annual report annual report annual report annual report 2005

22
RADIATION CHEMISTRY AND PHYSICS, RADIATION TECHNOLOGIES
via different mechanism than the analogous radicals
in Met-Gly. The decay kinetics of the
(S∴N) + Met-Tyr radical matches the formation kinetics
of the Met-TyrO • radical (Fig.3, inset). These
kinetic results confirm the hypothesis that an intramolecular
electron transfer proceeds between the
intact tyrosine residue and the (S∴N) + Met-Tyr
radical.
In a solution containing Met-Tyr at pH 1.0
(Fig.4), the absorption spectrum recorded 5 µs after
the electron pulse was similar to the absorption
spectrum recorded in a solution of Tyr-Met at pH
1.0, excluding a small absorption of (S∴N) + Met
radical in the spectrum recorded in the Met-Tyr
solution.
Conclusions
A variety of transient products was formed during
• OH oxidation of Met-Tyr and Tyr-Met. The
main difference in the transient-products pattern
concerns the (S∴N) + Met radical.
The observed slow decay of dihydroxycyclohexadienyl
radicals excluded them as a source of
tyrosyl radicals in both peptides, on the short time
scale at pH~6. The remaining fraction of hydroxyl
radicals reacted with methionine residues forming
hydroxysulfuranyl radicals Met(>S • -OH).
These radicals were transformed into sulfur-centered
radical cations Met(>S +• ) via proton-catalyzed
dehydration.
Due to a large difference in redox potentials of
the Met(>S +• ) radical E 0 (Met(>S +• )/Met)~1.6 V
[3] and the TyrO • radical E 0 (TyrO • )/Tyr)~0.94 V
[4], fast electron transfer likely occurred, making
the observation of Met(>S +• ) radicals unfeasible.
Hence, we assume a mechanism of the electron
transfer for both dipeptides at pH~1 – reactions
(3a) and (3b):
Met(>S +• )-Tyr → Met-TyrO • (3a)
Tyr-Met(>S +• ) → TyrO • -Met (3b)
The hydroxysulfuranyl radical Tyr-Met(>S • -OH)
was excluded as a precursor of the TyrO • radical at
pH~6, because its redox potential E 0 (Met(>S • -OH)
/Met)~1.43 V [3] is lower than the potential of
Met(>S +• ). Other reaction pathways involving the
Met(>S +• ) radicals (e.g. decarboxylation) were experimentally
excluded.
Another reaction pathway was observed during
the oxidation of Met-Tyr at pH~6. The H 3
N + -Met
(>S • -OH)Tyr radical underwent deprotonation
using a proton from the protonated amino group.
As a result, the sulfur-nitrogen bonded radical cation
(S∴N) + Met-Tyr was formed – reaction (4).
H 3 N + -Met(>S • -OH)Tyr → (S∴N) + Met-Tyr (4)
Subsequently, an intramolecular electron transfer
occurred between the intact tyrosine and the
(S∴N) + Met-Tyr radical – reaction (5):
(S∴N) + Met-Tyr → Met-TyrO • + H + (5)
Reaction (5) proceeded with slower rate compared
to reaction (3) which is in line with lower
redox potential of the (S∴N) + Met radical at pH~6
(E 0 ((S∴N) + Met/Met)~1.44 V [5]).
References
[1]. Prütz W.A., Butler J., Land E.J.: Int. J. Radiat. Biol.,
47, 149-156 (1985).
[2]. Bobrowski K., Wierzchowski K.L., Holcman J., Ciurak
M.: Int. J. Radiat. Biol., 57, 919-932 (1990).
[3]. Merényi G., Lind J., Engman L.: J. Phys. Chem., 100,
8875-8881 (1996).
[4]. DeFelippis M.R., Murthy C.P., Faraggi M., Klapper
M.H.: Biochemistry, 28, 4847 (1989).
[5]. Prütz W.A., Butler J., Land E.J., Swallow A.J.: Int. J.
Radiat. Biol., 55, 539-556 (1989).
EPR STUDY OF DIPEPTIDES CONTAINING TYROSINE
Grażyna Strzelczak, Krzysztof Bobrowski, Jacek Michalik
Tyrosine radicals play an essential role in a number
of biological processes. They are postulated in
mediation of long-distance electron-transfer reactions
in a number of enzymes including galactose
oxidase [1], ribonucleotide reductase [2], prostaglandin
H synthase [3] and photosystem II [4]. Previous
work on tyrosyl radicals in polycrystalline
tyrosine-containing peptides by means of FT-IR
(Fourier-transform infrared) spectroscopy provided
evidence for a migration of unpaired spin
density from the phenoxyl ring to the terminal
amino group suggesting an interaction between the
π system of the tyrosyl radical and the amino group
[5].
The aim of our study was to test that such an
interaction occurs and whether it has an effect on
the character of forming radicals. Therefore,
tyrosyl radicals were generated in dipeptides with
various neighbouring amino acid residues located
at the C-terminus of tyrosine.
Polycrystalline dipeptides Tyr-Gly, Tyr-Leu,
Tyr-Met were irradiated in a 60 Co-gamma source
Fig.1. EPR spectrum of tyrosyl radical detected at 77 K in
Tyr-Gly.
with doses of 3 kGy in liquid nitrogen. Radicals
were identified by the electron paramagnetic resonance
(EPR) spectroscopy method. EPR experiments
were performed using a Bruker ESP-300