Adsorption of euglobulin on agglutinating milk fat globules

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Adsorption of euglobulin on agglutinating milk fat globules The relatively high rate of creaming of the fat emulsion in milk at low temperatures, has been explained satisfactorily by the formation of clusters of the fat globules 1,~. HEKMAet al. ~,4 and BROUWER5 demonstrated that the clustering of the fat globules could be enhanced by addition of blood serum, the active constituent being the euglobulin. I)UNKLEY AND SOMMER2 likewise showed that clustering and creaming are promoted specifically by milk euglobulin. By analogy with the clustering of bacteria by specific antibodies, the clustering of the fat globules in milk is often called agglutination 6. It is generally accepted, that the euglobulin exerts its action after adsorption on to the surface of the fat globules, although this has never been proved unequivocally. More information on the euglobulin adsorption would be important for our understanding of the mechanism of the agglutination and in the present communication we report the determination of the adsorption of 13tI-labelled euglobulin, and of some other main milk proteins, on to the globules under different experimental conditions. Both raw bulk milk from Frisian cows and "agglutinin-depleted" milk2, ~ were used. Euglobulin was isolated from colostral cow's milk according to the method of SMITH8./3-Lactoglobulin was prepared as described by LARSON AND JENNESS9; casein was precipitated from skim milk at pH 4.6. The proteins were mildly labelled with 18tI by the technique to be described by VERINGA AND KERKHOF MOGOTTM. It was checked that iodination did not affect the clustering properties of the euglobulin (cf. Fig. I, Curves o and o). The sedimentation coefficients and electrophoretic patterns of all the iodinated proteins likewise were unchanged.

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"2 E 60 ~40

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600 1000 14.00 ' ~51[~Euglobulin added (mg/kg milk)

Fig. i. Clustering times of milk-fat globules a n d adsorption of 131I-labelled euglobulin on t h e surface o f t h e fat globules of "agglutinin-depleted" milks. A, e x p e r i m e n t s at IO°; O, e x p e r i m e n t s a t 5 ° ; O, clustering times w i t h u n i o d i n a t e d euglobulin at 5 °.

13iochim. Biophys. Acta, 94

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Clustering times of the fat globules were determined b y visual inspection of a thin layer (0.5 mm) of the milk as described b y DUNKLEY AND SOMMER~. The proteins were added to the milk dissolved in a salt solution simulating milk ultrafiltrate n. In most cases their distribution between the fat and plasma phases was determined after spontaneous creaming of the milk fat for 24 h at 4 °. In a few experiments concerning the effect of temperature on the adsorption, the milk was gently centrifuged for 5 min at 3000 × g to promote the separation which was poor at the higher temperatures. The partition of the proteins was calculated from the radioactivities and the fat contents of the milk and the cream TM. Let x a n d y be the amounts of protein present per IOO g of fat and plasma phase, respectively. Then for the milk IOO A ---- p x + (IOO - - p ) y , where A is the amount of protein added to IOO g milk and p is the percentage of fat in the milk. Similarly for the cream; 1:oo B ~ qx + ( I O O - q)y, where B is the amount of protein measured in IOO g cream and q is the percentage of fat in the cream. The values of x calculated from these equations are presented in the last column of Table I. TABLE I PARTITION OF SOME 131I-LABELLED NORMAL SAMFLES OF MILK

MILK

PROTEINS

BETWEEN

Expt. Added (mg/kg of milk) No. Euglofl-Lacto- Casein bulin globulin

Treatment of the milk

I 2

sp. cr.* 32.6 3 h, 4 °, centr.** 63.6 0. 5 h, 45 °, centr. 56.o sp. cr. 76.0 sp. cr. 287.0 2 h, 4 °, centr. 271.o 2 h, 2o °, centr. 284.o sp. cr. 44.4 sp. cr. 62.3

3 4 5 6

26.1 61. 3 61.3 60.2 239.8 239.8 239.8 58.1 78.1

THE

Protein in cream (mg/kg)

FAT

AND

PLASMA

Fat content (%)

PHASES OF

Milk

Cream

Protein on fat globules (rag/zoo g)

3.78 4.67 4,67 4,o6 4.38 4,38 4,38 3.45 3,75

25.2 12.2 20.o 32.8 19.9 16.9 18.9 23.7 23.4

5.6 9.0 2.8 11. 3 53.0 47.8 53.1 o o

* sp. cr. = s p o n t a n e o u s creaming, ** centr. = centrifuged.

Some typical results are collected in Fig. I and Table I. In accordance with the generally accepted view, the figure shows that increased clustering of the fat globules is indeed accompanied b y enhanced adsorption of euglobulin. I t appears that the amount adsorbed is strongly dependent on the temperature, as is shown b y comparison of the adsorption isotherms at 5 ° and IO °. At surface concentrations of about 2 mg of euglobulin per g of fat, the clustering time becomes constant. Expt. 2 of Table I clearly demonstrates that at 45 ° the adsorption of euglobulin has decreased considerably, accounting for the well-known effect of temperature on the rate of creaming TM. Expts. 3-6 of the same table show, further, that among the other milk proteins investigated, about 8 % of fl-lactoglobulin is adsorbed, whereas casein is not adsorbed at all. It should be stressed, however, that the adsorption of fl-lactoglobulin does not lead to an increased agglutination of the milk fat globules 14. I t is of interest to assess the specific area occupied b y the adsorbed proteins Biochim. Biophys. Acta, 94 (I965) 576-57 8

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under conditions normally prevailing in milk. Normal milk contains about 4oo mg of euglobulin per kg (ref. 15). With the reasonable assumption that from this quantity the same percentage is adsorbed as was found with the small addition of 131I-labelled euglobulin (cf. Expt. I, Table I), we can calculate the concentration at the surface to be 0.8 mg/g of fat. For/3-1actoglobulin, owing to its high content of 4 g/kg of milk 1~, a similar calculation leads to a surface concentration even as high as 8 mg/g fat. For Frisian cows the total surface of the fat globules is estimated at 1.8 m2/g of fat ~3. Consequently the area available for the adsorbed euglobulin and/3-1actoglobulin, is about 0.2 m2/mg protein. This figure should be compared to the values recorded for close-packed, unfolded protein monolayers, which are of the order of o.8-I.o m2/mg (ref. 16). Obviously the adsorbed proteins cannot form a single unfolded monolayer at the fat surface. Either several of such monolayers are superimposed, or the polypeptide chains are only partly attached to the surface, their free ends projecting into the plasma phase. It has been suggested previously ~, that the adsorbed euglobulin would cause agglutination by re-inforcing the the London-Van der Waals attraction between the fat globulins. In view of the small amount of euglobulin adsorbed, it is evident that this assumption is no longer tenable. It has further been demonstrated2,14, that the addition of euglobulin does not affect the C-potential of the fat globules. Thus it seems unlikely that euglobulin acts by reducing the electrical surface potential below a critical level 2. Rather the data presented above suggest that agglutination is brought about by the formation of euglobulin bridges between the fat globules. The model of the surface layer with the peptide chains projecting into the plasma phase would fit best into this picture.

Netherlands Institute for Dairy Research, Ede (The Netherlands)

T. A. J. PAYENS J. KooPs M. F. KERKHOF MOGOT

I W. VAN DAM AND H. A. SIRKS, Verslag. Landbouwk. Onderzoek., 26 (1922) lO6. 2 W. L. DUNKLEY AND H. H. SOMMER, The creaming of milk, Res. Bull. Agr. Expt. Slat. Univ.

Wisc., No. 151, Madison, lO44. W. VAN DAM, E. HRKMA AND H. A. SIRKS, Verslag. Landbouwk. Onderzoek., 28 (1923) IOO. E. HEKMA AND H. A. SIRKS, Verslag Landbouwk. Onderzoek., 29 (1924) 94. E. BROUWER, Verslag Landbouwk. Onderzoek., 3 ° (1925) 261. S. ORLA-JENSEN, C. LE DOUS, J. JACOBSEN AND M. C. OTTE, Lair, 9 (1929) 724 • P. F. SHARP AND V. N. KRUKOVSKY, J. Dairy Sci., 22 (1939) 743E. L. SMITH, J. Biol. Chem., 165 (1946) 665 . B. L. LARSON AND R. JENNESS, Biochem. Preps., 4 (1955) 23. H. A. VERINGA AND M. F. KERKHOF MOGOT, tO be p u b l i s h e d . R. JENNESS AND J. KOOPS, Neth. Milk Dairy J., 16 (1962) 153. H. MOLDER, Neth. Milk Dairy J., i i (1957) 197. R. JENNESS AND S. PATTON, Principles of Dairy Chemistr% W i l e y , N e w Y ork, C h a p m a n a n d H a l l , L o n d o n , 1959, p. 269. 14 T. A. J. PAYENS, Kiel. Milchwirtsch. Forschungsber., in t h e press. 15 T. L. McMEEKIN, in H. NEORATH AND K. BAILEY, The Proteins, Vol. 2A, A c a d e m i c Press, N e w Y o r k , 1954, p. 389. 16 H. B. BULL, Advan. Protein Chem., 3 (1947) 95. 3 4 5 6 7 8 9 IO II 12 13

Received October I5th, 1964 Biochim. Biophys. Acta, 94 (1965) 576-578