BiochemicalSociety Transactions (1995) 23 2378
Modification of aromatic amino acids by reactive
nitrogen species.
ALBERT VAN DER VLIET, JASON P. EISERICH, BARRY
HALLIWELL and CARROLL E. CROSS.
metal ions, so they are not likely to participate in our
studies. We postulate that ONOO- nitrates tyrosine via
formation of tyrosyl radicals, which explains the formation
of dityrosine. These tyrosyl radicals then combine with the
simultaneously generated 'NO, to form 3-nitrotyrosine.
Division of PulmonarylCritical Care Medicine, Department
of Internal Medicine, University of California, Davis, UCD
Medical Center, Sacramento, CA 95817, USA.
FIGURE 1: Formation of nitrotyrosine by reactive nitrogen
species.
Reactive nitrogen species are becoming increasingly
recognized as important mediators of damage in biological
systems [ l ] . Nitrogen monoxide ('NO) is a radical species
with widespread biological functions [2], but excessive
levels of *NO can be toxic, either via autoxidation, forming
reactive intermediates such as nitrogen dioxide ('NO,) and
dinitrogen trioxide (N203) [3], or, more importantly, by
reaction with superoxide (02*-),
forming peroxynitrite
(ONOO-) [4], a powerful oxidant. At physiological pH
ONOO- is protonated and decomposes rapidly, forming an
intermediate with the reactivity of hydroxyl radical ('OH)
and 'NO, 15). Hence, ONOO- is capable of oxidizing
various biomolecules, leading to lipid peroxidation and
oxidative damage to proteins and nucleic acids. Specific
protein modifications include hydroxylation and nitration of
aromatic amino acids [6,7], and tyrosine nitration in
proteins in various disease states is regarded as evidence
for the involvement of ONOO- in vivo.
Some inconsistency exists in the literature as to the
mechanisms involved in aromatic hydroxylation and
nitration. Peroxynitrite reacts with free phenylalanine (or
phenylalanine residues in proteins) to form 0-,m- and ptyrosine [6], and this was taken as evidence for the
formation of 'OH during decomposition of peroxynitrite.
However, as shown in Table I, the hydroxyl radical
scavenger mannitol (added at large excess over
phenylalanine)
only
partially
inhibited
aromatic
hydroxylation, which does not appear consistent with
involvement of free 'OH. Peroxynitrite also reacts rapidly
with tyrosine residues to form 3-nitrotyrosine and dityrosine
as major products [6]. No significant formation of
dihydroxyphenylalanine (DOPA) was detected, however,
which also argues against formation of free 'OH.
TABLE I: Effect of mannitol (100 mM) on hydroxylation of
phenylalanine (5 mM) by ONOO- (1 mM).
ONOO- (pH 7.4)
5.3 f 0.4
6.0 f 0.2
5.9 f 0.8
+ mannitol
2.2
2.0
2.6
Nitration of tyrosine by ONOO- was proposed to
occur via formation of a nitronium species (NO,+) [7]. This
species is formed by heterolytic scission of ONOO- in the
presence of transition metal ions. We treated our reaction
media with Dowex chelating resin to remove transition
4
5
6
7
8
9
10
PH
DL-Tyrosine (1 mM) was treated with ONOO- (1 mM) or with
'NO, (14 ppm for 2 hrs) at various pH values, and 3nitrotyrosine was measured by HPLC [6].
0: Nitration by ONOO-; 0 : Nitration by 'NO2.
Qualitatively similar results were obtained upon reaction of
tyrosine with 'NO2 in a controlled environment exposure
chamber. As shown in Fig l.,
nitration of tyrosine by both
'NO2 and ONOO- increases with pH. This is most likely a
result of increased presence of tyrosine as tyrosyl anion,
which is more susceptible to oxidation. The rapid decrease
in nitration by ONOO- at pH higher than 7.5 is caused by
decreased formation of peroxynitrous acid, which is
responsible for nitration.
Our results thus suggest that ONOO- nitrates tyrosine
residues via tyrosyl radical formation and intermediate
formation of 'NO,.
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