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doi:10.1016/j.id

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ment, Zagazig U

International Dairy Journal 15 (2005) 17–27

www.elsevier.com/locate/idairyj

Peptic hydrolysis of ovine b-lactoglobulin and a-lactalbuminExceptional susceptibility of native ovine

b-lactoglobulin to pepsinolysis

Khaled El-Zahar, Mahmoud Sitohy1, Yvan Choiset, Franc-ois Metro,Thomas Haertle, Jean-Marc Chobert�

Institut National de la Recherche Agronomique, Laboratoire d’Etude des Interactions des Molecules Alimentaires, rue de la Geraudiere,

BP 71627, 44316 Nantes Cedex 3, France

Received 22 January 2004; accepted 3 June 2004

Abstract

Ovine b-lactoglobulin (BLG, a mixture of variants A and B at a ratio of 46/54) and a-lactalbumin (ALA) were subjected to pepsinactivity. The degree of peptic hydrolysis of native whole BLG reached 63%, 74%, 82% and 87% after 2, 4, 8 and 20 h hydrolysis,

respectively. BLG variant B was degraded completely after 2 h of pepsin digestion while variant A was degraded gradually showing

19%, 44%, 61% and 73% hydrolysis after 2, 4, 8 and 20 h, respectively. The main factors responsible for the exceptional pepsin

susceptibility of ovine BLG are the slightly different tertiary structure of ovine BLG (compared with bovine BLG) as perceived from

near circular dichroism spectra at pH 2, and its higher surface hydrophobicity, as demonstrated by a higher binding activity to 1-

anilinonaphthalene-8-sulphonate. Reversed phase-high performance liquid chromatograms (RP-HPLC) profiles of the peptic

hydrolysates of BLG showed the production of hydrophobic peptides at the early stages of hydrolysis, while more hydrophilic

peptides appeared only at a later stage of hydrolysis. Mass spectroscopy analysis allowed the characterisation of 17 and 13 peptides

after 2 and 20 h hydrolysis, respectively. Most of the enzyme activity was oriented first towards the N-terminal part of the molecule

and later towards the C-terminal part of the protein; little or no activity was observed in the central region of the molecule even after

20 h hydrolysis. Native ovine ALA was almost completely degraded by pepsin, yielding 93%, 94%, 95% and 98% hydrolysis after 2,

4, 8 and 24 h, respectively. The RP-HPLC profile of the ALA hydrolysate showed 5 major hydrophobic peptides and 7 minor more

hydrophilic peptides, which did not change with the time of hydrolysis.

r 2004 Elsevier Ltd. All rights reserved.

Keywords: Ovine milk proteins; a-Lactalbumin; b-Lactoglobulin; Pepsin; Hydrolysis

1. Introduction

b-Lactoglobulin (BLG) is a major whey proteinsecreted in the milk of ruminants. Because of itsabundance and ease of purification, BLG was one of

e front matter r 2004 Elsevier Ltd. All rights reserved.

airyj.2004.06.002

ing author. Tel.: +33-2-4067-5085; fax: +33-2-4067-

ess: chobert@nantes.inra.fr (J.-M. Chobert).

ddress: Faculty of Agriculture, Biochemistry Depart-

niversity, Zagazig, Egypt.

the first proteins for which both amino acid compositionand sequence were established. Binding studies carriedout on BLG indicated its involvement in stronginteractions with small hydrophobic ligands, such asfatty acids or retinoids (Brown, 1984). Various protei-nases have been used as tools for elucidating conforma-tion changes of this protein. Ample structural andbiophysical data available for the bovine BLG moleculeprovide the basis for a study of the correlation of itsstructural transformations with its susceptibility to thehydrolysis by proteinases. Native bovine BLG is highly

ARTICLE IN PRESSK. El-Zahar et al. / International Dairy Journal 15 (2005) 17–2718

resistant to peptic digestion (Reddy, Kella, & Kinsella,1988), less than 1% of it is hydrolysed over extendedhydrolysis times (Asselin, Herbert, & Amiot, 1989). Itwas demonstrated that bovine BLG could not behydrolysed by pepsin from different sources (Kinekawa& Kitabatake, 1996). The structural basis of theresistance of BLG to peptic digestion have been studiedpreviously (Reddy et al., 1988). The particular folding ofBLG makes it resistant to peptic hydrolysis (Dalgalar-rondo, Dufour, Chobert, Bertrand-Harb, & Haertle,1995), as its peptic cleavage sites (hydrophobic oraromatic amino acid side chains) are buried well insidethe b-barrel, forming a strong hydrophobic core. Thecore of the molecule is made up of a very short a-helixsegment and eight strands of anti-parallel b-sheets,which wrap around to form an anti-parallel b-barrel.Bovine BLG shows a remarkable stability at low pH,resisting denaturation at pH 2.0 and hydrolysis bypepsin at the optimum pH (2.5) of the enzyme(Stapelfeldt, Petersen, Kristiansen, Qvist, & Skibstes,1996).Native a-lactalbumin (ALA) is sensitive to pepsin

hydrolysis and, its digestion yields several polypeptides(Pellegrini, Thomas, Bramaz, Hunziker, & von Fell-enberg, 1999). It was shown that while native BLG wasalmost insensitive to the action of pepsin, ALA wasalmost completely hydrolysed into small peptides in30min (Schmidt & Poll, 1991; Miranda & Pelissier,1983; Reddy et al., 1988).Physical or chemical modifications of BLG denature

it and consequently, enhance its susceptibility to pepsinhydrolysis. Solvent-induced structural transformationmay trigger BLG susceptibility to peptic action (Dalga-larrondo et al., 1995). Heat-induced structural changeswere also found to increase BLG aggregation, thusincreasing the exposure of the peptic cleavage sites andhence the susceptibility of the protein to pepsin action(Bertrand-Harb, Baday, Dalgalarrondo, Chobert, &Haertle, 2002; Maeda, Abe, Watanabe, & Arai, 1987;Guo, Fox, & Flynn, 1995; Schmidt & van Markwijk,1993). Cleavage of one or more disulphide bondsresponsible for the stabilisation of the tertiary structureof BLG may also increase its accessibility to proteolyticenzymes. Sulphitolysis of BLG was useful chemicalmodification enhancing its susceptibility to pepsin(Reddy et al., 1988; Klemaszewski & Kinsella, 1991;Kananen et al., 2000). Esterification of bovine milkproteins also increases their susceptibility to pepsinaction as a result of introducing new hydrophobic pepticcleavage sites (Briand, Chobert, & Haertle, 1995;Chobert, Briand, Grinberg, & Haertle, 1995; Sitohy,Chobert, & Haertle, 2001).In spite of the similarity between ovine and bovine

whey proteins, fine structural differences may changetheir susceptibility to pepsinolysis. Tryptic and peptichydrolysis of ovine and caprine whole wheys have been

recently studied (Pintado & Malcata, 2000) withoutcharacterisation of the resulting peptides. The aims ofthis study were to investigate the initial hydrolysis ofnative ovine BLG, containing a mixture of variants Aand B, and ALA by pepsin, and to characterise theprincipal liberated peptides.

2. Materials and methods

2.1. Preparation of ALA and BLG

Fresh ovine milk whey was prepared from skimmedmilk (Lacaune breed, dairy farm of INRA, Rennes,France) by precipitation of caseins at pH 4.6 with 1 N

HCl. Caseins were removed by centrifugation at 10,000g

for 30min at 4 1C. The supernatant containing wheyproteins was dialysed against distilled water for 48 h,frozen and lyophilised. Bovine BLG and ALA wereprepared according to Mailliart and Ribadeau Dumas(1988). As revealed from reversed phase-high perfor-mance liquid chromatograms (RP-HPLC) and SDS-polyacrylamide gel electrophoresis (PAGE), bovineBLG is a mixture of genetic variants A and B (ratio1:1), more than 95% pure.

2.2. Anion-exchange chromatography

Ovine ALA and BLG were isolated from fresh wheyusing the anion-exchange chromatography on DEAE-Sepharose Fast Flow column (300� 50mm; Amersham-Pharmacia, Orsay, France). Elution was made in 25mM

Tris, pH 8.0, containing 5mM CaCl2, using a linearNaCl (0–1M) gradient. Homogeneity of the proteinpreparation was checked by high performance gelpermeation (GP) chromatography and SDS-tricinePAGE according to the method of Schagger and vonJagow (1987).

2.3. Gel-permeation chromatography

Ovine BLG and ALA obtained by anion-exchangechromatography were further purified by GP chroma-tography on a TSK-G3000 SWXL column(300� 7.8mm; Tosohaas, Montgomeryville, PA). Theelution was carried out at a flow rate of 0.8mLmin�1

with 30mM sodium phosphate, pH 6.7 or with 30mM

glycine, pH 2.5. ALA and BLG were over 95% pure aspreviously described (El-Zahar et al., 2004).

2.4. Pepsin hydrolysis

Samples of ALA and BLG of ovine milk were dilutedin 20mM sodium citrate buffer, pH 2.6, to obtain a finalconcentration of 2mgmL�1. Appropriate aliquots ofporcine pepsin (EC 3.4.23.1; activity: 3200–4500 units

ARTICLE IN PRESSK. El-Zahar et al. / International Dairy Journal 15 (2005) 17–27 19

per mg of protein) solution (2mgmL�1 in distilledwater, Sigma, St Louis, MO, USA) were added to givean enzyme/substrate (E=S) ratio of 2.5% (w/w). Thereaction mixture was incubated at 37 1C for 24 h. At theend of reaction, the pH was adjusted to 7.0 by adding anappropriate amount of 1M Tris–HCl buffer, pH 8.9.Sampling was made at appropriate time intervalsduring the peptic hydrolysis of ALA and BLG. Sampleswere then kept frozen at �20 1C until analysis byRP-HPLC.

2.5. Separation of peptic peptides by RP-HPLC

A modification of the method of Visser, Slangen,Lagerwerf, van Dongen, and Haverkamp (1995) wasused to separate and identify peptides in the pepsinhydrolysates. Chromatographies were carried out on aWaters 2695 separation module equipped with a Waters996 photodiode array detector using the Milleniumsoftware (Waters, Millford, MA, USA). RP-HPLC wasrun on a Nucleosil C18 column (250� 4mm; MachereyNagel, France), equilibrated with solvent A (0.11%, v/v,trifluoroacetic acid (TFA) in H2O). Elution wasperformed by using a gradient from 10% solvent B(80% acetonitrile, 19.91% H2O, 0.09% TFA, v/v/v) to100% solvent B in 23min. The temperature of thecolumn was maintained at 301C and the flow rate was0.6mLmin�1. The absorbance of the eluted fractionswas recorded at 220 nm.

2.6. Circular dichroism (CD)

CD spectra were measured using a CD6 dichrographand recorded with Cdmax software (Jobin Yvon,Longjumeau, France). All spectra were obtained at25 1C using a protein concentration of 2mgmL�1 in a10mM glycine-HCl buffer at pH 2.5 or in a 30mM

sodium phosphate-NaOH buffer at pH 7.0. Near-UV(250–320 nm) spectra were measured in a 5mmpath length cylindrical cell. Five spectra were accumu-lated with 1 nm steps and 1 s integration time. Thespectra were corrected by subtracting the bufferspectrum before calculating the molar ellipticity.For all experiments, the BLG concentration wasdetermined spectrophotometrically assuming �278 ¼17; 600M

�1 cm�1.

2.7. Surface hydrophobicity

Protein surface hydrophobicity was determined ac-cording to the method of Moro, Gatti, and Delorenzi(2001), with slight modifications. Fluorescence experi-ments were carried out in triplicate at 20 1C using anAminco SLM 4800C spectrofluorimeter (Thermo Spec-tronic, Rochester, NY, USA) in the ratio mode. Analiquot (1.3mL) of whey protein dispersion (20 mM;

0.31mgmL�1, adjusted to pH 2 with HCl) was placed inthe cell of the spectrophotometer and titrated with10mM ANS (1-anilinonaphthalene-8-sulphonate) dis-solved in DMF (N,N,dimethylformamide) added suc-cessively in small aliquots. The emitted fluorescence(lem ¼ 480 nm) was monitored using excitation wave-length, lex ¼ 390 nm. The relative fluorescence intensitywas adjusted to 1 when 20 mL of ANS was added to1.3mL of deionised water at pH 2.0 in the absence ofprotein. Analysis of binding data was performed toobtain the maximum fluorescence at saturated ANSconcentration (Fmax), which is a function of the numberof hydrophobic sites per mg of protein sample accessibleto the marker.

2.8. Nanoscale capillary liquid chromatography-tandem

mass spectrometric (LC-MS/MS) analysis

LC-MS/MS analysis of the digested ovine BLG wereperformed using an UltiMate capillary LC system (LCPackings, Dionex, Amsterdam, The Netherlands)coupled to a hybrid quadrupole orthogonal accelerationtime-of-flight tandem mass spectrometer (Q-TOF Glo-bal, Micromass, Manchester, UK). The LC-MS unionwas made with a PicoTip (New Objective, Woburn,MA, USA) fitted on a Z spray (Micromass) interface.Chromatographic separations were performed on areversed phase (RP) capillary column (C18, 3 mm,100 A, 15 cm length, 75 mm i.d., LC Packings) with aflow of 170 nLmin�1. The gradient profile used con-sisted of a linear gradient from 98% solvent A (H2O/acetonitrile/formic acid, 80/20/0.1, v/v/v) to 60%solvent B (H2O/acetonitrile/formic acid, 5/95/0.1, v/v/v) in 70min.Mass data acquisitions were obtained by Mass Lynx

Software (Micromass) using automatic switching be-tween MS and MS/MS modes. Peptides eluted from thechromatographic column were detected for 1 s; whentheir signal reached a defined threshold (4 counts s�1)they could be selected for fragmentation. An MS/MSscan (1 s) was then performed on the three most intensepeptide ions detected. MS/MS scans of each selected ionwere summed until the total fragmentation timeattributed to one selected precursor had been reachedor the signal in an MS/MS scan had fallen to 0.Acquisitions were performed with the dynamic exclu-sions of m/z ratios of already fragmented ions (exclusionof a 70.3Da mass window around the m/z ratio ofpreviously selected precursors). Fragmentation wasperformed using argon as the collision gas and thecollision energy profile was optimised.The software used for data acquisition was Mass

Lynx 4.0 (Micromass) and the treatment of data wasperformed by using the Protein Lynx global serverVersion 2 (Micromass).

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Retention time (min)

Abs

orba

nce

at 2

14 n

m 0.0

0.5

1.0

1.5

2.0

3 6 9 12 15 18

B A

0.0

0.5

1.0

1.5

2.0

A

0.0

0.4

0.8

1.2

3 6 9 12 15 18

0.0

0.4

0.8

1.2

3 6 9 12 15 18

3 6 9 12 15 18

AA

(a) (b)

(c) (d)

Fig. 1. RP-HPLC of ewes’ milk b-lactoglobulin following hydrolysis with pepsin for 0 (a, control), 2 (b), 4 (c) or 20 (d) h at pH 2.6, using an

enzyme–substrate ratio of 2.5% (w/w). Details of the chromatographic conditions are given in Section 2.5.

K. El-Zahar et al. / International Dairy Journal 15 (2005) 17–2720

3. Results and discussion

3.1. Pepsin hydrolysis of BLG

RP-HPLC profiles of BLG peptic hydrolysates(Fig. 1) show that BLG was fragmented into differentpeptides during the course of hydrolysis. The degree ofhydrolysis was 63%, 74%, 82% and 88% after 2, 4, 8and 20 h, respectively. The peak corresponding to BLGvariant B fraction completely disappeared after 2 hhydrolysis while that of variant A gradually decreased.The non-hydrolysed protein, which was comprised ofvariant A only, accounted for 37%, 26%, 18% and 13%of the whole BLG, (equivalent to 81%, 56%, 39% and27% of the starting amount of variant A) after 2, 4, 8and 20 h peptic hydrolysis, respectively.The RP-HPLC chromatograms of the hydrolysates of

BLG variants A and B show very similar peptide profilesafter 2 and 4 h during which time most of the liberatedpeptides were eluted within the range 9–13min, demon-strating their relatively high hydrophobicity. On theother hand, RP-HPLC chromatograms of the 20 hpeptic hydrolysate of BLG showed peptide profileswhere the majority of the released peptides were elutedwithin 7–9min, indicating their hydrophilicity. Hydro-phobic peptides initially released at the start of thehydrolysis were further cleaved at the later stages ofproteolysis, yielding smaller and more hydrophilicpeptides. This phenomenon was previously observed inthe case of bovine BLG hydrolysed with pepsin in 40%ethanol (Dalgalarrondo et al., 1995).The changes in the levels of peptides with different

retention times as calculated from peak areas during

hydrolysis are shown in Fig. 2. The level of peptides withlow elution times (4.9–7.25min), which are morehydrophilic, continued to increase with the progress ofhydrolysis. In contrast, the peptides with relativelyhigher retention times (8.4–11.15min) and which aremore hydrophobic in nature, reached a maximum after2–8 h hydrolysis and then began to decrease. Thispattern shows the degradation of the latter hydrophobicpeptides into smaller more hydrophilic peptides. Gen-erally, pepsin hydrolysis of BLG for a short time (2–4 h)produces hydrolysates with a more hydrophobic naturewhile extensive pepsin hydrolysis (20 h) produces hydro-lysates with a more hydrophilic nature.The measure of the changes in surface hydrophobicity

of BLG during its pepsin hydrolysis for 2 h (data notshown) indicated a decrease in hydrophobicity after 30,45 and 60min hydrolysis, which might have originatedfrom the exposed cleaved sites. However, after 2 hhydrolysis, the surface hydrophobicity had increasedmarkedly. This increase might be due to the exposure ofa previously hidden hydrophobic region close to the coreof globular protein. This would give rise to furtherpepsin digestion as pepsin preferentially hydrolysespeptide bonds between hydrophobic and aromaticamino acid residues (Antonov, 1977).

3.2. Factors causing pepsin susceptibility of ovine BLG

Comparison of the amino acid sequences of bovineand ovine BLG variant B shows that Tyr 20, Asp 53,Ser 150, Glu 158 and Ile 162 in bovine BLG arereplaced by His, Asn, Ala, Gly and Val in ovine BLG,respectively (Swiss-Prot P02757; www.expasy.org/

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Pea

k ar

ea (

arbi

trar

y un

its)

Hydrolysis time (h)

02468

10

0 5 10 15 20

02468

10

0 5 10 15 20

0

10

20

30

0 5 10 15 20

0

10

20

30

0 5 10 15 20

0

10

20

30

0 5 10 15 20

0

10

20

30

0 5 10 15 20(a) (d)

(b) (e)

(c) (f)

Fig. 2. Changes in the concentrations of different peptide categories, based on retention times during RP-HPLC, during the hydrolysis of ewes’ milk

b-lactoglobulin with pepsin. The retention times, in min, for the different peptide categories were. 4.9 (a), 6.5 (b), 7.25 (c), 8.4 (d), 9.8 (e) and 11.15 (f).Details of the chromatographic conditions are given in Section 2.5.

K. El-Zahar et al. / International Dairy Journal 15 (2005) 17–27 21

cgi-bin/niceprot.pI LACB-SHEEP, 1988). This changegenerally favours an increased protein hydrophobicity.The change occurring in three amino acids located in thelast (C-terminal) 20 amino acids of the molecule maymake this portion of the ovine BLG variant Bmolecule more hydrophobic than the correspondingregion in bovine BLG variant B. This phenomenon wasfurther evidenced by calculating the hydrophobicity ofthe amino acid residues of the BLG variant Bpolypeptide chain according to the method of Kyteand Doolittle (1982) and on the base of the hydro-phobicity data recorded in Swiss-Prot P02757 (LACB-SHEEP, 1988). The resulting profiles showed that the 25C-terminal amino acids in ovine BLG variant B weremore hydrophobic than in bovine BLGvariant B. BLG variant A showed a comparablebehaviour (Fig. 3) due to the similar but smallerdifferences in the primary structures between the ovineand bovine BLGs. Consequently, the increased hydro-phobicity in ovine BLG variant B may be responsiblefor its susceptibility to hydrolysis by pepsin. Thesurface hydrophobicity of both ovine and bovineBLGs was compared by measuring their interactionwith ANS. Data in Fig. 4(a) show that thesurface hydrophobicity of ovine BLG at pH 2 wasmuch higher (184%) than that of bovine BLG. It may beconcluded that the increased surface hydrophobicity ofovine BLG may play a crucial role in enhancing itssusceptibility to pepsin hydrolysis since pepsin prefer-

entially cleaves peptide bonds, which are hydrophobic innature.The difference in surface hydrophobicity between

native ovine and bovine BLGs, based on their binding toANS, exists also at pH 7 (data not shown) buthydrophobicity at acid pH is higher than at neutralpH. The relatively higher binding of ANS to BLG atacid pH may reflect a partially folded BLG structurewith a highly structured b-sheet core and smalleramount of unstructured regions (Molinari et al., 1996).The less ordered, unstructured regions of BLG at pH 2may expose hydrophobic amino acid side chains(Hamdan, Curcuruto, Molinari, Zetta, & Ragona,1996) making the protein molecule more prone to pepticaction. The increase in ANS fluorescence intensity atacid pH in the presence of BLG might be due toenhanced electrostatic interactions between the nega-tively charged ANS and the positively charged BLG(Matulis & Lovrien, 1998; Alizadeh-Pasdar & Li-Chan,2000). However, the difference between the two BLGs(ovine and bovine) is mainly due to adifferent hydrophobic environment, since the electro-static interactions should be the same for both proteinsas their contents of basic amino acids are nearlyidentical.The slight changes in the primary structure of ovine

BLG resulting from the replacement of 5 amino acids,compared with its bovine form, may have an impact onits tertiary structure also, as deduced from their near-

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Fig. 3. Theoretical hydrophobicity of the genetic variants A (a) and B (b) of b-lactoglobulin from ewes’ milk (—) or bovine milk (– – –). Details of the

calculation of the theoretical hydrophobicity are given in Section 3.2.

K. El-Zahar et al. / International Dairy Journal 15 (2005) 17–2722

UV CD profile at pH 2 (Fig. 4(b)). The CD profiles weremore or less similar for both BLGs except for lowerintensity of the signal at 285 nm and the absence of thesignal at 267 nm in the case of ovine BLG. Thisdifference is observed at pH 2 only since the signal at267 nm was present at pH 7 in the two BLGs (data notshown). This slight conformational change, which mayarise from the small changes in the primary structureand hence, the hydrophobic environment, may in turnexplain the difference in peptic susceptibility betweenovine and bovine BLGs at pH 2.0. Some of thehydrophobic regions may be more exposed at thesurface of the molecule. Particularly, the more hydro-phobic C-terminal end of ovine BLG may be moreexposed at the surface of the molecule making itsusceptible to pepsin hydrolysis.

3.3. Characterisation of the BLG released peptides by

mass spectroscopy

Data in Table 1 represent the composition of the 17peptides released from BLG after 2 h hydrolysis withpepsin and their position on the original polypeptidechain. Five peptides with a molecular mass 42 kDa and4 peptides with a molecular mass very close to 2 kDawere released after 2 h hydrolysis. Two peptides with amolecular mass around 1000–1100Da and six peptidesof medium sizes (1200–1600Da) could be detected.After 20 h peptic hydrolysis (Table 2) the total

number of peptides had decreased to 13. The disap-pearance of 4 peptides at this stage of hydrolysis is dueto their complete hydrolysis. Only three peptides had amolecular mass 42 kDa and 2 other peptides showed a

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Fig. 4. Comparison of the fluorescence (a) and near-ultraviolet

circular dichroism (b) spectra of ovine (—) and bovine (- - -) b-lactoglobulin. See Sections 2.6 and 2.7 for experimental details.

Table 1

Primary structure and molecular mass of b-lactoglobulin peptides obtained

Charge (z) Position Molecular ma

2+ 1–11 1217.67

2+ 1–19 2101.16

3+ 1–24 2640.41

2+ 10–19 1129.61

2+ 23–41 1928.00

2+ 25–41 1725.92

2+ 29–41 1381.79

2+ 32–41 1068.59

2+ 42–54 1487.75

2+ 43–54 1324.69

3+ 83–104 2607.45

2+ 95–104 1270.45

4+ 132–149 2093.22

4+ 133–149 2022.18

4+ 134–149 1909.10

4+ 135–149 1780.05

3+ 137–149 1504.89

aSee text Sections 2.4 and 2.8 for details of hydrolysis and sequence analy

K. El-Zahar et al. / International Dairy Journal 15 (2005) 17–27 23

molecular mass close to this value. Four peptides had alow molecular mass of p1 kDa, the remaining 5peptides were of medium sizes (1200–1600Da).The higher concentration of large peptides at the early

stage of hydrolysis (2 h) than after 20 h is supported byRP-HPLC, which showed mainly peptides which werehydrophobic and of large molecular mass after 2 hhydrolysis. Some of the large peptides released after ashort time of hydrolysis were digested further. Forexample, peptides Ile1–Asp11, Ile1–Trp19 and Ile1–-Met24, which appeared after 2 h, disappeared when thehydrolysis was extended to 20 h. At this stage ofhydrolysis, peptide Ile12–Ala23 appeared. Similarly,the peptides Ala23–Val41, Ala25–Val41 and Ile29–-Val41 obtained after 2 h hydrolysis, disappeared after20 h. Likewise, the peptide Lys83–Leu104 appearingafter 2 h hydrolysis may give rise to the peptideAsp96–Leu104 when the hydrolysis was extended to20 h. Some peptides remained intact during the wholeperiod of hydrolysis, for example, peptides Leu32–-Val41, Val43–Leu54 and Ala132–Leu149.Although most of the peptides appearing after 20 h

were derived from larger peptides liberated at the earlystages of hydrolysis, one large sized new peptide(Cys106–Glu131) appeared after 20 h only, without theappearance of a possible precursor. This peptide,located in the middle of the BLG molecule, probablyin the interior of the hydrophobic core, was inaccessibleat the early stages of hydrolysis. It became susceptible topepsin hydrolysis only after removal of the adjacentpeptides from its C- and N-terminal regions.Generally, the pepsin cleavage sites were concentrated

around the N- and C-termini of the BLG, at least at thebeginning of hydrolysis. The N-terminal part was moreaccessible to the enzymatic action than the C-terminal

after two hours of hydrolysis with pepsina

ss (m/z) Sequences

IIVTQTMKGLD

IIVTQTMKGLDIQKVAGTW

IIVTQTMKGLDIQKVAGTWHSLAM

LDIQKVAGTW

AMAASDISLLDAQSAPLRV

AASDISLLDAQSAPLRV

ISLLDAQSAPLRV

LLDAQSAPLRV

YVEELKPTPEGNL

VEELKPTPEGNL

KIDALNENKVLVLDTDYKKYLL

LDTDYKKYLL

ALEKFDKALKALPMHIRL

LEKFDKALKALPMHIRL

EKFDKALKALPMHIRL

KFDKALKALPMHIRL

DKALKALPMHIRL

sis.

ARTICLE IN PRESS

Table 2

Primary structure and molecular mass of b-lactoglobulin peptides obtained after 20 h of hydrolysis with pepsina

Charge (z) Position Molecular mass (m/z) Sequences

2+ 10–19 1129.61 LDIQKVAGTW

2+ 12–23 1309.71 IQKVAGTWHSLA

2+ 13–19 789.27 QKVAGTW

2+ xx–26–32–xx 1928.01b xx–ASDISLL–xx

2+ 32–41 1068.59 LDAQSAPLRV

2+ 42–54 1487.75 YVEELKPTPEGNL

2+ 43–54 1324.69 VEELKPTPEGNL

2+ 43–57 1681.39 VEELKPTPEGNLEIL

3+ 94–114 2508.88 VLDTDYKKYLLFCMENSAEPE

2+ 96–104 1157.60 DTDYKKYLL

3+ 106–131 2896.23 CMENSAEPEQSLACQCLVRTPEVDNE

3+ 132–146 1710.95 ALEKFDKALKALPMH

4+ 132–149 2093.22 ALEKFDKALKALPMHIRL

aSee text Sections 2.4 and 2.8 for details of hydrolysis and sequence analysis.bPeptic peptide with a molecular mass of 1928.01 including the peptide A26–L32 with a molecular mass of 717.82.

Retention time (min)

Abs

orba

nce

at 2

14 n

m

0.0

1.0

2.0

3.0

9 12 15 18 210.0

0.5

1.0

1.5

3 6 9 12

0.0

0.5

1.0

1.5

3 6 9 120.0

0.5

1.0

1.5

3 6 9 1 2

(a) (b)

(c) (d)

Fig. 5. RP-HPLC of ewes’ milk a-lactalbumin following hydrolysis with pepsin for 0 (a, control), 2 (b), 4 (c) or 24 (d) h at pH 2.6, using an enzyme-

substrate ratio of 2.5% (w/w). Details of the chromatographic conditions are given in Section 2.5.

K. El-Zahar et al. / International Dairy Journal 15 (2005) 17–2724

part (Tables 1 and 2). A very limited hydrolysis occurredin the middle of the molecule, especially betweenpositions 56 and 131, as reflected by the release ofonly two peptides from this region. It may be concludedthat cleavage sites in the centre of the BLG molecule arewell buried inside the globule, and are, therefore,inaccessible to pepsin. Some of these sites, especially inthe segment 58–93, remained out of the reach of theenzyme action until the end of the 20 h hydrolysisperiod.Heat denatured bovine BLG showed 31 cleavage sites

after 24 h peptic hydrolysis (Maeda et al., 1987), whileDalgalarrondo et al. (1995) reported 17 cleavage siteswhen pepsinolysis of bovine BLG was performed in40% ethanol. In the present study, native ovine BLGhydrolysed by pepsin showed �30 cleavage sites after 2and 20 h of hydrolysis (Tables 1 and 2). At the later

stage of hydrolysis, new cleavage sites appeared as aresult of unshielding of previously hidden hydrophobicsegments of the BLG molecule. For example, thefollowing cleavage sites were observed only after 20 hof hydrolysis: Ile12–Gln13, Ala23–Met24, Leu57–-Leu58, Leu93–Val94, Leu95–Asp96, Phe105–Cys106,Glu114–Gln115 and His146–Ile147.Although pepsin preferentially cleaves peptide bonds

involving hydrophobic amino acid residues (Leu, Ile,Phe, Val and Trp) it has a rather broad specificity. Itshowed a preferential tendency towards cleavage of Leu-X bonds, which agrees with the results of Dalgalarrondoet al. (1995) in the case of bovine BLG. The intermediateregion Leu58–Phe82 of the BLG molecule remaineduncleaved even after 20 h hydrolysis in spite of theexistence of potential hydrophobic cleavages sites (e.g.,Leu58, Trp61, Ile71, Ile72, Ile78, Val81).

ARTICLE IN PRESS

Hydrolysis time (h)

Pea

k ar

ea (

arbi

trar

y un

its)

0

2

4

6

8

0

2

4

6

8

0

10

20

30

0

10

20

30

40

0

10

20

30

40

0

2

4

6

8

0

2

4

6

8

0

2

4

6

8

0 10 20 0 10 20

0 10 20 0 10 20

0 10 20 0 10 20

0 10 20 0 10 20

(a) (e)

(b) (f)

(c) (g)

(d) (h)

Fig. 6. Changes in the concentrations of different peptide categories, based on retention times during RP-HPLC, during the hydrolysis of ewes’ milk

a-lactalbumin with pepsin. The retention times, in min, for the different peptide categories were: 4.6 (a), 5.3 (b), 6.0 (c), 6.4 (d), 7.9 (e), 8.8 (f), 9.3 (g)and 9.8 (h). Details of the chromatographic conditions are given in Section 2.5.

K. El-Zahar et al. / International Dairy Journal 15 (2005) 17–27 25

3.4. Pepsin hydrolysis of ovine ALA

Native ovine ALA was subjected to pepsin hydrolysis(E=S ¼ 0:5%, W/W, 37 1C and pH 2.6) for differenttime periods. The degree of hydrolysis was estimated tobe 93%, 94%, 95% and 98% after 2, 4, 8 and 24 h,respectively, based on the changes in the relative peakareas corresponding to the intact ALA during hydro-lysis.RP-HPLC analysis of the released peptic peptides of

ovine ALA shows that they reach the final stage ofhydrolysis rapidly (Fig. 5). The majority of the liberatedpeptides (5 main peptide peaks) eluted after relativelylong retention times (8–12min) due to their hydrophobicproperties, while 7 minor more hydrophilic peptideseluted earlier. The stability of the peptide profilesshowing only minor transformations, in contrast towhat was observed in the case of ovine BLG,demonstrates the simultaneous availability of most ofthe cleavage sites on the ALA to pepsin activity once theenzyme is in contact with the protein. The RP-HPLCprofile agrees well with the results of Pellegrini et al.(1999).

The changes in yield of the peptides released duringhydrolysis are presented in Fig. 6. The concentration ofmost of the peptides increased with the advance ofhydrolysis and only very few peptides showed a decreasein concentration. This confirms that the molten globularstate of ALA at pH 2 displays most of the cleavage sitessimultaneously.The susceptibility of ALA to pepsin is widely reported

and is due to the conformation of the protein at theacidic, pH optimum of pepsin. At acid pH (E2) and inthe apo-state obtained at elevated temperature, ALA isin a canonic molten globular state (Dolgikh et al., 1981;Kuwajima, 1996). Binding of calcium to the proteinresults in a pronounced change in its tertiary structure(Permyakov, Morozova, & Burstein, 1985); Ca2+-induced effects have been observed using fluorescenceemission of tryptophanyl residues (Ostrovsky, Kalini-chenko, Emelyanenko, Klimanov, & Permyakov, 1988).Calcium binding strengthens the tertiary structure ofALA, and hence, its stability to denaturing agents suchas urea or guanidine hydrochloride (Permyakov et al.,1985). Hence, in the absence of calcium ions, themolecule becomes susceptible to denaturation or

ARTICLE IN PRESSK. El-Zahar et al. / International Dairy Journal 15 (2005) 17–2726

conformational changes, leading to the molten globularstate. At pHp2, protons expel Ca2+ from their bindingsites. Consequently, ALA loses its stability, and adoptsthe state of a molten globule, which is more prone tohydrolysis by pepsin. The molten globule of ALA stillretains a globular shape, but which, being highlyhydrated (about 270 bound water molecules/ALA)(Kharakoz & Bychkova, 1997), is more swollen com-pared to the native shape. Griko, Freire, and Privalov(1994) reported that ALA is in a compact intermediatestate or molten globule state at pH 3.At room temperature, apo-ALA (without Ca2+)

showed a higher number of ANS binding sites comparedto the halo-form (with Ca2+) (Eynard, Lametti, Relkin,& Bonomi, 1992).

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