Preparation of Phosphopeptides Derived from αs-Casein and β-Casein Using Immobilized Glutamic Acid-Specific Endopeptidase and Characterization of Their Calcium Binding

Preparation of Phosphopeptides Derived from as-Casein and b-Casein Using Immobilized Glutamic Acid-Specific Endopeptidase and Characterization of Their Calcium Binding O. PARK and J. C. ALLEN1 Southeast Dairy Foods Research Center, Department of Food Science, North Carolina State University, Raleigh 27695-7624

ABSTRACT Phosphopeptides that were derived from as-CN or b-CN were prepared with immobilized glutamic acidspecific endopeptidase, and their Ca2+ binding was characterized. as-Casein or b-CN was hydrolyzed in a fluidized bed bioreactor containing 2 ml of immobilized glutamic acid-specific endopeptidase by recirculating 20 ml of as-CN or b-CN solution (10 mg/ml in 50 mM Tris·HCl and 0.02% NaN3, pH 8.0) for 3 h at 20°C. The molecular masses of casein peptides were monitored by SDS-PAGE. Each hydrolysate was applied to an anion-exchange column using stepwise elution with various concentrations of KCl to separate peptides. The casein phosphopeptide content of the elution profile was monitored by analysis of protein and P concentrations. Calcium binding in phosphopeptide-enriched fractions was determined by CaCl2 titration and measurement of free Ca2+ with a Caselective electrode. The electrophoresis patterns showed four major peptides having molecular masses of 10.8, 9.0, 6.6, and 3.6 kDa in the as-CN hydrolysate and 9.3, 8.2, and 6.2 kDa in the b-CN hydrolysate. The highest concentrations of P were detected in the fractions that eluted with 0.4 and 0.5 M KCl for the as-CN hydrolysate and with 0.4 M KCl for the b-CN hydrolysate. The calcium-binding ability was found only in the fraction that was eluted with 0.4 M KCl; the maximum Ca2+ binding and the apparent binding constant were 0.24 mmol/mg of protein and 75 M–1, and 0.14 mmol/mg of protein and 148 M–1, respectively. as-Casein phosphopeptides had different patterns for Ca2+ binding than did b-CN phosphopeptides as the total Ca concentration was increased. Calcium binding to these casein phosphopeptides differed from that previously characterized for the tryptic peptides. ( Key words: casein phosphopeptides, immobilized

Received August 4, 1997. Accepted July 13, 1998. 1Author to whom correspondence should be addressed. 1998 J Dairy Sci 81:2858–2865

enzyme, glutamic acid-specific endopeptidase, calcium binding) Abbreviation key: CPP = casein phosphopeptides, GSE = glutamic acid-specific endopeptidase. INTRODUCTION Casein is the major protein component of bovine milk. An important feature of its structure is that casein is a phosphoprotein composed of as1-CN, as2CN, b-CN, and k-CN in the ratio of 3:0.8:3:1 (28). Each fraction differs considerably in its P content: 8 to 9, 10 to 13, 5, and 1 residues, respectively. These phosphorylated proteins contain clusters of phosphoserine that have been shown to stabilize amorphous calcium phosphate and casein micelle structures. Some of the biological and technological functions that recently have been attributed to the phosphopeptides derived from casein include Ca absorption, Ca retention, and bone calcification (17, 19, 25, 30, 35); hypotensive effect (16, 36); anticariogenicity (24); milk curdling (37); and stability of cream liqueurs (11). These functional properties are mainly related to the physicochemical properties of casein phosphopeptides ( CPP) that enable the chelation of various bivalemt and trivalent minerals, thereby enhancing mineral solubility (15). The physicochemical forms of Ca in milk have been shown to have an important function in the stability of the casein micelle as well as possibly contributing to the high bioavailability of Ca. These unique properties of CPP have led to much interest in the isolation of phosphopeptide fractions and individual phosphopeptides from enzymatic digests of whole casein and purified casein components (6, 10, 14, 34). Much of this work has been done with the use of trypsin. Tryptic hydrolysis yielded a mixture of peptides with homogeneous phosphopeptides that were obtained from b-CN ( 1 8 ) and a mixture of P-containing peptides in the case of as1CN (20). However, a commercial process for the production of CPP would be more effective if an indus-

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trial or food-grade enzyme were used. Adamson and Reynolds ( 1 ) isolated and characterized CPP prepared by alcalase, showing a number of homologous peptides of various lengths resulting from the broad specificity of alcalase. Glutamic acid-specific endopeptidases ( GSE) have been newly developed and isolated from a commercial extract obtained by fermentation with Bacillus licheniformis (27). The hydrolytic specificity of GSE is at the carboxyl side of acidic amino acid residues, preferentially at glutamic acid (5, 27). This enzyme is completely inhibited by diisopropylfluorophosphate, suggesting that GSE are serine endopeptidases. The GSE could be useful for commercial preparation of CPP because the bacterial enzyme may be produced less expensively than some other proteases. However, the acceptability of the CPP preparation derived from GSE hydrolysis in regard to mineral binding and other properties has not been investigated. The objective of this study was to prepare CPP that were derived from as-CN or b-CN using immobilized GSE and to characterize the Ca2+-binding ability, which is the main function of CPP. MATERIALS AND METHODS Chemicals Reagent-grade chemicals and distilled-deionized water were used throughout. The GSE (SP446) was provided by Novo Nordisk (Franklinton, NC). Immobilization of GSE The GSE was immobilized on succinamidopropyl glass beads by the method of Janolino and Swaisgood (13). Controlled-pore glass beads (CPG-2000 A, 120/ 200 mesh size; CPG Inc., Fairfield, NJ) were used for this purpose. Degassed succinamidopropyl beads were activated for 30 min with 1-ethyl-3-[3dimethylamino)propyl]carbodiimide by stirring with N2 gas at 20°C. The activated beads were rapidly (<2 min) washed with pH 7.0 phosphate buffer at 0°C. Then, a dilute enzyme solution ( 6 mg/ml in 0.1N phosphate buffer, pH 7.0) was recirculated for 24 h at 4°C. After reacting, the immobilized enzyme was washed with 2 M urea, followed by 2 M NaCl and Tris·HCl buffer containing 0.02% NaN3 to remove noncovalently bound enzyme. The immobilized enzyme was stored at 4°C. The activity of the immobilized GSE was determined according to the method of Breddam and Meldal ( 5 ) . The hydrolysis of substrates, based on intramolecular quenching, containing the anthraniloyl group in 50 mM bicine, pH 8.0, was assayed by

monitoring the fluorescence emission at 420 nm upon excitation at 320 nm using a scanning spectrofluorometer (Optical Technology Devices, Inc., Elmsford, NY) at 25°C. The activity was 19 U/g of beads. One unit was the change in fluorescence of 0.3 per min with anthranilyl-Ala-Phe-Ala-Phe-Glu-ValPhe-Nitro-Tyr-Asp as a substrate at pH 8.0 at 25°C. Proteolysis of as-CN or b-CN by Immobilized GSE The hydrolysis of purified as-CN or b-CN ( 2 1 ) was carried out in 50 mM Tris·HCl, pH 8.0, containing 0.02% NaN3 at 20°C. A fluidized bed bioreactor containing 2 ml of immobilized GSE beads was used to recirculate 20 ml of as-CN or b-CN solution (10 mg/ ml buffer) for 3 h. SDS-PAGE as-Casein or b-CN hydrolysates were analyzed by SDS-PAGE (Hoefer Scientific Instruments, San Francisco, CA) using a 1.5 mm thick and 16.5% T ([acrylamide (grams) + bis-acrylamide (grams)] × 100/100 ml) and 3% C (bis-acrylamide (grams) × 100/([acrylamide (grams) + bis-acrylamide (grams)]) polyacrylamide slab gel according to the discontinuous procedure of Schagger and Von Jagow (26). Protein bands were visualized by brilliant blue G staining. Molecular masses were estimated from a standard curve that was prepared with molecular mass markers of 16,950 (myoglobin, 1–153), 14,440 (myoglobin, 1–131), 10,600 (myoglobin, 56–153), 8160 (myoglobin, 56–131), 6210 (myoglobin, 1–55), 3480 (glucagon), and 2510 kDa (myoglobin, 132–153). The relative mobilities ( R f ) were fit to the equation log ( M M ) = a + b × Rf, where MM represents molecular mass, and a and b are constants. Separation of CPP A column (QAE Sephadex A 25; Pharmacia Fine Chemicals, Piscataway, NJ) was equilibrated with 50 mM Tris·HCl containing 0.02% NaN3, pH 8.0. Hydrolysates of as-CN or b-CN were loaded onto the column at pH 8.0 with a flow rate of 3 ml/min. Unbound peptides were eluted with equilibration buffer. Peptide fractions were obtained by stepwise elution with 0.1, 0.2, 0.4, and 0.5 M KCl containing 50 mM Tris·HCl buffer, pH 8.0. The eluate was continuously monitored at 280 nm. When the last fraction had emerged from the column, material remaining on the column was removed by elution with 2 M KCl, and then the column was regenerated with elution buffer. Journal of Dairy Science Vol. 81, No. 11, 1998

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Figure 1. The SDS-PAGE patterns for hydrolysates of as-CN prepared with a fluidized bed bioreactor containing 2 ml of immobilized glutamic acid-specific endopeptidase beads with increasing times of hydrolysis. Hydrolysis was performed in 50 mM Tris·HCl containing 0.02% NaN3, pH 8.0 at 20°C. STD = Standard molecular mass markers.

Figure 2. The SDS-PAGE patterns for hydrolysates of b-CN prepared with a fluidized bed bioreactor containing 2 ml of immobilized glutamic acid-specific endopeptidase beads with increasing times (minutes) of hydrolysis. Hydrolysis was performed in 50 mM Tris·HCl containing 0.02% NaN3, pH 8.0 at 20°C. STD = Standard molecular mass markers.

Protein and P contents for each fraction were determined by micro-Kjeldahl ( N × 6.38) ( 3 ) and a microdetermination method ( 7 ) , respectively.

for 3 h. The hydrolysis patterns of as-CN or b-CN treated with immobilized GSE are shown in Figures 1 and 2. The SDS-PAGE for as-CN hydrolysates gave four well-defined bands that indicate molecular sizes from 3.6 to 10.8 kDa. The hydrolysis occurred rapidly after 5 min of incubation. The electrophoresis showed

Ca2+-binding Measurement The Ca2+-binding isotherms for each fraction that contains P from as- or b-CN were obtained from measurements of free Ca2+ with a Ca2+ selective electrode (model 93-20; Orion, Boston, MA) connected to an Orion ion analyzer (model 290 A). Protein solutions (10 ml) were titrated with 0.1 M CaCl2 at 20°C. The pH changes through entire titration were less than 1.0 pH unit. Free Ca2+ was measured after each 100-ml addition of CaCl2 solution, and the concentration of total Ca2+ was corrected for dilution. The reaction mixture was stirred for approximately 1 min before the millivolt measurement was taken. The bound Ca was calculated by subtracting the free Ca2+ from the total Ca and dividing by ligand concentration. The apparent association constants and the maximum Ca that was bound per milligram of protein were calculated using a Scatchard plot analysis. RESULTS

TABLE 1. Protein and phosphorus contents for each peptide fraction obtained from QAE Sephadex A 25 chromatography1 of as-CN or b-CN hydrolysates prepared by immobilized glutamic acidspecific endopeptidase.2 Fraction number3

Purified as-CN (91.7%) or b-CN (95%) was hydrolyzed with the immobilized GSE bioreactor at 20°C Journal of Dairy Science Vol. 81, No. 11, 1998

P5

(mg/ml) as-CN Unbound I II III IV b-CN Unbound I II III

X

SE

(mmol/mg of protein) X SE

0.507 ... 0.293 0.855 0.335

0.1 ... 0.2 0.0010 0.0000

... ... 0.033 0.217 0.028

... ... 0.001 0.051 0.001

0.278 0.178 0.724 0.615

0.049 0.0000 0.0000 0.0030

... ... ... 0.183

... ... ... 0.0071

1Pharmacia

Fine Chemicals (Piscataway, NJ). expressed as means of duplicate determinations. 3Equal volume fractions eluted with 0 (unbound), 0.1 ( I ) , 0.2 (II), 0.4 (III), or 0.5 ( I V ) M KCl in 50 mM Tris·HCl, pH 8.0. 4Protein measured by micro-Kjeldahl ( N × 6.38). 5Phosphorus by microdetermination method (820 nm). 2Data

Proteolysis of as-CN or b-CN

Protein4

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three major bands of 9.3, 8.2, and 6.2 kDa in the b-CN hydrolysates. However, peptides with <6.2-kDa molecular mass could not be seen on the gel (Figure 2). Separation of CPP The GSE hydrolysates that were derived from asCN or b-CN were separated by chromatography on QAE Sephadex A 25 (Figure 3). The chromatographic patterns of as-CN and b-CN hydrolysates were different. For as-CN, once unbound peptides were eluted with 50 mM Tris·HCl, pH 8.0, no additional peaks appeared until the elution buffer ionic strength was increased to 0.4 M KCl. For b-CN hydrolysates, four different peaks appeared, one with each change of elution buffer concentration (0, 0.1, 0.2, and 0.4 M KCl in Tris equilibration buffer). Both as-CN and b-CN peptide fractions contained the greatest amount of P in the peak that was eluted with 0.4 M KCl, 0.217 ± 0.051 mmol of P/mg of protein, and 0.183 ± 0.007 mmol of P/mg of protein, respectively (Table 1).

Ca2+-binding Properties The binding isotherms (Figure 4 ) obtained for the phosphopeptide-enriched fraction derived from as-CN showed that bound Ca increased as the total Ca concentration increased. The b-CN phosphopeptide fraction showed increasing Ca2+ binding until a plateau appeared at approximately 6 mM total Ca titration. The Ca2+-binding pattern of the b-CN fraction was similar to that of the as-CN fraction at low total Ca concentration, but the as-CN fraction showed greater Ca2+ binding than that of the b-CN fraction at high Ca concentration. The Scatchard plots calculated from these two isotherms are shown in Figure 5. The as-CN phosphopeptide fraction gave a straight line, indicating a defined number of equivalent binding sites showing a low affinity for Ca2+ with an apparent association constant, 75 M–1, and a maximum of bound Ca of 0.24 mmol/mg of protein. However, the maximum of bound Ca and the apparent association constant calculated for the b-CN phosphopeptide fraction were 0.14 mmol/ mg of protein and 148 M–1, respectively.

Figure 3. Chromatographic patterns of as-CN or b-CN hydrolysates obtained with immobilized glutamic acid-specific endopeptidases on QAE Sephadex A 25 column (Pharmacia Fine Chemicals, Piscataway, NJ). Peptides absorbance at 280 nm (left scale) is shown by the solid line. The dotted line shows the stepwise changes in KCl concentration in elution buffer. Journal of Dairy Science Vol. 81, No. 11, 1998

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Figure 4. Binding of Ca2+ to phosphopeptides derived from asCN ( ◊) and b-CN ( ◊) prepared using glutamic acid-specific endopeptidases. Only fractions eluted with 0.4 M KCl containing 50 mM Tris·HCl, pH 8.0 at 20°C, which contain the highest phosphopeptide concentration, were characterized.

DISCUSSION The CPP can be prepared by hydrolysis of casein using various proteolytic enzymes. Trypsin cleavage at the carboxyl side of Arg and Lys ( 3 2 ) has been widely used for this purpose and has yielded a mixture of peptides containing all of the P of b-CN (6, 18) and a mixture of peptides containing amino acid residues f 43–58, f 59–79, and f 43–79 from as1-CN (34). More recently, Adamson and Reynolds ( 1 ) used an alcalase enzyme with a broad specificity to digest whole casein to prepare CPP. The CPP that were released by alcalase were truncated relative to those released by the action of trypsin, containing the cluster sequences as well as a group of tri-, di-, and monophosphorylated peptides. The GSE that was isolated from B. licheniformis (5, 27) consists of one peptide chain of 222 amino acid residues and has a calculated molecular mass of 23,589 Da. This enzyme is completely inhibited by diisopropylfluorophosphate, suggesting that it is a serine endopeptidase. The amino acid sequences show similarity to the Glu-specific and Asp-specific enzymes isolated from Staphylococcus aureus V8 (27). The substrate preference appears to be essentially specific for all types of proteins with Glu-Xaa and Asp-Xaa; there is a strong preference for the former, Journal of Dairy Science Vol. 81, No. 11, 1998

although those proteins with Xaa as Asp and, in particular, Xaa as Pro, are hydrolyzed at very low rates ( 5 ) . Considering these characteristics of GSE, we prepared CPP derived from as-CN or b-CN with a continuous process using immobilized GSE. The results were different from those of the previous study using trypsin (22). The hydrolysis patterns for each protein (Figures 1 and 2 ) gave different molecular size distributions. Their theoretical, limited peptides are 2051 Da from as1-CN and 4846 Da from b-CN, which correspond to f 158–175 of as1-CN B and f 48–91 of b-CN A2, respectively (Figure 6). The limited peptides that were derived from the GSE hydrolysis of as-CN or b-CN were found on SDSPAGE. The CPP that were derived from as-CN and bCN were not detected on SDS-PAGE (Figures 1 and 2 and Table 2). Although peptides were expected to be smaller than those of tryptic hydrolysates, because of the substrate specificity of the enzymes, the bands below 3 kDa were not detectable by SDS-PAGE. Ionexchange chromatography showed only two peaks by stepwise elution with KCl. For as-CN, these peaks contained less P than did the fractions of tryptic hydrolysates that were enriched by CPP. Possibly, the GSE hydrolysis produced smaller CPP fractions relative to tryptic hydrolysis that could not be separated well by QAE Sephadex A 25 chromatography.

Figure 5. Scatchard analysis for the binding of Ca2+ to phosphopeptides derived from as-CN ( ◊) and b-CN ( o) prepared using glutamic acid-specific endopeptidases. The fractions characterized were eluted with 0.4 M KCl containing 50 mM Tris·HCl, pH 8.0 at 20°C.

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The CPP have been reported to be bioactive peptides with unique properties, especially in respect to increasing Ca bioavailability (16). Proposed mechanisms are a slowing of the rate of gastric emptying, facilitating the formation of soluble Ca complexes, and enhancing the exposure of intestinal mucosal absorption sites to the available Ca. The primary structures of the CPP residues have been described (28). The Ca-chelating activity of CPP in vitro (23, 34) was attributed to the role of component phosphoserine (SerP) residues, which resulted in a highly polar acidic domain. Other studies (2, 4, 9, 12) supported the hypothesis that the Ca2+-binding ability of CPP is primarily due to SerP groups (2, 4, 9, 12). However, it has been suggested that other side chains, such as glutamic and aspartic acids, may contribute to the metal ion binding. Dickson and Perkins ( 8 ) reported that the free carboxyl groups of the proteins are potential binding sites for metal ions, although of relatively minor importance. Those researchers also found that, at a low concentration of

TABLE 2. Comparison of experimental mass with theoretical mass based on primary structures of peptides from as1-CN B and b-CN A2 using immobilized glutamic acid-specific endopeptidases. Molecular mass Sequence

Theoretical

Experimental (Da)

as1-CN f 52–141 f 44–117 f 86–141 f 90–118 f 158–175 (limited) f 64–692 f 44–472 f 48–512 b-CN f 12–91 f 48–121 f 130–184 f 48–91 (limited) f 15–202

10,805 9005 6606 3552 2051 919 482 413

10,800 9000 6600 3600 ND3 ND ND ND

9369 8257 6243 4846 920

9300 8250 6210 ND ND

1According

to the review article of Swaisgood (28). phosphopeptides. 3Not detectable using SDS-PAGE. 2Casein

the cation, interactions are predominantly with the phosphate groups. Van Dijk ( 3 1 ) suggested that a Ca bridge may be formed between SerP and Glu. He took into account the hypothesis that Glu has an important function in the interaction between SerP and metal ions, which is followed by the formation of protein complexes in the micelle formation. The glutamic acid may catalyze the formation of the larger complex by forming a Ca bridge with SerP. The adjacent glutamic acid residue retains this Ca2+ in a weak bond, after which CaPO–4 takes over the Ca2+.

Figure 6. Primary sequences of as1-CN and b-CN A2 with the sites of proteolysis using immobilized glutamic acid-specific endopeptidases indicated. Sequences were according to Swaisgood (28). Arrows indicate the theoretical cleavage sites for glutamic acidspecific endopeptidase.

In our results, the phosphopeptide-enriched fraction of as-CN showed greater the maximum Ca2+binding ability (0.24 mmol/mg of protein) than that (0.14 mmol/mg of protein) of the b-CN fraction. However, Ca2+-binding affinity was approximately two times weaker for the as-CN phosphopeptide fraction (75 M–1) than for the b-CN fraction (147 M–1) . The data indicate that SerP groups were primarily responsible for Ca2+ binding because the binding was proportional to the amount of P. However, both fractions of as- and b-CN phosphopeptides made with GSE showed less Ca2+-binding ability than those made by tryptic hydrolysis. The CPP prepared by GSE hydrolysis are shorter than those of tryptic hydrolysis and have fewer SerP clusters. Therefore, CPP made using this enzyme may not be long enough to bind Ca with high affinity. According to the theory of Van Dijk (31), the elimination of glutamic acid Journal of Dairy Science Vol. 81, No. 11, 1998

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residues that are adjacent to SerP residues might cause a loss of the ability of the peptide to form Ca complexes. In the present study, even though the bCN phosphopeptide fraction made with GSE contained greater amounts of P (0.183 ± 0.007 mmol/mg of protein) than the analogous tryptic fraction (0.080 ± 0.016 mmol/mg of protein) (22), the Ca2+-binding ability of GSE peptides was less than that of tryptic peptides; the maximum amount of bound Ca was 0.14 mmol/mg of protein and 0.51 mmol/mg of protein, respectively. This hypothesis was also supported by the results of Dickson and Perkins ( 8 ) who found that additional phosphorylation of as1-CN gave a lower apparent association constant for complexes with Ca2+ than did untreated as1-CN. An explanation may be that a larger proportion of the SerP groups in untreated as1-CN is accompanied by Glu, which promotes the binding of Ca2+. Walstra and Jenness ( 3 3 ) also demonstrated that a large proportion of the SerP groups (88, 73, 80, and 100% of the groups in as1-, as2-, b-, and k-CN, respectively) is accompanied by a glutamic acid or SerP residue in the adjacent or the next amino acid position, resulting in the cluster sequences (SerP-SerP-SerP-Glu-Glu) (29). Overall, these results indicate that the Glu residue may affect the Ca2+-binding to SerP groups. The GSE may not be useful for the preparation of CPP if the desired function is Ca2+ binding because the terminal Glu cannot serve the same important function as Glu embedded in the peptide chain. However, further research on the molecular characteristics of the peptides generated during GSE proteolysis in regard to other functional properties and their influence on metabolic processes may suggest other reasons for the utilization of this material in food systems. ACKNOWLEDGMENTS This research was supported by the Southeast Dairy Foods Research Center (Raleigh, NC), North Carolina Dairy Foundation (Raleigh), and DMV International Nutritionals (Fraser, NY). The authors thank Novo Nordisk (Franklinton, NC) for providing the enzyme. REFERENCES 1 Adamson, N. J., and E. C. Reynolds. 1996. Characterization of casein phosphopeptides prepared using alcalase: determination of enzyme specificity. Enzyme Microbiol. Technol. 19:202–207. 2 Aoki, T., Y. Kako, and T. Imamura. 1986. Separation of casein aggregates cross-linked by colloidal calcium phosphate from bovine casein micelles by high performance gel chromatography in the presence of urea. J. Dairy Res. 53:53–59. 3 Association of Official Analytical Chemists. 1980. Official Methods of Analysis. 13th ed. AOAC, Washington, DC. Journal of Dairy Science Vol. 81, No. 11, 1998

4 Berrocal, R., S. Chanton, M. A. Juillerat, B. Pavillard, J.-C. Scherz, and R. Jost. 1989. Tryptic phosphopeptides from whole casein. II. Physicochemical properties related to the solubilization of calcium. J. Dairy Res. 56:335–341. 5 Breddam, K., and M. Meldal. 1992. Substrate preferences of glutamic-acid-specific endopeptidases assessed by synthetic peptide substrates based on intramolecular fluorescence quenching. Eur. J. Biochem. 206:103–107. 6 Carles, C., and B. Ribadeau-Dumas. 1986. Determination of gradient elution conditions for the separation of peptide mixtures by reverse-phase high-performance liquid chromatography: bovine b-casein tryptic digest. J. Dairy Res. 53:595–600. 7 Chen, P. S., Jr., T. Y. Toribara, and H. Warner. 1956. Microdetermination of phosphorus. Anal. Chem. 28:1756–1758. 8 Dickson, I. R., and D. J. Perkins. 1971. Studies on the interactions between purified bovine caseins and alkaline-earthmetal ions. Biochem. J. 124:235–240. 9 Farrell, H. M., Jr., and M. P. Thompson. 1988. The caseins of milk as calcium-binding proteins. Pages 117–137 in Calcium Binding Proteins. Vol. II. M. P. Thompson, ed. CRC Press, Boca Raton, FL. 10 Gagnaire, V., A. Pierre, D. Molle, and J. Leonil. 1996. Phosphopeptides interact with colloidal calcium phosphate isolated by tryptic hydrolysis of bovine casein micelles. J. Dairy Res. 63: 405–422. 11 Horne, D. S., and T. G. Parker. 1981. Factors affecting the ethanol stability of bovine milk. II. The origin of the pH transition. J. Dairy Res. 48:285–291. 12 Jang, H. D., and H. E. Swaisgood. 1990. Characteristics of the interaction of calcium with casein submicelles as determined by analytical affinity chromatography. Arch. Biochem. Biophys. 283:318–325. 13 Janolino, V. G., and H. E. Swaisgood. 1982. Analysis and optimization of methods using water-soluble carbodiimide for immobilization of biomolecules to porous glass. Biotechnol. Bioeng. 24:1069–1080. 14 Juillerat, M. A., R. Baechler, R. Berrocal, S. Chanton, J.-C. Scherz, and R. Jost. 1989. Tryptic phosphopeptides from whole casein. 1. Preparation and analysis by fast protein liquid chromatography. J. Dairy Res. 56:603–611. 15 Kitts, D. D., and Y. V. Yuan. 1992. Caseinophosphopeptides and calcium bioavailability. Trends Food Sci. Technol. 3:31–35. 16 Kitts, D. D., Y. V. Yuan, T. Nagasawa, and Y. Moriyama. 1992. Effect of casein phosphopeptides and calcium intake on ileal 45Ca disappearance and temporal systolic blood pressure in spontaneously hypertensive rats. Br. J. Nutr. 68:765–781. 17 Kopra, N., K.-E. Scholz-Ahrens, and C. A. Barth. 1992. Effect of casein phosphopeptides on utilization of calcium in vitamin Dreplete and vitamin D-deficient rats. Milchwissenschaft 47: 488–492. 18 Manson, W., and W. D. Annan. 1971. The structure of a phosphopeptide derived from b-casein. Arch. Biochem. Biophys. 145: 16–26. 19 Meisel, H., and H. Frister. 1989. Chemical characterization of bioactive peptides from in vivo digests of casein. J. Dairy Res. 56:343–349. 20 Osterberg, R. 1960. Isolation of a phosphopeptide as magnesium complex from a trypsin hydrolysate of a-casein by anion exchange chromatography. Biochem. Biophys. Acta 42:312–315. 21 Park, O., H. E. Swaisgood, and J. C. Allen. 1993. Comparison of ion exchange methods for fractionation of casein from milk. J. Dairy Sci. 76(Suppl. 1):109.(Abstr.) 22 Park, O., H. E. Swaisgood, and J. C. Allen. 1998. Calciumbinding of phosphopeptides derived from hydrolysis of ascasein or b-casein using immobilized trypsin. J. Dairy Sci. 81: 81:2850–2857. 23 Reeves, R. E., and N. G. Latour. 1958. Calcium phosphate sequestering phosphopeptide from casein. Science (Washington, DC) 128:472. 24 Reynolds, E. C. 1987. The prevention of sub-surface demineralization of bovine enamel and change in plague composition by casein in an intra-oral model. J. Dental Res. 66:1120–1127.

CASEIN PHOSPHOPEPTIDES 25 Sato, R., M. Shindo, H. Gunshin, T. Noguchi, and H. Naito. 1991. Characterization of phosphopeptide derived from bovine b-casein: an inhibitor to intra-intestinal precipitation of calcium phosphate. Biochem. Biophys. Acta 1077:413–415. 26 Schagger, H., and G. Von Jagow. 1987. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166: 368–379. 27 Svendsen, I., and K. Breddam. 1992. Isolation and amino acid sequence of a glutamic acid specific endopeptidase from Bacillus licheniformis. Eur. J. Biochem. 204:165–171. 28 Swaisgood, H. E. 1992. Chemistry of the caseins. Pages 63–118 in Advanced Dairy Chemistry. Vol. 1. Proteins. P. F. Fox, ed. Elsevier Appl. Sci., New York, NY. 29 Swaisgood, H. E. 1993. Comparative studies and nomenclature. Review and update of casein chemistry. J. Dairy Sci. 76: 3054–3061. 30 Tsuchita, H., T. Goto, T. Shimizu, Y. Yonehara, and T. Kuwata. 1996. Dietary casein phosphopeptides prevent bone loss in aged ovariectomized rats. J. Nutr. 126:86–93.

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31 Van Dijk, H.J.M. 1990. The properties of casein micelles. 2. Formation and degradation of the micellar calcium phosphate. Neth. Milk Dairy J. 44:111–124. 32 Voet, D., and J. G. Voet. 1990. Enzymatic catalysis. Ch. 14. Pages 373–382 in Biochemistry. John Wiley & Sons, Inc., New York, NY. 33 Walstra, P., and R. Jenness. 1984. Dairy Chemistry and Physics. John Wiley & Sons, New York, NY. 34 West, D. W. 1977. A simple method for the isolation of a phosphopeptide from bovine as1-casein. J. Dairy Res. 44: 373–376. 35 West, D. W. 1986. Structure and function of the phosphorylated residues of casein. J. Dairy Res. 53:333–352. 36 Yuan, Y. V., and D. D. Kitts. 1991. Confirmation of calcium absorption and femoral utilization in spontaneously hypertensive rats fed casein phosphopeptide supplemented diets. Nutr. Res. 11:1257–1272. 37 Yun, S.-E., K. Ohmiya, and S. Shimizu. 1982. Role of the phosphoryl group of b-casein in milk curdling. Agric. Biol. Chem. 46:1505–1511.

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