The Relationship of Physicochemical Properties to the Antioxidative

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The relationship of physicochemical properties to the antioxidative activity of free amino acids in Fenton system Sonja Milic, Jelena Bogdanovic Pristov, Dragosav Mutavdži#, Aleksandar Savic, Mihajlo Spasic, and Ivan Spasojevic Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es5053396 • Publication Date (Web): 12 Mar 2015 Downloaded from http://pubs.acs.org on March 19, 2015

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Environmental Science & Technology

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The relationship of physicochemical properties to

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the antioxidative activity of free amino acids in

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Fenton system

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Sonja Milić,† Jelena Bogdanović Pristov,† Dragosav Mutavdžić,† Aleksandar Savić,†,§ Mihajlo

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Spasić,‡ Ivan Spasojević†,*

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Department of Life Sciences, Institute for Multidisciplinary Research, University of Belgrade,

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Belgrade, Serbia ‡

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Department of Physiology, Institute for Biological Research 'Siniša Stanković', University of

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Belgrade, Belgrade, Serbia

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*Corresponding author: Department of Life Sciences, Institute for Multidisciplinary Research, Kneza Višeslava 1, 11030 Belgrade, Serbia. Phone: +381 11 2078459; Fax: +381 11 3055289; E-mail: [email protected] §

Current address: LASIR CNRS UMR 8516, Université Lille 1, Sciences et Technologies, 59655

Villeneuve d’Ascq Cedex, France

ACS Paragon Plus Environment


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ABSTRACT

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Herein we compared antioxidative activities (AA) of 25 free L-amino acids (FAA) against

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Fenton system-mediated hydroxyl radical (HO•) production in aqueous solution, and examined

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the relation between AA and a set of physicochemical properties. The rank order according to

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AA was: Trp > norleucine > Phe, Leu > Ile > His >3,4-dihydroxyphenylalanine, Arg > Val >

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Lys, Tyr, Pro > hydroxyproline > α-aminobutyric acid > Gln, Thr, Ser > Glu, Ala, Gly, Asn,

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Asp. Sulfur-containing FAA generated different secondary reactive products, which were

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discriminated by the means of electron paramagnetic resonance spin-trapping spectroscopy. AA

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showed a general positive correlation with hydrophobicity. However, when taken separately,

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uncharged FAA exhibited strong positive correlation of AA with hydrophobicity whereas

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charged FAA showed negative or no significant correlation depending on the scale applied. A

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general strong negative correlation was found between AA and polarity. Steric parameters and

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hydration numbers correlated positively with AA of non-polar side-chain FAA. In addition, a

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decrease of temperature which promotes hydrophobic hydration resulted in increased AA. This

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implies that HO•-provoked oxidation of FAA is strongly affected by hydrophobic hydration. Our

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findings are important for the understanding of oxidation processes in natural and waste waters.

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Word count: 4596

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Number of figures: 6 (4 small and 2 large)

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Total word count: 6996

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TOC Art

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INTRODUCTION

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Free amino acids (FAA) are present in natural and waste waters.1,2 Their concentrations vary

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from 15 µg/L in seawater up to 0.2 mg/L in untreated wastewaters and 5 mg/L in eutrophic

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lakes.1,3 FAA represent bioavailable subclass of dissolved organic nitrogen that supports growth

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of bacteria and algae.3,4 Some FAA are known to interfere with wastewater processing and might

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lead to generation of harmful byproducts.5 Hydroxyl radical (HO•) that is produced via Fenton

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reaction (Fe2+ + H2O2 → Fe3+ + OH- + HO•) is involved in modification/degradation of organic

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compounds in aqueous systems.6–8 Fenton reaction is one of the key oxidation reactions in

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natural waters, and it is applied as advanced oxidation process in the industry of remediation of

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contaminated and waste waters.6 Rate constants for reactions of FAA and other compounds with

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HO• are commonly calculated using water radiolysis as HO•-generating system.9,10 However, the

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interactions of FAA with Fenton system are far more complex and might also involve the

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sequestration of iron, pro-oxidative activity of FAA and their derivatives via iron reduction (Fe3+

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→ Fe2+), and interactions with Fenton reaction intermediates. Data on antioxidative activity

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(AA) of FAA against HO• production in Fenton system are scarce, whereas comparative analysis

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is missing. Further, mechanistic details about the reactions between HO• and FAA are still being

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elucidated. E.g. we have just recently described the production of CH3SH and several reactive

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intermediates in Met + HO• reaction.11 Importantly, there are no reports on structure-activity

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relationship (SAR) that could tell which physicochemical parameters define/affect the ability of

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FAA to scavenge HO• in aqueous solution. SAR analysis of FAA might be important for

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understanding the interactions of proteins with HO• as well. For example, two SAR studies

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showed a positive correlation between the number of highly hydrophobic amino acids in protein

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fragments and fractions and their antioxidative performance against HO•.12,13 4


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Here we utilize electron paramagnetic resonance (EPR) spin-trapping spectroscopy to

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determine and compare AA of 25 FAA against Fenton system. The spin-trapping technique is

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based on the reaction of 'EPR silent' spin-trap with free radical which yields a more persistent

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EPR active nitroxide spin-adduct.14–16 DEPMPO, a sophisticated EPR spin-trap reagent, is

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applied in order to analyze reactive products of reaction of HO• with sulfur-containing FAA –

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Cys, homocysteine (HCY), and Met. In the next step, the relationship between AA and a variety

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of physicochemical properties – hydrophobicity, stericity, polarity, and hydration number, and

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contribution of functional groups to AA, are examined. We took into account that in spite of their

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common zwitterionic structure, FAA represent a very versatile group of compounds, and that the

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properties and behavior of amino acids in water are diversified by side-chains.17 Therefore, FAA

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(referred as all FAA) were divided into four subgroups, according to the key properties of side-

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chains: FAA with charged side-chains (referred as charged FAA), FAA with uncharged side-

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chains (uncharged FAA), FAA with uncharged non-polar side-chains (non-polar FAA), and FAA

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with uncharged polar side-chains (polar FAA).

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EXPERIMENTAL

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Reagents. Twenty proteinogenic and five non-proteinogenic L-amino acids – norleucine (Nle),

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α-aminobutyric acid (AABA), hydroxyproline (HYP), 3,4-dihydroxyphenylalanine (DOPA), and

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HCY (≥98% purity) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fenton reaction

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was performed by combining 1 mM H2O2 (Carlo Erba Reagents, Milano, Italy) and 0.2 mM

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FeSO4 (Merck, Darmstadt, Germany) with(out) FAA (5 mM). DEPMPO (5-diethoxyphosphoryl-

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5-methyl-1-pyrroline-N-oxide; Enzo Life Sciences International, Plymouth Meeting, PA, USA)

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was added prior to the reaction initiation at a final concentration of 5 mM. All experiments were

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performed using bidistilled deionized ultrapure (18 MΩ) water. The pH value of the system 5



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composed of Fenton reaction reagents and FAA was in 4.5–4.7 range. Static oxidation-reduction

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potential (ORP) of FAA (5 mM) solutions in water was measured using RedoxSYSTM Analyzer

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(Luoxis Diagnostics, Englewood, CO, USA). All experiments were performed at 293 K. For

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selected FAA, the experiments were also conducted at 274 K. For this purpose all solutions and

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mixtures were kept, prepared, and incubated (for 2 min) on ice. Experiments with sulfur-

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containing FAA were also performed under anoxic conditions (Ar atmosphere; all solutions were

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purged with Ar).

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EPR spectroscopy. Samples were drawn into 10 cm-long gas-permeable Teflon tubes (wall

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thickness, 0.025 mm; internal diameter, 0.6 mm; Zeus industries, Raritan, NJ, USA) to maintain

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constant O2 level in the sample (oxygen might affect the width of spectral lines), and placed in

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quartz capillaries. EPR spectra were recorded using a Varian E104-A EPR spectrometer

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operating at X-band (9.572 GHz) with the following settings: modulation amplitude, 0.2 mT,

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except for HCY (0.1 mT); modulation frequency, 100 kHz; microwave power, 20 mW; time

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constant, 32 ms; scanning time, 2 min. Recordings were performed using EW software

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(Scientific Software, Bloomington, IL, USA), 2 min after the reaction had started, except for

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sulfur-containing FAA (1 min; in air or in Ar). Spectral simulations were performed using

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WINEPR SimFonia computer program (Bruker Analytische Messtechnik GmbH, Darmstadt,

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Germany) to establish the intensity of signal of DEPMPO adduct with HO• (DEPMPO/OH;

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simulation parameters: aN = 1.40 mT, aH = 1.32 mT, aHγ = 0.03 mT (3H), and aP = 4.73 mT), and

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to determine the presence of other adducts. AA values were calculated using the following

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equation: 1 - IFAA/IFenton (IFAA - signal intensity in the system with FAA; IFenton - mean intensity

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for control on the same experimental day). Maximal AA is 1, meaning that all HO• is removed

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and no signal of DEPMPO/OH adduct could be detected. In the FAA-free Fenton system, AA is 6


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zero. EC50 value (mM) is the effective concentration at which AA was 0.5. It was determined

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from concentration-AA curves by interpolation. Total rate constants for AA of FAA in the

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Fenton system were calculated according the following equation: kFenton = (IFenton-IFAA)/IFAA ×

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kDEPMPO × [DEPMPO]0/[FAA]0.18,19 Rate constant for reaction between DEPMPO and HO•

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(kDEPMPO) is approximately 7.5 × 109 M-1s-1.20 The initial concentration ratio [DEPMPO]0/[FAA]0

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here was 1.

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Parameters. Correlation analysis was performed using a set of 15 physicochemical parameters

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(Table S1).21–32 The relation between AA and hydrophobicity was intuitive from the results

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obtained in the first part of the study, so the focus was on this parameter. Nine different

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hydrophobicity/hydrophilicity scales were applied, taking care to include up-to-date scales,

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widely applied scales, scales established using different approaches (experimental, theoretical,

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consensus), and scales with hydrophobicity parameters for non-proteinogenic amino acids.21–29

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Of note, only a few reports have addressed hydrophobicity of non-proteinogenic amino acids.23,28

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Pertinent to this, Log P scale (P is octanol/water partition coefficient), a widely applied

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parameter of hydrophobicity, was established for all FAA using DruLiTo (NIPER, Nagar, India).

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Structures were converted to SMILES format and Log P was calculated using additivity

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approach – summation of each group contribution. His was not included in SAR analysis,

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because its side-chain has pKa≈ 6, and most scales were established at neutral pH. Pertinent to

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this, Cowan and Whittaker have found that the hydrophobicity of amino acids with uncharged

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side-chains does not differ significantly at pH 3 and pH 7.5.22

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Group contribution method. The contribution of each functional group to AA was calculated

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according to Benson’s thermochemical group additivity principle.33 FAA structures were

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fragmented into the following groups: NH3+CHCOO-, CH, CH2, CH3, OH, benzene ring, COO-, 7



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CONH2, and specific groups for Trp (indole), Lys (amino), Arg (guanidino), and His

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(imidazole). Using this principle, a system of linear equations was built [for example AA(Val) =

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AA(NH3+CHCOO-) + AA(CH) + 2×AA(CH3)], from which group contributions were calculated

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in Matlab 2010a (Matworks, Natick, MA, USA), with least squares minimization of error.

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Negative values for group contributions to AA were allowed, since the production of HO• might

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be promoted in the Fenton system via iron reduction or interactions with the reaction

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intermediates. The same approach was used to calculate the contribution of functional groups to

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hydrophobicity/hydrophilicity (imidazole was excluded).

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Correlation analysis. Pearson's correlation coefficient was used to test the degree of

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correlation between different parameters and AA, as well as between contributions of functional

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groups to AA and to hydrophobicity/hydrophilicity. If two groups of FAA show a different level

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of AA changes with the unit change of specific physicochemical parameter, different regression

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slopes are observed. The significance of the difference between slopes was assessed by the test

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of homogeneity of regression slopes (Chow test). The significance level for this test was 0.05

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(two-tailed). Further, a series of correlation coefficients were calculated in order to establish

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which FAA significantly decrease the correlation between AA and rate constants previously

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determined for reactions of FAA with radiolitically-produced HO• (kradiolysis;9,10,19 last column in

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Table S2). Calculus was repeated two times: (i) One FAA was excluded in each series; in this

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way 21 R values were obtained; (ii) DOPA (the first upper outlier) + one FAA were excluded,

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and 20 R values were obtained. Results are presented as box plots.

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Statistical analysis. All experiments with FAA were performed at least in quadruplicate on

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separate experimental days. Each day, control experiments were performed in triplicate. The data

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are presented as mean ± SE. Statistical differences were evaluated by the means of non8


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parametric two-tailed Mann-Whitney test (P Leu, Nle, Phe, Ile > His

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> DOPA, Arg > Val > Lys, Tyr, Pro > HYP > AABA >Gln, Thr, Ser > Glu, Ala, Gly, Asn, Asp

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(Figure 2a). The last eight FAA did not show significant AA compared to control values. 9



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Figure 2. Antioxidative activity (AA) of free amino acids (FAA) against Fenton reaction-

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generated HO• radical in water. (a) AA values of 22 FAA. AA represents a relative decrease of

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yield of DEPMPO/OH adduct and pertinent EPR spectrum intensity in the presence of particular 10


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FAA. In control system (FAA-free Fenton reaction) AA = 0 ± SE (dashed lines). Of note, His

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side-chain is not charged at pH ~4.5, it is only polar. * significantly different compared to

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control (P < 0.05). (b) Coefficients of correlation between AA and rate constants for reactions of

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FAA and radiolitically-produced HO•. In each series one FAA (left; 21 R values were obtained)

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or DOPA + one FAA (right; 20 R values) were excluded. Boxes represent the median R and the

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25th and 75th percentiles; whiskers represent the non-outlier range. Outliers (circles) –values

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that are more than 1.5 × interquartile range outside the box. (c) Contribution of FAA functional

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groups to AA.

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Total rate constants (kFenton) are presented in Table S2. For almost all FAA (except Tyr) kFenton

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was higher compared to kradiolysis. This implies that AA of FAA in the Fenton system involves

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additional mechanisms besides HO• scavenging. AA values in the Fenton system showed

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positive correlation (R = 0.461; P = 0.018) with kradiolysis. Figure 2b shows the variability of R

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with exclusion of each of 22 FAA. Significantly higher R values were obtained when DOPA and

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Tyr were excluded from the calculus. In other words, DOPA and Tyr significantly decreased the

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correlation. According to group contribution method, rank order of contributions to AA was:

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indole > CH > imidazole > benzene > CH2 > CH3 > NH3+CHCOO- > guanidino > OH > CONH2

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> COO- > amino (Figure 2c). In general, non-polar structures – aromatic rings and alkyl groups

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showed positive contributions, whereas polar/charged groups had negative contributions. The

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contribution of NH3+CHCOO- was next to zero. AA values that were calculated using

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contributions of groups fitted well with experimental AA (R = 0.990; P < 0.001). Trp, Phe, Leu,

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Ile, and Nle almost completely removed HO• (AA was nearly 1). In order to eliminate the effects

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of saturation, EC50 values were determined (Figure S1). Rank order according to EC50 was: Trp >

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Nle > Phe, Leu > Ile. Static ORP values of 5 mM solutions of most FAA were similar and higher 11



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than 200 mV (Table S3). DOPA, Cys, and HCY were exceptions with considerably lower ORP,

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which implies higher reducing capacity compared to other FAA.

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Figure 3. Reactive products of Fenton system in the presence of sulfur-containing FAA. (a) EPR

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signal of three DEPMPO adducts in Cys + Fenton. Simulation (gray) parameters: DEPMPO/S-

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Cys (83%; aN = 1.41 mT, aH = 1.49 mT, aP = 4.58 mT), DEPMPO/X (10%; aN = 1.55 mT, aH =

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1.73 mT, aP = 5.26 mT), DEPMPO/OH (7%). (b) Characteristic EPR signal of DEPMPO/S-HCY

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in HCY + Fenton. Simulation: same parameters as for DEPMPO/S-Cys. (c) EPR signal of

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DEPMPO adducts produced in Met + Fenton . Simulation: DEPMPO/OH (67%), adducts of two

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carbon-centered radicals – DEPMPO/C (16%; aN = 1.45 mT, aH = 2.15 mT, aP = 4.59 mT; and aN 12


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= 1.48 mT, aH = 2.07 mT, aP = 4.78 mT), DEPMPO/N (10%; aN = 1.54 mT, aN = 0.19 mT, aH =

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1.79 mT, aP = 5.14 mT), DEPMPO/SR (7%; aN = 1.41 mT, aH = 1.49 mT, aP = 4.58 mT). (d)

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EPR signal of DEPMPO adducts produced in Met + Fenton system under anoxic conditions.

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Simulation: DEPMPO/OH (38%), DEPMPO/C (38%; aN = 1.45 mT, aH = 2.15 mT, aP = 4.59

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mT), DEPMPO/N (19%; aN = 1.45 mT, aN = 0.19 mT, aH = 2.02 mT, aP = 4.88 mT),

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DEPMPO/SR (5%).

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The reaction of sulfur-containing FAA with HO• generated a number of spin-adducts, other

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than DEPMPO/OH (Figure 3). It is important to note that Cys and HCY showed the ability to

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reduce spin-adducts, which led to a rapid decrease of intensity of EPR spectra (Figure S2),

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making the estimation of AA against HO• impracticable. The steeper slope for HCY-mediated

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spin-adduct reduction compared to Cys is in accordance with the higher reducing potential (i.e.

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lower static ORP) of HCY. In the presence of Cys, EPR signal was composed of spectra of three

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spin-adducts (Figure 3a). The predominant species was Cys thiyl radical (Cys-S•). Besides this,

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DEPMPO/OH and another adduct of uncertain origin were present. The latter adduct does not

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originate from some derivative of Cys-S• and molecular oxygen since the spectrum of Cys +

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Fenton system was not altered under anoxic conditions (not shown). Figure 3b shows a

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drastically simpler setup for HCY. Namely, HO•-mediated oxidation of HCY resulted only in the

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production of HCY thiyl radical (HCY-S•). The spectrum did not show changes under anoxic

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settings (not shown). It is noteworthy that EPR spectra for Cys and HCY show slight asymmetry

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that could arise from the reduction of spin-adduct during the scan time or due to slow tumbling

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of a relatively large spin-adduct. Finally, the oxidation of Met gave rise to five different

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paramagnetic species: HO•, two carbon-centered, and one nitrogen- and one sulfur-centered

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radical (Figure 3c). Significant alterations emerged in the signal from Met + Fenton system 13



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under anoxic conditions (Figure 3d). The spectrum was composed of signals of spin-adducts of

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four reactive species: HO•, only one carbon-, one nitrogen- (different than in the experiments

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performed in air), and sulfur-centered radical. These changes imply that O2 is involved in the

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formation of products of Met oxidation.

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Figure 4. Relationship between AA and hydrophobicity/hydrophilicity. (a) Side-chain

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hydrophobicity. AA-hydrophobicity relationship was different for FAA with uncharged and

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charged side-chains. (PU/C = 0.001);21 (b) Residue hydrophobicity normalized to Gly (PU/C =

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0.025);22 (c) Hydrophilicity (z1-scale) (PU/C = 0.001);23 (d) Side-chain hydrophobicity (PU/C = 14


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0.326);24 (e) Calculated hydrophobicity (PU/C = 0.037);25 (f) Calculated descriptor of

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hydrophilicity (z1) (PU/C = 0.003);26 (g) Calculated descriptor of hydrophilicity (F4) (PU/C =

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0.001);27 (h) Hydrophobicity parameter Log P normalized to Log P (Gly) (PU/C = 0.001);28 (i)

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Consensus hydrophobicity scale (PU/C < 0.001);29 (j) Hydrophobicity parameter Log P (this

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paper) (PU/C = 0.005). R – coefficients of correlation, * significant at 0.05 level; ** significant at

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0.01 level.

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AA values and hydrophobicity for all FAA showed positive correlation, regardless of

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hydrophobicity scale applied (Figure 4). It is important to note that some scales describe

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hydrophilicity (Figure 4c, f, g), which is reciprocally proportional to hydrophobicity. For these

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scales, negative R values were obtained. When FAA were divided to charged and uncharged

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FAA, a different result emerged. Namely, AA of charged FAA showed negative or no significant

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correlation with hydrophobicity, with an exception of one scale (Figure 4b). On the other hand, R

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values for uncharged FAA were higher compared to all FAA. Homogeneity test showed that

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AA-hydrophobicity relationships for uncharged and charged FAA are different, with an

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exception of one hydrophobicity scale (Figure 4d). Relationships can be described also by linear

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equations, but these are not comparable because hydrophobicity scales differ (e.g. some are

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normalized to Gly, in some Gly was appointed with 0 hydrophobicity, etc). R values for non-

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polar and polar FAA were not higher compared to uncharged FAA taken together (data not

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shown). Slopes were similar for all (sub)groups and homogeneity test showed that AA-

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hydrophobicity relationships for non-polar and polar FAA are not different (P > 0.05), except for

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two scales.22,23 Further, contributions of functional groups to hydrophobicity/hydrophilicity of all

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FAA were established. From this, the relationships between group contribution to

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hydrophobicity/hydrophilicity and to AA were calculated (Table S4). A very strong positive

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(negative for hydrophilicity) correlation was obtained for all 10 scales.

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A significant positive correlation was found between AA and different steric parameters

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(Figure 5a–c). Correlation coefficients were higher for uncharged and charged FAA taken

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separately, compared to all FAA. Homogeneity test showed that AA-stericity relationships for

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these two groups do not differ.

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Figure 5. Relationship between AA and steric parameters and polarity. FAA with uncharged

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side-chain were divided to non-polar and polar (right column). (a) Length of the side-chain,

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measured in the direction in which it is attached to the Gly backbone;28 (b) Normalized van der

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Waals volume of side-chain;28 (c) Molar volume (cm3/mol) of FAA in the solvent at neutral

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pH;30 (d) Polarity.31 R – coefficients of correlation, * significant at 0.05 level, ** significant at

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0.01 level. PN/P < 0.05 – relationships for FAA with non-polar and polar side-chains are different.

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AA-stericity/polarity relationships were not different for FAA with uncharged and charged side-

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chains (PU/C > 0.05).

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AA of polar FAA did not correlate with two steric parameters – side-chain length and molar

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volume. Correlation coefficients for non-polar FAA were higher compared to R values for

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uncharged FAA. Homogeneity test did not show a difference in AA-stericity relationships

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between non-polar and polar FAA. We noted that Tyr and DOPA had a strong impact on

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calculus for steric parameters. These two FAA show dual hydrophobic (due to aromatic ring) and

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polar (due to hydroxyl group) nature,28 which apparently complicates their interactions and

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consequently the analysis. Negative correlation between AA and polarity was observed for all

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FAA subgroups (Figure 5d). In addition to hydrophobicity, polarity appears to be the strongest

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correlator with AA. Non-polar and polar FAA showed significantly different AA-polarity

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relationships according to homogeneity test.

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A strong positive correlation was found between AA and hydration numbers (Figure 6a). The

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analysis included non-polar FAA and Ser. It appears that Ser did not affect the relationship.

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Decrease of temperature is known to promote hydration of amino acids.34–36 Figure 6b shows AA

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for non-polar FAA at T = 274 K. AA values were higher compared to AA at 293 K (Table S2),

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with an exception of Gly. For Ala and Phe, an increasing trend could be observed although it was 17



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not statistically significant. We have also tried to perform experiments at high T (323 K and 333

286

K), but this was hampered by temperature-sensitivity of DEPMPO adducts.

287 288

289 290

Figure 6. Relationship between antioxidative activity (AA) and hydration and the effects of

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temperature decrease on AA. (a) Correlation between AA and hydration number. Only one polar

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side-chain FAA (Ser) was included into the calculus. Results without Ser: R = 0.978 (P< 0.001);

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y = 0.216x - 1.320. (b) AA of non-polar FAA at 274 K. In control system (FAA-free Fenton

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reaction) AA = 0 ± SE (dashed lines). * Significantly different compared to control (P < 0.05). a

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Significantly different compared to AA of the same FAA at 293 K (P < 0.05)

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DISCUSSION

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Trp, Phe, Leu, Ile, His, and Arg showed the highest AA against Fenton system among

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proteinogenic FAA. These results are in general agreement with kradiolysis (M-1s-1): Trp (13 × 109),

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Tyr (13 × 109), Phe (6.9 × 109), His (4.8 × 109), Arg (3.5 × 109), Ile (1.8 × 109), and Leu (1.7 ×

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109).9,10 Tyr is obviously an exception, showing lower activity in the Fenton reaction than in

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water radiolysis system. DOPA also shows discrepancy between AA (lower than Ile and Leu)

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and rate constant in water radiolysis system (2.6 × 109 M-1s-1 for L-DOPA; 4.1 × 1010 M-1s-1 for

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DL-DOPA).19 Different activity of Tyr and DOPA in these two systems is further confirmed by

304

their negative impact on correlation between AA and kradiolysis. Dual antioxidative/pro-oxidative

305

nature of DOPA most likely accounts for these effects. Namely, DOPA can reduce Fe3+ back to

306

Fe2+ thus promoting Fenton chemistry.37 In line with this is our observation that DOPA solution

307

shows considerably lower static ORP compared to other FAA (except HCY). One of the main

308

products of reaction of Tyr with HO• is DOPA,38 which in turn decreases the value of AA of Tyr.

309

The scheme of pro-oxidative activity of Tyr and DOPA in the Fenton system is presented in

310

Figure S3. It is noteworthy that aromatic FAA are involved in the production of genotoxins and

311

other harmful byproducts of wastewater treatment.5 It appears that Fenton chemistry is highly

312

efficient in their degradation, so it might be applicable in decreasing pertinent biohazard risk.

313

Gly, Ala, Glu, Asn, Asp, Ser, Thr, and Gln did not show (almost) any AA here. This could be

314

explained by much slower reactions of these FAA compared to spin-trap DEPMPO, which

315

competes with FAA for HO•. The outlined group of FAA is at the bottom of the rank order of

316

kradiolysis, which range from 1.7 × 107 M-1s-1 for Gly to 5.4 × 108 M-1s-1 for Gln,10 whereas the rate 19



Page 20 of 32

317

constant for the trapping of HO• radical by DEPMPO is estimated to be between 7.1–7.8 × 109

318

M-1s-1.20 On the other hand, Lys, which exhibited substantial AA here, shows kradiolysis (3.5 × 108

319

M-1s-1) that is lower compared to Gln. This discrepancy could be explained by the ability of Lys

320

to sequester iron,39 which reflected positively on AA in the Fenton system. Observed negative

321

AA values for Gly, Ala, Asp, Asn, and Glu, although statistically non-significant, require some

322

explanation. Static ORP of these FAA were similar to most other examined FAA, so a possibility

323

is that they are capable of reducing Fe3+ can be excluded. On the other hand, structures of these

324

FAA are dominated by carboxyl and amino groups, either because their side-chains are small

325

(Gly and Ala) or because those groups are also present in the side-chain (Asp, Asn, and Glu).

326

Carboxyl and amino groups can form coordinate bonds with iron,40 and might potentially affect

327

Fenton reaction intermediates in a way that promotes HO•-producing or Fe3+-recycling branch of

328

the system.41.42 Finally, it appears that the composition of FAA pools in natural waters is 'shaped'

329

by susceptibility of FAA to oxidation/degradation via Fenton reaction. Namely, Glu, Gly, Asp,

330

and Ser are the most abundant in river waters, whereas Gly, Ser, Ala, Lys, Glu, and His are most

331

abundant FAA in seawater.1,43 All these FAA, except Lys and His, showed weak AA in the

332

Fenton system. In addition, analyses of FAA pools in wastewater have shown a similar trend of

333

prevalence of FAA that are not prone to Fenton-provoked oxidation, such as Ser, Gln, and Thr,44

334

and Glu, Ala, and Gly.45

335

Cys and HCY exhibited different redox properties. The reaction of HCY with HO• gave rise

336

exclusively to thiyl radical (HCY-S•), which was not the case with Cys. In addition, HCY

337

showed considerably lower static ORP than Cys. Although HCY differs from Cys by only one -

338

CH2- group in the side-chain, this could significantly impact its properties. One potentially

339

important factor is the inductive electron-withdrawing effect of -COO- and -NH3+, which 20


Page 21 of 32


340

weakens down the side-chain and is known to affect reactions with free radicals.46 It is important

341

to point out here that thiyl radical is involved in the formation of different end-products in

342

aqueous systems.38 The product of Cys oxidation – Cys-SO2H is abundant in seawater, whereas

343

Cys is present only in traces.1 Finally, the results on HO•-mediated oxidation of Met confirmed

344

our previous findings that have been obtained using another spin-trap (BMPO). Proposed

345

structures of paramagnetic products have been discussed previously.11 In contrast to Cys and

346

HCY, derivatives of Met in the Fenton system appear to be affected by molecular oxygen. Redox

347

chemistry of sulfur-containing FAA and thiyl radical in aqueous systems requires further

348

examination.

349

We found a general positive correlation between hydrophobicity and AA. The strategy of most

350

previous SAR studies has been to concurrently examine a wide spectrum of compounds in order

351

to come to some general conclusion/rule. However, the analysis of correlation between

352

physicochemical properties and AA of FAA showed be more informative when essential

353

characteristics of FAA – the presence of charged and polar groups on the side-chain, were taken

354

into consideration. Coefficients of correlation between AA and hydrophobicity, stericity, and

355

polarity (14 different scales) were larger when uncharged and charged FAA were considered

356

separately, than in the setup where all FAA were taken together. This strongly implies that the

357

division of all FAA to uncharged and charged was justified. It should be pointed out that in

358

comparison to other (sub)groups, FAA with charged side-chains showed higher R values for AA-

359

stericity and AA-hydrophobicity relationships. However, our calculus included only four FAA

360

with charged side-chains, so there might be some overfitting present. In addition, dibasic FAA

361

can sequester Fe2+ from reacting with H2O2,39 which most likely contributed to higher AA

362

compared to FAA with negatively charged side-chains. On the other hand, Arg and Lys show 21



Page 22 of 32

363

higher hydrophobicity as well as longer side-chains and larger volumes compared to Asp and

364

Glu. Hence, high correlation coefficients for charged FAA might represent a consequence of a

365

chance match of two sets of vaguely related properties and should be taken with caution.

366

AA of uncharged FAA showed positive correlation with hydrophobicity and steric parameters,

367

and negative correlation with polarity. In line with this is the positive contribution of

368

hydrophobic functional groups – aromatic rings, CH (branching), and CH2 (chain length) to AA,

369

and the negative contribution of polar groups. A similar trend for group contribution in reactions

370

with HO• has been observed previously via meta-analysis of a larger number of different

371

compounds.33 It is important to note that the contribution of functional groups to AA is strongly

372

positively linked to their contribution to hydrophobicity. Non-polar and polar FAA showed

373

similar AA-hydrophobicity relationships, which implies that the hydrophobicity of FAA defines

374

their AA regardless of the presence of polar groups in the side-chain. A very strong positive

375

correlation (R = 0.977) was found between AA and hydration numbers of non-polar FAA. We

376

cannot be sure whether this is the case for other FAA, since hydration number of only one polar

377

FAA was available.32 There is a number of other hydration scales, some of which include a wider

378

range of amino acids, but these apparently suffer from different shortcomings.32 Hydrophobic

379

hydration is the key contributor to the hydration of FAA with non-polar side-chains. Water shell

380

is formed around non-polar side-chains, and the number of hydrating water molecules increases

381

with hydrophobicity/length of the side-chain.47,48 Hydration number is temperature-sensitive, and

382

the change applied here is known to promote hydrophobic hydration.34–36 According to our

383

results, increased hydrophobic hydration leads to higher AA. The only FAA that did not exhibit

384

higher AA at lower temperature was Gly, which does not have side-chain that could be a subject

385

of hydrophobic hydration. Hydrophobic hydration might make FAA a 'larger target', thus 22


Page 23 of 32


386

increasing the probability of collision with HO•. Importantly, the movement of HO• in water

387

involves hydrogen exchange chain-reaction with water molecules acting as bridges via reaction:

388

HO• + H2O → H2O + HO•, but transfer appears not to go through hydrogen bonds.49,50 Water

389

molecules surrounding non-polar side-chains show higher number of hydrogen bonds compared

390

to bulk water,18 so the number of hydrogen bonds that are formed between the shell and bulk

391

water is decreased. Transfer of HO• from the bulk water to the water molecules in the

392

hydrophobic hydration shell and further to FAA might be promoted by the lower density of

393

hydrogen bonds down that route. It appears that in addition to the size of (hydrated) target, the

394

structure of water around FAA might be important for AA. Hydrophobic and polar (and ionic)

395

hydration show substantial differences.34,35 According to our findings that AA increases with

396

hydrophobicity and decreases with polarity, water organized around FAA in the manner of

397

hydrophobic hydration might have a critical role in 'catching' HO•. Blom and colleagues have

398

noted that it only appears simple to determine the number of water molecules that are needed to

399

solvate an amino acid.51 In relation to this, it is tempting to speculate that hydration numbers for

400

some other FAA could be estimated from rate constants for reactions of FAA with HO• at

401

different temperatures.

402

Finally, it is noteworthy that natural and waste waters contain another larger amino acids pool

403

known as dissolved combined amino acids. This pool consists of small peptides, protein

404

fragments, amino acids linked to sugars, and amino acids adsorbed onto humic acids and other

405

materials.1,3 Our results imply that the reactivity of FAA with HO• is defined by the properties of

406

their side-chains (for example, AA of Gly and the contribution of NH3+CHCOO- group were

407

next to zero). This has been also reported for short peptides and protein fragments.12,13 Goshe

408

and co-authors have shown that peptide backbone is rarely a target for HO• and that this radical 23



Page 24 of 32

409

prevalently reacts with side-chains.52 Altogether, it appears that in addition to FAA, our findings

410

might be relevant for understanding the process of oxidation/modification of dissolved combined

411

amino acids in water systems.

412

ACKNOWLEDGMENTS

413

This work was supported by the Ministry of Education, Science and Technological

414

Development of the Republic of Serbia, Grant numbers 173017, III43010 and 173014.

415 416

Supporting Information Available

417

Scales of physicochemical properties of amino acids, tables listing AA, k, ORP values, and

418

group contribution to hydrophobicity, concentration-AA curves for Leu, Ile, Nle, Trp, and Phe,

419

curves of decrease of EPR signal intensity in the presence of Cys and HCY, and scheme of pro-

420

oxidative activity of Tyr and DOPA. This information is available free of charge via the Internet

421

at http://pubs.acs.org/.

422

24


Page 25 of 32


423

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