Characterization of Caseins from Mongolian Yak, Khainak, and Bactrian Camel B Ochirkhuyag, Jm Chobert, M Dalgalarrondo, Y Choiset, T Haertlé

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Characterization of caseins from Mongolian yak, khainak, and bactrian camel B Ochirkhuyag, Jm Chobert, M Dalgalarrondo, Y Choiset, T Haertlé

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B Ochirkhuyag, Jm Chobert, M Dalgalarrondo, Y Choiset, T Haertlé. Characterization of caseins from Mongolian yak, khainak, and bactrian camel. Le Lait, INRA Editions, 1997, 77 (5), pp.601-613. hal-00929550

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Original article

Characterization of caseins from Mongolian yak, khainak, and bactrian cam el

B Ochirkhuyag 2, lM Chobert 1*, M Dalgalarrondo 1, Y Choiset 1, T Haertlé '

1 Laboratoire d'étude des interactions des molécules alimentaires, Inra, rue de la Géraudière, BP 71627, 44316 Nantes cedex 03, France; 2 Institute of Chemistry, Academy of Sciences, Vlan Bator, Mongolia

(Received 25 November 1996; accepted 5 May 1997)

Summary - The composition of acid-precipitated caseins from ruminant Mongolian domestic ani- maIs was analyzed and a comparative study between camel (Camelus bactrianus) and dromedary (Camelus dromedarius) was realized. Acid-precipitated whole caseins were analyzed for ami no acid composition, separated by anion exchange chromatography and identified by alkaline urea-PAGE. Elution profiles and electrophoretic mobilities of the main components of yak and khainak caseins were nearly identical to their cow counterparts. However, the main part of a:;l-casein of yak was eluted in lower molarity in NaCI. Characterization by PAGE, ami no acid composition and N-terminal sequence of individual caseins from camel (Camelus bactrianus) indicated that milk of this ruminant contains dominantly aS1-' aS2-' and ~-casein and small amounts of x-casein as is the case for the milk of dromedary (Camelus dromedarius). caseins / yak / khainak / camel

Résumé - Caractérisation des caséines de ruminants de Mongolie: yak, khainak et chameau bactrien, La composition en acides aminés des caséines totales de deux ruminants de Mongolie (yak, khainak) a été déterminée. Les différentes caséines ont été séparées par chromatographie sur échangeur d'ions et analysées par électrophorèse en milieu urée et à pH alcalin. Les caséines des deux espèces étudiées ont un comportement voisin, proche de celui des caséines de vache. La seule différence notable réside dans le fait que chez le yak, la caséine aSI est éluée à une molarité inférieure à celle utilisée lors de la séparation des caséines de vache et de khainak. Une étude comparative a été réalisée entre le chameau (Camelus bactrianus) et le dromadaire (Camelus dromedarius). Ces deux espèces renferment principalement les caséines aS l' aS2 et B, dont la séquence N-terminale a été déterminée. La caséine K n'est que faiblement représentée. caséines / yak / khainak / camélidés

* Correspondence and reprints 602 B Ochirkhuyag et al

INTRODUCTION sent a more systematic description of the major milk proteins of principal Mongol Many populations of Central Asia, and Mon- stock animais and to compensate for this gols in particular, have thousands of years of gap. This paper describes the separation of old traditions of using milk and milk prod- the caseins from Mongolian cow, yak, ucts for nutritional purposes. Sometimes, khainak, and Camelus bactrianus by dif- they use also particular dairy products as ferent ion exchange chromatographies and curative agents (Kadirova, 1985). The first their further purification by reversed-phase European written mentions of the use of HPLC. For comparison, the caseins from milk as main staple of nomadic tribes origi- Camelus dromedarius (Arabi an dromedary) nating from Asia by ancient Greeks are due were separated using the same set of purifi- to Homer (-800 BC), Herodot (-500 BC) cation methods. The major casein compo- and Strabo (-100 BC) in their descriptions nents were characterized by urea and SDS of barbarian populations termed ya.Àa.K- polyacrylamide gel electrophoresis, and by 'tO<\>a.yot (galactophagi). Antique Greeks determination of their amino acid compo- attributed to this type of diet the particular sition and N-terminal sequence. strength and fierceness of described nomads. The earliest conserved written Chine se records of the mare' s milk use for prepara- MATERIALS AND METHODS tion of koumys can be traced to almost 2000 BC, to the descriptions of northem nomadic Preparation of milk sampi es tribes by Han historian Ssu-ma Ch'ien (sum- marizing ail preexisting information around Milk was collected from weil identified single the 1st century BC) and to conserved runic Mongolian domestic animais. The milk was and Ouigur petroglyphs on still existing stel- Iyophilized in the Food Industry Research Insti- lae. Nowadays, dairy products derived from tute of Ulan Bator, Mongolia. The lyophilized samples were kept frozen at -4 "C until further milk of animais herded on the Mongolian processing. The dromedary milk was a kind gift steps and deserts constitute still a major ele- of Professor Mahmoud Sitohy and was also pre- ment of indigenous diet. In 1995, Mongolia, pared from the single dromedary from Egyptian counting about 2.5 millions inhabitants, had desert animal stock. 416 000 cows, 708 000 yaks and 58 000 khainaks (hybrid between cow and yak), and had also 367 000 bactrian camels. Preparation and separation of caseins Unfortunately, despite the studies of Grosc1aude et al (1982), despite long ali- The whole casein was obtained from skim milk mentary practice, information about milk by precipitation at the isoelectric point (pH 4.6) proteins of Mongolian domestic animais is using 1 N hydrochloric acid. The precipitate was rather fragmentary. Moreover, available washed with distilled water, solubilised at pH 7 by addition of sodium hydroxide, precipitated information concerning dromedary milk again at pH 4.6 and washed three times with dis- (Farah and Farah-Riesen, 1985; Beg et al, tilled water. Finally, the whole casein was solu- 1984, 1986a and b, 1987; Abdel Rahim, bilised at pH 7, freeze-dried and stored at -20 oc. 1987; Mehaia, 1987a and b; Mohammed The individual caseins were separated by ion and Larsson- Raznikiewicz, 1989, 1991; exchange chromatography on QAE-Sepharose Farah, 1993) is related mainly to the Ara- (Pharmacia, Orsay, France), by applying a gra- bian dromedary Camelus dromedarius dient from buffer A (0.02 mol/L imidazole, pH 7.0, 3.3 mol/L urea, 0.3% (v/v) 2-mercap- species, and very scarce for the camel toethanol) to buffer B (0.02 mol/L imidazole, Camelus bactrianus species. The present pH 7.0, 3.3 mol/L urea, 0.3% (v/v) 2-mercap- work has been carried out in order to pre- toethanol, 1 mol/L NaCI), at room temperature, Caseins from Mongolian ruminants 603 with a flow rate of 200 mUh. The ion exchange solvent N40% solvent B (80% acetonitrile/20% chromatography was performed on an Econo H20IO.085% TFA) to 30% solvent A/70% system (Bio-Rad, Ivry sur Seine, France) equip- solvent B during 40 min. The flow rate was ment. 1 mL/min and absorbency was recorded at 214 nm.

Polyacrylamide gel electrophoresis Amino acid analysis Polyacrylamide gel electrophoresis (PAGE) was performed in a vertical mini slab gel apparatus The proteins were hydrolyzed under vacuum in Protean II (Bio-Rad). The acrylamide gel (10% the presence of constant boiling 6 N HCI for 24 h in 4 rnol/L urea, Tris-HCl, pH 8.8 buffer solu- at 110°C in a Pico-Tag station (Waters). After tion) was prepared according to Davis (1962). acid hydrolysis, the ami no acids were deriva- Electrophoresis was performed at constant tised with phenyl isothiocyanate (PITe) accord- amperage (15 mA during 7 min in the stacking ing to Bidlingmeyer et al (1984) and quantified gel, and 30 mA for about 30 min in the running by RP-HPLC on a Pico-Tag Cl8 column (3.9 mm gel). The SDS-PAGE was carried out according id x 15 cm). Speed-vac dried samples were dis- to the method of Laemmli (1970). The running solved in 95% 2 mmollL Na2HP04, pH 7.4, 5% gel (8.0 x 6.0 x 0.075 cm) contained 15% and acetonitrile. The column was equilibrated in sol- the stacking gel 4% acrylamide. Casein ratios vent A (94% 0.14 mollL CH3COONa, 0.5 mL were calculated by scanning the electrophoretic TEAIL, pH 6.4, 6% acetonitrile): the elution was gel, by using a Bioprofil station Program. performed with a gradient from solvent A to solvent B (40% H20 / 60% acetonitrile) according to Dalgalarrondo et al (1990). Both the column and solvent were maintained at 38 oc. The flow Chymosin test rate was 1.0 mL /min and absorbency was recorded at 254 nm. Chymosin of calf stomach (EC 3.4.23.4.), 23.6 Ulmg ofprotein (Sigma Chemical Co), was dissolved in distilled water (1 mg/mL). An aliquot N-terminal sequencing (5 ug) of the enzyme was supplied to 1 mg of bovine lC-, and p-casein, and to 10 mg of camel and dromedary caseins issued from fraction 3 of The N-terminal ami no acid sequence analysis ion exchange chromatography (see Results), dis- was performed on an Applied Biosystems model solved in 1 mL of a 50 mmol/L imidazole/ HCI 477 A sequencer with an on-line identification buffer (pH 6.5). The resulting solution was incu- of the phenyl thiohydantoin derivatives. Reagents bated at 37 "C for 20 min. The reaction was used for sequencing were purchased from Perkin stopped by adding solid urea until 3 mollL. Then, Elmer (Paris Nord II, France). The amino acid the solution was submitted to alkaline urea sequences obtained were searched at the NCBI PAGE, as described above. using the BLAST network service.

Reversed-phase high performance RESUL TS AND DISCUSSION Iiquid chromatography (RP-HPLC) Acid-precipitated whole caseins from cow, RP-HPLC preparative purification (LiChroCART yak, khainak, camel (Camelus bactrianus) 100 C'8 column, 10 mm id x 250 mm) was car- and dromedary (Camelus dromedarius) ried out on a Waters instrument (Waters Asso- were separated by anion-ex change chro- ciates, Millford, MA, USA) equipped with an matography on QAE-Sepharose column interface module system assisted by a chro- (fig 1). As already known, classical anion matography work station Maxima 820. The col- exchange chromatography on DEAE-celIu- umn was equilibrated with solvent A (0.1 % TFA lose column resolved whole bovine casein in H20, pH 2.5). The separation of caseins was achieved by applying a linear gradient from 60% into different fractions containing y-, K-, 604 8 Ochirkhuyag et al

/3-, as2- + as1-' and as1-casein, respectively that obtained from cow casein (fig 1A). (Mercier et al, 1968). As shown in figure Eight peaks were obtained for yak and 1Band C, the separation of khainak and yak khainak caseins, and nine for cow casein. caseins gave an elution pattern similar to Under the conditions used, caseins of

NaCI mol/L

A '...... 1.0 0.50

1 Z J 5 6 ~ 0.5 0.25

...... ,"

o 50 100 150 200 250 min

NaCI mof/L B 0.50 1.0

0.5 0.25 , " ';~:: ...J\j\ ...... ".. ",) o 50 100 ------150 200 250 min

A2BO NaCI mol/L

0.50 C 1.0

4 5

0.25 0.5

0.00 0.0 0 50 100 150 200 250 min

Fig 1. Fractionation of acid precipitated total caseins of cow (A), khainak (H), yak (C), Came/us dromedarius (D), and Came/us bactrianus (E) by ion- exchange chromatography on QAE-Sepharose. 6.8 mL fractions were collected at the flow rate of 200 mL/h. Fractionnement des caséines entières de vache (A), khainak (8), yak (C), Camelus dromedarius (D) et Came lus bactrianus (E) par chromatographie d'échange d'ions sur colonne de QAE-Sepharose. Le volume de chaque fraction était de 6,8 mL et /e débit de 200 mUh. Caseins from Mongolian ruminants 605

NaCI[M]

0.50 D 1.0

0.25 0.5

0.00 t..;-:-:.:.:.:::..:..:.:---r------r---,----,------r-...cJ 0.0 a 50 100 150 200 250 min

NaCI[M]

0.50 E 1.0

0.25 0.5

a 50 100 150 200 250 min

Fig 1. (continued)

Came/us species (fig ID, E) gave a different 3, although this casein was also found as a elution pattern than those obtained with yak contaminant in fractions 4 and 5 of yak, and and khainak caseins. 5 and 6 of khainak caseins. The main part of the protein eluted at 0.16 mol/L NaCI and corresponding to peaks 4 and 5 of yak, and Caseins of yak and khainak to peaks 5 and 6 of khainak casein migrated by electrophoresis near the region of migra- The alkaline urea-P AGE shows (fig 2) that tion of cow ~-casein. Proteins obtained from the first two peaks eluted by ion exchange fractions 6 and 7 of yak, and 7 and 8 of chromatography of yak and khainak whole khainak (elution beginning at 0.26 mol/L casein did not contain any protein band. Elu- NaCI) gave bands with electrophoretic tion of casein components began at a rnolar- mobilities similar to those of cow as -casein. ity of 0.12 mol/L in NaCI. The elec- Fraction 8 of yak casein contained a very trophoretic mobilities of proteins issued small amount of protein and, despite sev- from peak 3 of yak and 3 and 4 of khainak eral analyses, did not show any visible band. (eluted at 0.12 mol/L NaCI) were nearly Yak milk predominantly consists of Œs1C- identical with electrophoretic mobility of casein (Grosclaude et al, 1982), what may cow x-casein. A test with chymosin has con- explain that almost ail ŒsCcasein was eluted firmed that the major part of yak and in peak 7, at 0.27 mol/L NaCI. On the con- khainak x-casein was contained in fraction trary, fractions 9 of cow and 8 of khainak, 606 B Ochirkhuyag et al

Fig 2. Urea-PAGE patterns of fractions issued from ion exchange chromatography on QAE-Sepharose of khainak (A), yak (B), Camelus bactrianus and Camelus dromedarius (C) caseins. A. as' ~, and K are as' ~, and K -cow caseins, respectively; t is total yak casein; 1 ta 8 are fractions 1 ta 8, respectively. B. as' ~, and K are a ,~, and K -cow caseins, respectivety; t is total khainak casein; 1 ta 8 are fractions 1 ta 8, respectively. C. as'~' and K are as' ~, and K -cow caseins, respectively; t is total Camelus bactrianus casein; B T is total Camelus dromedarius casein; 1 ta 5 are fractions 1 ta 5 of Camelus bactrianus casein, respectively; 6 ta 10 are fractions 1 ta 5 of Camelus dromedarius casein, respectively. Électrophorégramme (milieu urée) des fractions issues de la chromatographie d'échange d'ions sur colonne de QAE-Sepharose des caséines de khainak (A), yak (B), Camelus bactrianus et Camelus dromedarius (C). A. as f3, et x: caséines as {J, et x de vache, respectivement; t : caséine entière de yak; J à 8 : fractions J à 8 respectivement, issues de la chromatographie sur échan- geur d'ions. B. as {J, et x: caséines as/ {J, et x de vache, respectivement; c t : caséine entière de khainak ; J à 8 : fractions J à 8 respectivement, issues de la chromatographie sur échangeur d'ions. C. as' {J, et x: caséines as {J, et /( de vache, respectivement; t : caséine entière de Camelus bactrianus; T: caséine entière de Camelus dromedarius ; J à 5 :fractions J à 5 respectivement, issues de la chromatographie sur échangeur d'ions de la caséine entière de Camelus bactrianus; 6 à JO : fractions J à 5 respectivement, issues de la chromatographie sur échangeur d'ions de la caséine entière de Camelus dromedarius. Caseins from Mongolian ruminants 607 which were eluted at the same molarity of Caseins of camel (Camelus bactrianus) NaCI, above 0.3 mol/L, contained signifi- and dromedary (Camelus dromedarius) cant amounts of lXsl-casein. At this molarity (0.3 mol/L NaCI), fraction 8 of yak con- Samples of caseins of Came/us bactrianus tained only small quantities of proteins. and Came/us dromedarius, as weil as their Urea-PAGE of whole casein from yak and fractions obtained by ion-exchange chro- khainak (fig 5) shows a similar composi- matography, were examined in order to tion in individual caseins, ie, 43.2 and 47.0% determine whether they present differences lXs-casein, 35.4 and 33.8% ~-casein, and or have a similar composition as caseins 16.4 and 14.2% x-casein, respectively. from Bovidae (see above). The separation Based on urea-PAGE and on the amino acid of whole caseins of Came/us species by composition of total casein (table 1), one anion-exchange chromatography on QAE- can assume that caseins of yak and khainak Sepharose column gave elution profiles dif- are nearly identical with those of cow. ferent from those obtained with bovine casein. Caseins from each species of camels were eluted in five peaks (fig ID, E). The electrophoretic pattern of acid-precipitated Table I. Amino acid composition of total caseins. whole caseins from bactrian and dromedary Composition en acides aminés des caséines camels (fig 2C: t, Came/us bactrianus, T, entières. Came/us dromedarius) shows that they contained two weil defined sharp bands and a Cow" COIvi,2 Yak!'2 Khainakëè diffuse one. The electrophoretic pattern of each peak obtained by anion exchange chro- Asx 7.9 7.1 6.2 6.8 matography indicated that peak 1 contained Glx 21.8 22.9 22.4 23.2 a prote in with a low mobility which has a Ser 5.6 5.5 5.8 5.9 molecular mass of about 63000 (as deter- mined by SDS-PAGE) and that peak 2 was Gly 2.1 1.8 1.8 2.0 deprived of any protein. Casein components His 2.8 2.8 2.6 2.8 were obtained in peaks 3, 4 and 5, which Arg 3.7 4.2 3.7 4.0 were eluted at 0.21, 0.26 and above Thr 5.1 4.3 5.0 4.6 0.30 mol/L NaCI, respectively. After urea- Ala 3.5 3.2 3.1 3.0 PAGE (fig 5), casein ratios were calculated Tyr 5.3 5.0 4.9 4.9 by scanning the electrophoretic gel, by using Val 6.8 5.9 6.1 6.2 a Bioprofil station Program. The whole Met 2.7 1.3 1.3 1.3 camel casein contained about 38% lXs1-' Cys 0.7 nd nd nd 21 %, lXs2-and 41 % ~-casein, respectively. Ile 6.4 4.9 4.9 4.8 Among the ~-casein fraction, 12% were con- Leu 10.4 8.8 9.0 9.2 stituted by x-casein which was co-eluted Phe 5.2 4.9 4.4 3.9 with ~-casein (see below). Lys 8.3 7.3 6.5 6.6 The different fractions issued from ion Pro 10.0 9.8 11.3 10.0 exchange chromatography of both Came/us Trp 1.4 nd nd nd species have been further purified by RP- HPLC (data not shown), before determin- a From Renner, 1991. bThis study. 1 Values are gllOO g ing their ami no acid composition (table II) total casein. 2 Values are number of residues/lOO and N-terminal sequence (table III). A 20-24 residues. amino acid long N-terminal sequence of a Données Renner, 1991. b Cette étude. J Valeurs en g/lOO g de caséine totale. 2 Nombre de résidus/lOO fractions 4, 5 and 3 of Came/us dromedar- résidus. ius and Came/us bactrianus has been deter- 608 8 Ochirkhuyag et al

'd~ ~ o () -s M~~~~~M~~~ ~OOO~MM~O "~ "":oO~NN """:N'l:5 o\NO"":O"":~O~ e o ";:

~~ ~"1~~ "'O~"100M"'O~ viM'D ~<'iévi <'i'l:5ér-: N ... ~OO'D~~~~O~OO~"'O"1~~~"'O"1 MOvi ~<'i~NoOMé~ M~é~ N N

"1'DO~~~~~"1~"1~0"1"10~~'D ~o\oO"":NMN"":OO~~NO~OO"":OO <'i N

OO~ 'DOOOO~~ "1~"'O~'DOO~"'OM o\N'l:5<'iN"":"":N~"":~ évioON'l:5é"": N M

~~'DN~~O'D~N~ "'O~N'D~"'O~ 0\ 'l:5<'iN~"":<'i~"":~Né~o\N'l:5é~ N M

N~~N~OO~~~'D~~"'O~M~~"'OO r-:M'l:5NNN~M'D"":~ é~'l:5"":o\é'l:5 N N

OO~~~~O'D ~ ~~"'O~~~~"'OM 'l:5"":~ NNoON~vi~ é~~~ é'l:5 N N

M o X ~~~~~O~~O~~~~~~~~~~ ~6~6~~~~~~>~èd~~~~~ Caseins frorn Mongolian ruminants 609

Table III. N-terrninal sequence of asJ-' asr and p-caseins of Came/us bactrianus and Came/us dromedarius; cornparison with asJ-' as2 and p-caseins of cow, goat, mou se and pig. Séquence N-termina/e des caséines as" as2 et f3 de Carnelus bactrianus et Carnelus drornedarius; com- paraison avec les caséines as l' as2 et f3 de vache, chèvre, souris et porc.

5 10 15 20 COW R - P-K-H-P-I-K-H-Q-G-L-P-Q-E-V-L-N-E-N-L- Goat R - P-K-H-P-I-N-H-R-G-L-S-P-E- V-P-N-E-N-L- Mouse R - L-H-S-R-N-A-V-S-S-Q-T-Q-Q-Q-H-S-S-S-E- Pig R - P-K-P-P-L-R-H-Q-E-H-L-Q-N-E-P-D-S-R-E- Carne1 B RlK-P-K- y-P-L-R- Y-P-E- V-F-Q-N-E-P-D-S-I-E- Cam el D RlK-P-K- y-P-L-R- Y-P-E- V-F-Q-N-E-P-D-S-I-E- aS2 Cow K-N-T-M-E-H- V-S-S-S-E-E-S-I-I-S-Q-E- T- y-K- Goat K-H-K-M-E-H- V-S-S-S-E-E-P-I-N-I-F-Q-E-I- Y- Mouse K-Q-R-M-E-Q- Y-I-S-S-E-E-S-M-D-N-S-Q-E-N-F- Pig K-H-E-M-E-H- V-S-S-S-E-E-S-I-N-I-S-Q-E-K- Y- Carnel B K-H-E-M-D-Q-G-X-X -X- E-E-Q-N-I- V-P-Q-K-X -K- Carnel D K-H-E-M- D-Q-G-X- X-X-E-E-Q-N-I- V-P-Q- K-X- K- P Cow R-E-L-E-E-L-N- V-P-G-E-I- V-E-S-L-S-S-S-E-E-S-I- T- Goat R-E-Q-E-E-L-N- V-V -G-E-T- V-E-S-L-S-S-S-E-E-S-I- T- Mouse R-E- T-T-F-T- VcS-S-E- T-D-S-I-S-S-E-E-S- V-E-H-I-N- Pig R-A-K-E-E-L-N-A-S-G-E- T-V-E-S-L-S-S-S-E-E-S-I- T- Carnel B R- E- K- E- E- F- K-T-A-G- E- A-L-E- X-I-X-X -X-X -X-X -X-X- Carnel D R-E-K-E-E-F- K-T-A-G-E-A-L- E-S- I-X-X-X-E-E-Q- T-H-

mined in order to confirm their identification heterozygous for this locus. In the sequence as asl-' as2-' and ~-caseins. A comparative of p-like casein of Camelus dromedarius study (ami no acid composition and N-ter- and Came lus bactrianus, sorne amino acids minaI sequence) of the different caseins could not be identified with sufficient accu- obtained from the two species of camels racy. In their bovine counterpart, they cor- (Came/us dromedarius and Camelus bac- respond to phosphoseryl residues. trianus), showed that they are identical. Alkaline urea-PAGE (fig 2C), amino acid Interestingly, the N-terminal sequence of compositions (table II) and N-terminal caseins from the two species of Came lus sequence (table III) of proteins obtained in showed more homologies to the N-termi- peaks 3, 4 and 5 suggested that they were nal region of caseins of pig than to other camel po, asJ- and as2-like caseins, respec- animais studied (cow, goat, mouse). How- tively. The camel as2-like and asJ-like ever, they preserved the strictly conserva- caseins presented a higher and a lower tive amino acid positions. Two N-terminal mobility, respectively, than that of their amino acid residues (arginine and lysine) bovine counterparts. This is probably have been obtained in equal amount in the depending on the degree of their phospho- sequence of as1-like casein of the two rylation (Mohammed and Larsson- species of Camelus. The animaIs could be Raznikiewicz, 1991). Camel p-like casein 610 B Ochirkhuyag et al presented a band with a migration similar action. Under the same conditions, ~-casein to that of cow x-casein, which agrees wel1 was resistant to chymosin action. The con- with the result of Larsson-Raznikiewicz and centration of the solution of x-like camel Mohammed (1986). casein loaded on the gel needed to be high Larsson-Raznikiewicz and Mohammed (untillO mg/mL), otherwise it was impos- (1986) have obtained dromedary x-Iike sible to observe its characteristic bands and casein, as a little peak between ~- and (Xsl- to see their disappearance upon action of caseins only once during several separations chymosin. Consequently, it can be supposed of dromedary whole casein by anion that camel milk contains very little amounts exchange chromatography on DEAE-cel1u- of x-casein. Since its cow counterpart con- lose. According to our results, camel x-Iike tains cysteyl residue, the anion-exchange casein co-eluted with camel ~-like casein, chromatography fraction containing camel eg, in fraction 3. As could be seen by alka- x-like casein was further passed through an line urea-PAGE, camel x-Iike casein affinity chromatography column contain- migrated just below the electrophoretic band ing thiol-activated Affi-Gel SOI (Pharma- of camel ~-like casein and it separated in cia). Unfortunately, this proved to be unsuc- three smal1 bands. cessful since no thiol containing protein was retained. In order to confirm this result, a test with chymosin was performed on fraction 3. The SDS-PAGE patterns of camel whole carnel x-like casein was incubated with chy- casein and of its fractions issued from ion- mosin and then submitted to alkaline urea- exchange chromatography are presented in PAGE. The results showed (fig 3) that the figure 4. Al1 the bands observed by SDS- three little bands disappeared after chymosin PAGE of whole camel casein were recov-

Fig 3. Action of chymosin as evidenced by 10 Urea-PAGE. Lanes: l, cow x-casein: 2, cow x-casein after chymosin action; 3, 4, fraction 2 issued from ion exchange chromatography of Came lus dromedarius casein, before and after chymosin action, respectively; 5, 6, fraction 2 issued from ion exchange chromatography of Camelus bactrianus casein, before and after chymosin action, respec- ti vely; 7, 8, fraction 3 issued from ion exchange chromatography of Camelus dromedarius casein, before and after chymosin action, respectively; 9, 10, fraction 3 issued from ion exchange chromatography of Camelus bactrianus casein, before and after chymosin action, respectively. Mise en évidence de l'action de la chymosine par électrophorèse sur gel de polyacrylamide, en pré- sence d'urée. 1: caséine x de vache ..2 : caséine «de vache après action de la chymosine .. 3 et 4: fraction 2, issue de la chromatographie sur échangeur d'ions de la caséine entière de Camelus dromedarius, respectivement avant et après action de la chymosine .. 5 et 6 : fraction 2, issue de la chromatographie sur échangeur d'ions de la caséine entière de Camelus bactrianus, respectivement avant et après action de la chymosine .. 7 et 8 :fraction 3. issue de la chromatographie sur échangeur d'ions de la caséine entière de Camelus dromedarius, respectivement avant et après action de la chymosine ..9 et 10 :fraction 3, issue de la chromatographie sur échangeur d'ions de la caséine entière de Camelus bactrianus, respectivement avant et après action de la chymosine. Caseins from Mongolian ruminants 611

T 2 3 4 5 6 7 Fig 4. SDS-PAGE of camel caseins. t is whole Camelus -94.0 kD bactrianus casein; T is -67.0 kD whole Camelus dromedar- -43.0 kD ius casein; 1, 2, and 3 are fractions 3, 4, and 5 issued -30.0 kD from ion exchange chromatography of Camelus bactrianus casein, respec- -20.1 kD tively; 4, 5, and 6 are fractions 3, 4, and 5 issued -14.4 kD from ion exchange chromatography of Camelus dromedarius casein, respectively; 7 is molecular mass standard. Électrophorèse sur gel de polyacrylamide, en présence de SDS des caséines de Camelus bactrianus et Camelus dromedarius. t: caséine entière de Camelus bactrianus .. T: caséine entière de Camelus dromedarius 1, 2 et 3 :fractions 3, 4 et 5 respectivement, issues de la chromatographie sur échangeur d'ions de la caséine entière de Camelus bactrianus ; 4, 5 et 6 : fractions 3, 4 et 5 respectivement, issues de la chromatographie sur échangeur d'ions de la caséine entière de Camelus dromedarius ; 7 : marqueurs de poids moléculaires.

a 1< 2 4 5

Fig S. Urea-PAGE patterns of total caseins. 1, Came lus bactrianus; 2, Camelus dromedarius; 3, yak; 4, khainak; 5, bovine. Électrophorèse sur gel de poly- jas acrylamide, en présence d'urée des caséines totales de: 1, Came- lus bactrianus ; 2, Camelus dromedarius ; 3, yak ..4, khainak ..5, vache.

ered after fractionation by ion-exchange (Came/us bac tria nus) are 27 400, 34 300 chromatography. The molecular masses of and 26 000, respectively. Sorne bands with casein components were estimated through lower molecular masses were present in calculation with Bioprofil station program. fraction 4 issued from ion exchange chro- The molecular masses of p-, o.sl- and o.s2- matography, possibly resulting from a degra- like case in of dromedary (Came/us dation. The determination of the molecular dromedarius) are 27 500, 35 300, and masses of strongly hydrophilic or hydropho- 26 300, respectively, and those for camel bie proteins results sometimes in under- or 612 B Ochirkhuyag et al overestimated values (Trieu-Cuot and ACKNOWLEDGMENTS Gripon, 1981; Creamer and Richardson, 1984). Bovine caseins contain strongly B Ochirkhuyagis a recipientof a fellowshipfrom hydrophilic (such as the N-terminal domain the Ministère des Affaires Etrangères, as an of ~-casein, as weIl as 8-12, 56-63, exchange between France and Mongolia. 129-199 residues of as2-casein) and hydrophobie (1-44, 90-113, 132-199 REFERENCES residues of asl-casein, as weIl as C-terminal domain of ~-casein) regions (Swaisgood, Abdel Rahim AG (1987) The chemical composition 1992), and the existence of the similar frag- and nutrition al value of camel (Came lus dromedar- ments in camel caseins can be expected too. ius) and goat (Capra bireus) milk. World Animal Rev Prod 23, 9-12 Beg OU, von Bahr-Lindstrom H, Zaidi ZH, Jornval H (1984) A small camel-milk protein rich in cys- CONCLUSIONS teine/half-cysteine. Biosci Rep 4, 1065-1070 Beg OU, von Bahr-Lindstrom H, Zaidi ZH, Jornval H (1986a) A camel milk whey protein rich in half- The amino acid compositions of whole cysteine. Primary structure, assessment of varia- tions, internaI repeat patterns, and relationships caseins from yak and khainak are quite sim- with neurophysin and other active polypeptides. ilar and do not differ significantly from the Eur J Biochem 159, 195-201 amino acid composition of bovine whole Beg OU, von Bahr-Lindstrom H, Zaidi ZH, Jornval H casein. As shown by ion-exchange chro- (1986b) Characterization of a camel milk protein rich in proline identifies a new J3-casein fragment. matography and urea-PAGE, yak and RegulPept 15,55-62 khainak caseins present similar chromato- Beg OU, von Bahr-Lindstrom H, Zaidi ZH, Jornval H graphie and electrophoretic patterns. (1987) Characterization of a heterogeneous camel milk whey non-casein protein. Eur J Biochem 216, Camel and dromedary milks contain aIl 270-274 four caseins homologous with cow a -' Bidlingmeyer BA, Cohen SA, Tarvin TL (1984) Rapid si analysis of aminoacids using pre-column derivati- aS2-' ~-, and x-caseins. The existence of x- zation. J Chromatogr 336,93-104 like casein in camel was demonstrated by Brignon G, Ribadeau Dumas B, Mercier JC, Pélissier alkaline urea-PAGE and chymosin diges- JP, Das BC (1977) Complete amino acid sequence tion. Itis migrating a bit faster than camel ~- of bovine asz-casein. FEBS Leu 76,274-279 like casein and it is resolving in three bands. Creamer LK, Richardson T (1984) Anomalous behaviour of bovine as 1- and J3-caseins on gel elec- As suggested by the results of anion trophoresis in sodium dodecyl sulphate buffers. exchange chromatography on QAE- Arch Biochem Biophys 234, 476-486 Sepharose column, which show that camel Dalgalarrondo M, Chobert JM, Dufour E, Bertrand- x-like casein elutes in higher NaCI molarity Harb C, Dumont JP, Haertlé T (1990) Characteri- zation of bovine J3-lactoglobulin B tryptic peptides (0.21 mollL) than its bovine counterpart, by reversed-phase high performance liquid chro- camel x-like casein has a higher positive matography. Milchwissenschaft 45,212-216 net charge. According to their amino acid Davis BJ (1962) Dise electrophoresis II. Method and composition and their N-terminal sequence, application to human serum proteins. Ann New York the caseins from milks of two camel species Acad Sei 121,404-427 Farah Z, Farah-Riesen M (1985) Separation and char- (Came lus dromedarius and Came lus bac- acterization of major components of camel milk trianus) do not present many dissimilari- casein. Milchwissenschaft 40, 669-671 ties. The observed differences of their Farah Z (1993) Composition and characteristics of molecular masses are either due to few vari- camel milk. J Dairy Res 60, 603-626 ations in their primary sequences or to the Grosclaude F, Mahé MF, Accolas JP (1982) Note sur le polymorphisme génétique des lactoprotéines de divergence of their glycosylation and/or bovins et des Yaks Mongols. Ann Génét Sél Anim phosphorylation. 14,545-550 Caseins from Mongolian ruminants 613

Homer (- 800 BC) ln: Iliad 13.6 Mercier JC, Brignon G, Ribadeau Dumas B (1973) Herodot (-500 BC) ln: Histories, 1.216.4; 4.2.1; 4.2.2 Structure primaire de la caséine K B bovine. and 4.23.3 Séquence complète. Eur J Biochem 35, 222-235 Mohammed MA, Larsson-Raznikiewicz M (1989) Sep- Kadyrova PH (1985) Came/ and mare milk in medic- aration of a camel milk casein fraction and its rela- ina/ nutrition (Kadyrova PH, ed) Kazakhstan Pub- tion to the coagulation properties of fresh milk. lisher, Alma-Ata, Kazakhstan Milchwissenschaft 44, 278-280 Laemmli UK (1970) Cleavage of structural proteins Mohammed MA, Larsson-Raznikiewicz M (1991) Heat during the assembly of the head of bacteriophage treatment of camel milk. Effects upon casein frac- T4. Nature 227,680-685 tion. Milchwissenschaft 46,562-565 Larsson-Raznikiewicz M, Mohammed MA (1986) Renner E (1991) Dictionary of milk and dairying, Analysis of the casein content in camel (Came/us Munchen, Volkswirtschaftliche Verlag. dromedarius) milk. Swedish J Agric Res 16, 13-18 Ribadeau Dumas B, Brignon G, Grosclaude F, Mercier Mehaia MA (1987a) Studies of camel casein micelles: JC (1972) Structure primaire de la caséine 13 bovine. Treatment with soluble and immobilized Neu- Séquence complète. Eur J Biochem 25, 505-514 roamidase. Carbohydr Po/ym 7, 361-369 Strabo, (-100 BC) ln: Geography, 7.3.7 and 7.3.9 Mehaia MA (1987b) Studies of camel milk case in Ssu-ma Ch'ien (-100 BC) ln: Records of the grand micelles: Treatment with soluble and immobilized historian. Qin dynasty. Hong Kong, Research Cen- chymosin. Milchwissenschaft 42,706-708 tre for Translation (eds), Chinese University of Mercier JC, Maubois JL, Poznanski S, Ribadeau Dumas Hong Kong; New York, Columbia University Press. B (1968) Fractionnement préparatif des caséines Translation published in 1993 de vache et de brebis par chromatographie sur Swaisgood HE (1992) Chemistry of the caseins. In: DEAE cellulose en milieu urée et 2-mercap- Advanced Dairy Chemistry. vol 1 (Fox PF, ed) toéthanol. Bull Soc Chim Bio/50, 521-530 Elsevier, London, 63-1 10 Mercier JC, Grosclaude F, Ribadeau Dumas B (1971) Trieu-Cuot P, Gripon JC (1981) Electrofocusing and Structure primaire de la caséine as I-bovine. two dimensional electrophoresis of bovine casein. Séquence complète. Eur J Biochem 23, 41-51 Eur J Biochem 25, 505-514