between Cobalamin(II)
and Dehydroascorbic Acid
I. A. Dereven’kov, Thu Thuy Bui Thi, D. S. Salnikov, and S. V. Makarov
Ivanovo State University of Chemistry and Technology, Ivanovo, 153000 Russia
e-mail: derevenkov@gmail.com
Received April 2, 2015
Abstract―The kinetics of the reaction between one-electron-reduced cobalamin (cobalamin(II), Cb(II))
and the two-electron-oxidized form of vitamin C (dehydroascorbic acid, DHA) with amino acids in an acidic
medium is studied by conventional UV–Vis spectroscopy. It is shown that the oxidation of Cbl(II) by dehy-
droascorbic acid proceeds only in the presence of sulfur-containing amino acids (cysteine, acetylcysteine).
A proposed reaction mechanism includes the step of amino acid coordination on the Co(II)-center through
the sulfur atom, along with that of the interaction between this complex and DHA molecules, which results
in the formation of ascorbyl radical and the corresponding Co(III) thiolate complex.
Keywords: reaction mechanism, kinetics, cobalamin, dehydroascorbic acid, ascorbic acid, thiols.
DOI: 10.1134/S0036024416030080
INTRODUCTION
the oxidation of Co(II) to Co(III) and the formation
of HAA– are observed in the presence of biological
ligands (GSH, SCN–).
Cobalamins (vitamin B12, Cbl) are the vitamins with
the most complex structures. They are cobalt com-
plexes with corrin macrocycle (equatorial ligands),
5,6-dimethylbenzimidazole nucleotide (lower axial
In this work, we studied the kinetics of the reaction
of cobalamin(II) in a weakly acidic medium with
ligand; DMBI), and different X groups (X = CN–, amino acids and the effect of the type of functional
groups on the mechanism of the process.
H O,
2
−, and others; upper axial ligand) [1].
CH3
Pentacoordinated one-electron reduced cobala-
min (cobalamin(II), Cbl(II)) is an important biologi-
cal form of the complex [2, 3]. It is formed by the reac-
tions between Cbl(III) and a variety of reducing
agents: ascorbic acid [4], monosaccharides [5], for-
mate [6], and others. Cobalamin(II) reacts at a high
EXPERIMENTAL
Hydroxocobalamin hydrochloride (Fluka; ≥95%),
L-cysteine (CySH), N-acetyl-L-cysteine (NACSH),
L-methionine, L-asparagine, L-aspartic acid, L-glu-
tamine, L-glutamic acid, L-tyrosine, L-serine
(Sigma-Aldrich), and sodium borohydride were used
without additional purification; other substances were
of chemical grade. Argon was used to maintain anaer-
obic conditions. Cbl(II) was obtained via the anaero-
bic reduction of hydroxocobalamin with sodium boro-
hydride. The excess of the reducing agent was removed
by adding acetone [23].
DHA was synthesized from HAA– according to the
procedure reported in [24]. DHA concentrations were
determined by its reduction with an excess of CySH in
the pH range of 6.0 to 6.5 to HAA–, the extinction
coefficients of which are well known [25].
rate with such free radicals as NO [7], NO [8], − [9],
O2
2
SO2−
[10], and others.
Dehydroascorbic acid (DHA) is a product of the
two-electron oxidation of ascorbic acid (HAA–) or the
one-electron oxidation of ascorbyl radical (AR–). The
reduction of DHA to HAA– in vivo is performed by
NADPH- [11, 12] and glutathione- [13–16] (GSH)
dependent enzymes. It is known that thiols (cysteine,
homocysteine, glutathione, and others [17, 18]),
hydrogen sulfide [19], and tris(2-carboxyethyl)phos-
phine [20] are also able to reduce DHA.
Unfortunately, the kinetics of reactions of metal
complexes with DHA remains poorly studied, in con-
trast to reactions involving ascorbic acid. It is known cally in sealed quartz cuvettes 1 cm thick on a Cary 50
that DHA oxidizes nitrosylhemoglobin to methemo- spectrophotometer equipped with cryothermostat.
globin and nitric oxide [21]. It was found in [22] that The reaction rate was controlled at 372 nm (for DHA
Cbl(II) does not react directly with DHA; however, reduction by cobalamin(II) with NACSH and CySH)
Kinetic measurements were performed anaerobi-
596

EFFECT OF AMINO ACIDS
597
Abs.
1.2
A372
0.6
0.5
1.0
0.8
0.6
0.4
0.2
0.4
0
2
4
6
8
10
τ, min
300
400
500
600
700
λ, nm
Fig. 2. Typical kinetic curve of the reaction between
Cbl(II) and DHA in the presence of CySH (dots) and its
Fig. 1. UV–Vis spectrum of Cbl(II) (solid line) and product
of the oxidation of Cbl(II) by dehydroascorbic acid in the
presence of CySH (dashed line) and NACSH (dotted line);
fitting to the exponent equation (line); [Cbl(II)] = 5 ×
0
–5
–3
10 mol/L; [DHA] = 1.7 × 10 mol/L; [CySH] = 6 ×
–4
10 mol/L; pH 5.47; 25°C.
–5
–3
[Cbl(II)] = 5 × 10 mol/L; [DHA] = 1 × 10 mol/L;
0
–3
[RSH] = 1 × 10 mol/L; pH 5.0; 25°C.
apparently due to the growing concentration of thio-
late anion (RS–). It should be noted that the highest
rate was observed for the reaction proceeding in the
presence of GSH; the lowest one was for the reaction
with NACSH. Microscopic pKa values of the dissoci-
ation of the SH-group are 8.9 for GSH with the pro-
tonated amino group, 9.1 for GSH with the deproton-
ated amino group [29], 8.5 for CySH with the proton-
ated amino group, 10.2 for CySH with deprotonated
amino group [30], and 9.6 for NACSH [31] at 25°C. It
seems that the negligible difference in the rates of the
reaction proceeding with CySH and GSH was due to
the similarity between the ionization constant values of
and 265 nm (for DHA reduction by thiol amino acids).
The data were analyzed using the Origin 7.5 program.
RESULTS AND DISCUSSION
It is known that Cbl(II) does not react with DHA
in the pH range of 1.5 to 7.0 [22]. The reaction was not
investigated at higher pH values because of the insta-
bility of DHA [26]. It was found that there was no
interaction between Cbl(II) and DHA in the presence
of such amino acids as glutamine, asparagine, glu-
tamic and aspartic acids, tyrosine, serine, and methi-
onine. With CySH and NACSH, the growth of the
maxima at 372 and 558 nm and their reduction at 312
and 474 nm are observed in the UV–Vis spectrum
(Fig. 1). The UV–Vis spectra of the reaction products
correspond to Cbl(III) complexes with cysteine [27]
and acetylcysteine [28]. A similar reaction proceeds
when GSH is present in the system [22].
The kinetics of the reaction between Cbl(II) and an
excess of DHA in the presence of CySH and NACSH
excesses was studied. A typical kinetic curve of the
process is described by a first order equation (Fig. 2).
The dependence of the observed rate constant (kobs)
on [DHA] is linear (Fig. 3), indicating the first order
of the reaction with respect to the oxidant. In addition,
there is a positive intercept on the Y-axis. The reaction
rate depends on [RSH]: the calculated slopes (k′) of
the kobs dependences on [DHA] increase linearly along
with [RSH] (Fig. 4).
kobs, 1/min
0.6
0.5
0.4
0.3
0.2
0.1
0
0.5
1.0
1.5
2.0
2.5
[DHA] × 103, mol/L
The effect of pH on the rate of reaction was studied.
It was shown that with CySH and NACSH, the reac-
tion rate grew along with pH, as was observed in the
presence of GSH in [22]. The increase in the rate was
Fig. 3. Dependence of the observed reaction rate constant
–5
(k ) on [DHA]; [Cbl(II)] = 5 × 10 mol/L; [CySH] =
obs
0
–4
3 × 10 mol/L; pH 5.47; 25°C.
RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY A Vol. 90 No. 3 2016

598
k, 1/min
DEREVEN’KOV et al.
kobs, 1/min
0.8
0.6
0.4
0.2
12
2
1
8
4
3
0
0
0.2
0.4
0.6
0.8
1.0
1.2
[CySH] × 103, mol/L
4
5
6
pH
Fig. 5. Dependence of k on pH for the reaction between
Fig. 4. Dependence of the observed reaction rate constant
obs
–5
Cbl(II) and DHA in the presence of (1) CySH, (2) GSH,
(k′) on [CySH]; [Cbl(II)] = 5 × 10 mol/L; pH 5.47;
0
–5
and (3) NACSH; [Cbl(II)] = 5 × 10 mol/L; [DHA] =
25°C.
0
–3
–3
1 × 10 mol/L; [RSH] = 1 × 10 mol/L; 25°C.
Abs.
1.2
A560
0.30
kobs, 1/min
1.8
0.24
0.18
0.12
1
0.8
1.2
0.6
0
0.4
0.8
1.2
0.4
0
[DHA]0/[Cbl(II)]0
2
300
400
500
600
700
λ, nm
0
20
40
60
80
100
[RSH] × 102, mol/L
Fig. 6. Spectral changes observed during the titration of
cobalamin(II) with dehydroascorbic acid. Insert: depen-
Fig. 7. Dependences of the observed rate constants (k′
)
obs
dence of absorption at 560 nm on [DHA] /[Cbl(II)];
for the reduction of dehydroascorbic acid with (1) cysteine
0
–5
–3
[Cbl(II)] = 5 × 10 mol/L; [CySH] = 1 × 10 mol/L;
and (2) acetylcysteine on the concentration of these amino
0
–5
pH 6.8; 25°C.
acids; [DHA] = 1 × 10 mol/L; pH 6.45; 25°C.
0
their thiol groups, while the higher value of the ioniza-
tion constant of NACSH thiol group relative to other
amino acids explains the lowest reaction rate for this
system at the same pH value (Fig. 5).
NACSH. This indicates the two-electron reduction of
DHA and formation of ascorbic acid in the course of
the reaction.
It is known that CySH, NACSH, and GSH are
able to reduce DHA to HAA– [17]. In this work, we
investigated the kinetics of the reaction in the presence
of CySH and NACSH. The reaction involving GSH
was studied in [22]. It was found that the first order of
To determine the stoichiometry of the reaction, we
conducted the titration of cobalamin(II) with dehy-
droascorbic acid in the presence of CySH and
NACSH (Fig. 6). It was found that the oxidation of
1 mol of Cbl(II) required 0.6 mol of DHA in the pres-
ence of CySH, and 0.4 mol of DHA in the presence of the reaction with respect to DHA was observed with
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EFFECT OF AMINO ACIDS
599
an excess of the reducing agent (Fig. 7). The observed lower than that of the reduction of dehydroascorbic
acid in the presence of both Cbl(II) and thiols (table).
rate constant (ko'bs) depends linearly on [RSH], con-
firming the first order of the reaction with respect to
Based on the obtained data, we propose the follow-
ing scheme of the reduction of dehydroascorbic acid
the abovementioned amino acids was considerably by cobalamin(II) in the presence of thiol amino acids:
RSH. However, the rate of the reaction of DHA with
RS-
RS-
RS-
AR-
H2O
(+H+)
+DHA
Co2+
N
Co2+
Co2+
Co3+
N
Co3+
N
+ RS-
RS—+
(-H+)
N(H+)
DHA
N(H+)
+H+
RSH
2AR- + H+
HAA- + DHA
RS∙
Co2+
N(H+)
+ AR-
neutral media and rapidly disproportionates to HAA–
The crucial step in this mechanism is the coordina-
tion of amino acids on the Co2+ center, which likely and DHA [36].
occurs through the sulfur atom. The binding of sulfur
amino acids by cobalamin(II) was proved previously
by electron paramagnetic resonance [32], while the
coordination of the ligands in the upper position of
cobalamin(II) through the nitrogen atom was not con-
firmed experimentally [33]. This could explain the
need for thiol amino acids for the reaction to proceed.
The presence of the methyl group in the methionine
molecule prevents the formation of the
Co(II)−methionine complex.
Coordination of the ligand in the upper position of
cobalamin(II) weakens the Co2+−N(DMBI) bond
[32, 34]. It should be noted that the binding of ligands
by the Co2+ center is a very fast process [35]. The reac-
tion of the resulting Co2+-thiolate complex with the
DHA molecule probably leads to electron transfer and
the formation of AR– and the corresponding thiolate
cobalamin(III) complex. AR– is unstable in acidic and
Let us consider the reasons for the origin of Y-inter-
cepts on the concentration dependences (Fig. 3). The
reverse reaction of the reduction of Cbl(III) thiolate
complexes to Cbl(II) by thiol amino acids or HAA–
formed in the reaction proceeds much slower than the
oxidation of Cbl(II) by the dehydroascorbic acid in the
presence of CySH, NACSH, and GSH. The origin of
the Y-intercepts (Fig. 3) is presumably due to reactions
with free radicals (ascorbyl- and thiyl), since the oxi-
dation of Co2+–thiolate complex can proceed on both
Co2+-center and thiolate anions.
ACKNOWLEDGMENTS
This work was supported by the Russian Science
Foundation, project no. 14-23-00204.
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