A STUDY OF WHEY PROTEINS FROM THE MILK OF VARIOUS ANIMALS*

BY H. F. DEUTSCH

(From the Departments of Physiological Chemistry and Physical Chemaktry, University of Wisconsin, Madison)

(Received for publication, April 11, 1947)

The proteins of the plasma and sera of various animals as studied by the electrophoretic technique show extreme species variation but are character- istic and reproducible for a given species (1, 2). It appeared desirable to determine whether the milk proteins of various mammals would show simi- lar marked variation in electrophoretic patterns and whether any marked relation between the proteins of milk sera and blood plasma exists. An electrophoretic and ultracentrifugal study of the milk serum proteins of different animals at various times post partum has revealed characteristic deviations in these proteins, but few if any apparent relationships to the homologous plasma proteins were found except for milk samples taken directly post partum.

EXPERIMENTAL

Fat was removed from the various milk samples by centrifugation, follow- ing which the skim milk was adjusted to pH 6.2 (f0.1) by the addition of dilute hydrochloric acid. For reasons to be discussed, the casein was re- moved by rennin, a small amount of a commercial preparation sufficient to precipitate the casein within 60 minutes at 35” being used. Following removal of the casein by centrifugation and filtration, the whey was dialyzed against cold running tap water for 16 to 20 hours to remove the major part of the lactose. The dialyzed protein was frozen and dried in vacua.

Electrophoretic analyses of these proteins were carried out in a bar- biturate-citrate buffer of pH 8.6 and ionic strength of 0.088, in which the sodium citrate contributed 48 per cent of the ionic strength. The samples were dialyzed against several changes of buffer for 30 to 60 hours at 1”. Since most samples showed varying degrees of turbidity, they were filtered through a thin Seitz pad prior to the final dialysis period. The electro- phoretic experiments were carried out in a long, single section cell of 11 ml. capacity at a constant potential gradient of approximately 8.5 volts per cm. and at a temperature of lo. Electrophoretic mobilities were measured by using the center of the initial boundary as the reference point. Com-

* A portion of this material was presented before the Division of Biological Chem- istry of the American Chemical Society at Chicago, September 9-13, 1946.

437

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438 WHEY PROTEINS

ponenta were designated numerically in the order of increasing mobility, since usually they bore no general relationship to the plasma proteins of the homologous species.

Sedimentation analyses were carried out in the high velocity, oil turbine Svedberg ultracentrifuge. The speed of the rotor was 50,400 R.P.M. The positions of the moving boundaries as a function of time were recorded by the cylindrical lens schlieren method in conjunction with a diagonal knife- edge.

Nitrogen analyses were performed on the skim milk and whey samples and the amount of casein was obtained by difference.

2

RENNIN

ACID

FIG. 1. Comparison of descending electrophoretic boundaries of goat whey aft,er removal of casein by isoelectric precipitation and by rennin.

Results

No differences in the electrophoretic composition of cow whey proteins were observed when the casein was removed by isoelectric precipitation or by treatment with rennin. However, in the case of pooled goat milk, a considerable part of the whey proteins of lower isoelectric point was re- moved by the acid precipitation, It was found necessary to lower the pH to 4.3 to 4.4 to remove effectively goat casein from these proteins, a pH somewhat lower than that required in the case of the bovine milk. The electrophoretic patterns of goat wheys from the same milk from which casein was removed by rennin and by acid treatment are shown in Fig. 1.

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Alb2.1

cow

Goat

Pig

Human

Sheep

Horse

H. F. DEUTSCH 439

Considerable amounts of the goat whey proteins of the lower isoelectric point were removed by acid precipitation and, since a similar effect might occur with milk from other species, all casein removals were performed with rennin. In the case of human milk, rennin treatment followed by adjust-

TABLE I

Distribution of Nitrogen in Fractions from Milk of Several Animals at Various Times Post Partum

Days after parturition

0 1

2 Aged*

0 2 4

Aged*

ot 03 21 3t 5$ gt 131 l-2* 6-9* 14* 40 90 7 35 11 90

Nitrogen -

skfmmik Whey

per cd pn cent

2.32 1.58 1.05 0.51 0.57 0.13 0.47 0.12 1.76 1.22 0.73 0.28 0.68 0.22 0.63 0.17 2.50 1.74 2.31 1.81 2.00 1.66 0.60 0.32 0.99 0.56 0.65 0.39 1.04 0.73 0.29 0.255 0.28 0.17

0.20 0.15 0.20 0.13 0.23 0.13 1.61 0.54 0.78 0.20 0.80 0.40 0.40 0.21

-

- .-

-

caseif

&e? cent 0.74 0.54 0.43 0.35 0.54 0.45 0.46 0.46 0.76 0.50 0.34 0.28 0.43 0.26 0.31 0.04 0.11

0.05 0.07 0.10 1.07

0.58 0.40 0.19

* Pooled samples; remainder individual. t Poland-China sow. f Chester white BOW. 3 31.3 per cent of whey nitrogen dialyeable.

ment of the pH to 4.8 was found necessary to precipitate the casein. The first postpartum milk of swine also offered some difhculties in this respect.

To determine whether appreciable amounts of casein remained with the whey proteins, a sample of casein was prepared by acid precipitation of bovine skim milk and electrophoretic analyses were carried out. In

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440 WHEY PROTEINS

TABLE II

Electrophoretic and Ultracentrifugal Analysis of Whey Proteins

Species

Bovine

Goat

Pig (Cheste white)

Pig (Po- land- China)

Human

Sheep

Horse

Days

pazn

0 1 2

AgedS u* 0 2 4

AwdS u 0 3 6 14 u 0 4 9 u l-2$

&St: 14 40 90 u 7 35 u

ll§ 90 u

- 1 Per cent and electrophoretic mobility,*

constituent No.

1 2 3 4 5 6 7 -------

69.5 5.110.312.7 2.4 55.7 8.2 17.2 16.9 2.0 26.3 14.522.534.6 2.1 10.2 18.0 20.2 48.3 3.3 2.2 3.9 5.0 6.1 7.3

52.8 31.6 5.2 4.0 6.4 11.0 63.0 9.2 9.6 7.1 9.2 64.8 9.310.1 6.5 9.5 53.7 8.6 15.9 12.3 1.7 3.9 5.6 6.5 7.3

39.6 9.3 11.2 12.5 7.120 32.520.6 9.8 14.2 7.5 15

7.429.0 16.8 8.4 7.930. 7.8 4.7 18.4 15.3 15.6 6.631 0.8 1.6 3.6 3.5 4.3 5.2 6

45.213.9 8.1 6.310.416 9.025.617.5 6.213.229

1.6 11.7 26.3 17.4 5.2 13.3 24 0.9 1.8 2.7 3.7 4.5 5.7 6

33.6 23.530.0 6.0 2.8 4.1 35.0 16.543.0 1.9 1.0 2.6 28.7 18.040.5 3.5 2.0’ 7.3 26.9 5.845.3 5.3 6.110.6 25.7 11.947.8 4.9 9.7

2.4 3.7 4.8 5.7 6.3 7.4 8.2 6.3 5.746.811.61 9.410

5.8 2.663.3 10.8 11.8 4 1.6 2.2 2.7 3.8 5.1 6.1 6

10.7 5.619.2 6.744.0 6.8 7 14.0 5.918.310.740.9 4.2 6 0.7 1.9 3.1 3.81 5.6, 7.8 9

- 8 -

1.5 1.4 7.:

-

. -

1 L i

-

Per cent sedimentation

-

$1

22

75 93

39 89

7 68

25 7

59 8

38 62

87 6

75 10

80 15

91 9 99 1

92 81

- -

- --

I

-

12

2

-

310 -

10

2 3

- * Average mobilities (u) in 1 X 10-s cm.2 volt-1 sec.-l. The mobilities are nega-

tive. t Sedimentation velocities (~~0~) in 1 X lo+ cm. sec.-’ unit field-‘. 3 Pooled samples. J Two animals. 11 Sedimentation constant 7.6

agreement with previous workers (3, 4), two electrophoretic components were observed. Their mobilities did not agree with any of those of the components of the whey, which suggests that the bovine whey samples studied did not contain significant amounts of casein.

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H. F. DEUTSCEX 441

Samples of the various milks showed characteristic changes at various times post partum. In general, these changes were characterized by pro- gressive decrease with time post partum in the nitrogen content of the whey and the components of low electrophoretic mobility. The higher molecular weight components showed similar decreases as revealed by sedimentation analysis. Such observations are compatible with numerous data in the

,

0 TIME

I DAY

2 DAY

POOLED AGED

FIG. 2. Descending electrophoretic boundaries of bovine whey at various times post partum.

literature which show marked increases in the albumin-globulin ratio of milk proteins with increase in postpartum time. The amounts of nitrogen found in the three milk fractions of the animals studied at various times following delivery are shown in Table I. The electrophoretic analyses for the same samples are shown in Table II. Numbers by which the com- ponents for a given species are designated bear no relationship to the same numbers used with components of other species, but are used merely as a

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442 WHEY PROTEINS

matter of convenience. Sedimentation analyses of the first and latest postpartum whey samples for each species are also included with the other data in Table II.

ii marked change in the concentration of various protein constituents of bovine whey will be apparent from an inspection of Tables I and II. The electrophoretic and sedimentation patterns reflecting these changes are shown in Figs. 2 and 8. In addition to the sedimentation components recorded in Fig. 8, there is present a component with sedimentation con-

0 TIME

2 DAYS

FIG. 3. Desrending eleckophoretic boundaries of goat whey at 0 and 48 hours post parbm .

stant szh. = 20 Svedberg units. This component represents about 10 per cent of the t,otal protein. After 2 days, the milk constituents began to approach those of normal pooled milk. The electrophoretic composition of bovine colostrum is analagous to that recorded by Smith (4). The glob- ulins of slower electrophoretic mobility are relatively heterogeneous. They possess a mobility within the range of the r-globulins separated from bovine serum by Smith (4) and by Hess and Deutsch (5). The elec- trophoretic pattern of the various bovine milk samples shown in Fig. 2

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H. F. DEUTSCH 443

strongly suggests t,hat several proteins are present in the component desig- nated Component 1.

Relatively rapid changes in t,he whey proteins of the goat are indicated hi the nitrogen analysis. These proteins mere essentially normal as indicated by nitrogen a.nd electrophoretic analysis for a sample taken a.t 48 hours

2-3 DAYS

I4 DAYS

90 WAYS

FIG. -I

WLAND - CHINA

7.

7

AL

1 L

’ I

0 TME

9 DAYS

CHESTER- WHITE

;

0 TIME

3 DAYS

14 DAYS

FIG. 5

FIG. 4. Descending electrophoretic boundaries of human whey at various times post partum.

FIG. 5. Descending electrophoretie boundaries of whey of Chester white and Poland-China pigs at various times post partum.

post partum (Fig. 3). On the basis of chemical analyses, Bergman and Turner (6) have reported that the goat milk constituent,s tend to approach a stable condition on about the 3rd to 4th day post partum. The electro- phoretic mobility of the fraction of slow movement was close to that re- ported for the analagous constituent of goat blood plasma (1, 7). More-

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444 WHEY PROTEINS

over this component was relatively homogeneous in the electrophoretic field. The sedimentation studies (Fig. 8) show changes similar to those recorded for the cow whey.

Relatively slight changes fith postpartum time in the electrophoretic and sedimentation diagrams were found in the case of the proteins of human milk whey. However, it can be seen from Fig. 4 that the amount of the electrophoretic Component 1 is largest in samples taken soon after

4

7 DAY

35 DAY

FIG. 6. Descending electrophoretic boundaries of sheep whey at various times post partum.

FIG. 7. Descending electrophoretic boundary of horse whey 11 days post partum.

birth. The sedimentation patterns for human milk whey are more com- plex compared to those observed for the other species; also smaller amounts of the proteins of sedimentation constants s2b = 1 to 3 Svedberg units are present.

The electrophoretic patterns of the milk plasma proteins of two breeds of pigs revealed slight differences even within the species, as is shown in Fig. 5. However, postpartum changes similar to those of the other animals studied, as reflected by the electrophoretic and sedimentation analyses

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H. F. DEUTSCH 445

(Fig. 8), are also evident. The whey prepared from milk collected from the Chester white sow 14 days post partum contained a component of lower electrophoretic mobility than had been previously noted. The sedimenta- tion patterns of the pig wheys reveal two definite heterogeneous areas for the proteins of lower sedimentation constant.

GOAT 1-1 3 6

PIG 5’1 3 6

HUMAN

SHEEP s-1 3 6

4

FIG. 8. Sedimentation diagrams of milk wheys at various times post partum. The earliest samples shown at the top of series and times post partum for various animals are as follows: cow, 0 and 2 days, aged; horse, 11 days; goat, 0 and 2 days; pig (Poland-China), 0 and 9 days; human, 1, 2, and 90 days; sheep, 7 and 35 days. Sedimentation constants in Svedberg units form the abscissae.

Two samples of sheep milk were studied. The sample taken 7 days post partum gave a whey which showed larger amounts of the globulin constituent compared to the later sample. The electrophoretic patterns of the whey proteins of this species, Fig. 6, are quite similar to those of the goat wheys. The sedimentation patterns of the sheep wheys shown in Fig. 8 reveal the presence of slightly larger amounts of protein of higher sedimentation constant in the sample taken at the earlier postpartum time.

The wheys from two samples of horse milk taken relatively late post

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446 WHEY PROTEINS

partum showed almost identical electrophoretic composition, as is seen by the results in Table II. However, the electrophorrbic patt,ern shown in Fig. 7 is distinctly different from that of the other animals. There is an electrophoretic component of very low mobility present in these wh ys. A component in t#he whey of a Chester white sow (Fig. 5), taken 1-i days post partum, has a similar mobility. The sedimentation diagram for horse whey (Fig. 8) reveals less heterogeneity in the prot.eins of low sedimentation constant, than is characteristic of the ot,her animal ~+eys.

DISCUSSIOA’

The changes in the nitrogen content found in samples of milk of various animals at different times post partum correspond in general with values reported in the literature. A port,ion of the nit)rogen of milk wheys is known to be dialyzable and to be made up of nitrogenous constituents other than protein. Thus, in the human milk sample collect.ed 1 t,o 2 days post partum, 31.3 per cent of the whey nit,rogen was found to b(l dialyzablt?. This is in agreement with the high non-protein nitrogen value of 15 to 25 per cent reported for normal human whole milk by various investigators (8-11). Cow’s milk has been reported to have much lower non-protein nitrogen values than human milk (9).

Colostrum wheys are characterized by the presence of large amounts of proteins of low electrophoretic mobility. In these wheys there is also found a marked increase in the amount of the protein component, of sedimentation constant s20u! = 6 Svedberg units. Smith (12) has found that the immune globulins of bovine colostrum which he has separated by fractionation and which correspond to our Component 1 consist largely of molecular kinetic units having a sediment’ation constant of spew = 7 Svedberg units. The corresponding immune globulins of our ucfractionated wheys have been found to give somewhat lower sedimentat,ion constants. Pedersen (13) has reported a normal lactoglobulin of milk to have a sedimentation con- stant of s20tu = 7.0 to 7.4 Svedberg units, although a fraction made up largely of this protein was found to give a somewhat lower value. The greater part of normal bovine whey proteins consists of the lactoglobulin of Palmer and the albumin of Kekwick, which have sedimentation constants (~20~) equal to approximately 3 and 2 Svedberg units respectively. A component not shown in Fig. 8 and possessing a sedimentation of s2h = 20 Svedberg units makes up approximately 10 per cent of the proteins of bovine colos- trum whey. So heavy a protein molecule has not been found in the serum r-globulins of normal (5) or hyperimmunized (12) cows. However, Smith (12) has recognized such a component in the euglobulin fraction of t.he immune lactoglobulins of bovine milk and colostrum.

The electrophoretic patterns of the whey proteins are characteristic

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H. F. DEUTSCH 447

of the species, as is true of the blood serum and plasma proteins (1, 2). Although some of the mobilities of the electrophoretic constituents of a given whey agree with t,hose of certain plasma constituents of the same animal, as determined previously (l), there seems to be little relation be- tween the plasma and whey proteins from an electrophoretic standpoint. The large amounts of protein in the wheys from the first postpartum milk samples, which we have designated Component 1 do, however, possess an electrophoretic mobility much like that of the immune globulins of the blood plasma.

The young of various species receive antibodies from different sources. The placental structure of humans is essentially one cell thick and allows the antibody protein to pass from the maternal blood to the young in utero. However, it appears that young foals, kids, lambs, pigs, and calves acquire these proteins from t,he colostrum (14-17). In these animals the placental membranes contain three or more cell la.yers (18). This difference may be reflected in the failure of human whey to show the high level of protein Component 1 which is observed in the early postpartum wheys of the pig, goat, and cow.

SUMMARY

The whey proteins of various animals show marked differences in elec- tophoretic composition and, as in the case of the plasma proteins, are characteristic for a given species. Marked changes in the protein content of the milk at various times post partum are indicated by changes in the electrophoretic and sedimentation patterns.

The author wishes to acknowledge t.he technical assistance of Mr. E. H. Hanson, Mrs. Alice McGilvery, and Miss M. S. Morris. The helpful suggestions of Dr. J. W. Williams are greatly appreciated. Various samples of milk were received through the courtesy of Dr. R. H. Grummer and Dr. R. C. Herrin. The work was supported in part by grants from the Wis- consin Alumni Research Foundation and the United States Public Health Service.

BIBLIOGRAPHY

1. Deutsch, H. I’., and Goodloe, M. B., J. Biol. Chem., 161.1 (1945). 2. Moore, 1). H., J. Riol. Chem., 161, 21 (1945). 3. Warner, R. C., J. Am. Chem. Sot., 66, 1725 (1944). 4. Smith, E. L., J. Biol. Chem., 164, 345 (1946). 5. Hess, E., and Deutsch, H. F., J. Am: Chem. Sot., in press. 6. Bergman, A. J., and Turner, C. W., J. Dairy SC., 20, 37 (1937). 7. Nichol, J. C., and Deutsch, H. F., J. Am. Chem. SIX., in press. 8. Macy, I. G., Outhouse, J., Long, RI. I,., Brown, M., Hunscher, H., and Hoobler,

B. R., Proc. Am. Sot. Riol. Chem., J. Hiol. Chwn., 74, p. xxxi (1927).

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448 WHEY PROTEINS

9. Courtney, A. M., and Brown, A., Arch. Dis. Childhood, 6,36 (1930). 10. Denis, W., Talbot, F. B., and Minot, A. S., J. Biol. Chem., 39, 47 (1919). 11. Frehn, A., 2. physiol. Chem., 66, 256 (1910). 12. Smith, E. L., J. Biol. Chem., 166,665 (1946). 13. Pedersen, K. O., Biochem. J., 30,948 (1936). 14. Howe, P. E., J. Biol. C/tern., 49, 115 (1921). 15. Earle, I. P., J. Agr. Res., 61, 479 (1935). 16. Little, R. B., and Orcutt, M. L., J. Exp. Med., 36,161 11922). 17. San Clemente, C. L., and Huddleson, I. F., Michigan State Agr. Ezp. Station,

Tech. Bull. 186, 3 (1943). 18. Ratner, B., Jackson, H. C., and Gruehl, H. L., J. Immunol., 14,249 (1927).

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H. F. DeutschTHE MILK OF VARIOUS ANIMALS

A STUDY OF WHEY PROTEINS FROM

1947, 169:437-448.J. Biol. Chem. 

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