Type I Photosensitized Oxidation of Methionine?

Article abstract of DOI:10.1111/php.13314

Methionine (Met) is an essential sulfur-containing amino acid, sensitive to oxidation. The oxidation of Met can occur by numerous pathways, including enzymatic modifications and oxidative stress, being able to cause relevant alterations in protein functio

Full text of DOI:10.1111/php.13314

DR. ANDRÉS HÉCTOR THOMAS (Orcid ID : 0000-0002-8054-7799)
DR. CAROLINA LORENTE (Orcid ID : 0000-0001-8723-3795)
Article type : Special Issue Research Article
SPECIAL ISSUE RESEARCH ARTICLE
Type I Photosensitized Oxidation of Methionine†
Carolina Castaño, Andrés H. Thomas, and Carolina Lorente*
Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas (INIFTA), Departamento
de Química, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, CCT-La
Plata-CONICET, Boulevard 113 y 64, 1900 La Plata, Argentina.
* Corresponding author e-mail: clorente@inifta.unlp.edu.ar
†This article is part of a Special Issue commemorating the XIV ELAFOT Conference held
from November 11th to 14th, 2019 in Viña del Mar, Chile.
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article
as doi: 10.1111/php.13314
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ABSTRACT
Methionine (Met) is an essential sulphur-containing amino acid, sensitive to oxidation. The
oxidation of Met can occur by numerous pathways, including enzymatic modifications and
oxidative stress, being able to cause relevant alterations in protein functionality. Under UV
radiation, Met may be oxidized by direct absorption (below 250 nm) or by photosensitized
reactions. Herein, kinetics of the reaction and identification of products during
photosensitized oxidation were analyzed to elucidate the mechanism for the degradation of
Met under UVA irradiation using pterines, pterin (Ptr) and 6-methylpterin (Mep), as
sensitizers. The process begins with an electron transfer from Met to the triplet excited state
of the photosensitizer (Ptr or Mep), to yield the corresponding pair of radicals, Met radical
cation (Met•+) and the radical anion of the sensitizer (Sens•-). In air-equilibrated solutions,
Met•+ incorporates one or two atoms of oxygen to yield methionine sulfoxide (MetO) and
methionine sulfone (MetO2), whereas Sens•- reacts with O2 to recover the photosensitizer
•-
and generate superoxide anion (O2 ). In anaerobic conditions, further free-radical reactions
lead to the formation of the corresponding dihydropterin derivatives (H2Ptr or H2Mep).
KEYWORDS. Photosensitization; Methionine; Electron-transfer; Type I/Type II
photooxidation; Pterins
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INTRODUCTION
Proteins have been shown to represent important targets for photodamage under UV
and visible irradiation, and tryptophan (Trp), tyrosine (Tyr), methionine (Met) and histidine
(His) are amino acids particularly susceptible to a variety of oxidizing agents (1). UV-A
(320-400 nm) and visible radiation induce oxidation reactions through photosensitized
processes. These processes involve excitation of the photosensitizers to yield singlet and
triplet excited states (2). The latter, having longer lifetimes, may undergo both electron
transfer with different substrates, mechanism known as type I photooxidation, or energy
1
transfer to molecular oxygen (O2) forming singlet oxygen (1O2(1g), denoted as O2)
responsible for type II photooxidation reactions (3).
Met, a non-polar sulphur-containing -amino acid (Scheme 1), is more easily
oxidized than the other amino acids and highly susceptible to oxidation by reactive oxygen
species (ROS) (4,5,6). Met is an essential amino acid and its deficiency leads to a failure in
the synthesis of critical proteins. Depending on the redox environment, Met oxidation
undergo by one- or two-electron transfer to yield Met radical cations. After one-electron
oxidation of Met, an unstable radical cation is formed, which is later converted in several
products, some of them irreversible. Met oxidation products are mainly methionine
sulfoxide (MetO) or methionine sulfone (MetO2) (Scheme 1). In vivo, MetO may be
enzymatically reduced by methionine sulfoxide reductase, and then the oxidation of Met is
repair (7). The accumulation of MetO occurs over the years or in some pathologies as
Alzheimer's disease (8). On the contrary, MetO2 is an irreversible product which cannot be
repaired enzymatically (7).
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<Scheme 1>
Typical type I photosensitized oxidation of Met has been reported in the presence of
a suitable sensitizer and UV or visible radiation (9, 10,11). Pterins are a family of
heterocyclic compounds widespread in biological systems, which have been identified as
efficient photosensitizers by type I and II mechanisms (12,13). Under UV-A excitation
(320-400) pterins photoinduce photodegradation of various biomolecules such as DNA
(14,15), nucleotides (16,17), proteins (18), amino acids (19,20,21) and lipids enriched in
polyunsaturated fatty acids (PUFAs) (22). Pterins are present in human epidermis in a low
concentration at normal physiological conditions, but during skin disorders, such as vitiligo,
pterins concentration is much higher (23).
In the present study, we evaluated the capability of two pterin derivatives, pterin
(Ptr) and 6-methylpterin (Mep), to photosensitize the degradation of Met in aqueous
solutions exposed to UV-A irradiation under different experimental conditions. In this
spectral region, pterins are the species that absorb radiation, whereas Met does not (Fig. 1).
The study was performed in the pH range 5.5-6.0, so that more than 99% of the pteridine
(pKa ~ 8) (24) was in the acid form, the predominant form at physiological pH. In this
work, UV/Visible spectrophotometry, HPLC with UV-visible and mass detection, and an
enzymatic method for H2O2 determination were used to follow the photochemical reactions,
and a mechanism for the photosensitized oxidation is proposed.
<Figure 1>
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MATERIALS AND METHODS
General
Ptr and Mep (purity > 99%, Schircks Laboratories, Switzerland and Sigma-Aldrich) was
used without further purification after checking for impurities by HPLC. Met and
ammonium acetate (NH4OAc) (Sigma Chemical Co) were of the highest purity available (>
98 %) and were used without further purification. Methanol (MeOH) and potasium iodide
(KI) were purchased from J. T. Baker and Laboratorios Cicarelli, respectively. Other
chemicals were from Sigma Chemical Co.
Solutions were prepared dissolving the pterine (Ptr or Mep) and Met in water. The
final pH of the solutions was adjusted by adding drops of HCl or NaOH solutions (0.1–0.2
M) with a micropipette. The ionic strength was ca. 10-3 M in all experiments. Concentration
ranges used for the experiments were 50-150 M and 600-800 M for the pterine and Met,
respectively.
Steady-state irradiation.
Irradiation set-up. Aqueous solutions containing a pterine (Ptr or Mep) and Met (pH
5.5-6.0) were irradiated in 1 cm path length quartz cells at room temperature with a
Rayonet RPR3500 lamps, with emission centered at 350 nm (fwhm bandwidth of ca 20
nm, Southern N.E. Ultraviolet Co.). The experiments were performed in the presence
and in the absence of O2. O2-free solutions were obtained by bubbling with Ar during
20 min. The measurements were carried out under conditions of reduced environmental
light.
Actinometry. Aberchrome 540 (Aberchromics Ltd.), the anhydride form of the (E)-R-
(2,5-dimethyl-3-furylethylidene)(isopropylidene)-succinic acid, was used as an
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actinometer for the measurements of the incident photon flux (qP,0) at the excitation
wavelength. The method for the determination of qP,0 has been described in detail
elsewhere (25,26). Values of the photon flux absorbed (qP,a), were calculated from qP,0
(qP,0 = 1.0 x 10-5 einstein L-1 min-1 at 350 nm) according to the Lambert-Beer law (qP,a
qP,0 (1-10-A), where A is the absorbance of the sensitizer at the excitation wavelength).
=
UV/Visible Analysis. UV-visible absorption spectra were registered on a Shimadzu UV-
1800 spectrophotometer. Measurements were made in quartz cells of 0.4 and 1 cm
optical path length.
High Performance Liquid Chromatography (HPLC). A Prominence equipment from
Shimadzu (solvent delivery module LC-20AT, on-line degasser DGU-20A5,
communications bus module CBM-20, auto sampler SIL-20A HT, column oven CTO-
10AS VP, photodiode array detector SPD-M20A), was used to monitor and quantify the
reactants and the photoproducts. Separation of the photosensitizer, the substrate and the
products was performed on a Sinergy Polar-RP coPtrn column (ether-linked phenyl phase
with polar endcapping, 150 x 4.6 mm, 5 m; Phenomenex), using as mobile phase solutions
containing 10 mM NH4OAc aqueous solution (pH 6.8). HPLC runs were monitored by
UV/Vis spectroscopy at different wavelengths. Absorption spectra of the reactants and
products were registered at each retention time with the photodiode array detector of the
equipment.
Data analysis. Determination of concentrations related to a given experiment were
performed on the same day. Kinetic analyses were carried out for each experiment. Error of
individual data is related to the detection methodology.
Detection and quantification of H2O2. H2O2 was determined by its reaction with 4-
aminophenazone and phenol catalyzed by the enzyme peroxidase to yield 4-(p-
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benzoquinone monoimino)phenazone, which is detected by its absorbance in the visible
region (27,28). This assay has high sensitivity and specificity due to the intense absorbance
of the product at 505 nm and the enzymatic catalysis, respectively. The reactants were
purchased from Wiener Laboratorios SAIC (cholesterol kit), and it was used as previously
described elsewhere (19).
Mass spectrometry analysis
The analysis was performed using an UPLC chromatograph (ACQUITY UPLC from
Waters) coupled to a quadrupole time-of-flight mass spectrometer (Xevo G2-QTof-MS
from Waters) (UPLC-QTof-MS), described elsewhere (22). UPLC analyses were
performed using an Acquity UPLC® BEH C18 (1.7 m; 2.1 x 50 mm) column (150 mm,
Waters), and isocratic elution with 0.1 % formic acid at a flow rate of 0.6 mL min-1.
RESULTS
Irradiation of solutions containing Ptr and Met
Aqueous solutions containing Met ( 600 M) and the photosensitizer Ptr (
100M), equilibrated at different O2 concentrations, were exposed to UV-A radiation
(350 nm) for different periods of time. Considerable changes in the absorption spectra
of the solutions were registered during irradiation even in the absence of O2.
Concentration profiles of reactants were obtained by HPLC. In aerated solutions, a
decrease of Met concentration was observed as a function of the irradiation time,
whereas Ptr concentration remained constant (Fig. 2a). On the other hand, in the
absence of O2, both Met and Ptr consumption were observed (Fig. 2b). Taking into
account previous studies carried out with Ptr and other amino acids (19,20,21), the
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behavior observed in the presence of O2 was expected, but the results obtained in
anaerobic conditions were surprising. In fact, studies carried out under anaerobic
conditions with Trp and Tyr showed that neither the substrate nor the photosensitizer
are consumed upon irradiation due to the recombination of radicals, which leads to the
recovering of the reactants (19,20).
<Figure 2>
In air-equilibrated solutions H2O2 was detected as a product. In addition, HPLC
analysis revealed the formation of a main product (P1), at a retention time (tR) 3.6 min.
On the other hand, in argon equilibrated solution, a different photoproduct (P2, tR 16.5
min) was detected, being its absorption spectra similar to those reports for 7.8-
dihyropterin derivatives (29), thus suggesting the reduction of Ptr in the photochemical
process.
7,8-Dihydropterin (H2Ptr) is not commercially available, and therefore it is not
possible to confirm its formation. It was previously reported that, in anaerobic
conditions, pteridines are photo-reduced to the corresponding 7,8-dihydroderivative in
the presence of a suitable electron donor (30,31).
Although Ptr is the most studied pterin derivative in terms of photosensitizing
properties, we changed to 6-methylpterin (Mep) in order to investigate a possible
photoreduction of the photosensitizer. Mep is a compound with photochemical
properties similar to Ptr (12), but its corresponding dihydroderivative, 6-methyl-7,8-
dihydropterin (H2Mep), is commercially available, stable, and well characterized by
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chromatography. Therefore, after having observed the general behavior with Ptr, we
performed the rest of the studies using Mep as photosensitizer.
Irradiation of solutions containing Mep and Met
Experiments similar to those described in the previous section were performed
using Mep instead of Ptr as a photosensitizer. In air-equilibrated solutions, the behavior
observed upon irradiation was equivalent to that observed with Ptr, that is, consumption
of the amino acid, no consumption of the photosensitizer, formation of H2O2 (Fig. 3a)
and formation of a product at 3.6 minutes of retention time (Fig. 3b). The addition of
commercial MetO to irradiated solutions results in an increase in the peak at 3.6
minutes (Fig. 3b), thus indicating that P1 is this Met oxidation product. According to
previous studies, MetO is a common product of thermal and photochemical oxidation of
Met.(8,9,32,33,34)
<Figure 3>
In deaerated solutions, both Met and Mep, as in the case of Ptr, were also
consumed, and a peak at retention time 16.1 min, and similar spectroscopic
characteristics as P2 (tR 16.5 min) was detected whose concentration increased as a
function of irradiation time (Fig. 4). The addition of commercial H2Mep as a standard,
resulted in an increase of the area of the peak at 16.1 min (Fig. 4, dash red line).
Moreover, the absorption spectra recorded at 16.1 min (Fig. 4, inset c) was coincident
with the absorption spectra of commercial H2Mep (Fig. 4, inset b). These results
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strongly suggest the reduction of Mep upon UVA irradiation in the presence of Met. In
this case, as expected, no MetO was detected.
<Figure 4>
In addition, control experiments showed that reactions between a pterine (Ptr or
Mep) and Met in the dark could be discarded. Moreover, no chemical modification of the
amino acid was detected when Met (600 M) solutions were irradiated at 350 nm in the
absence of the photosensitizer, under aerobic or anaerobic conditions, thus excluding
spurious effects of light absorption by Met.
Mechanistic analysis
Kinetic analyses on the Mep-photosensitized degradation of Met were performed at
different experimental conditions in order to determine the mechanism. Iodide (I−) is an
efficient and selective quencher of triplet-excited states of pterins at micromolar
concentrations (31,35). Thus, to evaluate the participation of triplet-excited state of Mep
(3Mep*), photosensitization experiments were carried out in aqueous solutions containing
Met and Mep at pH 5.5 in the presence of 400 M KI, both in air-equilibrated and argon–
equilibrated solutions. At this KI concentration, the quenching of the singlet excited state of
Mep (1Mep*) is less than 3% (F0 = 8.9 10-9 s, kF = 6.2 x 109 M-1s-1 (35)), and therefore any
1
change in the rate of Met consumption is not due to deactivation of Mep*. The rate of
consumption of Met, both in the presence and in the absence of O2, was lower in the
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3
presence of I− (Fig. 5), indicating that Mep* is involved in the mechanism of
photodegradation of Met.
<Figure 5>
1
1
Since Met is oxidized by O2 (33) and pterins are relatively good O2-
photosensitizers (12), we investigated the role of this ROS in the oxidation of Met
photosensitized by Mep in aerobic conditions. Therefore, experiments using D2O as solvent
were carried out and the results were compared to those obtained in H2O. The 1O2 lifetime
() is much longer in D2O than in H2O (c.a. 60 s and 4 s, respectively) (36). Therefore,
if 1O2 is involved in the oxidation of Met, the rate of consumption should be about 20-times
faster in D2O than in H2O. The determined rate of Met consumption was higher in D2O
(Fig. 6), but lower than expected considering a pure type II mechanism. Previously, it has
1
been reported the rate constant value of the total quenching (kT) of O2 by Met (1.3 x 10-7
M-1 s-1) and that the quenching is almost completely a chemical process (kr ~ kT) (37).
1
Accordingly, in a given experiment, the contribution of O2 to the Mep-photosensitized
oxidation of Met can be evaluated by comparing the experimental initial rate of Met
1
consumption ((d[Met]/dt)0,exp) to the calculated initial rate of the reaction between O2 and
Met ((d[Met]/dt)0,calc, equation 1).
-(d[Met]/dt)0,calc = kr [Met] [1O2]
(1)
1
The steady-state concentration of O2 during irradiation of a solution containing Mep and
Met is given by:
[1O2] = qP,a Φ∆ / (kd + ktMep [Mep] + ktMet [Met])
(2)
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where qP,a (einstein L-1 s-1) is the photon flux absorbed by Mep; Φ∆ is the quantum yield of
1
1O2 production by Mep (Φ∆ = 0.10 ± 0.02) (12); kd is the rate constant of O2 deactivation
by the solvent (2.6 x 105 s-1); ktMep and ktMet are the overall rate constants of 1O2 quenching
by Mep (8.0 x 106 M-1 s-1 (12)) and Met (1.3 x 107 M-1 s-1 (37)), respectively. Considering
the experimental conditions of Fig. 6 (120 M Mep, 700 M Met), (d[Met]/dt)0,calc, was 1.5
M min-1, considerably lower than (d[Met]/dt)0,exp (5.7M min-1), calculated by HPLC
analysis. The difference observed between (d[Met]/dt)0,calc and (d[Met]/dt)0,exp indicates that
1
the chemical reaction between Met and O2 does not contribute significantly to the
photosensitized oxidation of Met. This behavior was already observed for the oxidation of
Trp and Tyr sensitized by Ptr (19,20) which is consequence of the significant difference
between the rate constants of the reaction of Met with 1O2 (1.3 x 107 M-1 s-1 (37)), and with
3Ptr* (3 x 109 M-1s-1 (35)), that is, Met reacts faster with 3Ptr*, to yield Met•+, than with 1O2.
<Figure 6>
Further experiments were carried out in the presence of superoxide dismutase
•-
(SOD), an enzyme that catalyzes the conversion of O2 into H2O2 and O2 (38). The
experiments revealed an increase in the rate of Met consumption when SOD was present in
•-
the solution (Fig. 7) indicating that O2 is involved in the mechanism.
<Figure 7>
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Considering all these kinetic results, and previous articles on photosensitization of
biomolecules with pterins, we propose a mechanism (Scheme 2) for Met degradation
during UV-A irradiation in the presence of Mep (or Ptr). Upon UV-A excitation Mep yields
a singlet excited state (1Mep*, Reaction 1) which, in turn, by intersystem crossing (ISC),
3
leads to the formation of the reactive triplet excited state (3Mep*, Reaction 2). Mep*
undergoes ISC to the ground state (Reaction 3) or reacts with Met in an electron transfer
(ET) reaction yielding the corresponding radical ions, Mep•- and Met•+ (Reaction 4, type I
mechanism).
<Scheme 2>
Based on the behavior of Met•+, the investigation of photoreduction of pterins (30)
and the results observed in the absence of O2, we proposed that a second electron transfer
reaction takes place in which H2Ptr is formed (Reaction 6). Considering previous reports on
photosensitized degradation of methionine by flavins, a molecular mechanism could be
proposed (Scheme 3) (9).
<Scheme 3>
In air-equilibrated solutions, energy transfer to O2 leads to the regeneration of Ptr
1
and the formation O2 (Reaction 9) (39,40), which competes with reactions 3 and 4. It was
•-
previously published (19), that during irradiation of solutions containing Ptr, O2 is
generated due to the reaction between Ptr•- and O2 (Reaction 8), and Ptr is recovered. Later,
•-
•
O2 may disproportionate with its conjugated acid HO2 to form H2O2 (Reactions 9). Also,
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•-
O2 may react with Met•+ to regenerate Met (Reaction 10), preventing its further oxidation,
•-
which is evidence by the increase of Met oxidation while O2 is eliminated from the
solution. Finally, Met or Met•+ is oxidized to different products by both type I and type II
Mechanism (Reactions 11 and 12, respectively), but in our reaction system type I
mechanism predominates.
Analysis of products by mass spectrometry
In UPLC-QTof-MS analysis of non-irradiated solutions, performed in positive
ion mode (ESI+), the intact molecular ions of Met and Mep as [M + H]+ species at tR
1.95 and 6.31 min, with m/z 150.0589 Da and 178.0732 Da were detected, in agreement
with their molar weights, 149 and 177, respectively.
In aerated irradiated solutions, as well as the reactants, two products were detected.
One product was detected at tR = 1.42 min, with a mass of 166.0538 Da coincident the
molecular ion [Met + O + H]+ , which corresponds to the incorporation of an atom of
oxygen to Met to yield MetO. It was also observed the molecular ion [Met + O + Na]+ at
m/z = 188.0361 Da. In addition, at longer irradiation times, a less intense peak was detected
at tR = 1,61 min, with m/z 182.0309 Da, which corresponds to the molecular ion [Met + 2O
+ H]+, that is the incorporation of two atoms of oxygen to Met, coincident with methionine
sulfone (MetO2).
In O2-free irradiated solutions, the corresponding dihydro-reduced pterin was
detected (H2Mep) at tR = 4,56, with a mass at 180.1011, identical to the commercial
H2Mep. No methionine oxidation products could be detected, although considering
previous reports on both photosensitized degradation and enzymatic fermentation of
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Met, Met•+ undergoes decarboxylation followed by a deamination to yield 3-
(methylthio)propanal or methional (Scheme 3).(9,41)
CONCLUSIONS
The photosensitized oxidation of methionine (Met) by pterin (Ptr) and 6-
methylpterin (Mep) was studied, both in aerated and deaerated aqueous solutions.
Despite Met reacts with singlet oxygen (1O2, photosensitized type II mechanism), the
mechanism of photosensitization takes place mainly by electron transfer mechanism
(type I) even in the presence of O2.
Summarizing, the process begins with the transfer of an electron from Met to the
triplet excited state of the photosensitizer (Ptr or Mep), to yield the corresponding pair
of radicals, Met radical cation (Met•+) and pteridine radical anion (Ptr•- or Mep•-). The
products of the amino acid are different depending on the availability of O2. In the
presence of O2, Met•+ incorporates one or two atoms of oxygen to yield methionine
sulfoxide (MetO) and methionine sulfone (MetO2), and the pteridine radical anion (Ptr•-
or Mep•-) reacts with O2 to recover the photosensitizer and generates superoxide anion
•-
(O2 ). In the absence of O2, pteridine radical anion is reduced to the corresponding
dihydroderivative (H2Ptr or H2Mep) by a radical formed after decarboxilation of Met•+.
ACKNOWLEDGEMENTS. The present work was partially supported by Agencia de
Promoción Científica y Tecnológica (ANPCyT-Grants PICT 2015-1988, PICT 2017-0925)
and the Universidad Nacional de La Plata (UNLP, Grant X712). The authors thank the
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Centre National de la Recherche Scientifique (CNRS) and CONICET for supporting their
collaboration through a Programme de Coopération Scientifique (CONICET-CNRS/PICS
No05920). The authors also thank Nathalie Martins-Froment (Service Commun de
Spectrométrie de Masse, FR2599) and Dr. Patricia Vicendo (Laboratoire des
InteractionsMoléculaires et Réactivité Chimique et Photochimique, IMRCP), from
Université de Toulouse III (Paul Sabatier) for their valuable help with the mass
spectrometry measurements. D C.C. thanks the CONICET for graduate research
fellowships. A.H.T and C.L. are research members of CONICET.
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Figure and Scheme Captions
Figure 1. Molecular structure of pterin (Ptr), 6-methylpterin (Mep) and methionine
(Met), and their corresponding absorption spectra in air-equilibrated aqueous solutions
(pH = 5.5). Solid line: acid form of Ptr; dotted line: acid form of Mep; dashed-dotted
lines: Met.
Figure 2. Concentration evolution of (a) Ptr and (b) Met during irradiation of aqueous
solution, equilibrated in air or argon, containing Met and Ptr ([Met]0 = 600 µM, [Ptr]0 =
100 µM, IR = 350 nm, pH = 5.5). Errors on individual experimental points are ~ ± 4
M.
Figure 3. UV-A irradiation of air-equilibrated solutions ([Met]0 = 800 µM, [Mep]0 =
120 µM, IR = 350 nm, pH = 5.5) (a) Concentration of Met, Mep and H2O2 (inset) as
function of irradiation time (errors on individual experimental points are ~ ± 4 M). (b)
Chromatograms obtained in HPLC, analysis at 220 nm, from irradiated solutions
containing Met and Mep, without (solid black line) and with (solid red line) the
additions of commercial MetO. Upper inset: evolution of MetO concentration; lower
inset: absorption spectra at tR =3.6 min (black line) and absorption spectra of
commercial MetO (red line).
Figure 4. UV-A irradiation of Ar-equilibrated solutions ([Met]0 = 610 µM, [Mep]0 =
120 µM, IR = 350 nm, pH = 5.5). Chromatograms obtained in HPLC, analysis at 330
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nm from irradiated solutions containing Met and Mep, without (solid black line) and
with (dash red line) the addition of commercial H2Mep. Insets: (a): concentration
evolution of Met, Mep and H2Mep during irradiation time (errors on individual
experimental points are ~ ± 4 M); (b): absorption spectra of commercial H2Mep; (c):
absorption spectra of the peak at tR =16,1 min.
Figure 5. Concentration evolution of Met, Mep and photoproducts during irradiation of air-
and Ar-equilibrated aqueous solutions, containing Met and Mep in the absence and in the
presence of KI ([Met]0 = 600 µM, [Mep]0 = 100 µM, IR = 350 nm, pH = 5.5). Errors on
individual experimental points are ~ ± 4 M.
Figure 6. Concentration evolution of Met, Mep and photoproduct MetO during irradiation
of D2O and H2O aerated solutions containing Met and Mep (IR = 350 nm, pH = 5.5.
([Met]0 ~ 700 µM; [Mep]0 = 120 µM. Errors on individual experimental points are ~ ± 4
M.
Figure 7. Concentration evolution of Met, Mep and photoproduct MetO during irradiation
of aerated aqueous solutions containing Met and Mep (IR = 350 nm; pH = 5.5; [Met]0 ~
670 µM; [Mep]0 = 110 µM, in the absence and in the presence of SOD (50 U/ml). Errors on
individual experimental points are ~ ± 4 M.
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Scheme 1: Mechanism of Met oxidation
Scheme 2: Proposed mechanism for Ptr-photosensitized Met oxidation
Scheme 3: Proposed mechanism of Met degradation in O2-free aqueous solutions
(according to the mechanism proposed in Ref (9))
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This article is protected by copyright. All rights reserved

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