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Isolation and Characterization of Lactoferrin Peptides with Stimulatory Effect on Osteoblast Proliferation Fengjiao Fan, Maolin Tu, Meng Liu, Pujie Shi, Yun Wang, Di Wu, and Ming Du J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 21 Jul 2017 Downloaded from http://pubs.acs.org on July 21, 2017

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Journal of Agricultural and Food Chemistry

Isolation and Characterization of Lactoferrin Peptides with Stimulatory Effect on Osteoblast Proliferation

Fengjiao Fana, Maolin Tua, Meng Liua, Pujie Shia, Yun Wanga, Di Wub, Ming Dub,*

a

Department of Food Science and Engineering, Harbin Institute of Technology, Harbin 150090, China;

b

School of Food Science and Technology, National Engineering Research Center of Seafood, Dalian Polytechnic University, Dalian 116034, China.

*

Corresponding author: Tel: +86-411-86332275; Fax: +86-411-86323262; E-mail:

[email protected].

1

ACS Paragon Plus Environment


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1

ABSTRACT Lactoferrin is reported to be a potential food protein with osteogenic

2

activity. However, the activity of lactoferrin peptides is questionable. In the present

3

study, we isolated and characterized peptides from lactoferrin with stimulatory effect

4

on osteoblast proliferation. Peptides from the lactoferrin pepsin hydrolysate were

5

purified using cation-exchange and gel-filtration chromatography. Effects of different

6

hydrolysates and peptides on the proliferation of osteoblast MC3T3-E1 cells were

7

compared by MTT assay. Results showed that fraction P5-a from Superdex Peptide

8

10/300 GL gel chromatography showed better activity. Tricine-sodium dodecyl sulfate

9

polyacrylamide gel electrophoresis (Tricine-SDS-PAGE) and high-performance liquid

10

chromatography coupled to electrospray ionization tandem mass spectrometry

11

(HPLC-ESI–MS/MS) confirmed that two peptides components of P5-a corresponded

12

to fractions of 20-78 and 191-277 amino acids in Bos taurus lactoferrin molecule (GI:

13

221706349). These results will provide some theoretical and practical data for the

14

preparation and application of osteogenic peptides in functional food industry.

15

KEYWORDS:

16

Proliferation of osteoblast, HPLC-ESI–MS/MS

Lactoferrin,

Peptides,

Enzymatic

hydrolysis,

2


Identification,

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17

INTRODUCTION

18

Bone remodeling, an ongoing process in skeletal tissue at all stages of life,

19

involves bone formation by osteoblasts and bone resorption by osteoclasts. During the

20

biological process of bone remodeling, an imbalance wherein bone resorption

21

predominates over bone formation will result in osteoporosis. Osteoporosis,

22

characterized by reduced bone mineral density (BMD), is one of the major public health

23

problems that enhance susceptibility to bone fractures and affect more than 200 million

24

people.1,2 Osteoblast plays an important role in bone rebuilding; hence, proteins with

25

osteogenic activity are attracting more attention owing to the prevalence and importance

26

of osteoporosis.

27

Bone morphogenetic proteins (BMPs) are members of the transforming growth

28

factor-β superfamily, known to induce the formation of bone and cartilage in vivo.3 The

29

most widely studied BMPs are BMP-2, BMP-3 (osteogenin), BMP-4, and BMP-7

30

(osteogenic protein-1). A synthetic peptide derived from BMP-2 is reported to induce

31

ectopic bone formation and promote repair of tibial bone defects.4,5

32

Studies have shown that lactoferrin, bovine angiogenin, and lactoperoxidase—the

33

three components of milk basic protein—modulate bone metabolism,6 stimulate

34

osteoblast proliferation and collagen production, and suppress osteoclast activity.7-9

35

Lactoferrin is a multi-functional protein from various secretions of organisms and has

36

been associated with almost 20 different physiological functions10 such as antimicrobial,

37

antitumor, antioxidative, osteogenic, and immunoregulatory activities.11-13 In recent

38

years, studies have highlighted the osteogenic activity of lactoferrin; Lactoferrin 3



39

improved BMD of C3H-ovariectomized mice and stimulated growth of osteoblastic

40

cells while inhibiting preosteoclastic cell growth in vitro.14 In addition, it acted as a

41

growth factor and showed in vitro and in vivo anabolic activity in bone.15-17 The

42

mitogen-activated protein kinase (MAPK) and osteoprotegerin (OPG)/receptor activator

43

of nuclear factor kappa-B ligand (RANKL)/receptor activator of nuclear factor kappa-B

44

(RANK) signaling pathways were involved in the effects of lactoferrin on bone

45

metabolism.18-20

46

Lactoferricin, a peptide derived from the N-terminal region of lactoferrin (fraction

47

17-41) has been reported to exhibit bactericidal activity higher than that of

48

lactoferrin.21,22 Some pepsin-digested bovine lactoferrin peptides showed antitumor

49

activity against human oral squamous cell carcinoma and murine tumor.23,24 In addition,

50

some lactoferrin peptides (< 3 kD) displayed antihypertensive effect and lowered blood

51

pressure in spontaneously hypertensive rats.25,26 Studies have shown that oral

52

administration of lactoferrin results in its degradation into bioactive peptides.27,28

53

However, very few studies have focused on the osteogenic activity of lactoferrin

54

peptides, thereby limiting their applications.

55

The objective of the present study was to isolate and identify peptides with

56

stimulatory effect on osteoblast proliferation from the pepsin hydrolysate of lactoferrin

57

using column chromatography and high-performance liquid chromatography (HPLC)

58

coupled to electrospray ionization tandem mass spectrometry (ESI-MS/MS). The select

59

peptides can be used as potential health-promoting functional foods for prevention of

60

osteoporosis. 4


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61

MATERIALS AND METHODS

62

Cell Line and Materials

63

MC3T3-E1 cells purchased from the Cell Bank of Chinese Academy of Sciences

64

(Shanghai, China) were cultured in α-modified Eagle’s medium (α-MEM) containing

65

10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (HyClone, Logan, UT,

66

USA).

67

Bovine lactoferrin (95.41% purity) used in this study was obtained from New

68

Zealand. Pepsin (EC 3.4.23.1, 1:3000) was purchased from AMRESCO (Cleveland, OH,

69

USA). SP Sepharose Fast Flow and Superdex Peptide 10/300 GL were supplied by GE

70

Healthcare (Beijing, China). All other reagents used were of analytical grade.

71

Preparation of Crude Peptides

72

Lactoferrin was dissolved in distilled deionized water at 5% (W/V), while pepsin

73

was dissolved at a concentration of 26.125 mg/L in 150 mM sodium chloride

74

(NaCl)/hydrochloric (HCl, pH 2.0). The pepsin solution (912 mL) was pre-warmed to

75

37 °C and mixed with 48 mL lactoferrin solution to initiate hydrolysis at 37 °C for

76

0-240 min. The ratio of pepsin to lactoferrin was 238 U/g. Aliquots of 50 mL of

77

pepsin-digested lactoferrin were taken at 5, 10, 30, 60, 90, 120, 180, and 240 min and

78

treated with 25 mL sodium bicarbonate (NaHCO3, 25 mM) to terminate the reaction in

79

an ice bath and the corresponding lactoferrin hydrolysates (LFH) were named as LFH5,

80

LFH10, LFH30, LFH60, LFH90, LFH120, LFH180, and LFH240, respectively.

81

Supernatants were separated by centrifugation for 10 min at 16000 ×g and the crude

82

peptides were obtained by dialysis with 3 kD cut-off membrane for complete removal of 5



83

salt.

84

Purification of Peptides

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85

Lactoferrin hydrolysate with highest stimulatory activity on osteoblast proliferation

86

(LFH5) was filtered using a 0.22 µm syringe filter and loaded onto an SP Sepharose

87

Fast Flow column (1.6 cm × 10 cm) pre-equilibrated with 0.05 mol/L

88

phosphate-buffered saline (PBS, pH 7.4) buffer. Elution was performed using 0.05

89

mol/L PBS buffer (pH 7.4) and 0.05 mol/L PBS buffer (with 1 mol/L NaCl, pH 7.4) as

90

mobile phase at a flow rate of 3 mL/min. Elution was operated at 4 °C and monitored at

91

280 nm wavelength. All peaks were analyzed for their effect on osteoblast proliferation.

92

The peak with the strongest activity was freeze-dried and loaded onto a Superdex

93

Peptide 10/300 GL column (1.0 cm × 300 mm). The column was equilibrated and eluted

94

with 0.02 mol/L PBS buffer (pH 7.6) at a flow rate of 0.5 mL/min. Elution was operated

95

at 4 °C and monitored at 280 nm wavelength. All fractions were analyzed for their

96

molecular masses and effects on osteoblast proliferation. The peak with the strongest

97

activity was freeze-dried and subjected to HPLC-ESI–MS/MS for the determination of

98

purified peptide sequence.

99

Sodium

100

Dodecyl

Sulfate

Polyacrylamide

Gel

Electrophoresis

(SDS-PAGE) Analysis

101

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was

102

carried out in a modified Laemmli system to investigate purity and molecular masses of

103

peptides using 5% and 12% acrylamide in stacking and separating gels, respectively.29

104

Protein bands were visualized by staining with Coomassie R250 brilliant blue. Standard 6


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105

proteins (Thermo Scientific, Waltham, MA, USA) used for molecular mass

106

determination were as follows: β-galactosidase (116 kD), bovine serum albumin (66.2

107

kD), ovalbumin (45 kD), lactate dehydrogenase (35 kD), REase Bsp981 (25 kD),

108

β-lactoglobulin (18.4 kD), and lysozyme (14.4 kD). Gels were scanned using Bio-Rad

109

Gel Doc XR system (Bio-Rad, Hercules, CA, USA) and analyzed by Quantity One

110

software Version 4.6.2 (Bio-Rad). Tricine–SDS-PAGE was preferred electrophoretic

111

system for separation of low molecular mass proteins.30 We selected 4% stacking and

112

16% separating gels according to the method used by Hermann Schägger.31 Low-range

113

protein marker (Thermo Scientific) used was a mixture of six recombinant proteins and

114

synthetic peptides (1.7 to 42 kD).

115

Cell Viability Assay

116

The

proliferation

activity

of

osteoblast

tetrazolium

was

measured

bromide

(MTT)

by

3-(4,

method.32,33

117

5-dimethylthiazol-2-yl)-2,5-diphenyl

118

MC3T3-E1 cells (l × 104 cells) were seeded in a 96-well plate and cultured in α-MEM

119

medium containing 10% FBS for 24 h in a humidified atmosphere of 5% CO2 at 37 °C.

120

Following incubation, the medium was dispensed and cells were washed with 0.01

121

mol/L PBS buffer (pH 7.2). Cells were further incubated for 24, 48, and 72 h at 37 °C in

122

presence of 95 µL α-MEM and 5 µL sample, followed by treatment with MTT solution

123

(0.5 mg/mL in PBS). After 4 h, the medium was removed and 150 µL of dimethyl

124

sulfoxide (DMSO) was added to dissolve MTT formazan crystals by shaking for 10 min.

125

The absorbance was measured at 490 nm wavelength on a microplate reader (Eon,

126

BioTek, USA). 7



127

Enzymatic Hydrolysis of Isolated Peptides by Trypsin

128

The lyophilized powder (about 30 µg) of the isolated peptide with the strongest

129

activity (named P5-a under Results and Discussion) was solubilized in 30 µL SDT

130

buffer（4% SDS, 100 mM dithiothreitol [DTT], and 150 mM Tris-HCl pH 8.0）at 90 °C

131

for 5 min. SDS, DTT, and other low molecular weight components were removed using

132

200 µL UA buffer (8 M urea and 150 mM Tris-HCl pH 8.0) by repeated ultrafiltration

133

(Microcon units, 30 kD). The retentate was treated with 100 µL of 0.05 M

134

iodoacetamide in UA buffer for 30 min in darkness. The filter was washed three times

135

with 100 µL UA buffer and treated twice with 100 µL ammonium bicarbonate

136

(NH4HCO3, 25 mM). The protein suspension was digested with 2 µg trypsin in 40 µL

137

NH4HCO3 (25 mM) overnight at 37 °C and the resulting peptides were collected as a

138

filtrate.34

139

Identification of Peptides by HPLC-ESI–MS/MS

140

The peptides of P5-a were identified by HPLC-ESI–MS/MS with some

141

modification in the method described by Xie.35 Chromatography was performed using

142

an Easy nLC system (Thermo Scientific). A sample volume of 6 µL was injected for

143

nanoLC-MS/MS analysis. Buffer A (0.1% formic acid in water) and buffer B (80%

144

acetonitrile and 0.1% formic acid) were used as mobile phases for gradient separation.

145

The column was equilibrated with 95% buffer A before sample injection, and samples

146

were automatically loaded onto a C18-reversed phase column (2 cm × 100 µm, 5 µm

147

resin, Thermo scientific) and eluted onto a C18-reversed phase analytical column (75

148

µm × 100 mm, 3 µm resin, Thermo Scientific). A 60-min gradient was run as follows: 8


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149

50 min from 4% to 50% B, 4 min linear gradient to 100% B, and 6 min at 100% B.

150

Peptides eluted from the C18 column were pumped through a capillary tip for

151

electrospray and analyzed by a Q-Exactive mass spectrometer (Thermo Scientific). Full

152

scans were acquired in Orbitrap mass analyzer over m/z 300-1800 range with resolution

153

of 70,000. Normalized collision energy was 30 eV and the underfill ratio was defined as

154

0.1%. The instrument was run with peptide recognition mode enabled. Data were

155

acquired using a data-dependent top10 method dynamically choosing the most abundant

156

precursor ions. Mascot 2.2 was used for searching against corresponding database.

157

Statistical Analysis

158

Data represent results of three independent experiments each performed in

159

triplicate and are expressed as mean ± standard deviation (SD). Statistical analyses were

160

performed using the statistical software package SPSS 19.0. One-way analysis of

161

variance (ANOVA) was employed to determine the significant difference between the

162

means at p < 0.05.

163

RESULTS AND DISCUSSION

164

Preparation of Lactoferrin Hydrolysates with Pepsin Digestion

165

Peptides from lactoferrin were obtained after hydrolysis with pepsin for 5-240 min.

166

Different digestion time resulted in different protein degradation profiles, owing to

167

differences in the degree of hydrolysis. As shown in Figure 1A, pepsin at pH 2.0

168

showed significant effects on the proteolysis of lactoferrin; no intact lactoferrin was

169

detected in all hydrolysates, as analyzed by SDS-PAGE. An increase in hydrolysis time

170

resulted in gradual lysis of lactoferrin into peptides with low molecular weight. This 9



171

observation is in line with a previous study reporting only 6% intact lactoferrin after

172

10-min treatment and no intact lactoferrin after 30-min treatment with porcine pepsin

173

A .36 Small differences observed may be attributed to various experimental conditions

174

and enzymes sources. After 90 min of hydrolysis, hydrolysate profiles were unchanged.

175

As molecular masses of peptides in all hydrolysates were less than 14.4 kD,

176

Tricine-SDS-PAGE analysis was performed. As shown in Figure 1B, LFH5 and LFH10

177

showed more peptides as compared to other hydrolysates. The peptide with the lowest

178

molecular mass in these hydrolysates was about 5 kD, while molecular masses of

179

peptides in LFH5 and LFH10 ranged from 10 to 17 kD.

180

Effects of Peptides on Proliferation of Osteoblast

181

MC3T3-E1 cells were cultured for 24, 48, and 72 h in presence of 100 µg/mL LFH

182

lyophilized powder. Many studies have reported that 100 µg/mL of lactoferrin exhibited

183

better osteogenic activity,37,38 which was consistent with the results of the present study.

184

Therefore, we investigated the activity of 100 µg/mL lactoferrin. Lactoferrin, LFH5, and

185

LFH10 increased the survival of MC3T3-E1 cells, as demonstrated by the MTT assay

186

(Figure 2). This result suggests that lactoferrin promoted the proliferation of primary

187

osteoblast and osteoblastic cell lines.37,39 However, the activity of LFH5 was

188

significantly higher than that of lactoferrin at 24 h, indicating that lactoferrin took

189

longer time to stimulate osteoblast proliferation via receptor and signaling pathways40,15

190

and that hydrolysates with small peptides may stimulate osteoblast proliferation at a

191

shorter time. LFH5 showed highest activity at 48 and 72 h, although the difference

192

between LFH5 and lactoferrin activities was not significant. 10


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193

In comparison with the control group, LFH5 significantly stimulated proliferation

194

of osteoblast cells after 24, 48, and 72 h of treatment, while LFH10 significantly

195

stimulated osteoblast cell proliferation after 24 and 48 h of treatment (Figure 2). An

196

increase of 15.29%, 29.73%, and 41.61% in cell growth was observed after 24, 48, and

197

72 h of LFH5 treatment, respectively. Although the proliferation ratio of

198

LFH30-LFH240 hydrolysates at 24 h and LFH120-LFH240 hydrolysates at 72 h seem

199

to be decreased, these hydrolysates showed no significant difference as compared with

200

the control group. Natural and synthetic peptides derived from different sources have

201

shown stimulatory effect on cell proliferation at 24-72 h.41,42 Therefore, LFH5 was

202

enriched with promising bioactive peptides and can be subjected to purification because

203

of its multiple constituents.

204

Isolation of Peptides with Stimulatory Effect on Proliferation of

205

Osteoblast

206

As shown in Figure 3A, we chose five fractions P1-P5 eluted from SP Sepharose

207

Fast Flow column and investigated their effect on osteoblast proliferation at 100 µg/mL

208

using MTT assay. As shown in Figure 4, P1, P4, and P5 significantly stimulated

209

proliferation of cells. Of the five fractions, P5 showed the highest activity after 24, 48,

210

and 72 h of treatment. An increase of 31.78%, 29.76%, and 33.73% in cell growth was

211

observed after 24, 48, and 72 h, respectively.

212

P5 fraction from several runs were pooled, concentrated by ultrafiltration, and used

213

for the subsequent gel-filtration chromatography. As shown in Figure 3B, two peptide

214

fractions P5-a and P5-b were isolated and their stimulatory effect on osteoblast 11



215

proliferation analyzed at 100 µg/mL using MTT assay. As shown in Figure 5, after

216

24~72 h of treatment, stimulatory effect of P5-a on osteoblast proliferation was higher

217

than that of P5-b and P5. An increase of 23.42 %, 13.24%, and 10.59% in cell growth

218

was observed after P5-a treatment for 24, 48, and 72 h, respectively. The purity and

219

molecular mass of P5-a was analyzed by electrophoresis. Tricine-SDS-PAGE analysis

220

showed that P5-a comprised two different peptides with molecular masses of 9.35 and

221

6.95 kD (Figure 6).

222

Identification of Peptides by HPLC-ESI–MS/MS

223

As shown in Table 1, amino acid sequences of peptides from P5-a were identified

224

by HPLC-ESI–MS/MS. Six peptides were identified based on the ESI-MS/MS and

225

Mascot analysis, combining with the selection standard of scores more than 40. These

226

peptides derived from different fractions of lactoferrin displayed sequence as follows:

227

WCTISQPEWFK (fraction 27-37aa), LGAPSITCVR (fraction 48-57aa), AFALECIR

228

(fraction 59-66aa), GEGENQCACSSR (fraction 194-205aa), CLQDGAGDVAFVKE

229

(fraction 217-229aa), and ECHLAQVPSHAVVAR (fraction 263-277aa).

230

As P5-a was isolated from pepsin hydrolysate of lactoferrin using chromatography,

231

the theoretical pepsin cleavage sites in lactoferrin molecule were determined by

232

bioinformatics based on the BIOPEP database.43 At pH 1.3 and > 2.0, lactoferrin

233

molecule displayed 102 and 274 pepsin cleavage sites, respectively (Figure 7). The

234

composition and size of peptides produced may vary with conditions such as enzyme

235

concentration, hydrolysis period, pH, and temperature. As P5-a comprised peptides

236

isolated from LFH5, it may include peptides with relatively longer amino acid chains. 12


Page 12 of 34

Page 13 of 34


237

BIOPEP analysis suggested that WCTISQPEWFK (fraction 27-37aa), LGAPSITCVR

238

(fraction 48-57aa), and AFALECIR (fraction 59-66aa) are derived from Peptide 1 with

239

calculated Mr. = 6976.36 Da (fraction 20-78), while GEGENQCACSSR (fraction

240

194-205aa), CLQDGAGDVAFVKE (fraction 217-229aa), and ECHLAQVPSHAVVAR

241

(fraction 263-277aa) are derived from Peptide 2 with calculated Mr. = 9544.71 Da

242

(fraction 191-277). These two peptides were observed as two bands corresponding to

243

6.95 and 9.53 kD on the SDS-PAGE gel (Figure 6). Thus, P5-a comprised two peptides,

244

APRKNVRWCTISQPEWFKCRRWQWRMKKLGAPSITCVRRAFALECIRAIAEKK

245

ADAVTL (Peptide 1) and LCKGEGENQCACSSREPYFGYSGAFKCLQDGAGDVAF

246

VKETTVFENLPEKADRDQYELLCLNNSRAPVDAFKECHLAQVPSHAVVAR

247

(Peptide 2). The identified peptide profile is shown in Figure 7. Sequences in blue

248

letters indicate these six peptides identified by HPLC-ESI–MS/MS (Table 1), while

249

those highlighted in yellow and green indicate Peptide 1 and 2, respectively.

250

In the present study, lactoferrin was hydrolyzed by pepsin to obtain fractions with

251

stimulatory effect on cell proliferation. The active fraction P5-a from lactoferrin

252

hydrolysate

253

cation-exchange chromatography and Superdex Peptide 10/300 GL gel chromatography.

254

P5-a was shown to significantly stimulate proliferation of osteoblastic cell line

255

MC3T3-E1 at 100 µg/mL concentration and comprised two long peptide components:

256

APRKNVRWCTISQPEWFKCRRWQWRMKKLGAPSITCVRRAFALECIRAIAEKK

257

ADAVTL (fraction 20-78) and LCKGEGENQCACSSREPYFGYSGAFKCLQDGAGD

258

VAFVKETTVFENLPEKADRDQYELLCLNNSRAPVDAFKECHLAQVPSHAVVAR

was subjected to purification using SP Sepharose Fast Flow

13



259

Page 14 of 34

(fraction 191-277).

260 261

ACKNOWLEDGEMENTS

262

We would like to thank Applied Protein Technology in Shanghai for determining the

263

sequence of peptides.

264

FUNDING SOURCES

265

This study was financially supported by the National Natural Science Foundation of

266

China

267

(2013BAD18B06-03).

(31371805),

the

National

Science

&

Technology

268 269

14


Pillar

Program

Page 15 of 34


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20


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Page 21 of 34


400

FIGURE CAPTIONS

401

Figure 1. Protein profiles of lactoferrin peptides. (A) SDS-PAGE. 1: Marker; 2:

402

Lactoferrin; 3: LFH5; 4: LFH10; 5: LFH30; 6: LFH60; 7: LFH90; 8: LFH120; 9:

403

LFH180; and 10: LFH240. (B) Tricine-SDS-PAGE. 1: Marker; 2: LFH5; 3: LFH10; 4:

404

LFH30; 5: LFH60; 6: LFH90; 7: LFH120; 8: LFH180; and 9: LFH240.

405

Figure 2. Effects of lactoferrin peptides on the proliferation of osteoblast. Error bars

406

indicate standard deviation. *p < 0.05 versus control; #p < 0.05 versus LFH5.

407

Figure 3. Purification of LFH5 by column chromatography. (A) Elution diagram of SP

408

Sepharose Fast Flow column. (B) Elution diagram of Superdex Peptide 10/300 GL

409

column.

410

Figure 4. Effects of peaks collected by cation-exchange chromatography on the

411

proliferation of osteoblast. Error bars indicate standard deviation. *p < 0.05 versus

412

control; &p < 0.05 versus P5.

413

Figure 5. Effects of fractions collected by gel-filtration chromatography on the

414

proliferation of osteoblast. Error bars indicate standard deviation. *p < 0.05 versus

415

control; $p < 0.05 versus P5-a.

416

Figure 6. Tricine-SDS-PAGE of P5-a. Analysis was carried out using 4% stacking and

417

16% separating gels. Low-range protein marker was a mixture of six recombinant

418

proteins and synthetic peptides (1.7 to 42 kD).

419

Figure 7. Peptides distribution in the partial amino acid sequence of lactoferrin (1-360).

420

The six peptides indicated in blue letters were identified by HPLC-ESI–MS/MS. The

421

peptides highlighted in yellow and green are Peptide 1 and Peptide 2, respectively. The 21



422

vertical lines indicate pepsin cleavage sites in lactoferrin.

22


Page 22 of 34

Page 23 of 34


TABLES Table 1. Identification of Peptides from Hydrolysate of P5-a by Trypsin. Position in No.

Peptide mass

Sequence lactoferrin

(Da)

pI

Hydrophilicity

Scores

1

WCTISQPEWFK

27-37

1481.69

6.4

-0.5

43.64

2

LGAPSITCVR

48-57

1073.58

9.0

-0.3

57.82

3

AFALECIR

59-66

979.50

6.2

-0.2

54.63

4

GEGENQCACSSR

194-205

1354.51

4.3

0.6

42.77

5

CLQDGAGDVAFVK

217-229

1379.662

3.9

0.0

80.71

6

ECHLAQVPSHAVVAR

263-277

1673.85

7.4

-0.2

72.92

23


FIGURE GRAPHICS

24

Page 25 of 34


24h

*

1.2 #

#

#

#

1.0

b

#

#

#

#

#

0.8

0.6 ol ntr Co

LF FH5 H10 H30 H60 H90 120 180 240 L LF LF LF LF LFH LFH LFH

(A)

48h

1.4

Ratio of proliferation


1.4

* *

1.2

*

*

#

* #

#

*

*

1.0 0.8 0.6 ol LF FH5 H10 H30 H60 H90 120 180 240 ntr o L L F L F L F L F FH FH FH C L L L

(B)

25




1.5

*

*

72h

*

1.2

#

#

#

# #

#

#

0.9

0.6 nt Co

rol LF FH5 H10 H30 H60 H90 120 180 240 L L F L F L F L F L FH L FH L FH

(C)

Figure 2

26


Page 26 of 34

Absorbance at 280 nm Electrical conductivity

P0

2000

100 80

1600

60

1200 P1

800

40 P4 P5

400

20

P2 P3

0

0 0

100

200

300

400

500

600

700

Elution volume (mL)

Absorbance at 280 nm (mAU)

(A)

P5-b

1080

P5-a

810 540 270 0 0

5

10

15

20

25

30

Elution volume (mL) (B)

Figure 3

27


35

40

Electrical conductivity (mS/cm)


Absorbance at 280 nm (mAU)

Page 27 of 34


24h


1.4

* *&

1.2 &

&

Control

P0

*&

&

1.0

&

0.8 0.6 P1

P2

P3

P4

P5

(A)

48h

1.4


* *&

1.2 *&

*&

&

&

&

1.0 0.8 0.6 Control

P0

P1

P2

P3

P4

(B)

28


P5

Page 28 of 34

Page 29 of 34


72h

1.4

*


*&

*&

*&

1.2

&

&

&

1.0 0.8 0.6 Control

P0

P1

P2

P3

P4

(C)

Figure 4

29


P5


24h


1.4 *

1.2 $ $

1.0

$

0.8 0.6 Control

P5

P5-a

P5-b

(A)

48h


1.4

1.2

* $

$

1.0

0.8

0.6 Control

P5

P5-a

P5-b

(B)

30


Page 30 of 34

Page 31 of 34


72h


1.4 1.2 $

1.0

*$

0.8 0.6 Control

P5

P5-a

P5-b

(C)

Figure 5

31



Figure 6

32


Page 32 of 34

Page 33 of 34


Figure 7

33



Graphic for Table of Contents

34


Page 34 of 34

Isolation and Characterization of Lactoferrin ... - ACS Publications

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