HAL Id: hal-00929176https://hal.archives-ouvertes.fr/hal-00929176

Submitted on 1 Jan 1989

HAL is a multi-disciplinary open accessarchive for the deposit and dissemination of sci-entific research documents, whether they are pub-lished or not. The documents may come fromteaching and research institutions in France orabroad, or from public or private research centers.

L’archive ouverte pluridisciplinaire HAL, estdestinée au dépôt et à la diffusion de documentsscientifiques de niveau recherche, publiés ou non,émanant des établissements d’enseignement et derecherche français ou étrangers, des laboratoirespublics ou privés.

Hydrolysis of α-lactalbumin by chymosin and pepsin.Effect of conformation and pH

G. Miranda, G. Hazé, Non Renseigné

To cite this version:G. Miranda, G. Hazé, Non Renseigné. Hydrolysis of α-lactalbumin by chymosin and pepsin. Effect ofconformation and pH. Le Lait, INRA Editions, 1989, 69 (6), pp.451-459. �hal-00929176�

Lait (1989) 69. 451-459© Elsevier/INRA

451

Original article

Hydrolysis of œ-lactalburnin by chymosin and pepsin.Effect of conformation and pH

G. Miranda, G. Hazé, P.Scanff and J.P.Pélissier

INRA, station de recherches laitières, 78350 Jouy-en-Josas, France

(received 21 March 1989. accepted 26 June 1989)

Summary - The correlation between change of conformation of a-Iactalbumin and its degradationby gastric enzymes was verified. With citrate buffer (0.1 M), the modification of a-Iactalbumin confor-mation occurred when the pH value was below pH 4.0. This conformational change was influencedby buffer composition and ionic strength. However, the presence of EDTA in the butter did not modi-fy the pH value at which the change of conformation of the protein occurred. Concomitantly, hydroly-sis of a-Iactalbumin by bovine and porcine pepsins A and rennet was observed at pH values corre-sponding to the change in conformation of the protein. However, with chymosin, under the same pHand ionie strength conditions there was no significant hydrolysis of a-Iactalbumin.

a-Iactalbumin (bovine) - conformation - hydrolysis - pepsin - chymosin - rennet

Résumé - Hydrolyse de l'a-lactalbumine par la chymosine et la pepsine: effet de la confor-mation et du pH. Nous avons vérifié la corrélation entre le changement de conformation de l'a-lactalbumine et sa dégradation par les protéases gastriques. En présence de tampon citrate (0,01M) le changement de conformation de l'a-lactalbumine se produit à des valeurs de pH inférieures à4,0. Le changement de conformation est influencé par la nature du tampon et par la force ionique dece dernier. Toutefois la présence d'EDTA dans le tampon ne modifie pas la valeur du pH à laquellese produit le changement de conformation de la protéine. Parallèlement, nous avons observé unehydrolyse de l'a-lactalbumine par les pepsines A bovine et porcine et par la présure aux valeurs dupH auxquelles le changement de conformation de l'a-lactalbumine intervient. Cependant avec lachymosine bovine, aux mêmes conditions de pH et de force ionique, nous n'avons pas observé d'hy-drolyse significative de l'a-lactalbumine.

a-lactalbumine (bovine) - conformation - hydrolyse - pepsine - chymosine - présure

452

different pH values: 2.50--6.50 at different ionicstrengths (0, 0.51 and 0.86 M NaCI).

G. Miranda et al.

INTRODUCTION

a-Iactalbumin is a calcium binding protein.This protein, in conjunction with a galacto-syl transferase, forms the lactose synthe-tase (E.C. 2.4.1.22) which is involved inthe biosynthesis of lactose. The primarystructure of bovine a-Iactalbumin has beenestablished (Brew et al., 1970). The con-formation of this protein is pH-dependent(Kronman et al., 1965; Kuwajima, 1977;Contaxis & Bigelow, 1981). Bovine a-

o lactalbumin is resistant to in vitro proteoly-sis by pepsin at pH 6.0, which is the pH ofcalf abomasum contents immediately afterfeeding milk, but is degraded at pH 2.0(Jenkins et al., 1980). In vivo experimentson pre-ruminant calves (Yvon et al., 1984)and on rats (Fushiki et al., 1986) have alsoshown that a-Iactalbumin is hydrolyzed bygastric proteinases concomitantly with theacidification of the stomach's content.However, "" 50% of the ingested a-lactalbumin leaves the stomach before be-ing proteolyzed by gastric enzymes (Yvonetai., 1984).

The aim of our study was to ascertainthat hydrolysis of a-Iactalbumin was corre-lated to the modification of its conforma-tion.

MATERIAL AND METHODS

a-Iactalbumin preparation

Bovine a-lactalbumin was prepared by themethod of Aschaffenburg & Drewry (1957) andthen purified by chromatography on an UltrogelAcA 54 column (Gaye et al., 1982). To eliminateeffects of possible association, a-lactalbuminwas dissolved at a concentration of 0.08 g/100ml (Kronman & Andreotti, 1964). Citrate or cit-

o rate-phosphate buffers (0.01 M) were used at

Gastric proteinases

Bovine pepsin A was purified from commercialpowder (Chr. Hansen's Laboratory L1d.)accord-ing to Martin et al. (1980). The proteolytic activi-ty of the preparation was measured on a syn-thetic hexapeptide : Leu-Ser-Phe(N02)-Nle-Ala-LeuOMe (Bachem, CA, USA) at 30 "C, in a sodi-um acetate buffer 0.1 M, pH 4.7 (Martin et al.,1980). The result, expressed as hydrolyzed pep-tide (prnol mg-1 S-1), was 2 540 ± 40. Porcinepepsin A (ref : P6887) and bovine chymosin(rennin) (ref : P7751) were obtained from theSigma Chemical Co. (USA). Liquid rennet (520mg chymosin, 290 mg pepsin/I) was obtainedfrom Boil-Hansen (France).

1

1

1

1

1

1

Hydrolysis of a-lactalbumin by gastric proteinas- 1

es ~as performed at 37 oC with an È/S ratio of ~1/2{500 (w/w) and stopped by adjusting the pH Vilto '9.0 with NH40H. Hydrolysis of the proteinwas verified by SD&-gradient polyacrylamidegel electrophoresis (SDs-GPGE) (Trieu-Cuotand Gripon, 1981). The kinetics of disappear-ance of the protein were checked on a FPLCsystem (Pharmacia, Uppsala, Sweden) by anionexchange chromatography on a Mono Q column(Pharmacia, Uppsala, Sweden). The columnwas equilibrated with Tris-HCI buffer, 10-2 M,

Change of conformation

Modification of the conformation of 0.-

lactalbumin was observed by measurement at291 nm of the difference absorption of trypto-phan residues at the different pH values and re-ferred to the absorbance of the reference solu-tion at pH 6.50 (Kuwajima, 1977). Theabsorbance was measured in citrate or citrate-phosphate buffers (0.01 M) with or withoutEDTA (0.001 and 0.01 M).

Hydrolysis of a-Iactalbumin

1

1

1

1

1

1

1

Hydrolysis of ex-Iactalbumin 453

pH 8.0. The peptides were eluted in a 20 min lin-ear gradient of NaCI from 0.05 to 0.22 M. Elu-tion rate was 1 ml/min.

Sorne particular peptides, soluble in 2% tri-chloroacetic acid (TCA), liberated during hydrol-ysis were purified on a Spectra Physics LC sys-tem. The column (u-Bondapack C18 column,Waters) was equilibrated in solvent A (0.15%TFA), and the peptides were eluted by a lineargradient of 0-100% solvent B (0.10% TFA,60% CH3CN). Peptides were detected at 220nm. The column was kept at 40 oC in a waterbath. The elution rate was 2 ml/min. These pep-tides were identified by their amino acid compo-sition, after acid hydrolysis (110°C, 24 h, 5.7 NHCI, under vacuum) according to Spackman etal. (1958). The N-terminal amino acids wereidentified by recurring Edman degradation usingthe technique of Tarr (1982).

RESUL TS AND DISCUSSION

Modification of the conformation of a-lactalbumin

ln citrate buffer (Fig. 1a) the absorbance ofa-Iactalbumin solution decreased at pHvalues below pH 4.0, which indicated achange of conformation of the protein be-low this pH value. With citrate-phosphatebuffer (Fig. 1a) the decrease of the absor-bance occurred below pH 3.5. With citratebuffer, in presence of EDTA (Fig. 1b) therewas no modification of the pH value atwhich change of conformation occurred.When the ionic strength of the citrate but-fer was increased by adding NaCI (0 to0.86 M) the change of conformation oc-curred at a higher pH value (Fig. 1c) (pH4.50 with 0.17 M of NaCI). With 0.51 and0.86 M of NaCI at the pH value near theisoelectric pH of a-Iactalbumin (Kronmanet al., 1965), partial precipitation of the pro-tein occurred. These results showed thatthe pH at which the protein changed con-

-0.05

a. Effect of bufferDifference of absorbance at 291nm

0.1

0.05

-o. ,

-0.15L-~-~-~~-~-~~-~~2 2.5

-0.06

3.5 4 4.5 5 5.5

pH of buffer solution6.5

b. Effect of EDTADifference of absorbance at 291nm

0.1

0.06

- OMEDTA

-+- 0.0141 EDrA

-+ O.001M EDTA

-0.1

-0.I5L-~-~-~~-~-~~-~~2 2.5 3 3.5 4.5 5 5.5 6.5

pH of buffer solution

c. Effect of ionie strenghtDifference of absorbance at 291nm

0.2

0.15

0.05

-+- D.tTM N.CI

..... O.5IM N.CI

~ O.B6UN.CI0.1

-0.05

-0.1

-O.f5L-~-~-~~-~-~~-~~2 2.5 3.5 " 4.5 5 5.5

pH of buffer solution6.5

Fig. 1. Effect of buffer (a), EDTA (b) and ionicstrength (c) on the change of conformation of ex-lactalbumin (0.08 mg/100 ml) at different pH va-lues.Effet de la nature du tampon (a), de la présenced'EDTA (b) et de la force ionique (c) sur le chan-gement de conformation de l'a-lactalbumine (0,08mgl100 ml) en fonction de la valeur du pH.

454

less active proteinase compared to pep-sins, in the pH range between 3.0-4.0,chymosin shows maximum proteolytic ac-tivity, but minimum stability which may pre-sumably be accounted for by autolysis ofthe enzyme (Foltmann, 1966). Similar au-toiYtic decomposition of proteinase isknown in the case of pepsin "" pH 2.0 (Folt-man, 1966). These properties could ex-plain why at pH values < 4.0 only a little a-lactalbumin could be proteolyzed by chy-mosin in spite of its conformationalchange. With rennet (Fig. 3d) the ob-served kinetics of hydrolysis were similarto those for bovine pepsin A.

G. Mirandaet al.

formation depended on the nature and onthe concentration of the salts in the bufferand that the presence or the absence ofCa2+ did not influence the pH value atwhich change of conformation occurred.

Electrophoretic study of a-Iactalbuminhydrolysis

Using SDs-GPGE it was observed that(X-Iactalbuminwas proteolyzed in vitro bybovine pepsin A when the pH of the solu-tion was below pH 4.0 (Fig. 2a). However,in the presence of 0.51 M NaCI, (X-lactalbumin was hydrolyzed by pepsin be-tween pH 2.5 and 4.5 (Fig. 2b). These re-sults show that there is a correlation be-tween the change of conformation of theprotein and its ability to be proteolyzed bybovine pepsin A.

Kinetics of hydrolysis of a-Iactalbumin

The hydrolytic action of bovine and por-cine pepsin A, bovine chymosin and ren-net were tested on (X-Iactalbuminsolutionin citrate buffers (pH 2.5-6.5). With bo-vine and porcine pepsin A (Fig. 3a, b), thekinetic curves obtained at different pH val-ues confirmed that pepsin A completelyhydrolyzed (X-Iactalbuminat pH values <pH 4.0. At these pH values, both enzymesare near their optimum for activity. The dis-appearance of a-Iactalbumin was faster atpH 3.0 than at pH 2.5 or pH 3.5. pH 3.0corresponds to the activity maxima of bothpepsins A on this protein. However, underthe same conditions, much faster hydroly-sis by porcine pepsin A was observed.With chymosin (Fig. 3c), only a small de-gree of proteolysis ("" 10%) occurred when

·the pH was < 4.0. Although chymosin is a

Characterization of some hydrolysisproducts

During hydrolysis of (X-Iactalbuminby bo-vine pepsin A, 3 peptides, soluble in 2%TCA, appeared rapidly (Fig. 4a) and re-mained as major products until the totaldisappearance of the protein (Fig. 4b).These peptides were analyzed to identifytheir sequence (Table 1). They correspond-ed to the sequences 41-52, 40-52 and 41-53 of the protein.

1

1

1

1

1

1

1

1

1

1

1

1

1

CONCLUSION

Our results show that the modification ofa-Iactalbumin conformation is pH-dependent and occurs at pH values < 4.0.However, this conformational change is in-fluenced by buffer composition and ionicstrength but is not influenced by the pres-ence or the absence of Ca2+. (X-lactalbumin hydrolysis by porcine and bo-vine pepsins A and rennet also occurredonly at pH values < 4.0. At higher pH, al-

Hydrolysis of a-Iactalbumin

a. Citrate buffer

b. Citrate buffer in presence of D.5IM NaCl

Fig. 2. SOS-GPGE of a-Iactalbumin hydrolysed by bovine pepsin A at different pH values with and wi-thout NaCI (0.51 M). S = molecular weight standard solution; A = a-Iactalbumin; 1,Â. 3 = ln vitro hydre-Iysate of a-Iactalbumin by bovine pepsin A (citrate buffer 0.01 M, 37 oC, E/S = 1/2(500) at different pHvalues for 40. ao and 120 min hydrolysis, respectively. YElectrophorèse SOS d'hydrolysats d'a-lactalbumine par la pepsine bovine A, à différents pH, en pré-sence et en absence de NaCI (0,51 M). S = standard de poids moléculaires. A = a-lactalbumine. 1,2,3 =_f1Ydrolysats in vitro d'a-lactalbumine par la pepsine bovine A (tampon citrate 0,01 M, 37 oC, EIS =112:500) à différents pH, après respectivement 40, 80 et 120 min d'hydrolyse.

\-

455

456 G. Miranda et al.

a. Bovine pepsin A b. Porcine pepsin A

Percent of residustcc-tsctetbumin Percent of residualCX-lactalbumin120 120

pH 6.0I~::;:~~~~~~;="",""pH 5.0'1 pH 4.5

b-...,..--8-'"'----,:r-_--EJ pH 4.0 f5~~~::=;PH6.0100pH 4.5pH 5.5pH 5.0

pH 4.080

60

pH 3.5pH 2.5 pH 3.5

pH 3.0

20 40 60 80 100 120 140 20 40 60 80 100 t20 140

Time of hydrolysis (min) Time of hydrolysis (min)

C. Chymosin d. Rennet

Percent of reskiuetcc-teotetbumin Percent of residuslcc-tectelbumln120 120

X pH 6.0

100pH 6.0 pH 5.5

t pH 5.0

0pH 4.5

pH 2.5

80pH 4.0

60

40

pH 3.5

20pH 3.0

pH 2.5

0 00 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140

Time of hydrolysis (min) Time of hydrolysis (min)

/'

(/Fig. 3. In vitro hydrolysis (citrate buffer 0.01 M, 37 oC, E/S = 1/2 500) of a-Iactalbumin solution (o.oamg/100 ml) at different pH values by bovine (a) and porcine (b) pepsins A, chymosin (c) and rennet(d). Results were expressed as percent of residual a-Iactalbumin during hydrolysis.Hydrolyse in vitro (tampon citrate 0,01 M, 37 "O, E/S = 1/2500) de l'a-lactalbumine à différents pH,par les pepsines A bovine (a) et porcine (b), la chymosine (c) et la présure (d). Les résultats sont ex-primés en pourcentage d'a-lactalbumine résiduelle au cours de l'hydrolyse.

/J

Hydrolysis of a-Iactalbumin

2

3

a. 40 min

1 2

3

b. 120 min

Fig. 4. RP-HPLC of the 2% TCA soluble fraction of a-Iactalbumin hydrolysed by bovine pepsin A (ci-trate buffer 0.01 M, pH 3.2, 37 oC, E/S = 1/21500)for 40 (a) and 120 (b) min of hydrolysis. RP-HPLCconditions: solvent A : TFAlH20 (1.15/1000); solvent B : TFAlH20/CH3CN (1/400/600); gradient: 0-100% B in 20 min; temp; 40 "C: À = 220 nm. 1 = peptide 41-52; 2 = peptide 40-52; 3 = peptide 41-53.RP-HPLC de la fraction soluble en TCA 2% final d'un hydrolysat d'a-lactalbumine par la pepsine bo-vine A (tampon citrate 0,01 M, pH 3,2, 37 oC, E/S = 1/2)500) après 40 (a) et 120 (b) min d'hydrolyse.Conditions en RP-HPLC : solvant A : TFAlH20 (1,15/1000); solvant B : TFAlH~/CH3CN (1/400/600);gradient: 0 à 100% B en 20 min; temp: 40 "C; À = 220 nm. 1 = peptide 41-52; 2 = peptide 40-52; 3 =peptide 41-53.

though they are not at their optimal of ac-tivity, these proteinases are always activeand hydrolyse proteins such as caseins(Pélissier, 1984). This is also the case forchymosin. These results suggest that thechange of conformation of a-Iactalbumin isresponsible for the susceptibility of a-lactalbumin at these pH values and thatthe native protein is resistant to proteoly-sis. In contrast with chymosin, there wasno significant hydrolysis of a-Iactalbuminregardless of the protein conformation.These results can be explained by a lower

activity of chymosin compared to pepsins,and by a loss of activity at a pH at whichchymosin has a minimum stability. Alithese results suggest that the in vivo hy-drolysis of a-Iactalbumin observed in thecalf only wh en the ~H value of the stom-ach's content is < 1;(Yvon et al., 1984), oc-curs specifically by the action of pepsins.ln other animal species, a-Iactalbumincould also be degraded by pepsins. But,as these enzymes may only act when thestomach pH is low, a large part of the a-lactalbumin could arrive intact in the gut.

457

j .f)

458 G. Miranda et al.

2 3

2.2 (2) 2.3 (3)0.7 (1) 0.7 (1)1.0 (1) 0.9 (1)2.0 (2) 2.0 (2)

0.9 (1) 1.3 (1)0.6 (1)0.6 (1) 0.6 (1)

0.6 (1) 0.9 (1)0.9 (1) 1.1 (1)0.7 (1) 0.6 (1)

1.2 (1)

Aia-lle40-52 41..,53

Table 1. Amino acid compositions and N-terminal amino acid sequences of peptides 1, 2 and 3 (seeFig. 4). Results (mol/mor) were obtained after 24 h acid hydrolysis. Figures in parentheses are theoret-ical values.Compositions en acides aminés et séquences N-terminales des peptides 1,2 et 3 (voir Fig. 4). Les ré-sultats (mole/mole) ont été obtenus après hydrolyse acide de 24 h. Les chiffres entre parenthèses cor-respondent aux valeurs théoriques.

Peptides

AsxThrSerGlxProGlyAlaValMetIleLeuTyrPheHisLysArgN-terOrigin

2.3 (3)0.8 (1)0.9 (1)2.0 (2)

0.9 (1)

0.6 (1)

0.6 (1)1.0 (1)0.6 (1)

41-52

REFERENCES

Aschaffenburg R. & Drewry J. (1957) Improvedmethod for the preparation of cristalline /3-lactoglobulin and œ-lactalbumln from cow's milk.Biochemistry 65, 273-277

Brew K., Castellino F.J., Vanaman T.C. & HillR.L.(1970) The complete amino acid sequenceof bovine œ-lactalburnin. J. Biol. Chem. 245,4570-4582

Contaxis C.C. & Bigelow C.C. (1981) Free ener-gy changes in œ-lactalbumin denaturation. Bio-chemistry20, 1618-1622

Foitmann B. (1966) A review on prorennin andrennin. CR Trav. Lab. Carlsberg 35, 143-231

Fushiki T., Yamamoto N., Naeshiro 1.& Iwai K.(1986) Digestion of œ-lactalburnln in rat gastroin-testinal tracts. Agric. Biol. Chem. 56, 95-100

Gaye P., Hué O., Raymond M.N., Hazé G. &Mercier J.C. (1982) Cell-free synthesis, protee-Iytic processing, core glycosylation and aminoterminal sequence of rabbit prs-u-lactalbu-min.Biochemistry 64, 173-184Jenkins K.J., Mahadevan S. & Emmons D.B.(1980) Susceptibility of proteins used in calf milkreplacers to hydrolysis by various proteolytic en-zymes. Cano J. Anim. Sei. 60, 907-914Kronman M.J. & Andreotti R.E. (1964) Inter andintramolecular interactions of œ-lactalbumin. 1.The apparent heterogeneity at acid pH. Bio-chemistry3,1145-1151Kronman M.J., Cerankowski L. & Holmes L.G.(1965) Inter and intramolecular interactions ofœ-lactalburnln, III. Spectral changes at acid pH.Biochemistry 4, 518-525Kuwajima K. (1977) A folding model of Cl-

lactalbumin deduced from the three-state dena-turation mechanism. J. Mol. Biol. 114, 241-258

Hydrolysis of a-Iactalbumin 459

Martin P., Raymond M.N., Bricas E. & RibadeauDumas B. (1980) Kinetic studies on the actiol)!of Mucor pusillus, Mucor miehei acid proteasesand chymosins A and B on a synthetic chromo-phoric hexapeptide. Biochim. Biophys. Acta 612,410-420

Pélissier J.P. (1984) Proteolysis of caseins. Areview. Sci. Aliments 4, 1-35

Spackman D.H., Stein W.H. & Moore S. (1958)Automatic recording apparatus for use in thechromatography of amino acids. Anal. Chem.30, 1190-1206

Tarr G.E. (1982) Manual batchwise sequencingmethods. In : Methods in Protein SequencesAnalysis (M. Elzinga ed.), Clifton, NJ, pp. 223-232

Trieu-Cuot P. & Gripon J.C. (1981) Electrofocus-ing and two-dimensional electrophoresis of bo-vine caseins. J. Dairy Res. 48, 303-310

Yvon M., Van Hille 1., Pélissier J.-P., GuilloteauP. & Toullec R. (1984) ln vivo milk digestion inthe calf abomasum. Il. Milk and whey proteoly-sis. Reprod. Nutr. Dev. 24, 835-843