Influence of the Oxidation States of 4-Methylcatechol and Catechin on

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Influence of oxidation state of 4-methyl catechol and catechin on the oxidative stability of #-lactoglobulin Sisse Jongberg, Mariana Utrera, David Morcuende , Marianne N. Lund, Leif H. Skibsted, and Mario Estévez J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b02551 • Publication Date (Web): 08 Sep 2015 Downloaded from http://pubs.acs.org on September 10, 2015

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Journal of Agricultural and Food Chemistry

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Influence of oxidation state of 4-methyl catechol and catechin on the

2

oxidative stability of β-lactoglobulin

3 4 5

Sisse Jongberg a*, Mariana Utrera b, David Morcuende b, Marianne N. Lund a, Leif H. Skibsted a,

6

Mario Estévez b

7 8

a

9

1958 Frederiksberg, Denmark.

Department of Food Science, Faculty of Science, University of Copenhagen, Rolighedsvej 30,

10

b

11

University of Extremadura, 10003, Cáceres, Spain. Present address: IPROCAR Research Institute,

12

Food Technology, University of Extremadura, 10003, Cáceres, Spain

Animal Production and Food Science Department, Food Technology, Veterinary Faculty,

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e-mails: Sisse Jongberg: [email protected]; Mariana Utrera: [email protected]; David

15

Morcuende: [email protected]; Marianne N. Lund: [email protected]; Leif H. Skibsted:

16

[email protected]; Mario Estévez: [email protected].

17 18

*

Corresponding author: Sisse Jongberg, phone: +45 3533 2181, fax: +45 3533 3344.

19 20

Table of Contents categories:

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1 ACS Paragon Plus Environment


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Abstract

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Chemical interactions between proteins and phenols affect the overall oxidative stability of a given

24

biological system. To investigate the effect of protein-phenol adduct formation on the oxidative

25

stability of β-lactoglobulin (β-LG), the protein was left to react with equimolar concentration of 4-

26

methyl catechol (4MC), catechin (Cat), or their respective quinone forms, 4-methylbenzoquinone

27

(4MBQ) and CatQ, and subsequently subjected to metal catalyzed oxidation by Fe(II)/H2O2 for 20

28

days at 37 °C. Reaction with 4MBQ resulted in 60 % thiol loss and 22 % loss of amino groups,

29

whereas addition of 4MC resulted in 12 % thiol loss. Reaction with Cat or CatQ resulted in no

30

apparent modification of β-LG. The oxidative stability of β-LG after reaction with each of 4MC,

31

4MBQ, Cat, or CatQ was impaired. Especially 4MC and 4MBQ were found to be pro-oxidative

32

towards α-aminoadipic semialdehyde and γ-glutamic semialdehyde formation, as well as the

33

generation of fluorescent Schiff base products. The changes observed were ascribed to the

34

redirection of oxidation due to the blocking of thiol groups, but also to the oxidative deamination

35

pathway accelerating the production of semialdehydes and subsequently Schiff base structures.

36 37 38

Keywords

39

β-Lactoglobulin, 4-methylcatechol, catechin, quinone, protein oxidation, metal-catalyzed oxidation

40 41 42


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1

Introduction

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Plant phenolics are used in food products and pharmaceuticals for their bioactivity with

45

regards to protection against primarily lipid oxidation 1. Lately, phenolic antioxidants have also

46

been applied to various products for the protection against protein oxidation. However, in

47

combination with proteins, phenolic compounds result in different consequences to the redox status

48

of the protein, including an increase in protein oxidation where the phenols act as pro-oxidants 2,

49

but also with synergistic antioxidative effects that protect lipids 3. The ambiguous effects are likely

50

associated with the binding affinity of the phenols towards proteins which includes both covalent

51

and non-covalent bonds

52

polyphenols. The binding between proteins and phenols are controlled by a number of factors

53

including the oxidative status of the biological system, the structure of the phenol and the amino

54

acid composition and conformation of the protein. Hence, it is difficult to estimate the effect of a

55

given antioxidant in a specific system 5.

4

and in differences in antioxidant mechanisms between different

56

As recently reviewed by Le Bourvellec & Renard 4, the covalent interactions between

57

proteins and phenols include nucleophilic addition, which yields chemical bonds that may result in

58

polymerized reaction products composed of both components. Oxidation of the phenol to its

59

oxidized quinone is a prerequisite for the nucleophilic addition, and requires a one-electron transfer

60

to generate a semiquinone radical followed by a second electron transfer to yield the quinone 5. The

61

oxidation occurs spontaneously in the presence of molecular oxygen under alkaline conditions (pH

62

> 9.0) 6, or can be catalyzed by polyphenol oxidases (PPO) 7, or metallic cations 8. Scheme 1 shows

63

the two-step metal catalyzed oxidation of phenols to quinones.

64

Quinones are electrophilic compounds and react rapidly with nucleophilic side chains,

65

especially amino or thiol groups 9. Scheme 2 shows the reaction between a quinone and a protein

66

thiol group to yield a thiol-quinone (TQ) adduct (Reaction A), and between a quinone and a protein



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amino group to yield amino-quinone (AQ) adducts (Reaction B1 and B2). The thiol and amino

68

group may react with the quinone via a Michael-type addition, where a carbon atom in the aromatic

69

ring is substituted with the protein residue to yield a carbon substituted protein-phenol adduct

70

(Reaction A and B1). Additionally, amino groups may also react through a nucleophilic reaction

71

with the oxo-group of the quinone to generate iminoquinones (Schiff base-like structures) (Reaction

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B2).

73

Many previous reports have focused on the structure-affinity relationship of binding between

74

phenols and proteins and the effect of such covalent interactions on the physicochemical properties

75

and digestibility of diverse food proteins 5, 10. However, only few works have studied the oxidative

76

stability of the protein-phenol adducts 11, 12, and rarely any considered the antioxidative capacity of

77

the modified protein and its own oxidation status.

78

A better understanding of the nature and redox consequences of the protein-phenol

79

interactions is required to comprehend the chemistry behind their biological effects and apply these

80

phytochemicals in food systems so that reliable and predictable antioxidant effects are gained. The

81

aim of the present study was to determine how protein-phenol adduct formation between the

82

quinones of 4-methylcatechol or catechin affects the metal-catalyzed oxidation of β-lactoglobulin

83

(β-LG). The two phenolic compounds were added either in their reduced phenolic form or in their

84

oxidized quinoic form in order to identify possible changes in the oxidation pattern of β-LG

85

depending on the redox status of the phenolic compounds. The oxidation was followed for

86

prolonged incubation times to investigate both early and later stages of oxidation.

87 88


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2

90

2.1

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Methods and Materials Reagents β-Lactoglobulin (Mw ~18,300) consisting of a mixture of the variants A and B was isolated 13

92

from bovine milk as described by Kristiansen, Otte, Ipsen, & Qvist

93

from Scharlau SL, Sentmenat, Spain. Catechin, 4-methyl catechol, Trizma® base, 5,5 dithiobis(2-

94

nitrobenzoic acid (DTNB), N-ethylmaleimide (NEM), Trolox, Neocuproine, Amberlyst® A26, and

95

fluorescamine

96

chemicals and double-deionized (MilliQ) water were used throughout.

was

obtained

from

Sigma-Aldrich,

St.

. Malic acid was obtained

Louis,

MO.

Reagent-grade

97 98

2.2

Peroxidation of phenols by periodate resin

99

Periodate resin was prepared according to Harrison and Hodge 14 with slight modifications as

100

recently described by Jongberg, Otte, and Lund 15. 4-methylcatechol (4MC) and catechin (Cat) was

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oxidized by mixing 158 mg periodate resin with 12.4 mg 4MC or 14.5 mg Cat dissolved in

102

acetonitrile for 3 minutes during stirring. The amount of 4MC converted to 4-methylbenzoquinone

103

(4MBQ) was estimated to be ~ 95 % by HPLC-UV/Vis

104

95 mM 4MBQ or 47.5 mM catechin quinone (Cat-Q). The quinone solutions were used

105

immediately for the preparation of samples.

16

giving an estimated concentration of ~

106 107

2.3

Preparation of samples

108

Samples were prepared in 0.1 malic acid buffer (pH 5.8) to contain 5.0 mg/mL (273 µM) β-

109

lactoglobulin (β-LG) alone, or with addition of 273 µM 4MC or Cat or their corresponding

110

quinones, 4MBQ or CatQ and left to react for 10 minutes at room temperature (20 °C). In parallel,

111

blank phenol and quinone samples were prepared in the same concentrations without addition of β-

112

LG. N-ethylmaleimide (NEM)-derivatized β-LG was prepared by letting 273 µM β-LG react with



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7.2 mM NEM for 90 minutes at room temperature (20 °C). A corresponding blank NEM sample

114

was prepared in parallel without addition of protein. In total, 11 samples were prepared in triplicate,

115

six samples with protein: β-LG, β-LG-4MC, β-LG-4MBQ, β-LG-Cat, β-LG-CatQ, and β-LG-NEM,

116

and five samples without protein: 4MC, 4MBQ, Cat, CatQ, NEM.

117 118

2.4

Metal-catalyzed oxidation

119

Oxidation of all 11 samples was initiated by addition of 50 µM FeSO4 and 5 mM H2O2 both

120

dissolved in MilliQ water. The samples were oxidized in the dark at 37 °C for up to 20 days with

121

constant stirring. Prior to initiation, day-0 samples were collected for thiol and Schiff base analyses,

122

which were carried out on the same day. Samples for AAS and GGS quantification were frozen at -

123

80 °C until analysis. Onwards, samples were collected at day 1, 3, 5, 8, 12, and 20, and the same

124

procedure as for the day-0 samples was followed.

125 126

2.5

Antioxidative activity by the CUPRAC assay

127

Samples were prepared as described above (Section 2.3 Preparation of samples), though

128

including additional concentrations of 4MC, 4MBQ, Cat, and CatQ. For each, three concentration

129

levels were added to the 273 µM β-LG in 0.1 M malic acid buffer (pH 5.8), namely 137, 273, or

130

546 µM corresponding to the molar ratio between β-LG and the phenol or quinone of 1:0.5, 1:1, or

131

1:2. In parallel, blank samples without addition of β-LG were prepared, containing only 137, 273,

132

or 546 µM 4MC, Cat, 4MBQ, or CatQ, respectively. Samples containing β-LG-NEM and its

133

corresponding blank NEM was prepared as described above. An aliquot of 0.1 mL sample was

134

added to a test tube containing 1.0 mL 10 mM CuCl2 dissolved in water, 1.0 mL 1 M ammonium

135

acetate buffer (pH 7.0), 1.0 mL 7.5 mM Neucoproine in ethanol, and 1.0 mL water. The mixture

136

was mixed and left to react at room temperature (20 °C) for 30 minutes before the absorbance at


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450 nm was read on a HITACHI U-2000 Spectrophotometer. Trolox dissolved in 80 % ethanol was

138

used as standard, and data are given as trolox equivalent antioxidant capacity (TEAC) and presented

139

as mM Trolox equivalents (mean ± sd) of three independent replicates (n=3). Three different TEAC

140

values are presented; one of the model system containing β-LG, one with of the blank model system

141

without β-LG, and finally a summarized TEAC, which represents the sum of the TEAC of the blank

142

model system plus the TEAC of β-LG alone.

143 144

2.6

Quantification of thiol groups

145

Protein thiol groups in samples prior to oxidation by FeSO4/H2O2 were determined by

146

derivatization with DTNB (5,5 dithiobis(2-nitrobenzoic acid) 17 and recently described by Jongberg

147

et al.

148

three independent replicates (n=3). All protein-containing samples were subtracted the contribution

149

measured in the corresponding blank sample without protein.

15

. The thiol concentrations given as nmol thiol/mg protein are presented as means ± sd of

150 151

2.7

Quantification of free amino groups

152

Free amino groups in samples prior to oxidation by FeSO4/H2O2 were determined as

153

described by Weigele, DeBernardo, Tengi, & Leimgruber 18 and Strauss & Gibson 19. An aliquot of

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850 µL sample containing ~0.01 mg/mL β-LG was added to 2.0 mL 0.05 M sodium tetraborate (pH

155

8.5) in a 4 mL quartz spectrofluorometer cell and 150 µL of 0.7 mM fluorescamine solution in

156

acetone was added. The cell was inverted four times, and the resulting fluorescence was measured

157

using 390/485 nm for excitation and emission on a Perkin Elmer LS45 Fluorescence spectrometer

158

(Llantrisant, UK). The contribution from malic acid buffer (pH 5.8) was recorded under the same

159

conditions and subtracted all samples. The free amino group concentration was calculated based on

160

a standard curve prepared from lysine diluted in malic acid buffer (pH 5.8). The concentration is



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given as µmol amino groups/mg protein and are presented as means ± sd of three independent

162

replicates (n=3). All protein-containing samples were subtracted the contribution measured in the

163

corresponding blank sample without protein.

164 165

2.8

Quantification of the semialdehydes AAS and GGS

166

The protein oxidation products α-aminoadipic and γ-glutamic semialdehyde, AAS and GGS,

167

respectively, were quantified in samples from the samples containing protein according to the

168

method described by Utrera et al.

169

ice-cold 10 % trichloroacetic acid (TCA), centrifuged at 5000 rpm for 30 minutes, and the

170

supernatant discarded. The pellet was precipitated again with 1.5 mL ice-cold 5 % TCA,

171

centrifuged for 5 minutes and the supernatant discarded. The pellet was derivatized by addition of

172

0.5 mL SDS/DTPA (1 % SDS and 1 mM di-ethylene-triamine-penta-acetic acid in 0.25 M MES

173

buffer, pH 6.0), 0.5 mL ABA (50 mM aminobenzoic acid in MES buffer), and 0.25 mL 100 mM

174

NaBH3CN in MES buffer. The mixture was incubated at 37 °C for 90 minutes with stirring every

175

30 minutes. Subsequently, the proteins were precipitated twice, first with 0.25 mL ice-cold 50 %

176

TCA and then with 1.5 mL 5 % ice-cold TCA. The precipitates were hydrolyzed in 6 N HCl at 110

177

°C for 18 hours and subsequently dried in vacuo at 40 °C using a Savant speed-vac concentrator.

178

Hydrolysates were reconstituted with 0.2 mL Milli-Q water and filtered through hydrophilic

179

polypropylene GH Polypro (GHP) syringe filters (0.45 µm pore size, Pall Corp., USA). Samples

180

were analyzed using UHPLC (Shimadzu USA manufacturing Inc., Canby, OR, USA) equipped

181

with a COSMOSIL 5 C18-AR-II RP-HPLC column (15 cm × 4.6 mm × 5 µm) coupled to a RF-10A

182

XL Shimadzu Fluorescence detector (excitation and emission wavelength of 283 and 350 nm,

183

respectively). The mobile phase was a mixture of 50 mM sodium acetate buffer (pH 5.4) and

184

acetonitrile, increasing gradually with a flow rate of 1.0 ml/min the acetonitrile concentration from

20

. An aliquot of 250 µL sample were precipitated with 1.3 mL


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0% to 8% between 0-20 minutes, then from 8-90 % between 20-30 minutes, and finally ending up

186

at 100 % buffer after 40 minutes. Identification of both ABA-derivatized semialdehydes in the FLD

187

chromatograms was carried out by comparing their retention times with those from standard AAS

188

and GGS (synthesized in vitro according to Akagawa et al.

189

carbonyls/mg protein as quantified using an ABA standard curve.

21

). Data are expressed as nmol

190 191

2.9

Quantification of Schiff base structures

192

The emission of fluorescence by protein oxidation products (Schiff base structures) was

193

assessed by use of fluorescence spectroscopy 22. Samples were diluted 50 fold in malic acid buffer

194

and then dispensed in a 4 mL quartz spectrofluorometer cell. The excitation wavelength was set at

195

350 and emission spectra were recorded from 400 to 500 nm (LS45 Perkin-Elmer fluorescence

196

spectrometer, Llantrisant, UK). Emission spectra of the malic acid buffer (pH 5.8) were recorded

197

under the same conditions and subtracted all sample spectra. Excitation and emission slit widths

198

were set at 10 nm, and data were collected at 500 nm per minute in both measurements. The content

199

of Schiff base structures (AU) was expressed as fluorescence intensity units emitted at 440 nm and

200

presented as mean ± sd of three independent replicates (n=3). All protein-containing samples were

201

subtracted the contribution measured in the corresponding blank sample without protein.

202 203 204 205

2.10 Statistical data analysis Statistical analysis were performed by analysis of variance (ANOVA) using R© version 2.12.1, The R Foundation for Statistical Computing (ISBN 3-900051-07-0).

206 207



208

3

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3.1

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Results Phenol- or quinone-derived modifications of β-LG

210

In order to evaluate the extent of protein-phenol adduct formation, the concentration of amino

211

and thiol groups of β-LG were evaluated before initiation of oxidation by Fe/H2O2. The samples

212

without β-LG were used as blank samples in the analyses to compensate for any interference from

213

the phenols or quinones in the assays. The results showed a significant decrease of 22 % amino

214

groups by addition of 4MBQ to the β-LG (Figure 1, upper panel). Higher reactivity of the phenols

215

and quinones were observed for the reaction with thiol groups, where addition of 4MBQ and 4MC

216

significantly reduced the thiol group concentration by 60% and 12%, respectively, (Figure 1, lower

217

panel). As a control, the thiol concentration was also determined in the NEM-treated β-LG, and

218

NEM was found to block 98% of the thiol groups.

219 220

3.2

Antioxidative capacity of the β-LG samples

221

The antioxidative capacity of the samples prior to oxidation by Fe(II)/H2O2 was evaluated by

222

the CUPRAC assay (CUPric Reducing Antioxidant Capacity) (Figure 2). The overall antioxidative

223

capacity was determined for all samples with or without β-LG, including not only the 1:1 molar

224

ratio between the protein and phenol or quinone, but also the ratios 1:0.5 and 1:2. The trolox

225

equivalent antioxidant capacity (TEAC) obtained for all samples increased proportionally with the

226

concentration of phenol or quinone (Figure 2, B-E). Comparing the measured TEAC of the sample

227

with the summarized TEAC indicate whether the combination of protein and phenol or quinone

228

interact to provide an additive, synergistic, or antagonistic antioxidative capacity. A statistically

229

significant difference (marked by * in Figure 2) between the measured TEAC and the summarized

230

TEAC indicates a synergistic or antagonistic effect, whereas the effect is additive for the samples

231

with no significant difference between the measured and summarized TEAC. In this way, additive


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effects were observed in the β-LG samples containing 4MC in the ratios 1:1 and 1:2 and in the β-

233

LG sample containing Cat in the ratio 1:2 (Figure 2, B and D). All β-LG samples containing

234

quinones (Figure 2, C and E) as well as the low ratios of 4MC (1:05) and Cat (1:0.5; 1:1) resulted in

235

antagonistic antioxidative capacities, which means that the combination of β-LG and phenols or

236

quinones in most cases reduces the overall antioxidative capacity. Also, derivatization of β-LG with

237

NEM (Figure 2, A) led to a significant decrease of TEAC as compared to β-LG alone.

238 239

3.3

Thiol loss during in vitro oxidation

240

The samples were oxidized by Fe/H2O2 at 37 °C for up to 20 days. The results showed that

241

thiols were rapidly lost and reached steady state already at day 1 after initiation for most samples

242

(Figure 3). The samples containing only β-LG reached ~15 nmol thiols per mg protein at day 1, and

243

this level was not significantly different from the level at day 20 (Figure 3). The same pattern was

244

observed in the β-LG samples containing 4MBQ, Cat, and CatQ (Figure 3), though, with a lower

245

starting point for the 4MBQ due to the modifications induced to the protein thiols as shown in

246

Figure 1. In contrast, a significant increase in thiol level from day 1 to 8 was found in the β-LG

247

sample containing 4MC, which unfortunately cannot be definitely explained based on the

248

experimental design of the present study.

249 250

3.4

AAS and GGS formation during in vitro oxidation

251

Both AAS and GGS were quantified in the β-LG samples and results showed that limited, but

252

significant formation took place up to day 12 in the samples containing only β-LG (Figure 4). In the

253

β-LG treated with NEM, where all thiol groups were blocked, a rapid and significant increase in

254

AAS was observed until day 5, where the concentration of the carbonyl peaked to decrease

255

afterwards. For the β-LG containing 4MC, 4MB, Cat, or CatQ, the concentration of AAS increased



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256

significantly until day 12, and then subsequently decreased (Figure 4, upper panel). The presence of

257

4MC or 4MBQ significantly increased the concentration of GGS already at day 1, which continued

258

to be significantly higher until day 12, as compared to the β-LG alone, whereas Cat and CatQ had

259

no significant effect on GGS formation in β-LG (Figure 4, lower panel). For the β-LG treated with

260

NEM, no GGS was found.

261 262

3.5

Formation of Schiff base structures during in vitro oxidation

263

Reaction between various carbonyl groups and amino groups leads to the formation of

264

unspecific Schiff base-structures that can be quantified by fluorescence spectroscopy. In the sample

265

containing only β-LG, the Schiff base-structures increased significantly after 12 days of storage,

266

whereas in the β-LG treated with NEM, the structures increased significantly already from day 1

267

(Figure 5). In the present study, the emission spectra for the sample containing only β-LG or β-LG

268

and NEM showed a maximum at 420 nm. However, the maximum shifted towards a higher

269

wavelength (440 nm) by addition of phenols or quinones. Both emission wavelengths were

270

monitored, but as no differences in the overall results were observed, only the data from 440 nm are

271

presented. In the β-LG samples containing 4MC or 4MBQ, significantly elevated fluorescence was

272

observed at day 3 as compared to the β-LG alone. In contrast, in β-LG samples containing Cat or

273

CatQ, the Schiff base-structures increased significantly after day 8 (Figure 5). No apparent

274

differences were observed between Cat and CatQ with regards to Schiff-base structures.

275 276


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4

278

4.1

Discussion β-LG oxidation and effect of NEM-blocking of thiol groups

279

The overall antioxidative capacity of β-LG as determined by CUPRAC was significantly

280

reduced after treatment with NEM indicating that β-LG was less resistant towards oxidation when

281

the thiols were blocked. The CUPRAC assay was selected as marker of overall antioxidative

282

activity as it also includes the reducing effect of protein thiols

283

with a recent report showing that thiol groups in a myofibrillar protein isolate (MPI) are important

284

for the scavenging of perferrylmyoglobin radicals 24. In the present study the thiols were rapidly lost

285

during the first day of storage in the presence of Fe(II) and hydrogen peroxide generating hydroxyl

286

radicals (Figure 3). NEM effectively blocked the thiol groups of β-LG, and consequently no thiols

287

were available for oxidation in the β-LG treated with NEM, which changed the target of the

288

oxidants. This is evident from the rapid formation of α-aminoadipic semialdehyde (AAS) during the

289

first days of storage in the β-LG treated with NEM as opposed to a slower formation of AAS in

290

untreated β-LG. Similarly, the formation of Schiff base-structures also increased more rapidly in

291

NEM-treated b-LG compared to untreated b-LG. These data shows that thiols are oxidized more

292

rapidly in our system than alkaline amino acids and are therefore better markers of early oxidation

293

events in systems without polyphenols, which is consistent with the higher rate constants reported

294

for the reaction of hydroxyl radicals with thiols than with alkaline amino acids (Davies, 2005).

295

Sulfur containing amino acid residues, such as Cys and Met may act as intramolecular antioxidants

296

in proteins by scavenging radical species 25. Elias et al. 26 found no loss of Met during oxidation of

297

β-LG, but a rather rapid initial loss of thiols, followed by a plateau during continuing oxidation.

298

This sacrificial protection by the protein thiols was not possible in the β-LG treated with NEM and

299

therefore other susceptible amino acid residues such as alkaline amino acids were subsequently

300

attacked by the oxidants.

23

, and the result is in accordance



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301

Concomitantly, in the β-LG treated with NEM, α-aminoadipic semialdehyde (AAS) increased

302

rapidly during the first days of storage, whereas γ-glutamic semialdehyde (GGS) increased slowly

303

until day 12 together with the formation of Schiff base-structures, which continued to increase

304

throughout the reaction period. This finding emphasizes the role of metal-catalyzed oxidation in the

305

carbonylation of β-LG when thiols are not available for oxidation. AAS, GGS, and other protein

306

oxidation products may engage in reactions involving a carbonyl group and an amino group leading

307

to advanced protein modifications similar to Maillard reactions with generation of Schiff base-

308

structures

309

systems is in agreement with previous reports and may be derived from the higher concentration

310

and susceptibility of lysine in β-LG towards metal-catalyzed oxidation compared to other alkaline

311

amino acids 29.

27, 28

. The overall higher concentration of AAS compared to that of GGS in the β-LG

312 313

4.2

Effect of protein-phenol adducts on β-LG oxidation

314

In the present study, quantification of amino and thiols groups prior to initiation of oxidation

315

showed that addition of 4MBQ decreased the concentration of protein amino and thiol groups,

316

suggesting formation of covalent thiol-quinone (TQ) and amino-quinone (AQ) adducts for β-LG in

317

the presence of 4MBQ (Scheme 2). In a previous study it was shown by LC-MS analysis that > 20

318

% of b-LG was found as protein-phenol adducts when 4MBQ was added in a equimolar ratio at pH

319

8.0, while 40 % of the thiols were lost 15. In the present study, 60 % thiols were lost by addition of

320

equimolar concentration of 4MBQ at pH 5.8. However, the concentration in the present study was

321

five times higher and pH was lower than in the aforementioned study, which may have led to an

322

increased reaction rate and changes in the protein structure that may have altered the reactivity of

323

specific amino acid side chains due to changed exposure to the environment 30. The smaller loss of

324

amino groups (ca. 20%) suggests that the reaction with quinones was slower than for thiols. This


Page 15 of 36


325

may be explained by the difference in pKa of the nucleophilic sites (e.g. pKa for Ɛ-amino group of

326

Lys is 10.79 and pKa for thiol group of Cys is 8.37) since a larger proportion of the thiols will be

327

deprotonated and thereby more reactive at pH 5.8 compared to the amines. On the basis of these

328

data it is not possible to clarify whether the thiol loss is caused by pro-oxidative behavior oxidation

329

or adduct formation. During the in vitro oxidation, the thiol level at day 1 was found to be similar in

330

the β-LG independent on the presence of 4MC or 4MBQ.

331

Addition of both 4MC and 4MBQ also caused an increase in the formation of AAS, GGS, and

332

Schiff bases. This apparent pro-oxidative effect was similar to the observation of the NEM-blocked

333

β-LG, and may be explained by three different mechanisms. Firstly, it may likely be caused by the

334

elimination of the thiols as oxidation targets or radical scavengers in the β-LG, exposing other

335

amino acids to oxidation. Secondly, phenols may also act as pro-oxidants forming hydrogen

336

peroxide in the presence of transition metals (Scheme 1). Zhou & Elias 31 showed that 400 µM (-)-

337

epigallocatechin-3-gallate (EGCG) produced 350 µM hydrogen peroxide during 4 days of storage

338

of emulsions in the presence of 25 µM Fe3+ (pH 7.0). In the present study, the phenols or quinones

339

(273 µM) and Fe(II) (50 µM) may in a similar manner generate hydrogen peroxide during oxidation

340

as part of a pro-oxidant mechanism. However, 5 mM hydrogen peroxide was added initially as

341

oxidation initiator, and this concentration exceeds indeed any possible hydrogen peroxide formation

342

by oxidation of the phenols. Previous studies have shown that a similar concentration of whey

343

protein was able to quench 0.45 mM hydrogen peroxide during three days storage 32. This indicates

344

that any hydrogen peroxide generated by phenol oxidation may be considered insignificant

345

compared to the excess hydrogen peroxide added as initiator. Finally, besides additions to Cys,

346

quinones may also react as already mentioned with other nucleophiles, and both Lys and Trp have

347

previously been found as targets 10. Reaction with amino groups from Lys to yield AQ adducts can

348

occur by two mechanisms, i) substitution of an aromatic carbon by the 1,4-Michael addition or ii)



Page 16 of 36

349

by formation of a Schiff base, where the oxo group of the quinone reacts with the amino group to

350

yield iminoquinones and the release of water (Scheme 2). As depicted in Scheme 3, the

351

iminoquinone may transform to the iminophenol, which through an oxidative deamination

352

mechanism releases an aminophenol and the corresponding semialdehyde, AAS

353

carbonyl, in turn, may engage further in advanced reaction pathways to yield Schiff-base structures

354

with other amino groups. Extracts from green tea, black tea, and coffee were found to oxidatively

355

deaminate lysine residues of bovine serum albumin in the presence of metal ions, indicating a lysyl

356

oxidase-like activity of the phenolic compounds in the extracts 34. This pro-oxidant mechanism of

357

certain phenolics has also been described for myofibrillar proteins oxidized in vitro by a hydroxyl-

358

radical generating system 2, 35 and also in complex muscle foods 36. Scheme 3 shows how AAS may

359

be generated through either metal-catalyzed oxidation of Lys (reaction A) or as described above by

360

the oxidative deamination pathway (reaction B). In the present study, the latter pathway seem to be

361

most pronounced, as the presence of phenols and quinones significantly increased AAS and the

362

Schiff base structures in β-LG. Taking these three mechanisms into consideration, the ability of

363

4MC and 4MBQ to eliminate thiols as oxidation target and to promote the oxidative deamination

364

pathway may be the major mechanisms behind the apparent pro-oxidative effect of these species

365

towards carbonylation of β-LG.

33, 34

. This

366

The chemical structure of 4MC is similar to the B-ring of catechin, indicating that the

367

reactivity of the two compounds may be similar. However, the effects of Cat or CatQ were not as

368

clear as that of 4MC or 4MBQ. Neither Cat nor CatQ were found to decrease the concentration of

369

amino groups or thiol groups prior to oxidation. However, significantly lower levels of thiols were

370

detected from day 1 and throughout the in vitro oxidation, suggesting other pro-oxidative activities

371

than the TQ-adduct formation. The formation of AAS in the presence of Cat or CatQ was found to

372

be higher than for β-LG alone indicating again a pro-oxidative activity, but no significant effect was


Page 17 of 36


373

observed for the formation of GGS, and Schiff bases were only significantly higher at day 12.

374

Utrera & Estévez

375

expressions of the oxidative damage caused in proteins by a hydroxyl-radical generating system.

376

These findings reflect the different chemistry (factors, catalysts, pathways) behind each of the

377

chemical changes induced in food proteins by oxidants (reviewed by Lund, Heinonen, Baron, &

378

Estévez

379

interaction mechanisms. For instance, the lysyl oxidase activity of certain phenols is only applicable

380

to alkaline amino acids and promote carbonylation (pro-oxidant effect) while the effect of the same

381

phenolic compound on thiol residues may lead to different outcome (i.e. antioxidant, pro-oxidant,

382

or no effect at all).

37

35

also found diverse effects of the same phenolic compounds on the different

). On the other hand, these results emphasize the specificity of certain phenol-protein

383 384

4.3

Redox chemistry of phenols and quinones

385

No difference was observed between the addition of Cat or CatQ for all protein oxidation

386

products evaluated throughout 20 days of in vitro oxidation, indicating that the redox state of

387

catechin did not affect the oxidation of β-LG. This is interesting, as the overall antioxidative

388

activity as determined by the CUPRAC assay showed a large variation between the Cat and CatQ

389

(Figure 2, B-E, dark grey bars). Indeed, the reducing capacity of Cat was found to be 4-fold higher

390

than the reducing capacity of CatQ, as one mole Cat or CatQ corresponded to 2.4 or 0.6 moles

391

Trolox, respectively. In contrast, the reducing capacity for 4MC was 1.7-fold higher than for

392

4MBQ. The reducing capacity observed for catechin is close to previously published data showing

393

that one mole of catechin corresponded to 3.1 moles of Trolox

394

present study was not found to influence these ratios, but the results indicate that the oxidation to

395

the quinoic structures had larger impact on Cat than on 4MC.

38

. The presence of β-LG in the



Page 18 of 36

396

The lack of coherence between the results of the CUPRAC assay and the determination of

397

oxidation products in vitro demonstrates the drawback of antioxidant assays and demonstrates how

398

they rarely provide exact information of the antioxidant capacity of a given system, and that the

399

reducing capacity or antioxidant activity is highly dependent on the oxidation substrate and the

400

actual conditions. However, in the present study the antioxidant assay may provide information on

401

the reaction between the protein and quinones, and the effect their interactions may have on the

402

given system. For the equimolar concentrations applied during the oxidation of β-LG, the presence

403

of 4MBQ, Cat, and CatQ resulted in antagonistic antioxidative effects in the β-LG, whereas 4MC

404

showed an additive affect. Antagonistic effects has previously been observed for quercetin-

405

derivatised BSA where covalent attachment between the phenol to the protein reduced TEAC as

406

compared to free quercetin 12.

407

The formation of covalent TQ or AQ adducts may explain the antagonistic effects as the

408

reaction between the quinone and a nucleophile results in the substitution at a carbon in the

409

aromatic ring of the quinone. The substitution reduces the quinone to the hydroquinone, and this

410

substitution is commonly considered as the mechanism by which quinones regenerates in cycles of

411

polymerization reactions with other phenols to yield polymeric compounds. In aqueous media, if no

412

nucleophiles or other phenols are available, quinones are reduced spontaneously in a single step

413

two-electron two-proton process to yield the phenol 39, and this may explain why a reducing activity

414

was observed for the quinoic compounds, 4MBQ and CatQ, in the CUPRAC assay in the present

415

study. For each polymerization cycle or nucleophile addition reaction, the ability of the quinone to

416

regenerate is reduced, which leads, in turn, to a less antioxidative activity of the compound. The

417

larger difference in reducing capacity observed between Cat and CatQ (4-fold) than that between

418

4MC and 4MBQ (1.7-fold) may result from different reduction or polymerisation mechanisms. It

419

may be hypothesized that Cat preferably is reduced through polymerization and 4MBQ is reduced


Page 19 of 36


420

through spontaneous reduction in the aqueous media. Spontaneous reduction will not compromise

421

the antioxidative capacity in contrast to polymerization, and hence, less difference between the

422

antioxidant capacity of the phenol and quinone may be observed for 4MBQ.

423

In the present study, the nucleophile reaction between β-LG and the quinones were simulated

424

by the NEM reaction with the thiol groups, which indeed resulted in a change in oxidation targets in

425

the β-LG by promoting AAS and Schiff base formation. The CUPRAC assay also showed that the

426

reducing capacity of β-LG was reduced by blocking the thiol groups with NEM. The decrease in the

427

overall reducing capacity of NEM-blocked β-LG may also explain the antagonistic effect observed

428

for the combination of β-LG and 4MBQ, as TQ adducts were generated in this sample. However,

429

this does not explain the antagonistic effects of β-LG combined with Cat or CatQ as neither amino

430

groups nor thiols were lost in those samples prior to oxidation.

431

In conclusion, after reaction with 4MC and 4MBQ, β-LG had a decrease of thiol groups by 12

432

and 60 %, respectively, and a 22 % decrease in amino groups was found by addition of 4MBQ.

433

Addition of Cat or CatQ did not lead to any instant decrease in either thiol or amino groups. The

434

oxidative stability of β-LG after reaction with 4MC, 4MBQ, Cat, or CatQ was impaired during 20

435

days storage in the presence of Fe(II) and hydrogen peroxide. Especially the presence of 4MC and

436

4MBQ served as apparent pro-oxidants, and the changes observed were ascribed to the redirection

437

of oxidation due to the blocking of thiol groups, but also to the oxidative deamination pathway

438

accelerating the production of semialdehydes and subsequently Schiff base structures. The complex

439

chemistry of the interaction mechanisms between phenols and different protein residues urges the

440

necessity of more mechanistic studies in this field. The pro-oxidant effect and hence, potential

441

harmful effect of certain phenolic compounds on proteins advices towards a more detailed

442

toxicological evaluation prior to use of phenolic compounds for food protection and in dietary

443

supplements.



444

5

Page 20 of 36

Acknowledgements

445

The authors acknowledge The Danish Council for Independent Research Technology and

446

Production within The Danish Agency for Science Technology and Innovation for granting the

447

project entitled: “Antioxidant mechanisms of natural phenolic compounds against protein cross-link

448

formation in meat and meat systems” (11-117033).

449


Page 21 of 36

450


6

References

451 452 453

1. Tomas-Barberan, F. A.; Andres-Lacueva, C. Polyphenols and Health: Current State and Progress. J. Agric. Food Chem. 2012, 60 (36), 8773-8775.

454

2. Estévez, M.; Heinonen, M. Effect of phenolic compounds on the formation of alpha-

455

aminoadipic and gamma-glutamic semialdehydes from myofibrillar proteins oxidized

456

by copper, iron, and myoglobin. J. Agric. Food Chem. 2010, 58 (7), 4448-4455.

457

3. Bonoli-Carbognin, M.; Cerretani, L.; Bendini, A.; Almajano, M. P.; Gordon, M. H. Bovine

458

Serum Albumin Produces a Synergistic Increase in the Antioxidant Activity of Virgin

459

Olive Oil Phenolic Compounds in Oil-in-Water Emulsions. J. Agric. Food Chem.

460

2008, 56 (16), 7076-7081.

461

4. Le Bourvellec, C.; Renard, C. M. G. C. Interactions between Polyphenols and

462

Macromolecules: Quantification Methods and Mechanisms. Crit. Rev. Food Sci. Nutr.

463

2012, 52 (1-3), 213-248.

464

5. Kroll, N. G.; Rawel, H. M.; Rohn, S. Reactions of plant phenolics with food proteins and

465

enzymes under special consideration of covalent bonds. Food Sci. Technol. Int. 2003,

466

9 (3), 205-218.

467

6. Cilliers, J. J. L.; Singleton, V. L. Characterization of the Products of Nonenzymic

468

Autoxidative Phenolic Reactions in A Caffeic Acid Model System. J. Agric. Food

469

Chem. 1991, 39 (7), 1298-1303.

470

7. Richard-Forget, F. C.; Rouet-Mayer, M. A.; Goupy, P. M.; Philippon, J.; Nicolas, J. J.

471

Oxidation of chlorogenic acid, catechins, and 4-methylcatechol in model solutions by

472

apple polyphenol oxidase. J. Agric. Food Chem. 1992, 40 (11), 2114-2122.



Page 22 of 36

473

8. Kalyanaraman, B.; Felix, C. C.; Sealy, R. C. Semiquinone Anion Radicals of

474

Catechol(Amine)S, Catechol Estrogens, and Their Metal-Ion Complexes. Environ.

475

Health Perspect. 1985, 64, 185-198.

476 477 478 479

9. Pierpoint, W. S. o-Quinones formed in plant extracts - their reactions with amino acids and peptides. Biochem. J. 1969, 112 (5), 609-616. 10. Rawel, H. M.; Kroll, J.; Hohl, U. C. Model studies on reactions of plant phenols with whey proteins. Nahrung-Food 2001, 45 (2), 72-81.

480

11. Almajano, M. P.; Delgado, M. E.; Gordon, M. H. Changes in the antioxidant properties of

481

protein solutions in the presence of epigallocatechin gallate. Food Chem. 2007, 101

482

(1), 126-130.

483 484

12. Rohn, S.; Rawel, H. M.; Kroll, J. Antioxidant activity of protein-bound quercetin. J. Agric. Food Chem. 2004, 52 (15), 4725-4729.

485

13. Kristiansen, K. R.; Otte, J.; Ipsen, R.; Qvist, K. B. Large-scale preparation of b-lactoglobulin

486

A and B by ultrafiltration and ion-exchange chromatography. Int. Dairy J. 1998, 8,

487

113-118.

488 489

14. Harrison, C. R.; Hodge, P. Polymer-supported periodate and iodate as oxidizing-agents. J. Chem. Soc. , Perkin Trans. 1 1982, (2), 509-511.

490

15. Jongberg, S.; Lund, M. N.; Otte, J. Dissociation and reduction of covalent b-lactoglobulin-

491

quinone adducts by dithiothreitol, tris(2-carboxyethyl)phosphine, or sodium sulfite.

492

Anal. Biochem. 2015, 478 (0), 40-48.

493

16. Jongberg, S.; Gislason, N. E.; Lund, M. N.; Skibsted, L. H.; Waterhouse, A. L. Thiol-quinone

494

adduct formation in myofibrillar proteins detected by LC-MS. J. Agric. Food Chem.

495

2011, 59 (13), 6900-6905.

496

17. Ellman, G. L. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 1959, 82 (1), 70-77.


Page 23 of 36

497 498 499 500


18. Weigele, M.; DeBernardo, S. L.; Tengi, J. P.; Leimgruber, W. Novel reagent for the fluorometric assay of primary amines. J. Am. Chem. Soc. 1972, 94 (16), 5927-5928. 19. Strauss, G.; Gibson, S. M. Plant phenolics as cross-linkers of gelatin gels and gelatin-based coacervates for use as food ingredients. Food Hydrocolloids 2004, 18 (1), 81-89.

501

20. Utrera, M.; Morcuende, D.; Rodríguez-Carpena, J. G.; Estévez, M. Fluorescent HPLC for the

502

detection of specific protein oxidation carbonyls - [alpha]-aminoadipic and [gamma]-

503

glutamic semialdehydes - in meat systems. Meat Sci. 2011, 89 (4), 500-506.

504

21. Akagawa, M.; Sasaki, D.; Ishii, Y.; Kurota, Y.; Yotsu-Yamashita, M.; Uchida, K.; Suyama, K.

505

New method for the quantitative determination of major protein carbonyls, alpha-

506

aminoadipic and gamma-glutamic semialdehydes: Investigation of the formation

507

mechanism and chemical nature in vitro and in vivo. Chem. Res. Toxicol. 2006, 19 (8),

508

1059-1065.

509

22. Estévez, M.; Kylli, P.; Puolanne, E.; Kivikari, R.; Heinonen, M. Fluorescence spectroscopy as

510

a novel approach for the assessment of myofibrillar protein oxidation in oil-in-water

511

emulsions. Meat Sci. 2008, 80 (4), 1290-1296.

512

23. Prior, R. L.; Wu, X. L.; Schaich, K. Standardized methods for the determination of

513

antioxidant capacity and phenolics in foods and dietary supplements. J. Agric. Food

514

Chem. 2005, 53 (10), 4290-4302.

515

24. Jongberg, S.; Lund, M. N.; Skibsted, L. H.; Davies, M. J. Competitive reduction of

516

perferrylmyoglobin radicals by protein thiols and plant phenols. J. Agric. Food Chem.

517

2014, 62 (46), 11279-11288.

518

25. Levine, R. L.; Berlett, B. S.; Moskovitz, J.; Mosoni, L.; Stadtman, E. R. Methionine residues

519

may protect proteins from critical oxidative damage. Mech. Ageing Dev. 1999, 107 (3),

520

323-332.



Page 24 of 36

521

26. Elias, R. J.; McClements, D. J.; Decker, E. A. Antioxidant activity of cysteine, tryptophan,

522

and methionine residues in continuous phase beta-lactoglobulin in oil-in-water

523

emulsions. J. Agric. Food Chem. 2005, 53 (26), 10248-10253.

524 525 526 527 528 529

27. Eyre, D. R.; Paz, M. A.; Gallop, P. M. Cross-Linking in Collagen and Elastin. Annu. Rev. Biochem. 1984, 53, 717-748. 28. Dolz, R.; Heidemann, E. Reactivity of the allysine aldehyde group. Connect. Tissue Res. 1989, 18 (4), 255-268. 29. Stadtman, E. R.; Berlett, B. S. Fenton Chemistry - Amino-Acid Oxidation. J. Biol. Chem. 1991, 266 (26), 17201-17211.

530

30. Casal, H. L.; Kohler, U.; Mantsch, H. H. Structural and Conformational-Changes of Beta-

531

Lactoglobulin-B - An Infrared Spectroscopic Study of the Effect of Ph and

532

Temperature. Biochim. Biophys. Acta 1988, 957 (1), 11-20.

533

31. Zhou, L.; Elias, R. J. Influence of cysteine and methionine availability on protein peroxide

534

scavenging activity and phenolic stability in emulsions. Food Chem. 2014, (146), 521-

535

530.

536 537 538 539

32. Zhou, L.; Elias, R. J. Investigating the hydrogen peroxide quenching capacity of proteins in polyphenol-rich foods. J. Agric. Food Chem. 2011, 59 (16), 8915-8922. 33. Akagawa, M.; Suyama, K. Amine oxidase-like activity of polyphenols - Mechanism and properties. Eur. J. Biochem. 2001, 268 (7), 1953-1963.

540

34. Akagawa, M.; Shigemitsu, T.; Suyama, K. Oxidative deamination of benzylamine and lysine

541

residue in bovine serum albumin by green tea, black tea, and coffee. J. Agric. Food

542

Chem. 2005, 53 (20), 8019-8024.


Page 25 of 36


543

35. Utrera, M.; Estevez, M. Impact of trolox, quercetin, genistein and gallic acid on the oxidative

544

damage to myofibrillar proteins: The carbonylation pathway. Food Chem. 2013, 141

545

(4), 4000-4009.

546

36. Cando, D.; Morcuende, D.; Utrera, M.; Estevez, M. Phenolic-rich extracts from Willowherb

547

(Epilobium hirsutum L.) inhibit lipid oxidation but accelerate protein carbonylation

548

and discoloration of beef patties. Eur. Food Res. Technol. 2014, 238 (5), 741-751.

549 550

37. Lund, M. N.; Heinonen, M.; Baron, C. P.; Estévez, M. Protein oxidation in muscle foods: A review. Mol. Nutr. Food Res. 2011, 55 (1), 83-95.

551

38. Ozyurek, M.; Guclu, K.; Tutem, E.; Baskan, K. S.; Ercag, E.; Celik, S. E.; Baki, S.; Yildiz, L.;

552

Karaman, S.; Apak, R. A comprehensive review of CUPRAC methodology. Anal.

553

Methods 2011, 3 (11), 2439-2453.

554 555

39. Guin, P. S.; Das, S.; Mandal, P. C. Electrochemical reduction of quinones in different media: A review. Int. J. Electrochem. 2011, 2011, 1-22.

556 557



Page 26 of 36

Figure captions

Figure 1. Free amino and thiol groups (mean±sd, n=3) in samples (pH 5.8, no oxidants added) containing 273 µM β-lactoglobulin alone (β-LG) or after reaction with 273 µM 4-methylcatechol (4MC), 4-methylbenzoquinone (4MBQ), catechin (Cat), catechin-quinone (CatQ) for 10 minutes or after treatment with excess N-ethylmaleimide (NEM) for 90 minutes at room temperature. Asterix (*) indicate that the sample is significantly different (P < 0.05) from the model system containing only β-LG.

Figure 2. Antioxidative capacity as determined by TEAC (mM Trolox equivalents, mean±sd, n=3) in samples (pH 5.8, no oxidants added) containing 273 µM β-lactoglobulin (β-LG) A) alone (1:0) or after treatment with 27.3 mM NEM, or after 10 minutes reaction at room temperature with 137 µM (1:0.5), 273 µM (1:1), or 546 µM (1:2) B) 4-methylcatechol (4MC), C) 4-methylbenzoquinone (4MBQ), D) catechin (Cat), or E) catechin-quinone (CatQ). Black bars ( the model systems containing β-LG. Grey bars ( without β-LG. Light grey bars (

) represent TEAC of

) represent TEAC of the blank model systems

) represent the summarized TEAC of the model systems

without β-LG added the TEAC of 273 µM β-LG alone (Panel A, 1:0). Asterix (*) denote a statistical significant difference (P < 0.05) between the measured TEAC and the summarized TEAC.

Figure 3. Thiol group concentration (mean±sd, n=3) during in vitro oxidation by Fe(II)/H2O2 of samples (pH 5.8, 37 °C) containing 273 µM β-lactoglobulin alone (β-LG) or after treatment with 27.3 mM N-ethylmaleimide (β-LG-NEM), or in the presence of equal molar concentration of 4methylcatechol (β-LG-4MC), 4-methylbenzoquinone (β-LG-4MBQ), catechin (β-LG-Cat) or catechin-quinone (β-LG-CatQ). Data from Figure 1 is included as t=0 day.


Page 27 of 36


Figure 4. α-Aminoadipic semialdehyde (AAS; Upper panel) and γ-glutamic semialdehyde (GGS; Lower panel) (mean±sd, n=3) during in vitro oxidation by Fe(II)/H2O2 of samples (pH 5.8, 37 °C) containing 273 µM β-lactoglobin alone (β-LG) or after treatment with 27.3 mM N-ethylmaleimide (β-LG-NEM), or in the presence of equal molar concentration of 4-methylcatechol (β-LG-4MC), 4methylbenzoquinone (β-LG-4MBQ), catechin (β-LG-Cat) or catechin-quinone (β-LG-CatQ).

Figure 5. Schiff base structures (mean±sd, n=3) during in vitro oxidation by Fe(II)/H2O2 of samples (pH 5.8, 37 °C) containing 273 µM β-lactoglobulin alone (β-LG) or after treatment with 27.3 mM N-ethylmaleimide (β-LG-NEM), or in the presence of equal molar concentration of 4methylcatechol (β-LG-4MC), 4-methylbenzoquinone (β-LG-4MBQ), catechin (β-LG-Cat) or catechin-quinone (β-LG-CatQ).



Page 28 of 36

Schemes Scheme 1. Two-step metal catalyzed oxidation of a phenol to yield the intermediate semiquinone radical and subsequently the quinone and hydrogen peroxide.


Page 29 of 36


Scheme 2. Nucleophilic addition between a quinone and a protein thiol group (A) to yield a thiolquinone (TQ) adduct or between a quinone and a protein amino group to yield amino-quinone (AQ) adducts either by Michael addition (B1) or by formation of Schiff base-like structures (B2).



Page 30 of 36

Scheme 3. Formation of AAS and the Schiff-base product azomethine via (A) metal catalyzed oxidation of a protein lysine residue or (B) oxidative deamination reaction of the amino-quinone adduct iminoquinone. R represents the methyl of 4-methyl catechol or the A and C ring of catechin.


Page 31 of 36


Figures Figure 1



Page 32 of 36

Figure 2


Page 33 of 36


Figure 3



Page 34 of 36

Figure 4


Page 35 of 36


Figure 5



Page 36 of 36

Graphical abstract


Influence of the Oxidation States of 4-Methylcatechol and Catechin on

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