Calcium-binding Peptides Derived from Tryptic Hydrolysates of Cheese Whey Protein

The purpose of this research was to investigate the potential use of cheese whey protein (CWP), a cheese by-product. The physiological activity of calcium-binding peptides in CWP may be used as a food additive that prevents bone disorders. This research also examined the characteristics of calcium-binding peptides. After the CWP was heat treated, it was hydrolyzed by trypsin. Then calcium-binding peptides were separated and purified by ion-exchange chromatography and reverse phase HPLC, respectively. To examine the characteristics of the purified calcium-binding peptides, amino acid composition and amino acid sequence were analyzed. Calcium-binding peptides with a small molecular weight of about 1.4 to 3.4 kDa were identified in the fraction that was flowed out from 0.25 M NaCl step gradient by ion-exchange chromatography of tryptic hydrolysates. The results of the amino acid analysis revealed that glutamic acid in a calcium-binding site took up most part of the amino acids including a quantity of proline, leucine and lysine. The amino acid sequence of calcium-binding peptides showed Phe-Leu-Asp-Asp-Asp-Leu-Thr-Asp and Ile-Leu-Asp-Lys from α-LA and IlePro-Ala-Val-Phe-Lys and Val-Tyr-Val-Glu-Glu-Leu-Lys from β-LG. (Asian-Aust. J. Anim. Sci. 2004. Vol 17, No. 10 : 1459-1464)


INTRODUCTION
Milk proteins and milk protein hydrolysis products are an excellent source of calcium because they are capable of the absorption and bioavailability of calcium (Wong et al., 1978;Harwalkar and McMahon, 1993).Recently, with the increasing number of the aged, there is a growing interest in calcium supplementary medicine to treat and prevent bone diseases.Intake of calcium increases the bone density of small children, and calcium also is essential among middleaged people to prevent osteoporosis (Renner, 1994).Calcium deficiency often causes osteoporosis, hypertension, and large intestine cancer (Avioli, 1984;Lipkin and Newmark, 1985;Spencer and Kramer, 1986;Osborne et al., 1996).
Studies showed that calmodulin and vitamin Ddependent calcium-binding protein are the main source of calcium supply (Wasserman et al., 1978;Wang and Waisman, 1979).However, CN and whey protein (WP) also seem to have calcium-binding sites since they are the main components of milk protein (Stuart et al., 1986;Jeyarajah and Allen, 1994;Bennett et al., 2000).
There has been much research about purifying and separating CPP (Reynolds, 1997;Vegarud et al., 2000).However, there are many phases to pass through to purify and separate them and it is very expensive.
The objectives of this research were to separate and purify calcium-binding peptides from the peptide derived from tryptic hydrolysates of CWP and investigate their characteristics.methods described by Adamson and Reynolds (1996).Trypsin was added to the reaction mixture of a enzyme to the substrate (E:S) ratio of 1:100 (wt/wt, protein basis).Hydrolysis was performed at 50°C for 4 h with the pH maintained at 8.0±0.1 by the addition of 0.5 N NaOH using a pH-stat (Metrohm Ltd., Herisan, Switzerland).The enzyme was inactivated by heating for 10 min at 90°C.The calcium-binding peptide was precipitated from the hydrolysate by the addition of 10% (wt/vol) calcium chloride to a 100 mM final concentration and then ethanol to a 50% (vol/vol) final concentration.The suspension was centrifuged (12,000×g) and the supernatant discarded.The precipitate was freeze-dried and stored at -20°C.

Separation of calcium-binding peptides
Separation of calcium-binding peptides in tryptic hydrolysates adopted the method of Rose et al. (1969).DEAE-cellulose (Sigma Co., USA) was equilibrated at 500 mL of 20 mM Tris-HCl buffer (pH 7.8), and the slurry of equilibrated DEAE-cellulose was packed in a column (20×2.5 cm).The tryptic hydrolysates were dissolved in the same buffer (pH 7.8) and loaded on a column and then eluted by a step gradient with the same buffer containing 0.25 M, 0.5 M, 0.75 M and 1 M NaCl.The flow rate was 3 mL/min, fraction volume 15 mL/tube, and elution was monitored at 280 nm.The injection volume was 40 mL, containing about 200 mg of protein.The samples were filtered through 0.5 µm syringe filters prior to application to the column.

Reverse phase HPLC
Peptides in heated-CWP hydrolysates were analyzed by RP-HPLC on a Nucleosil (Nucleosil C-18 5 Micron, Alltech Associates, Inc., USA) C-18 column (4.6×250 mm), equilibrated with solvent A [0.1% trifluoroacetic acid (TFA) in H 2 O] and eluted with a linear gradient to solvent B (0.1% TFA in acetonitrile) for 40 min.Runs were conducted at room temperature using a Dionex HPLC system (ASI 100, Dionex Co., USA) at a flow rate of 1.0 mL/min, and the absorbance of the column elute was monitored at 214 nm.The injection volume was generally 10 µL, and the concentration of peptide material applied was approximately equivalent to 0.5 mg/mL protein.The samples were filtered through a 0.2 µm syringe filters prior to application to the C-18 column.

Preparative HPLC
A large-scale separation of the fractions was performed by preparative HPLC (Delta Prep 4000, Waters Co., USA) using a C-18 column (Cosmosil 15C18-AR II, 28×250 mm) at a flow rate of 150 mL/min.The column was equilibrated with solvent A (0.1% TFA in H 2 O) and eluted with a linear gradient to solvent B (0.1% TFA in acetonitrile) for 40 min.The injection volume was 15 mL, and the absorbance was measured at 214 nm.

Calcium-binding ability
The calcium-binding abilities of tryptic hydrolysate fractions were determined by Ion chromatography on an IonPac CS12A column.Runs were conducted at room temperature using a Dionex IC system (DX120, Dionex Co., USA) at a flow rate of 1.0 mL/min and eluted with 18 mM of methane-based sulfuric acid.The injection volume was 25 µL, and six cations (DIONEX P/N 43162) were used as a standard.All samples were filtered through a 0.2 µm syringe filter prior to application to the column.The calcium-binding abilities of tryptic hydrolysate fractions were expressed as mM of calcium bound in mg of protein.

Amino acid analysis
The amino acid analysis was performed by the method of Moore et al. (1958).The purified calcium-binding peptides (exactly 1 mg of protein) were dialyzed exhaustively against distilled water and lyophilized, and then hydrolyzed in 1 mL of 6 N HCl in evacuated tubes at 110°C for 24 h.After being concentrated by speed vacuum concentration (MAXI-DRY PLUS, Heto-Holten A/S., Denmark), the sample was dissolved in 0.2 M sodium citrate loading buffer (pH 2.2), and then filted through a 0.2 µm syringe filters.The analysis of amino acid was carried out on an amino acid analyzer (Biochem 20, Pharmacia, Sweden).

Amino acid sequence
The N-terminal sequence of the purified calciumbinding peptides was identified by sequence analysis with a protein sequencer (962592A, Perkin-Elmer, USA).If the Nterminus of a protein was blocked and could not be sequenced, the protein was subjected to trypsin digestion followed by peptide sequencing (Shaw et al., 1993).

Protein determination
Protein concentration in the tryptic hydrolysates and fractions was determined by the dye-binding method of Bradford (1976).BSA was used as the standard.

Separation, identification, and purification of calciumbinding peptides
Figure 1 shows the ion-exchange chromatogram of tryptic hydrolysates.The eluted F-1, F-2 and F-3 from 0 M, 0.25 M and 0.5 M NaCl step gradient all had respective peaks.
The diatomic calcium electrolyte of F-1, F-2 and F-3 was measured by ion chromatography tagged with calcium affinity column to confirm calcium-binding abilities as shown in Figure 2. The calcium-binding ability of F-2 increased rapidly beginning at CaCl 2 2 mM.F-3 appeared to increase steadily, and F-1 showed no changes.In this experiment, calcium-binding ability was highest in F-2 from 0.25 M NaCl step gradient.These results are similar to those of Friedlander and Norman (1980) who reported on calcium-binding abilities in fraction from 0.22 M NaCl gradient by ion-exchange chromatography.F-2, with calcium-binding ability purified by RP-HPLC, was fractionated into 6 fractions (Figure 3).After carrying out a large-scale separation, the calcium-binding ability was identified by ion chromatography.As a result, fractions 3, 4, and 5 seemed to have superb calcium-binding ability (Figure 4).
The fractions that have superb calcium-binding abilities were cut off to the size under 5 kDa [using by concentrator (Sartorius, Membrane 5 kDa MWCO PES., USA)]-the size in which physiological activity appears.Figure 5 shows that the molecules have a small molecular weight of about 1.4 to 3.4 kDa.

Amino acid composition of calcium-binding peptides
Table 1 shows the amino acid compositions of calciumbinding peptides.This experimental material CWP had the highest amount of Glu (17.80%) and the lowest amount of Cys (0.68%).Other amino acids were distributed evenly.These results are similar to the reports of Turgeon and Gauthier (1990) and Pintado et al. (1999) on amino acid compositions of CWP.In addition, the amino acid composition of calcium-binding peptides had the most Glu (30.08%) but did not any Thr, Ser and Met.These results are similar to the N-terminal amino acid composition of calcium-binding proteins in bovine intestines, which was researched by Fullmer and Wasserman (1981).Their studies show that there are no Lys, Met, Trp, His and Arg.
The high percentage of Glu in calcium-binding peptides, in comparison with CWP, would be predicted that because trypsin cannot isolate amino acids with many branches.Glu, Pro and Leu were abundant, and Lys (7.12%) also taked up a high percentage.
Bovine α-LA is known to have strong calcium-binding sites with Asp and Lys as binding residues (Anderson et al., 1997;Veprintsev et al., 1999;Noyelle and van Dael, 2002).The results of this experiment are similar to those in the report of Tamura et al. (1982) who stated that Asp, Glu, Leu and Lys were abundant in the two materials, which showed calcium-binding ability in milk protein.Similarly, Tuan et al. (1978) has also reported on the acidic amino acids (Asp and Glu), basic amino acids (Lys and Arg) being the most abundant in calcium-binding proteins of chick chorioallantoic membrane.In general, proteins having the ability to interact with calcium ion have side chains composed of carbonyl, carboxyl, and alcohol functional groups (e.g., Asp, Asn, Glu, Ser and Thr) (Nemirovskiy and Gross, 2000).

Amino acid sequence of calcium-binding peptides
Calcium-binding peptides appeared to have 6 peaks by RP-HPLC (Figure 6).Each was separately fractionated, and the amino acid sequence was examined starting from the Nterminus (Table 2).
In peak 3, the strong calcium-binding sites with Asp (82, 84, 87), which are originated from α-LA, were detected.Peaks 1, 4, and 6 also appeared to have calcium-binding sites with Lys, Leu and Glu.New sites were found in peaks  1 and 6 that have calcium-binding abilities, which were originated from β-LG.This shows the possibilities that β- LG may also have calcium-binding sites.

CONCLUSIONS
This research has revealed the characteristics of calcium-binding peptides originated from tryptic hydrolysates in cheese whey protein after separating and purifying them.Our results show that calcium-binding peptides are applicable in therapeutic formula concerning osteoporosis.They are also good for food additives that utilize cheese by-product to enhance calcium supply within the human body.

Table 1 .
Amino acid composition of CWP and calcium-binding peptides 1 1 Results from acid hydrolysis for 24 h are given in molar ratios (%). 2 Below the 5 kDa. 3 Not determined.

Table 2 .
Amino acid sequence of calcium-binding peptides Peak no.