Subscriber access provided by UNIV OF CAMBRIDGE
Article
Degradation of Amino Acids and Structure in Model Proteins and Bacteriophage MS2 by Chlorine, Bromine and Ozone Jong Kwon Choe, David H. Richards, Corey J. Wilson, and William A. Mitch Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b03813 • Publication Date (Web): 21 Oct 2015 Downloaded from http://pubs.acs.org on October 22, 2015
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 28
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Environmental Science & Technology
Degradation of Amino Acids and Structure in Model Proteins and Bacteriophage MS2 by Chlorine, Bromine and Ozone Jong Kwon Choe1, David H. Richards2, Corey J. Wilson2, William A. Mitch1,* 1. Department of Civil and Environmental Engineering, Stanford University, Stanford, CA, 94305, United States 2. Department of Chemical and Environmental Engineering, Yale University, New Haven, CT, 06520, United States
*Corresponding Author: phone (650) 725-9298; fax (650) 723-7058; e-mail:
[email protected] 1 ACS Paragon Plus Environment
Environmental Science & Technology
17 18 19
ABSTRACT
20
disinfectant-protein reactions, this study characterized the disinfectant:protein molar ratios at
21
which 50% degradation of oxidizable amino acids (i.e., Met, Tyr, Trp, His, Lys) and structure
22
were observed during HOCl, HOBr, and O3 treatment of three well-characterized model proteins
23
and bacteriophage MS2. A critical question is the extent to which the targeting of amino acids is
24
driven by their disinfectant rate constants rather than their geometrical arrangement. Across the
25
model proteins and bacteriophage MS2 (coat protein), differing widely in structure, methionine
26
was preferentially targeted, forming predominantly methionine sulfoxide. This targeting concurs
27
with its high disinfectant rate constants and supports its hypothesized role as a sacrificial
28
antioxidant. Despite higher HOCl and HOBr rate constants with histidine and lysine than for
29
tyrosine, tyrosine generally was degraded in preference to histidine, and to a lesser extent, lysine.
30
These results concur with the prevalence of geometrical motifs featuring histidines or lysines
31
near tyrosines, facilitating histidine and lysine regeneration upon Cl[+1] transfer from their
32
chloramines to tyrosines. Lysine nitrile formation occurred at or above oxidant doses where 3,5-
33
dihalotyrosine products began to degrade. For O3, which lacks a similar oxidant transfer
34
pathway, histidine, tyrosine and lysine degradation followed their relative O3 rate constants.
35
Except for its low reactivity with lysine, the O3 doses required to degrade amino acids were as
36
low as or lower than for HOCl or HOBr, indicating its oxidative efficiency. Loss of structure did
37
not correlate with loss of particular amino acids, suggesting the need to characterize the
38
oxidation of specific geometric motifs to understand structural degradation.
39 40 41
INTRODUCTION
Proteins are important targets of chemical disinfectants. To improve the understanding of
2 ACS Paragon Plus Environment
Page 2 of 28
Page 3 of 28
42
Environmental Science & Technology
Chemical oxidants (e.g., hypochlorous acid (HOCl), hypobromous acid (HOBr), and
43
ozone (O3)) are commonly applied to drinking waters, wastewaters, and recreational waters to
44
inactivate pathogens. In addition, neutrophils and eosinophils produce HOCl and HOBr,
45
respectively, as part of the immune response to pathogens in the human body.1,2 While pathogen
46
inactivation kinetics have been characterized,3–11 the mechanistic understanding of how
47
disinfectant reactions with biomolecules within pathogens results in pathogen inactivation needs
48
improvement. Among pathogens, viruses represent a relatively simple case. First, compared to
49
the array of biomolecular targets in other pathogens, viruses consist predominantly of a genome
50
wrapped in capsid proteins. Second, outside of their hosts, viruses lack mechanisms to repair
51
oxidative damage.
52
Research has drawn different conclusions regarding the relative importance of oxidative
53
damage to the genome or capsid proteins for driving virus inactivation.4,8,12–17 Previous work
54
evaluated the relative importance of capsid and genome damage by HOCl, singlet oxygen,
55
chlorine dioxide and UV light (254 nm) for inactivation of MS2, fr, and GA phages, members of
56
the Leviviridae phage family, exhibiting a high degree of genome and protein sequence
57
similarity.4,8 For singlet oxygen and UV light, genomic damage was the predominant driver of
58
inactivation, resulting in replication inhibition. For chlorine dioxide, protein capsid damage
59
dominated, inhibiting phage binding to the host cell. For HOCl, damage to the genome and
60
capsid proteins both were important for inactivation.
61
Protein structure ultimately is governed by both the amino acid sequence (i.e., primary
62
structure) and side chain interactions (i.e., secondary and tertiary structures). Observed rate
63
constants near neutral pH for HOCl, HOBr and O3 reactions with protein constituents are higher
64
with a subset of amino acid side chains (“oxidizable amino acids”) than with peptide bonds
3 ACS Paragon Plus Environment
Environmental Science & Technology
65
(Table 1). Accordingly, protein exposure to disinfectants results in covalent modifications to
66
these side chains,10-18 with the associated alterations in side chain interactions potentially
67
modifying protein structure. Higher disinfectant exposures could cleave peptide bonds, resulting
68
in protein fragmentation.11
69
Previous research on oxidant-mediated protein damage has focused on kinetic models
70
employing disinfectant reaction rate constants with individual amino acids to predict those
71
initially targeted by oxidants.18–20 However, some studies have suggested the importance of the
72
three-dimensional arrangement of amino acids for determining their susceptibility to disinfectant
73
attack. First, while kinetic models combining HOCl rate constants largely matched experimental
74
results for parent amino acid loss in mixtures of free N-acetyl amino acids, they did not match
75
results for HOCl treatment of proteins.21 For example, for lysozyme treatment with a 25 molar
76
excess of HOCl, ~60% loss of lysine and tyrosine were observed with the protein, but only ~10%
77
loss was predicted by the kinetic model or observed with the same residues constituting
78
lysozyme as a N-acetyl amino acid mixture.21 Second, in free amino acid mixtures, lysine
79
inhibited the halogenation of tyrosine by scavenging HOCl or HOBr.22 However, previous
80
research hypothesized that when lysines are constrained to be located near tyrosines within
81
peptides, the lysine-ε-haloamines formed by HOCl or HOBr reactions with lysine side chains
82
serve as halogen transfer agents, promoting halogenation of proximal tyrosines (Scheme 1).21,23–
83
28
84
the model protein, adenylate kinase, promoted tyrosine bromination during HOBr treatment, but
85
reduced the tyrosine halogenation during HOCl treatment.22 Third, Lundeen and McNeill29
86
demonstrated that singlet oxygen oxidation of histidine increased with histidine’s proximity to
87
the surface of glyceraldehyde-3-phosphate dehydrogenase. Fourth, the finding that genomic and
Highlighting the potential complexities associated with three-dimensional structures, lysines in
4 ACS Paragon Plus Environment
Page 4 of 28
Page 5 of 28
Environmental Science & Technology
88
viral capsid damage were comparable for bacteriophage MS2 inactivation by HOCl despite
89
significantly higher rate constants for HOCl reaction with oxidizable amino acids than with
90
nucleotides also suggests the potential importance of the three-dimensional arrangement of
91
biomolecules within viruses.4 Lastly, singlet oxygen preferentially oxidized a specific
92
methionine (Met88) in MS2, but not in the structurally similar fr phage; the difference was
93
attributed to this residue’s greater solvent accessibility in MS2.8
94
The long-term goal of our research is to understand the importance of the three-
95
dimensional arrangement of amino acids for disinfectant-mediated protein damage to better
96
characterize pathogen deactivation mechanisms. However, characterization of site-specific
97
damage to proteins is complex. Generally, the process involves enzymatic cleavage (e.g., trypsin
98
digest) of proteins into ~5-20 residue oligomers and mass spectral characterization of parent
99
oligomer loss and daughter oligomer formation vs. oxidant exposure. While this problem is
100
tractable for disinfectants that react with high specificity with amino acids (e.g., singlet oxygen
101
with histidine29), application to disinfectants, such as HOCl, that react with an array of residues
102
is problematic. For a single 20-residue oligomer containing 4 residues oxidizable by HOCl to
103
unique products, the process would involve monitoring the loss of 1 parent oligomer and
104
production of 14 product oligomers. For the 129-residue MS2 coat protein, about 90 oligomers
105
would need to be tracked. Although previous research has indicated that amino acid geometry
106
can affect the reactivity of specific residues, the importance of these site-specific observations
107
relative to whole protein degradation is unclear. Because these geometrical effects likely are
108
highly variable across proteins, it is critical to understand their importance before engaging in a
109
labor-intensive, protein-by-protein characterization of protein degradation processes.
5 ACS Paragon Plus Environment
Environmental Science & Technology
110
The first objective of the research presented herein was to evaluate the extent to which
111
oxidizable residue degradation by HOCl, HOBr and O3 can be predicted by rate constants. A
112
range of oxidant doses was applied to adenylate kinase, lysozyme, ribose binding protein and
113
MS2; the first three proteins served as well-characterized model proteins. After disinfectant
114
treatment, proteins were digested to liberate individual amino acids. While amino acid location
115
within the protein was lost, the quantification of parent amino acids and oxidized daughter
116
products was facilitated. The results allowed us to evaluate whether the order with which amino
117
acids are targeted is common across proteins and predictable by rate constants, or whether the
118
geometrical arrangement of amino acids affects their susceptibility to oxidant reactions. A
119
second objective was to understand how the three oxidants differ in targeting amino acids. A
120
third objective was to evaluate whether structural loss is associated with degradation of specific
121
amino acids. A final objective was to measure the formation of oxidation products that have
122
previously been characterized in the literature as a function of oxidant exposure. Product
123
characterization is desirable because the interactions between these modified side chains and
124
nearby residues affect structure. Additionally, like other byproducts of disinfection,30,31 these
125
products may be associated with toxicity. While previous research has identified specific
126
oxidation products (e.g., 3-chlorotyrosine, methionine sulfoxide22,32), their production and
127
stability vs. disinfectant exposure in proteins generally has not been characterized.
128 129 130
MATERIALS AND METHODS The Supporting Information provides reagent sources.
131
Model proteins and MS2 virus preparation and purification: Lysozyme (LZ), adenylate
132
kinase (AdK), and ribose binding protein (RBP) served as model proteins because their 6 ACS Paragon Plus Environment
Page 6 of 28
Page 7 of 28
Environmental Science & Technology
133
structures are well-characterized (Table 2), including by X-ray crystallography, not because of
134
their relevance to pathogens. Lysozyme (chicken egg white; 90%) was purchased from Sigma-
135
Aldrich. Computational protein design (CPD) was employed to redesign ADK removing specific
136
oxidizable residues. The AdK, redesigned AdKs, and RBP production and purification
137
procedures, described previously,22,33 are summarized in the SI.
138
The MS2 capsid consists of 180 coat protein copies and 1 maturation protein. The coat
139
protein is well-characterized (Table 2). MS2 bacteriophage and Escherichia coli were obtained
140
from the American Type Culture Collection. MS2 was propagated and purified as described
141
previously,4 and summarized in the SI. Only a fraction of MS2 may be enumerated via plaque
142
assays. To estimate the molar ratio of applied oxidants to coat proteins, the MS2 coat protein
143
concentration in the stock solution was quantified by a different procedure. A stock solution
144
aliquot was treated by strong acid digestion to hydrolyze the proteins (see below), releasing free
145
amino acids. The free amino acid concentrations were quantified (see below) and divided by
146
their number in the MS2 coat protein (which dominates the protein content vs. the maturation
147
protein 180-fold) to estimate the MS2 coat protein concentration. The ratio of the concentrations
148
of amino acids measured to those expected based upon their prevalence within MS2 coat protein
149
was 1.0 (± 0.3 standard deviation) across six amino acids (glycine, alanine, lysine, methionine,
150
tyrosine and tryptophan), suggesting purity of the MS2 stock.
151
Protein oxidation. Model proteins and whole bacteriophage MS2 were treated with HOCl,
152
HOBr, or O3 in centrifuge tubes. While whole bacteriophage MS2 was treated, the results are
153
discussed based upon the oxidant ratio relative to coat protein, because the coat protein is in 180-
154
fold excess relative to maturation protein. Preparation and standardization of oxidant stock
155
solutions is described in the SI. Briefly, O3 stocks were prepared by sparging O3 generated from
7 ACS Paragon Plus Environment
Environmental Science & Technology
156
oxygen gas through chilled, deionized water. For HOCl and HOBr, 30 µM proteins were dosed
157
with 0-10,800 µM HOCl or HOBr (oxidant:protein molar ratios of 0-360) at pH 7.4. For
158
ozonation, 15 µM of proteins were dosed with 0-540 µM O3 (ozone:protein molar ratios of 0-36)
159
at pH 7.4; the O3 dose was limited by the ability to generate concentrated O3 stocks. While these
160
absolute oxidant concentrations exceed those applied for water disinfection, our purpose here
161
was to evaluate amino acid transformations with respect to oxidant:protein molar ratio. While we
162
employed high protein concentrations here, high oxidant:protein molar ratios are expected in
163
natural waters because of their low pathogen concentrations. After 24 h, residual oxidants were
164
quenched with glutathione at 1.2 times the molar concentration of oxidants applied. Glutathione
165
was selected because it did not interfere with the amino acid analysis and because initial
166
experiments demonstrated that it does not reduce amino acid oxidation products (e.g.,
167
methionine sulfoxide).
168
Protein digestion and analysis. Free amino acids were liberated from proteins by acid-catalyzed
169
hydrolysis (strong acid digestion) using methanesulfonic acid.34 However, because strong acid
170
digestion can destroy some amino acid oxidation products, an enzymatic digestion procedure
171
was used to quantify these products35,36. Enzymatic digestion was accomplished using Pronase E
172
(Protease type XIV; Sigma-Aldrich), an enzyme cocktail derived from Streptomyces griseus.
173
Details are provided in the SI. Liberated amino acids were derivatized using 6-aminoquinolyl-N-
174
hydroxysuccinimidyl carbamate (AQC, Chemodex Ltd., Switzerland, 95%),37 and analyzed by
175
HPLC-MS. Protein structural decay upon oxidative challenge was assessed by collecting Far-UV
176
circular dichroism spectra. The SI provides additional details.
177 178
RESULTS AND DISCUSSION
8 ACS Paragon Plus Environment
Page 8 of 28
Page 9 of 28
179
Environmental Science & Technology
Protein characteristics. Oxidizable residues, featuring HOCl rate constants at least as
180
high as tyrosine (and higher than peptide bonds), include methionine, cysteine, histidine,
181
tryptophan, lysine and tyrosine (Table 1). Table 2 provides the numbers of each oxidizable
182
residue, and other structural data for the model proteins and MS2 coat protein. The total number
183
of oxidizable residues per protein was fairly similar (30-35) across the model proteins, but the
184
MS2 coat protein featured only 16 oxidizable residues. The fraction of total residues that were
185
oxidizable ranged from 12.4% (MS2 coat protein) to 23.3% (LZ). Lysine generally was the most
186
prevalent oxidizable residue, and tryptophan, histidine and cysteine (except LZ) were the least
187
prevalent. Except for cysteine, the majority of oxidizable residues in the model proteins were
188
solvent-accessible, defined as having side chains with >10 Å2 surface exposure (as calculated by
189
GETAREA software38); solvent-accessibility was not calculated for MS2 coat protein because
190
the GETAREA software does not consider accessibility restrictions related to interfaces with
191
other coat proteins or the genome. Although each protein contained α-helices and β-sheets, α-
192
helices were more prevalent in the model proteins, while β-sheets were more prevalent in the
193
MS2 coat protein.
194
Targeting of oxidizable amino acids. Each protein was exposed to an array of
195
disinfectant:protein molar ratios in triplicate. For MS2, the entire virus was treated with
196
disinfectant, but the oxidant:protein molar ratio was calculated using the measured total protein
197
concentration, considering that the coat protein concentration exceeded the maturation protein
198
concentration 180-fold. Although the lower molar ratios where structural damage occurred were
199
preferentially sampled, higher molar ratios were evaluated because, even after pathogen
200
inactivation, protein detritus can contribute to disinfection byproduct formation. Figures 1 (LZ),
201
2 (MS2), S2 (RBP) and S3 (AdK) provide the loss of 5 of the 6 oxidizable amino acids in each
9 ACS Paragon Plus Environment
Environmental Science & Technology
202
protein vs. oxidant:protein molar ratio. Loss of the other oxidizable amino acid, cysteine, could
203
not be quantified because it oxidizes during digestion. We confirmed that there was insignificant
204
loss of other amino acids (e.g., alanine). The oxidant dosage where 50% loss of each oxidizable
205
amino acid was observed (denoted as AA50 (e.g., Met50)) was used as the principle metric for its
206
oxidation susceptibility. Residue losses generally followed sigmoidal relationships with oxidant
207
dosage. The AA50 values were determined via linear regression for 3-4 experimental values
208
surrounding the 50% value (Table 3; Figures 1, 2, S2, S3). The error represents the 95%
209
confidence interval from this regression. The relative error increased for high AA50 values,
210
partially due to the wider spacing of oxidant:protein molar ratios evaluated; regardless, for these
211
scenarios, these amino acids were poorly targeted by the oxidants. Comparisons between AA50
212
values were reported only if significant at the p < 0.05 level based on Welch’s t-test.
213
For HOCl treatment, the AA50 values generally followed the order: Met50 < Tyr50 ≤ Lys50
214
< His50, indicating that methionine was the most reactive with HOCl. For MS2 coat protein, and
215
to a lesser extent RBP, Lys50 > Tyr50. Only LZ and MS2 coat protein contained tryptophan
216
(Table 2); for both proteins Trp50 was comparable to Tyr50. For LZ, oxidizable residue loss was
217
similar to that observed previously for HOCl:LZ molar ratios 0-25.21 Based on observed HOCl
218
rate constants with N-acetylated amino acids at pH 7.4, the order of reactivity was Met > His >
219
Trp ≈ Lys > Tyr, and the rate constants varied by 6 orders of magnitude (Table 1). For
220
methionine, tryptophan and lysine (for AdK and LZ), the similarity between the order in which
221
amino acids degraded and their HOCl rate constants across four proteins varying in structure
222
suggests that relative reactivity with HOCl is more important than geometry for driving
223
degradation.
10 ACS Paragon Plus Environment
Page 10 of 28
Page 11 of 28
224
Environmental Science & Technology
However, the results for histidine, tyrosine, and lysine (for MS2 coat protein and to a
225
lesser degree, RBP) suggest the potential importance of three-dimensional structure. Although
226
their rate constants indicate that HOCl is 3 and 2 orders of magnitude more reactive with
227
histidine and lysine than tyrosine, respectively, tyrosine was degraded prior to histidine in all
228
model proteins (MS2 does not contain histidine) and prior to lysine for MS2 coat protein and
229
RBP. Additionally, the low reactivity of these histidines and lysines occurs even though all of the
230
histidines in the model proteins, and lysines in MS2 coat protein and RBP are surface-accessible
231
(Table 2). Note that the HOCl rate constants with the amino acids (Table 1) relate to HOCl’s
232
initial attack on the amino acid. Histidine and lysine would form N-chloramines.23,39 Previous
233
research suggested that these N-chloramines could promote tyrosine chlorination by transferring
234
Cl[+1] to tyrosine, regenerating the parent amino acid21,23,35 (Scheme 1). For HOCl-treated AdK,
235
we found that lysine chloramines formed lysine nitrile by hydrochloric acid eliminations.22 By
236
scavenging HOCl, lysine thereby partially protected tyrosine from chlorination, and, like
237
methionine, served as an antioxidant. Although chlorine transfer from histidine N-chloramines to
238
tyrosine has been hypothesized21,39, it has not been evaluated experimentally in proteins. If
239
chlorine transfer is important, histidine or lysine degradation may not be observed until
240
degradation of proximal tyrosines prevents this transfer (and histidine or lysine regeneration).
241
For AdK and LZ, loss of lysine and tyrosine and lysine nitrile formation occurred over similar
242
oxidant dosages, suggesting only partial protection of tyrosine by lysine’s antioxidant activity.
243
In contrast, for all model proteins, histidine loss occurred predominantly at HOCl:protein molar
244
ratios above where significant tyrosine transformation to 3,5-dichlorotyrosine occurred. For
245
MS2 coat protein, which does not contain histidines, and to a lesser degree for RBP, tyrosine loss
246
preceded lysine loss, suggesting that Cl[+1] transfer from lysine to tyrosine may be important.
11 ACS Paragon Plus Environment
Environmental Science & Technology
247
Additional support for Cl[+1] transfer between histidine/lysine and tyrosine is provided
248
by the protein crystal structures. AdK’s two histidines and LZ’s single histidine are located
249
within 15 angstroms of tyrosines (Table S2). For comparison, single bond lengths are ~2
250
angstroms. Thus, these histidine N-chloramines would be well-positioned to transfer Cl[+1] to
251
tyrosines, protecting histidine at the expense of tyrosine such that His50 > Tyr50. In contrast, only
252
4 of AdK’s 14 lysines and none of LZ’s 6 lysines are near tyrosines, inhibiting Cl[+1] transfer to
253
tyrosines such that Lys50 ≈ Tyr50.
254
None of RBP’s histidines are located near tyrosines, although all are located near other
255
oxidizable residues (i.e., lysine or methionine), potentially enabling histidine preservation by
256
Cl[+1] transfer from histidine N-chloramines. While His50 > Tyr50, the difference was less than
257
for AdK and LZ. Lysine promotion of tyrosine chlorination may be possible, because RBP’s 3
258
tyrosines are located near lysines. However, with only 3 of the 24 lysines adjacent to tyrosines, it
259
would be difficult to validate this hypothesis based upon a decrease in lysine degradation (due to
260
its regeneration via Cl[+1] transfer). While MS2 coat protein does not contain histidine, 3 of the
261
6 lysines are located near tyrosines. The fact that Lys50 was significantly higher than Tyr50
262
suggests that the proximity of lysines and tyrosines protects lysine by Cl[+1] transfer to tyrosine.
263
For HOBr treatment, the order of oxidizable amino acid degradation was Met ≈ Trp ≈ Tyr
264
≈ His > Lys across the model proteins (Table 3). The order was similar for MS2 coat protein,
265
although the Met50 value determined for the two Met residues was very uncertain. A rapid ~30-
266
45% loss in Met over the first 4 molar equivalents of HOBr was followed by much slower
267
(~20%) additional loss over 72 HOBr molar equivalents (Figure 2), suggesting that one Met
268
residue was degraded more readily than the other. The observed HOBr reaction rate constants
269
with the oxidizable amino acids at pH 7.4 were in the order Trp ~ Met ~ His > Lys ~ Tyr, but
12 ACS Paragon Plus Environment
Page 12 of 28
Page 13 of 28
Environmental Science & Technology
270
their range was only ~1 order of magnitude (Table 1).20 The similarities between the AA50 values
271
likely reflects the low range in rate constants and again suggests the predominant effect of HOBr
272
rate constants over geometry for determining residue oxidation. However, although the HOBr
273
rate constant with histidine is ten-fold higher than with lysine or tyrosine, tyrosines were
274
degraded as readily as histidines and more readily than lysines. As for HOCl, these results may
275
indicate the importance of histidine-tyrosine or lysine-tyrosine interactions associated with their
276
proximity within protein structures. In previous research with AdK, lysine promoted tyrosine
277
bromination.22 Five of AdK’s 9 tyrosines are proximal to lysines and an additional 2 are near
278
histidines (Table S2).
279
For O3 treatment, the AA50 values were comparable for methionine, tryptophan, histidine
280
and tyrosine, while lysine was unreactive (Table 3). Compared to HOCl and HOBr, O3 exhibited
281
the lowest percentage differences in AA50 values for different amino acids (excluding lysine).
282
For instance, for RBP, the ratios between the highest and lowest AA50 values were 9.3 for HOCl
283
(His50:Met50), 5.2 for HOBr (Lys50:Met50), but only 2.0 for O3 (Tyr50:Met50). Again, these results
284
suggest the importance of O3 rate constants with the amino acids. These rate constants differed
285
less than 3-fold among the oxidizable amino acids, except lysine, which was 4 orders of
286
magnitude less reactive (Table 1). Except for lysine, AA50 values for O3 generally were as low as
287
or lower than those for HOCl or HOBr, suggesting a high efficiency for amino acid degradation
288
by O3. In contrast to HOCl and HOBr, tyrosine degradation was not enhanced, and histidine
289
degradation was not hindered relative to the reactivities predicted by reaction rate constants. A
290
reaction analogous to Cl[+1] transfer from histidine N-chloramines to tyrosine does not exist for
291
O3.
13 ACS Paragon Plus Environment
Environmental Science & Technology
292
Product formation. Methionine was preferentially targeted by all three oxidants.
293
Oxidation produced first methionine sulfoxide at yields ranging from ~15% (e.g., HOBr
294
treatment of LZ or O3 treatment of AdK) to nearly stoichiometric (e.g, all oxidants with RBP or
295
O3 with MS2 coat protein) over oxidant:protein molar ratios up through ~24 (Figures 1, 2, S1
296
(for product structures), S2, and S3). At higher oxidant:protein molar ratios, methionine
297
sulfoxide concentrations leveled off or declined. Less than stoichiometric yields suggest that
298
other, uncharacterized products of methionine form, or that methionine sulfoxide is further
299
oxidized to other products. At the highest oxidant:protein molar ratios (i.e., 48-360) for HOBr
300
and particularly for HOCl, traces of methionine sulfone were detected, likely due to oxidation of
301
methionine sulfoxide. Due to the difficulty of generating concentrated O3 stocks, O3:protein
302
molar ratios > 36 were not evaluated, so methionine sulfone formation during ozonation could
303
not be confirmed. With Met50 generally lower than the AA50 values for the other oxidizable
304
residues, oxidant scavenging associated with methionine oxidation to methionine sulfoxide or
305
methionine sulfone indicates that methionine serves as a sacrificial antioxidant.
306
Tyrosine degradation occurred at higher oxidant dosages than methionine. For HOCl or
307
HOBr, low yields ( AdK_H-free (48.0 °C) > AdK_Kfree (44.5 °C) > AdK_Mfree (40.5 °C) > AdK_Yfree (31.3
351
°C) (Table S3). The replacement of tyrosines resulted in by far the greatest loss in protein
352
stability even though there were more lysines than tyrosines. While these results might suggest a
353
global importance for tyrosines, one alternative is that interactions of only a few of the tyrosines
354
are of paramount importance to AdK’s structural stability. To test the importance of individual
355
tyrosines, computational design could be used to replace individual tyrosines. However, this
356
approach is highly labor-intensive and the results may be specific only to AdK, a situation
357
analogous to the use of high-resolution mass spectrometry to characterize oxidative
358
transformations of the oligopeptides generated by single enzyme digestion of proteins.
359 360
As an alternative, we sought first to further evaluate the hypothesis that particular oxidizable amino acids (e.g., tyrosines) were globally important for protein structural stability
16 ACS Paragon Plus Environment
Page 16 of 28
Page 17 of 28
Environmental Science & Technology
361
across different proteins. The oxidant dosage associated with 50% loss of the far-UV circular
362
dichroism signal at 222 nm (CD50) was used as the primary metric of the loss of structure via
363
unfolding, fragmentation, and aggregation. CD50 values were determined for each of the model
364
proteins for treatment with each of the three oxidants (Figure S4 and Table 3); no CD signal was
365
attainable for intact bacteriophage MS2. If loss of a particular amino acid drives structural loss,
366
its AA50 value should correlate with the CD50 value.
367
No such broad correlations were observed. For all three oxidants, AdK was the most
368
susceptible to structural loss (i.e., low CD50), even though its oxidizable amino acids were the
369
most resistant to oxidative transformation (e.g., highest AA50 values). Indeed, AdK structural
370
loss preceded loss of even methionine (i.e., CD50 < Met50). For AdK, the CD50 values (3.1-5.8)
371
suggest that oxidative damage to fewer than 6 of the 35 oxidizable amino acids can drive
372
structural damage. While the melting temperature data suggest that oxidative transformation of a
373
particular tyrosine may be critical for structural loss, testing this hypothesis would require higher
374
resolution evaluations of each tyrosine.
375
Implications: Oxidative degradation of enzymatic function or structure (e.g., viral capsids) can
376
play an important role in pathogen inactivation during disinfection. The alteration in amino acid
377
side chain interactions resulting from oxidative modifications ultimately are believed to be
378
responsible for degradation of protein structure and function. While protein oxidative
379
transformations would appear to require a characterization of the three-dimensional geometrical
380
interactions among amino acids, previous research had suggested that protein oxidation could be
381
understood largely by the relative reactivity of amino acids with disinfectants. Due to the
382
complexity involved in accounting for geometry, this research sought to first evaluate whether
383
there are trends observable across proteins that trump specific geometries.
17 ACS Paragon Plus Environment
Environmental Science & Technology
384
The results were mixed. Across all three model proteins and MS2 coat protein, many
385
oxidizable amino acids were degraded in the order predictable based upon their relative
386
reactivity with disinfectants (e.g., methionine was degraded first for all disinfectants, and lysine
387
was hardly degraded by ozone). However, even without high resolution evaluations of specific
388
geometries, the potential importance of geometry was indicated for Cl[+1] transfer between
389
histidine and lysine chloramines and tyrosines, likely because of the prevalence of geometrical
390
motifs featuring these residues in close proximity. The methionine data provide additional
391
suggestions for the importance of geometry. Although methionines were preferentially degraded
392
on average (i.e., lowest AA50 values), certain methionines resisted oxidative damage, perhaps
393
because they were less oxidant-accessible. For example, although half of LZ’s 6 methionines
394
were degraded at a 5.2 HOCl:protein molar ratio, one of the methionines appeared to persist at
395
up to 180 HOCl:LZ (i.e., 5 µM out of an initial 30 µM methionine in Figure 1), a dose far above
396
the other AA50 values.
397
In contrast, our inability to detect similar broad trends linking oxidation of specific
398
residues with structural damage suggests that individual geometries may play a dominant role in
399
driving structural damage. Although not evaluated here, general trends related to loss of
400
enzymatic activity are even less likely be observed across proteins, because the active site
401
constitutes only a subset of the protein.
402
Findings from this study suggest that a detailed characterization of geometrical effects
403
involving high-resolution methods likely is needed to fully characterize oxidative protein
404
damage. This process will require proceeding protein-by-protein. However, to interpret the data
405
it would be helpful to use model oligopeptides to understand the interactions of oxidizable
406
residues within specific geometrical motifs. Previous work using model oligopeptides
18 ACS Paragon Plus Environment
Page 18 of 28
Page 19 of 28
Environmental Science & Technology
407
demonstrated the potential for lysine chloramines to promote tyrosine chlorination by Cl[+1]
408
transfer, particularly when lysine was separated from tyrosine by two residues (i.e., Y-x-x-K)
409
within α-helical arrangements due to their geometric proximity.23 Work is ongoing in our
410
laboratory to validate the effect of geometry on histidine-tyrosine interactions using
411
oligopeptides, and similar work is needed for other geometries and oxidizable amino acid
412
combinations. Additionally, the implications of the final transformation products on protein
413
structure must be understood. For example, how, if at all, does the 3-chlorotyrosine product
414
formed within Y-x-x-K motifs alter the structure of α-helices? Characterizing such mechanisms
415
will contribute to our understanding of how certain pathogens may be more resistant to
416
inactivation by disinfectants.
417 418
ASSOCIATED CONTENT
419
Supporting Information
420
Chemical reagents and additional analytical details, distance between oxidizable amino
421
acids in studied proteins, and chemical structures of oxidized amino acid products are provided.
422
This information is available free of charge via the Internet at http://pubs.acs.org/.
423 424
ACKNOWLEDGEMENT
425
This work was supported by NSF Chemical, Bioengineering, Environmental, and Transport
426
Systems (CBET#1066526). Niveen Ismail is acknowledged for help with the MS2 plaque assay.
427
We also thank Ofelia Romero for insightful discussion and technical feedback.
428 429
References 19 ACS Paragon Plus Environment
Environmental Science & Technology
430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474
(1) (2) (3)
(4)
(5) (6)
(7)
(8)
(9) (10)
(11) (12) (13) (14) (15) (16) (17)
(18) (19)
Weiss, S. J. Tissue Destruction by Neutrophils. N. Engl. J. Med. 1989, 320 (6), 365–376. Weiss, S. J.; Test, S. T.; Eckmann, C. M.; Roos, D.; Regiani, S. Brominating oxidants generated by human eosinophils. Science 1986, 234 (4773), 200–203. Sattar, S. A.; Raphael, R. A.; Lochnan, H.; Springthorpe, V. S. Rotavirus inactivation by chemical disinfectants and antiseptics used in hospitals. Can. J. Microbiol. 1983, 29 (10), 1464–1469. Wigginton, K. R.; Pecson, B. M.; Sigstam, T.; Bosshard, F.; Kohn, T. Virus Inactivation Mechanisms: Impact of Disinfectants on Virus Function and Structural Integrity. Environ. Sci. Technol. 2012, 46 (21), 12069–12078. Katzenelson, E.; Kletter, B.; Shuval, H. I. Inactivation Kinetics of Viruses and Bacteria in Water by Use of Ozone. J. Am. Water Works Assoc. 1974, 66 (12), 725–729. Sobsey, M. D.; Fuji, T.; Shields, P. A. Inactivation of Hepatitis A Virus and Model Viruses in Water by Free Chlorine and Monochloramine. Water Sci. Technol. 2011, 20, 385-391. Kahler, A. M.; Cromeans, T. L.; Roberts, J. M.; Hill, V. R. Effects of source water quality on chlorine inactivation of adenovirus, coxsackievirus, echovirus, and murine norovirus. Appl. Environ. Microbiol. 2010, 76 (15), 5159–5164. Sigstam, T.; Gannon, G.; Cascella, M.; Pecson, B. M.; Wigginton, K. R.; Kohn, T. Subtle Differences in Virus Composition Affect Disinfection Kinetics and Mechanisms. Appl. Environ. Microbiol. 2013, 79 (11), 3455–3467. Luh, J.; Mariñas, B. J. Inactivation of Mycobacterium avium with Free Chlorine. Environ. Sci. Technol. 2007, 41 (14), 5096–5102. Rennecker, J. L.; Kim, J.-H.; Corona-Vasquez, B.; Marinas, B. J. Role of Disinfectant Concentration and pH in the Inactivation Kinetics of Cryptosporidium parvum Oocysts with Ozone and Monochloramine. Environ. Sci. Technol. 2001, 35 (13), 2752–2757. U.S. Environmental Protection Agency. EPA Guidance Manual: Disinfection Profiling and Benchmarking; 1999. O’Brien, R. T.; Newman, J. Structural and compositional changes associated with chlorine inactivation of polioviruses. Appl. Environ. Microbiol. 1979, 38 (6), 1034–1039. Li, J. W.; Xin, Z. T.; Wang, X. W.; Zheng, J. L.; Chao, F. H. Mechanisms of Inactivation of Hepatitis A Virus by Chlorine. Appl. Environ. Microbiol. 2002, 68 (10), 4951–4955. Kim, C. K.; Gentile, D. M.; Sproul, O. J. Mechanism of Ozone Inactivation of Bacteriophage f2. Appl. Environ. Microbiol. 1980, 39 (1), 210–218. Roy, D.; Wong, P. K.; Engelbrecht, R. S.; Chian, E. S. Mechanism of enteroviral inactivation by ozone. Appl. Environ. Microbiol. 1981, 41 (3), 718–723. Page, M. A.; Shisler, J. L.; Mariñas, B. J. Mechanistic Aspects of Adenovirus Serotype 2 Inactivation with Free Chlorine. Appl. Environ. Microbiol. 2010, 76 (9), 2946–2954. Gall, A. M.; Shisler, J. L.; Mariñas, B. J. Analysis of the Viral Replication Cycle of Adenovirus Serotype 2 after Inactivation by Free Chlorine. Environ. Sci. Technol. 2015, 49 (7), 4584–4590. Hawkins, C. L.; Pattison, D. I.; Davies, M. J. Hypochlorite-induced oxidation of amino acids, peptides and proteins. Amino Acids 2003, 25 (3-4), 259–274. Pattison, D. I.; Davies, M. J. Absolute Rate Constants for the Reaction of Hypochlorous Acid with Protein Side Chains and Peptide Bonds. Chem. Res. Toxicol. 2001, 14 (10), 1453–1464.
20 ACS Paragon Plus Environment
Page 20 of 28
Page 21 of 28
475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520
Environmental Science & Technology
(20) Pattison, D. I.; Davies, M. J. Kinetic Analysis of the Reactions of Hypobromous Acid with Protein Components: Implications for Cellular Damage and Use of 3-Bromotyrosine as a Marker of Oxidative Stress. Biochemistry 2004, 43 (16), 4799–4809. (21) Pattison, D. I.; Hawkins, C. L.; Davies, M. J. Hypochlorous Acid-Mediated Protein Oxidation: How Important Are Chloramine Transfer Reactions and Protein Tertiary Structure? Biochemistry 2007, 46 (34), 9853–9864. (22) Sivey, J. D.; Howell, S. C.; Bean, D. J.; McCurry, D. L.; Mitch, W. A.; Wilson, C. J. Role of Lysine during Protein Modification by HOCl and HOBr: Halogen-Transfer Agent or Sacrificial Antioxidant? Biochemistry 2013, 52 (7), 1260–1271. (23) Bergt, C.; Fu, X.; Huq, N. P.; Kao, J.; Heinecke, J. W. Lysine Residues Direct the Chlorination of Tyrosines in YXXK Motifs of Apolipoprotein A-I When Hypochlorous Acid Oxidizes High Density Lipoprotein. J. Biol. Chem. 2004, 279 (9), 7856–7866. (24) Nightingale, Z. D.; Lancha Jr., A. H.; Handelman, S. K.; Dolnikowski, G. G.; Busse, S. C.; Dratz, E. A.; Blumberg, J. B.; Handelman, G. J. Relative reactivity of lysine and other peptide-bound amino acids to oxidation by hypochlorite. Free Radic. Biol. Med. 2000, 29 (5), 425–433. (25) Wu, W.; Chen, Y.; d’ Avignon, A.; Hazen, S. L. 3-Bromotyrosine and 3,5Dibromotyrosine Are Major Products of Protein Oxidation by Eosinophil Peroxidase: Potential Markers for Eosinophil-Dependent Tissue Injury in Vivo. Biochemistry 1999, 38 (12), 3538–3548. (26) Kang, J., Joseph I.; Neidigh, J. W. Hypochlorous Acid Damages Histone Proteins Forming 3-Chlorotyrosine and 3,5-Dichlorotyrosine. Chem. Res. Toxicol. 2008, 21 (5), 1028–1038. (27) Curtis, M. P.; Hicks, A. J.; Neidigh, J. W. Kinetics of 3-Chlorotyrosine Formation and Loss due to Hypochlorous Acid and Chloramines. Chem. Res. Toxicol. 2011, 24 (3), 418– 428. (28) Shah, A. D.; Mitch, W. A. Halonitroalkanes, Halonitriles, Haloamides, and NNitrosamines: A Critical Review of Nitrogenous Disinfection Byproduct Formation Pathways. Environ. Sci. Technol. 2012, 46 (1), 119–131. (29) Lundeen, R. A.; McNeill, K. Reactivity Differences of Combined and Free Amino Acids: Quantifying the Relationship between Three-Dimensional Protein Structure and Singlet Oxygen Reaction Rates. Environ. Sci. Technol. 2013, 47 (24), 14215–14223. (30) Plewa, M. J.; Muellner, M. G.; Richardson, S. D.; Fasano, F.; Buettner, K. M.; Woo, Y.T.; McKague, A. B.; Wagner, E. D. Occurrence, Synthesis, and Mammalian Cell Cytotoxicity and Genotoxicity of Haloacetamides: An Emerging Class of Nitrogenous Drinking Water Disinfection Byproducts. Environ. Sci. Technol. 2008, 42 (3), 955–961. (31) Plewa, M. J.; Wagner, E. D.; Jazwierska, P.; Richardson, S. D.; Chen, P. H.; McKague, A. B. Halonitromethane Drinking Water Disinfection Byproducts: Chemical Characterization and Mammalian Cell Cytotoxicity and Genotoxicity. Environ. Sci. Technol. 2004, 38 (1), 62–68. (32) Sharma, V. K.; Graham, N. J. D. Oxidation of Amino Acids, Peptides and Proteins by Ozone: A Review. Ozone Sci. Eng. 2010, 32 (2), 81–90. (33) Howell, S. C.; Inampudi, K. K.; Bean, D. P.; Wilson, C.J. Understanding thermal adaptation of enzymes through the multistate rational design and stability prediction of 100 adenylate kinases, Structure 2014, 22, 218-229. (34) Fountoulakis, M.; Lahm, H.-W. Hydrolysis and amino acid composition analysis of proteins. J. Chromatogr. A 1998, 826 (2), 109–134. 21 ACS Paragon Plus Environment
Environmental Science & Technology
521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542
(35) Walse, S. S.; Plewa, M. J.; Mitch, W. A. Exploring Amino Acid Side Chain Decomposition Using Enzymatic Digestion and HPLC-MS: Combined Lysine Transformations in Chlorinated Waters. Anal. Chem. 2009, 81 (18), 7650–7659. (36) Brisbane, P. G.; Amato, M.; Ladd, J. N. Gas chromatographic analysis of amino acids from the action of proteolytic enzymes on soil humic acids. Soil Biol. Biochem. 1972, 4 (1), 51–61. (37) Cohen, S. A.; Michaud, D. P. Synthesis of a Fluorescent Derivatizing Reagent, 6Aminoquinolyl-N-Hydroxysuccinimidyl Carbamate, and Its Application for the Analysis of Hydrolysate Amino Acids via High-Performance Liquid Chromatography. Anal. Biochem. 1993, 211 (2), 279–287. (38) Fraczkiewicz, R.; Braun, W. Exact and efficient analytical calculation of the accessible surface areas and their gradients for macromolecules. J. Comput. Chem. 1998, 19 (3), 319–333. (39) Pattison, D. I.; Davies, M. J. Kinetic Analysis of the Role of Histidine Chloramines in Hypochlorous Acid Mediated Protein Oxidation. Biochemistry 2005, 44 (19), 7378–7387. (40) Hazen, S. L.; Heinecke, J. W. 3-Chlorotyrosine, a specific marker of myeloperoxidasecatalyzed oxidation, is markedly elevated in low density lipoprotein isolated from human atherosclerotic intima. J. Clin. Invest. 1997, 99 (9), 2075–2081. (41) Gallard, H.; von Gunten, U. Chlorination of Phenols: Kinetics and Formation of Chloroform. Environ. Sci. Technol. 2002, 36 (5), 884–890.
22 ACS Paragon Plus Environment
Page 22 of 28
Page 23 of 28
543
Environmental Science & Technology
Scheme 1. Transfer of Cl[+1] from histidine or lysine chloramines to tyrosine.
544 545
23 ACS Paragon Plus Environment
Environmental Science & Technology
546 547 548 549
550 551 552 553 554 555 556 557 558
Table 1. Literature values for observed second order reaction rate constants (M-1s-1) for oxidizable residues with chemical oxidants at pH 7.4. HOCl HOBr O3 Met 3.8 x 107 3.6 x 106 4.0 x 106 Cys 3.0 x 107 1.2 x 107 4.4 x 109 His 1.0 x 105 3.0 x 106 5.3 x 106 Trp 1.1 x 104 3.7 x 106 7.0 x 106 3 5 Lys 5.0 x 10 2.9 x 10 5.2 x 102 Tyr 4.4 x 101 2.3 x 105 2.8 x 106 -3 1 3 Peptide Bond 10 -10 10 6.0 x 10-1 For HOCl and HOBr, rate constants were measured at pH 7.419,20; for O3, rate constants were calculated for pH 7.4 based upon the speciation of the amino acids at pH 7.4 and literature rate constants measured for the different amino acid species.45
Table 2. Summary of oxidizable residues and secondary structural characteristics for lysozyme (LZ; PDB entry 1LYZ), adenylate kinase (AdK; PDB entry 1P3J), ribose binding protein (RBP; 2DRI), and MS2 bacteriophage coat protein (PDB entry 1MSC).
LZ
ADK
RBP
MS2
Met Tyr His
Number of residues (Number of solvent-accessible residues) 6 (6) 6 (5) 4 (2) 3 (3) 9 (8) 3 (3) 1 (1) 2 (2) 3 (3)
2 4 0
Lys
6 (6)
14 (14)
24 (24)
6
Trp
6 (5)
0 (0)
0 (0)
2
Cys 8 (1) Oxidizable AA 30 (22) Total AA 129
559 560 561
Page 24 of 28
α-helix β-sheet Τm
29% 10% 74 ˚C
4 (1) 0 (0) 35 (30) 34 (32) 212 271 Percentage of secondary structure 47% 45% 16% 22% 51 ˚C 58 ˚C
24 ACS Paragon Plus Environment
2 16 129 17% 34% ___
Page 25 of 28
562 563
Environmental Science & Technology
Table 3. Oxidant:protein molar ratios for 50% loss of oxidizable residues (Tyr50, Met50, Lys50, His50, Trp50) and structure (CD50) for lysozyme (LZ), adenylate kinase (ADK), ribose binding protein (RBP), and MS2 coat proteins.
564 565 566
a
Met50 value for HOBr treatment of MS2 coat proteins was between 4-72 molar equivalents. The error value represents the 95% confidence value.
b
25 ACS Paragon Plus Environment
Environmental Science & Technology
Page 26 of 28
567 568 569 570 HOCl Tyrosine
HOBr 3,5-DichloroTyrosine
Tyrosine
50
25
40 30 20 10
40 30 20 10
0
15 10 5
2
0
2 4 8 12 16 24 36 72 96 180 360 Molar Equivalents of Oxidant Molar Ratio of Oxidant:Protein Methionine Methionine Sulfoxide Methionine Sulfone
8 12 16 24 48 72 96 180 360 Molar Equivalents Ratio of Oxidant:Protein Molar of Oxidant Methionine Methionine Sulfoxide Methionine Sulfone
4
4
35
18
30
30
15
25 20 15 10
25 20 15 10
5
5
0
0 2
Concentration, µ M
35 Concentration, µ M
Concentration, µ M
8 12 16 24 48 72 96 180 360 Molar Ratio Equivalents of Oxidant Molar of Oxidant:Protein Lysine Lysine Nitrile Histidine Tryptophan
12 9 6 3 0
0
4
2
4
Lysine
0
8 12 16 24 48 72 96 180 360 Molar of Oxidant Molar Equivalents Ratio of Oxidant:Protein Lysine Nitrile
Histidine
Tryptophan 50
80
80
40
40 20 0
Concentration, µ M
100
60
60 40 20
2
4
8 12 16 24 48 72 96 180 360 Equivalents of Oxidant Molar Ratio of Oxidant:Protein
4
8 12 16 24 36 72 96 180 360 Molar Equivalents of Oxidant Molar Ratio of Oxidant:Protein Lysine Nitrile
Histidine
Tryptophan
30 20 10 0
0 0
2
Lysine
100
Concentration, µ M
Concentration, µ M
2
20
0
0
0
572 573 574
Concentration, µ M
50
8 12 16 24 48 72 96 180 360 Equivalents of Oxidant Molar Ratio of Oxidant:Protein Methionine Methionine Sulfoxide Methionine Sulfone
571
Tyrosine 30
0
C
O3 3,5-DibromoTyrosine
60
0
B
3-BromoTyrosine
60 Concentration, µ M
Concentration, µ M
A
3-ChloroTyrosine
0
2
4
8 12 16 24 48 72 96 180 360 Molar Molar Equivalents Ratio of Oxidant:Protein of Oxidant
0
2
4 8 12 16 24 36 72 96 180 360 Molar Equivalents of Oxidant Molar Ratio of Oxidant:Protein
Figure 1. Changes in the distribution of oxidizable amino acid residues and their oxidation products following treatment of lysozyme protein (LZ) with HOCl, HOBr, or O3 for 24 h.
26 ACS Paragon Plus Environment
Page 27 of 28
Environmental Science & Technology
575 HOCl Tyrosine
3-ChloroTyrosine
HOBr 3,5-DichloroTyrosine
Tyrosine
3-BromoTyrosine
O3
3,5-DibromoTyrosine
Tyrosine 25
20
10
20
15
4 8 12 16 24 48 72 96 180 360 Molar MolarRatio Equivalents of Oxidant:Protein of Oxidant Methionine Methionine Sulfoxide Methionine Sulfone
2
8 12 16 24 48 72 96 180 360 MolarEquivalents Ratio of Oxidant:Protein Molar of Oxidant Methionine Methionine Sulfoxide Methionine Sulfone
2
0
8 6 4 2
4 8 12 16 24 48 72 96 180 360 MolarRatio Equivalents of Oxidant Molar of Oxidant:Protein Lysine Lysine nitrile Tryptophan
2 0
0
2
4
8 12 16 24 48 72 96 180 360 Molar of Oxidant MolarEquivalents Ratio of Oxidant:Protein Lysine Lysine Nitrile Tryptophan 35
Concentration, µ M
40
10
30 25 20 15 10 5
5 0
2
4 8 12 16 24 48 72 96 180 360 MolarRatio Equivalents of Oxidant Molar of Oxidant:Protein
0
2
4 8 12 16 24 36 72 96 180 360 MolarRatio Equivalents of Oxidant Molar of Oxidant:Protein Lysine Lysine Nitrile Tryptophan
0
2
4 8 12 16 24 48 72 96 180 360 MolarRatio Equivalents of Oxidant Molar of Oxidant:Protein
30 25 20 15 10 5
0
0
Methionine Sulfone
6
35
15
Methionine Sulfoxide
4
40
20
8 12 16 24 48 72 96 180 360 Molar of Oxidant:Protein MolarRatio Equivalents of Oxidant
8
35 25
4
Methionine
40 30
2
10
0 2
Concentration, µ M
Concentration, µ M
4
Concentration, µ M
Concentration, µ M
6 4
0
577 578 579 580
2
10 Concentration, µ M
10 8
5 0
0
0
576
10
0 0
C
20
10
0
B
Concentration, µ M
Concentration, µ M
Concentration, µ M
A
0 0
2
4
8 12 16 24 48 72 96 180 360 Molar Ratio Equivalents of Oxidant Molar of Oxidant:Protein
Figure 2. Changes in the distribution of oxidizable amino acid residues and their oxidation products following treatment of bacteriophage MS2 coat protein with HOCl, HOBr, or O3 for 24 h.
581 582
27 ACS Paragon Plus Environment
Environmental Science & Technology
583 584
TOC Art
585 586
28 ACS Paragon Plus Environment
Page 28 of 28