The Composition, Structure and Origin of Proteose-peptone Component 8F of Bovine Milk

Transcription

1 Eur. J. Biochem. YO, (1978) The Composition, Structure and Origin of Proteose-peptone Component 8F of Bovine Milk Anthony T. ANDREWS Chemistry Department, National Institute for Research in Dairying, Shinfield, Reading (Received March 28, 1978) Proteose-peptone component 8F (or %-fast ) has been prepared from bovine milk. Sedimentation equilibrium analysis, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate and gel filtration in urea-containing buffers all gave molecular weight values between 33 and 39. The N-terminal sequence was found to be Arg-Glu- by dansylation and Edman degradation. Hydrazinolysis released lysine from the C-terminus. A mixture of carboxypeptidases A and B showed that the C-terminal sequence was -Thr-(Arg,Ile,Asn)-Lys. The phosphate content was 3.8 mol/mol and was completely released by a short alkaline hydrolysis indicating linkage to serine. This and all other aspects of the composition were entirely consistent with the identification of this proteosepeptone as residues 1-28 of the p-casein molecule. This identity was confirmed by a peptide mapping procedure. Thus proteose-peptone component 8F represents the N-terminal fragment when the yl -caseins are formed by proteolysis of B-casein. When milk is heated for several minutes at temperatures close to boiling point the major whey proteins, and undoubtedly many minor constituents as well, are denatured and on subsequent adjustment of the ph to 4.6 are precipitated together with the caseins. After their removal, other protein components can be precipitated by the addition of protein-precipitating agents such as ammonium sulphate or trichloroacetic acid ; they represent the proteose-peptone fraction, which therefore consists of all the proteins which are not rendered insoluble at ph 4.6 by the heat treatment. Since it is merely defined in such simple operational terms, the proteose-peptone fraction contains a great many, largely undefined, components; as it is largely free of the major milk proteins, it provides a good starting point in a search for the breakdown fragments of these major proteins. We have recently been examining proteolytic activity in bovine milk and in the preceding paper [l] we have shown that what has been classically referred to [2] as proteose-peptone component 5 does in fact represent the N-terminal parts of the B-casein molecule corresponding to a proteolytic cleavage which leads to the formation of the yz and y3-caseins as the C- terminal segments. The object of this paper is to show that proteose-peptone component 8F (or %fast ) Enzymes. Carboxypeptidase A (EC ); carboxypeptidase B (EC ). [2,3] represents the N-terminal28 amino acid residues which match formation of yl-casein (the C-terminal residues 29-29) from p-casein. This also provides further evidence for proteolysis as the most probable mechanism for the formation of the y-caseins. MATERIALS AND METHODS Preparation of Proteose-peptone Component 8F A crude mixture of proteose-peptone was prepared and fractionated by gel filtration on Sephadex G-75 as described in the preceding paper [I]. Those fractions which were found to be rich in component 8F were pooled and the protein precipitated by addition of trichloroacetic acid to 12.5 %. The precipitate was collected by centrifugation (2 x g for 15 min), dissolved in 2 ml of.2 M sodium phosphate buffer ph 6.5 containing 8 M urea and further purified by gel filtration on a column (22 x 575 mm) of Sephadex G-5 in the phosphate/urea buffer. Fractions (3 ml) were monitored for protein by absorbance at 235 nm and also by examining 1-p1 portions by polyacrylamide gel electrophoresis [4]. Fractions containing component 8F were pooled, dialysed overnight at 4 C versus HzO to remove the bulk of the urea and the protein precipitated by addition of trichloroacetic acid to 12.5%. The precipitate was washed once by

2 68 Proteose-peptone Component 8F suspension and centrifugation in 12.5 % trichloroacetic acid and twice with acetone and then air-dried. Anulytical Methods The molecular weight of component 8F was determined by low-speed equilibrium ultracentrifugation [5] using a value for the partial specific volume (UZ) calculated from the composition [6,7] and also by gel filtration on a Sephadex G-75 column in.2 M sodium phosphate buffer ph 6.5 containing 8 M urea as described previously [I]. Standard proteins (with molecular weights in parentheses) used for column calibration included bovine serum albumin (67), p-lactoglobulin (1 8 ), a-lactalbumin (14 3), cytochrome c (124), lima bean trypsin inhibitor (9), glucagon (35) and bacitracin (147). The last four of these were also used as molecular weight standards in polyacrylamide gel electrophoresis in the presence of dodecyl sulphate [8]. Amino acid analysis, phosphate content, dansylation and Edman degradation of the N-terminal amino acid sequence, hydrazinolysis for identification of the C-terminal amino acid and peptide mapping (for which 3 mg of fl-casein or 4 mg of component 8F were used) were all performed by methods described or referred to previously [l]. The C-terminal sequence was confirmed by treatment with a mixture of carboxypeptidase A and carboxypeptidase B. For this, component 8F in.2 M N-ethylmorpholine acetate buffer ph 8. was first treated at 37 "C for 4 min with carboxypeptidase B (Sigma Chemical Co. Ltd, 185 units/ mg) at a substrate/enzyme ratio of 5/1 and then carboxypeptidase A (Sigma, containing 2 mg protein/ ml and 5 units/mg) solution was added (5 units/mg of component 8F) and incubation continued. Amino acids released were measured with an LKB Biochrome model 412 amino acid analyser. RESULTS Isolation of Component 8F The results of the preparation of a crude mixture of proteose-peptone components and the initial gel filtration step on Sephadex G-75 in.2 M sodium phosphate buffer ph 6.5 are described in the preceding paper [l]. Component 8F was found in lowmolecular-weight fractions indicated by the pool F shown in Fig.1 of [l] and the polyacrylamide gel electrophoresis patterns of relevant fractions are shown in Fig.2 of [l]. Component 8F represented between 8 % and 12 % of the total proteose-peptone fraction. Pool F material was rechromatographed on the Sephadex G-5 column in.2 M sodium phosphate ph 6.5 plus 8 M urea buffer (Fig. 1). Fractions shown.25 - / r I 2! Fraction number Fig. 1. Rechromatography of component RFon u column (22 x 575 mm) of Sephadex G-5 in.2 M sodium phosphate huj'er ph 6.5. Fractions of' 3 ml were collected at a flow rate of about 1 ml/h. Fractions shown by polyacrylamide gel electrophoresis to contain component 8F were pooled, as indicated by the bar by gel electrophoresis to contain component 8F were pooled as indicated by the bar, and yielded 6.6 mg from 15.5 mg of pool F material. Molecular Weight As was the case with proteose-peptone component 5, component 8F also appeared to have an anomalously high molecular weight when studied by gel filtration in aqueous buffers, probably for similar reasons [l]. Values of 1-13 were obtained on a Sephadex G-5 column [l] in.2 M NH4HC3 buffers. Gel filtration in the presence of urea [l], however, gave a molecular weight of 33 t 2 for component 8F. This was supported by polyacrylamide gel electrophoresis in the presence of dodecyl sulphate from which a value of 39 & 7 was obtained. Ultracentrifugal analysis in the non-dissociating.2 M sodium phosphate buffer ph 6.5 gave a mean value of 36 i 4, using u2 =.693 ml/g, as calculated from the composition. Composition of Component 8F The overall composition (corrected for 14.9 % moisture content) is shown in Table 1. Component 8F was devoid of all sulphur-containing amino acids and also all aromatic amino acids. Because of this, the ultraviolet spectrum showed no maximum in the region of nm and no minimum between 25 and 26nm. Above 25nm the absorption was very low (E = 26 M-' cm-' at 28 nm). The phosphorus content was 3.52%, equivalent to nearly

3 ~~ ~ ~. ~~ ~~~ ~ A. T. Andrews 69 Table 1. The composition ofproteose-peptone component 8F Samples of component 8F (approximately.15 pmol) were hydrolysed under Nz in 1.O-ml portions of 6 M HC1 containing.68 % thioglycollic acid for 24, 48 and 72 h at 11 "C. Recoveries were within the range %. Values for threonine, serine and ammonia are extrapolated ones (ammonia in /%casein A' comes from amide groupings of asparagine). Phosphate was determined colorimetrically [9] Amino acid Amount Nearest j-casein A* integer residues 1-28 mo1/3469 g Aspartic acid Threonine 1.oo 1 1 Serine Glutamic acid 7.2 I I Proline Glycine A 1 an i n e.39 Valine Methionine.25 Isoleucine Leu c i n e Tyrosine trace Phenylalanine.33 Histidine.27 Lysine Arginine NHs Phosphate mol/mol (Table 1) and it was found to be completely released in an inorganic form by treatment with.2 M NaOH at 1 "C for 2 h which indicated that it was probably linked to serine. Using the anthrone procedure only small amounts of hexose were measured (less than.7 mol/mol) and no glucosamine or galactosamine was detectable with the amino acid analyser, so it was concluded that component 8F was not a glycoprotein. Terminal Amino Acid Analyses and Peptide Maps Arginine was found to be the N-terminal amino acid by dansylation, the dansyl derivative being obtained in good yield and identified unambiguously [lo]. After one cycle of Edman degradation and further dansylation only dansyl-glutamic acid was identified, although yields were poor. No further dansyl derivatives were identifiable after two further reaction cycles, so these results suggested arginine linked to glutamic acid as the N-terminal amino acid sequence. Hydrazinolysis released lysine from the C-terminal position as the only free amino acid in quite good yield (.625 mol/mol). The C-terminal location of lysine was confirmed by treatment with carboxypeptidase B which released lysine most rapidly, followed by much smaller amounts of asparagine, isoleucine Time (h) Fig. 2. Time courses of the release of C-terminal amino acids by a mixture of carboxypeptidases A and B establishing the C-terminul seq~en~e-thr-(arg,ile,asn)-lys. Release of lyshe (-) asparagine (O----D), isoleucine (A..... A), arginine (-) and threonine ( x ~ x ). Arrow indicates point of addition of carboxypeptidase A and arginine. When coupled with carboxypeptidase A, as described in Materials and Methods, amino acids were released as shown in Fig. 2. After the liberation of lysine in accordance with its terminal location, asparagine, isoleucine and arginine were released at rather similar rates so that it was not possible to deduce an unequivocal sequence. The quantity of asparagine and threonine was rather uncertain since they were not well resolved on the amino acid analyser with the standard buffer system employed, but it appeared that after about 2-h incubation the amount of isoleucine released was greater than that of asparagine, while the amount of arginine was less. Also after 2 h, some threonine and traces of several other amino acids became evident. Thus from Fig. 2 one may conclude that the most probable C-terminal sequence for component 8F was -Thr-(Arg,Ile,Asn)-Lys. After about 3-h incubation the amount of isoleucine released exceeded that of lysine and this would be best explained by the presence of a further isoleucine residue beyond the above sequence. The data in Fig. 2 is thus entirely consistent with residues ofp-casein in which the sequence -1le-Thr-Arg-Ile-Asn-Lys- occurs [ Peptide maps of trypsin digests of /?-casein and of component 8F are shown in Fig.3. Component 8F gave only three major peptides and a number of other faint spots which were probably due to contaminants or may be derived from trypsin autodigestion. Comparison with Fig.3A showed that all the peptides from component 8F corresponded to peptides which were also produced by trypsin digestion of p-casein.

4 7 Proteose-peptone Component 8F A --- L-2 B m g Q O + l - Electrophoresis - Electrophoresis Fig. 3. Pepiide maps on thin-layer crllulose[ikrtrs of tr yptic digests of (A) [krsein rmd (B) component 8F. Plates were prepared by electrophoresis in 1 M acetic acid adjusted to ph 3.6 with pyridine in the horizontal dimension and chromatography in the organic phase of butan-1-1, acetic acid/hzo (4/1/5, by vol.) in the vertical dimension Arg-Glu-Leu-Glu-Glu-Leu-Asn-Val-Pro-Gly-Glu-lle-Val-Glu-5erP-Leu SerP-5erP-SerP-Glu-Glu-Ser-l le-thr-arg-l le-asn-lys-lys-l le..... Fig.4. Partial amino acid st yurnce of the N-twminal region of b- cnsein [Ill. The arrow marks the position of cleavage when y ~ - casein (residues 29-29) is formed from the C-terminal portion and component XF from the N-terminal fragment This was confirmed by applying component 8F digest and fl-casein digest to the same plate and this indicated that component 8F represented part of the 8-casein molecule. DISCUSSION The partial amino acid sequence of the N-terminal region of/?-casein is shown in Fig. 4. The data presented in this paper demonstrates that component 8F represents the N-terminal segment (residues 1-28), matching the formation of the yl-caseins from the C-terminal residues The only discrepancies in the composition of component 8F compared to that expected for residues 1-28 are the slightly low values (Table 1) for serine and isoleucine; however, as has been discussed previously [l 1, the serine is extensively phosphorylated which in our experience leads to low recoveries on acid hydrolysis ; also isoleucine, together with leucine and valine, is sometimes rather resistant to hydrolysis. Recoveries of all three of these amino acids are in fact a little low, probably for this reason, although the discrepancies are quite small. There are no genetic substitutions in this part of the /?-casein molecule so that all genetic variants of fl-casein will give the same component 8F material. The original classification of the proteose-peptones into the major components 3, 5 and 8 was based on the free-boundary electrophoresis [2] of whey in which eight identifiable peaks were seen. The present demonstration that components 5 [l] and 8F both represent part of the &casein molecule and are most probably formed from /?-casein by proteolytic breakdown, calls into question the nomenclature of these components and also that of the y-caseins. Indeed, the original classification of the proteose-peptones has now become completely untenable since we have detected at least 25 bands on polyacrylamide electrophoresis gels of a crude proteose-peptone mixture. The classical definition of the caseins as phosphoproteins with a high proline content insoluble at ph 4.6, while clearly only a convenient operational definition, also is now becoming somewhat strained, since components 5 and 8 are both phosphoproteins containing proline, while yz-casein and y3-casein are not phosphoproteins. It seems desirable that minor proteins (including the y-caseins) which are breakdown products of larger casein molecules should not be referred to as caseins. However, since rapid progress on the identification of naturally occurring fragments of the major milk proteins can be expected within the next few years, it is probably best to retain existing nomenclature for the time being.

5 A. T. Andrews 71 Kaminogawa and colleagues [12,13] have investigated the properties of a protease in bovine milk and have compared it to plasmin [14]. This has led Eigel to study the effects of plasmin in vitro on p-casein [15] and on a,l-casein and x-casein [16]. He found [15] that plasmin digestion of p-casein resulted in the formation of a mixture of yl, y2 and y3-caseins, a finding that we have confirmed. Plasmin has a trypsin-like specificity and splits polypeptide chains immediately after lysine or arginine residues. Since p-casein A2 contains 11 lysine and four arginine residues there are therefore 15 potential cleavage sites. It was found [IS] that the y-caseins are rapidly formed and then subsequently are further broken down rather more slowly ; thus the y-caseins represent a relatively stable intermediate step in the breakdown of p-casein to small peptides by plasmin. As expected, both components 5 and 8F possess the required C-terminal lysine and so also may be regarded as stable intermediates in such a proteolytic breakdown. The fact that these proteose-peptones and the y-caseins are found in quite large amounts in milk is consistent with their rapid formation by low levels of protease and only a slow rate of further breakdown. Inspection of the amino acid sequence of p-casein (e.g. Fig.7 of [l]) shows that -Lys-Lys- is present at position and -Lys-His-Lys- at It appears that these two highly basic regions represent preferred sites of hydrolysis by plasmin or a plasmin-like milk protease. Some aspects of the chemistry of milk proteins including their primary structures have recently been reviewed [17] and it can be seen that similar basic regions occur in the polypeptide chains of other major milk proteins. The sequence -Lys-Lys-Tyr-Lys- occurs at position of asl-casein, with Arg-Pro-Lys- His- at position 1-4 and Lys-His at 7-8 and In x-casein, Lys-Lys is present at with Arg- His at The sequence Lys-Lys also occurs at positions 69-7 and 1-11 of p-lactoglobulin and at of a-lactalbumin. It is possible that such points may represent the most preferred cleavage sites for these proteins also, although confirmation of this must await the necessary experimental evidence. In this context it is worth noting that the plasmin digestion of a,l-casein results [16] in the formation of a small number of well-defined electrophoretic bands, for two ofwhich molecular weights of 12 3 and 13 were reported. Calculations based on the sequence of scsl-casein B show that if the molecule was split initially between the two lysine residues at positions 12 and 13 then the N-terminal portion would have a molecular weight of and the C-terminal part While Eigel [15,16] also reports that plasmin hydrolyses caseins in the order p > u,1 > x, these experiments were carried out in true solution in borate buffers of ph 8.4. In milk, where most of the casein is present in a micellar form it is probable that the kinetics of the proteolysis will be different, although being also hydrophilic areas the basic regions of the casein molecules are likely to be exposed to solvent and thus relatively easily accessible to proteolytic enzymes. Such an exposed location may further enhance the preference for attack at these sites on the polypeptide chains relative to other less-favoured susceptible bonds. The comparatively large amounts of components 5 and 8F and the y-caseins found in milk indicate that b-casein is broken down more rapidly than a,l-casein or x-casein and this may be at least partly explained by the fact that a relatively larger proportion of the p-casein exists in a nonmicellar soluble form. The author thanks Mrs D. J. Knight for performing the amino acid analyses, Mrs V. A. Hill for the ultracentrifuge runs and Mr M. D. Taylor for his excellent technical assistance. REFERENCES 1. Andrews, A. T. (1978) Eur. J. Biochem. 9, Larson. B. L. & Rolleri, G. D. (1955) J. Dairy Sci. 38, Kolar, C. W. & Brunner, J. R. (197) J. Dairy Sci. 53, Hillier, R. M. (1976) J. Dairy Res. 43, Van Holde, K. E. (1967) Fractions, no. 1, Beckman Instruments Inc., Palo Alto. 6. Charlewood, P. A. (1967) J. Am. Chem. Soc. 79, McMeekin, T. L., Groves, M. L. & Hipp, N..I. (1949) J. Am. Chon. Soc. 71, Swank, K. T. & Munkres, K. D. (1971) Anal. Biod7m7. 39, Chen, P. S., Toribara, T. Y. & Warner, H. (1956) Anal. Chem. 28, Seiler, N. (197) Mrthod.s Biochem. Ancil Ribadeau-Dumas, B., Rrignon, G., Groscleude, F. & Mercier, J.-C. (1972) Eur. J. Biochem. 25, Kaminogawa, S., Sato, F. & Yamaguchi, K. (1971) Agr. Bid. Chem. 35, Kaniinogawa, S. & Yamauchi, K. (1972) Agr. Bid. Chrm. 36, Kaminogawa, S., Mizobuchi, H. & Yamauchi, K. (1972) Agr. Biol. Chem. 3fi* Eigel, W. N. (1977) Int..I. Biochem. 8, Eigel. W. N. (1977) J. Dairy Sci. 6, Whitney, R. McL., Brunner, J. R., Ebner, K. E., Farrcll, H. M., Josephson, R. V., Morr, C. V. &Swaisgood, H. E. (1976) J. Dairy Sci. 59, A. T. Andrews, National Institute for Research in Dairying, Shinfield, Reading, Great Britain, RG2 9AT