Degradation of Amino Acids and Structure in Model Proteins and

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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

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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


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ABSTRACT

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disinfectant-protein reactions, this study characterized the disinfectant:protein molar ratios at

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which 50% degradation of oxidizable amino acids (i.e., Met, Tyr, Trp, His, Lys) and structure

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were observed during HOCl, HOBr, and O3 treatment of three well-characterized model proteins

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and bacteriophage MS2. A critical question is the extent to which the targeting of amino acids is

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driven by their disinfectant rate constants rather than their geometrical arrangement. Across the

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model proteins and bacteriophage MS2 (coat protein), differing widely in structure, methionine

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was preferentially targeted, forming predominantly methionine sulfoxide. This targeting concurs

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with its high disinfectant rate constants and supports its hypothesized role as a sacrificial

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antioxidant. Despite higher HOCl and HOBr rate constants with histidine and lysine than for

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tyrosine, tyrosine generally was degraded in preference to histidine, and to a lesser extent, lysine.

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These results concur with the prevalence of geometrical motifs featuring histidines or lysines

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near tyrosines, facilitating histidine and lysine regeneration upon Cl[+1] transfer from their

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chloramines to tyrosines. Lysine nitrile formation occurred at or above oxidant doses where 3,5-

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dihalotyrosine products began to degrade. For O3, which lacks a similar oxidant transfer

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pathway, histidine, tyrosine and lysine degradation followed their relative O3 rate constants.

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Except for its low reactivity with lysine, the O3 doses required to degrade amino acids were as

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low as or lower than for HOCl or HOBr, indicating its oxidative efficiency. Loss of structure did

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not correlate with loss of particular amino acids, suggesting the need to characterize the

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oxidation of specific geometric motifs to understand structural degradation.

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INTRODUCTION

Proteins are important targets of chemical disinfectants. To improve the understanding of


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Chemical oxidants (e.g., hypochlorous acid (HOCl), hypobromous acid (HOBr), and

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ozone (O3)) are commonly applied to drinking waters, wastewaters, and recreational waters to

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inactivate pathogens. In addition, neutrophils and eosinophils produce HOCl and HOBr,

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respectively, as part of the immune response to pathogens in the human body.1,2 While pathogen

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inactivation kinetics have been characterized,3–11 the mechanistic understanding of how

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disinfectant reactions with biomolecules within pathogens results in pathogen inactivation needs

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improvement. Among pathogens, viruses represent a relatively simple case. First, compared to

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the array of biomolecular targets in other pathogens, viruses consist predominantly of a genome

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wrapped in capsid proteins. Second, outside of their hosts, viruses lack mechanisms to repair

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oxidative damage.

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Research has drawn different conclusions regarding the relative importance of oxidative

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damage to the genome or capsid proteins for driving virus inactivation.4,8,12–17 Previous work

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evaluated the relative importance of capsid and genome damage by HOCl, singlet oxygen,

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chlorine dioxide and UV light (254 nm) for inactivation of MS2, fr, and GA phages, members of

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the Leviviridae phage family, exhibiting a high degree of genome and protein sequence

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similarity.4,8 For singlet oxygen and UV light, genomic damage was the predominant driver of

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inactivation, resulting in replication inhibition. For chlorine dioxide, protein capsid damage

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dominated, inhibiting phage binding to the host cell. For HOCl, damage to the genome and

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capsid proteins both were important for inactivation.

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Protein structure ultimately is governed by both the amino acid sequence (i.e., primary

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structure) and side chain interactions (i.e., secondary and tertiary structures). Observed rate

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constants near neutral pH for HOCl, HOBr and O3 reactions with protein constituents are higher

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with a subset of amino acid side chains (“oxidizable amino acids”) than with peptide bonds



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(Table 1). Accordingly, protein exposure to disinfectants results in covalent modifications to

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these side chains,10-18 with the associated alterations in side chain interactions potentially

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modifying protein structure. Higher disinfectant exposures could cleave peptide bonds, resulting

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in protein fragmentation.11

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Previous research on oxidant-mediated protein damage has focused on kinetic models

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employing disinfectant reaction rate constants with individual amino acids to predict those

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initially targeted by oxidants.18–20 However, some studies have suggested the importance of the

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three-dimensional arrangement of amino acids for determining their susceptibility to disinfectant

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attack. First, while kinetic models combining HOCl rate constants largely matched experimental

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results for parent amino acid loss in mixtures of free N-acetyl amino acids, they did not match

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results for HOCl treatment of proteins.21 For example, for lysozyme treatment with a 25 molar

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excess of HOCl, ~60% loss of lysine and tyrosine were observed with the protein, but only ~10%

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loss was predicted by the kinetic model or observed with the same residues constituting

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lysozyme as a N-acetyl amino acid mixture.21 Second, in free amino acid mixtures, lysine

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inhibited the halogenation of tyrosine by scavenging HOCl or HOBr.22 However, previous

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research hypothesized that when lysines are constrained to be located near tyrosines within

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peptides, the lysine-ε-haloamines formed by HOCl or HOBr reactions with lysine side chains

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serve as halogen transfer agents, promoting halogenation of proximal tyrosines (Scheme 1).21,23–

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the model protein, adenylate kinase, promoted tyrosine bromination during HOBr treatment, but

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reduced the tyrosine halogenation during HOCl treatment.22 Third, Lundeen and McNeill29

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demonstrated that singlet oxygen oxidation of histidine increased with histidine’s proximity to

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the surface of glyceraldehyde-3-phosphate dehydrogenase. Fourth, the finding that genomic and

Highlighting the potential complexities associated with three-dimensional structures, lysines in


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viral capsid damage were comparable for bacteriophage MS2 inactivation by HOCl despite

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significantly higher rate constants for HOCl reaction with oxidizable amino acids than with

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nucleotides also suggests the potential importance of the three-dimensional arrangement of

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biomolecules within viruses.4 Lastly, singlet oxygen preferentially oxidized a specific

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methionine (Met88) in MS2, but not in the structurally similar fr phage; the difference was

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attributed to this residue’s greater solvent accessibility in MS2.8

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The long-term goal of our research is to understand the importance of the three-

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dimensional arrangement of amino acids for disinfectant-mediated protein damage to better

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characterize pathogen deactivation mechanisms. However, characterization of site-specific

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damage to proteins is complex. Generally, the process involves enzymatic cleavage (e.g., trypsin

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digest) of proteins into ~5-20 residue oligomers and mass spectral characterization of parent

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oligomer loss and daughter oligomer formation vs. oxidant exposure. While this problem is

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tractable for disinfectants that react with high specificity with amino acids (e.g., singlet oxygen

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with histidine29), application to disinfectants, such as HOCl, that react with an array of residues

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is problematic. For a single 20-residue oligomer containing 4 residues oxidizable by HOCl to

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unique products, the process would involve monitoring the loss of 1 parent oligomer and

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production of 14 product oligomers. For the 129-residue MS2 coat protein, about 90 oligomers

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would need to be tracked. Although previous research has indicated that amino acid geometry

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can affect the reactivity of specific residues, the importance of these site-specific observations

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relative to whole protein degradation is unclear. Because these geometrical effects likely are

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highly variable across proteins, it is critical to understand their importance before engaging in a

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labor-intensive, protein-by-protein characterization of protein degradation processes.



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The first objective of the research presented herein was to evaluate the extent to which

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oxidizable residue degradation by HOCl, HOBr and O3 can be predicted by rate constants. A

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range of oxidant doses was applied to adenylate kinase, lysozyme, ribose binding protein and

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MS2; the first three proteins served as well-characterized model proteins. After disinfectant

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treatment, proteins were digested to liberate individual amino acids. While amino acid location

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within the protein was lost, the quantification of parent amino acids and oxidized daughter

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products was facilitated. The results allowed us to evaluate whether the order with which amino

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acids are targeted is common across proteins and predictable by rate constants, or whether the

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geometrical arrangement of amino acids affects their susceptibility to oxidant reactions. A

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second objective was to understand how the three oxidants differ in targeting amino acids. A

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third objective was to evaluate whether structural loss is associated with degradation of specific

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amino acids. A final objective was to measure the formation of oxidation products that have

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previously been characterized in the literature as a function of oxidant exposure. Product

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characterization is desirable because the interactions between these modified side chains and

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nearby residues affect structure. Additionally, like other byproducts of disinfection,30,31 these

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products may be associated with toxicity. While previous research has identified specific

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oxidation products (e.g., 3-chlorotyrosine, methionine sulfoxide22,32), their production and

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stability vs. disinfectant exposure in proteins generally has not been characterized.

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MATERIALS AND METHODS The Supporting Information provides reagent sources.

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Model proteins and MS2 virus preparation and purification: Lysozyme (LZ), adenylate

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kinase (AdK), and ribose binding protein (RBP) served as model proteins because their 6 ACS Paragon Plus Environment

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structures are well-characterized (Table 2), including by X-ray crystallography, not because of

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their relevance to pathogens. Lysozyme (chicken egg white; 90%) was purchased from Sigma-

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Aldrich. Computational protein design (CPD) was employed to redesign ADK removing specific

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oxidizable residues. The AdK, redesigned AdKs, and RBP production and purification

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procedures, described previously,22,33 are summarized in the SI.

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The MS2 capsid consists of 180 coat protein copies and 1 maturation protein. The coat

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protein is well-characterized (Table 2). MS2 bacteriophage and Escherichia coli were obtained

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from the American Type Culture Collection. MS2 was propagated and purified as described

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previously,4 and summarized in the SI. Only a fraction of MS2 may be enumerated via plaque

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assays. To estimate the molar ratio of applied oxidants to coat proteins, the MS2 coat protein

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concentration in the stock solution was quantified by a different procedure. A stock solution

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aliquot was treated by strong acid digestion to hydrolyze the proteins (see below), releasing free

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amino acids. The free amino acid concentrations were quantified (see below) and divided by

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their number in the MS2 coat protein (which dominates the protein content vs. the maturation

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protein 180-fold) to estimate the MS2 coat protein concentration. The ratio of the concentrations

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of amino acids measured to those expected based upon their prevalence within MS2 coat protein

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was 1.0 (± 0.3 standard deviation) across six amino acids (glycine, alanine, lysine, methionine,

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tyrosine and tryptophan), suggesting purity of the MS2 stock.

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Protein oxidation. Model proteins and whole bacteriophage MS2 were treated with HOCl,

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HOBr, or O3 in centrifuge tubes. While whole bacteriophage MS2 was treated, the results are

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discussed based upon the oxidant ratio relative to coat protein, because the coat protein is in 180-

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fold excess relative to maturation protein. Preparation and standardization of oxidant stock

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solutions is described in the SI. Briefly, O3 stocks were prepared by sparging O3 generated from



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oxygen gas through chilled, deionized water. For HOCl and HOBr, 30 µM proteins were dosed

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with 0-10,800 µM HOCl or HOBr (oxidant:protein molar ratios of 0-360) at pH 7.4. For

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ozonation, 15 µM of proteins were dosed with 0-540 µM O3 (ozone:protein molar ratios of 0-36)

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at pH 7.4; the O3 dose was limited by the ability to generate concentrated O3 stocks. While these

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absolute oxidant concentrations exceed those applied for water disinfection, our purpose here

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was to evaluate amino acid transformations with respect to oxidant:protein molar ratio. While we

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employed high protein concentrations here, high oxidant:protein molar ratios are expected in

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natural waters because of their low pathogen concentrations. After 24 h, residual oxidants were

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quenched with glutathione at 1.2 times the molar concentration of oxidants applied. Glutathione

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was selected because it did not interfere with the amino acid analysis and because initial

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experiments demonstrated that it does not reduce amino acid oxidation products (e.g.,

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methionine sulfoxide).

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Protein digestion and analysis. Free amino acids were liberated from proteins by acid-catalyzed

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hydrolysis (strong acid digestion) using methanesulfonic acid.34 However, because strong acid

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digestion can destroy some amino acid oxidation products, an enzymatic digestion procedure

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was used to quantify these products35,36. Enzymatic digestion was accomplished using Pronase E

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(Protease type XIV; Sigma-Aldrich), an enzyme cocktail derived from Streptomyces griseus.

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Details are provided in the SI. Liberated amino acids were derivatized using 6-aminoquinolyl-N-

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hydroxysuccinimidyl carbamate (AQC, Chemodex Ltd., Switzerland, 95%),37 and analyzed by

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HPLC-MS. Protein structural decay upon oxidative challenge was assessed by collecting Far-UV

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circular dichroism spectra. The SI provides additional details.

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RESULTS AND DISCUSSION


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Protein characteristics. Oxidizable residues, featuring HOCl rate constants at least as

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high as tyrosine (and higher than peptide bonds), include methionine, cysteine, histidine,

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tryptophan, lysine and tyrosine (Table 1). Table 2 provides the numbers of each oxidizable

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residue, and other structural data for the model proteins and MS2 coat protein. The total number

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of oxidizable residues per protein was fairly similar (30-35) across the model proteins, but the

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MS2 coat protein featured only 16 oxidizable residues. The fraction of total residues that were

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oxidizable ranged from 12.4% (MS2 coat protein) to 23.3% (LZ). Lysine generally was the most

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prevalent oxidizable residue, and tryptophan, histidine and cysteine (except LZ) were the least

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prevalent. Except for cysteine, the majority of oxidizable residues in the model proteins were

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solvent-accessible, defined as having side chains with >10 Å2 surface exposure (as calculated by

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GETAREA software38); solvent-accessibility was not calculated for MS2 coat protein because

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the GETAREA software does not consider accessibility restrictions related to interfaces with

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other coat proteins or the genome. Although each protein contained α-helices and β-sheets, α-

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helices were more prevalent in the model proteins, while β-sheets were more prevalent in the

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MS2 coat protein.

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Targeting of oxidizable amino acids. Each protein was exposed to an array of

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disinfectant:protein molar ratios in triplicate. For MS2, the entire virus was treated with

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disinfectant, but the oxidant:protein molar ratio was calculated using the measured total protein

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concentration, considering that the coat protein concentration exceeded the maturation protein

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concentration 180-fold. Although the lower molar ratios where structural damage occurred were

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preferentially sampled, higher molar ratios were evaluated because, even after pathogen

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inactivation, protein detritus can contribute to disinfection byproduct formation. Figures 1 (LZ),

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2 (MS2), S2 (RBP) and S3 (AdK) provide the loss of 5 of the 6 oxidizable amino acids in each



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protein vs. oxidant:protein molar ratio. Loss of the other oxidizable amino acid, cysteine, could

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not be quantified because it oxidizes during digestion. We confirmed that there was insignificant

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loss of other amino acids (e.g., alanine). The oxidant dosage where 50% loss of each oxidizable

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amino acid was observed (denoted as AA50 (e.g., Met50)) was used as the principle metric for its

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oxidation susceptibility. Residue losses generally followed sigmoidal relationships with oxidant

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dosage. The AA50 values were determined via linear regression for 3-4 experimental values

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surrounding the 50% value (Table 3; Figures 1, 2, S2, S3). The error represents the 95%

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confidence interval from this regression. The relative error increased for high AA50 values,

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partially due to the wider spacing of oxidant:protein molar ratios evaluated; regardless, for these

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scenarios, these amino acids were poorly targeted by the oxidants. Comparisons between AA50

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values were reported only if significant at the p < 0.05 level based on Welch’s t-test.

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For HOCl treatment, the AA50 values generally followed the order: Met50 < Tyr50 ≤ Lys50

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< His50, indicating that methionine was the most reactive with HOCl. For MS2 coat protein, and

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to a lesser extent RBP, Lys50 > Tyr50. Only LZ and MS2 coat protein contained tryptophan

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(Table 2); for both proteins Trp50 was comparable to Tyr50. For LZ, oxidizable residue loss was

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similar to that observed previously for HOCl:LZ molar ratios 0-25.21 Based on observed HOCl

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rate constants with N-acetylated amino acids at pH 7.4, the order of reactivity was Met > His >

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Trp ≈ Lys > Tyr, and the rate constants varied by 6 orders of magnitude (Table 1). For

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methionine, tryptophan and lysine (for AdK and LZ), the similarity between the order in which

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amino acids degraded and their HOCl rate constants across four proteins varying in structure

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suggests that relative reactivity with HOCl is more important than geometry for driving

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degradation.


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However, the results for histidine, tyrosine, and lysine (for MS2 coat protein and to a

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lesser degree, RBP) suggest the potential importance of three-dimensional structure. Although

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their rate constants indicate that HOCl is 3 and 2 orders of magnitude more reactive with

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histidine and lysine than tyrosine, respectively, tyrosine was degraded prior to histidine in all

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model proteins (MS2 does not contain histidine) and prior to lysine for MS2 coat protein and

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RBP. Additionally, the low reactivity of these histidines and lysines occurs even though all of the

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histidines in the model proteins, and lysines in MS2 coat protein and RBP are surface-accessible

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(Table 2). Note that the HOCl rate constants with the amino acids (Table 1) relate to HOCl’s

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initial attack on the amino acid. Histidine and lysine would form N-chloramines.23,39 Previous

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research suggested that these N-chloramines could promote tyrosine chlorination by transferring

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Cl[+1] to tyrosine, regenerating the parent amino acid21,23,35 (Scheme 1). For HOCl-treated AdK,

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we found that lysine chloramines formed lysine nitrile by hydrochloric acid eliminations.22 By

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scavenging HOCl, lysine thereby partially protected tyrosine from chlorination, and, like

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methionine, served as an antioxidant. Although chlorine transfer from histidine N-chloramines to

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tyrosine has been hypothesized21,39, it has not been evaluated experimentally in proteins. If

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chlorine transfer is important, histidine or lysine degradation may not be observed until

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degradation of proximal tyrosines prevents this transfer (and histidine or lysine regeneration).

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For AdK and LZ, loss of lysine and tyrosine and lysine nitrile formation occurred over similar

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oxidant dosages, suggesting only partial protection of tyrosine by lysine’s antioxidant activity.

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In contrast, for all model proteins, histidine loss occurred predominantly at HOCl:protein molar

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ratios above where significant tyrosine transformation to 3,5-dichlorotyrosine occurred. For

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MS2 coat protein, which does not contain histidines, and to a lesser degree for RBP, tyrosine loss

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preceded lysine loss, suggesting that Cl[+1] transfer from lysine to tyrosine may be important.



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Additional support for Cl[+1] transfer between histidine/lysine and tyrosine is provided

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by the protein crystal structures. AdK’s two histidines and LZ’s single histidine are located

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within 15 angstroms of tyrosines (Table S2). For comparison, single bond lengths are ~2

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angstroms. Thus, these histidine N-chloramines would be well-positioned to transfer Cl[+1] to

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tyrosines, protecting histidine at the expense of tyrosine such that His50 > Tyr50. In contrast, only

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4 of AdK’s 14 lysines and none of LZ’s 6 lysines are near tyrosines, inhibiting Cl[+1] transfer to

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tyrosines such that Lys50 ≈ Tyr50.

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None of RBP’s histidines are located near tyrosines, although all are located near other

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oxidizable residues (i.e., lysine or methionine), potentially enabling histidine preservation by

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Cl[+1] transfer from histidine N-chloramines. While His50 > Tyr50, the difference was less than

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for AdK and LZ. Lysine promotion of tyrosine chlorination may be possible, because RBP’s 3

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tyrosines are located near lysines. However, with only 3 of the 24 lysines adjacent to tyrosines, it

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would be difficult to validate this hypothesis based upon a decrease in lysine degradation (due to

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its regeneration via Cl[+1] transfer). While MS2 coat protein does not contain histidine, 3 of the

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6 lysines are located near tyrosines. The fact that Lys50 was significantly higher than Tyr50

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suggests that the proximity of lysines and tyrosines protects lysine by Cl[+1] transfer to tyrosine.

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For HOBr treatment, the order of oxidizable amino acid degradation was Met ≈ Trp ≈ Tyr

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≈ His > Lys across the model proteins (Table 3). The order was similar for MS2 coat protein,

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although the Met50 value determined for the two Met residues was very uncertain. A rapid ~30-

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45% loss in Met over the first 4 molar equivalents of HOBr was followed by much slower

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(~20%) additional loss over 72 HOBr molar equivalents (Figure 2), suggesting that one Met

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residue was degraded more readily than the other. The observed HOBr reaction rate constants

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with the oxidizable amino acids at pH 7.4 were in the order Trp ~ Met ~ His > Lys ~ Tyr, but


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their range was only ~1 order of magnitude (Table 1).20 The similarities between the AA50 values

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likely reflects the low range in rate constants and again suggests the predominant effect of HOBr

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rate constants over geometry for determining residue oxidation. However, although the HOBr

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rate constant with histidine is ten-fold higher than with lysine or tyrosine, tyrosines were

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degraded as readily as histidines and more readily than lysines. As for HOCl, these results may

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indicate the importance of histidine-tyrosine or lysine-tyrosine interactions associated with their

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proximity within protein structures. In previous research with AdK, lysine promoted tyrosine

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bromination.22 Five of AdK’s 9 tyrosines are proximal to lysines and an additional 2 are near

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histidines (Table S2).

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For O3 treatment, the AA50 values were comparable for methionine, tryptophan, histidine

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and tyrosine, while lysine was unreactive (Table 3). Compared to HOCl and HOBr, O3 exhibited

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the lowest percentage differences in AA50 values for different amino acids (excluding lysine).

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For instance, for RBP, the ratios between the highest and lowest AA50 values were 9.3 for HOCl

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(His50:Met50), 5.2 for HOBr (Lys50:Met50), but only 2.0 for O3 (Tyr50:Met50). Again, these results

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suggest the importance of O3 rate constants with the amino acids. These rate constants differed

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less than 3-fold among the oxidizable amino acids, except lysine, which was 4 orders of

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magnitude less reactive (Table 1). Except for lysine, AA50 values for O3 generally were as low as

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or lower than those for HOCl or HOBr, suggesting a high efficiency for amino acid degradation

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by O3. In contrast to HOCl and HOBr, tyrosine degradation was not enhanced, and histidine

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degradation was not hindered relative to the reactivities predicted by reaction rate constants. A

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reaction analogous to Cl[+1] transfer from histidine N-chloramines to tyrosine does not exist for

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O3.



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Product formation. Methionine was preferentially targeted by all three oxidants.

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Oxidation produced first methionine sulfoxide at yields ranging from ~15% (e.g., HOBr

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treatment of LZ or O3 treatment of AdK) to nearly stoichiometric (e.g, all oxidants with RBP or

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O3 with MS2 coat protein) over oxidant:protein molar ratios up through ~24 (Figures 1, 2, S1

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(for product structures), S2, and S3). At higher oxidant:protein molar ratios, methionine

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sulfoxide concentrations leveled off or declined. Less than stoichiometric yields suggest that

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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

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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

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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

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residues, oxidant scavenging associated with methionine oxidation to methionine sulfoxide or

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methionine sulfone indicates that methionine serves as a sacrificial antioxidant.

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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

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°C) (Table S3). The replacement of tyrosines resulted in by far the greatest loss in protein

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stability even though there were more lysines than tyrosines. While these results might suggest a

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global importance for tyrosines, one alternative is that interactions of only a few of the tyrosines

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are of paramount importance to AdK’s structural stability. To test the importance of individual

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tyrosines, computational design could be used to replace individual tyrosines. However, this

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approach is highly labor-intensive and the results may be specific only to AdK, a situation

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analogous to the use of high-resolution mass spectrometry to characterize oxidative

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transformations of the oligopeptides generated by single enzyme digestion of proteins.

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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


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across different proteins. The oxidant dosage associated with 50% loss of the far-UV circular

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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

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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,

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its AA50 value should correlate with the CD50 value.

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No such broad correlations were observed. For all three oxidants, AdK was the most

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susceptible to structural loss (i.e., low CD50), even though its oxidizable amino acids were the

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most resistant to oxidative transformation (e.g., highest AA50 values). Indeed, AdK structural

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loss preceded loss of even methionine (i.e., CD50 < Met50). For AdK, the CD50 values (3.1-5.8)

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suggest that oxidative damage to fewer than 6 of the 35 oxidizable amino acids can drive

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structural damage. While the melting temperature data suggest that oxidative transformation of a

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particular tyrosine may be critical for structural loss, testing this hypothesis would require higher

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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.



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


Page 18 of 28

Page 19 of 28


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


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)

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(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.


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(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


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(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.


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Page 23 of 28

543


Scheme 1. Transfer of Cl[+1] from histidine or lysine chloramines to tyrosine.

544 545



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


2 16 129 17% 34% ___

Page 25 of 28

562 563


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



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


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.


Page 27 of 28


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 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



583 584

TOC Art

585 586


Page 28 of 28

Degradation of Amino Acids and Structure in Model Proteins and

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