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Glycation and phosphorylation of -lactalbumin by dry heating: Effect on protein structure and physiological functions

H. Enomoto^*, Y. Hayashi^*, C. P. Li, S. Ohki, H. Ohtomo, M. Shiokawa and T. Aoki^,1

^* United Chair of Applied Resource Chemistry, Course of Bioresource Science for Processing, United Graduate School of Agricultural Sciences, Kagoshima University, Kagoshima 890-0065, Japan
Department of Food and Pharmacy Engineering, School of Chemistry Science and Technology, Yunnan University, Kunming 650091, China
Food Technology Research Institute, Division of Research and Development, Meiji Dairies Corporation, 540 Naruda, Odawara, Kanagawa 250-0862, Japan
Department of Biochemical Science and Technology, Faculty of Agriculture, Kagoshima University, Kagoshima 890-0065, Japan

¹ Corresponding author: aoki{at}chem.agri.kagoshima-u.ac.jp

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISSCUSSION CONCLUSIONS ACKNOWLEDGMENTS REFERENCES

 
-Lactalbumin (-LA) was glycated with maltopentaose (MP) through the Maillard reaction (MP--LA) and subsequently phosphorylated by dry heating in the presence of pyrophosphate to investigate its structure and physiological functions. Glycation occurred effectively, and the sugar content of -LA increased by approximately 22.3% through the Maillard reaction. The phosphorylation of MP--LA was enhanced with an increase in the dry-heating time from 1 to 5 d, and the phosphorous content of MP--LA increased by approximately 1.01% by dry heating at pH 4.0 and 85°C for 5 d in the presence of pyrophosphate. The electrophoretic mobility of -LA increased with an increase in the phosphorylation level. The circular dichroism spectra showed that the change in the secondary structure of the -LA molecule by glycation and subsequent phosphorylation was slight. However, the Trp fluorescence intensity was increased by phosphorylation after glycation. In addition, the differential scanning calorimetry thermograms of -LA showed that the denaturation temperature of MP--LA was decreased by phosphorylation. These results indicated that molten (partially unfolded) conformations of -LA were formed by dry heating in the presence of pyrophosphate after glycation. The anti--LA antibody response was significantly reduced by glycation and subsequent phosphorylation. The suppressive effect of -LA on the production of proinflammatory cytokines such as IL-6 and tumor necrosis factor- from THP-1 cells after stimulation with lipopolysaccharide was significantly enhanced by glycation with MP and was further enhanced by phosphorylation after glycation. The Ca phosphate-solubilizing ability of -LA was enhanced by phosphorylation. The apoptotic activity of -LA was reduced by glycation and subsequent phosphorylation. These results suggest that phosphorylation by dry heating in the presence of pyrophosphate after glycation with MP through the Maillard reaction is a useful method for improvement of the physiological functions of -LA.

Key Words: -lactalbumin • glycation • phosphorylation • physiological function

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISSCUSSION CONCLUSIONS ACKNOWLEDGMENTS REFERENCES

 
Phosphorylation has been proved to be a useful method for improving the functional properties of food proteins. The functional properties of some phosphorylated proteins have been studied and reviewed by Matheis and Whitaker (1984). Over the past few decades, several phosphorylation methods have been reported by researchers (Seguro and Motoki, 1989; Aoki et al., 1994, 1997; Kato et al., 1995; Sitohy et al., 1995; Vojdani and Whitaker, 1996; Chobert, 2003). However, these phosphorylation methods have posed some problems (Li et al., 2003), making them very difficult to put to practical use. Li et al. (2003, 2004) phosphorylated egg white protein by dry heating in the presence of phosphate, significantly improving some functional properties. However, whey protein isolate (WPI) showed a lower phosphorylation level than egg white protein by dry heating under the same conditions, presumably because of a lower sugar content of WPI (Li et al., 2003). We then attempted to prepare phosphorylated WPI by glycation with maltopentaose (MP) through the Maillard reaction and subsequent phosphorylation by dry heating in the presence of pyrophosphate, with the result that some functional properties, such as heat stability, emulsifying properties, and gelling properties, were improved by glycation and subsequent phosphorylation (Li et al., 2005a). Furthermore, the Ca phosphate-solubilizing ability of WPI was enhanced by phosphorylation, and the antigenicity of β-LG and BSA, major allergens in WPI, was significantly reduced by glycation and subsequent phosphorylation (Enomoto et al., 2007, 2008).

-Lactalbumin is a 14.2-kDa Ca-binding protein with 4 disulfide bonds (Permyakov and Berliner, 2000). The structure of -LA is divided into the -domain and the β-domain by a deep cleft (Permyakov and Berliner, 2000). Calcium binding to the strong Ca-binding site increases the stability of the native form of -LA (Permyakov and Berliner, 2000). -Lactalbumin undergoes partial unfolding to form a molten globule at low pH or by the removal of Ca at neutral pH and slightly denaturing conditions (Permyakov and Berliner, 2000). Regarding the physiological functions, it is well-known that -LA is one of the 2 components of lactose synthase, which catalyzes the final step in lactose biosynthesis in the lactating mammary gland (Brodbeck et al., 1967). Recently, it has been reported that -LA and its hydrolysate have many physiological functions, such as reduction of stress (Markus et al., 2000), antimicrobial activity (Pellegrini et al., 1999), opioid activity (Nagendra, 2000), antihypertensive action (FitzGerald et al., 2004), regulation of cell growth (Sternhagen and Allen, 2001), antiulcer activity (Matsumoto et al., 2001), and immunomodulation (Cross and Gill, 2000). Svensson et al. (2000) showed that -LA derived from human whey could be converted to the apoptotic-inducing form when removing bound Ca, by treatment with EDTA, and passing it through an anion-exchange column previously conditioned with oleic acid. The active form of the protein, called "human -LA made lethal to tumor cells" (HAMLET), was described as a complex formed by apo--LA and oleic acid (Svensson et al., 2000). The HAMLET induces apoptosis (programmed cell death) in tumor cells but spares mature cells (Svensson et al., 2000). Bovine -LA or the -LA of other species, together with oleic acid, can be converted to HAMLET-like complexes by the same method (Pettersson et al., 2006). It was also demonstrated that whey protein or -LA had a marked suppressive effect against the increased release of proinflammatory cytokines, such as IL-1, IL-6, and tumor necrosis factor- (TNF-), from the D-galactosamine-induced liver injury rat model or ischemia/reperfusion-induced intestinal injury rat model (Kume et al., 2006; Yamaguchi and Uchida, 2007). -Lactalbumin is a second major whey protein in WPI, and its behavior affects the functional properties of WPI.

In the present study, we phosphorylated -LA by dry heating in the presence of pyrophosphate after glycation with MP through the Maillard reaction to investigate protein structure and physiological functions of phosphorylated -LA. Because -LA is well-known as a major allergen in milk (Maynard et al., 1997), the effects of glycation and subsequent phosphorylation on the antigenicity of -LA were also examined.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISSCUSSION CONCLUSIONS ACKNOWLEDGMENTS REFERENCES

 
Materials
Quaternary aminoethyl-Toyopearl was purchased from Tosoh Co. Inc. (Tokyo, Japan). An adult male Japanese white rabbit (JW/CSK) was purchased from Charles River Japan Inc. (Yokohama, Japan). Interferon-, phorbol 12-myristate 13-acetate, RPMI-1640 medium, and LPS from Salmonella Minnesota were purchased from Sigma-Aldrich (St. Louis, MO). Fetal bovine serum (FBS) was purchased from Equitech-Bio Inc. (Kerrville, TX). The FBS was inactivated by heating before the experiment. Maltopentaose, sodium pyruvate, gentamicin, gelatin, oleic acid, and trypan blue solution were purchased from Nacalai Tesque Co. Inc. (Kyoto, Japan). All other reagents were of analytical grade.

The crude -LA was isolated from raw skim milk according to the method of Armstrong et al. (1967) and purified by column chromatography with quaternary aminoethyl-Toyopearl. To make the -LA into holo-form, CaCl₂ was added in the crude -LA solution and dialyzed against 50 mM Tris-HCl buffer (pH 8.5). The crude -LA solution was applied to the column and eluted by 0 to 300 mM NaCl linear gradient in the same buffer at 1.0 mL/min according to the modified method of Ye et al. (2000). The major fraction was dialyzed against deionized water and then lyophilized. The purity of -LA was confirmed by SDS-PAGE according to the method of Laemmli (1970); the prepared -LA was holo-form.

Preparation of Glycated and Phosphorylated -LA
Native -LA (N--LA) and MP (1:0.5 wt/wt) were dissolved in deionized water at a protein concentration of 20 g/L, and the solution pH was adjusted to 8.0 with 1 M NaOH, followed by lyophilization. The dried sample was kept at 50°C and 65% relative humidity (RH) for 3 d using a saturated KI solution in a desiccator according to a previously published method (Aoki et al., 2001), and was then dissolved in 0.1 M sodium pyrophosphate buffer at pH 4.0. The lyophilized sample was incubated at 85°C for 1 and 5 d, and the dry-heated samples were then dissolved in deionized water. The solution was dialyzed to remove free MP and pyrophosphate for 3 d against deionized water and then lyophilized (PP–MP--LA).

In comparison with PP–MP--LA, MP-conjugated -LA (MP--LA) and dry-heated -LA (DH--LA) were prepared. To prepare MP--LA, N--LA and MP (1:0.5 wt/wt) were dissolved in deionized water at a protein concentration of 20 g/L, and the pH value of the solution was adjusted to 8.0 with 1 M NaOH, followed by lyophilization. The dried sample was kept at 50°C (65% RH) for 3 d using a saturated KI solution in a desiccator, and then dialyzed against deionized water for 3 d, after which the solution was lyophilized. To prepare DH--LA, N--LA was dissolved in deionized water at a concentration of 20 g/L, and the pH was adjusted to pH 8.0 with 1 M NaOH, followed by lyophilization. The dried sample was kept at 50°C (65% RH) for 3 d using a saturated KI solution in a desiccator, and was then dissolved in deionized water at a concentration of 20 g/L, and the pH was adjusted to 4.0 with 1M HCl, followed by lyophilization. The lyophilized sample was incubated at 85°C for 5 d, after which the solution was lyophilized.

Determination of Sugar Content
The total sugar contents of N-, DH-, MP-, and PP–MP--LA were determined by the phenol-sulfuric acid method (Dubois et al., 1956). For the determination of free sugar, 2 mL of a 2 g/L sample solution was ultrafiltered through Centrisalt I (AG-W-3400, Sartorius, Goettingen, Germany; molecular mass cutoff = 10,000). The sugar content in the ultrafiltrate was regarded as free sugar. The sugar bound to -LA was estimated by the difference between the total and free sugar content.

Determination of P Content
Protein samples were digested in perchloric acid. Phosphorus in the digest was regarded as the total P of protein. For the determination of inorganic P (Pi), 2 mL of 2 g/L sample solution was ultrafiltered through Centrisalt I (AG-W-3400, Sartorius; molecular mass cut off = 10,000). The P content in the ultrafiltrate was regarded as Pi. The P content was determined by the method of Chen et al. (1956). The amount of P bound to proteins was estimated by the difference between the total P and Pi content.

Measurement of Solubility
Protein samples were dissolved at a protein concentration of 1 g/L in 50 mM Tris-HCl buffer (pH 7.0), and then centrifuged at 1,000 x g for 15 min. The concentration of protein in the supernatant was determined using the method of Lowry et al. (1951).

Electrophoresis
Native PAGE was performed using 15.0% gels in the absence of SDS, and SDS-PAGE was performed using 15.0% gels under both reducing and nonreducing conditions in the presence and absence of 2-mercaptoethanol (2-ME) according to the method of Laemmli (1970). The gels were stained in Coomassie Brilliant Blue R-250 for 1 h.

Circular Dichroism Spectra
Circular dichroism (CD) spectra were measured at 190 to 250 nm with a Jasco J-820 spectropolarimeter (Jasco Co., Tokyo, Japan) using a cell with a 1.0-mm path length, and the digitized data were transferred to a microcomputer and processed. An average of 5 scans was recorded. Samples were dissolved in 50 mM phosphate buffer (pH 7.0) at a protein concentration of 0.1 g/L. Circular dichroism spectra were represented in terms of mean residue ellipticity (degrees cm²/dmol). The protein concentration in the solution was determined using the method of Lowry et al. (1951).

Trp Fluorescence Spectra
Tryptophan fluorescence intensity (FI) of protein samples was scanned at emissions from 300 to 400 nm excited at a wavelength of 280 nm by an FP-6600 fluorescence spectrophotometer (Jasco Co.) at 25°C. Each sample was dissolved in 50 mM phosphate buffer (pH 7.0) at a protein concentration of 0.1 g/L. The protein concentration in the solution was determined using the method of Lowry et al. (1951).

Differential Scanning Calorimetry
Differential scanning calorimetry (DSC) was performed in a VP-DSC Microcalorimeter (MicroCal, Northampton, MA). Before the DSC experiments, samples were dialyzed against 20 mM phosphate buffer (pH 7.4). After being filtered through a 0.22-µm filter, samples and reference solutions were properly degassed and loaded into the calorimeter. The experiments were carried out under an extra pressure of 1 atm to avoid degassing during heating. The calorimetric data were analyzed using Origin software provided with the calorimeter. The protein concentration was 1 g/L and was heated in the calorimeter at a scan rate of 1°C/min over a range of 30 to 80°C. The protein concentration in the solution was determined using the method of Lowry et al. (1951).

Immunization
An adult male JW/CSK rabbit was immunized subcutaneously with -LA emulsified in Freund’s complete adjuvant (Difco Laboratories, Detroit, MI). One month after the primary immunization, the rabbit was boosted with -LA emulsified in Freund’s incomplete adjuvant (Difco Laboratories). Blood samples were collected 1 wk after the secondary immunizations and stored at 4°C for 24 h to form a clot. Antiserum was prepared from the sample after clot formation and verified by the double-diffusion test of Ouchterlony (1949).

ELISA
A noncompetitive ELISA was carried out according to the method in a previous paper (Enomoto et al., 2007). -Lactalbumin samples dissolved in PBS at a protein concentration of 0.1 g/L (100 µL) were added to the wells of a polystyrene microtitration plate (Maxisorp, Nunc A/S, Roskilde, Denmark), and the plate was incubated at 4°C overnight to coat the wells with each antigen. After removal of the solution, each well was washed 3 times with 120 µL of PBS-Tween (PBS containing 0.5 g/L of Tween 20). A 120-µL quantity of a 10 g/L gelatin/PBS solution was added to each well and the plate was incubated at 25°C for 2 h, after which it was washed 3 times. One hundred microliters of an antibody (antisera) diluted with PBS was added to each well, and the plate was incubated at 25°C for 2 h. After 3 washings, 100 µL of alkaline phosphatase-labeled goat anti-rabbit Ig (Dako A/S, Glostrup, Denmark) diluted with PBS-Tween was added to each well. The plate was incubated at 25°C for 2 h, and the wells were then washed 3 times. One hundred microliters of 1 g/L of sodium p-nitrophenyl phosphate disodium/diethanolamine hydrochloride buffer (pH 9.8) was added to each well, and the plate was incubated at 25°C for 30 min. After the addition of 5 M NaOH solution (20 µL) to each well to stop the reaction, the absorbance at 405 nm was measured using a Bio-Rad 550 microplate reader (Bio-Rad Laboratories Inc., Hercules, CA).

Cell Culture
A human monocytic leukemia cell line (THP-1) was purchased from the Riken cell bank (Tsukuba, Japan). Cells were grown in RPMI-1640 medium supplemented with 10% FBS, 100 U/mL of penicillin, and 100 U/mL of streptomycin (Gibco, BCL, Burlington, Ontario, CA) at 37°C under 5% CO₂ in air. A murine leukemia cell line (L1210) was purchased from the American Type Culture Collection (ATCC, Manassas, VA). Cells were grown in RPMI-1640 medium supplemented with 10% FBS, 1 mM sodium pyruvate, and 50 U/mL of gentamicin at 37°C under 5% CO₂ in air. Exponentially growing cells were used in the following experiments.

Measurement of IL-6
The effect of -LA samples on the LPS-induced IL-6 response in THP-1 cells was investigated according to the method of Mattsby-Baltzer et al. (1996). The THP-1 cells were pretreated by adding IFN- (Sigma-Aldrich Inc.) at a concentration of 200 U/mL for 18 h before the cell experiment. Interferon- increases the sensitivity for stimulation with LPS (Vey et al., 1992). Cells were washed by centrifugation at 200 x g for 10 min at room temperature. The cell density was adjusted to 1.25 x 10⁶ cells/mL using fresh complete medium (RPMI-1640 supplemented with 5% FBS). The cells (800 µL) were added to the wells of a 24-well plate (144530, Nunc A/S), and incubated for 2 h in the absence and presence of protein samples of different protein concentrations (10 or 100 µg/mL). The cells were further incubated for 24 h in the absence and presence of LPS (0.5 µg/mL). The final volume of the media in each well was 1 mL. After the incubation period, the cell culture media were removed and centrifuged at 400 x g for 10 min at room temperature. Concentration of IL-6 in the supernatant was determined by an IL-6 Human, Biotrak ELISA System (GE Healthcare UK Limited, Buckinghamshire, UK), according to the manufacturer’s instructions. The LPS alone served as a control.

Measurement of TNF-
The THP-1 cell density was adjusted to 0.2 x 10⁶ cells/mL by using fresh complete medium (RPMI-1640 medium supplemented with 10% FBS). The cells (1 mL) were added to the wells of a 24-well plate (142475, Nunc A/S) and were differentiated into adherent macrophage-like cells in the presence of phorbol 12-myristate 13-acetate (100 ng/mL). After incubation for 3 d, nonadherent cells were washed away, and the remaining adherent macrophage-like cells were incubated for 2 h in the absence and presence of protein samples of different concentrations (10 or 100 µg/mL). The cells were further incubated for 6 h in the absence and presence of LPS (100 ng/mL). The final volume of the media in each well was 1 mL. Concentration of TNF- in the supernatant was determined by a Biotrak ELISA System (GE Healthcare UK Limited), according to the manufacturer’s instructions. The LPS alone served as a control.

Measurement of Solubilization of Ca Phosphate
The preparation of test solutions was conducted according to the procedures for artificial CN micelles (Aoki, 1989). Forty microliters of 1.0 M potassium citrate, 200 µL of 0.2 M CaCl₂, and 240 µL of 0.2 M K₂HPO₄ were added to 2 mL of 4% protein solution, followed by the addition of 200 µL of 0.2 M CaCl₂, and 100 µL of 0.2 M K₂HPO₄. The addition of 200 µL of 0.2 M CaCl₂, and 100 µL of 0.2 M K₂HPO₄ was repeated to yield Ca and Pi concentrations of 30 and 22 mM, respectively. The interval set for the addition was 15 min, and all additions were accompanied by stirring at pH 6.7. The volume was adjusted to 4 mL by measuring the weight of the solutions. The prepared solutions were allowed to stand for 20 h at 25°C, and then centrifuged at 1,000 x g for 15 min at 25°C. The Ca and Pi in the supernatant were then determined (the former by using a Hitachi Z-600 atomic absorption spectrophotometer; Hitachi Inc., Tokyo, Japan).

Formation of HAMLET-Like Complex on Oleic Acid-Conditioned Matrices
The HAMLET-like complex from bovine -LA is called "bovine -LA made lethal to tumor cells" (BAMLET). The BAMLET-like complexes from -LA samples were prepared according to the method of Svensson et al. (2000). A column (11.3 x 1.5 cm) packed with DEAE-Trisacryl M (BioSepra, Villeneuve la Garenne, France) was attached to an HPLC system (pump: PU-2080 plus, UV detector: UV-2075 plus, Jasco) and eluted with a NaCl gradient (buffer A: 10 mM Tris-HCl, pH 8.5, 0.1 M NaCl; buffer B: 10 mM Tris-HCl, pH 8.5, 1 M NaCl). First, the matrix was conditioned with oleic acid. Twenty milligrams of oleic acid was dissolved in 1 mL of 99.5% ethanol by sonication for 3 min (Branson 3510 bath sonicator, Branson, CT). After addition of 20 mL of buffer A, the lipid solution was applied to the column and dispersed throughout the matrix by using a NaCl:60-mL linear gradient (100 to 0% A, 0 to 100% B). Twenty milligrams of protein sample was dissolved in 40 mL of buffer A containing 0.08 mM EDTA. The protein solution was applied to the column and eluted with NaCl:80-mL linear gradient (100 to 85% A, 0 to 15% B), 40 mL (85% A, 15% B), 20 mL (20% A, 80% B), and 40 mL (100% A). The protein fraction eluted with high salt content was desalted by dialysis against distilled water and lyophilized.

Apoptosis Assay
The apoptosis assay was conducted according to the method of Svensson et al. (2000). The L1210 cells were harvested by centrifugation (400 x g, 10 min, room temperature), washed in PBS, resuspended in cell culture medium without FBS, seeded into a 24-well microplate (144530, Nunc A/S) at a density of 2 x 10⁶ cells/well, and incubated at 37°C under 5% CO₂ in air. The lyophilized samples were suspended in PBS, and 100-µL quantities of these solutions were added to wells at a final protein concentration of 0.03 to 0.06 mg/mL. The protein concentration in the solution was determined using the method of Lowry et al. (1951). After 1 h, 100 µL of FBS was added to each well at a final concentration of 10%. The final volume of the media in each well was 1 mL. The PBS without BAMLET samples served as a control.

Cell viability was determined by trypan blue exclusion after 5 h of incubation. For analysis, 50 µL of the cell suspension was mixed with 50 µL of a 0.5% trypan blue solution, and the number of stained cells (dead cells) per 100 cells was determined by interference contrast microscopy (Nikon Co. Inc., Tokyo, Japan).

Statistical Analysis
The data are expressed as mean values with its SD. Significant differences between mean values are determined by Student’s t-test at the 5% significance level.

	RESULTS AND DISSCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISSCUSSION CONCLUSIONS ACKNOWLEDGMENTS REFERENCES

 
Characteristics of Glycated and Phosphorylated -LA
-Lactalbumin was conjugated with MP (MP--LA) at pH 8.0 and 50°C (65% RH) for 3 d through the Maillard reaction, and the MP--LA was then phosphorylated by dry heating in the presence of MP and pyrophosphate. Table 1 shows some characteristics of the various -LA samples. No sugar was detected in -LA, whereas the sugar content of -LA increased to 12.3% after incubation with MP at 50°C (65% RH) for 3 d, and then further still to 22.3% by dry heating at pH 4.0 and 85°C for 5 d in the presence of MP and pyrophosphate. The number of introduced MP estimated by sugar content (Table 1) was approximately 2.4 for MP--LA, and 4.9 for PP–MP--LA-5d, respectively. In most foods, the -amino group of the Lys residues in proteins is the primary source of reactive amino groups (Ames, 1992). When it was assumed that only the -amino group of the Lys residue was modified, the modified Lys residue was 21.8% for MP--LA, and 44.5% for PP–MP--LA-5d.

View this table: [in this window] [in a new window]   Table 1. Some characteristics of -lactalbumins evaluated

The solubility of -LA samples was measured at pH 7.0. No significant effect of dry heating in the absence or presence of MP and pyrophosphate on the solubility of -LA was observed, and even when dry heated for 5 d in the presence of MP and pyrophosphate after glycation, the solubility of -LA was 98.9%.

Native PAGE was performed to elucidate the changes of charge in protein by glycation and subsequent phosphorylation. Figure 1A shows the native PAGE patterns of N-, DH-, MP-, and PP–MP--LA. In the present study, a single band was observed in N--LA. In the absence of MP and pyrophosphate, there were almost no changes in the mobility of the band, whereas it decreased by glycation with MP. As glycation substitutes basic AA side-chains, it induces a slight loss of basicity, and consequently, a moderate acidification of the -LA. However, the mobility of MP--LA decreased, which might have been caused by the introduction of MP to the -LA and the subsequent increase in the molecular mass. On the other hand, compared with MP--LA, the mobility of protein increased with an increase in dry-heating time from 1 to 5 d in the presence of MP and pyrophosphate, and that mobility increase was in agreement with the phosphorylation level (Table 1).

View larger version (37K): [in this window] [in a new window]   Figure 1. Electrophoretic patterns of native (N), dry-heated (DH), maltopentaose-conjugated (MP), and phosphorylated, maltopentaose-conjugated (PP–MP) -LA: (A) native PAGE (15% polyacrylamide gel in the absence of SDS); (B) SDS-PAGE (15% polyacrylamide gel in the presence of 1.7% SDS) with (+) and without (–) 5% of 2-mercaptoethanol (2-ME). Mr = marker protein.

Structure of Glycated and Phosphorylated -LA
We used CD spectroscopy to determine the respective impact of glycation with MP and subsequent phosphorylation by dry heating in the presence of MP and pyrophosphate on the structure of the protein at the secondary folding level. Figure 2 shows the CD spectra of the -LA samples. The CD spectrum of N--LA showed double minima, at 208 and 222 nm, indicative of a predominant -helical structure, and these minima were unchanged by glycation alone. However, the minima were slightly increased by dry heating alone, and were further slightly increased by phosphorylation after glycation with the increase of dry-heating time from 1 to 5 d, suggesting that the amount of the -helical secondary structure of -LA was slightly increased by phosphorylation after glycation. These results indicated that the secondary structure of -LA was not significantly affected by glycation and subsequent phosphorylation.

View larger version (23K): [in this window] [in a new window]   Figure 2. Circular dichroism spectra of native (N), dry-heated (DH), maltopentaose-conjugated (MP), and phosphorylated, maltopentaose-conjugated (PP–MP) -LA. Protein samples were 0.1 g/L in 50 mM phosphate buffer (pH 7.0). Circular dichroism spectra of -LA samples were measured from 190 to 250 nm.

View larger version (29K): [in this window] [in a new window]   Figure 3. Tryptophan fluorescence spectra of native (N), dry-heated (DH), maltopentaose-conjugated (MP), and phosphorylated, maltopentaose-conjugated (PP–MP) -LA. The excitation wavelength was 280 nm, and the emission was scanned from 300 to 400 nm. Fluorescence spectra of -LA samples were measured at 0.1 g/L in triplicate.

View larger version (24K): [in this window] [in a new window]   Figure 4. Differential scanning calorimetry profiles of native (N), dry-heated (DH), maltopentaose-conjugated (MP), and phosphorylated, maltopentaose-conjugated (PP–MP) -LA. Differential scanning calorimetry scans were performed with a protein solution of 1 g/L in 20 mM phosphate buffer (pH 7.4). These samples were heated in the calorimeter at a scan rate of 1°C/min over a range of 30 to 80°C.

Physiological Functions of Glycated and Phosphorylated -LA

View larger version (16K): [in this window] [in a new window]   Figure 5. Antigenicity of native (N), dry-heated (DH), maltopentaose-conjugated (MP), and phosphorylated, maltopentaose-conjugated (PP–MP) -LA in an adult male Japanese white (JW/CSK) rabbit. The anti--LA response after secondary immunization of the rabbit was evaluated by noncompetitive ELISA, and the results are shown as ELISA values (absorbance at 405 nm). Each value represents the mean ± SD (n = 5). Values with different letters are significantly different at P < 0.05 as determined by Student’s t-test.

View larger version (36K): [in this window] [in a new window]   Figure 6. Effect of native (N), dry-heated (DH), maltopentaose-conjugated (MP), and phosphorylated, maltopentaose-conjugated (PP–MP) -LA on the (A) IL-6 response of THP-1 monocytes and (B) tumor necrosis factor (TNF)- response of THP-1 macrophages after stimulation with LPS. The control is media with LPS and without protein. Each datum is expressed as a percentage of the control value. Each value shows the mean ± SD (n = 5). Values with different letters are significantly different at P < 0.05 as determined by Student’s t-test.

View larger version (26K): [in this window] [in a new window]   Figure 7. Calcium phosphate-solubilizing ability of native (N), dry-heated (DH), maltopentaose-conjugated (MP), and phosphorylated, maltopentaose-conjugated (PP–MP) -LA. The test solution contained 20 g/L of protein, 30 mM Ca, 22 mM inorganic P (Pi), and 10 mM citrate, with pH adjusted to 6.7 with 1 M KOH. Each column shows the mean values ± SD (n = 3).

View this table: [in this window] [in a new window]   Table 2. The viability1 of L1210 cells after exposure to bovine -LA made lethal to tumor cells (BAMLET) at different concentrations (0.03 to 0.06 mg/mL) from different -LA

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISSCUSSION CONCLUSIONS ACKNOWLEDGMENTS REFERENCES

	ACKNOWLEDGMENTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISSCUSSION CONCLUSIONS ACKNOWLEDGMENTS REFERENCES

 
This work was supported by a grant-in-aid (17580238) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. We thank Kentaro Morizane (Nihon Shokken Co., Ehime, Japan) for conducting the DSC experiment.

Received for publication January 5, 2009. Accepted for publication February 27, 2009.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISSCUSSION CONCLUSIONS ACKNOWLEDGMENTS REFERENCES

 


Ames, J. M. 1992. The Maillard reaction. Pages 99–153 in Biochemistry of Food Proteins. B. J. F. Hudson, ed. Elsevier Science Publishers, London, UK.

Aoki, T. 1989. Incorporation of individual casein constituents into casein aggregates cross-linked by colloidal calcium phosphate in artificial casein micelles. J. Dairy Res. 56:613–618.[CrossRef]

Aoki, T., Fukumoto, T., Kimura, T., Kato, Y. and Matsuda, T.. 1994. Whey protein- and egg white protein-glucose 6-phosphate conjugates with calcium phosphate-solubilizing properties. Biosci. Biotechnol. Biochem. 58:1727–1728.

Aoki, T., Hiidome, Y., Sugimoto, Y., Ibrahim, H. R. and Kato, Y.. 2001. Modification of ovalbumin with oligogalacturonic acid through the Maillard reaction. Food Res. Int. 34:127–132.[CrossRef]

Aoki, T., Kitahata, K., Fukumoto, T., Sugimoto, Y., Ibrahim, H. R., Kimura, T., Kato, Y. and Matsuda, T.. 1997. Improvement of functional properties of β-lactoglobulin by conjugation with glucose-6-phosphate through the Maillard reaction. Food Res. Int. 30:401–406.[CrossRef]

Armstrong, J. M., McKenzie, H. A. and Sawyer, W. H.. 1967. On the fractionation of β-lactoglobulin and -lactalbumin. Biochim. Biophys. Acta 147:60–72.[Medline]

Auger, M. J., and J. A. Ross. 1992. The biology of the macrophage. Pages 3–74 in The macrophage. C. E. Lewis and J. O. McGee, ed. IRL Press, New York, NY.

Brodbeck, U., Denton, W. L., Tanahashi, N. and Ebner, K. E.. 1967. The isolation and identification of the B protein of lactose synthetase as -lactalbumin. J. Biol. Chem. 242:1391–1397.[Abstract/Free Full Text]

Chen, P. S., Toribara, T. Y. and Warner, H.. 1956. Microdetermination of phosphorus. Anal. Chem. 28:1756–1758.

Chevalier, F., Chobert, J. M., Mollé, D. and Haertlé, T.. 2001. Maillard glycation of β-lactoglobulin with several sugars: Comparative study of the properties of the obtained polymers and of the substituted sites. Lait 81:651–666.[CrossRef]

Chobert, J. M. 2003. Milk protein modification to improve functional and biological properties. Pages 1–60 in Advances in Food and Nutrition Research. Vol. 47. S. L. Taylor, ed. Academic Press, New York, NY.

Cross, M. L. and Gill, H. S.. 2000. Immunomodulatory properties of milk. Br. J. Nutr. 84:S81–S89.[Medline]

Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A. and Smith, F.. 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28:350–356.

Enomoto, H., Li, C. P., Morizane, K., Ibrahim, H. R., Sugimoto, Y., Ohki, S., Ohtomo, H. and Aoki, T.. 2007. Glycation and phosphorylation of β-lactoglobulin by dry-heating: Effect on protein structure and some properties. J. Agric. Food Chem. 55:2392–2398.[CrossRef][Medline]

Enomoto, H., Li, C. P., Morizane, K., Ibrahim, H. R., Sugimoto, Y., Ohki, S., Ohtomo, H. and Aoki, T.. 2008. Improvement of functional properties of bovine serum albumin through phosphorylation by dry-heating in the presence of pyrophosphate. J. Food Sci. 73:C84–C91.[CrossRef][Medline]

Enomoto, H., Nagae, S., Hayashi, Y., Li, C. P., Ibrahim, H. R., Sugimoto, Y. and Aoki, T.. 2009. Improvement of functional properties of egg white protein through glycation and phosphorylation by dry-heating. Asian-australas. J. Anim. Sci. 22:591–597.

Feeney, R. E. 1975. Chemical changes in food proteins. Pages 233–254 in Evaluation of Proteins for Humans. C. E. Bodwell, ed. AVI Publishers, Westport, CT.

FitzGerald, R. J., Murray, B. A. and Walsh, D. J.. 2004. Hypotensive peptides from milk proteins. J. Nutr. 134:980S–988S.[Abstract/Free Full Text]

Fox, P. F. 2003. Milk proteins: General and historical aspects. Pages 1–41 in Advanced Dairy Chemistry. Vol. 1. P. F. Fox and P. L. H. McSweeney, ed. Plenum Publishers, New York, NY.

Kato, A., Ibrahim, H. R., Watanabe, H., Honma, K. and Kobayashi, K.. 1989. New approach to improve the gelling and surface functional properties of dried egg white by heating in dry state. J. Agric. Food Chem. 37:433–437.[CrossRef]

Kato, Y., Aoki, T., Kato, N., Nakamura, R. and Matsuda, T.. 1995. Modification of ovalbumin with glucose-6-phosphate by amino-carbonyl reaction. Improvement of protein heat stability and emulsifying activity. J. Agric. Food Chem. 43:301–305.[CrossRef]

Kume, H., Okazaki, K. and Sasaki, H.. 2006. Hepatoprotective effects of whey protein on _D-galactosamine-induced hepatitis and liver fibrosis in rats. Biosci. Biotechnol. Biochem. 70:1281–1285.[CrossRef][Medline]

Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685.[CrossRef][Medline]

Li, C. P., Enomoto, H., Ohki, S., Ohtomo, H. and Aoki, T.. 2005a. Improvement of functional properties of whey protein isolate through glycation and phosphorylation by dry heating. J. Dairy Sci. 88:4137–4145.[Abstract/Free Full Text]

Li, C. P., Hayashi, Y., Shinohara, H., Ibrahim, H. R., Sugimoto, Y., Kurawaki, J., Matsudomi, N. and Aoki, T.. 2005b. Phosphorylation of ovalbumin by dry-heating in the presence of pyrophosphate: Effect on protein structure and some properties. J. Agric. Food Chem. 53:4962–4967.[CrossRef][Medline]

Li, C. P., Ibrahim, H. R., Sugimoto, Y., Hatta, H. and Aoki, T.. 2004. Improvement of functional properties of egg white protein through phosphorylation by dry-heating in the presence of pyrophosphate. J. Agric. Food Chem. 52:5752–5758.[CrossRef][Medline]

Li, C. P., Salvador, A. S., Ibrahim, H. R., Sugimoto, Y. and Aoki, T.. 2003. Phosphorylation of egg white proteins by dry-heating in the presence of phosphate. J. Agric. Food Chem. 51:6808–6815.[CrossRef][Medline]

Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J.. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265–275.[Free Full Text]

Markus, C. R., Olivier, B., Panhuysen, G. E. M., Gugten, J. V. D., Alles, M. S., Truiten, A., Westenberg, H. G. M., Fekkes, D., Koppeschaar, H. F. and de Haan, E. E. H. F.. 2000. The bovine protein -lactalbumin increases the plasma ratio of tryptophan to the other large neutral amino acids, and in vulnerable subjects raises brain serotonin activity, reduces cortisol concentration, and improves mood under stress. Am. J. Clin. Nutr. 71:1536–1544.[Abstract/Free Full Text]

Matheis, G. and Whitaker, J. R.. 1984. Chemical phosphorylation of food proteins: An overview and a prospectus. J. Agric. Food Chem. 32:699–705.[CrossRef]

Matsuda, N. and Hattori, Y.. 2006. Systemic inflammatory response syndrome (SIRS): Molecular pathophysiology and gene therapy. J. Pharmacol. Sci. 101:189–198.[CrossRef][Medline]

Matsumoto, H., Shimokawa, Y., Ushida, Y., Toida, T. and Hayasawa, H.. 2001. New biological function of bovine alpha-lactalbumin: Protective effect against ethanol- and stress-induced gastric mucosal injury in rats. Biosci. Biotechnol. Biochem. 65:1104–1111.[CrossRef][Medline]

Mattsby-Baltzer, I., Roseanu, A., Motas, C., Elverfors, J., Engberg, I. and Hanson, L. Å.. 1996. Lactoferrin or a fragment thereof inhibits the endotoxin-induced interleukin-6 response in human monocytic cells. Pediatr. Res. 40:257–262.[Medline]

Maynard, F., Jost, R. and Wal, J.-M.. 1997. Human IgE binding capacity of tryptic peptides from bovine -lactalbumin. Int. Arch. Allergy Immunol. 113:478–488.[Medline]

Möller, B. and Villiger, P. M.. 2006. Inhibition of IL-1, IL-6, and TNF- in immune-mediated inflammatory diseases. Springer Semin. Immunopathol. 27:391–408.[CrossRef][Medline]

Ouchterlony, O. 1949. Antigen-antibody reactions in gels. Acta Pathol. Microbiol. Scand. 26:507–515.[Medline]

Pellegrini, A., Thomas, U., Bramaz, N., Hunziker, P. and von Fellenberg, R.. 1999. Isolation and identification of three bactericidal domains in the bovine -lactalbumin molecule. Biochim. Biophys. Acta 1426:439–448.[Medline]

Permyakov, E. A. and Berliner, L. J.. 2000. -Lactalbumin: Structure and function. FEBS Lett. 473:269–274.[CrossRef][Medline]

Pettersson, J., Mossoberg, A.-K. and Svanborg, C.. 2006. -Lactalbumin species variation, HAMLET formation, and tumor cell death. Biochem. Biophys. Res. Commun. 345:260–270.[CrossRef][Medline]

Seguro, K. and Motoki, M.. 1989. Enzymatic phosphorylation of soybean proteins by protein kinase. Agric. Biol. Chem. 53:3263–3268.

Shah, N. P. 2000. Effects of milk-derived bioactives: An overview. Br. J. Nutr. 84(Suppl. 1):S3–S10.[Medline]

Sitohy, M., Chobert, J. M. and Haertlé, T.. 1995. Phosphorylation of β-lactoglobulin under mild conditions. J. Agric. Food Chem. 43:59–62.[CrossRef]

Sternhagen, L. G. and Allen, J. C.. 2001. Growth rates of a human colon adenocarcinoma cell line are regulated by the milk protein -lactalbumin. Adv. Exp. Med. Biol. 501:115–120.[Medline]

Svensson, M., Håkansson, A., Mossberg, A. K., Linse, S. and Svanborg, C.. 2000. Conversion of alpha-lactalbumin to a protein inducing apoptosis. Proc. Natl. Acad. Sci. USA 97:4221–4226.[Abstract/Free Full Text]

Vey, E., Zhang, J.-M. and Dayer, J.-M.. 1992. IFN- and 1,25(OH)₂D₃ induce on THP-1 cells distinct patterns of cell surface antigen expression, cytokine production, and responsiveness to contact with activated T cells. J. Immunol. 149:2040–2046.[Abstract]

Vojdani, F. and Whitaker, J. R.. 1996. Phosphorylation of proteins and their functional and structural properties. ACS Symp. Ser. 650:210–229.

Watanabe, K., Xu, J. Q. and Shimoyamada, M.. 1999. Inhibiting effects of egg white dry-heated at 120°C on heat aggregation and coagulation of egg white and characteristics of dry-heated egg white. J. Agric. Food Chem. 47:4083–4088.[CrossRef][Medline]

Wijesinha-Bettoni, R., Gao, C., Jenkins, J. A., Mackie, A. R., Wilde, P. J., Mills, E. N. C. and Smith, L. J.. 2007. Heat treatment of bovine -lactalbumin results in partially folded, disulfide bond shuffled states with enhanced surface activity. Biochemistry 46:9774–9784.[CrossRef][Medline]

Yamaguchi, M. and Uchida, M.. 2007. -Lactalbumin suppresses interleukin-6 release after intestinal ischemia/reperfusion via nitric oxide in rats. Inflammopharmacology 15:43–47.[CrossRef][Medline]

Yamamoto, Y. and Gaynor, R. B.. 2001. Therapeutic potential of inhibition of the NF-B pathway in the treatment of inflammation and cancer. J. Clin. Invest. 107:135–142.[Medline]

Ye, X., Yoshida, S. and Ng, T. B.. 2000. Isolation of lactoperoxidase, lactoferrin, -lactalbumin, β-lactoglobulin A from bovine rennet whey using ion exchange chromatography. Int. J. Biochem. Cell Biol. 32:1143–1150.[CrossRef][Medline]

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