317

Biochimica et Biophysica Acta, 578 (1979) 317--324 © Elsevier/North-Holland Biomedical Press

BBA 38168

ISOLATION AND CHARACTERIZATION OF PORCINE I-CASEIN

D.M. MULVIHILL and P.F. FOX

Department of Food Chemistry, University College, Cork (Republic of Ireland) (Received August 25th, 1978)

Key words: [J-Casein; Amino acid composition; (Porcine)

Summary Porcine l-casein was isolated by chromatography on DEAE-cellulose. The protein had a molecular weight of 24 900 as determined by gel filtration on Sephadex G-100 in guanidine-HC1. Its amino acid composition differed from bovine l-casein especially in respect to serine, alanine and leucine. In c o m m o n with bovine t-casein the N-terminal amino acid was arginine; the C-terminal was either alanine or valine, while the C-terminal of bovine l-casein is valine. At any temperature porcine /]-casein was more sensitive to Ca 2÷ than bovine/]-casein, while at a fixed Ca 2÷ concentration porcine/]-casein aggregated at a lower temperature than bovine t-casein. Porcine l-casein was susceptible to hydrolysis by calf chymosin but the proteolytic specificity differed from that of calf chymosin on bovine l-casein.

Introduction All mammalian milks contain protein which precipitates at pH 4.6 and 20°C [1] and which can therefore be classified as casein [2]. The caseins of all species examined by Sloan et al. [3] were heterogeneous but the milks of seven c o m m o n species contain a protein of approximately similar electrophoretic mobility to bovine l-casein [4]. The primary sequence of bovine l-casein has been established [5] and the l-caseins o f ovine and caprine milks differ only slightly from bovine t-casein with respect to molecular weight, amino acid composition, phosphorus content and Ca 2÷ sensitivity [6,7]. This paper outlines the isolation and characterization of the l-casein-like protein from porcine milk which apart from preliminary studies [8], has not been characterized. For simplicity this protein is referred to throughout as porcine l-casein.

318 Materials and Methods

Isolation of porcine [J-casein. Isoelectric casein was prepared from porcine milk, obtained from animals in mid lactation, by acidification at pH 4.6 with 2 M HC1. The precipitated protein was washed thoroughly with distilled water, dissolved at pH 7.0 by small additions of 2 M NaOH and lyophilized. Sodium caseinate (1 g) was dissolved in 50 ml 0.02 M sodium phosphate buffer~ pH 6.5 and chromatographed on an 80 X 2.2 cm column of DEAE-cellulose in the same buffer. The casein was eluted from the column with a 1 1 linear NaC1 gradient (0--0.5 M); 10-ml fractions were collected at a rate of 30 ml/h and the protein content of the eluate monitored at 280 nm. Casein-rich fractions were dialysed against distilled water, lyophilized and examined by electrophoresis in polyacrylamide gels [9]. Amino acid analysis. Duplicate samples of porcine [j-casein were hydrolysed in vacuo in 6 M HC1 at 110 + l°C for 24 or 48 h. The amino acid composition of the hydrolysate was determined on a Locarte Automatic Amino Acid Analyser (2, Wendell Rd., London W12 9RT, England) calibrated using standard amino acid solutions supplied by the Pierce Chemical Co. (Rockford, IL, U.S.A.). Tryptophan content was determined spectrophotometrically [10] on a 1.24 • 10 -s M (0.31 mg/ml) solution of [j-casein in 0.02 M sodium phosphate buffer, pH 6.5, containing 6 M guanidine-HCl. Phosphorus was determined as described by Allen [11]. Terminal amino acid analysis. Porcine [J-casein (10 mg/ml) dissolved in Nethylmorpholine acetate, pH 8.5, was incubated with diisopropylfluorophosphate (DFP)-treated carboxypeptidase A (Sigma Chemical Co. Ltd., London) as described by Ambler [12] for 0, 0.25, 0.5 and 1 h. The released amino acids were extracted into ethanol/water (70 : 30, v/v) and identified by gas-liquid chromatography on 0.65% ethylene glycol adipate on 801100 Chromosorb W {acid washed, AW) (Supelco) as described by Zumwalt et al. [13]. [j-Casein (10 mg/ml) dissolved in 0.005 M Tris buffer, pH 8.5, containing 0.005 M MgC12 was treated with leucine aminopeptidase (Sigma) as described b y Blackburn [14]. The liberated amino acids were identified by t w o dimensional paper chromatography using butanol/acetic acid/water (12 : 3 : 5, v/v) followed by phenol (160 g + 40 ml H20)/ethanol/water/ammonia (150 : 40 : 10 : 1, v/v), as described b y Smith [15]. Molecular weight estimation. The molecular weight of porcine [j-casein was estimated by gel filtration on a column (80 X 2.2 cm, Vo = 76 ml) of Sephadex G-100 (Pharmacia, Uppsala, Sweden) in 0.02 M Tris]0.01 M EDTA (disodium salt) buffer, pH 8.2, containing 6 M guanidine-HC1. A callibration curve, Fig. 1, was constructed by the method of Richardson and Creamer [16] using ovalbumin (mol. wt. 43 000), bovine [J-casein (mol. w t . 24 000), [J-lactoglobulin (mol. wt. 18 300), ~-lactalbumin (mol. wt. 14 200), cytochrome c (mol. wt. 12 500) and insulin (mol. wt. 5700) as molecular weight standards. Sensitivity of porcine [J-casein to Ca 2÷. Porcine or bovine fl-casein was dissolved in 0.05 M sodium cacodylate buffer, pH 6.8, containing 0.05 M NaC1, at a concentration of approx. 0.75 mg/ml, samples (2 ml) in cuvettes were equilibrated in a water bath at 6°C. Solutions (1 ml) of CaC12 in the same buffer were

319

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2

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3 ¸

60

~..4o

9b

70 ,

~o

~o

( Moleculor

~io

~o

~o

2Go

weight )1/2

Fig. 1. Calibration curve for the c o l u m n of S e p h a d e x G - 1 0 0 in guanidine-HCl u s e d for m o l e c u l a r w e i g h t e s t i m a t i o n . 1, insulin; 2, c y t o c h r o m e c; 3, ~-lactalbumin; 4,/3-1actoglobulin: 5, b o v i n e /~-casein; 6, ovalbumin.

mixed into the protein solutions to give [Ca 2+] in the mixture ranging from 2 to 30 mM. The samples were heated to 42°C at 2°C increments, and the turbidities measured at 440 nm, allowing a 5 min equilibration period at each temperature. Hydrolysis of porcine #J-casein by calf chymosin. Aqueous solutions of porcine or bovine fl-casein (1% protein, w/v) at pH 6.0 were treated with calf chymosin (preparation used by Mulvihill and Fox [17]) and incubated at 30°C for 12 h. Electrophoresis on polyacrylamide gels [9] Was carried out on the incubated and control samples. Results

Isolation of porcine #J-casein The elution profile of porcine sodium caseinate from DEAE-cellulose is

3C

20

05

o.4 ~. 1C

o2 .~ o.,

o

20

4o

do

;o

~o

Froct Kbn N O

Fig. 2. C h r o m a t o g r a p h y of p o r c i n e s o d i u m caseinate (1 g) o n a c o l u m n ( 8 0 X 2.2 cm) o f D E A E - c e l l u l o s e in 0 . 0 2 M s o d i u m p h o s p h a t e buffer, pH 6 . 5 . T h e casein was eluted using a 1 1 linear NaC1 gradient ( 0 - 0.5 M); at a f l o w rate o f 3 0 m l / h . F r a c t i o n size: 1 0 ml.

320 shown in Fig. 2. Protein-rich fractions were dialysed and lyophilized. Gel electrophoresis showed that fractions 49--51, contained homogeneous 3-casein {Fig. 4, slot 1).

Molecular weight The molecular weight of porcine 3-casein was estimated from the calibration curve, Fig. 1, to be 24 900; the accuracy of the determination is probably within the range +2000 but is likely to be better because of the closeness of the elution volumes for bovine (Mr = 24 000 [18]) and porcine 3-caseins. Amino acid composition The amino acid composition of porcine 3-casein was determined using samples that had been acid hydrolysed for 24--48 h. Duplicate analyses were averaged. The number o f residues per mol of protein was calculated initially on the basis that the protein had a molecular weight of 24 900. The results, together with the reported composition of bovine 3-casein, Table

TABLE I AMINO ACID ANALYSIS OF PORCINE ~-CASEIN (RESIDUES AMINO ACID/mol PROTEIN) P o r c i n e ~-casein

B o v i n e ~-casein A 2

[5,18] Calculated number of residues

Probable number of residues

Number of residues

9.8 8.0 12.3 40.9 37.2 7.3 11.0 17.2 4.7 S .9 26.6 4.4 7.7 5.0 I0.I 5.6 0.9 7.8

10 8 12 41 37 7 11 17 5 9 27 4 8 5 10 6 I 8

9 9 16 39 35 5 5 19 6 10 22 4 9 5 11 4 1 5

C-terminal N-terminal

AIa/V~ Arg

V~ Azg

Number of residues

218

209

Aspartic acid Threonine a Seine b Glutamic acid Proline Glycine Alanine Valine c Methionine Isoleucine c Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Tryptophan d Phosphorus

Molecular weight Sequence Amino acid analysis Sephadex G-100 column a b c d

23 982 25 427 24 900

2 4 h h y d r o l y s i s d a t a i n c r e a s e d b y 5% t o c o m p e n s a t e f o r d e s t r u c t i o n . 24 h hydrolysis data increased by 10%. 48 h hydrolysis data. D e r i v e d f r o m s p e c t r a l m e a s u r e m e n t s , see t e x t ,

321

16

12 i

E Oa

04

0

4

8

1~

16

20

24

28

30

CoCI 2 coric, m M

Fig. 3. Ca 2+ sensitivity of B-caseins (0.5 mg/ml) in a 0.05 M sodium eaeodylate buffer, pH 6.8, containing 0.05 M NaCL Porcine B-casein: 16°C (a), 20°C (o), 24°C (u); bovine B-casein: 16°C (A), 20°C (e), 24°C (=).

I, show that there are some major differences in the amino acid composition of the two caseins. The n u m b e r of serine residues was significantly lower in porcine fl-casein, while glycine, alanine, leucine and arginine were higher than in bovine ~-casein. The total number of residues in porcine ~-casein was higher than bovine fl-casein and it contained three phosphate residues/mol more than bovine ~-casein.

Terminal amino acid analysis Carboxypeptidase A liberated alanine and valine from porcine ~-casein at approximately equal rates indicating that either of these amino acids may be the C-terminal residue; the C-terminal sequence of bovine ~-casein was found to be Ile-Val-OH in agreement with Ribadeau-Dumas et al. [5]. Leucine aminopeptidase treatment of porcine fi~casein liberated only arginine indicating that, like bovine ~-casein, arginine is the N-terminal amino acid of porcine ~-casein. Ca 2+sensitivity o f porcine ~-casein The increase in turbidities of porcine and bovine fl-casein solutions with increasing concentration of CaC12 at 16, 20 and 24°C in 0.05 M sodium cacodylate, pH 6.8, containing 0.05 M NaC1 are shown in Fig. 3. At any given temperature porcine /3-casein b e c a m e turbid at a lower [Ca 2÷] than did bovine ~casein. Also at any given [Ca 2÷] porcine ~-casein became turbid at a lower temperature than did bovine fl-casein; porcine ~-casein aggregated even in the presence of 2 mM Ca2+> 42°C whereas the turbidity of bovine /]-casein remained unchanged under these conditions. Ca 2÷ aggregates of both caseins redispersed on cooling. Sensitivity o f porcine (J-casein to hydrolysis by calf chymosin Electrophoretograms of samples of porcine and bovine ~-caseins hydrolysed by calf chymosin are shown in Fig. 4. Bovine ~-casein was hydrolysed to fl-I, fl-II and fl-III [19--21] (Fig. 4, slot 2) while porcine fl-casein was hydrolysed to

322

porc

%-31~ f~-]£ porci porcine t~

(~ _casein

1

2

3

4

Fig. 4. Polyacrylamide gel electrophorcsis (pH 9.1, 4.5 M urea) of purified porcine/3-casein (1), chymosinhydrolysed porcine ~ a s e i n (2), bovine ~-casein (3) and bovine ~-casein hydrolysed by chymosin (4).

~-I, which was further hydrolysed to a high mobility peptide doublet with an electrophoretic mobility similar to bovine fi-III (Fig. 4, slot 2). Discussion

The milks of all major species examined contain a protein with an electrophoretic mobility similar to bovine fi-casein. In addition to bovine fi-casein, which has been characterized in detail, caprine [6], ovine [7], buffalo [22] and human (cf. ref. 23); /3-caseins have been isolated and shown to be similar to bovine ~-casein with respect to many chemical and physical properties. Human ~-casein consists of a mixture of six proteins with a c o m m o n polypeptide chain but containing zero to five phosphate residues per mol; ovine and caprine ~caseins each consists of two proteins differing in phosphorus content.

323

The fl-casein-like protein of porcine milk was isolated by chromatography on DEAE-cellulose; it also had m a n y properties typical of/~-caseins. Its molecular weight (24 900) was slightly larger than that of bovine fl-casein (24 000) as are caprine and human fl-caseins while ovine fl-casein is slightly smaller. In c o m m o n with other fl-caseins, porcine fl-casein was very low in t r y p t o p h a n and very rich in proline. Bovine and porcine fl-caseins differed by two or less residues with respect to 14 of the 17 amino acids present but differed considerably with respect to serine, alanine and leucine (12, 11, 27 and 16, 5, 22 for porcine and bovine fl-caseins, respectively). Ovine, caprine and buffalo ~-caseins are nearly similar to bovine fl-casein with respect to these three residues while human fl-casein contains 9, 7 and 26 seryl, alanyl and leucyl residues, respectively [24]. Like bovine and buffalo fl-caseins, the N-terminal residue of porcine flcasein is arginine, but its C-terminal residues were AIa-Val-OH or Val-Ala-OH while those of bovine and buffalo ~-caseins are Ile-Val-OH. Porcine fl-casein was considerably richer in phosPhorus than any fl-casein so far characterized: bovine, buffalo, caprine f12 and bovine f12 each contains 5 residues of phosphorus per mol, caprine/31 contains 6, ovine fl, contains 4 while human fl-casein contains 0--5 residues. In bovine fl-casein all the phosphorus is esterified to serine, 31% of which are phosphorylated. The lower level of serine in porcine ~-casein together with its higher phosphorus content means that if all the phosphorus is esterfied to serine, 66% of the serines in that protein are phosphorylated. Porcine fl-casein might be expected to have a higher electrophoretic mobility than other ~-caseins in view of its higher phosphorus content; the additional negative charge on these groups is apparently offset possibly by higher arginine (porcine, 6; bovine, 4; ovine, caprine, human and buffalo, 3), a higher content of hydrophobic residues especially leucine and a slightly higher molecular weight. A distinguishing characteristic of fl-caseins visa via other caseins is their solubility in the presence of Ca2÷: the K-caseins of all species examined are soluble in Ca 2÷ up to at least 0.4 M at 0--100°C; all as-caseins are insoluble at [Ca 2÷] > 6 mM independent of temperature while fl-caseins are soluble in 0.4 M Ca2÷< 20°C but solubility decreases abruptly at ~20°C above which ~-caseins are very insoluble > 6 mM Ca 2÷. Porcine fl-casein conforms to these general characteristics but at any temperature porcine fl-casein was more sensitive to Ca ~÷ than bovine /3-casein and at any given [Ca 2÷] it aggregated at a lower temperature. Caprine and ov.ine fl,-caseins are slightly more Ca 2÷ sensitive than bovine /~casein [7]. The greater sensitivity of porcine fl-casein to Ca 2÷ may be due to its higher phosphorus content. Bovine fl-casein is rapidly hydrolysed at bonds 189--190 and/or 192--193, 163--164 and~or 1 6 5 - 1 6 6 and/or 167/168 and 139/140 to yield/3-I,/3-II and fl-III, respectively. Ovine and caprine ~-caseins are also hydrolysed to three principal peptides with electrophoretic mobilities similar to those from bovine fl~asein [25]. Porcine/~casein was rapidly hydrolysed to fl-I corresponding in electrophoretic mobility to bovine ~-I suggesting that a similar bond was cleaved in both caseins. However no peptide corresponding to bovine fl-II was evident in porcine casein hydrolyzates; instead porcine/3-I was hydrolysed to a peptide doublet electrophoretically similar to bovine fl-III. Apparently the amino acid sequence of porcine ~-casein in the region 163--168 differs from

324

that of bovine ~-casein which has a cluster of four non-phosphorylated serine residues in that region.

References 1 Woodward. D.R. (1976) Dairy Sci. Abstr. 38, No. 3 , 1 3 7 - - 1 5 0 2 T h o m p s o n , M.P., Tarassuk, N.P., Jenness. R., Lillevik, H.A., Ashworth, U.S. and Rose, D. (1965). J. Dairy Sci. 48, 159--169 3 Sloan, R.E., Jenness, R., Kenyon, A.L. and Regehr, E.A. (1961) Comp, Biochem. Physiol. 4, 47--62 4 0 ' C o n n e r , P. and Fox, P.F. (1973) Neth. Milk Dairy J. 2 7 , 1 9 9 - - 2 1 6 5 Ribadeau-Dumas, B., Brignon, G., Grosclaude, F. and Mercier, J.C. (1972) Eur. J. Biochem. 25, 505-514 6 Richardson, B.C. and Creamer, L.K. (1974) Biochim. Biophys. A c t a 3 6 5 , 1 3 3 - - 1 3 7 7 Richardson, B.C. and Creamer, L.K. (1976) N . Z . J . Dairy Sci. Technol. 11, 46--53 8 Woychik, J.H. and Wondolowski, M.V. (1969) J, Dairy Sci. 5 2 , 9 0 1 9 T h o m p s o n , M.P., Kiddy, C.A., Johnson. J.O. and Weinberg, R.M. (1964) J. Dairy Sei. 4 7 , 3 7 8 - - 3 8 1 10 Edelhoch. H. (1967) Biochemistry 6, 1948--1954 11 Allen, R.J.L. (1940) Biochem. J. 34, 858--865 12 Ambler, R.P. (1972) in Methods Enzymol. (Hits, C.H.W. and Timasheff, S.N., eds.), Vol. 25, p. 262, Academic Press, New York 13 Zumwalt, R.W., Roach, D. and Gehrke, C.W. (1970) J. Chromatogr. 5 3 , 1 7 1 - - 1 9 3 14 Blackburn, S. (1970) Protein Sequence Determination, Marcel Dekker, Inc., New Y ork 15 Smith, I. (1962) in Chromatographic and Electrophoretic Techniques, (Smith, I., ed.), Vol. 1 pp. 82-118, Interscience Publishers Inc., New York 16 Richardson, B,C. and Creamer, L.K. (1975) Biochim. Biophys. Acta 393, 37--47 17 Mulvihill, D.M. and Fox, P.F. (1977) J. Dairy Res. 44, 533--540 18 Mercier, J.C., Grosclaude, F. and Ribadeau-Dumas. B. (1972) Milchwissenschaft 27,402---406 19 Creamer, L.K., Mills, O.E. and Richards, E.L. (1971) J. Dairy Res. 38, 269--280 20 Pelissier, J.P., Mercier, J.C. and Ribadeau-Dumas, R. (1974) Ann. Biol. Anim. Biochim. Biophys. 14, 343--362 21 Visser, S. and Slanger, K.J. (1977) Neth. Milk Dairy J. 31, 16--30 22 Addeo, F., Mercier, J.C. and Ribadeau-Dumas, B. (1977) J. Dairy Res. 4 4 , 4 5 5 23 Bezkorovainy, A. (1977) J. Dairy Sci. 60, 1023 24 Groves, M.L. and Gordon. W.G. (1970) Arch. Biochem. Biophys. 140, 47 25 Mulvihill, D.M. (1978) Ph.D. Thesis, National University of Ireland