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(Received for publication, June 26,
1995; and in revised form, November 2, 1995) From the
During exposure to ozone, the methionine and aromatic amino acid
residues of Escherichia coli glutamine synthetase (GS) and
bovine serum albumin (BSA) are oxidized rapidly in the order Met >
Trp > Tyr
Ozone is one of the most toxic pollutants in the atmosphere.
Brief exposure to concentrations of ozone below 1 ppm leads to damage
of the lung, especially to regional damage of bronchiolar ciliated
cells and alveolar epithelial type I cells. Prolonged exposure leads to
inhibition of ciliagenesis and type 2 cell maturation, to inflammation,
fibrosis, enhanced collagen synthesis, and to increased sensitivity to
bacterial infection (for review, see Mehlman and Borek(1) ,
Menzel(2) , and Stokinger(3) ). Pryor (4) has
argued that ozone reacts within the alveolar epithelial type I cells so
rapidly that it cannot cross the lung-lining fluid layer except in
patchy areas where the lower airways are virtually uncovered. This
accounts for the observation that tissue damage by ozone is largely
restricted to the lung. Nevertheless, exposure of rats to ozone has
been shown to elicit a number of extrapulmonary effects, including a
decrease in visual acuity(5) , defects in the desaturation of
oxyhemoglobin in skin capillaries(6) , changes in cardiac
protein metabolism(7) , increased lipid peroxidation in heart
and brain tissue, and the elevation of peroxide scavengers in these
tissues(8) . Because of its pronounced cytotoxicity, recent
studies have focused on the fact that ozone can give rise to a number
of other reactive oxygen species including OH,
H The rates at which the aromatic and sulfur amino acid residues of
proteins are oxidized by ozone varies widely from one protein to
another, suggesting that the susceptibility to oxidation is dependent
upon the size and amino acid composition as well as the secondary and
tertiary structure. In the present study, we compare the ability of
ozone to oxidize amino acid residues in Escherichia coli glutamine synthetase (GS) (
From the data in Fig. 1and other data not shown, it
is evident that the modification of a given kind of aromatic amino acid
residue is a nearly linear function of time until at least 60 to 90% of
the residues have been oxidized. Also, as shown in Table 1, the
specific rate constant k = (number of residues modified
per min)/(total number of residues present in the protein) for each
aromatic amino acid is about 1.5-2.0 times higher for residues in
BSA than in GS. Since GS and BSA contain nearly equal amounts of each
of the aromatic amino acids (Table 1), this difference in
susceptibility to oxidation by ozone likely reflects differences in the
primary, secondary, tertiary, or quaternary structures. In this regard,
it is noteworthy that each of the 12 identical subunits of GS are
arranged in two superimposed hexaganol arrays, whereas BSA exists as a
monomer.
Figure 1:
Ozone-induced changes in the number of
tyrosine, histidine, and aspartic acid residues of BSA and GS. Mixtures
(2.5 ml) containing 10 mM potassium phosphate buffer, pH 7.4,
and 0.75 mg/ml BSA (closed symbols) or GS (open
symbols) were bubbled with an ozone/oxygen mixture yielding 545
nmol of ozone/min. At times indicated, aliquots were removed and the
amino acid composition of the proteins was determined. The
time-dependent changes in the number of tyrosine (triangles)
and histidine (squares) residues per subunit is indicated in
the lower half of the figure. The time-dependent increase in
the number of aspartic acid residues in BSA (closed circles)
and GS (open circles) is indicated in the upper portion of the figure.
To confirm that ozone promotes conversion
of histidine residues to aspartic acid or to a derivative that yields
aspartic acid upon acid hydrolysis, a protein preparation in which all
of the histidine residues were uniformly labeled with
Figure 2:
Ozone-elicited conversion of
[
Figure 3:
Effect of histidine location in
tripeptides on its oxidation. Tripeptides, 1.6 mM in 1.3 ml of
10 mM potassium phosphate buffer, pH 7.4, were treated with
ozone at a rate of 242 nmol per min. At the times indicated, 0.01-ml
aliquots were removed, and amino acid composition was determined as
described under ``Experimental Procedures.'' Symbols are as follows: squares, HAA; circles, AAH; triangles, AHA. A, broken lines and open
symbols refer to the amount of histidine residue present at the
times indicated. Solid lines and closed symbols refer
to the amounts of aspartic acid formed. B, relationship
between the amount of histidine residues lost and the amount of
aspartic acid formed.
The fact that a histidine
residue in the internal position of the peptide is stoichiometrically
recovered as aspartic acid explains why the histidine residues in BSA
and GS are converted almost entirely to aspartic acid. In contrast,
aspartic acid accounts for only 32% of the products formed from a
histidine residue occupying the N-terminal position of the peptide. An
examination of the ultraviolet absorption spectra of the peptides shows
that ozone treatment leads, in the case of HAA, to products possessing
much higher absorption in the range of 360 nm than products obtained
from either AHA or AAH (Fig. 4). Finally, it appears significant
that little or no carbonyl compounds could be detected among the
products obtained from any of the histidine-containing tripeptides.
Figure 4:
Time-dependent changes in spectrum of
tripeptides during ozone exposure. Conditions were as in Fig. 3.
At the times indicated, 0.1 ml of reaction mixture was diluted to 1.0
ml and the spectrum was recorded. Curves 1-7 counting
from the bottom up refer to spectra taken at 0, 2, 5, 10, 15, 20, and
30 min, respectively.
Figure 5:
Effect of ozone on histidine content and
catalytic activity of glutamine synthetase. GS, 0.75 mg/ml in 2.5 ml of
10 mM potassium phosphate buffer, pH 7.4, was treated with
ozone, 545 nmol/min, as described in Fig. 1. At the times
indicated, aliquots were assayed for histidine content of the protein (circles) and GS
Figure 6:
Ozone-mediated generation of carbonyl
groups in GS and BSA. The conditions were as described in Fig. 1. At the times indicated, 0.1 ml of reaction mixtures was
assayed for carbonyl content.
Figure 7:
Relationship between tyrosine modification
and quinoprotein formed. Mixtures (2.5 ml) containing 0.75 mg/ml BSA (circles) or GS (triangles) were bubbled with ozone
at a rate of 500 nmol/min. At the times indicated, aliquots were
assayed for tyrosine (open symbols) or quinoproteins (closed symbols) as described under ``Experimental
Procedures.'' The inset is a plot of quinoidal substances (+Q) formed versus the amount of tyrosine (Tyr)
lost, both expressed as nanomoles of residues/nmol of protein
subunit.
The demonstration that several kinds of amino acid residues
in BSA are more rapidly oxidized by ozone than they are in GS confirms
results of earlier workers (21) showing that the sensitivity of
a given kind of amino acid residue to oxidation by ozone varies from
one protein to another. However, the conclusion that this variability
is due to differences in the primary, secondary, tertiary, and
quaternary structures of the proteins is strengthened by the results
presented here, because the subunits of BSA and GS are of comparable
size and contain nearly equal numbers of each of the aromatic amino
acids. Three observations support the conclusion that the histidine
residues in protein and peptides are converted to aspartyl residues or
to derivatives that are converted to aspartic acid upon acid
hydrolysis. 1) The ozone-induced loss of histidine residues in BSA and
GS is accompanied by a nearly stoichiometric increase in the amount of
aspartic acid present in acid hydrolysis of the ozone-treated proteins.
2) The histidine lost during oxidation of the tripeptide, Ala-His-Ala,
is recovered in acid hydrolysates as aspartic acid. 3) When proteins in
which all histidine residues are uniformly labeled with Of particular interest is the
finding that the ozone-dependent loss of GS activity is directly
proportional to the total number of histidine residues that are
modified (Fig. 5), over a range of histidine residues
representing 75% of all histidine residues in the protein. This is in
sharp contrast to the fact that, when GS is exposed to the
ascorbate/FeIII/O In earlier studies, Farber and Levine (37) presented evidence indicating that in the oxidation of GS
by the ascorbate/FeIII/O
Volume 271,
Number 8,
Issue of February 23, 1996 pp. 4177-4182
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
His > Phe. The loss of His is matched by a nearly
equivalent formation of aspartate or of a derivative that is converted
to aspartic acid upon acid hydrolysis. Conversion of His to aspartate
was confirmed by showing that the oxidation of E. coli protein
in which all His residues were uniformly labeled with 
C
gave rise to C-labeled aspartic acid in 80% yield and also
by the demonstration that His residues in the tripeptides Ala-His-Ala
or Ala-Ala-His gave rise to nearly stoichiometric amounts of aspartic
acid whereas oxidation of His-Ala-Ala yielded only 36% aspartate. The
oxidation of BSA and GS led to formation, respectively, of 11 and 3.3
eq of carbonyl groups and 0.5 and 0.3 eq of quinoprotein per subunit.
Although BSA and GS contain nearly identical amounts of each kind of
aromatic amino acid residues, oxidation of these residues in BSA was
about 1.5-2.0 times faster than in GS indicating that the
susceptibility to oxidation is dependent on the primary, secondary,
tertiary, and quaternary structure of the protein.
O, O,
RCOO, O
, and singlet
oxygen(9, 10, 11, 12, 13, 14, 15) .
In some circumstances, these secondary reactive oxygen species might be
more damaging than ozone itself. Thus, the reaction of ozone with
polyunsaturated fatty acids in membrane lipids leads to the generation
of relatively stable lipid peroxides, which can subsequently give rise
to tissue-damaging alkoxy radicals and alkyl
radicals(12, 15, 16) . Lipid-independent
mechanisms must also be considered since ozone reacts directly with
some amino acids(17, 18) , with proteins (see (1) for review), and with nucleic
acids(19, 20) . In the case of free amino acids and
amino acid residues in proteins, cysteine, methionine, tryptophan,
phenylalanine, and histidine, residues are particularly sensitive to
oxidation by ozone; other amino acids are fairly resistant to
ozone(17, 21) . Kynurenine and N-formylkynurenine have been identified as major products of
free tryptophan and of tryptophan residues in proteins(22) .
Cysteic acid and cystine are major products of cysteine
oxidation(2, 22, 23) . Methionine sulfoxide
is a major product of methionine oxidation(17) .
Dihydroxyphenylalanine was produced in low yields during the oxidation
of tyrosine by ozone. Ammonia and an amino acid tentatively identified
as proline are products of histidine oxidation at pH 4.6(17) . 
)and bovine serum albumin
(BSA). These two proteins are similar with respect to subunit size (50 versus 67 kDa), they contain nearly equal numbers of Trp, Tyr,
His, and Phe residues per subunit, and x-ray crystallographic data are
available for GS (24) and human serum albumin (25) ,
which is highly homologous to BSA. The GS was selected also because the
results could be compared with those derived from extensive studies on
the oxidation of the enzyme by metal-catalyzed oxidation (MCO) systems.
In addition, we investigated the ozone-mediated oxidation of a series
of tripeptides, each of which contained two alanine residues and one
histidine residue that was present in either the N-terminal, middle, or
C-terminal position.
Materials
Bovine serum albumin (BSA) was
obtained from Sigma. Glutamine synthetase (GS) was from E. coli YMC
10/pgln6, which overproduces the enzyme. The enzyme was purified
by the zinc-induced aggregation procedure as described(26) . GS
activity was measured by the -glutamyl transferase
assay(27) . L-[U-
C]histidine,
0.38 mCi/µmol, was from DuPont NEN.Carbonyl Group Assay
The presence of carbonyl
groups was measured by the 2,4-dinitrophenylhydrazine technique as
modified for analysis using reverse phase high performance liquid
chromatography to separate excess reagent from the protein hydrazone
derivatives (28) .Ozonolysis
Prior to ozonolysis, protein solutions
were buffer-exchanged by passage through a PD-10 gel filtration column
(Sephadex G-25) from Pharmacia, eluted with 10 mM potassium
phosphate buffer, pH 7.4, and adjusted to a final concentration of 3.0
mg/ml. Ozone was produced by passing 100% oxygen through a Sander model
25 ozonizer purchased at a local aquarium shop. Quantitation of the
ozone was accomplished by titrametric measurement of the I produced when the ozone-oxygen mixture was passed through a
solution of 1 M KI at pH 7.4, using 0.001 M NaSO as titrant, or by the
bleaching of indigotrisulfonate(29) . The rate of bubbling of
the ozone-oxygen mixture through the reaction mixture was adjusted to
yield a constant supply of ozone for the duration of the experiment.
The supply of ozone was varied from 50 to 500 nmol/min depending upon
the experiment. As standard procedure, 1.875 ml of the 10 mM phosphate buffer, pH 7.4, was pregassed with the ozone/oxygen
mixture for 10 min; then, 0.625 ml of the protein stock solution was
added to yield a mixture containing 0.75 mg of protein/ml, and the
bubbling was continued at a constant rate. At various times, aliquots
were removed and analyzed for spectral changes, total amino acid
composition, quinoprotein content, and carbonyl content.Amino Acid Analysis
Aliquots of reaction mixtures
were subjected to acid hydrolysis as described previously(30) .
Amino acids in the hydrolysate were converted to their o-phthaldialdehyde (OPA) derivatives, and these were separated
by high pressure liquid chromatography on a Jones 15-cm C
column and quantified as described previously(30) .
Tryptophan is destroyed by acid hydrolysis; therefore, alkaline
hydrolysates were used for measurement of tryptophan(31) .
Quantitation of proline was achieved by first derivatizing the primary
amino acids with OPA, then the secondary amino acids were converted to
their Fmoc (N-(9-fluorenyl)methoxycarbonyl) derivatives, prior
to separation by HPLC(32) . The HPLC was performed on a
Hewlett-Packard model 1090 instrument equipped with a Hewlett-Packard
1046 programmable fluorescence detector.D- and L-Aspartate
D-
and L-Aspartate were determined as their chiral adducts with o-phthaldialdehyde as described by Aswad(33) . Some
racemization of aspartate occurs during the acid hydrolysis of peptides
and proteins, and the rate might differ from that of free aspartate.
However, under the conditions used here, the rate of racemization was
found to be the same for both free L-aspartate and for
aspartyl residues released from either GS or BSA, 1.4 
10 min
.
Preparation of
[
Protein in
which all histidine residues were uniformly labeled with C]Histidine-labeled ProteinC
was prepared by growing E. coli on a minimal medium that was
supplemented with [U-C]histidine as follows: 400
ml of culture medium containing 2 g glucose, 4 mM glutamine,
and L-[U-C]histidine (100 µCi, 0.65
µM) was inoculated with 7.0 ml of an overnight culture of E. coli, strain W. Cultures were incubated at 37 °C for 17
h. Cells were harvested by centrifugation and immediately frozen in
liquid nitrogen. The cells (0.28 g) were suspended in 2.0 ml of 0.1 M potassium phosphate buffer, pH 7.2, and were disrupted by
passing twice through a French press at 20,000 p.s.i. The broken cell
suspension was centrifuged at 13,000 g (SS34 rotor)
for 30 min. The debris was discarded. The cell-free supernatant
fraction was adjusted to pH 5.85, 10 mg of protein per ml, total volume
3.1 ml. Then, one-tenth volume of a 10% solution of streptomycin
sulfate was added and the pH was readjusted to 5.85. After 15 min, the
precipitated nucleic acid was removed by centrifugation, 15 min at
20,000 g. The clear supernatant solution was adjusted
to pH 7.6 and passed through a PD gel filtration column
that had been equilibrated with 0.1 M potassium phosphate
buffer, pH 7.6. The protein fraction was supplemented with a mixture of
protease inhibitors to final concentrations of: leupeptin, 0.5
µM; pepstatin, 0.7 µg/ml; phenylmethylsulfonyl
fluoride, 400 µg/ml; aprotinin, 5 µg/ml. The final preparation
contained 13.2 mg of histidine-labeled protein (1,032,000 cpm/mg). By
means of acid hydrolysis and amino acid analysis, it was established
that virtually all of the radioactivity of the protein was in the
histidine residues.
Quinoprotein
The presence of quinoidal derivatives
of aromatic amino acid residues in proteins was determined by the
method of Paz et al.(34) , which takes advantage of
the fact that, at alkaline pH, quinones and bis-carbonyl compounds
catalyze redox cycling using glycine as the electron donor and nitro
blue tetrazolium (NBT) as the electron acceptor. In our experiments,
0.2 ml of protein solution (0.75 mg/ml) was mixed with 0.2 ml of 2 M glycine (adjusted to pH 10 with KOH) in 12 75 mm
test tubes; then, 1.0 ml of 0.24 mM NBT (in the pH 10 glycine
solution) was added. After incubating at 37 °C for 20 min, the
absorbance of the reduced NBT was measured at 530 nm. The relative
concentration of quinoprotein was estimated by comparison with standard
values obtained with 3,4-dihydroxyphenylalanine (DOPA) incubated under
the same conditions. The absorbance of the DOPA-catalyzed reactions is
a linear function of DOPA concentration from 0 to 1.0 µM.
Effect of Ozone on the Aromatic and Sulfur-containing
Amino Acid Residues
As found in previous studies (35) with other proteins, the aromatic and sulfur-containing
amino acid residues of BSA and GS are most sensitive to ozone
modification. In both proteins, the order of susceptibility to
oxidation is Met > Trp > His > Tyr > Phe (Table 1).
In addition, as noted by Mudd et al.(17) , some
cysteine residues are converted to cysteic acid residues. Under our
conditions, only 0.6 out of 4 residues in GS and 3 of 35 residues in
BSA were converted to cysteic acid residues during a 120-min exposure
to ozone at a rate of 500 nmol per min. None of the other amino acid
residues were modified at a significant rate upon exposure of the
proteins to ozone at levels varying from 70 to 800 nmol/min (data not
shown).
Histidine Residues Are Converted to Aspartic Acid
Residues
The ozone-mediated oxidation of BSA and GS leads to an
increase in the number of aspartic acid residues or to residues that
are converted to aspartic acid during acid hydrolysis (Fig. 1).
The possibility that the aspartic acid is derived from histidine
residues is suggested by the fact that there is a nearly stoichiometric
relationship between the number of histidine residues lost and the
number of aspartic residues formed. This probably involves conversion
of the histidine residues to asparagine or to 2-oxohistidine residues,
which may be converted to aspartic acid upon acid hydrolysis (see
``Discussion'').C
was prepared by growing a histidine-requiring auxotrope of E. coli on a medium containing uniformly labeled
[C]histidine as the sole histidine source. The
[C]histidine-labeled protein fraction from
cell-free extracts of the organism was then exposed to ozone under our
standard conditions. As shown in Fig. 2, the treatment with
ozone led to a nearly linear, time-dependent decrease in the amount of C-labeled histidine and was accompanied by a nearly
stoichiometric (approximately 80%) increase in the amount of C-labeled aspartate that was present following acid
hydrolysis of the ozone-treated protein. Conversion of histidine
residues to aspartate was confirmed also by the demonstration that the
histidine residue in a tripeptide containing one His residue and two
Ala residues was converted to aspartic acid following exposure to ozone
and acid hydrolysis.
C]histidine residues in E. coli protein to [C]aspartic acid. 4.0 mg of
histidine-labeled protein from E. coli containing 1,032,000
cpm/mg in a total volume of 2.2 ml was bubbled with an ozone-oxygen
mixture at a rate corresponding to 545 nmol/min. At the times
indicated, 0.1-ml aliquots were removed, hydrolyzed in 6 N HCl, and amino acids in the hydrolysate were separated by HPLC of
their OPA derivatives. The histidine and aspartic acid fractions were
collected, and the C content was determined by the
scintillation counting technique. For plotting, the radioactivity of
the aspartic acid fraction was multiplied by 1.5 to compensate for the
fact that 2 of the 6 carbon atoms of histidine are lost during its
conversion to aspartic acid. The dashed line represents the
expected relationship if all of the histidine that disappeared was
recovered as aspartic acid. The inset shows how the amount of
histidine declines with time of ozone
exposure.
Oxidation of Tripeptides
To determine how the
location of histidine residues in the polypeptide chain affects its
susceptibility to oxidation by ozone and the kinds of products formed,
we investigated the oxidation of tripeptides containing two residues of
alanine (A) and a single residue of histidine (H) that was present in
either the C-terminal, middle, or N-terminal position. As shown in Fig. 3A (open symbols), the rate of histidine
loss was independent of its position in the tripeptide. However, the
fraction of modified histidine that was recovered as aspartic acid in
acid hydrolysates of the ozone-treated peptide varied depending upon
its location in the peptide. Thus, the yields of aspartic acid from
HAA, AAH, and AHA were 32, 88, and 88%, respectively. Moreover, for
each tripeptide, the fraction of histidine that was converted to
aspartic acid was constant throughout the time course of ozone exposure (Fig. 3B). These results confirm that ozone promotes
conversion of histidine residues to a derivative that appears as
aspartic acid following acid hydrolysis.
Conversion of Histidyl Residues to D-Aspartate
Racemization of L-aspartyl or L-asparaginyl residues occurs in mammalian proteins with the
content of the D-isomer increasing as a function of animal
age(36) . Since ozonolysis of histidyl residues of proteins and
of histidyl peptides gave rise to aspartate, we examined the
possibility that some of the aspartate formed might be the D-isomer. No D-aspartic acid was detected in BSA or
GS exposed to ozone for 10 min, except for the basal level due to acid
hydrolysis. Moreover, no D-aspartic acid was recovered in acid
hydrolysates of the ozone-treated AHA tripeptide. However, D-aspartic acid accounted for 8 and 17%, respectively, of the
aspartic acid derived from the ozone-treated HAA and AAH tripeptides.
The effect of time of exposure to ozone was examined for Ala-Ala-His.
Racemization was 17% regardless of the length of exposure (5-10
min).Effect of Ozone Treatment on GS Activity
Under our
conditions, when GS is exposed to a constant supply of ozone (545
nmol/min), the loss of catalytic activity and the number of histidine
residues lost are both linear functions of time, and complete loss of
activity is not obtained until 65% of the histidine residues (10
residues/subunit) have been modified (Fig. 5). This is in
contrast to the site-specific inactivation of GS by metal-catalyzed
oxidation systems where the loss of activity is associated with the
oxidation of only one histidine residue (His-269) that is present at
the catalytic site(37, 38) . It is, therefore, evident
that the histidine residue at the catalytic site is no more sensitive
to ozone oxidation than at least 10 other histidine residues in the
enzyme.
-glutamyltransferase activity (squares). The inset illustrates the relationship
between the loss of histidine residues and the loss of GS activity,
expressed as percentages of the amount originally
present.
Generation of Carbonyl Groups
When proteins are
exposed to ionizing radiation (39, 40, 41) or
to MCO systems(38, 42, 43) , the side chains
of some amino acids are converted to carbonyl derivatives. Ozone reacts
readily with compounds possessing ethylenic double bonds to form
ozonides, which upon decomposition may lead to carbon-carbon bond
cleavage yielding either two aldehydic or one aldehyde and a carboxyl
group. Aldehydes have, in fact, been identified among the proteolytic
degradation products of protein fractions from lungs of rabbits
following their exposure to ozone(44) . It is therefore not
surprising that carbonyl groups are generated during the exposure of
BSA and GS to ozone (Fig. 6). Under our conditions, the rate of
carbonyl group formation in BSA is 3 to 4 times faster than in GS.
After 10 min, the yield of carbonyl groups in BSA and GS was 11 and 3.3
eq per subunit, respectively. The sources of these carbonyl groups were
not determined. Ozone-induced generation of carbonyl groups in human
serum albumin was reported previously by Cross et al.(45) .
Generation of DOPA-like Derivatives
As illustrated
in Fig. 7, during exposure of GS and BSA to ozone there is a
time-dependent increase in the level of quinoprotein formation as
determined by the NBT assay. Both the rate and amplitude of
quinoprotein formation are greater with BSA than with GS.
Significantly, the level of quinoprotein reaches a maximum that
coincides with complete destruction of tyrosine, suggesting that the
quinoprotein formation involves conversion of tyrosine residues to DOPA
derivatives. The level of such derivatives corresponds to about 0.3 to
0.5 mol of quinoprotein per mol of GS and BSA, respectively. In fact,
there is an almost linear relationship between the loss of tyrosine
residues and the amount of quinoprotein formed (inset, Fig. 7).
C
are treated with ozone, the labeled histidine which is lost is almost
quantitatively (80%) recovered in acid hydrolysates as C-labeled aspartic acid. MCO system, the loss of activity is
correlated with the loss of a single histidine residue
(His-269)(36, 38) . Thus, the inactivation of GS by
ozone involves a more or less random attack of histidine residues;
His-269 is no more susceptible to ozone attack than at least 10 other
histidine residues in the molecule (and possibly even less so if
oxidation of any of the other residues causes any loss of catalytic
activity). MCO system His-269 is converted to
an asparaginyl residue, which upon acid hydrolysis would be converted
to aspartic acid. In the meantime, prompted by the report of Uchida et al.(47) that the histidine moiety of N-benzoylhistidine is converted to 2-oxohistidine by the
CuII/HO/MCO system, Levine and co-workers ()reinvestigated the metal-catalyzed oxidation of GS using
more advanced technology and have confirmed that the oxidation of
His-269 does indeed give rise to 2-oxohistidine, which under some
conditions can be converted to aspartic acid by acid hydrolysis.
Accordingly, we suspected that the oxidation of histidine residues by
ozone would also yield 2-oxohistidine as the primary product. Thus, if
2-oxohistidine is formed, it can only be a transitory intermediate that
is rapidly converted further to asparagine or aspartate.
))
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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