Isolation and Identification of Tyrosinase-Inhibitory and Copper

Subscriber access provided by TUFTS UNIV

Food and Beverage Chemistry/Biochemistry

Isolation and Identification of Tyrosinase Inhibitory and CopperChelating Peptides from Hydrolyzed Rice Bran-Derived Albumin Supatcha Kubglomsong, Chockchai - Theerakulkait, Ralph L. Reed, Liping Yang, Claudia S Maier, and Jan F. Stevens J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01849 • Publication Date (Web): 17 Jul 2018 Downloaded from http://pubs.acs.org on July 19, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 40

Journal of Agricultural and Food Chemistry

Isolation and Identification of Tyrosinase Inhibitory and CopperChelating Peptides from Hydrolyzed Rice Bran-Derived Albumin

Supatcha Kubglomsong a, Chockchai Theerakulkait b, Ralph L. Reed c, Liping Yang d, Claudia S. Maier d, Jan F. Stevens c

a

School of Human Ecology (Program in Food, Nutrition and Applications),

Sukhothai Thammathirat Open University, Chaengwattana Rd., Bangpood, Pakkret, Nonthaburi, 11120 Thailand b

Department of Food Science and Technology, Faculty of Agro-Industry, Kasetsart University, Chatuchak, Bangkok, 10900, Thailand

c

Department of Pharmaceutical Sciences, College of Pharmacy and the Linus Pauling Institute, Oregon State University, Corvallis, OR 97331, USA d

Department of Chemistry, Oregon State University, Corvallis, OR 97331, USA

* Corresponding author, Tel: +66-2-562-5032, Fax: +66-2-562-5021, E-mail: [email protected] (C. Theerakulkait)

ACS Paragon Plus Environment

1


1

Page 2 of 40

ABSTRACT

2

Rice bran albumin (RBAlb), which showed higher tyrosinase inhibitory

3

activity than other protein fractions, was hydrolyzed with papain to improve

4

the bioactivity. The obtained RBAlb hydrolysate (RBAlbH) was separated into

5

11 peptide fractions by RP-HPLC. Tyrosinase inhibition and copper chelation

6

activities decreased with increasing retention time of the peptide fractions.

7

RBAlbH fraction 1, which exhibited the greatest activity, contained 13 peptides

8

whose sequences were determined by using LC-MS/MS. Most of the peptide

9

sequences contained features of previously reported tyrosinase inhibitory and

10

metal chelating peptides, especially peptide SSEYYGGEGSSSEQGYYGEG.

11

RBAlbH fraction 1 showed more effective tyrosinase inhibition (IC50 1.31

12

mg/mL) than citric acid (IC50 9.38 mg/mL), but less than ascorbic acid (IC50

13

0.03 mg/mL) (P ≤ 0.05). It showed copper chelating activity (IC50 0.62 mg/mL),

14

stronger than EDTA (IC50 1.06 mg/mL) (P ≤ 0.05). These results suggest that

15

RBAlbH has potential as a natural tyrosinase inhibitor and copper chelator for

16

application in the food and cosmetic industries.

17 rice

bran

18

KEYWORDS:

19

inhibition, copper chelation

albumin,

enzymatic

hydrolysates,

tyrosinase

20 21 22 23 24 25


2

Page 3 of 40


26

INTRODUCTION

27

Tyrosinase (EC 1.14.18.1) is a binuclear copper enzyme belonging to the

28

polyphenol oxidases (PPO) family that is widely distributed among plants,

29

animals, and microorganisms. It catalyzes two different reactions in the

30

presence of oxygen: the hydroxylation of monophenols to o-diphenols and the

31

subsequent oxidation of o-diphenols to the corresponding o-quinones. These

32

o-quinones are unstable and can undergo polymerization to form undesirable

33

brown pigments called melanins.1-5 Tyrosinase is responsible for the

34

enzymatic browning in many vegetables and fruits during preparation process

35

and long-term storage, leading to nutritional and economic loss.1,2,6 In

36

addition, tyrosinase is involved with some hyperpigmentation disorders of the

37

skin such as melasma and age spots. It may also be related to Parkinson’s

38

disease and cancer.4,7 Thus, tyrosinase inhibition is of interest in the food,

39

medicine and cosmetics fields.

40

Several potent substances have been applied for the purpose of

41

tyrosinase inhibition. Ascorbic acid has been widely used as an antibrowning

42

agent;3,8 however, its browning inhibitory effect is only temporary due to its

43

rapid consumption during redox processes.2,9 Hydroquinone, kojic acid and

44

sulfite are effective compounds that have been used to inhibit tyrosinase.

45

Hydroquinone and kojic acid are used as a skin-whitening agents to reduce

46

melanin production, while sulfiting agents are used as enzymatic browning

47

inhibitors in many fruits and vegetable products. However, these compounds

48

have adverse effects on human health.4,10 Moreover, the U.S. Food and Drug

49

Administration has prohibited the use of sulfite in most fresh fruits and

50

vegetables.11 In addition, many consumers prefer to use natural substances


3


Page 4 of 40

51

as opposed to synthetic alternatives. Therefore, various tyrosinase inhibitors

52

from natural sources have been widely investigated.

53

Rice (Oryza sativa L.) is one of the world’s most important food crops with

54

a production of about 740 million metric tons,12 resulting in commensurately

55

huge amounts of rice bran as a primary co-product derived from the outer

56

layers of rice caryopsis. Rice bran comprises about 5 to 8% of paddy rice

57

weight and is removed during the milling process. It is mostly used as raw

58

material in rice bran oil industry or animal feed. However, rice bran is an

59

excellent source of phytochemicals, vitamins, minerals, dietary fibers and

60

unsaturated fats as well as high quality proteins.13,14 Rice bran contains about

61

10 to 15% of protein.13 Rice bran protein is hypoallergenic and it also shows

62

anticancer and antioxidant properties.13,15 Thus, rice bran protein has been

63

extensively explored as a potential source of alternative ingredients in food

64

and nutraceutical industries.

65

Several researchers have reported the tyrosinase inhibitory effect and

66

copper chelating activity of proteins, protein hydrolysates and peptides from

67

natural sources such as rice bran,2,16 silk,17 sunflower,18 zein,19 chickpea,20,21

68

cowpea22 and red seaweed.23 Some synthetic short peptides appear to show

69

inhibitory activity against tyrosinase.4,7,24 Some oligopeptides25,26 and squid

70

collagen hydrolysate27 that showed tyrosinase inhibitory effect have been

71

suggested to be applied as cosmetic agents. Bioactive peptides are small

72

protein fragments that have biological activity after they are released from

73

proteins by hydrolytic treatment.20,23 Hydrolytic cleavage involves unfolding of

74

native protein molecules; active moieties become more exposed or they are

75

newly formed by hydrolysis. Protein hydrolysates from zein,19 casein28 and


4

Page 5 of 40


76

rice starch by-product29 have been found to possess tyrosinase inhibitory or

77

copper chelating activity greater than the protein they originate from. Papain is

78

a commercial enzyme that has been used for production bioactive peptides.

79

Liu et al.30 reported that Camellia oleifera seed-cake papain hydrolysate

80

exhibited excellent antioxidant activities and copper chelating activity.

81

Moreover, hydrolysates of casein31 and palm kernel cake protein32 produced

82

by papain digestion demonstrated strong antioxidative activities and metal

83

chelating activity. Moreover, there have been some reports that the

84

hydrolysates of rice bran protein fractions33 and brown rice protein fractions34

85

exhibit antioxidant activities. Wattanasiritham et al.15 isolated and identified

86

the antioxidative peptides from hydrolyzed rice bran albumin. However, there

87

is limited information about the tyrosinase inhibition and copper chelating

88

activity of rice bran protein fraction papain hydrolysates and their peptide

89

structures. Therefore, the peptides in hydrolyzed rice bran albumin fractions

90

were separated. The tyrosinase inhibitory effect and copper chelating activity

91

of these hydrolyzed rice bran albumin fractions were investigated and the

92

peptide sequences identified by mass spectrometry (MS/MS) in this study.

93 94

MATERIALS AND METHODS

95

Materials. The potential for controlling of tyrosinase varies with the

96

source of rice cultivars and tyrosinase source; thus, fresh rice bran from Khao

97

Dawk Mali 105 (KDML 105) rice (Oryza sativa L.), the most popular Thai

98

aromatic rice variety, and the variety that exhibited the highest PPO inhibitory

99

efficiency among other commercially consumed rice varieties from our

100

previous study were selected for this study. KDML 105 was purchased from


5


Page 6 of 40

101

Surin Taveepol Rice Mill, Surin, Thailand. Tyrosinase from mushroom (EC

102

1.14.18.1) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium

103

potassium tartrate, bovine serum albumin, papain, trifluoroacetic acid, 3,4-

104

Dihydroxy-L-phenylalanine (L-DOPA) and pyrocatechol violet were purchased

105

from Sigma-Aldrich (St. Louis, MO, USA). Copper sulfate was purchased from

106

Mallinckrodt Chemical (Paris, KY, USA). Potassium iodide was purchased

107

from Mallinckrodt Baker (Phillipsburg, NJ, USA). Acetonitrile was purchased

108

from VWR Analytical (Radnor, PA, USA).

109

Preparation of Rice Bran Protein Fractions. Full-fat rice bran was

110

initially screened by passing through a 50-mesh sieve, and then defatted with

111

three volumes of hexane according to the procedures of Kubglomsong and

112

Theerakulkait.2 The obtained defatted rice bran was packed in an aluminum

113

foil bag and kept frozen at -20 ºC.

114

Rice bran protein fractions were prepared by the modified method of

115

Agboola et al.35 with some modifications. The defatted rice bran was first

116

extracted with distilled water (DW) in a 1:5 (w/v) ratio of rice bran to DW using

117

an overhead stirrer at 500 rpm for 60 min. After centrifugation (10,000xg, 30

118

min) at 25 ºC, the supernatant was collected to obtain the albumin fraction.

119

The residue from this step was similarly extracted with 5% NaCl, 0.1 M NaOH,

120

and 70% ethanol to obtain globulin, glutelin, and prolamin fraction,

121

respectively. The supernatant from each fraction was filtered through nylon

122

cloth (100 mesh), and the pH was adjusted with 1.0 N HCl to 4.1, 4.3, 4.8, and

123

5.0, respectively. Rice bran protein fractions: albumin (RBAlb), globulin

124

(RBGlo), glutelin (RBGlu) and prolamin (RBPro) were obtained after

125

centrifugation.


6

Page 7 of 40


126

Rice bran protein fractions were dispersed in DW and adjusted to pH 7

127

with 1.0 N NaOH, then centrifuged (10,000xg, 30 min) at 25 ºC. The

128

supernatants were dialyzed using dialysis tubing with a molecular weight cut-

129

off of 6,000 Da against DW at 4 ºC overnight, and then centrifuged at the

130

same conditions. Protein content, molecular weight and tyrosinase inhibition

131

of rice bran protein fractions were determined as described below.

132

Determination of Protein Content. Biuret reagent was prepared by

133

dissolving 2.5 g potassium iodide, 4.5 g sodium potassium tartrate and 1.5 g

134

copper sulfate (CuSO4.5H2O) in 200 mL of 0.2 M NaOH, and then adjusting

135

the final volume to 500 mL with DW (a modified method of Chanput et al.).33

136

Thirty µL of sample was pipetted into a 96-well plate and mixed with 150 µL of

137

biuret reagent. The absorbance at 540 nm was read after 30 min incubation

138

against the reagent blank. Protein concentration was quantified using bovine

139

serum albumin (BSA) as a standard with concentration ranging from 1-

140

10 mg/mL.

141

Determination

of

the

Molecular

Weight

of

Protein

by

Gel

142

Electrophoresis. The molecular weight of rice bran protein fractions were

143

determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis

144

(SDS-PAGE) according to the modified procedure of Tang et al.36 with 12 and

145

4% (w/v) acrylamide separating gel and stacking gel, respectively. The

146

samples at protein concentration of 4 µg/mL were mixed with sample buffer

147

containing 0.5 M Tris-HCl pH 6.8, 10% (w/v) SDS, glycerol, 1% (w/v)

148

bromophenol blue, and 2-mercaptoethanol, then heated for 5 min in boiling

149

water. After cooling to room temperature, 10 µL of sample solutions were

150

loaded into the gel wells for electrophoresis with an electrode buffer (pH 8.3)


7


Page 8 of 40

151

consisting of 0.1% (w/v) SDS, 1.44% (w/v) glycine, and 0.3% (w/v) Tris base.

152

SDS-PAGE was run using a Mini-Protean® Tetra Vertical Electrophoresis Cell

153

and a model 3000XI power supply (Bio-Rad Laboratories, Hercules, CA,

154

USA). For protein visualization the gel was stained by immersing in a solution

155

consisting of 0.1% (w/v) Coomassie Brilliant Blue R-250 in a mixture of 40%

156

(v/v) methanol and 10% (v/v) acetic acid. The gel was destained in a solution

157

consisting of 40% (v/v) methanol and 10% (v/v) acetic acid. PageRulerTM Plus

158

Prestained Protein Ladder (Thermo Scientific, Rockford, IL, USA) was used

159

as the standard marker with a molecular weight range of 10-250 kDa.

160

Determination of Tyrosinase Inhibition. Tyrosinase inhibitory activity

161

was determined using a 96-well plate (a modified method of Masuda et al.).6

162

Tyrosinase was prepared at 100 unit/mL in 0.05 M sodium phosphate buffer

163

(pH 6.8). The wells were assigned the following mixtures: control [without

164

(w/o) sample], 120 µL of 0.05 M sodium phosphate buffer (pH 6.8) and 40 µL

165

of tyrosinase; blank (w/o sample, w/o tyrosinase), 160 µL of the same buffer;

166

sample, 80 µL of the same buffer, 40 µL of tyrosinase and 40 µL of the

167

sample; blank sample (w/o tyrosinase), 120 µL of the same buffer and 40 µL

168

of the sample solutions. The reaction contents of each well were mixed by the

169

microplate mixer and incubated at room temperature for 10 min, and then 40

170

µL of 2.5 mM L-DOPA prepared in the same buffer was added and incubated

171

at room temperature for 2 min. The absorbance (A) at 475 nm was measured

172

with a microplate reader (SpectraMax 190 Microplate Reader, Molecular

173

Devices, Sunnyvale, CA, USA). Percent tyrosinase inhibitory activity was

174

calculated as:

175


8

Page 9 of 40


176

% Tyrosinase inhibitory activity = [(AControl-ABlank) - (Asample-ABlank sample)] x 100 /

177

(AControl- ABlank)

178 179

Determination of Copper Chelating Activity. The copper chelating

180

activities of RBAlbH fractions were measured according to the modified

181

method of Carrasco-Castilla et al.37 RBAlbH fractions (10 µL) were mixed with

182

280 µL of 50 mM sodium acetate buffer (pH 6.0), 6 µL of 4 mM pyrocatechol

183

violet prepared in the same buffer, and 10 µL of 1 µg/µL CuSO4.5H2O. The

184

disappearance of the blue color was observed by measuring the absorbance

185

at 632 nm using a microplate reader (SpectraMax 190 Microplate Reader,

186

Molecular Devices, Sunnyvale, CA, USA). Water was used as a control

187

instead of a sample. Percent copper chelating activity was calculated from

188

absorbance (A) at 632 nm as follows:

189 190

% Copper chelating activity = (Acontrol - Asample) x 100 / Acontrol

191 192

Effect of Rice Bran Protein Fractions on Tyrosinase Inhibition. Rice

193

bran protein fractions: RBAlb, RBGlo, RBGlu, and RBPro were adjusted to a

194

protein concentration of 2 mg/mL and investigated for mushroom tyrosinase

195

inhibition. The rice bran protein fraction that showed the highest tyrosinase

196

inhibitory activity (RBAlb) was selected for further study.

197

Effect of RBAlb Concentration on Tyrosinase Inhibition. The RBAlb

198

fraction was prepared at protein concentrations of 1-10 mg/mL, and then

199

tyrosinase inhibition was investigated. The protein concentration of RBAlb that

200

showed the highest tyrosinase inhibition (8 mg/mL) was selected for


9


Page 10 of 40

201

hydrolysis and the isolation and identification of tyrosinase inhibitory and

202

copper-chelating peptides.

203

Preparation of Rice Bran Albumin Hydrolysate (RBAlbH). RBAlb at a

204

protein concentration of 8 mg/mL was hydrolyzed with papain at conditions

205

optimized from our preliminary studies that led to the highest tyrosinase

206

inhibition at an enzyme to substrate ratio of 1:100 (w/w) at 37 ºC. The pH was

207

maintained at 8.0 throughout the hydrolysis time with 1.0 N NaOH. The

208

hydrolysis was carried out for 30 min, then terminated by immersing the

209

incubation container in boiling water for 5 min and cooling quickly with an ice

210

bath. The obtained RBAlbH was freeze-dried and kept in aluminum foil bags

211

at -20 ºC for further study.

212

Isolation of Peptides from RBAlbH. The lyophilized RBAlbH was mixed

213

with Milli-Q water (100 mg/mL) and centrifuged at 13,000xg at 25 ºC for 5 min.

214

The supernatant was filtered through a 0.45 µm nylon filter (Thermo Scientific,

215

Rockwood, TN, USA), and then 100 µL of the sample was fractionated by

216

reversed-phase liquid chromatography (RP-HPLC) using a Discovery® BIO

217

Wide Pore C18 HPLC Column (5 µm, 25 cm × 10 mm i.d.) (Supelco, Sigma

218

Aldrich, St. Louis, MO, USA) with a Discovery® BIO Wide Pore C18

219

Supelguard™ Cartridge (10 µm, 1 cm × 10 mm i.d.) (Supelco). The HPLC

220

system (Waters Delta 600, Waters Corporation, Milford, MA, USA) consisted

221

of a vacuum degasser, a quaternary solvent pump, a Waters 717 Plus

222

autosampler, a Waters 2996 photodiode array detector and a computer with

223

Empower software. Separation was performed using 0.1% (v/v) trifluoroacetic

224

acid (TFA) in water as eluent A and acetonitrile (ACN) as eluent B with the

225

flow rate at 4.0 mL/min. The solvent gradient was kept at 0% B for 2.5 min,


10

Page 11 of 40


226

then changed from 0 to 30% B over 20 min, then changed from 30 to 100% B

227

over 7.5 min, and then kept at 100% B for 6 min. Fractions eluting from the

228

column were manually collected by observation of the chromatogram

229

monitored at 215 and 280 nm. RBAlbH fractions were concentrated at 40 ºC

230

under vacuum using a rotary evaporator to remove most of the acetonitrile

231

and then the concentrate was freeze-dried. Each RBAlbH fraction was

232

reconstituted with water and adjusted to the same protein concentration of 1

233

mg/mL for determination of tyrosinase inhibition and copper chelating activity.

234

Identification of Amino Acid Sequences of RBAlbH Fractions by LC-

235

MS/MS. One µL of each RBAlbH fraction (fraction 1-11) isolated by RP-HPLC

236

was injected into a nanoLC-MS system (an Orbitrap FusionTM Lumos mass

237

spectrometer) with a Nano ESI source (Thermo Scientific, Waltham, MA)

238

coupled with a Waters nanoAcquityTM UPLC system (Waters, Milford, MA)).

239

Peptides were loaded on a 2G nanoAcquity UPLCTrap column (180 µm × 20

240

mm, 5 µm) for 5 min with solvent (0.1% formic acid in 3% ACN) at a flow rate

241

of 5 µL/min and separated by an Acquity UPLC Peptide BEH C18 column

242

(100 µm × 100 mm, 1.7 µm) following a 120 min gradient at a flow rate of 500

243

nL/min consisting of mobile phase A (0.1% formic acid in water) and mobile

244

phase B (0.1% formic acid in acetonitrile), where B was increased from 3-10%

245

over 3 min, from 10-30% over 102 min, from 30-90% over 3 min and held 4

246

min, and from 90-3% over 1 min and held 7 min. The nanoLC eluate was

247

directly electrosprayed into the mass spectrometer in the positive ion mode.

248

The spray voltage was 2400 V and the ion transfer tube temperature was 300

249

ºC. Full MS spectra were acquired in the Orbitrap at resolution settings of

250

120,000 at m/z 200 with a scan range from 400 to 1500, and automatic gain


11


Page 12 of 40

251

control (AGC) target was set to 4.0 × 105. Under top speed data-dependent

252

mode, the most intense parent ions peaks with charge state range 2-7 were

253

selected for fragmentation by collision induced dissociation (CID) with

254

normalized collision energy of 35%. MS/MS spectra were acquired in the ion

255

trap and the exclusion window was set at 1.6 Da and AGC was 104.

256

All raw data files were analyzed with Thermo ScientificTM Proteome

257

DiscovererTM 2.1 software and searched using Sequest HT against Uniprot

258

Oryza sativa protein database including Papain enzyme sequence. The

259

overall false discovery rate (FDR) for peptides was less than 1% and peptide

260

sequences

261

Carbamidomethylation of cysteine and oxidation of methionine were specified

262

as static modification and dynamic modification respectively. Mass tolerances

263

were set at ±10 ppm for precursor ions and 0.6 Da for fragments.

264

were

allowed

a

maximum

of

two

missed

cleavages.

Statistical Analysis. The experiments were performed with three

265

replications.

The data were subjected to one-way analysis of variance.

266

Duncan’s multiple range tests were applied for significant differences between

267

treatments (P ≤ 0.05).

268 269

RESULTS AND DISCUSSION

270

Effect of Rice Bran Protein Fractions on Tyrosinase Inhibition.

271

Percentages of tyrosinase inhibition of rice bran protein fractions are shown in

272

Figure 1. It was found that the tyrosinase inhibitory effect of RBAlb was higher

273

than that of RBGlu, RBGlo and RBPro (P ≤ 0.05). Therefore, RBAlb was

274

selected for further study. The different amino acid profiles of rice bran protein

275

fractions might establish the different tyrosinase inhibition. Padhye and


12

Page 13 of 40


276

Salunkhe38 found that rice albumin contained higher amounts of uncharged

277

polar amino acids than other fractions, whereas those of prolamin fraction had

278

the lowest. Schurink et al.24 reported that peptides containing polar,

279

uncharged amino acid residues such as serine and cysteine are good

280

tyrosinase inhibitors. This might be related to tyrosinase inhibition of RBAlb. In

281

addition, Wang et al.39 reported that rice bran glutelin fraction contained high

282

amounts of sulfur-containing amino acids such as cysteine that have been

283

reported to inhibit tyrosinase activity.

284

Molecular weights of rice bran protein fractions were determined by

285

sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

286

(Figure 2). It was found that molecular weights of RBAlb, RBGlo and RBGlu

287

were in the range of 10 to 60 kDa; while those of RBPro were in the range of

288

10 to 15 kDa. These results are in agreement with those of Chanput et al.33

289

who reported similar molecular weights for the same fractions. In addition,

290

Padhye and Salunkhe38 found that molecular weights of rice albumin, globulin,

291

glutelin and prolamin were between 7-135, 13-60, 8-29 and 7-13 kDa,

292

respectively. Rice bran proteins reported by Tang et al.36 were in the range of

293

6.5-66.2 kDa.

294

Effect of RBAlb Concentration on Tyrosinase Inhibition. Percentages

295

of tyrosinase inhibition of RBAlb at different protein concentrations are shown

296

in Figure 3. Increasing RBAlb protein concentration from 1 to 8 mg/mL

297

gradually increased tyrosinase inhibitory effect (P ≤ 0.05); however, the

298

inhibitory effect of RBAlb not increase at protein concentration of 9 and 10

299

mg/mL (P > 0.05). This result reveals that RBAlb showed tyrosinase inhibition

300

in a dose-dependent manner until saturation occurred at 9 mg/mL. This result


13


Page 14 of 40

301

was in line with Lee et al.40 work who found that tyrosinase inhibitory ability of

302

the synthetic hexapeptide (SFKLRY-NH2) increased with the increasing of

303

concentrations before a plateau was reached.

304

Tyrosinase Inhibition and Copper Chelating Activity of Peptide

305

Fractions from RBAlbH. An HPLC chromatogram showing the 11 peptide

306

fractions from RBAlbH is presented in Figure 4. The tyrosinase inhibitory and

307

copper chelating activity of each fraction are shown in Figure 5a and 5b,

308

respectively. For both tyrosinase inhibition (Figure 5a) and copper chelating

309

activity (Figure 5b), we found that the first eluted fraction had the highest

310

activities. The tyrosinase inhibition and copper chelating activity of the

311

fractions decreased with elution time and showed the lowest activity in fraction

312

11. The earlier eluting fractions contained peptides that exhibited greater

313

tyrosinase inhibition and copper chelating activity than the later eluting

314

fractions. In addition, the earlier eluting fractions are rich in serine, and serine

315

is a hydrophilic residue. These results are similar to Megías et al.18,41 who

316

reported that RP-HPLC fractions of chickpea and sunflower protein

317

hydrolysates that eluted first exhibited the greatest copper chelating activity.

318

Tyrosinase is an enzyme that contains a binuclear copper active site for

319

catalyzing the oxidation reaction. These two copper ions are essential for the

320

enzyme activities and are directly involved in the monophenolase and

321

diphenolase reactions of tyrosinase.4,5,10 Therefore, the chelation of copper

322

ions at the active site of tyrosinase could retard or interrupt the enzyme

323

activity.5,17 Kahn28 demonstrated that proteins, peptides and amino acids

324

could reduce tyrosinase activity by chelating the essential copper at the active

325

site. In addition, several researchers have reported the relation between


14

Page 15 of 40


326

tyrosinase inhibition and copper chelating activity in their research on squid

327

skin collagen hydrolysate,27 whey protein isolates,42 proteins, protein

328

hydrolysates and amino acids from milk,28 hydroxypyridinone derivatives,5 and

329

collagen peptide from jellyfish.43 Moreover, molecular docking was performed

330

by some researchers to understand the interaction between inhibitor and

331

binding site of tyrosinase. It was found that hydroxypyridinone derivatives5

332

and indole-containing octapeptides44 chelated with copper at the enzyme

333

active site, thereby influencing the tyrosinase inhibition. Therefore, we

334

supposed that the tyrosinase inhibitory mechanism of RBAlbH likely involves

335

copper chelating activity.

336

Table 1 shows IC50 values for RBAlbH fraction 1, commercial tyrosinase

337

inhibitors (ascorbic acid and citric acid)3,8 and a known strong metal ion

338

chelator (EDTA).17 RBAlbH fraction 1 effectively inhibited tyrosinase activity

339

with the IC50 of 1.31 mg/mL. Its inhibitory efficiency was greater than citric

340

acid, which showed IC50 of 9.38 mg/mL (P ≤ 0.05); however, its inhibitory

341

efficiency was lower than ascorbic acid, which showed IC50 of 0.03 mg/mL (P

342

≤ 0.05). In addition, RBAlbH fraction 1 showed copper chelating activity with

343

the IC50 of 0.62 mg/mL, while EDTA had IC50 of 1.06 mg/mL. This result

344

suggested that RBAlbH exhibited a stronger copper chelating activity than

345

EDTA on a mass basis (P ≤ 0.05). In addition, RBAlbH fraction 1 exhibited a

346

greater tyrosinase inhibition than that of Wu et al.17 who reported tyrosinase

347

inhibitory effect of sericin hydrolysate with IC50 of 8.71 mg/mL. Copper

348

chelating activity of RBAlbH fraction 1 was stronger than zein hydrolysate19

349

and whey protein isolate,42 which showed IC50 of about 16 and 6 mg/mL,

350

respectively. However, RBAlbH fraction 1 showed less inhibitory activity than


15


Page 16 of 40

351

collagen peptide from jellyfish43 that showed IC50 for tyrosinase inhibition and

352

copper chelating activity of 78.2 and 88.7 µg/mL, respectively.

353

Identification of Amino Acid Sequences of RBAlbH Fractions by LC-

354

MS/MS. The peptides in the RBAlbH fractions were analyzed by LC-MS/MS.

355

Thirteen peptides from the most active RBAlbH fraction 1 were identified and

356

are shown in Table 2. These peptides range from 14 to 50 residues and have

357

molecular weights ranging from 1327 to 4819 Da. The peptide size has been

358

reported to associate with biological activities. Zhuang et al.45 found that the

359

peptides from corn gluten meal at molecular weight

Isolation and Identification of Tyrosinase-Inhibitory and Copper

Recommend Documents