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Comparative micelle structure. III. The isolation and chemical characterization of caprine β1-casein and β2-casein
Biochimica et Biophysica Acta, 365 (1974) 133-137
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 36797 COMPARATIVE MICELLE STRUCTURE III. THE ISOLATION AND CHEMICAL CHARACTERIZATION OF CAPRINE fl~-CASEIN AND flz-CASEIN B. C. RICHARDSON and L. K. CREAMER New Zealand Dairy Research Institute, Palmerston North (New Zealand)
(Received April 2nd, 1974)
SUMMARY Two pure caprine (goat) fl-caseins were purified by DEAE- and CM-cellulose column chromatography in buffers containing urea and 2-mercaptoethanol. The molecular weight of each fl-casein was found to be about 24 500 as determined by gel filtration on Sepharose 6B in guanidine. HC1, and each had the same amino acid composition; Asp9, Thr~2, Seras, Glu4a, Proa3, Gly6, Alas, Val2~, Met6, Ile9, Leuzo, Tyr4, Phe9, Hiss, Lys~2, Arga, Trp. Phosphate determinations showed, however, that fl~casein (the more mobile component on electrophoresis at pH 8.6) contained six residues per molecule, whilst fl2-casein contained only five. This difference may have been caused by a gene duplication and mutation. Both caseins are similar to bovine fl-casein in molecular weight and net charge.
The caseins of caprine milk are present in subunits, which coalesce with Ca 2+ and phosphate to form micelles. These subunits and micelles are very similar in mineral composition and in appearance to their bovine or ovine (sheep) counterparts [1]. fl-Casein constitutes approx. 30 ~o bovine casein [2], whereas fl-caseins are the major components of both caprine [1, 3, 4] and human [5, 6] casein. Consequently the flcaseins are likely to play a more important role in the stability of the caprine micelle than in the stability of bovine casein micelles. Caprine casein has been reported [3] to contain two major fl-casein components with electrophoretic mobilities in alkaline buffers similar to that of bovine fl-casein. In the present investigation the purification and chemical characterization of these two caprine fl-caseins has been achieved. Whole caprine casein was isolated and then separated into individual caseins using the chromatographic procedures previously described [7]. Each fl-casein fraction was rechromatographed on Whatman CM-32 cellulose at pH 4.0 in a sodium formate buffer (0.01 M) which contained urea and 2-mercaptoethanol. A gradient 0-0.15 M NaC1 was used to elute the fl-casein. Bovine fl-casein B was isolated using the procedure of Mills and Creamer [8]. The procedures used for gel electrophoresis at pH 8.6 [7] and in a Tris-borate buffer containing Mg 2+ [2], amino acid analysis [7], phosphorus estimation [7], extinction coefficient determination [7], tryptophan analysis
134 (procedure W) [9], tyrosine estimation [10], and molecular weight determination [11, 12] followed published methods. Examination of milk samples from 25 goats by gel electrophoresis showed that they all contained two bands of about equal intensity with similar mobility to bovine {4-casein. Each of these was readily isolated from the whole casein (ref. 7, Fig. 1) with the more mobile component on alkaline gel electrophoresis (/:~l-casein) lzeing eluted less readily from the DEAE-cellulose column than the other component (/42-casein). Gel electrophoresis at pH 4 reversed the order of mobility so that/~'2-casein became more mobile than {/l-casein. in the presence of Mg z+ in the electrophoresis buffer the mobilities of {/~- and /4z-casein decrease and the two components merge (Fig. 1). This suggested that {/~and {/z-casein differed in the number of phosphate groups that each contained, since Mg 2+ associates strongly with the phosphate groups of the caseins, and largely neutralizes their charge. This conclusion is supported by the results t¥om acid gel electrophoresis which show that /3l-casein carries more negative charge than [42casein at both pH 4 and pH 8.6. At these pH values the phosphate groups would carry single and double negative charges, respectively.
Fig. I. Gel electrophoresis of bovine and caprine caseins in alkaline buffer and in the presence of Mg2+. Samples: 1, bovine skim milk; 2, caprine whole casein; 3, {/~-casein;4, /~-casein. The molecular weights of caprine ¢!tl- and {4z-casein and bovine {4-casein were determined on a calibrated column of Sepharose 6B in 6 M guanidine.HCI solution (Table I). In addition, a sample containing both {4x- and {/z-casein was chromatographed on the molecular weight column and the fractions collected as before. After weighing, each fraction was dialysed to remove salts and after gel electrophoresis and densitometry the ratio of {/1- to {42-casein was found to be constant, across the protein peak. This showed that the molecular weights of these two proteins were not distinguishable within the limits of this technique (±5C0). The amino acid compositions of caprine {/~- and {/z-casein and bovine {4-casein B were determined using acid-hydrolysed samples [7] (Table I). Tryptophan contents of {/1- and {/z-casein were 0.62 and 0.67 ~ , respectively. Inorganic phosphorus was determined for each {/-casein in the acid-hydrolysed samples, the average being 0.78 and 0.67 ~ for/3~- and /3z-casein, respectively. The absorbance of 1 cm solutions of
* S o . text.
Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine* Phenylalanine Histidine Lysine Arginine Tryptophan * Phosphorus residues Molecular weight (from Sepharose 6B column) Molecular weight (from amino acid composition) No. of residues
Residue
25 500
1.31 1.36 0.14 0.18 0.26 0.14 0.17 0.49 0.07 0.15 0.08 0.40 0.07
0.22
23 500
9.1 11.6 14.7 43.0 32.6 5.6 4.7 21.3 6.3 9.0 20.5 3.4 9.0 5.2 11.7 3.1 0.8 5.0
Mean No. residues
9.0 11.5 14.8 43.2 32.8 5.6 4.7 21.0 5.8 8.6 20.2 3.6 8.8 5.1 12.1 3.3 0. 7 6. l
fl2-casein
Mean No. residues
S.D.
ill-casein
Caprine
0.89 1.13 0.20 0.16 0.52 0.24 0.31 0.47 0.06 0.14 0.14 0.20 0.15
0.12
S.D.
25 100
---
9.3 8.9 15.2 39.8 35.0 5.1 5.7 18.4 5.6 9.0 21.8 4.0 8.8 6.1 11.0 4.4
residues
Bovine fl-casein B Mean No.
24 439 213
9 12 15 43 33 6 5 21 6 9 20 4 9 5 12 3 1 6
ill-casein Probable No. residues
Caprine
AMINO ACID COMPOSITION OF fl-CASEINS (RESIDUES A M I N O ACID/MOLE PROTEIN)
TABLE I
23 982 209
I
5
1
5
24 360 213
9 9 16 39 35 5 5 19 6 10 22 4 9 5 11 4
9 12 15 43 33 6 5 21 6 9 20 4 9 5 12 3
fl2-casein Probable No. residues
23 712-24 108 210
0-5
1
11 9 9 39 39 3 7 19 3 13 26 7 5 5 11 3
Bovine Human fl-casein A2 fl-casein (refs 14 (ref, 5) and 15)
136 /4~- and/J2-casein 1 ')£, w/v (pH 7.0) at 280 nm (corrected for Rayleigh scattering) were 4.2 and 4.5, respectively. These are similar to the literature values [13] for bovine/Jcasein (4.5-4.7) suggesting that the caprine/J-caseins and bovine/Z-casein contain the same number of tyrosine and tryptophan residues. The absorbance in 0.1 M N a O H solution showed the ratio of tyrosine to tryptophan to be 3.8 and 3.7 for {;~- and/Jzcasein, respectively [10]. Consequently, tryptophan was taken as one residue per molecule and tyrosine as four per molecule, despite the lower values obtained from the amino acid analysis (Table I). The mobility on polyacrylamide gels of bovine/J-casein B is similar to that of caprine /~2-casein, the molecular weight of the proteins is similar and laence the net charge is probably similar at both pH 4 and pH 8.6. The number of histidine residues is the same for the two proteins (5) whilst the number of lysine plus arginine residues is one more for the B variant of bovine casein [14]. Consequently, the flz-casein (or fl~-casein) should have one more carboxyl residue than bovine/Z-casein B, i.e. 23 aspartic acid and glutamic acid residues and 29 asparagine plus glutamine residues (the bovine casein contains 26 amide groups [15] and 22 carboxyl groups). As the only difference in charged residues between t h e / ~ - and [42caseins is the number of phosphate groups, the relative number of carboxyl and amide groups would be the same for the two caprine caseins. The amino acid composition of the caprine caseins is very close to that published for the bovine/J-casein Az (Table I). The sum of lysine and arginine residues is constant at 15, and the sum of leucine, isoleucine and valine residues is constant at 5 I. The only major difference between the two caprine/4-caseins is the different phosphate level with [J~- and/J2-caseins containing six and five phosphates per molecule, respectively. The different levels of phosphorylation found in bovine/J-casein is known to be the result of genetic variation in the sequence [14]. in contrast, human/4-casein, similar in composition and properties to its bovine counterpart [6], contains six /J-caseins varying only in their level of phosphorylation [5]. Since such proteins have not yet been reported to occur in caprine casein, the same mechanism of multiple phosphorylation may not occur in the synthesis of caprine []-casein. An alternative possibility, however, is that a gene duplication and a subsequent mutation occurred for one of these proteins such that one casein contains five phosphate residues whilst the other contains six. ACKNOWLEDGEMENT The authors wish to thank Dr B. Ribadeau-Dumas for discussing his results prior to their publication. REFERENCES 1 Richardson, B. C., Creamer, L. K., Pearce, K. N. and Munford, R. E. (1974) J. Dairy Res., in the press 2 Waugh, D. F., Creamer, L. K., Slattery, C. W. and Dresdner, S. W. (1970) Biochemistry 9, 786795 3 Zittle, C. A. and Custer, J. H. (1966) J. Dairy Sci. 49, 788-791 4 O'Connor, P. and Fox, P. F. (1973) Neth. Milk Dairy J. 27, 199-217 5 Groves, M. L. and Gordon, W. G. (1970) Arch. Biochem. Biophys. 140, 47-51 6 Nagasawa, T., Kiyosawa, I. and Kurwahara, K. (1970) J. Dairy Sci. 53, 136-145
137 7 Richardson, B. C., Creamer, L. K. and Munford, R. E. (1973) Biochim. Biophys. Acta 310, 111117 8 Mills, O. E. and Creamer, L. K. (1973) N . Z . J . Dairy Sci. Tech. 8, 8-13 9 Spies, J. R. (1967) Anal. Chem. 39, 1412-1416 10 Beaven, G. H. and Holiday, E. R. (1952) Adv. Protein Chem. 7, 319 11 Fish, W. W., Mann, K. G. and Tanford, C. (1969) J. Biol. Chem. 244, 4989-4994 12 Bryce, C. F. A. and Crichton, R. R. (1971) J. Chromatogr. 63, 267-280 13 Thompson, M. P. (1971) in Milk Proteins (McKenzie, H. A., ed.), Vol. II, p. 157, Academic Press, New York 14 Mercier, J.-C., Grosclaude, F. and Ribadeau-Dumas, B. (1972) Milchwissenschaft 27, 402-408 15 Grosclaude, F., Mah6, M.-F. and Ribadeau-Dumas, B. (1973) Eur. J. Biochem. 40, 323-324
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 36797 COMPARATIVE MICELLE STRUCTURE III. THE ISOLATION AND CHEMICAL CHARACTERIZATION OF CAPRINE fl~-CASEIN AND flz-CASEIN B. C. RICHARDSON and L. K. CREAMER New Zealand Dairy Research Institute, Palmerston North (New Zealand)
(Received April 2nd, 1974)
SUMMARY Two pure caprine (goat) fl-caseins were purified by DEAE- and CM-cellulose column chromatography in buffers containing urea and 2-mercaptoethanol. The molecular weight of each fl-casein was found to be about 24 500 as determined by gel filtration on Sepharose 6B in guanidine. HC1, and each had the same amino acid composition; Asp9, Thr~2, Seras, Glu4a, Proa3, Gly6, Alas, Val2~, Met6, Ile9, Leuzo, Tyr4, Phe9, Hiss, Lys~2, Arga, Trp. Phosphate determinations showed, however, that fl~casein (the more mobile component on electrophoresis at pH 8.6) contained six residues per molecule, whilst fl2-casein contained only five. This difference may have been caused by a gene duplication and mutation. Both caseins are similar to bovine fl-casein in molecular weight and net charge.
The caseins of caprine milk are present in subunits, which coalesce with Ca 2+ and phosphate to form micelles. These subunits and micelles are very similar in mineral composition and in appearance to their bovine or ovine (sheep) counterparts [1]. fl-Casein constitutes approx. 30 ~o bovine casein [2], whereas fl-caseins are the major components of both caprine [1, 3, 4] and human [5, 6] casein. Consequently the flcaseins are likely to play a more important role in the stability of the caprine micelle than in the stability of bovine casein micelles. Caprine casein has been reported [3] to contain two major fl-casein components with electrophoretic mobilities in alkaline buffers similar to that of bovine fl-casein. In the present investigation the purification and chemical characterization of these two caprine fl-caseins has been achieved. Whole caprine casein was isolated and then separated into individual caseins using the chromatographic procedures previously described [7]. Each fl-casein fraction was rechromatographed on Whatman CM-32 cellulose at pH 4.0 in a sodium formate buffer (0.01 M) which contained urea and 2-mercaptoethanol. A gradient 0-0.15 M NaC1 was used to elute the fl-casein. Bovine fl-casein B was isolated using the procedure of Mills and Creamer [8]. The procedures used for gel electrophoresis at pH 8.6 [7] and in a Tris-borate buffer containing Mg 2+ [2], amino acid analysis [7], phosphorus estimation [7], extinction coefficient determination [7], tryptophan analysis
134 (procedure W) [9], tyrosine estimation [10], and molecular weight determination [11, 12] followed published methods. Examination of milk samples from 25 goats by gel electrophoresis showed that they all contained two bands of about equal intensity with similar mobility to bovine {4-casein. Each of these was readily isolated from the whole casein (ref. 7, Fig. 1) with the more mobile component on alkaline gel electrophoresis (/:~l-casein) lzeing eluted less readily from the DEAE-cellulose column than the other component (/42-casein). Gel electrophoresis at pH 4 reversed the order of mobility so that/~'2-casein became more mobile than {/l-casein. in the presence of Mg z+ in the electrophoresis buffer the mobilities of {/~- and /4z-casein decrease and the two components merge (Fig. 1). This suggested that {/~and {/z-casein differed in the number of phosphate groups that each contained, since Mg 2+ associates strongly with the phosphate groups of the caseins, and largely neutralizes their charge. This conclusion is supported by the results t¥om acid gel electrophoresis which show that /3l-casein carries more negative charge than [42casein at both pH 4 and pH 8.6. At these pH values the phosphate groups would carry single and double negative charges, respectively.
Fig. I. Gel electrophoresis of bovine and caprine caseins in alkaline buffer and in the presence of Mg2+. Samples: 1, bovine skim milk; 2, caprine whole casein; 3, {/~-casein;4, /~-casein. The molecular weights of caprine ¢!tl- and {4z-casein and bovine {4-casein were determined on a calibrated column of Sepharose 6B in 6 M guanidine.HCI solution (Table I). In addition, a sample containing both {4x- and {/z-casein was chromatographed on the molecular weight column and the fractions collected as before. After weighing, each fraction was dialysed to remove salts and after gel electrophoresis and densitometry the ratio of {/1- to {42-casein was found to be constant, across the protein peak. This showed that the molecular weights of these two proteins were not distinguishable within the limits of this technique (±5C0). The amino acid compositions of caprine {/~- and {/z-casein and bovine {4-casein B were determined using acid-hydrolysed samples [7] (Table I). Tryptophan contents of {/1- and {/z-casein were 0.62 and 0.67 ~ , respectively. Inorganic phosphorus was determined for each {/-casein in the acid-hydrolysed samples, the average being 0.78 and 0.67 ~ for/3~- and /3z-casein, respectively. The absorbance of 1 cm solutions of
* S o . text.
Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine* Phenylalanine Histidine Lysine Arginine Tryptophan * Phosphorus residues Molecular weight (from Sepharose 6B column) Molecular weight (from amino acid composition) No. of residues
Residue
25 500
1.31 1.36 0.14 0.18 0.26 0.14 0.17 0.49 0.07 0.15 0.08 0.40 0.07
0.22
23 500
9.1 11.6 14.7 43.0 32.6 5.6 4.7 21.3 6.3 9.0 20.5 3.4 9.0 5.2 11.7 3.1 0.8 5.0
Mean No. residues
9.0 11.5 14.8 43.2 32.8 5.6 4.7 21.0 5.8 8.6 20.2 3.6 8.8 5.1 12.1 3.3 0. 7 6. l
fl2-casein
Mean No. residues
S.D.
ill-casein
Caprine
0.89 1.13 0.20 0.16 0.52 0.24 0.31 0.47 0.06 0.14 0.14 0.20 0.15
0.12
S.D.
25 100
---
9.3 8.9 15.2 39.8 35.0 5.1 5.7 18.4 5.6 9.0 21.8 4.0 8.8 6.1 11.0 4.4
residues
Bovine fl-casein B Mean No.
24 439 213
9 12 15 43 33 6 5 21 6 9 20 4 9 5 12 3 1 6
ill-casein Probable No. residues
Caprine
AMINO ACID COMPOSITION OF fl-CASEINS (RESIDUES A M I N O ACID/MOLE PROTEIN)
TABLE I
23 982 209
I
5
1
5
24 360 213
9 9 16 39 35 5 5 19 6 10 22 4 9 5 11 4
9 12 15 43 33 6 5 21 6 9 20 4 9 5 12 3
fl2-casein Probable No. residues
23 712-24 108 210
0-5
1
11 9 9 39 39 3 7 19 3 13 26 7 5 5 11 3
Bovine Human fl-casein A2 fl-casein (refs 14 (ref, 5) and 15)
136 /4~- and/J2-casein 1 ')£, w/v (pH 7.0) at 280 nm (corrected for Rayleigh scattering) were 4.2 and 4.5, respectively. These are similar to the literature values [13] for bovine/Jcasein (4.5-4.7) suggesting that the caprine/J-caseins and bovine/Z-casein contain the same number of tyrosine and tryptophan residues. The absorbance in 0.1 M N a O H solution showed the ratio of tyrosine to tryptophan to be 3.8 and 3.7 for {;~- and/Jzcasein, respectively [10]. Consequently, tryptophan was taken as one residue per molecule and tyrosine as four per molecule, despite the lower values obtained from the amino acid analysis (Table I). The mobility on polyacrylamide gels of bovine/J-casein B is similar to that of caprine /~2-casein, the molecular weight of the proteins is similar and laence the net charge is probably similar at both pH 4 and pH 8.6. The number of histidine residues is the same for the two proteins (5) whilst the number of lysine plus arginine residues is one more for the B variant of bovine casein [14]. Consequently, the flz-casein (or fl~-casein) should have one more carboxyl residue than bovine/Z-casein B, i.e. 23 aspartic acid and glutamic acid residues and 29 asparagine plus glutamine residues (the bovine casein contains 26 amide groups [15] and 22 carboxyl groups). As the only difference in charged residues between t h e / ~ - and [42caseins is the number of phosphate groups, the relative number of carboxyl and amide groups would be the same for the two caprine caseins. The amino acid composition of the caprine caseins is very close to that published for the bovine/J-casein Az (Table I). The sum of lysine and arginine residues is constant at 15, and the sum of leucine, isoleucine and valine residues is constant at 5 I. The only major difference between the two caprine/4-caseins is the different phosphate level with [J~- and/J2-caseins containing six and five phosphates per molecule, respectively. The different levels of phosphorylation found in bovine/J-casein is known to be the result of genetic variation in the sequence [14]. in contrast, human/4-casein, similar in composition and properties to its bovine counterpart [6], contains six /J-caseins varying only in their level of phosphorylation [5]. Since such proteins have not yet been reported to occur in caprine casein, the same mechanism of multiple phosphorylation may not occur in the synthesis of caprine []-casein. An alternative possibility, however, is that a gene duplication and a subsequent mutation occurred for one of these proteins such that one casein contains five phosphate residues whilst the other contains six. ACKNOWLEDGEMENT The authors wish to thank Dr B. Ribadeau-Dumas for discussing his results prior to their publication. REFERENCES 1 Richardson, B. C., Creamer, L. K., Pearce, K. N. and Munford, R. E. (1974) J. Dairy Res., in the press 2 Waugh, D. F., Creamer, L. K., Slattery, C. W. and Dresdner, S. W. (1970) Biochemistry 9, 786795 3 Zittle, C. A. and Custer, J. H. (1966) J. Dairy Sci. 49, 788-791 4 O'Connor, P. and Fox, P. F. (1973) Neth. Milk Dairy J. 27, 199-217 5 Groves, M. L. and Gordon, W. G. (1970) Arch. Biochem. Biophys. 140, 47-51 6 Nagasawa, T., Kiyosawa, I. and Kurwahara, K. (1970) J. Dairy Sci. 53, 136-145
137 7 Richardson, B. C., Creamer, L. K. and Munford, R. E. (1973) Biochim. Biophys. Acta 310, 111117 8 Mills, O. E. and Creamer, L. K. (1973) N . Z . J . Dairy Sci. Tech. 8, 8-13 9 Spies, J. R. (1967) Anal. Chem. 39, 1412-1416 10 Beaven, G. H. and Holiday, E. R. (1952) Adv. Protein Chem. 7, 319 11 Fish, W. W., Mann, K. G. and Tanford, C. (1969) J. Biol. Chem. 244, 4989-4994 12 Bryce, C. F. A. and Crichton, R. R. (1971) J. Chromatogr. 63, 267-280 13 Thompson, M. P. (1971) in Milk Proteins (McKenzie, H. A., ed.), Vol. II, p. 157, Academic Press, New York 14 Mercier, J.-C., Grosclaude, F. and Ribadeau-Dumas, B. (1972) Milchwissenschaft 27, 402-408 15 Grosclaude, F., Mah6, M.-F. and Ribadeau-Dumas, B. (1973) Eur. J. Biochem. 40, 323-324