I n t e r a c t i o n o f Bov ine a - L a c t a l b u m i n and

/~ -Lactog lobul in dur ing Hea t ing

A. A. ELFAGM and J. V. WHEELOCK School of Biological Sciences

University of Bradford Bradford, West Yorkshire, BD7 1 DP

ABSTRACT

The degree of thermodenaturation of a-lactalbumin is greater when heated in the presence of/3-1actoglobulin than when heated by itself. This effect increases with temperature and pH (range 6.4 to 7.2). a-Lactalbumin interacts with an aggre- gated form of/3-1actoglobulin, a-Lactalbu- rain does not affect the amount of undenatured/3-1actoglobulin at the end of heating.

INTRODUCTION

In previous reports (3, 6), it was postulated that there is an interaction between /3-1acto- globulin and ~qactalbumin when milk is heated. By using the isolated proteins, we show that there is an interaction between ~-lactalbumin and /3-1actoglobulin during heating. Further experiments show the effect of temperature, pH, and relative proportions of the proteins and sulfhydryl blocking agents on the interaction.

MATERIALS AND METHODS

Preparation of Whey Proteins

~-Lactalbumin was prepared by the method of Aschaffenburg (2)./3-Lactoglobulin was pur- chased from BDH Chemicals Ltd., Poole, En- gland. Both proteins were purified by gel filtration on Sephadex G-100 prior to use. The proteins were pure by gel electrophoresis.

Preparation and Treatment of Protein Solutions

fl-Lactoglobulin and/or a-lactalbumin solu- tion(s) were prepared by dissolving freeze-dried material (.2 and/or .1 g/100 ml) in synthetic milk ultra-filtrate (SMUF) (7). In Exp. 1, the denatured protein was precipitated from the heated solution by ammonium sulphate (264

Received June 8, 1977.

g/liter). The filtrate was dialyzed and freeze- dried. In the other experiments no attempt was made to precipitate the denatured proteins by ammonium sulphate; the heated mixture was dialyzed against phosphate buffer (pH 6.8), and this sample was fractionated.

Preparation of Phosphate Buffer (pH 6.8)

The phosphate buffer was prepared by mix, ing 51 ml of .2 M NaH2PO4 with 49 ml of .2 M Na2 HPO4 and diluting to 200 ml with distilled water. The buffer also contained .02% NAN3.

Fractionation of the Heated Sample

This was performed by gel filtration at room temperature on Sephadex G-100 (Pharmacia GB, Ltd., London, England). The flow rate was approximately 15 ml/h, and the volume of each fraction was 4 ml. The absorbance of each fraction was read independently.

Gel Electrophoresis

This was done at 250 V for 2 h in a vertical cell (E-C apparatus corporation, Philadelphia) by using a polyacrylamide slab gel. Solution of 9.5% polyacrylamide (Cyanogum "41", BDH Chemicals Ltd., Poole, England) was used for the gel preparation, and the running buffer was .110 M-Tris-.053 M glycine, pH 8.6.

R ESU LTS

Interaction Between/~-Lactoglobulin and a-Lactalbumin (Exp, 1, Fig. 1)

The results show that there was a reduction in the amount of/3-1actoglobulin and of a-lactal- bumin recovered by gel filtration after heating at 80 C for 20 min. However, the results also show that the amount of a-lactalbumin re- covered was reduced markedly by the presence of/3-1actoglobulin during heating. The amount of/3-1actoglobulin recovered from the mixture was similar to that when the protein was heated by itself. This result shows that/3-1actoglobulin

1978 J Dairy Sci 61:28--32 28

INTERACTION OF e-LACTALBUMIN AND/3-LACTOGLOBULIN 29

control

heated

I~- lactoglobulin

1.1 I

1.0

.9

.g

.7

.6

.5

.4

.2

o

.8

.7

.6

.5

.4

.2

30 60 90

a- lactalbumin B- lactoglobul in + =-lactalbumin

I I I /

i i

l I

I I

0 30 60 90 0 30 60 90

tube number

FIG. 1. Gel filtration profiles of #-lactoglobulin, a-lactalbumin, and a mixture of the two proteins recovered from heated (80 C, 20 min) solutions at pH 6.7. The heated mixture contained .2 g / 1 0 0 ml /3-1actogtobulin and/or .1 g / lO0 ml adactalbumin. A column (65 × 2.5 cm) of Sephadex G-100 was used for fractionation, and the running solution was phos- phate buffer pH 6.8. Peaks I and I1 are/3-1actogtobulin and aqactalbumin.

÷ +

7 7 8 7 ~ - - ® =

I I I I

.2 I I

0 I / I /

.2 I I

0

m~ .2 Aa I /

0 I / I /

I I I I

0 40 80 0 40 80 0 40 80 0 40 80 0 40 80 0 40 80

tube number

FIG. 2. Gel filtration profiles of solutions of l~-lactogtobulin, a-lactalbumin, and a mixture of the two proteins, heated (80 C, 20 min) at different pH's. The concentration of the proteins in the heated mixture, detail s of chromatography, and identification of peaks I and II are in Fig. 1. See the text for identification of peaks A 1 and A s .

affected the thermodenaturation of a-lactalbu- min and that there was some form of interac- tion between the two proteins.

Effect of pH (Exp. 2, Fig. 2)

This experiment showed that the effect of /3-1actoglobulin on the loss of ~-lactalbumin during heating increased with pH (columns 5 and 6, Fig. 2). However, the presence of ~-lactalbumin did not have any marked effect on the thermodenaturation of ~3-1actoglobulin (columns 4 and 6, Fig. 2). In this experiment 10-ml aliquots of/3-1actoglobulin and/or ~-lact- albumin solution were adjusted to the desired

pH, heated, dialyzed against phosphate buffer, and then applied directly to the column in an attempt to retain some of the products of heating for separation by gel filtration. The profiles (column 4, Fig. 2) show that when /3-1actoglobulin was heated, a new fraction (peak A2) was obtained which eluted in the void volume of Sephadex G-100. This indicated that the molecular weight of A2 was > 150,000 daltons. At pH 7.2 another product of inter- mediate size (peak A1 ) could be observed. Peak A2 also appeared in the mixture when the two proteins are heated together (column 6, Fig. 2). Fractionation of the heated samples on Sepha- rose 4B (results not shown) showed that peak A2 eluted behind the void volume. This sug-

Journal of Dairy Science Vol. 61, No. 1, 1978

0 ELFAGM AND WHEELOCK

gests tha t A~ has a molecular weight less than 20 × 10 6 daltons.

Effect of Heating Conditions (Exp. 3, Fig. 3)

As the t empera tu re increased f rom 70 to 85 C, there was a progressive decrease in the

heating temperatures

control

70 C

74 C

77 C

80 C

85 C

%

&

.2

.1

I o

E .1 I

A i A ou 0

.2

~.~ 0 -

.1

0 ' Az

.2

.1

0 ' 0 40 80

I I

1

II I II

i ~ AI ,

II II

I | i FA 0 40 80 0 40 80

tube number

FIG. 3. Gel filtration profiles of solutions of /3-1actoglobulin, ~qactalbumin, and a mixture of the two proteins heated (pH 6.8) for 20 min at different temperatures. The concentration of the proteins in the heated mixture, details of chromatography, and identi- fication of peaks I and II are in Fig. 1. See text for identification of peaks A~ and A~.

a m o u n t o f native/3-1actoglobulin recovered, and the a m o u n t of the aggregated fo rm (peak Az) increased. Heating caused a reduc t ion in the a m o u n t of a- lac ta lbumin recovered, bu t there was little change as the t empera tu re of hea t ing increased. The loss of a- lac ta lbumin associated wi th the presence of/3-1actoglobulin could n o t be observed at 70 and 74 C bu t increased progressively f rom 77 to 85 C. a -Lac ta lbumin was af fec ted only after the appearance o f the aggregated form(s) of/3-1actoglobulin at 77 C. The results d e m o n s t r a t e d tha t the dena tu ra t ion of e - lac ta lbumin did no t involve any de tec tab le in te rmedia te form.

Effect of the Ratios of/3-Lactoglobulin:~-Lactalbumin (Exp. 4, Fig, 4)

For the ratios used, variation in the a m o u n t of/3-1actoglobulin relative to a- lac ta lbumin did n o t have any e f fec t on the loss of ~-lactalbu- min. However, when there was a high propor- t ion of /3-1actoglobulin (3/3-1actoglobulin to

~E -==

o~ mixing ,65

2 ~ : 2 a .

0

. 3 i I I

.2

2B:la i .I

0

3B:la

~) ,10 30 0 ;0 ;33 1 4O eO u '~0 80 D 40 80 0 40 BO

tube number

FIG. 4. Gel filtration profiles of solutions of t34actoglobulin, a-lactalbumin, and a mixture of the two proteins heated at 80 C for 20 min (pH 6.8). The relative concentration of the proteins was varied. Details of chromatography and identification of peaks I and II are in Fig. 1. See text for identification of peaks A 1 and A 2.

Journal of Dairy Science Vol. 61, No. 1, 1978

INTERACTION OF a-LACTALBUMIN AND ~-LACTOGLOBULIN 3 1

la- lac ta lbumin) the a-lactalbumin had a marked effect in reducing the comparatively large amount of aggregated 3-1actoglobulin (peak A2 ).

Effect of Sulfhydryl Blocking Agents (Exp. 5, Fig. 5)

The presence of iodoacetate during heating did not have marked effect, and the elution profiles were similar to those for controls. However, a small amount of an aggregated form of 3-1actoglobulin was observed when iodoace- tate was included in the solution. In contrast, N-ethylmaleimide (NEM) prevented the appear- ance of any aggregated form of 3-1actoglobulin on heating. Furthermore, in the presence of NEM, 3-1actoglobulin did not reduce the amount of ~-lactalbumin recovered after heat- ing. p-Hydroxymercuribenzoate (PHMB) in- duced a marked change in the profile of the unheated /3-1actoglobulin. This probably was

agent added

. 2 ~ A I I

C)

•

i .2 II A2 I

0 40 80 0 40 80 0 40 80 0 40 80 0 40 80 0 40 80 T~be Numbe~

FIG. 5. Gel filtration profiles of solutions of 3-1actoglobulin, a-lactalbumin, and a mixture of the two proteins heated (80 C, 20 min) at pH 6.8 in the presence of iodoacetate (.052 g/lO0 rrd), NEM (.035 g/lO0 ml), and PHMB (.1 g/100 ml). The concentra- tion of the proteins in the heated mixture, details of chromatography, and identification of peaks I and II are in Fig. 1. See text for identification of peaks A 1 and A~.

due to an irreversible change in the form of the molecule. However, the effect of PHMB in preventing the formation of the large aggregate (A=) of 3-1actoglobulin was similar to that of NEM.

DISCUSSION

Thermodenaturat ion of 3-1actoglobulin pro- ceeds via 2 major steps (1, 4, 10). First, there is formation of a small aggregate of four mono- mers which is referred to as the primary denatured (aggregated) form. This aggregation involves sulfhydryl groups and occurs at tem- peratures above 70 C. Above this temperature the rate of formation of the primary denatured form increased sharply to a maximum between 80 and 85 C. Second, there is conversion of the small aggregate to a large one (s20 = 29 S). This process is referred to as secondary aggregation. Secondary aggregation does not involve sulfh- ydryl groups and occurs at temperatures lower than those needed for the primary. McKenzie (8) prefers the terms type I and type II aggregation instead of primary and secondary aggregations, respectively. On the basis of Saw- yer 's data (10), McKenzie also outlined a third type of aggregation (type l i d which occurs via the monomers and does not require SH/S-S interchange and/or interaction.

In the light of this information, the products of the thermodenaturat ion of 3-1actoglobulin in our experiments may be identified as follows: the void volume peak on Sephadex G-100 (A2) is apparendy the large aggregate produced by type II aggregations whereas the small aggregate (A1) would be produced by type I aggregation. Using gel filtration on Sephadex G-200, Sawyer (10) was unable to separate the products of type I aggregation in a distinct peak, and, therefore, he postulated that they are heteroge- neous. On the other hand, the products of type II aggregation were eluted in a sharp peak behind the void volume peak. Similar character- istics were exhibited by peak A2 in our experiments since it is eluted in the void volume in Sephadex G-100 and behind the void volume on Sepharose 4B. The results in Fig. 4 show that the formation of the large aggregate (Peak A 2 ) increased with temperature. These findings conflict wi th the results of Briggs and Hull (4) which indicated that formation of the large aggregate (the secondary stage of aggrega-

Journal of Dairy Science Vol. 61, No. 1, 1978

3 2 ELFAGM AND WHEELOCK

tion) was suppressed by increasing the tempera- ture above 75 C. The results with NEM (Fig. 5) support the conclusion that sulfhydryl groups are essential to initiate aggregation; blocking these groups would prevent the formation of type I, so type II aggregation would not occur. The effect of iodoacetate on the thermode- naturation of /3-1actoglobulin has not been studied. However, our results (Fig. 5) show that this compound has little effect in inhibiting conversion of Al into A2. Figure 2 shows that after heating, the amount of native 3-1acto- globulin recovered decreased as the pH in- creased above 6.8. These findings agree with the results of Anderson (1). Mills and Creamer (9) have suggested that changes in pH alter the conformation of the protein and open up the molecule so that the sulfhydryl groups which normally are buried inside become more acces- sible (5). The results in Fig. 1 show that /3-1actoglobulin reduces the amount of c~-lactal- bumin recovered after heating. This indicates that there is probably an interaction between the two proteins during heating. The results in Fig. 3 suggest that the formation of the small aggregate (A1) of 3qactoglobulin is necessary for this interaction. The results in Fig. 5 show that NEM prevents the interaction. However, it is not possible to determine whether this effect is due to the inhibition of the formation of Al or the inhibition of the interaction between monomers of the two proteins.

The results in Fig. 2 show that variation in pH has no effect in the thermodenaturat ion of c~-lactalbumin. However, when 3-1actoglobulin is present, the loss of c~-lactalbumin increases as the pH is increased. Since the denaturation of

3-1actoglobulin increases with pH, this result agrees with the view that ~-lactalbumin inter- acts with the aggregated form ( A I ) of 3-1acto- globulin.

These conclusions fully agree with (3, 6) on whole milk and provide support for the conclu- sion that there is an interaction between 3-1ac- toglobulin and a-lactalbumin in whole milk during heating.

REFERENCES

1 Anderson, C. A. 1955. Heat denaturation of 3-1actoglobulin and c~-lactalbumin. Ph.D. thesis, University of Minnesota, St. Paul.

2 Aschaffenburg, R. 1968. Preparation of c~-lactalbu- rain from cow's or goat 's milk: A method improv- ing the yield. J. Dairy Sci. 51:1295.

3 Baer, A., M. Oroz, and B. Blanc. 1976. Serological studies on heat-induced interactions of c~-lactalbu- min and milk proteins. J. Dairy Res. 43:419.

4 Briggs, D. R., and R. Hull. 1945. Studies on protein denaturation. I. Electrophoretic study kinetics at neutrality of heat denaturation of 3- lactoglobulin. J. Amer. Chem. Soc. 67:2007.

5 Dunill, P., and D. W. Green. 1966. Sulfhydryl groups and the N ~ R conformational change in 3-1actoglobulin. J./Viol. Biol. 15:147.

6 Elfagm, A. A., and J. V. Wheelock. 1977. Effect of heat on ~-lactalbumin and 3-1actoglobulin in bovine milk. J. Dairy Res. 44: 367.

7 McGann, T. C. A. 1960. Studies on the caseinate- phosphate complex of milk. Unpublished Ph.D. thesis, National University o f Ireland, Cork.

8 McKenzie, H. A. 1971. 3-Lactog]obulins. Page 257 in Milk Proteins. Vol. II. H. A. McKenzie, ed. Academic Press, New York.

9 Mills, O. E., and L. K. Creamer. 1975. A conforma- tional change in bovine 3-1actoglobulin at low pH. Biochim. Biophys. Acta. 379:618.

10 Sawyer, W. H. 1968. Heat denaturation of bovine 3-1actoglobulins and relevance of disulfhide aggre- gation. J. Dairy Sci. 51:323.

Journal of Dairy Science Vol. 61, No. 1, 1978