Radiation chemical studies of methionine in aqueous solution: Understanding the role of molecular oxygen

Article abstract of DOI:10.1021/tx900427d

The oxidation of methionine is an important reaction in the biological milieu. Despite a few decades of intense studies, several fundamental aspects remain to be defined. We have investigated in detail the γ-radiolysis of free methionine in the absence and presence of molecular oxygen followed by product characterization and quantification. The primary site of attack by HO^? radicals and H^? atoms is the sulfur atom of methionine. We have disclosed that HO^? radicals do not oxidize methionine to the corresponding sulfoxide in either the presence or the absence of oxygen; the oxidizing species is H₂O₂ derived either from the radiolysis of water or from the disproportionation of the byproduct O₂^?-. 3-Methylthiopropionaldehyde is the major product of HO^? radical attack in the presence of molecular oxygen. Together with the direct oxidation at sulfur as the major product, the potential of H^? atoms is also proven to be highly specific for sulfur atom attack under anoxic and aerobic conditions. The major products derived from the H^? atoms attack are found to be α-aminobutyric acid or homoserine, in the absence or presence of oxygen, respectively. All together, these results help clarify the fate of methionine related to a biological environment and offer a molecular basis for envisaging other possible pathways of in vivo degradation as well as other markers.

Full text of DOI:10.1021/tx900427d

258
Chem. Res. Toxicol. 2010, 23, 258–263
Radiation Chemical Studies of Methionine in Aqueous Solution:
Understanding the Role of Molecular Oxygen
Sebastia´n Barata-Vallejo,†,‡ Carla Ferreri,† Al Postigo,§ and Chryssostomos Chatgilialoglu*,†
ISOF, Consiglio Nazionale delle Ricerche, Via Piero Gobetti 101, 40129 Bologna, Italy, and Faculty of Science,
UniVersity of Belgrano-Conicet, VillanueVa 1324, 1426 Buenos Aires, Argentina
ReceiVed NoVember 29, 2009
The oxidation of methionine is an important reaction in the biological milieu. Despite a few decades
of intense studies, several fundamental aspects remain to be defined. We have investigated in detail the
γ-radiolysis of free methionine in the absence and presence of molecular oxygen followed by product
characterization and quantification. The primary site of attack by HO• radicals and H• atoms is the sulfur
atom of methionine. We have disclosed that HO• radicals do not oxidize methionine to the corresponding
sulfoxide in either the presence or the absence of oxygen; the oxidizing species is H2O2 derived either
from the radiolysis of water or from the disproportionation of the byproduct O2•-. 3-Methylthiopropi-
onaldehyde is the major product of HO• radical attack in the presence of molecular oxygen. Together
with the direct oxidation at sulfur as the major product, the potential of H• atoms is also proven to be
highly specific for sulfur atom attack under anoxic and aerobic conditions. The major products derived
from the H• atoms attack are found to be R-aminobutyric acid or homoserine, in the absence or presence
of oxygen, respectively. All together, these results help clarify the fate of methionine related to a biological
environment and offer a molecular basis for envisaging other possible pathways of in ViVo degradation
as well as other markers.
Scheme 1. Mechanism of Met (1) Attack by HO• Radicals in
Introduction
Neutral Solutions (13–15)
One of the most important damages in proteins by reactive
oxygen species (ROS) is the oxidation of methionine (Met, 1)
residues (1, 2). The major oxidation products are the two
epimeric forms of sulfoxide (i.e., R and S epimers) that can be
repaired enzymatically by methionine sulfoxide reductase (Mrs)
(3, 4). For each epimeric residue, a specific enzyme exists (MrsA
and MrsB for the S and R isomer, respectively). For this reason,
Met residues in proteins are suggested to act as an antioxidant
pool, and several studies support this vision (5, 6). The oxidation
of Met occurs either by nonradical oxidants (like H2O2,
HOONO, or HOCl) or by radical species (like HO•, CO3-•, or
•
N3 ) (7–11). Although the nonradical oxidation is site-specific
atom (13). Although the direct detection of radical 2 by time-
resolved spectroscopy is missing, the analogous sulfuranyl
radical has been observed in aqueous solutions by replacing
Met with CH3SCH2C(O)OMe or CH3SCH3 and found to react
with oxygen with a rate constant of 2 × 108 M-1 s-1 (16–18).
with the formation of methionine sulfoxide, Met(O), the radical-
based oxidation is believed to be more complex (12).
The reaction of HO• radicals with Met has been the subject
of several investigations. Asmus and co-workers in a series of
radiation chemical studies in the eighties disclosed the reaction
mechanism (13–15). The initial step (k1 ) 2.3 × 1010 M-1 s-1
)
Although aliphatic sulfides react with HO• radicals in the
presence of molecular oxygen affording the corresponding
sulfoxides, it is not clear whether the analogous reaction of Met
also affords the sulfoxide. Early works on radiolysis of Met
showed the formation of sulfoxide among other products under
deareated conditions, and more recently, the formation of
sulfoxide in the radiolytic study of model peptides under aerobic
conditions has been attributed to HO• radical attack (19, 20).
Several reviews clearly indicate or imply that oxidation of Met
occurs by HO• radicals (7–11). Is it possible that radical 2 in
Scheme 1 be trapped by oxygen prior to the unimolecular
rearrangement? On the basis of the above-mentioned rate
constants, the answer is no. If this is true, how does the oxidation
of Met by HO• radicals occur? It has become evident that the
reaction of HO• radicals with Met in the absence or in the
presence of molecular oxygen is not yet fully understood. In
is a competition process between addition of HO• radical to the
sulfur atom (Scheme 1) and hydrogen abstraction. The hydroxyl
adducts 2 immediately coordinate with nitrogen in a three-
electron bond to yield species 3. The optical spectra of this
intermediate has been recorded and found to decay (k2 ) 3.6 ×
106 s-1) directly into CO2 and R-aminoalkyl radical (4). It is
worth mentioning that the radiation chemical yield of CO2 was
found to be 0.49 ( 0.2 µmol J-1 in the pH range 6-8, which
accounts for about 90% of the HO• radical attack to the sulfur
* To whom correspondence should be addressed. E-mail: chrys@isof.
cnr.it.
† Consiglio Nazionale delle Ricerche.
‡ Doctoral candidate at the Faculty of Pharmacy and Biochemistry,
University of Buenos Aires, Argentina.
§ University of Belgrano-Conicet.
10.1021/tx900427d  2010 American Chemical Society
Published on Web 12/29/2009

Radiation Chemical Studies of Methionine
Chem. Res. Toxicol., Vol. 23, No. 1, 2010 259
this paper, we will show that HO• radicals do not oxidize Met
to Met(O) in either the absence or the presence of oxygen.
In the radiolysis of water, together with radical (HO•) and
nonradical oxidants (H2O2), primary reducing species like
hydrated electrons (eaq-) and H• atoms are produced. Reductive
radical stress has been less widely investigated than oxidative
radical stress (21). The reaction of these reducing species with
sulfur-containing residues in peptides and proteins has been
studied in connection with biomimetic chemistry on tandem
protein/lipid damages under reductive radical stress (22–24).
In this paper, the reaction of H• atoms with Met (k ) 1.1 × 109
M-1 s-1) will take part of the investigation, whereas the reaction
Figure 1. HPLC analyses of γ-irradiation of N2O-saturated solutions
of 10.16 mM Met (1) at natural pH (dose rate of ∼6.5 Gy min-1) after
OPA derivatization of the amino compounds. The consumption of 1
(green peaks) led to the formation of three products, the major one
being the amine 8 (violet peak at 20 min retention time). Inset:
expansion of the chromatogram between 9.5 and 14 min of the 4 kGy
irradiated sample; the red and black peaks correspond to Met(O) (7)
and Aba (6), and the splitting of the red peak is due to the R and S
epimers of sulfoxide.
-
of eaq with Met, which takes place with a much lower rate
constant (k ) 4.5 × 107 M-1 s-1), will not be considered (13).
Therefore, the main subject of this article is concerned with
the identification and quantification of final products upon the
reaction of HO• radicals or H• atoms (generated by γ-radiolysis
of water) on Met in the absence or presence of molecular
oxygen.
NMR spectroscopy and 2,4-dinitrophenylhydrazine (2,4-DNPH)
derivatization followed by HPLC analysis. For NMR analysis, the
irradiated solution was extracted with deuterated chloroform, and
the proton and carbon NMR spectra were recorded immediately
after (see the Supporting Information). For recognition and
quantification of the 2,4-dinitrophenylhydrazone of the aldehyde,
a published protocol was employed and adapted to our system (28).
The comparison was obtained from the same derivatization
procedure performed with the commercially available compound
followed by HPLC analysis and spike experiments.
Materials and Methods
Materials. The following commercially available starting materials
were obtained from Sigma-Aldrich Co. and used as received: Met,
Met(O), R-aminobutyric acid (Aba), 3-methylthiopropylamine, 3-me-
thylthiopropanaldehyde, and homoserine. Solvents were purchased
from Merck (HPLC grade) and used without further purification. Water
was purified with a Millipore system. All aqueous amino acid solutions
were prepared immediately before use.
Continuous Radiolyses. Irradiations were performed at room
temperature (22 ( 2 °C) using a 60Co-Gammacell at different dose
rates. The exact absorbed radiation dose was determined with the
Fricke chemical dosimeter, by taking G(Fe3+) ) 1.61 µmol J-1
(25). Mixtures of gases were obtained by an appropriate mixing,
controlled by flow meter. The gas stream was obtained by a line
connected with a needle inserted in the vessel, and the flow was
adjusted to get a continuous bubbling during irradiation. Two
hundred fifty milliliters of an Ar-purged aqueous solution of Met
(the standard concentration of experiments was about 10 mM, pH
5.80) was saturated with the desired gas or gas mixture prior to
γ-irradiation. The pH was registered for the solution at the final
irradiation dose. HPLC analyses were recorded on an Agilent 1100
Liquid Chromatograph, equipped with a quaternary pump delivery
system, a column thermostat, and a variable-wavelength detector.
RP18 5 µm columns were used as specified in each detection method.
OPA Derivatization of Irradiated Samples and HPLC
Analysis. Aliquots of the irradiated Met solution were withdrawn
at the specified doses for immediate conversion to the OPA
(orthophthaldialdehyde) derivatives by adapting some reported
procedures (26, 27). One hundred microliters of the sample was
diluted in 250 µL of borate solution (0.4 M H3BO3, pH 10.2 adjusted
with 4 M NaOH), 50 µL of the freshly prepared OPA reagent (10
mg/mL OPA and 10 mg/mL 2-mercaptoethanol) was added, and
the solution was vortexed for 2 min and finally diluted with 320
µL of 1.5% v/v concentrated H3PO4. After derivatization, 20 µL
of the reaction mixture was injected for HPLC analysis. A
GraceSmart RP 18 5 µm column (150 mm × 4.6 mm) was used at
40 °C, with detection at λ ) 338 nm. Mobile phase A was 10 mM
NaH2PO4, adjusted to pH 7.5 with 4 M NaOH, while mobile phase
B was acetonitrile/methanol/water (45/45/10 v/v/v). The separation
was obtained at a flow rate of 1 mL/min with the following gradient
program: 0.5 min at 5% B, a 16.0 min increase of eluent B to 47%
followed by a 21.0 min step increase of eluent B to 100%, then
washing at 100% B and equilibration at 5% B. The total time was
25 min.
Results and Discussion
Reaction of HO• Radical and H• Atom with Met in
Deaerated Aqueous Solutions. Radiolysis of neutral water leads
to the reactive species eaq-, HO•, and H• together with H+ and
H2O2 as shown in eq 1. The values in parentheses represent the
radiation chemical yields (G) in units of µmol J-1. In N2O-
-
saturated solution (∼0.02 M of N2O), eaq are efficiently
transformed into HO• radicals via eq 2 (k2 ) 9.1 × 109 M-1
s-1), affording G(HO•) ) 0.55 µmol J-1; that is, HO• radicals
and H• atoms account for 90 and 10%, respectively, of the
reactive species (29, 30).
H2O
9
γ8 eaq- (0.27), HO• (0.28), H• (0.06),
H+ (0.27), H2O2 (0.07)
(1)
(2)
-
eaq + N2O + H2O f HO• + N2 + HO-
Initially, γ-radiolysis experiments coupled with product
studies were carried out under deaerated conditions. N2O-
saturated solutions containing Met (10.16 mM) at natural pH
were irradiated under stationary state conditions with a dose
rate of ca. 6.5 Gy min-1 followed by OPA derivatization (26, 27)
of amino compounds and HPLC analysis. The consumption of
1 (green peaks, Figure 1) led to the formation of three new
products, a major one and two minor ones. The two minor
products that elute from the column before Met were identified
as Aba (6) and Met(O) (7) by comparison with the correspond-
ing OPA derivatives from the commercially available com-
pounds (inset of Figure 1, black and red peaks, respectively).
On the other hand, the major product was isolated from the
crude reaction mixture and characterized by spectroscopic means
to be the amine 8 (see Scheme 2). OPA derivatization of the
commercially available amine 8 and HPLC analysis confirmed
Recognition and Quantification of 3-(Methylthio)-propional-
dehyde. Ten millimolar aqueous solutions of Met were irradiated
for 500 Gy while flushing a very controlled stream of the
appropriate gas or gas mixture. The aldehyde was recognized by

260 Chem. Res. Toxicol., Vol. 23, No. 1, 2010
Barata-Vallejo et al.
Scheme 2. Reaction Mechanism for the γ-Irradiation of
at the sulfur atom, which is the same resulting from the radiation
chemical yield of CO2 (13).
N2O-Saturated Solution of Met (1)a
In summary, in the absence of molecular oxygen, (i) the
addition of HO• radicals to the sulfur atom of Met (the major
path) does not afford Met(O) but exclusively the radical 4,
whose fate depends on the reaction conditions, and (ii) the main
reaction of H• atoms with Met is the homolytic substitution at
sulfur with the formation of Aba (6).
Reaction of H2O2 and Superoxide with Met in
Oxygenated Aqueous Solutions. From the previous section, it
is noticeable that the amount of H2O2 produced from the
radiolysis of water corresponds exactly to the formation of
Met(O). It is well-known that H2O2 oxidizes Met to Met(O),
and a rate constant of ∼1 × 10-2 M-1 s-1 has been reported in
phosphate buffer at pH 6-8 (31), which fits very well with our
findings. Indeed, the half-life for the oxidation of 10 mM Met
is estimated to be t1/2 ≈ 2 h, and our irradiation time is ∼ 10 h
(dose rate ∼ 6.5 Gy min-1 for a total dose of 4 kGy, cf. Figure
1). To simulate the steady-state formation of H2O2, 500 µL of
1 M aqueous H2O2 was introduced by syringe pump (flow rate
of 100 µL/h) to an aqueous solution of Met (250 mL, 10.41
mM, pH 5.8) under stirring for 5 h (H2O2 final concentration )
2 mM). Samples were withdrawn each hour, followed by OPA
derivatization of amino compounds and HPLC analysis. The
oxidation of Met to Met(O) occurs readily and quantitatively
(see the Supporting Information).
a For details of radical 4 formation, see Scheme 1.
the identity of the product at 20 min of retention time (Figure
1). As expected, the concentration of 1 decreased as the
irradiation dose increased in the range of 0-4 kGy, whereas
the concentrations of 6, 7, and 8 increased. After 4 kGy of
irradiation, 27% of Met was consumed. The product yield (mol
kg-1) divided by the absorbed dose (1 Gy ) 1 J kg-1) gave the
radiation chemical yield. Analysis of the data in terms of
radiation chemical yield (G) gave G(-1) ) 0.70, G(6) ) 0.03,
G(7) ) 0.07, and G(8) ) 0.29 µmol J-1 when the lines are
extrapolated to zero dose (Figure 2).
Superoxide radical anions (O2-•) are generated as the
exclusive reactant upon O2-saturated solutions containing >0.1
M HCOONa at pH ∼ 7 (32). Indeed, in O2-saturated solution
-•
(1.34 mM of O2), eaq- and H• are efficiently converted into O2
via eqs 3 (k3 ) 1.9 × 1010 M-1 s-1) and 4 (k4 ) 2 × 1010 M-1
s-1), the pKa(HOO•) being 4.8 (eq 5). On the other hand, HO•
and H• are transformed first to CO2-• via eqs 6 (k6 ) 3.5 × 109
On the basis of the known rate constants for the reactions of
H• atom and HO• radical with Met (see above), the following
observations are underlined (cf. Scheme 2): First, taking into
account that G(HO•) + G(H•) + G(H2O2) ) 0.68 µmol J-1 is
similar to G(-1) ) 0.70, this suggests that all HO• radicals, H•
atoms, and H2O2 react with 1; second, the equality G(H2O2) )
G(7) ) 0.07 µmol J-1 indicates that H2O2 reacts quantitatively
with Met to give the corresponding sulfoxide (see the next
section); third, we note that G(6) ) 0.03 is the half of G(H•) )
0.06 and G(8) ) 0.29 is nearly half of G(HO•) ) 0.55,
attributing the lower value of G(8) to the radical-radical
termination of radical 4 to give 8 and 9, the latter promptly
hydrolyzed to aldehyde 10. The presence of aldehyde 10 was
confirmed in the reaction mixture by chloroform-D extraction
-•
M-1 s-1) and 7 (k7 ) 1.3 × 108 M-1 s-1) and then to O2 via
eq 8 (k3 ) 2.4 × 109 M-1 s-1).
-
-•
eaq + O2 f O2
(3)
(4)
(5)
(6)
(7)
(8)
H• + O2 f HOO•
-•
HOO• h O2 + H+
-
-•
HO• + HCO2 f CO2 + H2O
-
-•
H• + HCO2 f CO2 + H2
1
and analyses by H and 13C NMR and GC/MS in comparison
with the authentic sample. Moreover, by a sequence of extraction
from the crude mixture, 2,4-DNPH derivatization (28), and
HPLC analysis, the formation of aldehyde 10 was quantified,
and the radiation chemical yield, G(10) ) 0.26, was obtained.
Therefore, the sum of various identified products accounts for
∼93% of the consumed Met.
-•
-•
CO2 + O2 f CO2 + O2
An O2-saturated aqueous solution of 0.5 M HCOONa and
10.55 mM Met at pH 7.3 is γ-irradiated, while oxygen is
bubbling throughout the reaction time. Aliquots (4 mL) are
withdrawn at each specified dose for immediate OPA deriva-
tization and HPLC analysis (Figure 3). The pH registered for
the solution at the final dose (4 kGy) is 7.3. The concentrations
of Met (1) decreased, whereas that of Met(O) (7) increased, as
the dose increases in the range of 0-4 kGy. Analysis of the
data in terms of radiation chemical yields (G) affords G(-1) )
0.34 and G(4) ) 0.35 µmol J-1 when the lines are extrapolated
to zero dose (inset of Figure 3), indicating a quantitative
transformation. The analysis of these values is straightforward,
taking into account that G(H2O2) ) 0.07 and G(O2-•) ) 0.61
µmol J-1 and considering that two molecules of O2-• are needed
for each Met oxidation. Reported findings clearly argue against
Further support of the reaction path of HO• radical attack
depicted in Schemes 1 and 2 was obtained from an independent
experiment, where the reaction is repeated in the presence of
0.1 mM thiol to trap the intermediate radical 4 [k g 2 × 109
M-1 s-1 has been estimated for the reaction of radical 4 with
cysteine (13)]. In particular, 0.1 mM ꢀ-mercaptoethanol was
added at 0 and 0.5 kGy of irradiation, and the crude mixture
was analyzed after 0.5 and 1 kGy by the protocol reported above.
Analysis of the data in terms of radiation chemical yield (G)
gave G(-1) ) 0.68, G(6) ) 0.03, G(7) ) 0.05, and G(8) )
0.50 µmol J-1. It is gratifying to see that the formation of amine
8 corresponds to about 90% percentage of the HO• radical attack

Radiation Chemical Studies of Methionine
Chem. Res. Toxicol., Vol. 23, No. 1, 2010 261
Figure 2. Radiation chemical yields (G) of 1, 8, 7, and 6 vs dose;
G(-1) ) 0.70, G(8) ) 0.29, G(7) ) 0.07, and G(6) ) 0.03 µmol J-1
are obtained when the lines are extrapolated to zero dose.
Figure 4. HPLC analyses of γ-irradiated air-saturated solutions of 10.43
mM Met at natural pH (dose rate of ∼6.5 Gy min-1) after OPA
derivatization of amino compounds. The consumption of Met (green
peaks) led to the formation of Met(O) (red peaks) and homoserine (blue
peaks). Inset: Expansion of the chromatogram between 8.5-10 min of
the 3.5 kGy irradiated sample; the splitting of the red peak is due to
the R and S epimers of sulfoxide.
Figure 3. HPLC analyses of irradiated (dose rate of ∼6.5 Gy min-1
)
O2-saturated solutions of 0.5 M HCOONa and 10.55 mM Met (1) at
pH 7.3 after OPA derivatization of amino compounds. Inset: Radiation
chemical yields (G) of 1 and 7 vs dose; G(-1) ) 0.34 and G(7) )
0.35 µmol J-1 obtained when the lines are extrapolated to zero dose.
Figure 5. Radiation chemical yields (G) of Met (1), Met(O) (7), and
homoserine (12) vs dose obtained from the experiment reported in
Figure 4; G(-1) ) 0.78, G(7) ) 0.42, and G(12) ) 0.05 µmol J-1 are
obtained when the lines are extrapolated to zero dose.
a direct oxidation of Met by superoxide (33). On the other hand,
Table 1. Radiation Chemical Yields (G) Extrapolated to
Zero Dose from the Irradiation of Met 1 in Oxygenated
Aqueous Solutions Under Various Conditions
•
-•
the reactivity of HO2 /O2 in aqueous solution is fully under-
stood, and disproportionation is expected to produce H2O2 (eq
9) (32). Therefore, a total 0.37 µmol J-1 radiation chemical yield
of H2O2 is expected during the experiments, which fits very
well with the corresponding values of Met disappearance and
Met(O) formation (Figure 3).
Ga
N2O:O2 ) 90:10
N2:O2 ) 80:20b
O2
G(-1)
G(7)
1.10
0.43
0.034
0.63
0.78
0.42
0.05
0.31
0.79
0.43
0.027
0.33
G(12)
G(10)c
-•
•
O2 + HO2 + H2O f H2O2 + O2 + HO-
(9)
a Units of
G .
in µmol J-1 b Air. c Radiation chemical yields
determined after 0.5 kGy of irradiation by 2,4-DNPH derivatization
method (an average of two measurements).
Reaction of HO• Radicals with Met in Oxygenated
Aqueous Solutions. To investigate the reaction of HO• radicals
with Met in the presence of molecular oxygen, three different
experimental conditions were investigated. In particular, about
10 mM Met solutions were saturated by N2O/O2 (90:10, v/v)
or by air (N2/O2 ) 80:20, v/v) or by pure O2 during the
irradiation.
An air-saturated aqueous solution of 10.43 mM 1 at natural
pH (5.8) was irradiated under stationary state conditions with a
dose rate of ca. 6.5 Gy min-1, while air is continuously bubbled
throughout the reaction time. At different doses, 4 mL aliquots
were withdrawn followed immediately by OPA derivatization
of amino compounds and HPLC analysis. A pH of 7.8 was
registered for the solution at the final dose of 3.5 kGy. The
consumption of 1 (green peaks, Figure 4) led to the formation
of two new products, which elute at shorter retention times.
The major (red peak) and minor (blue) products were identified
as Met(O) (4) and homoserine (12) respectively in comparison
with the corresponding OPA derivatization of commercially
available compounds.
As usual, the data derived from the experiments in Figure 4
are treated in terms of radiation chemical yields (G). Figure 5
shows that linear correlations do exist between G values and
dose. By extrapolation of the lines to zero dose, the following
values were obtained G(-1) ) 0.78, G(7) ) 0.42, and G(12)
) 0.05 µmol J-1, which are reported in the third column of
Table 1. By replacing air with pure oxygen or N2O/O2 (90:10,
v/v) mixture, analogous experiments were performed, and the
details of these experiments are reported in the Supporting
Information. Also, in these cases, the consumption of Met led

262 Chem. Res. Toxicol., Vol. 23, No. 1, 2010
Barata-Vallejo et al.
process between addition of HO• radical to the sulfur atom
(∼90%) and hydrogen abstraction (∼10%). Therefore, G(HO•)
≈ 0.50 and G(HO•) ≈ 0.05 are associated with addition and
hydrogen abstraction. We notice that the radiation chemical yield
from disappearance of Met, G(-1) ) 1.10, matches very well
with that from the sum of primary reactions of HO• radicals,
H• atoms, and H2O2 with 1 [viz. G(HO•) + G(H•) + G(H2O2)
) 0.68 µmol J-1], plus secondary reactions derived from peroxyl
radicals with 1 (viz. G(11) + G(14) ≈ 0.10 µmol J-1), plus the
amount of H2O2 presumably derived from the disproportionation
of superoxide (half of aldehyde 10, viz. G(H2O2) ) 0.32 µmol
J-1). Our proposal is in excellent agreement with the available
rate constants from pulse radiolysis studies (13, 16), which
suggest that the formation of radical 4 will be more than 2 orders
of magnitude faster than the addition of sulfuranyl radical 2 to
molecular oxygen (see the Introduction). We also notice that
the amount of aldehyde 10 is higher than the amount of HO•
radicals produced, which suggests that the reaction of peroxyl
radicals 11 and 14 with Met contributes to the formation of
Scheme 3. Reaction Mechanism for the γ-Irradiation of
Oxygenated Solution of Met (1)
-•
more aldehyde 10 and presumably of extra O2 (34).
-
•-
(ii) In 1 atm of air, eaq are efficiently converted into O2
(eq 3), whereas ∼30% of H• are transformed into O2 (eqs 4
and 5) and ∼70% react with Met to give homoserine (12).
G(HO•) ≈ 0.25 and G(HO•) ≈ 0.03 µmol J-1 are associated
with addition and hydrogen abstraction. The addition of HO•
radicals to sulfur, G(HO•) ≈ 0.25, produces G(10) ≈ 0.25 and
G(O2•-) ≈ 0.25. Peroxyl radical (viz. G(11) + G(14) ≈ 0.08
µmol J-1) also accounts for the extra formation of aldehyde 10
-•
to the formation of Met(O) and homoserine, and the summary
of the corresponding radiation chemical yields is also reported
in Table 1.
The presence of aldehyde 10 in the reaction mixtures was
considered next. In all cases, after the reaction is over, the
presence of aldehyde 10 was identified by chloroform-D
extraction followed by 1H and 13C NMR and GC/MS analyses
and compared with the authentic sample. However, the quan-
tification is not straightforward because during the continuous
bubbling of gas throughout the reaction time, aldehyde may also
be partially removed by the gas stream. For example, after 4
kGy irradiation (time ∼ 10 h) of N2O/O2 (90:10)-saturated 10
mM Met sample, followed by the above-described 2,4-DNPH
derivatization and HPLC analysis, we were able to obtain a low
limit value, G(10) g 0.32. To get more favorable conditions
for evaluating the formation of aldehyde 10, new samples were
irradiated only for 0.5 kGy followed by the 2,4-DNPH deriva-
tization and HPLC analysis. The radiation chemical yields
reported in Table 1 (last line) are the average of two runs. It is
gratifying to see that the disappearance of Met equals the sum
of the identified products including the aldehyde, viz., for N2O/
O2 (90:10), G(-1) ) 1.10 vs G(7) + G(12) + G(10) ) 1.09
µmol J-1, and for air, G(-1) ) 0.78 vs G(7) + G(12) + G(10)
) 0.78.
-•
and O2 (see above). Therefore, the amount of H2O2 derived
from the disproportionation of superoxide oxidizes Met to
Met(O). Analogous consideration can be made for O2-saturated
experiments, where the only difference is the varied percentage
of H• atom splitting, that is, ∼70% of H• are transformed into
O2-•and ∼30% react with Met to afford homoserine (12). It is
satisfying to see that going from the air- to oxygen-saturated
experiments homoserine concentration is reduced by half.
In summary, in the presence of molecular oxygen, (i) the
addition of HO• radicals to the sulfur atom of Met (the major
path) does not afford Met(O) but exclusively aldehyde 10; (ii)
H2O2, derived either from radiolysis of water or the dispropor-
tionation of superoxide, affords Met(O); and (iii) the reaction
of H• atoms with Met via homolytic substitution at sulfur is
still effective, ensuring the formation of homoserine (12).
Conclusions
Table 1 shows that large amounts, and nearly the same
concentration of Met(O) is formed in all cases. Interestingly,
the results of air- or O2-saturated solutions are similar, whereas
much larger amounts of Met are consumed in the N2O/O2 (90:
10) experiment with the formation of a larger amount of
aldehyde 10. In other words, in the experiments with air or O2,
the G(-1) is about 15% larger than the sum of primary species
[viz. G(eaq-) + G(HO•) + G(H•) + G(H2O2) ) 0.68 µmol J-1],
whereas with N2O/O2 (90:10), it is about 62% higher. The main
difference between N2O/O2 and air or O2 is that in the former
In this paper, we disclosed that HO• radicals do not oxidize
Met to the corresponding sulfoxide in either the absence or the
presence of oxygen. H2O2 derived either from the γ-radiolysis
of water (in the absence or presence of O2) or from the
•-
disproportionation of the byproduct O2 (in the presence of
O2) is the oxidizing species. The reaction of H• atom (a primary
species from radiolysis of water) with Met via homolytic
substitution at sulfur is effective, ensuring the formation of Aba
or homoserine in either the absence or the presence of O2,
respectively. These results help clarify the fate of Met under
strictly biologically related conditions and offer molecular basis
for envisaging other markers of Met degradation to be evaluated
in ViVo.
-
the eaq are efficiently transformed to HO• radical (eq 2),
-
whereas in the latter two conditions, the eaq are trapped by
•-
oxygen to give O2 (eq 3).
The mechanism conceived from the results of Table 1 is
depicted in Scheme 3 for the following reasons:
-
(i) In 1 atm of N2O/O2 (90:10, v/v), eaq are efficiently
Acknowledgment. This research was carried out in the
context of bilateral CNR-CONICET project 2009-2010. The
support and sponsorship concerned by COST Action CM0603
on “Free Radicals in Chemical Biology (CHEMBIORADI-
converted into HO• (eq 2), whereas 20% of H• are transformed
-•
into O2 (eqs 4 and 5) and 80% react with Met. As we
mentioned above, the reaction Met + HO• is a competition

Radiation Chemical Studies of Methionine
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CAL)” are kindly acknowledged. We thank Dr. Massimo
Capobianco and Dr. Quinto G. Mulazzani for many useful
discussions.
Supporting Information Available: Detailed experimental
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