The Content of Carbohydrates in Caseins froin Different Species.

1 :>

BENGT JOHANSSON aud LARS SVENNERHOLM.

Recei\erl 9 J i i r i e 1%~;.

C'oniparative investigations have revealed striliing difference> Iwtn-een human and cow's casein concerning physico-rhcniical and biological properties (MELLASDER 1947). However. the dif- ferences in chemical composition so far reported have heen limited to a significantly higher phosphorus content in cow's casein (see XELLANDER 194'7). WILLIAMSON (1944) has also found a slight dissimilarity in amino acid composition. using microbiological techniques. Reinvestigations of the amino acid content hv mean+ of ion-exchange chromatography by the method of MOORE and STETS (1951 ) have also demonstrated minor differences (JOH ASS-

\OF. STRTT) and MELLAXIIER. unpublished experinicnts). In a preliminary communication we have drawn attcntioii t o

er carbohydrate content of liiinian c

ation of the carhohydrate content of ca~ciii ' has lieen performed earlier. Some researchers haw u s d con-*\ I'aseiii wlic~n applying F,r,sos and MORGAX'S method ( I 933) for lic~xoiarriiric determinations 011 protein hydrolvsates ( N i r 2 k \ o ~ 1936 J I A \ ~ v ~ N E and SM:A t 193T. A ? ; s s ~ a s ~ r s i ) r ~ a n t 1 ('(>ti-

53). ITexosc. was analywd in some c.aseiii prrparationi ll? E N a d I~ATT(:AARU in 1933. A ~ A S A X V N I C and J Ik r t r (1952)

prqmred a inucopolysaccharidc from a tryptic digest of C O N

c.as~in. I t contained glucosamine. galactose and mannose. Dcspitc

and SVENSERHOLX 1955).

CARBOHYDRATES I N CASEINS FROM DIFFERENT SPECIES. 325

thorough perusal of t he literature we have found 110 analysis of the carbohydrates in human casein.

In t h e present s tudy we have estimated the amount of hexose. hexosamine and sialic acid in caseins from bovine and human milk. On account of t he decidedly larger amount of carbohydrates found in human casein, we were interested t o see if human casein was unique in this respect. Therefore we have also included analyses of caseins from sheep, goat, horse, whale and reindeer.

Experimental. Materials. I n all cases the milks used for the preparation of the

caseins were as fresh as possible. The milk from whale arid reindeer was frozen and toluol added for preservation. Mature milk was used with the exception of horse where the casein was prepared from colostrum. No data were available for the whale milk.

Preparation of caseins. Caseins from all species except homo could be precipitated in the same way by merely acidifying the milk t o pH 4.0: with 0.1 N hydrochloric acid with vigorous stirring. For the sake of convenience, however, the milk from whale and reindeer was diluted twice with distilled water owing to their large amount of solids. I n the case of human milk the standard method for the preparation of casein could not be used. It was found necessary either to dilute the milk 5-10 times or to remove calcium ions by potassium oxalate before adding hydrochloric acid. The precipitates of human casein had a slight but obvious mucinous character.

The precipitated caseins were washed first with physiological saline and afterwards repeatedly with distilled water. The wash water was tested for reducing sugar. At least two washings were given after the wash water was found to be negative. The caseins were dried with ethanol and the ethanol removed by ether. They were dried to constant weight above phosphorus pentoxide in a vacuum desiccator. Weighed amounts of caseins were dissolved in 0.1 N sodium hydroxide and ali- p o t s taken for the carbohydrate analysis.

;Methods. Hexose was determined by an orcinol-sulphuric acid method as

described by VASSEGR (1948). As standard was used an equimolar solution of galactose-mannose. By means of a sample blank the in- fluence of sialic acid on the read absorbance could be eliminated (SVENNERHOLM 1956).

Hexosainine was determined by a modification of ELSOX and MOR- GAK’S method (Ss EXSERHOLM in manuscript), in which the influence on colour formation of interaction between amino acids and hexoses is negligible.

3% BEXG'P JOHANQSON ASU LARS SVENNERHOLM.

Szczlw a c i d wa5 determined with Bial's reagent (orcinol-hydrochlor ir &l - Fe3+) by the method of SVEYKERROLM (in manuscript). As ztandard substance was used sialic acid from bovine submaxillary inuciii. which wa4 R gift from Professor GVYXAR BLIX. Uppsala. to onc of I I ~ (L. S.).

Resirlts and Discussion.

*4 comparison between the carbohydrate content of hunian ant1 cow casein is giren 111 Table I. Ti1 human casein therP i \ R niarkedly greater aniourit of every component analysetl - t h r difference being most pronounced for hexose and hexosainiiw .

Table 1. C'urbohydmt~ Conteiit of H?iTn(l?k rind Cow Caseans. (Vnlttes g i r e i i ~v

per cent of d r y weight.) 1 I Sialic Acid Total ( " a r b

(CI~HLINO,O)H~O)l hydrate. I

1 Ca,ein HeLosamine , H I

The total carbohydrate content is no less than about five tiiiies greater in human than in bovine casein. The small deviations inside the two groups are apparent. The bovine milk was from the dairy. so tha t no conclusions can be drawn about individuai variations. but thre? of the five human caseins were prepared from the milk of different individual women. Here too the dif- ferent preparations contained about the same amounts. These results demonstrate tha t it is possible to prepare caseins with a fairly constant carbohydrate content and that the individual variations also seein to be small.

The agreement with earlier results for cow casein is also good. S ~ R E X ~ E N and HAL-GAARD (1933) found 0.31 yo hexose. but the\- used a casein which was reprecipitated twice. Wc have alro reprecipitated caseins several times as described for tht. prepara- tion of a-casein by ~ V A R N E R (1944). which led to a casein with twice the amount of hexose (0 .42 O h ) . TILLMAX a i d PHlrJrprr

Nenn value. J Nnmber of ~ttmple.. ' Range.

CARBOHYDRATES IN CASEINS FROM DIFFERENT SPECIES. 327

0.16

0.42

0 .44

0.23

Table 11.

Carbohydrate Content of Caseins from Different Species. ( C’alices y i c w in per cent of dry weight.)

0.22

0 .59

O..iS

0.44

Casein

Human cow Sheep Goat Whale Horse Reindeer

Sialic Acid Total Carbo- 1 111HplNO10’H20)~ hydrates I

0 . i6

0.39

0.11

0.30

0.3; 0.56

0.46

1 . 0 3 I 0.80

0.55

0.68

1 .36

1.22

1.13

(1929) also found a higher hexose value in caseins which had been reprecipitated several times (0 .36 yo and 0.49 yo). We think however that our method of preparation will give a truer picture of the carbohydrate content of caseins in the natural state.

The hexosamine content reported by MASAMUNE and N ~ c . 4 -

ZUMI (1937) was 0.20 yo-0.26 yo, although NILSSON’S (1936) figures were somewhat higher, 0.32 yo and 0 .40 yo. ANASTASSIADIS and COMMON (1953), on the other hand, could not demonstrate hexos- amine in bovine casein. We have not tried to isolate hexosamine from casein but its presence is indubitable. Chromatography on Dowex 50 of an acid hydrolysate of human casein by the method of GARDELL (1953) demonstrated two fractions, which gave a positive ELSON and MORGAN reaction for hexosamine. The faster- running fraction was eluted in the same position as crystalline glucosamine-HC1, and the slower-running in that of galactosamine- HCl. The ratio of glucosamine to galactosamine was found to be 2 to 1.

The chromogen reacting with Bial’s reagent to give a red violet colour was isolated from bovine colostrum by KUHN and BROSSMER (1954), who named it lactaminic acid. It has also been isolated by ZrLLIKEiv, BRAUN and GYORGY (1955) from the non protein part of human milk under the name of gynaminic acid. We have preferred to use the name sialic acid proposed by BLIX, SVENNERHOLM and WERNER (1952), because BLIX (1936) was the first to isolate a pure substance in this group of chromo- gens. The most thoroughly investigated sialic acids have been

32s 1,ENGT J O H A S S S O S A S D LARS SVENSERHOLM.

i w h t e d froin subrnaxillary niucin of different apecies ( 1 3 1 , n . I A S ~ B E R G . OUIN and KERNER l955), where they haw indicated tlifferent acyl-groups in the acid: in cow sialic acid ail 0-acetyl C I W U ~ J and a n N-acetyl group. in ovine sialic acid an N-acetyl group, and in porcine sialic acid an N-glycolyl group. Sialic aciclh iiolnted froin other sources contain only an K-acctyl group as o\iiie sialic acid (001s 1955). Gynarniriic acid isolated as tlc-

i l d by ZILLIKES, BRAUN arid GYORGY (1955) gave an X-ray po\t der diagram identical with ovine sialic acid (SVESSERHOLM. uiipublished results), which further supports the use of the nanic hialic acid for the ehromogen of caseins.

A11 values given for sialic acid refer t o a standard of boviiir G l i c acid isolated from cow submaxillary rnuciii arid containing t \ \ o acetyl groups. KUKN and BROSSMER (1956) liavr also isolated froin milk of homo, cow, goat, sheep and pig. a carbo2iyclrntc Iiioiety composed of sialic acid and lactose. in which the sialic acid contained an N-acetyl group and an 0-acetyl group.

The physiological importance of the carbohydrate components of milk is still unknown. Sialic acid containing mucoproteins and mucopolysaccharides from other sources, such as e. 9. urine (TAMM and HORSFALL 1950, 1952) and submaxillary mucin ( B r 13.54. KLEXK and FAILLARD 1954) has been denlonstratcd t Iir receptor substance of influenza virus. ZILLIKEN, BRACT and

)RGY (1935) have also demonstrated that saccharides coil- taiiiing sialic acid (pynaminic acid) are a growth factor for Lacto- licicillns bifidus var. Penn.

Siiiii m nr J'.

'aseins froin milks of lionio. row. goat ilieep i i o l

< I l i t1 whale have becn investigated for their content of l i e \ow lic~sosamine and sialic acid.

The total carbohydrate content does not exceed 2 O 0 except in tlir case of human casein, which contains about -2 O ( ) . Thc ;iiiioiiiit of all components investigated is greater in liuniaii casciii t h e difference in comparison with other species inrestigatrd twin2 in( )qt pronounced for hexose and hexosaminc..

\\-c arc indebted to the Head of the Departinelit. T'rofwsc>i OLOF MELLASDER, for the gift of samples of bovine and huniai~ raseins and valuable discussions and criticism during the pc'r-

CARBOHYDRATES IN CASEINS FROM DIFFERENT SPECIES. 329

formance of t he work. We also owe our thanks t o Professor JOHAN T. RUUD, Biologisk Laboratorium, University of Oslo, for a sample of whale milk.

We are also very grateful t o Mrs. BIRGIT JOHANSSON for valuable technical assistance.

References.

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BLIX, G., Hoppe-Seyler’s Z. Physiol. Chem. 1936. 240. 43. BLIX, G., E. LISDBERG, L. ODIN and I. WERNER, Nature 1955. 175.

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