Electrophoresis of Cottage Cheese W h e y Proteins and T h e i r P o l y m e r s

DORETTA N. LEE, EDWIN E. MOORE, and RICHARD L. MERSON 1 Department of Food Scienceand Technology University of California Davis 95616

appeared in whey at pH above 6.2 and at - 1 C.

ABSTRACT

Cottage cheese whey solutions containing sodium dodecyl sulfate were resolved electrophoretically on 5% sodium dodecyl sulfate polyacrylamide gels. Major bands were identified by comparing their electrophoretic mobilities to those of known whey components. Other components were identified principally from molecular weights determined from a calibration correlating mobility and molecular weight. Conditions which affect polymerization of proteins were studied in whey and solutions of purified/3-1actoglobulin. Formation of a number of polymers was induced by concentrating the whey samples, lowering the temperature, adjusting pH, or adding salts. The dimer, trimer, tetramer, and octamer of /3-1actoglobulin, the dimer and trimer of bovine serum albumin, and several unidentified components in the 100,000 to 300,000 molecular weight range were observed. The octamer state of J3-1actoglobulin was observed in whey at pH values between 5.1 and 8.0, at temperatures below 10 C, and with .2M addition of potassium thiocyanate, potassium iodide, calcium chloride, or sodium acetate. Similar polymer formation, and temperature and pH effects, were observed with solutions of purified ~-lactoglobulin, which contained dimers, trimers, and tetramers. The/3-lactoglobulin octamer in whey samples could be dissociated by the addition of acid. The bovine serum albumin dimer

INTRODUCTION

Polyacrylamide gel electrophoresis of proteins in sodium dodecyl sulfate (SDS) has gained increasing application as a simple and economical means of identifying and characterizing proteins. The binding of SDS to proteins minimizes charge differences among protein molecules and results in electrophoretic migration proportional to molecular size (10). The technique was introduced by Shapiro et al. (15) in 1967 in a study of muscle protein and was further refined by Weber and Osborn (25) and Dunker and Rueckert (4). Satisfactory results have been obtained when applying the technique to studies of various muscle proteins (6, 11, 14, 26) and in milk protein analysis (2, 5, 9). There is cumulative evidence that constituents of high molecular weight initially present or formed during ultrafiltration of whey are at least partly responsible for membrane fouling (7). This paper reports the use of SDS electrophoresis to observe formation of high molecular weight whey protein polymers that result from changes in concentration, pH, temperature, and addition of various neutral salts. Identification of native whey proteins was confirmed by comparing the electrophoretic mobilities with known whey protein standards. Molecular weights of whey proteins and polymers were determined from a calibration curve based on both whey and non-whey protein markers and were compared to those reported in the literature. In addition /3-1actoglobulin was purified and subjected to various environmental alterations to aid in identifying high molecular Received September 26, 1974. ~Supported in part by the Dairy Council of weight polymers which were observed in whey samples. California. 658

ELECTROPHORESIS OF WHEY PROTEINS

659

anhydrous methanol, and 875 ml of distilled water). After destaining, the gels were stored in 7.5% acetic acid solution.

MATERIALS A N D METHODS SDS Disc Gel Electrophoresis

The electrophoresis procedure followed that described by Weber and Osborn (25) with modifications. In the overnight incubation of protein samples at 40 C, .002M iodoacetamide (1% in SDS and .002M i o d o a c e t a m i d e ) w a s used instead of 2-mercaptoethanol to prevent disulfide interchange reactions of sulfhydrylcontaining proteins (16). Sucrose crystals were dissolved in the incubated protein samples before application to the gels to retard diffusion of samples into the upper buffer chamber. Because of an interest in high molecular weight components of whey which foul ultrafiltration membranes (7), relatively porous 5% gels were used. The acrylamide to methytene-bisacrylamide ratio of the 5% acrylamide solution was mahntained at 29:1 ( w / w ) a s recommended by Dunker and Rueckert (4). Electrophoresis was at room temperature at a constant current of 8 mA per gel for 5 h in phosphate buffer (3.9 g NaH2PO4, 19.3 g NaHPO 4 • 7 H20, 1 g SDS per liter, pH 7.0). The gels were stained in Coomassie brilliant blue solution (1.25 g Coomassie brilliant blue in 454 ml of 50% methanol and 46 ml of glacial acetic acid) for 5 h. They were destained electrophoretically for another 5 h in a gel electrophoresis apparatus using destaining solution (75 ml of glacial acetic acid, 50 ml

Molecular Size Calibration Graph

A molecular size calibration curve was prepared according to the internal calibration technique of Dunker and Rueckert (4). Both whey and nonwhey protein markers were used. Their molecular weights and sources are in Table 1. Preparation of Protein Samples for Polymerization Studies

Polymerization behavior of whey proteins was studied by comparing electrophoretic patterns of native whey proteins to those of proteins in whey that was subjected to various perturbations of the solvent environment. 1) Concentration. Commercial cottage cheese whey o b t a i n e d from a local cheese maker was concentrated (8) to one-fourth, one-fifth, or one-sixth its original volume on a PMIO (Amicon Corp.) membrane in a thinchannel ultrafiltration (UF) cell (Model CEC1, Amicon Corp.). The retentate was continually recycled to the feed tank. The feed whey, the bulk retentate (BR) left in the feed tank at the end of each of the three ultrafiltration experiments, the immediate retentate (IR) accumulated on the membrane in the UF cell, and the

TABLE 1. Proteins used for calibration curve in Fig. 2.

Proteins

Molecular weights (4, 25)

Whey protein markers Immunoglobin Bovine serum albumin

160,000 66,000

3-Lactoglobulin

18,500

a-Lactalbumin

16,200

Nonwhey protein markers fl-Galactosidase Ovalbumin Pepsin Trypsin

130,000 43,000 35,000 23,500

Source

Pentex Nutritional Biochemicals Corp. (Bovine Albumin Fraction V) Procedure of Aschaffenburg and Drewry (1) Procedure of Robbins and Kronman (12) Worthington Gift from Prof. R. E. Feeney, U.C. Davis Armour (porcine) Armour (bovine)

Journal of Dairy Science Vol. 58, No. 5

660

LEE ET AL.

ultrafiltrate which passed through the membrane were analyzed qualitatively by SDS electrophoresis. Sample sizes of 30/~1 of feed whey, 10 /al of bulk retentate, 10 /~1 of immediate retentate and 30/~1 of ultrafiltrate were used to minimize variations in sample loading on the SDS gels. 2) pH. Whey samples with pH adjusted to 1.9 to 10.7 were prepared with N HCL or N NaOH. 3) Temperature. Whey solutions were held overnight at -1, 2, 10, 50, and 80 C to allow equilibration at the various temperatures. Overnight incubation of samples in SDS and iodoacetamide before electrophoresis was also performed at these temperatures. Samples were applied directly to the SDS gels without any significant change in temperature before electrophoresis. 4) Salts. Effects of salts on macromolecular stability were induced by changing the ionic environment. Both "salting in" and "salting out" reagents of the Hofmeister series were added to whey in .2M concentration. Salting-in reagents were potassium thiocyanate, potassium iodide, and calcium chloride. Salting-out reagents were sodium sulfate and sodium acetate. 5) Purified ~-lactoglobulin. Based on the polymerization behavior in response to temper-

ature and pH adjustments, purified j3-1actoglobulin from two different sources was subjected to low temperature (2 C) and alkaline pH treatments. Beta-lactoglobulin was crystallized by the procedure of Aschaffenburg and Drewry (1). Results were checked by chromatographically purified /3-1actoglobulin (27). Both optimal and heavy sample loading of the /3-1actoglobulin were used to demonstrate that additional high molecular weight bands did not result from impurities in the samples.

RESULTS AND DISCUSSION Identification of Bands

Electrophoretic separations of whey proteins and individual whey protein standards on 5% SDS polyacrylamide gels are in Fig. 1. Molecular size calibration with bovine serum albumin as the reference for relative mobility is in Fig. 2. Protein bands in the SDS gels are identified by molecular weight in Table 2.

\ ZOO -

%

~ o v r n e serum albumin trin'~

X amma-jIIobulm

~bovine serum albumin elimar

1OO - .

Molecular Weight

~alactosiOase

m

~

b. . . . .

.Ib . . . . .

se,um

m0mer

199,000 160.000- ~ 1 ~ 132,000

--

~vllbumln

°

94.900 -~41~ ~ 84,000 ~

66.000 trypsin

]64:ooo-00O-~ t 29,S00---~ 23,000"~

-

,, I

18,500,~11~B

i

i ~

16.2oo~

20 - -

i

g ~o

a

b

c

d

e

f

g

h

FIG. 1. Sodium dodecyl sulfate electrophoretograms of a whey sample and seven individual protein standards: a, whey; b, gamma-globulin; c, bovine serum albumin; d, as-casein; e, 3-casein; f, K-casein; g, ~3-1actoglobulin; and h, a-lactalbumin. Journal of Dairy Science Vol. 58, No. S

p'lact°il°~ulin X q- laCtalbumln

I

Q.4

L 0,8

t 1,2

[ 1.6

I

2.0

RELATIVEId0BILITY

F'IG. 2. Standard curve for sodium dodecyl sulfate gel electrophoresis using known proteins as molecularweight markers: e, non-whey protein standards; o, whey protein standards.

ELECTROPHORESIS OF WHEY PROTEINS TABLE 2. Identification of bands in sodium dodecyl sulfate gels of whey samples. Molecular weight

Protein

160,000 94,000 84,000 66,000 36,000 34,000 29,5 O0 23,000 18,500 16,200

~,-globulin caseinate complex (tentative) caseinate complex (tentative) bovine serum albumin monomer 13-1actoglobulin dimer as-casein 13-casein casein monomer /3-1actoglobulin monomer a-lactalbumin

Major whey proteins, which consist of 7globulin (160,000 daltons), bovine serum albumin (BSA) (66,000 daltons), 13-1actoglobulin (18,500 daltons), and a-lactalbumin (16,200 daltons), are resolved as dark bands in Fig. la. These bands correspond to the principal bands of the whey protein standards Fig. lb, lc, lg, and lh. Under conditions in both whey and purified/3-1actoglobulin samples, 13-1actoglobulin resolves predominantly as the monomer (18,500 daltons) on SDS gels at room temperature and pH 4.5. The BSA standard, however, resolves into several components including bands at 66,000, 132,000, and 198,000 daltons corresponding to the BSA monomer, dimer, and trimer. Less intense protein bands in Fig. la appear consistently in whey at 94,000, 84,000, and 23,000 dahons but with variability in the region from 29,500 to 36,500 daltons. Intensity and consistency of bands in the latter region vary with whey batches and may be caused by variable residual casein content. The principal bands in the as-casein and 13-casein standards (Fig. ld, le) correspond to the 34,000 and 29,500 dalton bands, respectively, in whey. These values agree with SDS values for these caseins reported by Mullin and Wolfe (9). However, the principal components in the K-casein standard resolved as a doublet (Fig. lf) with bands at 34,000 and 32,000 daltons. The 34,000 band is believed to be as-casein and the 32,000 band K-casein, although the latter value is higher than the 28,000 molecular weight for mercaptoethanol-reduced K-casein reported by Swaisgood et al. (17) or the 26,000 SDS value

661

reported by Mullin and Wolfe (9). In whey, t3-casein (29,500 dahons) appeared more frequently than the 34,000 to 36,000 bands. At times dimeric ~3-1actoglobulin (36,000 daltons), K-casein and/or as-casein (34,000 daltons), and /3-casein (29,500 daltons) were in three discrete bands. Other times, it was possible that these proteins masked each other because of incomplete resolution. Regularly observed minor bands of 94,000, 84,000, and 23,000 daltons were not identified satisfactorily. Difficulties encountered in preparing pure casein samples and the indefinite, complicated nature of caseinate complexes hindered identification. However, from literature values of the molecular weights of casein and casein complexes (9, 13, 19), it is tentatively regarded that the 23,0oo dalton band is monomeric casein and the 94,000 and 84,000 dalton bands are caseinate complexes. C o n c e n t r a t e d Samples

Results of SDS electrophoresis of whey samples concentrated by ultrafiltration are in Fig. 3. High molecular weight proteins were formed with samples of increasing concentration. At 4-fold concentration, the immediate retentate sample (Fig. 3b) shows traces of octameric 13-1actoglobulin (144,000 daltons just below the regular ~/-globulin band) plus bands Molecular Weight

~,

265,000 ----.1~ 230.000

ooo ~ s3'.ooo

"'ji ~

i e

b

c

d

e

f

g

h

FIG. 3. Sodium dodecyl sulfate electrophoretograms of whey ultrafiltration samples concentrated 4-fold, 5-fold, and 6-fold: a, feed whey; b, 4-fold IR; c, 4-fold BR; d, 5-fold IR; e, 5-fold BR; f, 6-fold IR; g, 6-fold BR; and h, ultrafiltrate. Journal of Dairy Science Vol. 58, No. 5

662

LEE ET AL.

at 58,000 and 53,000 daltons (just below the BSA band). The multiplicity of high moleculal weight polymers increased markedly at 5-fold concentration as shown in Fig. 3d. In addition to the regularly observed bands, discrete 144,000 (~-lactoglobulin octamer), 132,000 (BSA dimer), and 122,000 dalton bands were present. There were even slight traces of higher molecular weight polymers: the top 265,000 dalton band, which may be the tetrameric form of BSA, and a 230,000 dalton band. Electrophoretic pattern was similar with the immediate retentate sample (Fig. 3f) at 6-fold concentration. There is an additional light band of 110,000 daltons. Resolution in the three bulk retentate samples (Fig. 3c, 3e, and 3g) is not as defined as that of the immediate retentate samples (Fig. 3b, 3d, 3f). In all three bulk retentate samples, the 110,000 dalton band appeared to be fairly consistent. The difference in resolution between immediate and bulk retentate samples may be explained by a different local environment for the proteins in the immediate vicinity of the membrane. Uhrafiltrate samples from the 4-fold, 5-fold, and 6-fold uhrafihration experiments contain traces of a-lactalbumin. Fig. 3h is an ultrafiltrate sample from the 5-fold uhrafihration experiment. Addition of Urea to Samples

Each of the retentate samples for Fig. 3 was suspended in an equal volume of 8M urea prior to electrophoresis to obtain sharper and more defined bands. Fig. 4 demonstrates the resolution of whey proteins of two different retentate samples treated with and without urea. Suspension of samples in urea overcomes diffuse resolution of proteins in concentrated samples. Addition of urea prior to SDS gel electrophoresis of regular whey samples produced no detectable change in resolution. However, with the retentate samples the final concentration of 4M urea rendered the highly concentrated proteins more soluble. It is well documented that urea can act as a hydrophobic solvent as well as hydrogen bond exchanger (10, 25). The multiplicity of high molecular weight bands in the retentate samples is not an artifact produced by urea. Although it is difficult, one can detect the same bands in nonurea-treated retentare samples by rotating the SDS gels. Journal of Dairy Science Vol. 58, No. 5

a

|

W

|

|

b

c

d

FIG. 4. Sodium dodecyl sulfate electrophoretograms of whey ultrafiltration samples treated with and without urea: a, 5-fold IR; b, 5-fold IR, urea-treated; c, 6-fold IR; and d, 6-fold IR, urea-treated.

Acidification of Samples

Retentate samples from the 5- and 6-fold uhrafihration experiments were acidified with N HC1 to test whether the high molecular weight polymers formed as a result of concentration are acid dissociable. SDS electrophoretograms of these acidified samples are in Fig. 5. Results were identical with acidified samples treated with urea. The acidified immediate retentate samples (Fig. 5b and 5d) resembled the reference whey sample. The acidified 5-fold bulk retentate sample (Fig. 5c) retained the 265,000 and 58,000 dalton bands. The 110,000 dalton band was still evident in the acidified 6-fold bulk retentate sample (Fig. 5e). Notably, the high molecular weight polymers originally present, particularly the 144,000, 132,000, and 122,000 bands (Fig. 3c to 3f), were not detectable in the acidified retentate samples (Fig. 5b to 5e). Acid dissociation of polymers formed during ultrafihration indicates the reversible nature of the interactions. The observed acid dissociation of the/3-1actoglobulin octamer is in accord with

663

ELECTROPHORESIS OF WHEY PROTEINS

Molecular Weight

Molecular Weight

265,000

-~;~ m ~ ~ " ,. . . . . 265,000 ,~'~ ~ 230,060

d

110,000~

~

~!~" ~ ' -

22,000

~ S Q S I O

)

58,000 - -

|

i

!

a

b

pH

|° c

d

e

FIG. 5. Sodium dodecyl sulfate electrophoretograms of acidified retentate samples: a, reference whey; b, 5-fold IR, pH 4.8 adjusted to pH 2.2; c, 5-fold BR, pH 4.6 adjusted to pH 2.5; d, 6-fold IR, pH 4.8 adjusted to pH 2.7; and e, 6-fold BR, pH 4.6 adjusted to pH 2.5.

reports of i3-1actoglobulin dissociation at low pH (21, 23). A difficulty with the acidified samples was that the stained bands faded upon storage and could be detected only for 1 wk in 7.5% acetic acid. Effect of pH

Resolution of whey proteins at various pH values is demonstrated in Fig. 6. The acidified whey samples (Fig. 6a, 6b, and 6c) resembled the unmodified control whey (Fig. 6d) except the bands were lighter because of the instability of the stain in acidified samples. At pH 5.1, which appears to be a transitional pH, the y-globulin region was diffuse and undefined (Fig. 6e). There was also a light trace of a 230,000 dalton band. A discrete octameric 13-1actoglobulin band (144,000 dahons) appeared at both pH 5.4 (Fig. 6f) and pH 5.7 (Fig. 6g). In addition to the octameric t3-1actoglobulin, the dimeric BSA band (132,000 daltons) appeared at pH 6.2 (Fig. 6h), pH 7.0 (Fig. 6i) and pH 8.0 (Fig. 6j). At these pH values the

36,000

|

a b c d e f g~h i j k ! t,9 2,8 3.6; 4.5 5.t 5.4 5.7 6.2 7,0 8,0 9 ~ 10,7

FIG. 6. Sodium dodecyl sulfate electrophoretograms of whey samples with pH adjusted in the range from 1.9 to 10.7: a, pH 1.9; b, pH 2.8; c, pH 3.6; d, pH 4.5 (control whey); e, pH 5.1; f, pH 5.4; g, pH 5.7; h, pH 6.2; i, pH 7.0; j, pH 8.0; k, pH 9.8; and 1, pH 10.7.

y-globulin band appears to be lighter suggesting that it may be unstable at alkaline pH. Protein bands in this region became diffuse at still higher pH. At pH 9.8, the octameric 13-1actoglobulin band was diffuse and a 122,000 dalton band appeared. At pH 10.7, there was a trace of an 185,000 dalton band, the y-globulin band was absent, and the intensity of the dimeric BSA (132,000) and the 122,000 bands increased. At pH 5.4, 5.7, 6.2, 7.0 (Fig. 6f, g, h, and i) the 265,000 dalton band was present. With increasing pH of whey samples, behavior was typical of association. The diffuse bands covering the 160,000 to 130,000 dalton region first appeared at the isoelectric pH of 13-1actoglobulin (Fig. 6e, pH 5.1). At the present stage of investigation, without coordinating with other measurements, it appears that pH 5.1 is a transition pH for the pH-dependent pattern of polymerization. According to Timasheff and Townend (21, 23), dissociation of dimeric /3-1actoglobulin into its monomers occurs at pH values below 3.0 and above 8.0. Electrophoretograms of cottage cheese whey (Fig. 6k, pH 9.8, and 61, pH 10.7) demonstrate decreasing intensity of the octameric 13-1actoglobulin coupled with increasing intensity of the dimeric state at pH above 8. A discrete octameric Journal of Dairy Science Vot. 58, No. 5

664

LEE ET AL.

13-1actoglobulin band appeared consistently in whey samples from pH 5.4 to 8.0. Dimers of BSA appeared from pH 6.2 to pH 10.7. At the extreme pH values of 9.8 and 10.7, a 122,000 dalton band also is present. Few changes in the constituents of whey appear at acid pH except, as pointed out earlier, the stain in acid whey sample gels fades on storage. Effect of Temperature In accord with the polymerization behavior of /3-1actoglobulin at low temperature reported by Timasbeff (19, 20) and Tang et al. (18), low temperature incubation of whey samples induced octamerization of /3-1actoglobulin. SDS electrophoretograms of whey samples incubated at different temperatures are in Fig. 7. The octameric /3-lactoglobulin band (144,000) was in whey incubated at -1, 2 and 4 C (Fig. 7a, b, and c). At 10 C, the whey sample showed traces of the octamer (Fig. 7d). Also in the -1 C whey sample, dimeric BSA (132,000) was present. The whey sample at 50 C (Fig. 7f) resembled the control whey (Fig. 7e). At 80 C (Fig. 7g) the absence of higher molecular weight bands, including those regularly observed, is due to precipitation of proteins at

Molecular Weight

such a high temperature. Dimeric and monomeric ~3-1actoglobulin were still in solution at this high temperature. Samples containing the /3-1actoglobulin octamer were acidified and the SDS electrophoretic results are in Fig. 7b' and 7c'. Again, the acidified samples did not show the octameric 13-1actoglobulin band originally present before acidification. Extensive study of association-dissociation behavior of /~-lactoglobulin protein by Timasheff and his coworkers (19, 20, 21, 22, 23) and Tang et al. (18), indicates that 13-1actoglobulin subunits have a strong tendency to associate to the octameric state at pH 4 and 5, especially at low temperature. Thus, the appearance of the 144,000 dalton band in whey samples at pH 4.3 to 4.6 incubated at -1, 2, 4 and 10 C (band intensity reduced) indirectly supports identification of this band as the /3-1actoglobulin octamer. The disassociation of this band under more acid conditions (pH 2.3) is further evidence in accord with known behavior of /3lactoglobulin. Addition of Salts Fig. 8 gives SDS electrophoretograms of whey suspended in various salt media to a final

Molecular Weight

144,000 --:_-i g g q 6 ,32,ooo

I "2

==i-li

?

J a

b c d e

f

i g

l

e

36,000

i~

b' C'

FIG. 7. Sodium dodecyl sulfate electrophoretograms of whey samples incubated at temperatures ranging from -1 C to 80 C: a, whey, -1 C; b, whey, 2 C; c, whey, 4 C; d, whey, 10 C; e, whey, 23 C (room temperature); f, whey, 50 C; g, whey, 80 C; b', whey at 2 C, acidified to pH 2.3; and c', whey at 4 C, acidified to pH 2.3. Journal of Dairy Science Vol. 58, No. 5

170 000 144,000

a

Jt

1i 3 b



' il c

d

e

f

FIG. 8. Sodium dodecyl sulfate electrophoretograms of whey samples with various salts added: a, whey (control); b, whey in 0.2M KSCN; c, whey in 0.2M KI; d, whey in 0.2M CaCI2; e, whey in 0.2M Na2SO4; and f, whey in 0.2M NaC2H302.

ELECTROPHORESIS OF WHEY PROTEINS concentration of .2M added salt. In all three samples with salting-in agents, KSCN, KI, and CaC12, the fi-lactoglobulin octamer band (144,000 daltons) was present (Fig. 8b, 8c, and 8d). At .1M, the octamer band was absent. (The latter SDS results are not in the figures.) An additional high molecular weight band of 170,000 daltons was also in these samples. This band is thought to be the more basic variant of bovine immunoglobulins, bovine IgG2 (3, 13) whereas the regularly observed 160,000 dalton band, which was called y-globulin earlier, is the less basic immunoglobulin, IgG1. In the .2M KI whey sample (Fig. 8c), there was a strong band of dimeric j3-1actoglobulin. The salting-out agents sodium sulfate and sodium acetate also were used at a concentration of .2M added salt. The electrophoretic pattern of the Na2SO 4 whey sample (Fig. 8e) resembled that of the control whey (Fig. 8a). The additional ~-lactoglobulin octamer band again appeared in the sodium acetate whey sample. Electrophoretic results indicate that at the salt concentration used, salt addition can induce protein polymerization, especially the octamerization of/3-1actoglobulin. Alteration of the ionic environment results in destabilization of the native state of proteins (24). The saltingqn ions, SCN-, I-, and Ca ++, may promote the unfolding or at least destabilize the folded native form of protein molecules. Thus, the observed ~-lactoglobulin octamer may be an intermolecular interaction product caused by increased exposure of protein-protein interaction sites resulting from the unfolding of molecules by the salting-in agents (24). However, the observed association seems to be in contradiction to Timasheff and Townend's report on ~-lactoglobulin octamerization (22). They stated that thiocyanate or calcium ions decreased the extent of ~3-1actoglobulin association while sulfate ions enhanced it. This discrepancy might be explained if more detailed information were available regarding the concentration of salts added in their experiments and the protein environment in whey. The effect of salt on protein depends on the type and concentration of salt, the type of protein, and the solvent environment. At the concentration of salts in these whey samples, the dividing line between salting-in and salting-out may not have been reached, and salts would have acted

665

as electrolytes in the initial salting-in stage. Further investigation is required to clarify the salt-addition effect on the association pattern. /3-1actoglobulin octamers in the acetate whey sample (Fig. 8t:) agrees with the association behavior of ~-lactoglobulin observed by Tang et al. (18).

Purified/~-Lactoglobulin The polymerization behavior of 13-1actoglobulin was studied by subjecting purified /3-1actoglobulin to conditions where polymerization had been observed with whey samples (low temperature and alkaline pH). The SDS results are in Fig. 9. The purity of the sample was checked by increasing sample loading (see Fig. 9c). The ~3-1actoglobulin (Fig. 9b, 9c) fractionated as described by Aschaffenburg and Drewry (1) contained mainly the monomeric ~3-1actoglobulin and a trace of the dimer in addition to a slight impurity of the 29,500 dalton band. Incubating the protein at 2 C (Fig. 9d) and adjusting the pH to 9.5 (Fig. 9e) caused an increase in intensity of the dimeric ~-lactoglobulin band and formation of trimeric

Molecular Weight

74.000 ~*~

~'~

54,000

+°,000 --

|+|jj a

b

c

d

e

29 +500

'l t ++ ;

h

FIG. 9. SDS electrophoretograms of purified 13-1actoglobulin: a, reference whey; b*, 13-1actoglobulin,pH 4.7 (control); c*, 13-1actoglobulin,pH 4.7 (double-volume sample loading, 2v); d*, 13-1actoglobulin at 2 C (2v); e*, 13-1actoglobulinadjusted to pH 9.5 (2v); f**, I3-1actoglobulin, pH 4.9; g**, 13-1actoglobulin at 2 C (2v); and h**, &lactoglobulin adjusted to pH 9.7 (2v). *t3-1actoglobulin crystallized by the method described by Aschaffenburg and Drewry (1). * *t3-1actoglobulin purified chromatographically (27). Journal of Dairy Science Vol. 58, No. 5

666

LEE ET AL.

(54,000 daltons) and tetramic (74,000 daltons) t3-1actoglobulin. SDS results were similar with t3-1actoglobulin samples prepared by Yaguchi et al. (27) (Fig. 9f, 9g, and 9h). In response to the cold temperature treatment (Fig. 9g), /3-1actoglobulin polymerized into dimeric, and trimeric, and traces of tetrameric forms. Polymerization into dimeric, trimeric, and tetrameric /3-1actoglobulin was again observed at the alkaline pH 9.7 (Fig. 9h). In agreement with polymerization results in the whey, multimers were in the ~3-1actoglobulin samples at low temperature and alkaline pH. The tetramer was the highest induced polymer state in the t3-1actoglobulin samples. The octamer band was not detected in the 13-1actoglobulin samples although it was in whey samples subjected to similar conditions of temperature and pH. This behavior could be explained by differences in environmental ionic and molecular constitution between the composite whey solution and the purified /3-1actoglobulin solution which undoubtedly affected the polymerization. On the other hand, the use of the Aschaffenburg and Drewry method of 13-1actoglobulin preparation (1) involves separation of /3-1actoglobulin from a-lactalbumin at pH 2.0. This low pH may have caused some irreversible changes in the protein molecules and hindered complete polymerization (octamerization) when /3-1actoglobulin was provided with conditions for octamerization. These studies support the postulation that low temperature and high pH promote polymerization of/3-1actoglobulin. CONCLUSION

SDS gel electrophoresis is a useful tool for studying protein changes in whey and in solutions of whey proteins. In contrast to other forms of electrophoresis the ability to estimate molecular weights with the SDS technique greatly facilitates identification of the protein bands. In particular, association of t3-1actoglobulin to the dimer, trimer, and tetramer state in aqueous solutions at pH 9.5 and at temperatures of 2 C has been observed. In whey the /3-1actoglobulin octamer appeared in the pH range from 5.1 to 8.0. Above pH 8 the octamer dissociated to the dimer and monomer. Cooling whey below 10 C, or concentrating it 4-fold or more, increased the tendency for 13-1actoglobulin to polymerize. Addition of potassium thiocyanate, potassium iodide, calcium chloride, or Journal of Dairy Science Vol. 58, No. 5

sodium acetate to a concentration of .2 M favored polymerization, but addition of sodium sulfate had no detectable effect. On the other hand acidifying whey below pH 3 promoted dissociation of the /3-1actoglobulin octamer. Dimers of bovine serum albumin appeared above pH 6.2 or at -1 C. SDS gel electrophoresis may be used effectively for monitoring protein changes during processing of milk or cheese wheys. The SDS results indicate that to minimize protein polymerization, low temperature storage should be avoided and further acidification should be helpful. REFERENCES

1. Aschaffenburg, R., and J. Drewry. 1952. Improved method for the preparation of crystalline 13-1actoglobulin and aqactalbumin from cow's milk. Biochem. J. 65:273. 2. Andrews, A. T. and G. C. Cheeseman. 1972. Properties of aseptically packed ultra-high-temperature milk. II. Molecular weight changes of the components during storage. J. Dairy Res. 39:395. 3. Butler, J. E. 1969. Bovine immunoglobins. A review. J. Dairy Sci. 52:1895. 4. Dunker, A. K. and R. R. Rueckert. 1969. Observations on molecular weight determinations on polyacrylamide gel. J. Biol. Chem. 244:5074. 5. Groves, M. L., W. G. Gordon, E. B. Kalan, and S. B. Jones. 1972. Composition of bovine 7-caseins A~ and A3, and further evidence for a relationship in biosynthesis of 7- and /3-caseins. J. Dairy Sci. 55:1041. 6. Jones, J. M. 1972. Studies on chicken actomyosin. 1. Effect of storage on muscle enzymic and physiochemical properties. J. Sci. Food Agr. 23 : 1009. 7. Lee, D. N. 1973. Reduction of membrane fouling during ultrafiltration of cottage cheese whey. MS Thesis, Univ. of California, Davis. 8. Lee, D. N., M. G. Miranda, and R. L. Merson. 1975. Scanning electron microscope studies of membrane deposits from whey ultrafiltration. J. Food Technol. (In press.) 9. Mullin, W. J., and F. H. Wolfe. 1974. Disc Gel Electrophoresis of caseins treated with proteolytic and glycolytic enzymes. J. Dairy Sci. 57:9. i0. Nelson, C.A. I971. The binding of detergents to proteins. J. Biol. Chem. 246:3895. 11. Penny, I. F. 1972. Conditioning of bovine muscle. 3. The a-actinin of bovine muscle. J. Sci. Fd. Agr. 23:403. 12. Robbins, F. M., and M. J. Kronman. 1964. A simplified method for preparing a-lactalbumin from cow's milk. Biochem. Biophys. Acta 82:186. 13. Rose, D., J. R. Brunner, E. B. Kalan, B. L. Larson, P. Melynchyn, H. E. Swaisgood, and D. F. Waugh. 1970. Nomenclature of the proteins of cow's milk: Third revision. J. Dairy Sci. 53:1.

ELECTROPHORESIS OF WHEY PROTEINS 14. Sender, P. M. 1971. Muscle fibrils: Solubilization and gel electrophoresis. FEBS Letters 17:106. 15. Shapiro, A. L., E. Vinuela, and J. V. Maizel. 1967. Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochem. Biophys. Res. Comm. 5:815. 16. Smithies, O. 1965. Disulfide bond cleavage and formation in proteins. Science 150:1595. 17. Swaisgood, H. E., J. R. Brunner, and H. A. Lillevik. 1964. Physical parameters of K-casein from cow's milk. Biochemistry 3 : 1616. 18. Tang, L. H., E. T. Adams, Jr., and G. Barlow. 1972. Temperature dependent self-association of j3-1actoglobulin A at pH 4.65. Fed. Proc. Fed. Amer. Soc. Exp. Biol. 21:913. 19. Timasheff, S. N. 1964. The nature of interactions in proteins derived from milk. Chapter 9 in Symposium on foods: Proteins and their reactions, H. W. Schultz and A. F. Anglemier, ed. Avi Publishing Co., Westport, CT. 20. Timasheff, S. N., and R. Towend. 1961. Molecular interactions in 13-1actoglobulin. V. The association of the genetic species below the isoelectric point. J. Amer. Chem. Soc. 83:464. 21. Timasheff, S. N., and R. Towend. 1961b. Molecular interactions in f3-1actoglobulin. The dissocia-

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tion of the genetic species of t3-1actoglobulin at acid pH's. J. Amer. Chem. Soc. 83:470. Timasheff, S. N., and R. Towend. 1969. j3-1actoglobulin as a model of subunit enzymes. Protides of the biological fluids. Proc. of the 16th Colloquium, Bruges 1968. Towend, R., L. Weinberger, and S. N. Timasheff. 1960. Molecular interactions in /3-1actoglobulin. IV. The dissociation of/3-1actoglobulin below pH 3.5. J. Amer. Chem. Soc. 82:3175. Von Hippel, P. H. 1968. Chapter 6 in Structure and stability of biological macromolecules, S. N. Timasheff and G. D. Fasman, ed. Dekker, New York. Weber, K., and O. Osborn. 1969. The reliability of molecular weight determinations by dodecyl sulfate polyacrylamide gel electrophoresis. J. Biol. Chem. 244:4406. Wolfe, F. H., J. D. Hay, and R. W. Currie. 1973. Polyacrylamide disc gel electrophoresis of fresh and aged chicken muscle proteins in sodium dodecyl sulfate. J. Food Sci. 38:987. Yaguchi, M., N. P. Tarassuk, and H. G. Hunziker. 1961. Chromatography of milk proteins on anion-exchange cellulose. J. Dairy Sci. 44:589.

Journal of Dairy Science Vol. 58, No. 5

Electrophoresis of cottage cheese whey proteins and their polymers.

Electrophoresis of Cottage Cheese W h e y Proteins and T h e i r P o l y m e r s DORETTA N. LEE, EDWIN E. MOORE, and RICHARD L. MERSON 1 Department o...
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