Hydrolysis of porcine b-casein by bovine plasmin and bovine chymosin

Z Lebensm Unters Forsch A (1999) 208 : 83 89 Q Springer-Verlag 1999 ORIGINAL PAPER Daniel P. Gallagher 7 Tanoj K. Singh Daniel M. Mulvihill Hydrolysis of porcine b-casein by bovine plasmin and bovine chymosin Received: 9 February 1998 / Revised version: 2 June 1998 Abstract The action of bovine chymosin and bovine plasmin on porcine b-casein was studied and compared with their effect on bovine b-casein in an attempt to elucidate the similarities in specificity of these enzymes on porcine and bovine b-caseins. Bovine plasmin rapidly hydrolysed porcine b-casein at Lys 106 -Arg 107, Lys 49 -Ile 50 and Lys 168 -Val 169 to yield g-caseins. Several peptides that had the same N-terminal amino acid sequence as porcine b-casein were also produced. Plasmin hydrolysis sites determined by separating the ph 4.6-soluble peptides formed on hydrolysis were Lys 33 - Leu 34, Lys 49 -Ile 50, Lys 95 -Asp 96, Lys 98 -Ala 99, Lys 106 - Arg 107, Lys 108 -Gly 109 and Lys 168 -Val 169. Porcine b-casein, like bovine b-casein, was hydrolysed by bovine chymosin into three distinct electrophoretic bands designated porcine b-i-, b-ii- and b-iii-casein, all of which had the N-terminal sequence of porcine b-casein. Two initial ph 4.6-soluble peptides formed on hydrolysis of porcine b-casein by chymosin had N-terminal amino acid sequences commencing at porcine b-casein amino acid 197, suggesting that porcine b-i-casein corresponds to fragment 1 196, while the sequences of two other small peptides commenced at amino acids 1 and 207. The C-terminal cleavage site(s) leading to formation of porcine b-ii and b-iii casein were not determined. Key words Porcine b-casein 7 Bovine b-casein 7 Hydrolysts 7 Bovine plasmin 7 Bovine chymosin Introduction D.P. Gallagher 7 T.K. Singh 7 D.M. Mulvihill (Y) Department of Food Chemistry, University College, Cork, Ireland The first report on the isolation of porcine b-casein was by Woychik and Wondolowski [1] but the protein was not characterised by them. Mulvihill and Fox [2] isolated porcine b-casein and reported that it contained 218 amino acids, had eight phosphorus residues per mole and a molecular weight of 24 900 Da. Bovine b- casein contains 209 amino acids, has five phosphorus residues per mole and has a molecular weight of 24 000 Da [3]. The full amino acid sequence of porcine b-casein has been derived from its cdna [4]; these authors reported that porcine b-casein contains 217 amino acid residues, has six potential phosphorylation sites and a molecular weight, in the non-phosphorylated form, of 24 397 Da. In agreement with Mulvihill and Fox [2], Alexander and Beattie [4] reported that porcine b-casein contains higher levels of Ser, Ala and Leu than its bovine counterpart, but they also reported higher levels of Lys and an absence of Trp relative to bovine b-casein. Porcine and bovine b-caseins contain similar levels of hydrophobic amino acids (46 and 44%, respectively) and Pro, and both proteins are devoid of Cys. Bovine b-casein in solution is hydrolysed sequentially by bovine chymosin at bonds Leu 192 -Tyr 193, Ala 189 - Phe 190, Leu 163 -Ser 164 and Leu 139 -Leu 140 to yield the peptides b-i 1, b-i 11, b-ii and b-iii casein, respectively; bonds Leu 165 -Ser 166 and Gln 167 -Ser 168 may also be hydrolysed to yield peptides indistinguishable electrophoretically from b-ii caseins [5, 6]. Bovine b-casein is hydrolysed by plasmin at bonds Lys 28 -Lys 29, Lys 105 - His 106 and Lys 107 -Glu 108 to yield g-caseins [g 1 (b-casein f29 209), g 2 (b-casein f106 209) and g 3 (b-casein f108 209)] and protease peptones (PP) [PP-5 (b-casein f1 105/107), PP-8 slow (b-casein f 29 105/107) and PP-8 fast (b-casein f1 28)] [7]. Visser et al. [8] identified sixteen peptides (1 25, 1 28, 2 25, 2 28, 29 48, 33 48, 33 97/99, 49 97, 49 99, 98 105, 100 105, 106 209, 114 169, 114 209, 117 209, 184 209) in plasmin hydrolysates of bovine b-casein; most Lys-X and Arg-X bonds were hydrolysed. The specificity of proteolytic enzymes on porcine b- casein has received little attention. In the present study, the action of bovine chymosin and bovine plasmin on porcine b-casein was studied, and compared with the

84 effect of these enzymes on bovine b-casein in an attempt to elucidate the similarities in specificity of these enzymes on porcine and bovine b-caseins. Materials and methods Preparation of porcine and bovine b-casein. Bulk porcine milk (from up to four sows) was obtained from the National Dairy Products Research Centre, Moorepark, Fermoy, Co. Cork, Ireland. The sows, between 8 and 25 days post-partum, were injected intraveneously with 10 IU of oxytocin to induce milk let-down; the milk was ejected manually. The milk was defatted by centrifugation at 2 500 g at 4 7C for 30 min and sodium azide (0.05%) was added as a preservative before storage at 4 7C. Bovine skim milk was obtained from the experimental creamery at University College, Cork. Porcine and bovine sodium caseinates were prepared essentially as described by Fox and Hoynes [9] except that the casein was re-dissolved and re-precipitated twice, before being dialysed against several changes of distilled water at 4 7C for 24 h, and freeze-dried. Porcine casein was fractionated essentially as described by Erhardt [10], but using a 0 0.4 M salt gradient. A sample (0.2 g) of the caseinate was dissolved in F8 ml of 10 mm Tris-imidazole buffer, ph 7.0, containing 3.3 mol/l urea and 0.01 mol/l 2-mercaptoethanol and chromatographed on a column of DEAE-cellulose (80!2.5 cm, DEAE-52, Whatman, Maidstone, UK) which was pre-equilibrated with the same buffer. Proteins were eluted from the ion-exchanger with a linear (0 0.4 M) NaCl gradient at a flow rate of 48 ml/h and the eluate monitored at 280 nm. The fractions corresponding to b-casein were pooled and dialysed exhaustively against several changes of distilled water at 4 7C for 24 h, and freeze-dried. A sample (0.5 g) of bovine sodium caseinate was chromatographed on a column of DEAE-cellulose (90!3.5 cm, DEAE-52, Whatman) equilibrated with 10 mm imidazole buffer, ph 7.0, containing 4.5 M urea and 0.1% 2-mercaptoethanol. Proteins were eluted with a linear NaCl (0 0.5 M) gradient at a flow rate of 48 ml/h and the eluate monitored at 280 nm. The fractions corresponding to b-casein were pooled and dialysed exhaustively against several changes of distilled water at 4 7C for 24 h, and freeze-dried. Hydrolysis of porcine and bovine b-caseins with bovine chymosin and plasmin. Bovine recombinant chymosin expressed in Kluveromyces marxianus var. lactis (Maxiren) was obtained from Gist Brocades, Delft, Netherlands. Prior to use, the enzyme preparation was dialysed against two 20-volume changes of distilled water at 4 7C for 24 h and freeze-dried. The freeze-dried powder was redispersed in 100 mm potassium phosphate buffer, ph 6.5. This solution had an activity of F70 chymosin units/ml (1 unit is the activity necessary to coagulate 10 ml of bovine milk, ph 6.5, in 100 s at 30 7C). Porcine or bovine b-casein (2 mg/ml) was dissolved in 0.2 M phosphate buffer, ph 6.5, and heated to 35 7C in a water bath; chymosin was then added (0.35 units/ml), the mixture maintained at 35 7Cand samples taken periodically and chymosin inactivated by heating in boiling water for 2 min. Bovine plasmin was obtained from Sigma (St. Louis, Mo., USA). Porcine or bovine b-casein (2 mg/ml) was dissolved in 50 mm ammonium bicarbonate buffer, ph 8.4, containing 0.05% sodium azide; 0.02 units (1 unit will produce a DA 275 nm of 1.0 from a-casein in 20 min at ph 7.5 at 35 7C when measuring perchloric acid-soluble products in a volume of 5 ml) of bovine plasmin were then added per ml and the solution incubated at 35 7C. Samples were taken periodically and the plasmin inactivated by heating in a boiling water bath for 2 min. Polyacrylamide gel electrophoresis (PAGE). PAGE was performed according to the method of Andrews [11]. The gels were stained using Coomassie Brilliant Blue G-250 [12]. Reversed-phase high performance liquid chromatography (RP- HPLC). Aliquots of hydrolysate were adjusted to ph 4.6, centrifuged at 10 000 g (Microcentaur centrifuge, Sanyo MSE Instruments, Leicester, UK) for 20 min. The supernatant was analysed by RP-HPLC using an automated Waters HPLC system (consisting of model 426 pump, model 600 S system controller, model 717 plus auto sampler; Millipore, Milford, Mass., USA) fitted with a Fig. 1 Urea-PAGE of porcine and bovine b-caseins following hydrolysis by bovine plasmin. Lanes 1 and 11 porcine sodium caseinate. Lanes 2 10 porcine b-casein following hydrolysis by bovine plasmin for 1, 2, 5, 10, 15, 30, 60, 120 and 180 min, respectively. The bands labelled a e were electroblotted and their N-terminal amino acid sequences determined (see Table 1). Lane 12 bovine sodium caseinate. Lanes 13 15 bovine b-casein following hydrolysis by bovine plasmin for 0, 30 and 180 min, respectively

85 Table 1 Identity of the peptides produced from porcine b-casein by bovine plasmin at ph 8.4 Electrophoretic HPLC N-terminal sequence Mass of peptide Position in porcine band a peak no. b b-casein sequence Exp. c Theor. d a Arg-Lys-Gly-Met-Pro P 107? b Arg-Lys-Gly-Met-Pro P 107? c Ile-His-Gln-Phe-Pro P 50? d Val-Leu-Pro-Val-Pro P 169? e Arg-Ala-Lys-Glu-Glu P 1? 1 Leu-Lys-Arg-Glu-Glu 2062.7 2058.2 34 49 2 Ala-Lys-Glu-Thr-Ile 1171.5 1169.4 99 108 885.7 885.7 99 106 3 Asp-Ser-Lys-Ala-Lys ND f 96? 4 Arg-Ala-Lys-Glu-Glu ND f 1? 5 Ile-His-Gln-Phe-Pro ND f 50? 6 Ile-His-Gln-Phe-Pro 6408.6 6417.3 50 106 7 Ile-His-Gln-Phe-Pro 5602.2 5541.6 e 50 98 8 Ile-His-Gln-Phe-Pro 5232.4 e 5211.2 50 95 9 Val-Leu-Pro-Val-Pro ND f 169? 10 Arg-Lys-Gly-Met-Pro 6851.1 6851.1 107 168 11 Val-Leu-Pro-Val-Pro ND f 169? a See Fig. 1 b See Fig. 3 c Experimental d Theoretical e Mass difference is due to addition of Na c, K c P or HCO 3 f Not determined Table 2 Identity of the peptides produced from porcine b-casein by bovine chymosin at ph 6.5 Electrophoretic band a HPLC N-terminal sequence Position in porcine peak no. b b-casein sequence a Arg-Ala-Lys-Glu-Glu 1? b Arg-Ala-Lys-Glu-Glu 1? c Arg-Ala-Lys-Glu-Glu 1? 1 Gly-Phe-Tyr-Pro-Val c 207 c? 2 Tyr-Gln-Asp-Pro-Leu 197? 3 Tyr-Gln-Asp-Pro-Leu 197? 4 Tyr-Gln-Asp-Pro-Leu 197? 5 Arg-Ala-Lys-Glu-Glu 1? a See Fig. 5 b See Fig. 6 c Alexander and Beattie [4] reported Ala instead of Val at amino acid residue 211 wide-pore C 8 column (5 mm particle size, 300 Å pore size, 280!4.6 mm; HPLC Technology, Macclesfield, UK) and guard column (20!4.6 mm). A 150 ml sample was injected onto the column and eluted using a gradient of two solvents: A, 0.1% trifluoroacetic acid (TFA; sequencing grade, Sigma) in deionised water; B, 0.1% TFA in acetonitrile [HPLC (far-uv) grade, Labscan, Dublin, Ireland]. Elution was initially with 100% A for 5 min, then with a linear gradient of 0 50% B over 55 min, B was maintained at 50% for a further 6 min and then increased to 60% over 4 min, and maintained at 60% for 3 min. The eluate was monitored at 214 nm using a Waters model 486 programmable detector (Millipore) interfaced with a personal computer controlled by Millenium 2010 software (Millipore). The flow rate was maintained at 0.75 ml/min. Eluted peptides were collected manually in polypropylene tubes and freeze-dried before further analysis. Electroblotting. Protein bands were transferred from a urea- PAGE gel to a poly (vinylidene difluoride) membrane (PVDF, pore size 0.22 mm ProBlott, Applied Biosystem, Foster City, Calif., USA) using a MiniTrans-Blott electrophoretic transfer cell (Bio-Rad, San Ramon, Calif., USA). Electrophoresis was performed at 90 V for 15 min in CAPS [3-(cyclohexylamino)-1-propanesulfonic acid] transfer buffer. The PVDF membranes were then stained using 0.2% (w/v) Coomassie Blue R-250 in 1% (v/v) acetic acid for 10 15 min, followed by destaining in distilled water. Individual protein bands were cut out from the membrane and sequenced directly. Peptide identification. Peptides were sequenced by an automated Edman degradation method using a pulsed-liquid phase protein/ peptide sequencer (Model 477A, Applied Biosystem). Liberated amino acids were detected as their phenylthiohydantoin derivatives by a model 120A microbore HPLC (Applied Biosystem). Mass analysis on isolated peptides was studied using a BioIon- 20 plasma desorption mass spectrometer (BioIon AB, Uppsala, Sweden). Samples were dissolved in 50% (v/v) methanol containing 1% TFA and applied to nitrocellulose-covered aluminised mylar targets. The samples were usually washed with small (F50 ml) volumes of 0.1 M ammonium bicarbonate to reduce the interference from Na c and/or K c.

86 Fig. 2 Primary sequence of bovine (B) [13] and porcine (P) [4] b-casein. Underlined amino acids indicate differences between the porcine and bovine proteins. Known plasmin hydrolysis sites are indicated by ( * ) while known chymosin hydrolysis sites are indicated by (c) Results and discussion Urea-PAGE (Fig. 1) showed that porcine and bovine b-caseins were hydrolysed rapidly by bovine plasmin; several electrophoretic bands migrating in the protease peptone region of the electrophoretic gel were evident in hydrolysates of both proteins. Bovine b-casein hydrolysates had three main bands in the g-casein region,

87 Fig. 3a, b RP-HPLC elution profile of ph 4.6-soluble peptides from porcine b-casein following hydrolysis by bovine plasmin for a 30 and b 180 min. The peptides in peaks labelled 1 11 were recovered and their N-terminal amino acid sequences and molecular weights determined (see Table 1) while the porcine b-casein hydrolysates exhibited four bands in this region. The four main porcine g-casein bands (a d) and one porcine proteose peptone band (e) were electroblotted from the electrophoretic gel and their N-terminal sequences determined (see Table 1). Porcine g-caseins a and b resulted from the cleavage of porcine b-casein at Lys 106 -Arg 107 (Fig. 2), while porcine g-caseins c and d resulted from the hydrolysis of Lys 49 -Ile 50 and Lys 168 -Val 169, respectively. Bovine g 1 - casein results from the hydrolysis of bovine b-casein at Lys 28 -Lys 29, but no similar hydrolysis site was found in porcine b-casein. Bovine g 2 - and g 3 -caseins result from the hydrolysis of Lys 105 -His 106 and Lys 107 -Glu 108, respectively. The N-terminal amino acid sequence of band e was the same as porcine b-casein, indicating that this band was a proteose peptone. Figure 3 shows the HPLC elution profile of the ph 4.6-soluble peptides formed on hydrolysis of porcine b-casein by bovine plasmin for 30 and 180 min. The peptides in peaks labelled 1 11 were isolated and their N-terminal sequences and molecular weights determined (Table 1). The main hydrolysis sites identified were Lys 33 -Leu 34, Lys 49 -Ile 50, Lys 95 -Asp 96, Lys 98 -Ala 99, Lys 106 -Arg 107, Lys 108 -Gly 109 and Lys 168 -Val 169. The plasmin hydrolysis sites in porcine b-casein and the identity of some of the peptides produced are illustrated schematically in Fig. 4. Like bovine b-casein, the N-terminal half of porcine b-casein appears to be more susceptible to hydrolysis by plasmin than the C-terminal region. Although plasmin cleaved porcine b-casein at Lys 106 - Arg 107 and Lys 108 -Gly 109, only one of these sites (Lys 106 -Arg 107 ) resulted in the production of a g-casein (Fig. 2); however, bovine g 2 - and g 3 -caseins resulted from the hydrolysis of two similar sites in bovine b- casein (i.e., Lys 105 -His 106 and Lys 107 -Glu 108 ). Urea-PAGE of porcine and bovine b-casein following hydrolysis by bovine chymosin is shown in Fig. 5. Bovine b-casein was hydrolysed to b-i, b-ii and b-iii casein over the 24-h period. Porcine b-casein was also hydrolysed to three distinct electrophoretic bands (a c) Fig. 4 Schematic of the identified sites of hydrolysis of porcine b-casein by bovine plasmin

88 Fig. 5 Urea-PAGE of porcine and bovine b-casein following hydrolysis by bovine chymosin. Lane 1 porcine sodium-caseinate. Lanes 2 12 porcine b- casein following hydrolysis by bovine chymosin for 0, 0.25. 0.5, 1, 2, 4, 8, 10, 12, 16 or 24 h, respectively. Lanes 13 15 bovine b-casein following hydrolysis by bovine chymosin for 0, 2 or 24 h, respectively. The bands labelled a, b and c were electroblotted and their N-terminal amino acid sequences determined (see Table 2) over the same time period. The first band (a) had a lower electrophoretic mobility than bovine b-i-casein, while band (b) had a higher electrophoretic mobility than bovine b-ii-casein; the electrophoretic mobility of band (c) was very similar to bovine b-iii-casein. Mulvihill and Fox [2] found that porcine b-casein was rapidly hydrolysed to b-i, which corresponded in electrophoretic mobility to bovine b-i casein, and suggested that a similar peptide bond was cleaved in both proteins. The authors stated that porcine b-i casein was further hydrolysed to a peptide doublet with a similar electrophoretic mobility to bovine b-iii casein and no peptide corresponding in mobility to bovine b-ii casein was evident in their porcine b-casein hydrolysate. The N-terminal sequence of the electrophoretic bands a, b and c (Table 2) was the same as porcine b-casein, indicating that the formation of these peptides involved hydrolysis towards the C-terminal of the protein. Figure 6 shows the resolution of the ph 4.6-soluble peptides formed from porcine b-casein by bovine chymosin over the 24-h period The peaks labelled 1 5 were isolated and their N-terminal sequences determined (Table 2). Peaks 3 and 4 were evident in chromatograms of the ph 4.6-soluble peptides formed after 2 h of hydrolysis and probably contained the small peptide(s) cleaved from porcine b-casein in the formation of band a in Fig. 5, which was the first large peptide formed from porcine b-casein. The N-terminal amino Fig. 6a, b RP-HPLC elution profile of ph 4.6-soluble peptides from porcine b-casein following hydrolysis by bovine chymosin for a 2 and b 24 h. The peptides in peaks labelled 1 5 were recovered and their N-terminal amino acid sequences determined (see Table 2)

89 acid sequences of peptides 2, 3 and 4 (Fig. 6) identified the hydrolysis site as Leu 196 -Tyr 197. These results suggest that porcine b-casein fragment 1 196 corresponds to band a (porcine b-i-casein), as the N-terminal amino acid sequence of this electrophoretic band was the same as that of porcine b-casein. This confirms the suggestion of Mulvihill and Fox [2] that a similar bond was cleaved in both porcine and bovine b-casein to yield porcine and bovine b-i-casein. The N-terminal amino acid sequence of the ph 4.6-soluble peptide 1 (Fig. 5) suggested that porcine b-casein was also cleaved at Gln 206 -Gly 207, while the N-terminal amino acid sequence of peptide 5 corresponded to that of porcine b- casein. The electrophoretic bands b and c (Fig. 5) had the same N-terminal amino acid sequence as porcine b- casein, and it is proposed that thay be deignated porcine b-ii- and b-iii-casein, respectively; the C-terminal amino acid sequences of the peptides were not determined, and at present the complete sequences of these peptides are not known. References 1. Woychik JH, Wondolowski MV (1969) J Dairy Sci 52: 901 2. Mulvihill DM, Fox PF (1979) Biochim Biophys Acta 578: 317 324 3. Stewart AF, Bonsing J, Beattie CW, Shah F, Willis IM, MacKinlay AG (1987) Mol Biol Evol 4:231 241 4. Alexander LJ, Beattie CW (1992) Anim Genet 23: 369 371 5. Pelissier JP, Mercier JC, Ribadeau-Dumas B (1974) Ann Biol Anim Biophys 14: 343 362 6. Visser S, Slangen KJ (1977) Neth Milk Dairy J 31:16 30 7. Eigel WN, Butler JE, Ernstrom CA, Farrell HM, Harwalkar VR, Jenness R, Whitney R McL (1984) J Dairy Sci 67: 1599 1631 8. Visser S, Slangen KJ, Alting AC, Vreeman HJ (1989) Milchwissenschaft 44: 335 339 9. Fox PF, Hoynes MCT (1975) J Dairy Res 42:427 435 10. Erhardt G (1989) Milchwissenschaft 44: 17 20 11. Andrews AT (1983) J Dairy Res 50:45 55 12. Blakesley RW, Boezi JA (1977) Anal Biochem 82: 580 581 13. Swaisgood HE (1992) Chemistry of the caseins. In: Fox PF (ed) Advanced dairy chemistry-proteins, vol 1. Elsevier, Amsterdam, pp 63 110 Acknowledgement Mass analysis on isolated peptides was carried out at the Department of Biochemistry, Faculty of Medicine, Nottingham University, Nottingham, UK.