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 of conformation and pH. Le Lait, INRA Editions, 1989, 69 (6), pp.451-459. hal-00929176 HAL Id: hal-00929176 https://hal.archives-ouvertes.fr/hal-00929176 Submitted on 1 Jan 1989 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Lait (1989) 69. 451-459 451 © Elsevier/INRA 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 degradation by 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 influenced by 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 pH and 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 (0,01 M) le changement de conformation de l'a-lactalbumine se produit à des valeurs de pH inférieures à 4,0. Le changement de conformation est influencé par la nature du tampon et par la force ionique de ce dernier. Toutefois la présence d'EDTA dans le tampon ne modifie pas la valeur du pH à laquelle se produit le changement de conformation de la protéine. Parallèlement, nous avons observé une hydrolyse de l'a-lactalbumine par les pepsines A bovine et porcine et par la présure aux valeurs du pH auxquelles le changement de conformation de l'a-lactalbumine intervient. Cependant avec la chymosine 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 G. Miranda et al. INTRODUCTION different pH values: 2.50--6.50 at different ionic strengths (0, 0.51 and 0.86 M NaCI). a-Iactalbumin is a calcium binding protein. This protein, in conjunction with a galacto- syl transferase, forms the lactose synthe- Gastric proteinases tase (E.C. 2.4.1.22) which is involved in the biosynthesis of lactose. The primary Bovine pepsin A was purified from commercial structure of bovine a-Iactalbumin has been powder (Chr. Hansen's Laboratory L1d.)accord- established (Brew et al., 1970). The con- ing to Martin et al. (1980). The proteolytic activi- ty of the preparation was measured on a syn- formation of this protein is pH-dependent thetic hexapeptide : Leu-Ser-Phe(N02)-Nle-Ala- (Kronman et al., 1965; Kuwajima, 1977; LeuOMe (Bachem, CA, USA) at 30 "C, in a sodi- Contaxis & Bigelow, 1981). Bovine a- um acetate buffer 0.1 M, pH 4.7 (Martin et al., o lactalbumin is resistant to in vitro proteoly- 1980). The result, expressed as hydrolyzed pep- sis by pepsin at pH 6.0, which is the pH of tide (prnol mg-1 S-1), was 2 540 ± 40. Porcine calf abomasum contents immediately after pepsin A (ref : P6887) and bovine chymosin (rennin) (ref : P7751) were obtained from the feeding milk, but is degraded at pH 2.0 Sigma Chemical Co. (USA). Liquid rennet (520 (Jenkins et al., 1980). In vivo experiments mg chymosin, 290 mg pepsin/I) was obtained on pre-ruminant calves (Yvon et al., 1984) from Boil-Hansen (France). and on rats (Fushiki et al., 1986) have also shown that a-Iactalbumin is hydrolyzed by gastric proteinases concomitantly with the Change of conformation acidification of the stomach's content. However, "" 50% of the ingested a- 1 lactalbumin leaves the stomach before be- Modification of the conformation of 0.- ing proteolyzed by gastric enzymes (Yvon lactalbumin was observed by measurement at 1 etai., 1984). 291 nm of the difference absorption of trypto- phan residues at the different pH values and re- The aim of our study was to ascertain ferred to the absorbance of the reference solu- 1 that hydrolysis of a-Iactalbumin was corre- tion at pH 6.50 (Kuwajima, 1977). The lated to the modification of its conforma- absorbance was measured in citrate or citrate- phosphate buffers (0.01 M) with or without 1 tion. EDTA (0.001 and 0.01 M). 1 Hydrolysis of a-Iactalbumin 1 MATERIAL AND METHODS Hydrolysis of a-lactalbumin by gastric proteinas- 1 es ~as performed at 37 oC with an È/S ratio of ~ a-Iactalbumin preparation 1/2{500 (w/w) and stopped by adjusting the pH Vil to '9.0 with NH40H. Hydrolysis of the protein Bovine a-lactalbumin was prepared by the was verified by SD&-gradient polyacrylamide method of Aschaffenburg & Drewry (1957) and gel electrophoresis (SDs-GPGE) (Trieu-Cuot then purified by chromatography on an Ultrogel and Gripon, 1981). The kinetics of disappear- 1 AcA 54 column (Gaye et al., 1982). To eliminate ance of the protein were checked on a FPLC effects of possible association, a-lactalbumin system (Pharmacia, Uppsala, Sweden) by anion 1 was dissolved at a concentration of 0.08 g/100 exchange chromatography on a Mono Q column ml (Kronman & Andreotti, 1964). Citrate or cit- (Pharmacia, Uppsala, Sweden). The column 2 1 o rate-phosphate buffers (0.01 M) were used at was equilibrated with Tris-HCI buffer, 10- M, 1 1 1 1 Hydrolysis of ex-Iactalbumin 453 pH 8.0. The peptides were eluted in a 20 min lin- a. Effect of buffer ear gradient of NaCI from 0.05 to 0.22 M. Elu- Difference of absorbance at 291nm tion rate was 1 ml/min. 0.1 Sorne particular peptides, soluble in 2% tri- chloroacetic acid (TCA), liberated during hydrol- 0.05 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 linear -0.05 gradient of 0-100% solvent B (0.10% TFA, -o. , 60% CH3CN). Peptides were detected at 220 nm. The column was kept at 40 oC in a water bath. The elution rate was 2 ml/min. These pep- -0.15L-~-~-~~-~-~~-~~ 2 2.5 3.5 4 4.5 5 5.5 6.5 tides were identified by their amino acid compo- pH of buffer solution sition, after acid hydrolysis (110°C, 24 h, 5.7 N HCI, under vacuum) according to Spackman et b. Effect of EDTA al. (1958). The N-terminal amino acids were Difference of absorbance at 291nm identified by recurring Edman degradation using 0.1 the technique of Tarr (1982). 0.06 RESUL TS AND DISCUSSION -0.06 - OMEDTA -0.1 -+- 0.0141 EDrA Modification of the conformation of a- -+ O.001M EDTA -0.I5L-~-~-~~-~-~~-~~ lactalbumin 2 2.5 3 3.5 4.5 5 5.5 6.5 pH of buffer solution c. Effect of ionie strenght ln citrate buffer (Fig. 1a) the absorbance of Difference of absorbance at 291nm a-Iactalbumin solution decreased at pH 0.2 values below pH 4.0, which indicated a 0.15 -+- D.tTM N.CI change of conformation of the protein be- ..... O.5IM N.CI 0.1 low this pH value. With citrate-phosphate ~ O.B6UN.CI buffer (Fig. 1a) the decrease of the absor- 0.05 bance occurred below pH 3.5. With citrate buffer, in presence of EDTA (Fig. 1b) there -0.05 was no modification of the pH value at -0.1 which change of conformation occurred. -O.f5L-~-~-~~-~-~~-~~ When the ionic strength of the citrate but- 2 2.5 3.5 " 4.5 5 5.5 6.5 fer was increased by adding NaCI (0 to pH of buffer solution 0.86 M) the change of conformation oc- curred at a higher pH value (Fig. 1c) (pH Fig. 1. Effect of buffer (a), EDTA (b) and ionic 4.50 with 0.17 M of NaCI). With 0.51 and strength (c) on the change of conformation of ex- 0.86 M of NaCI at the pH value near the lactalbumin (0.08 mg/100 ml) at different pH va- isoelectric pH of a-Iactalbumin (Kronman lues. Effet de la nature du tampon (a), de la présence et al., 1965), partial precipitation of the pro- d'EDTA (b) et de la force ionique (c) sur le chan- tein occurred. These results showed that gement de conformation de l'a-lactalbumine (0,08 the pH at which the protein changed con- mgl100 ml) en fonction de la valeur du pH.