Assessing the Indirect Photochemical Transformation of Dissolved

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Assessing the indirect photochemical transformation of dissolved combined amino acids through the use of systematically designed histidine-containing oligopeptides Chiheng Chu, Rachel A. Lundeen, Michael Sander, and Kristopher McNeill Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b03498 • Publication Date (Web): 01 Oct 2015 Downloaded from http://pubs.acs.org on October 12, 2015

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1

Environmental Science & Technology

Manuscript

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Assessing the indirect photochemical transformation of dissolved combined amino acids

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through the use of systematically designed histidine-containing oligopeptides

5 6

Chiheng Chu, Rachel A. Lundeen, Michael Sander, and Kristopher McNeill *

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Institute of Biogeochemistry and Pollutant Dynamics (IBP), Department of Environmental Systems Science, ETH Zurich, 8092 Zurich, Switzerland

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*Corresponding author

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Kristopher McNeill

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Tel. +41 (0)44 6324755; Fax. +41 (0)44 6321438

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Email: [email protected]

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Number of Figures:

4

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Number of Tables:

0

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Total word count:

6991

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1 ACS Paragon Plus Environment


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Abstract

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Photooxidation is an important abiotic transformation pathway for amino acids (AAs) in

23

sunlit waters. While dissolved free AAs are well studied, the photooxidation of dissolved

24

combined AAs (DCAAs) remains poorly investigated. This study was a systematic investigation

25

of the effect of neighboring photostable AA residues (i.e., aliphatic, cationic, anionic, aromatic

26

residues) on the environmental indirect photochemical transformation of histidine (His) in His-

27

containing oligopeptides. The pKa values of His in the peptides were found to be between 4.3

28

and 8.1. Accordingly, the phototransformation rate constants of the His-containing peptides were

29

highly pH-dependent in an environmentally relevant pH range with higher reactivity for neutral

30

His than for the protonated species. The photostable AA residues significantly modulated the

31

photoreactivity of oligopeptides either through altering the accessibility of His to

32

photochemically produced oxidants or through shifting the pKa values of His residues. In

33

addition, the influence of neighboring photostable AA residues on the sorption-enhanced

34

phototransformation of oligopeptides in solutions containing chromophoric dissolved organic

35

matter (CDOM) was assessed. The constituent photostable AA residues promoted sorption of

36

His-containing peptides to CDOM macromolecules, through electrostatic attraction, hydrophobic

37

effect, and/or low-barrier hydrogen bonds, and subsequently increased the apparent

38

phototransformation rate constants by up to two orders of magnitude.


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INTRODUCTION

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Dissolved free and combined amino acids (AAs) make up the largest fraction of the

41

identifiable pool of dissolved organic nitrogen1,2 and a major source of bioavailable nitrogen for

42

aquatic microorganisms.3,4 While biotic and abiotic transformation processes of dissolved AAs

43

are of great biogeochemical importance, these processes are currently not well understood.

44

Uptake into microbial cells is expected to be an important environmental fate for free AAs. By

45

comparison, dissolved combined AAs (DCAAs; e.g., peptides and proteins) are structurally

46

more complex5,6 and require further hydrolysis to liberate smaller AAs before they can be

47

assimilated by microorganisms. DCAAs are susceptible to abiotic transformation processes

48

occurring in surface waters, including photochemical transformation1 and sorption to inorganic

49

particles7 and natural organic matter.8,9

50

The photochemical transformation of photolabile free AAs, through either direct or indirect

51

photooxidation, has been assessed in sunlit waters.10-14 Yet few studies have examined the

52

susceptibility of these AAs to phototransformation when present as DCAAs.5,6 Recent studies on

53

dissolved AAs photochemistry have focused on examining the photooxidation of free histidine

54

(His)10,15 and His residues in intact proteins.16 His does not undergo direct phototransformation

55

by sunlight but rather the photoreactivity of His in natural waters is dominated by reaction with

56

singlet oxygen (1O2).10 In addition, the 1O2-mediated phototransformation of free His is pH-

57

dependent with higher reactivity of neutral His than for the protonated species.15,17 This pH-

58

dependence modulates His reactivity in natural waters because the imidazole moiety has a pKa of

59

6.0 and hence His undergoes protonation/deprotonation reactions in the environmentally relevant

60

pH range.



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Studies examining abiotic processes (e.g., photochemical transformation and sorption) of free

62

His provide a suitable foundation to further our understanding of His photooxidation in the

63

context of higher order structures (i.e., DCAAs). Previous studies suggested that the photolabile

64

residues exclusively dominated the photoreactivity of oligopeptides.18-20 However, other studies

65

argued that the neighboring photostable residues and the relative position of photolabile residues

66

within the sequence also influenced the reactivity of oligopeptides.16,21-24 For instance, the

67

neighboring photostable AA residues may induce a shift in the pKa value of a photolabile residue,

68

which subsequently affects the photoreactivity of DCAAs. Shifts in the pKa values of AAs and

69

the underlying mechanisms that result in these shifts have been extensively studied by

70

biochemists.25-29 The conformation, functionality and stability of many oligopeptides and

71

proteins are tied to structure-dependent alterations of AA pKa values.30-34 Likewise, the

72

environmental photoreactivities of DCAAs are expected to vary depending on the pKa values of

73

their constituent photolabile residues, especially for AAs that have environmentally relevant pKa

74

values, namely histidine.

75

The indirect phototransformation of DCAAs in chromophoric dissolved organic matter

76

(CDOM) solutions is of particular interest because their phototranformation rates might be

77

greatly enhanced through sorption to CDOM. In natural waters, CDOM acts both as the major

78

sensitizer of photochemically produced reactive intermediates (PPRI)35 and as an important

79

sorbent.15 PPRI play an important role in various biological and chemical transformation

80

processes. Molecules associated with CDOM have been shown to be exposed to high PPRI

81

concentrations within and near the CDOM macromolecules and undergo enhanced

82

phototransformation rates.36-42 For free His, moderate (3- to 4-fold) enhancements in apparent

83

1

O2 reaction rate constants were observed in CDOM solutions at pH values below 6.0, due to


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sorption of the protonated His to CDOM that overcompensated the lower reactivity of this

85

species with 1O2.15 The 1O2 reaction rate enhancements of His-containing DCAAs resulting from

86

sorption to CDOM are expected to be much higher if the His residue in the CDOM-associated

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DCAAs remains in its more reactive neutral form. The sorption-enhanced phototransformation

88

of DCAAs is of particular interest because sorption of DCAAs to CDOM occurs universally and

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via numerous association processes.43-45 To our knowledge, a systematic assessment of the

90

abiotic transformation processes of DCAAs has not been conducted.

91

In this study, we aim to assess the indirect photochemical transformation of His residues with

92

1

93

heptapeptides, as representative DCAAs having only primary protein structure (Figure 1). Each

94

oligopeptide contained one photooxidizable His residue that was combined with up to six

95

photostable AA residues with varying physicochemical properties in a variety of combinations.

96

We conducted 1O2-mediated photooxidation studies with the His-containing oligopeptides in

97

order (i) to examine the effects of neighboring residues (i.e., steric or charge interactions) on the

98

rates of His residue photochemical transformation and (ii) to determine how charged or

99

hydrophobic residues influence sorption-enhanced phototransformation of His-containing

100

oligopeptides in solutions containing CDOM. For aim (i), we examined a series of His-

101

containing oligopeptides that also contained either neighboring aliphatic, cationic, anionic, or

102

aromatic moieties (Figure 1) to help understand how the physicochemical properties of

103

neighboring AAs influence the photooxidation of His in DCAAs. For aim (ii), we systematically

104

designed His-containing oligopeptides with AAs that varied in charge or hydrophobicity (Figure

105

1) to help investigate the enhancements in phototransformation rates in CDOM solutions due to

106

sorption of the oligopeptides to the CDOM macromolecules via various interactions.

O2 in DCAAs. We designed His-containing oligopeptides, including di-, hexa- and



107

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MATERIALS AND METHODS

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Chemicals, CDOM and oligopeptides. Sources and preparation methods of chemicals,

109

CDOM and oligopeptides are detailed in the Supporting Information (SI). Suwannee River

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Natural Organic Matter (SRNOM, 1R101N) was chosen as the model CDOM.

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The studied His-containing oligopeptides were ascribed to five subgroups, depending on the

112

numbers and properties of constituent photostable AA residues (Figure 1): (i) charged

113

dipeptides, including HR and HH (H = histidine, R = arginine), (ii) hydrophobic dipeptides,

114

represented by HF (F = phenylalanine; F is aromatic and among the most hydrophobic amino

115

acids), (iii) charged heptapeptides, including DDDHDDD, AAAHAAA, RAAHAAR,

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RARHRAR, and RRRHRRR (A = alanine, D = aspartic acid), (iv) heptapeptides that varied in

117

hydrophobicity, including AAAHAAA, FAAHAAF, FAFHFAF, FFFHFFF (see Table S2 for

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pepetide hydrophobicity), and (v) polyfunctional peptides, including carnosine (β-AH),

119

FHGTVK and AGAHLK (K = lysine, G = glycine, T = threonine, V = valine, L = leucine).

120

Carnosine is highly concentrated in muscle and brain tissues where it acts as scavenger of

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reactive oxygen species46-48 and likely to be an important His-containing peptide in natural

122

waters. FHGTVK and AGAHLK were previously studied in our group as peptides generated by

123

trypsin digestion of glyceraldehyde-3-phosphate dehydrogenase (GAPDH).16

124

Photolysis of His-containing oligopeptides in lumichrome- and SRNOM-sensitized

125

systems. The sensitized photolyses of His-containing oligopeptides were carried out in separate

126

experimental setups over a wide pH range using either lumichrome (10 µM) or SRNOM (11.4

127

mg C/L) as 1O2-sensitizers, that resulted in homogeneous and microheterogeneous distributions

128

of 1O2 in solutions, respectively.15,37 Dipeptide solutions were prepared with the following pH-


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buffering species: sodium acetate (pH 4.0-6.0), phosphate (pH 6.0-7.8), or borate (above pH 7.8).

130

Hexapeptide and heptapeptide solutions were prepared with the following pH-buffering species:

131

ammonium acetate buffer (pH 4.0-6.0) or Tris-acetate buffer (pH 6.5-8.5). Each photolysis

132

solution contained one of the 15 oligopeptides studied at an initial concentration of 40 µM, a pH

133

buffering species (5 mM), furfuryl alcohol (FFA; initial concentrations of 40 µM), and either

134

lumichrome or SRNOM as the 1O2 sensitizer. Solutions containing oligopeptides and SRNOM

135

were stored overnight in the dark at 4 °C to allow for attainment of apparent sorption

136

equilibrium before starting the photolysis (see Section S7 for the evidence of sorption

137

equilibrium attainment). Sensitizer-free solutions prepared at pH 8 served as controls to assess

138

direct phototransformation of the oligopeptides. All photolyses were conducted in borosilicate

139

test tubes using a photochemical reactor (Rayonet) equipped with 365 nm bulbs (Southern New

140

England Ultraviolet Co., RPR-3500 Å). Aliquots were taken from the sample solution at certain

141

time points and split for quantification of FFA and oligopeptide (described below).

142

Analysis of oligopeptides. Aliquots from the photolysis experiments of individual synthetic

143

oligopeptides were sampled and the concentrations of oligopeptides were analyzed using one of

144

two methods: (i) Dipeptides were derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl

145

carbamate (AQC) using previous methods15,16,49 and subsequently analyzed by ultra high-

146

pressure liquid chromatography (UPLC, Waters ACQUITY) coupled to a fluorescence detector

147

(see SI for further details); (ii) Aliquots of hexa- or heptapeptides photolysis solutions were

148

immediately mixed with a mixture of peptide internal standards and subsequently analyzed on a

149

Waters ACQUITY nanoUPLC coupled to an Orbitrap high resolution mass spectrometry

150

(HRMS) detector equipped with electrospray ionization (ESI-HRMS, Thermo Exactive). All



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analyses were carried out in positive ionization mode. Previously published nanoUPLC-ESI-

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HRMS methods16 were followed, as detailed in the SI.

153

Determination of steady-state 1O2 concentration. FFA was used as a probe to determine

154

the steady-state 1O2 concentration in the bulk aqueous phase ([1O2]aq). FFA concentrations were

155

determined using a Waters ACQUITY UPLC coupled with a photodiode array detector. Detailed

156

information on sample preparation, separation by UPLC and detection are provided in the SI. A

157

steady-state 1O2 concentration was obtained by dividing the pseudo-first-order rate constant of

158

FFA FFA degradation by its reaction rate constant with 1O2 ( krxn = 8.3 × 107 M–1s–1).50 In SRNOM-

159

containing solutions with a microheterogeneous distribution of 1O2, the FFA-measured 1O2

160

concentration reflects the apparent 1O2 concentration in aqueous phase.

161

Calculation of 1O2 reaction rate constants of oligopeptides. The apparent reaction rate

162

constants (krxn, M–1s–1) of oligopeptides were calculated by dividing each pseudo-first-order

163

phototransformation rate constant by the FFA-measured 1O2 concentration. In lumichrome-

164

containing systems with homogenous 1O2 distributions, the determined second-order rate

165

constants for oligopeptides represent true bimolecular rate constants. In the SRNOM-containing

166

solutions with microheterogeneous distributions of 1O2, sorbed oligopeptides experienced higher

167

local 1O2 concentration than the FFA-measured 1O2 concentration in bulk solution. Thus, the

168

apparent krxn are higher than the intrinsic oligopeptide rate constants.

169

Calculation of pH-dependent 1O2 reaction rates of oligopeptides in lumichrome-

170

sensitized systems. The pH-dependent reaction rate constants in lumichrome-sensitized systems

171

( k aqpred ) were modeled considering the respective reaction rate constants of deprotonated and

172

protonated His residues in oligopeptides ( krxn and

His0

His+ rxn

k

), and their respective fractions (

f

0

His

and 8

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f

+ His

) (Equation 1). We adopted a 3700-fold higher 1O2 reaction rate for the neutral His than

174

protonated species in His-containing oligopeptides from a previous study (i.e.,

175

3700).15 His0

aq

0

His rxn

k

His+

krxn,calc = krxn fHis + krxn fHis 0

176 177

(૚)

+

The fractions of deprotonated or protonated His residues (

His+

/ krxn =

f

0

His

and

f

+ His

) were calculated

based on the pKa of imidazole sidechains and the solution pH (Equation 2 and 3).

fHis =

1 + 1+[H ]/Ka

(૛)

fHis =

1 + 1+Ka /[H ]

(૜)

0

+

178

The measured oligopeptide phototransformation rate constants in lumichrome-sensitized

179

systems ( k aqrxn ) at various pH values were fit to Equation 1. The resulting fit allowed for

180

aq calculation of oligopeptide reaction rate constants ( krxn,calc ) as a function of pH as well as the

181

His acquisition of krxn and the pKa values of His imidazoles in these oligopeptides (Figure 1).

0

182 183

RESULTS AND DISSUSSION

184

Phototransformation rates of His-containing oligopeptides in lumichrome-sensitized

185

systems. The photochemical transformation rate constants of His-containing oligopeptides in

186

lumichrome-sensitized systems were assessed as a function of solution pH. The steady-state

187

concentrations of 1O2 in lumichrome-sensitized systems are summarized in the SI (Table S3 and



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188

S4).

189

experiments, followed first-order kinetics at all investigated pH values (Figure 2a, shows

190

AAAHAAA as an example). The pseudo-first-order phototransformation rate constants (kobs, s–1)

191

of AAAHAAA increased strongly from pH 4.1 to pH 8.0. Control experiments with no 1O2

192

sensitizer showed no measurable direct phototransformation of AAAHAAA (Figure 2a). The

193

apparent 1O2 reaction rate constants ( k rxn , M–1s–1) of AAAHAAA were calculated by dividing

194

kobs by the FFA-measured 1O2 concentrations (Figure 2c and 2d).

195

The phototransformation of each His-containing oligopeptide, determined in separate

aq

Following the same approach as described above for AAAHAAA, the

aq rxn

k of each His-

196

containing oligopeptide was determined in lumichrome-sensitized systems at various pH values

197

(Figure 2). The

198

pH. The increases were well described by Equation 1 (Figure 2b-e) and therefore followed the

199

pH dependence of the reactivity of free His with 1O2, where the neutral imidazole in His0

200

exhibited much higher reactivity than the protonated imidazolium form, His+.15 Furthermore,

201

none of the His-containing oligopeptides underwent direct phototransformation in sensitizer-free

202

solutions (data shown only for AAAHAAA in Figure 2a), consistent with previous studies on

203

free His photochemistry.15 The good fits of experimental

204

investigated oligopeptides validate using

205

reaction rate constants in lumichrome-sensitized systems (Table S2 and S3).

k values of all His-containing oligopeptides with 1O2 increased with solution aq rxn

k

aq rxn

data by Equation 1 for

aq to represent the 1O2-mediated oligopeptide krxn,calc

206

Effect of neighboring photostable AAs on the photoreactivity of His in oligopeptides. The

207

data in Figure 2 show that the photoreactivity of His residues in the studied oligopeptides is

208

differentially modulated by neighboring residues. Neighboring photostable AAs may have three

209

effects on photoreactivity of His in oligopeptides: (i) shifts in the pKa of His alter the ratio of 10 ACS Paragon Plus Environment

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more reactive (His0) to less reactive (His+) forms; (ii) electronic effects may influence the

211

intrinsic reactivity of His toward 1O2; and (iii) the accessibility of His to 1O2 may be depressed

212

by aggregation or steric hinderance.

213

To assess the first possibility, the pKa values for the His imidazole residues were extracted for

214

each studied oligopeptide from fits of phototransformation rates to Equation 1. The inflections

215

in Figure 2 indicate the shifts of pKa values of His in the oligopeptides, which ranged from 4.3

216

(FFFHFFF) to 8.1 (DDDHDDD) (Figure 1).

217

The pKa values of His residues neighbored by negatively and positively charged AAs

218

increased and decreased, respectively, relative to free His (pKa = 6.0). The shifts in the pKa

219

values can be rationalized on the basis of electrostatic effects. For instance, the imidazolium

220

form of His in DDDHDDD (pKa = 8.1) was stabilized by the negatively charged carboxylate

221

groups of D, thereby requiring high pH values larger than 6.0 to achieve deprotonation. The

222

opposite was true for RRRHRRR in which the positively charged guanidinium in R

223

electrostatically hindered protonation of His (pKa = 4.7). The low pKa values of 4.6 and 4.3 for

224

FAFHFAF and FFFHFFF were likely due to formation of peptides aggregates (discussed in

225

more detail below). Protonated His is known to be stabilized by hydrogen bonding with water.51

226

This stabilization effect is diminished for His in aggregates due to limited ability of water to

227

interact with the imidazolium sidechain. We attribute the high pKa values of 7.4 and 7.6 for HR

228

and HF, respectively, to an internal hydrogen-bond between the imidazolium group and the C-

229

terminal carboxylate (see insert). While hydrogen-bond is, in principle, possible also for HH, the

230

observed pKa value of His in HH (6.2) is believed to depict only the C-terminal His imidazole

231

group, which cannot participate in internal hydrogen-bonding with the C-terminal carboxylate.



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Internal hydrogen-bond in HR, HF, and HH 232

The results suggest that the electrostatic effects and the microenvironment induced by

233

neighboring AA residues may significantly shift the pKa values of His in oligopeptides, thereby

234

also affecting the protonation equilibrium and thus its reactivity towards 1O2. For example, at pH

235

5.0, the

236

RAAHAAR, (2.4 ± 0.1) × 107 M–1s–1 for RARHRAR, and (4.7 ± 0.4) × 107 M–1s–1 for

237

RRRHRRR, which corresponds to a 23-fold range in the reactivity in this series (Figure 2c).

k values were (0.2 ± 0.1) × 107 M–1s–1 for DDDHDDD, (0.8 ± 0.1) × 107 M–1s–1 for aq rxn

238

In addition to changes in the reactivity induced by shifts in the His pKa value, there was also

239

evidence for electronic effects that modulated the intrinsic photoreactivity of the His residue in

240

the oligopeptides. To separate the electronic effects from the pKa effect,

241

for comparison (Figure 1). In this study, the four most reactive oligopeptides with

242

more than 50% greater than free His (6.5 × 107 M–1s–1) were all dipeptides: HH (1.3 × 108 M-1 s-

243

1

244

2e). The high 1O2 reactivity of HH can be rationalized by two photoreactive sites in HH;

245

however, this rationalization does not hold for the other dipeptides. The cause of the high

246

reactivity of these His-containing dipeptides is not known, nor is it known whether it is a general

247

phenomenon for other His-containing dipeptides.

248 249

0

His rxn

k

values were used 0

His rxn

k

values

), HF (1.3 × 108 M-1 s-1), HR (1.4 x 108 M-1 s-1), and carnosine (1.1 x 108 M-1 s-1) (Figure 1 and

The photochemical data also indicates evidence for steric effects reducing the reactivity of certain oligopeptides. In particular, variations of 1O2-mediated

0

His rxn

k

were observed in the 12

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0

His rxn

k

value of FAAHAAF (6.6 × 107 M–1s–1) was

250

hydrophobic heptapeptide series. While the

251

similar to free His (6.5 × 107 M–1s–1), FAFHFAF and FFFHFFF showed much lower reactivity

252

(1.3 × 107 M–1s–1 and 8.4 × 106 M–1s–1, respectively) (Figure 1 and 2d). We subsequently

253

hypothesized that intra- and interpeptide aggregation for FAFHFAF and FFFHFFF occurred

254

between the F residues when peptides were dissolved in water (as F is among the most

255

hydrophobic AAs). In an aggregate, the encapsulated His residues could be significantly

256

protected from 1O2 oxidation, similar to the protective effect observed where His residues were

257

buried within a folded protein structure.16

258

To test the hypothesis of aggregation-induced decrease in 1O2 reactivity, the 1O2-mediated

259

photolyses of AAAHAAA, FAAHAAF, FAFHFAF and FFFHFFF (separately) were further

260

conducted in ethanol, in which aggregation through the hydrophobic effect should be strongly

261

diminished. Photolysis results show that much higher

262

FFFHFFF ((5.5 ± 0.1) × 107 M–1s–1 and (5.4 ± 0.3) × 107 M–1s–1, respectively) in ethanol than in

263

water ((1.0 ± 0.1) × 107 M–1s–1 and (9.3 ± 0.1) × 106 M–1s–1, respectively), consistent with

264

aggregation-induced decrease in 1O2 reactivity in water. By comparison, AAAHAAA and

265

FAAHAAF showed no evidence of forming aggregates in water and their measured

266

were quite close in water ((7.0 ± 0.2) × 107 M–1s–1 and (6.7 ± 0.2) × 107 M–1s–1, respectively) and

267

in ethanol ((6.3 ± 0.8) × 107 M–1s–1 and (6.2 ± 0.1) × 107 M–1s–1, respectively). Interestingly,

268

while the

269

compared to water, they were still ~10% lower than the

270

suggesting that F residues directly adjacent to His in the oligopeptide sequence decreased the

values were found for FAFHFAF and

0

His rxn

k

values

0

His rxn

k

0

His rxn

k

values of FAFHFAF and FFFHFFF measured in ethanol were enhanced greatly 0

His rxn

k

of AAAHAAA and FAAHAAF,



Page 14 of 33

271

reactivity (Figure 2d). These slightly smaller values may reflect limited effect on accessibility of

272

His to 1O2 due to steric hindrance caused by the large neighboring AAs.

273

Sorption-enhanced phototransformation of His-containing oligopeptides in SRNOM1

274

sensitized systems. The pH-dependent

275

oligopeptides were established in lumichrome-sensitized systems. Based on those results, the

276

pH-dependent photochemistry of His-containing oligopeptides was investigated separately in

277

SRNOM-sensitized systems, in which sorption to the sensitizer could play a role. Below we

278

discuss the phototransformation results based on the type of constituent photostable AA residues

279

in the designed oligopeptides: aliphatic, cationic, anionic, aromatic, and polyfunctional (see

280

Figure 1 for definitions and Section S5).

281

Phototransformation

of

His

in

O2 reaction rate constants of His-containing

aliphatic

AA-containing

The

oligopeptides.

282

phototransformation of AAAHAAA was investigated to assess the effect of aliphatic residues on

283

peptide photoreactivity in SRNOM solutions. At solution pH above 6.0, the experimental

284

values for AAAHAAA obtained in SRNOM-sensitized systems were in good agreement with

285

the

286

AAAHAAA was experiencing the bulk 1O2 steady-state concentration and its reactivity was not

287

affected by the presence of SRNOM. At solution pH below 6.0, a large enhancement on the

288

AAAHAAA k CDOM was observed over rxn

289

constant is consistent with sorption of AAAHAAA to SRNOM. The pH-dependent enhancement

290

was in accordance with the protonation of His residue in AAAHAAA, suggesting that the

291

sorption was driven by electrostatic attraction between the positively charged His imidazole and

292

negatively charged sites in SRNOM (e.g., carboxylate moieties). This enhancement is

aq

krxn,calc in lumichrome-sensitized systems (c.f., Figure

k

CDOM rxn

2c and S1), strongly suggesting that

aq

krxn,calc (Figure S1). The enhanced apparent reaction rate


Page 15 of 33


293

CDOM aq particularly apparent when computing krxn / krxn,calc which increased from a ratio of one at pH

294

values above the pKa to a ratio of 53 at solution pH values below the pKa of the His residue

295

(Figure 3b and S1). The sorption and concomitantly subsequent enhanced peptide

296

phototransformation rates below pH 6.0 were likely caused by a combined effect of increasing

297

positive charge in the pepetide and decreasing negative charge of SRNOM, due to protonation of

298

carboxylate moieties.15

299

Phototransformation of His in cationic AA-containing oligopeptides. SRNOM-sensitized

300

phototransformation of His-containing oligopeptides that contain cationic residues was

301

investigated

302

phototransformation rates of dipeptides (Figure 3a) and heptapeptides (Figure 3b). The

303

15 CDOM aq dipeptide HH had a higher krxn / krxn,calc ratio than free His. This is particularly interesting

304

because it implies that one His in HH may be in the protonated form, which acts to promote

305

sorption to SRNOM, while the other His in HH may be in the neutral form, acting as the

306

photoreactive moiety. By contrast, free His protonation increased association with SRNOM but

307

decreased the 1O2 reactivity of His because the His+ moiety is much less reactive than His0. The

308

CDOM aq dual roles of His moieties in HH lead to a higher krxn / krxn,calc ratio than free His. The dipeptide

309

CDOM aq HR, which contains a positively charged guandinium moiety, also showed higher krxn / krxn,calc

310

ratios than free His (Figure 3a). Droge et al.52 showed that cationic amine heterocycles, in

311

which the positive change was delocalized, were superior to simple amine cations in promoting

312

sorption to natural organic matter. We hypothesize that the guanidinium group of arginine is

313

more similar to cationic heterocycles than simple amines in this regard. While the R sidechain

314

pKa values may be affected by other residues in peptides and shifted to lower pH values than that

to

assess

the

effect

of

electrostatic

interactions

with

SRNOM

on



Page 16 of 33

315

of free R (pKa = 12.0, Figure 1), they were likely to be higher than tested pH (4.1-7.9) and

316

arginine sidechains were protonated at all pH values. Thus, the enhanced

317

observed over the entire measured pH range.

k

CDOM rxn

values were

318

CDOM aq The krxn / krxn,calc ratios for heptapeptides with increasing number of cationic arginine

319

residues were compared at various solution pH values (Figure 3b). At solution pH above 6.0,

320

RARHRAR and RRRHRRR showed enhanced phototransformation in SRNOM-sensitized

321

systems relative to

322

higher than RARHRAR (i.e., 6.9 at pH 7.8), due to the higher number of positively charged

323

guanidinium groups in RRRHRRR. Conversely, no enhancement of the phototransformation rate

324

CDOM aq was observed for RAAHAAR; where, RAAHAAR had nearly identical krxn / krxn,calc ratios

325

CDOM aq with AAAHAAA. At pH values below 6.0, the krxn / krxn,calc ratio for RAAHAAR and

326

RARHRAR increased. While protonation decreased the intrinsic His reactivity, it increased the

327

CDOM aq association to SRNOM and resulted in a higher 1O2 rate enhancement. The krxn / krxn,calc ratio

328

of RRRHRRR was relatively stable over all investigated pH values even at low pH values. The

329

CDOM aq differences in the krxn / krxn,calc ratios among the heptapeptides were smaller at low pH

330

CDOM aq compared to the krxn / krxn,calc ratio variations at high pH. At pH 4.1, nearly identical

331

krxn

332

CDOM aq enhancements on phototransformation rates (Figure 3b). The pH independent krxn / krxn,calc

333

CDOM aq ratios for RRRHRRR and identical krxn / krxn,calc ratios for these heptapeptides at low pH

334

suggest a maximum (around 50) of rate enhancement caused by sorption to SRNOM. This

335

1 CDOM aq maximum krxn / krxn,calc ratio is in good agreement with the concentration gradient of O2 intra-

CDOM

aq

krxn,calc .

CDOM aq The krxn / krxn,calc ratio for RRRHRRR (i.e., 47 at pH 7.8) was

aq / krxn,calc ratios of around 50 were obtained for all heptapeptides, illustrating substantial


Page 17 of 33


336

CDOM macromolecules observed by Latch et al., where a 101-102 times higher concentration of

337

1

338

(estimated at CDOM concentrations around 10 mg C/L).37

O2 was generated in intra-CDOM aggregates than the bulk aqueous 1O2 concentration

339

Phototransformation of His in anionic AA-containing oligopeptide. Figure 3e shows the

340

photolysis data of DDDHDDD in SRNOM solutions at various pH values. At pH 7.9, the

341

krxn

342

CDOM aq SRNOM. Decreasing the solution pH resulted in pronounced increases in krxn / krxn,calc ,

343

suggesting that DDDHDDD associated with SRNOM despite its repulsive charge and polarity,

344

both of which were expected to hinder sorption. We propose that low barrier hydrogen bonds

345

(LBHB) may have resulted in significant association of DDDHDDD with SRNOM at low

346

solution pH. It is known that strong LBHB form when sorbent and sorbate have comparable pKa

347

values and when solution pH approaches their pKa values.53-56 In the case of DDDHDDD,

348

negative-charge-assisted LBHB (i.e., [—O•••H•••O—]-) may form between the carboxylate

349

groups in the aspartic acid residues (Figure 1) and analogous carboxylates in SRNOM at pH

350

values between 4.0 and 6.0. At higher pH values (e.g., at pH 7.9), both the carboxylate groups in

351

the D sidechain and SRNOM were deprotonated and thus, no enhancement on

352

phototransformation was observed. Further support for the LBHB-mediated sorption affinity of

353

DDDHDDD to SRNOM was demonstrated by observed reversible association of DDDHDDD to

354

carboxylate-terminated self-assembled monolayers using a quartz crystal microbalance, as

355

detailed in the SI.

CDOM

aq / krxn,calc ratio was close to unity, suggesting weak sorption (if any) of DDDHDDD to

356

Phototransformation of His in aromatic AA-containing oligopeptides. SRNOM-sensitized

357

phototransformation of His-containing dipeptide and heptapeptides that contain hydrophobic 17 ACS Paragon Plus Environment


Page 18 of 33

358

residues was investigated to assess the effect of hydrophobic binding on peptide

359

CDOM aq phototransformation rates (Figure 3c and 3d). The dipeptide HF shows similar krxn / krxn,calc

360

ratios as free His over the measured pH range, reaching a maximum ratio of 10 at the lowest

361

studied solution pH. This suggests one apolar AA is not enough to change the binding

362

mechanism and electrostatic interactions remain dominant (Figure 3c). At solution pH values

363

above 6.0, FAAHAAF and AAFHFAA showed no enhancement in 1O2 reaction rates in

364

SRNOM solutions, which was similar to the reactivity of AAAHAAA (Figure 3d). By contrast,

365

CDOM aq apparent enhancements ( krxn / krxn,calc > 1) were observed for FAFHFAF and FFFHFFF, these

366

enhancements were identical between pH 5.0 and pH 8.0. The observed enhancements on

367

reaction rates were likely due to a combination of effects, namely hydrophobic binding to

368

SRNOM promoted by the F residues and de-aggregation of the heptapeptides. In the later case,

369

association of hydrophobic peptides with SRNOM might facilitate de-aggregation because

370

SRNOM can act as a co-solvent, an analogous role to ethanol in lumichrome-sensitized

371

photolyses described above (Figure 2d). The photoreactivity of His residues might be either

372

enhanced with increased accessibility to reactants or depressed with raised pKa of His sidechain

373

upon de-aggregation.

374

CDOM aq The krxn / krxn,calc ratios of FAAHAAF and AAFHFAA increased as the solution pH

375

decreased below 6. This finding is consistent with the protonation of His imidazole sidechain

376

(the fitted pKa value for both FAAHAAF and AAFHFAA was 6.3), which induced electrostatic

377

attraction of FAAHAAF and AAFHFAA to SRNOM and subsequently enhanced

378

CDOM aq phototransformation. For FAFHFAF and FFFHFFF, the krxn / krxn,calc ratios increased at pH

379

values below 5.0 (pKa values were 4.6 for FAFHFAF and 4.3 for FFFHFFF). The increased


Page 19 of 33


380

ratios are also in accordance with the protonation of imidazole sidechain, suggesting the sorption

381

mechanisms to be a combination of electrostatic interaction (between positively charged

382

imidazole and SRNOM) and the hydrophobic effect (between hydrophobic F residues and

383

SRNOM). In the latter case, the hydrophobic effect was enhanced at low solution pH with higher

384

SRNOM hydrophobicity. Interestingly, similar to the results obtained for cationic heptapeptides,

385

CDOM aq a maximum value of the krxn / krxn,calc ratios around 80 was obtained for aromatic heptapeptides

386

at pH 4.1 (Figure 3b and 3d), lending further support to the idea of a maximum threshold on the

387

sorption-induced enhancement of oligopeptides phototransformation rates in SRNOM solutions.

388

Phototransformation of His in polyfunctional oligopeptides. Polyfunctional oligopeptides

389

were studied to assess the sorption-enhanced phototransformation in more representative

390

DCAAs. At pH values above 6.0, no enhancement on

391

oligopeptides (Figure 3f), indicating no sorption of oligopeptides to SRNOM despite the

392

presence of hydrophobic residues (F and V in FHGTVK and L in AGAHLK). As solution pH

393

CDOM aq decreased, increased krxn / krxn,calc ratios were again observed for the polyfunctional

394

oligopeptides in accordance with the protonation of His residue, indicating the electrostatic

395

attraction between the His imidazole sidechain and SRNOM. Notably, all studied polyfunctional

396

oligopeptides had identical magnitudes of enhancements on reaction rates, despite the fact that

397

FHGTVK and AGAHLK also contained a basic amine moiety in the K sidechain. Thus, the

398

enhanced

399

CDOM aq interaction of protonated His residue with SRNOM. The krxn / krxn,calc ratios obtained in

400

SRNOM solutions indicate that electrostatic interaction dominated the sorption of polyfunctional

401

oligopeptides to SRNOM.

k

CDOM rxn

k

CDOM rxn

was observed for the polyfunctional

of FHGTVK and AGAHLK were exclusively attributed to electrostatic



Page 20 of 33

402

Environmental implications. The goal of this study was to use these systematically designed

403

oligopeptides as model DCAAs to study the relationship between their physicochemical

404

properties and their photochemical reactivity. Herein we examined the effect of photostable

405

constituent AA residues (i.e., aliphatic, anionic, cationic, aromatic AA residues, Figure 1) on the

406

photoreacitivity of His-containing oligopeptides under various environment conditions (e.g.,

407

acidic and basic pH). A visualization of the magnitude of the reactivity differences observed

408

under different conditions is presented in Figure 4. In this figure, we compared the half-lives

409

(assuming [1O2]aq of 1 pM) of the studied heptapeptides, focusing on two pH values, pH 5.0 ±

410

0.1 and pH 7.9 ± 0.2 (Figure 4).

411

In general, neighboring amino acids affected the reactivity of the His sidechain in three ways.

412

First, they led to a shift in the pKa value of the protonated His+, which in turn shifted the

413

distribution of reactive His0 and unreactive His+ forms. Second, the neighboring residues

414

affected the intrinsic reactivity of the His, as evidenced by a shift in the reaction rate constant of

415

neutral His sidechain ( krxn ). Finally, the neighboring residues affected the affinity of the

416

oligopeptides for SRNOM, which led to an apparent enhancement in the reactivity. Apparent

417

phototransformation rate constants of His-containing oligopeptides were enhanced by up to two

418

orders of magnitude upon sorption to SRNOM, where the sorption mechanisms were

419

electrostatic interaction, hydrophobic effect and/or LBHB.

0

His

420

The end result of these effects was an observed factor of approximately 400 in the half-lives

421

of the heptapeptides under lumichrome-sensitized conditions (Figure 4). Under SRNOM-

422

sensitized conditions, a similar factor of approximately 500 in reactivity was observed, but the

423

heptapeptides were generally ten or more times more reactive than under lumichrome-sensitized


Page 21 of 33


424

conditions. All of the heptapeptides were significantly more reactive at pH 5.0 with SRNOM

425

than with lumichrome and four heptapeptides, namely RARHRAR, RRRHRRR, FAFHFAF, and

426

FFFHFFF, were also more reactive at pH 7.9 with SRNOM than with lumichrome. This result

427

has wide implications for nutrient availability in surface waters, illustrating that the

428

photochemical half-life of the same oligopeptide could vary by orders of magnitude depending

429

on environmental conditions. The results of this study also have implications for predicting the

430

environmental photochemical reactivity of AA-based molecules.

431

We expect CDOM-association to play a more important role for environmental

432

phototransformation of DCAAs in the context of higher order structures. Previous studies have

433

demonstrated that both hydrophobic effect and electrostatic interactions act as important sorption

434

mechanisms of proteins to lipid bilayers, nanoparticles and humic substances.7-9,57-61 Thus, we

435

expect sorption-enhanced photochemistry for larger peptides or proteins in natural waters to be

436

driven by various sorption mechanisms. In addition to sorption-enhanced photooxidation by

437

CDOM, photoreactive AA residues might be protected from photooxidation due to antioxidant

438

properties of CDOM.13,62 For both cases, we suggest that the sorptive affinity of biomolecules to

439

CDOM needs to be considered when studying the photochemistry of AA-based biomolecules in

440

aquatic environments.



Page 22 of 33

441

Supporting Information Available

442

Supporting figures, tables, detailed experimental methods and additional experiments described

443

within the manuscript are provided. This material is available free of charge via the Internet at

444

http://pubs.acs.org.

445

Acknowledgements

446 447 448 449 450

This work was financially supported by a grant from the Swiss National Science Foundation (Project numbers 200021_138008 and 200020_159809). The authors gratefully acknowledge Betsy L. Edhlund and Lauren C. Kennedy for conducting preliminary experiments. We thank Armanious Antonius and Elisabeth M.-L. Janssen for helpful discussions and experimental support.


Page 23 of 33


His

cationic

aliphatic NH

+H

+H

CO 2–

3N

+H

pKa 6.0

krxn = 6.5

x 10 7

CO 2– +H

His (H) His 0

NH

CO 2– Ala (A)

3N

CO 2-

3N

+H

3N

β−Ala (βA)

M -1s-1

R1

NH 3N

O

+H

CO 2-

CO 2 Arg (R)

Asp (D)

pK a 12.0

pK a 3.9

3N

R2

R3 N H

H N

CO 2–

3N

Phe (F)

R5 N H

O NH

O

H N

R6

R7 N H

CO 2-

N 0

peptide

pKa (His)

His krxn (10 7 M -1s-1)

-H -R -F

HH HR HF

6.2 ± 0.1 7.4 ± 0.1 7.6 ± 0.3

12.5 ± 0.7 14.4 ± 1.6 12.7 ± 3.9

His 0

pKa (His) krxn (10 7 M -1s-1)

βAH (carnosine) 6.6 ± 0.0 6.1 ± 0.1 FHGTVK 6.2 ± 0.1 AGAHLK

O

O

R2

polyfunctional peptides

451 452 453 454 455 456 457 458

O

H N O

R2

peptide

+H

heptapeptides

N

+H

CO 2–

3N

–

dipeptides

H N

aromatic

NH 2+

H 2N

N

anionic

10.6 ± 0.3 7.3 ± 0.8 7.8 ± 0.4

R1 R 2R 3 R 5R 6R7

peptide

0

His pKa (His) krxn (10 7 M -1s-1)

DDDAAARAARARRRR-

-DDD -AAA -AAR -RAR -RRR

DDDHDDD AAAHAAA RAAHAAR RARHRAR RRRHRRR

8.1 ± 6.6 ± 6.1 ± 5.3 ± 4.7 ±

0.2 0.1 0.1 0.0 0.0

AAAFAAAAFFAFFFF-

-AAA -AAF -FAA -FAF -FFF

AAAHAAA F AAHAAF AAF HF AA F AF HF AF F F F HF F F

6.6 ± 6.3 ± 6.3 ± 4.6 ± 4.3 ±

0.1 0.0 0.1 0.1 0.1

5.8 ± 1.1 7.3 ± 0.2 6.9 ± 0.4 6.8 ± 0.2 6.7 ± 0.1 7.3 ± 6.6 ± 6.0 ± 1.0 ± 0.95 ±

0.2 0.3 0.3 0.1 0.03

Figure 1. Structures, pKa values, and 1O2-mediated reaction rate constants for the histidine (His)-containing oligopeptides employed in this study. The heptapeptides are categorized by their incorporation of charged residues, including anionic D and cationic R residues, or hydrophobic F residues. 1O2-mediated reaction rate constants of neutral His residue ( krxnHis ) and the pKa values of the His residue for each His-containing oligopeptide were obtained from fitting their observed 1O2 photochemical kinetic data using Equation 1. 0



459 460 461

Page 24 of 33

Figure 2. (a) 1O2-mediated phototransformation of His-containing oligopeptides sensitized by lumichrome at pH 4.1-8.0 (showing AAAHAAA as an example). Oligopeptide 24 ACS Paragon Plus Environment

Page 25 of 33

462 463 464 465 466 467 468 469 470 471 472 473 474 475 476


phototransformation (natural logarithm of the ratio of oligopeptide concentration at time point t and initial concentration, ln([peptide]t/[peptide]0)) was normalized to a representative 1O2 steady state concentrations of 20.0 pM in the lumichrome-sensitized photolysis experiments and plotted versus photolysis time (t). Sensitizer-free photolysis of AAAHAAA, which served as a direct photolysis control at pH 8.0, is also plotted. Solid lines represent the first-order reaction fits of the experimental data (symbols). Error bars represent the range in ion intensities of AAAHAAA measured at two ionization states in the mass spectra (singly and doubly charged). (b)-(e) Plot of lumichrome-sensitized 1O2 reaction rate constants of His-containing (b) dipeptides, (c) charged heptapeptides, (d) heptapeptides varying in degrees of hydrophobicity, and (e) polyfunctional peptides as a function of solution pH. Solid lines represent the fits of the reaction rate constants using Equation 1. Peptide photolyses results conducted in ethanol (EtOH) at pH 8.0 were also plotted in panel (d) (grey insert). The phototransformation data of AAAHAAA is plotted twice in panels (c) and (d) for comparison purposes. Error bars represent the standard deviation of fitting curve of oligopeptide phototransformation. When error bars are not visible, they are contained within the marker symbols.

477



Page 26 of 33

478 26 ACS Paragon Plus Environment

Page 27 of 33

479 480 481 482 483 484 485 486 487 488


Figure 3. CDOM-sensitized phototransformation gave higher than calculated rate constants, herein visualized as 1O2-mediated reaction rate constants of His-containing oligopeptides in CDOM aq SRNOM-sensitized photolyses divided by their calculated rate constants, krxn , at / krxn,calc various pH values, for (a) positively charged dipeptides, (b) positively charged heptapeptides, (c) hydrophobic dipeptide, (d) heptapeptides that vary in degrees of hydrophobicity, (e) negatively charged heptapeptide, and (f) polyfunctional oligopeptides. Errors bars represent the ratio of the CDOM standard deviation of the fitted curve of the experimental krxn in SRNOM solutions to the aq calculated rate constants in lumichrome-sensitized systems ( krxn,calc ). When error bars are not visible, they are contained within the marker symbols. Free His photolysis data from Chu et al.15 were re-plotted (in panel (a) and (c)) for reference.

489



490 491 492 493 494 495 496 497 498 499

Page 28 of 33

Figure 4. Half-lives of 1O2-mediated (assuming [1O2]aq = 1 pM) phototransformation of Hiscontaining heptapeptides. The effects of photostable constituent neighboring AA residues (e.g., aliphatic, cationic, anionic, and aromatic AA residues) on half-lives of His-containing oligopeptides were assessed in groups at acid (pH 5.0 ± 0.1) and basic (pH 7.9 ± 0.2) solution pH in lumichrome-sensitized systems and SRNOM-sensitized systems. The 1O2 half-lives of free His at pH 5 and pH 8 under lumichrome-sensitized conditions are plotted for comparison (dashed lines). Errors bars represent the calculated half-life errors based on the errors of experimental reaction rate constants.


Page 29 of 33


500

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19. Bertolotti, S. G.; Garcia, N. A.; Arguello, G. A. Effect of the peptide-bond on the singletmolecular-oxygen-mediated sensitized photooxidation of tyrosine and tryptophan dipeptides - a kinetic study. J. Photochem. Photobiol., B 1991, 10(1-2), 57-70. 20. Michaeli, A.; Feitelson, J. Reactivity of singlet oxygen toward amino-acids and peptides. Photochem. Photobiol. 1994, 59(3), 284-289. 21. Lundeen, R. A.; Janssen, E. M.; Chu, C.; Mcneill, K. Environmental photochemistry of amino acids, peptides and proteins. Chimia 2014, 68(11), 812-817. 22. Criado, S.; Marioli, J. M.; Allegretti, P. E.; Furlong, J.; Nieto, F. J. R.; Martire, D. O.; Garcia, N. A. Oxidation of di- and tripeptides of tyrosine and valine mediated by singlet molecular oxygen, phosphate radicals and sulfate radicals. J. Photochem. Photobiol., B 2001, 65(1), 74-84. 23. Reis, A.; Fonseca, C.; Maciel, E.; Domingues, P.; Domingues, M. R. M. Influence of amino acid relative position on the oxidative modification of histidine and glycine peptides. Anal. Bioanal. Chem. 2011, 399(8), 2779-2794. 24. Michaeli, A.; Feitelson, J. Reactivity of singlet oxygen toward large peptides. Photochem Photobiol 1995, 61(3), 255-60. 25. Naor, M. M.; Jensen, J. H. Determinants of cysteine pK(a) values in creatine kinase and alpha 1-antitrypsin. Proteins-Structure Function and Bioinformatics 2004, 57(4), 799-803. 26. Harris, T. K.; Turner, G. J. Structural basis of perturbed pK(a) values of catalytic groups in enzyme active sites. Iubmb Life 2002, 53(2), 85-98. 27. Edgcomb, S. P.; Murphy, K. P. Variability in the pK(a) of histidine side-chains correlates with burial within proteins. Proteins 2002, 49(1), 1-6. 28. Wang, P. F.; McLeish, M. J.; Kneen, M. M.; Lee, G.; Kenyon, G. L. An unusually low pK(a) for Cys282 in the active site of human muscle creatine kinase. Biochemistry 2001, 40(39), 11698-11705. 29. Cederholm, M. T.; Stuckey, J. A.; Doscher, M. S.; Lee, L. Histidine pK(a) shifts accompanying the inactivating Asp121- Asn substitution in a semisynthetic bovine pancreatic ribonuclease. Proc. Natl. Acad. Sci. U.S.A 1991, 88(18), 8116-8120. 30. Poi, M. J.; Tomaszewski, J. W.; Yuan, C. H.; Dunlap, C. A.; Andersen, N. H.; Gelb, M. H.; Tsai, M. D. A low-barrier hydrogen bond between histidine of secreted phospholipase A(2) and a transition state analog inhibitor. J Mol Biol 2003, 329(5), 997-1009. 31. Ash, E. L.; Sudmeier, J. L.; DeFabo, E. C.; Bachovchin, W. W. A low-barrier hydrogen bond in the catalytic triad of serine proteases? Theory versus experiment. Science 1997, 278(5340), 1128-1132. 32. Tobin, J. B.; Whitt, S. A.; Cassidy, C. S.; Frey, P. A. Low-barrier hydrogen-bonding in molecular-complexes analogous to histidine and aspartate in the catalytic triad of serine proteases. Biochemistry 1995, 34(21), 6919-6924. 33. McDougal, O. M.; Granum, D. M.; Swartz, M.; Rohleder, C.; Maupin, C. M. pK(a) determination of histidine residues in alpha-Conotoxin MII peptides by H-1 NMR and constant pH molecular dynamics simulation. J Phys Chem B 2013, 117(9), 2653-2661. 34. Everhart, D.; Cartier, G. E.; Malhotra, A.; Gomes, A. V.; McIntosh, J. M.; Luetje, C. W. Determinants of potency on alpha-conotoxin MII, a peptide antagonist of neuronal nicotinic receptors. Biochemistry 2004, 43(10), 2732-2737. 35. Zepp, R. G., Environmental photoprocesses involving natural organic matter. Environmental Research Laboratory, Office of Research and Development, US Environmental Protection Agency: 1987. 30 ACS Paragon Plus Environment

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Assessing the Indirect Photochemical Transformation of Dissolved

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