Proteose-Peptone Fraction of Bovine Milk: Lacteal Serum Components 5 and 8—Casein-Associated Glycoproteins1

Proteose-Peptone Fraction of Bovine Milk: Lacteal Serum Components 5 and 8--Casein-Associated Glycoproteins I C. W. KOLAR ~ and J. R. BRUNNER Department of Food Science, Michigan State University East Lansing 48823

trate of heated skimmilk (95 C for 30 rain) following adjustment to p H 4.6 to 4.8. HowLacteal serum Components 5 and 8 were ever, in recent times, this fraction and compoisolated from both heated and unheated nents thereof have been identified in and isolated skimmilk. At p H 8.6 Component 5 migrated from unheated skimmilk (2, 6, 14, 18, 22). Thus, as a double zone in starch-urea-gels (disa sometimes stated hypothesis that the proteosecontinuous buffer) and as a single zone in peptone fraction consisted of "secondary" or polyacrylamide gels (continuous buffer). derived proteins is refuted. Apparently the Component 8 migrated in the ion front on stability of these proteins to heat and acid led starch gels but resolved into two principal investigators to conclude that they were conzones in the polyacrylamide gel system, fronting much smaller molecules. designated as 8-fast and 8-slow in ascending Larson and Rolleri (19) observed three disorder of relative mobility. Components cernible boundaries in Tiselius patterns of the 8-fast and 8-slow were fractionated by gelproteose-peptone fraction of heated skimmilk permeation chromatography on Bio-Gel designated as milk serum Components 3, 5, and P-10 polyacrylamide beads. 8 in ascending order of electrophoretic mobiliComponent 5 is characterized by its high ties. Jenness (13, 14) isolated a relatively pure proline (~-~ 10.5%) and relatively low carpreparation of Component 5 from unheated bohydrate contents ( ~ 1.5%). Components skimmilk by its coprecipitation with casein when 8-fast and 8-slow contain relatively high NaCI was added to saturation. Interestingly, concentrations of total phosphorus ( ~ 7.4 he noted that unheated preparations exhibited and 5.5% as H2P03) and intermediate a loaf-depressant property when incorporated amounts of carbohydrate (~-~ 2.07 and into bread doughs and that heating the prepa9.0%). All three components are void of ration negated this property. Further, he sugcysteine and cystine and contain only low gested that Component 5 was the principal concentrations of methionine. component of the proteose-peptone fraction and I n veronal buffer at pl=[ 8.6, r / 2 = 0.1, that it was unlike other milk proteins. Ganguli a sedimentation coefficient (S°,o.w) of 1.22 et al. (11) separated a "proteose" from the and a molecular weight (Mw) of ~ 14,300 proteose-peptone fraction by salting-out with were observed for Component 5. Similar (NI.I4)2SO 4 when added to one-half saturation. parameters for Components 8-fast and Because of similarities in the fractionation pro8-slow were S°20.w = 4,100 and 9,900. An cedures, the proteose thus obtained should be isoelectric p H of 3.3 is reported for Comcomparable to the sigma-proteose fraction reponent 8-fast and partial specific volumes ported earlier by Aschaffenburg (1). Extensive (V) of 0.736, 0.700, and 0.719 (ce/g) for composition data were reported for the proteoseComponent 5, 8-slow, and 8-fast were calpeptone fractions; their glycoproteinaceous naculated from composition data. ture was confirmed. Aschaffenburg and Drewry (2) detected 6 zones on paper electropherograms of proteoseThe "proteose-peptone" fraction of skimmilk peptone fractions prepared from both heated has been characterized as a mixture of heatand unheated skimmilk. They concluded that stable, acid-soluble glycoproteins (5, 6, 10, 11, the principal and fastest migrating zone cor13, 18, 19, 22, 24, 27). Classically, these proteins responded to Component 5 and that the 5 slower have been detected in and isolated from the illzones were constituents of Component 3. No indication was given relative to zones correReceived for publication March 2, 1970. sponding to Component 8. Previous reports 1 Published as Journal article no. 5011 from the from this laboratory described the preparation of proteose-peptones from both heated and unMichigan Agricultural Experiment Station. heated skimmilk as well as the distribution of 2 Present address: Ralston-Purina Corp., St. these proteins within the milk protein systems Louis, Missouri. Abstract

997

•{~[]8

KOLAR

AND BRUNNER

PROTEOSE-PEPTONE PREPARATIONS [Obtained from heated or unheated skimmilk (22)] Make up to 1 to 2% in deionized water Adjust to pH 7.0 Add (NH4)2SO 4 to 25% saturation Adjust to pH 4.9 Centrifuge at 8,000 × g for 30 rain !

I

SUPERNATANT $1

RESIDUE R1 (Enriched in Component 5) Add (Ntt4)2SO 4 to 40% saturation Disperse in water at pH 7.0 at pH 4.9 Centrifuge at 1,000 × g for 30 rain, Centrifuge at8,000 X g discard pellet for 30 min Repeat salting-out step 4 × at pH 4.6 Disperse in water and desalt on Bio-Gel P-2 Lyophilize and dry over P.,O 5 COMPONENT 5 !

SUPERNATANT $2

RESIDUE R2 (Components 3 and 5)

Adjust to pH 7.0 Add (NH4)=S04 to 50% saturation at pH 7.0 Centrifuge at 8,000 X g for 30 min I

RESIDUE R 3 (Enriched in Component 3) Add (NH4)2S04 to 65% saturation at pH 7.0 Centrifuge at 8,000 × g for 30 min

SUPERNATANT $3

!

SUPERNATANT $4

RESIDUE R 4 (Components 3 and 8)

Add (NH4)2SO 4 to 80% saturation at pH 7.0 Centrifuge at 8,000 × g . for 30 rain I

SUPERNATANT $5

RESIDUE R~ (Discard)

Concentrate and fractionate on Bio-Gel P-10 Desalt fractions on Bio-Gel P-2 Lyophilize and dry over P205 !

COMPONENT 8-FAST

COMPONENT 8-SLOW

FIo. 1. Sehematie representation of the procedures employed to fractionate proteose-peptone preparations into Components 3, 5, 8-fast, and 8-slow. JOURNAL OF ])AIRY SCIEI~C] ~ O L . 53, NO. 8

P R O T E O S E - P E P T O N E IN M I L K

(18, 22). Components 5 and 8 were isolated from both the whey and casein protein fractions, whereas Component 3 was found only in the whey fraction and was characterized by a carbohydrate content of about 17%. It seems appropriate to note the excellent studies of Bezkorovainy (3, 4), who isolated several phosphoglyeoprotein species from bovine sermn and the acid wheys of both cow's milk and colostrum by a combination of D E A E and CMC-cellulosic chromatography. The analytical data reported for these glycoproteins were qualitatively similar to those of the proteose-peptone fractions (6, 11, 27). The purpose of our study was to provide basic chemical and physical characteristics of the proteose-peptone components usually identified as milk serum Components 5 and 8. A preliminary report of the principal results was presented previously (17). Materials and Methods

Fresh, uncooled milk from the University dairy herd (Holstein cattle) was centrifugally separated to obtain the skimmilk portion from which all protein preparations were derived. The Component 5 preparation characterized in this study was isolated from heated (95 C for

999

30 min) skimmilk, whereas preparations of Component 8 were obtained from both lmated (95 C for 30 rain) and unheated skimmilk. PREPARATION OF C O M P O l g E I ~ S 5 At~D $

The basic procedure employed to fractionate proteose-peptone into its individual components is outlined in Figure 1. As indieated, the isolation process begins with a proteose-peptone preparation from heated skimmilk or a proteosepeptone-enriched preparation from unheated skimmilk. Procedures for obtaining these preparations were described in a report from this laboratory (22). Fractionation of the proteosepeptones was by salting-out at selected concentrations of (NH4)2S04 at controlled levels of pH. Residue-R1, a Component 5-rich precipitate, was further purified by repeated solution, centrifugation, and salting-out at 25% (w/v) saturation with (NH4)2SO4 . Residues R2, Rs, and R4 represent proteose-peptone fractions salted out with (NH4)2S04 at concentrations of 40, 50, and 65%, respectively, and contain varying proportions of Components 3, 5, and 8. Residue-R 3 consists essentially of Component 3, a whey glycoprotein (22). Supernatant-Ss, containing essentially the proteins attributed to Component 8, was sub-

FIG. 2. Starch urea gel electropherograms of proteose-peptone preparations from unheated and heated skimmilk. The preparation from unheated skimmilk shows thz presence of contaminating whey proteins. JOURNAL OF DAIRY SCIENCE ~'OL, 53, NO. S

1000

KOLAR

AND BRUNNER

~ractionated into two principal fractions--i.e., 8-fast and 8-slow--by gel permeation chromatography on polyacrylamide beads. Glass columns (2.8 em id and 40 cm long) were coated with dimethyldichlorosilane and filled with water-equilibrated, Bio-Gel P-10 polyacrylamide beads. A void volume of approximately 55 ml and a flow rate of about 150 ml/hour characterized the column operations. Five milliliters of Component 8-containing solution, concentrated to about 2% (w/v) protein, were added to the top of the column and eluted with deionized water. The eluate was monitored at 254 and 280 nm. Eluate fractions were collected at 25, 30, 40, and 50 ml, measured from the point of emergence of the initial peak. Each fraction was assayed by polyaerylamide gel eleetrophoresis to identify the fractions containing unresolved protein species. These fractions were combined and rechromatographed to achieve further separation of the principal components. Salt-free, lyophilized preparations of Components 5 and 8, suitable for subsequent chemical and physical analyses, were obtained by passing 2% (w/v) solutions of the protein preparations over Bio-Gel P-2 columns, followed by lyophili-

zation and exposure to P205 in a partial vacuum for 48 hours. The common technique of exhaustive dialysis to eliminate salt ions from protein systems was untenable for these proteins because of their molecular size. Up to one-haIf of the proteinaceous material passed through a Visking membrane in 24 hours. CHARACTERIZATION OF C O M P O N E N T S 5 AND 8

Amino acids. Amino acid compositions of the protein preparations were determined on 20and 70-hour acid hydrolysates (0.6 mg/ml of 6 N HCI at 110 C). Analyses were performed with a Beekman-Spinco Model 120-C Automatic Amino Acid Analyzer. Chromatograms were quantitated by comparison with a standard amino acid mixture. Nor-leucine was employed as an internal standard. All residue concentrations were corrected to reflect their maximum values (12). Tryptophan and eysteine contents were evaluated by the methods of Spies (25) and Ellman (8). Carbohydrates. Components of the carbohydrate moiety were identified, as described (22), by paper chromatography following their re-

FIG. 3. Starch urea gel and polyacrylamide gel electropherograms of fractions obtained from a proteose-peptone preparation (unheated) by the scheme outlined in Figure 1. Electrophoresis was performed in horizontal gels and stained with Amido B]ack for visualization of the protein zones. Fractions 1~, Rs, and $5 represent specimens of Components 5, 3, and 8. JO~rR~AL OF DAIRY SCIENCE VOL. 53, NO. S

~ROTEOSE-PEPTONE

lease from the protein by mild hydrolysis in 2 ~ HC1. Authentic glueosamine, galaetosamine, galactose, glucose, mannose, and fucose were used as reference materials for tentative identifications. Chromatograms were migrated with a butanol-pyridine-water ( 6 : 4 : 3 , v/v) mixture by descending chromatography. A solution of triphenyltetrazolium with NaOH was used for visualization of carbohydrate-containing zones. Qualitative identification of glucosamine and galactosamine was augmented by their identification in ehromatograms from the amino acid analysis. Quantitative estimates of the carbohydrate classes were achieved by spectrophotometric techniques. I-Iexose was determined by the phenol-sulfuric acid method of Dubois et al. (7). Hexosamine was determined by the method of Johansen et al. (15) in which the chromogen resulting from addition of acetylacetone was steam-distilled into Ehrlieh's pdimethylaminobenzaldehyde receiver. Sialic acid was determined by Warren's (30) thiobarbituric acid method as modified by Marier et al. (20). Elemental analyses. Protein nitrogen was determined by a micro-Kjeldahl procedure. Phosphorus was determined by the method of Smuher (26). Electrophoretic methods. Electrophoretic toobilities of the protein specimens were evaluated from Tiselius patterns with a Perkin-Elmer Model 38-A Electrophoresis Apparatus, employing a 2-ml cell. The protein specimens were carried in veronal buffer of p H 8.6 and 0.1 ionic strength. One specimen of Component 8 (i.e., 8-fast) was observed from p H 2.0 to 8.6. Mobilities were plotted versus p H to estimate its isoelectric pH. The concentration of protein was 15 mg/ml for all electrophoretie runs. A starch urea gel (SUG) zonal eleetrophoretie technique was adapted from the procedure described by W a k e and Baldwin (29). Electrophoresis was performed in a water-cooled gel bed at 15 C. The gel was connected to one-liter buffer tanks with paper-filter wicks (E and D 652). The usual discontinuous buffer system, consisting of Tris-citrate-urea ( p H 8.6) in the gel and boric a e i d - N a O t I ( p H 8.6) in the buffer tanks, was employed. Polyacrylamide gel (PAG) electrophoresis was performed in both horizontal (laboratoryconstructed) and vertical (E-C Apparatus Co.) water-cooled gel beds. A continuous buffer systern ( p H 8.6), consisting of boric acid (13.0 g / liter) and NaOH (2.1 g / l i t e r ) , was employed in these applications. Cyanogen 41, at an 8% concentration in the borate buffer and catalyzed with 0.03 ml/liter of N, N, N 1, Nl-tetramethyl-

1001

IN M I L K

ethylenediamine and 0.3 g/liter of ammonium persulfate, served as the gel medium. Urea was added to 4.5 ~I when its incorporation into the aerylamide gel was desired. Electrophoresis in the horizontal gels was performed at a constant current of approximately 15 mamp/cm 2 of cross-sectional area for about 15 hours. I n the vertical E-C cell, eleetrophoresis was performed at a constant cell current of about 100 mamp for 6 hours. Routinely, the resolved protein-containing zones were visualized by the Amido Black staining procedure. I n one set of experiments, involving the identification of glycoproteins in P A G patterns of proteose-peptone and related fractions, a periodic acid-Sehiff reagent was used (16). Ultrace~trifugal techniques. Sedimentationvelocity and sedimentation-equilibrium studies were performed in a Beckman-Spinco, Model E Analytical Ultracentrifuge. A capillary-type, synthetic-boundary cell operated at 59,780 rpm

FIG. 4. Polyacrylamide gel electropherograms showing the resolution of Component 8 (Fraction $5, Fig. 1) into its principal constituents of 8-fast and 8-slow by gel permeation chromatography on Bio-Gel P-10. Jou~

oF DAEaY SCXE~C~ 170L. 53, NO. 8

2002

KOLAR AND B R U N N E R

and 20 C was employed for the sedimentationvelocity studies. Protein concentrations ranging from 5 to 10 mg/ml in veronal buffer (pH 8.6, r / 2 : 0.1) were employed. Apparent-sedimentation coefficients were corrected for solvent effects and protein concentrations and expressed as S°2o.w at infinite dilution of the protein. A short-column, sedimentation-equilibrium technique by Van Holde and Baldwin (28) was employed for determination of molecular weights. Analyses were performed at 20 C and at speeds of about 25,000 rpm in a doublesector, synthetic-boundary cell fitted with a filled-Epon centerpiece. Apparent weight-average molecular weights of protein of from 5 to 10 mg/ml in veronal buffer were plotted versus protein concentration and extrapolated to a standard value at infinite dilution of the protein (i.e., M°w). Solvent densities were determined with 10-ml pyenometers equilibrated to 20 ± .01 C. Solution densities were calculated according to F u jita (9) from the expression psoln = psolv -{~sol,-) e, where e is the concentration of (1 -

-

:

the protein in g/ml. Relative viscosities were determined in a Cannon-Ubbelohde semimicroviscometer at 20 ± 0.01 C and partial specific volumes (V) were calculated from the compositional data (Table 1). Results and Discussion PREPARATION OF C O M P O N E N T S 5 A N D 8 FROM HEATED AND UNHEATED S K I M M I L K

Starch urea gel eleetropherograms of proteosepeptone-rich preparations obtained from heated and unheated skimmilk are shown in Figure 2. These patterns are essentially similar to data reported by Kolar and Brunner (18). Component 8 migrated as a single zone in the ion front, whereas Component 5 appeared as a double zone in the middle of the pattern and Component 3 was a single dominant zone of slower mobility. The preparation from unheated skimmilk contained other whey protein contaminants. The fractionation of these preparations into Components 3, 5, and 8 by the scheme outlined in Figure 1 was monitored by both horizontal

FRACTION

,::

Fro. 5. :Po]yacrylamide gel electropl~erograms of a preparation of proteose-peotone (P-P) iso]ated from unheated skimmilk and fractions obtained therefrom by the scheme outlined in Figure 1. The fraction designated as "8" consists of a purified specimen of 8-slow. The gel on the left side of the figure was stained with Amido t~lack to visualize the proteins; whereas, the gel to the right side of the figure was treated with Schiff periodic reagent to visualize the glycoproteins. Electrophoresis was performed in a vertical cell (E-C Apparatus Co.). JOURNAL OF ~A][RY SCIENCE ~rOL. 53, NO. 8,

PROTEOSE-PEPTONE

IN M I L K

1003

TABLE 1. Composition of Component 5, 8-fast, and 8-slow specimens isolated from heated and unheated skimmilk. Component 5

Component 8-fast

Residue

Heated

Heated

Lysine Histidine Arginine Aspartic Acid Threonine Serine Glutamic Acid Proline Glyeine Alanine 1/2 Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan b Cysteine e Unidentified

7.55 1.92 2.60 5.81 4.72 5.77 24.89 10.55 1.23 1.92 None 6.60 1.68 6.14 7.33 1.94 4.86 0.20 None

5.52 0.82 5.42 8.11 5.04 10.00 25.30 3.67 1.19 0.93 None 5.18 1.10 7.36 7.16 0.70 1.04 0.23 None 0.47

Waa a Hexose Hexosamine Sialic Acid

95.71 0.93 0.23 0.29

WCHO Phosphorus (H2PO 3) Wtotal Nitrogen

Component 8-slow

Unheated

Heated

Unheated

23.96 2.51 1.21 0.80 None 5.57 1.07 8.41 4.27 1.45 0.73 0.06 None 0.18

8.42 4.92 1.35 13.98 4.45 8.54 14.62 3.14 0.50 2.04 None 2.49 0.37 2.84 5.85 0.65 2.44 0.19 None 0.51

8.79 5.63 1.36 15.34 4.49 8.67 15.40 3.38 0.53 2.13 None 2.40 0.67 2.90 6.27 0.68 2.42 0.23 None 0.88

88.72 1.37 0.26 0.40

88.06 0.99 0.35 0.47

77.19 4.53 2.45 3.29

82.17 2.98 1.79 2.88

1.45 2.53

2.03 6.26

1.81 8.59

10.27 5.17

7.63 5.79

99.69 13.83

97.01 13.33

98.46 13.16

92.63 12.31

95.61 13.03

(g/100 g protein) 7.92 1.44 3.63 8.20 4.48 12.17

a Amino acid residues were determined from 20- and 70-hour hydrolysates (6 ~ HC1) and corrected for time of hydrolysis according to Hirs et al. (12). b Method of Spies (25). ¢ Method of Ellman (8). SUG and PAG eleetrophoresis (Fig. 3). The final supernatant-S5 contained materials which migrated in a sharp zone with the ion front in starch gels run with the discontinuous buffer system, but showed two principal and several minor zones in polyacrylamide gels run with the continuous borate buffer system. When the Component 8-rich fraction was subjected to electrophoresis in PAG, a second dimension-i.e., nornlal to the original direction--with the discontinuous buffer system, both of the principal zones moved with the ion front as a single zone. Thus, it was concluded that Component 8 was a mixture of at least two principal proteins, designated here as 8-fast and 8-slow in descending order of their rates of electropho-

retie migration. The purified preparation of Component 5 (Residue R 1 in Fig. 1) exhibited two closely migrating zones by SUG electro° phoresis and but one principal zone by P A G electrophoresis. However, because of the relatively high concentration (4% so]n) of sample applied to the PAG, resolution of these proteins may have been obscured. And, as reported previously, preparations of Component 3 (Residue R 3 in Fig. 1) showed a single zone in both SUG and PAG, employing both continuous and discontinuous buffer systems, electrophoresis when stained with Amido Black. Polyaerylamide gel (continuous) electrophoretie patterns of the 5 e]nate fractions eolleeted during the gel filtration (Bio-Gel P-10) of the JOURNAL OF DAIRY SCIENCE ~OL. 53, NO. S

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XOLAR AND B R U N N E R

Component 8-containing Supernatant-S 5 are shown in Figure 4. The first two fractions were predominantly Component 8-slow, whereas the last fraction contained Component 8-fast. The third and fourth fractions were composed of a mixture of 8-fast and 8-slow and were rechromatographed to yield the individual components. A proteose-peptone preparation and selected fractions obtained therefrom by the scheme outlined in Figure 1 were subjected to vertical (E-C cell) P A G eleetrophoresis in a continuous buffer system. Figure 5 shows two sets of electropherograms: one was stained with Amido Black to show the zones of migrating proteins; whereas, the second was treated with periodicSchiff reagent to show zones of glycoproteins. I n these patterns, Component 5 stained heavily with Amido Black but was barely visible when ( DESCENDING

2800 sec.

. ASCENDING ~,

F- IO.SQVcm Fro. 7. Photomicrograph (magnified 26×, l%inburg illumination) of "crystallized" Component 8fast. Crystals formed in veronal buffer (pH 8.6, F/2 = 0.10) at a protein concentration of 15 mg/ml.

3100 sec.

300Qsec,

F- 10. 87 V cm -1

F- 10.63V cm "1

°A..,A 3200 sec.

treated for the detection of glycoproteins. Similarly, Components 8-fast and 8-slow were more visible in the Amido Black-stained gel but showed additional zones in the carbohydratedeveloped gel. Component 3, although clearly visible in both gels, constituted the predominant glycoprotein zone. CO][POSITIO~AL CHARACTERISTICS OF COMPONENTS 5 AND 8

F - lff 79 V cm-1

E.A

F- 1O.79 V cm -I 4100 sec. FIG. 6. Electrophoretic patterns of the proteosepeptone fractions studied: A, Component 8-fast from unheated skimmilk; B, Component 8-fast from heated skimmilk; C, Component 8-slow from unheated skimmilk; D, Component 8-slow from heated skimmilk ; and E, Component 5 from heated skimmilk. Electrophoresis was performed in veronal buffer at pH 8.6, r / 2 = 0.1 and at ~ 20C. J O U R N A L OF DAIRY SCIENCE "VOL, 53, NO. 8

The amino acid, carbohydrate, phosphorus, and nitrogen compositions of proteose-peptone Components 5, 8-fast, and 8-slow are reported in Table 1. These proteins have common characteristics of low methionine content and the absence of cysteine and cystine. Component 5 contains significantly more proline ( ~ 10.5%) than either of the Component 8 fractions. Indeed, the proline and glutamie acid contents of Component 5 are quite similar to those reported for casein. The three proteose-peptone proteins contain about equal concentrations of aspartie acid plus glutamic acid ( ~ 30%). However, the distribution of these residues varies between

PROTEOSE-PEPTONE IN MILK the three species. The amino acid compositions reported are in general agreement with compositions reported by Bezkorovainy (4) for several lacteal phosphoglycoproteins, but they are quite dissimilar to those reported by Gangull et al. (10) for a proteose-peptone preparation. They (10) reported high values for eysteine (50%), methionine (5.0%), and tryptophan (4.2%), suggesting the possibility that the principal whey proteins were contaminants in their preparation. The total carbohydrate contents of Components 5, 8-fast, and 8-slow were approximately 1.5, 2.0, and 9.0%. The carbohydrate content of Component 3 was reported to be about 17% (22). This compositional feature reflects the results observed in the polyaerylamide gel developed for glycoproteins in which Component 3 was the dominant zone. The chromatographic studies demonstrated that sialic acid, galactose, mannose, galactosamine, and glucosamine were constituents of Components 5, 8-fast, and 8-slow but their relative compositions varied between proteins. The low nitrogen contents of these protein fractions (~-~ 12 to 14%) reflect the presence of carbohydrate moieties and the relatively high concentrations of phosphorus (0.97% for Component 5, 1.98 to 2.22% for Component 8-slow, and 2.40 to 3.29% for Component 8-fast). The analyses as presented in Table 1 account for

1005

approximately 99.7% of Component 5, 92.6 to 95.6% of Component 8-slow, and 97.0 to 98.5% of Component 8-fast. Based on the compositional data reported, partial specific volumes (V) of 0.736 ec/g for Component 5, 0.700 cc/g for Component 8-slow, and 0.719 cc/g for Component 8-fast were calculated. PI~YSlCAL PROPERTIES Tiselius electrophoretic patterns of Components 5, 8-slow, and 8-fast in veronal buffer at pH 8.6, F/2 = 0.1, showed single, rather diffuse boundaries with average mobilities of --4.9, --9.2, and --9.2 Tiselius units (Fig. 6). These mobilities are slightly higher than those reported previously for the proteose-peptone components in a mixed system, i.e., proteose-peptone or fractions therefrom (15, 19). This observation is rationalized by assuming some degree of molecular interaction between the individual components comprising the proteose-peptone system. Supporting this conjecture was the observation that purified specimens of Component 5 precipitated from a saline solution at p H 4.8, whereas a solution of proteose-peptone at the same conditions remained stable. Obviously, Components 8-fast and 8-slow cannot be distinguished by moving-boundary electrophoresis. An isoelectric p H of 3.3 (~ ---- 0) at an ionic strength of 0.1 was determined for Component 8-fast from a plot of electrophoretic

TABLE 2. Sedimentation and molecular weight characteristics of Components 5, 8-fast, and 8-slow. S~PP 2o,w

M--~,app

4.1 5.1 6.0 6.1

1.21 1.19 1.21 1.19 S°2o,w = 1.22

13,700 13,400

Component 8-fast e

5.2 6.1 7.0 10.1

0.74 ...... 0.73 0.70 S°~o,w ----0.78

4,000 4,100 3,500 3,500 M°w -- 4,100

Component 8-slow e

5.0 7.1 10.1

1.27 1.16 1.16 S°20,w = 1.35

8,700 7,100 7,000 Mow ~

Component

Protein a (mg/ml)

Component 5 b

.

.

.

.

.

.

.

.

.

.

.

.

13,400 M°w = 14,300

9,900

a I n veronal buffer, p H 8.6, r / 2 = 0.1. b Obtained from heated skimmilk. c Obtained from unheated skimmilk. JOURNAL OF DAIRY SCIENCE VOL. 53, NO. S

1006

K O L A R AND BRUNNEI~

mobilities at various values of p H to

( p H 2.0

s.6).

An interesting phenomenon was encountered when Component 8-fast was dissolved (15 m g / ml) in veronal buffer in preparing the freeboundary experiments. After approximately 2 hours of dialysis against the buffer, a dense precipitate was formed which appeared as irregular rhombiz crystals under a light microscope at 26 magnification (Fig. 7). Crystals of sodium veronal formed by evaporating a portion of the buffer were needle-like, with no resemblance to the crystals harvested from the protein-veronal solution. When observed in the presence of a small amount of crystal violet solution, the crystals appeared to take up the dye. Thus, it is suggested that their c~'stals consisted entirely of protein or at least a protein adjunct of sodium veronal. This phenomenon was not observed with either Components 5 or 8-slow.

:

16

Sedimentation coefficients and weight-average molecular weights for Components 5, 8-slow, and 8-fast are presented in Table 2. Photographic reproductions of typical ultracentrifugation data from which these values were calculated are shown in Figure 8. These data indicate that Components 5, 8-fast, and 8-slow are relatively small proteins possessing molecular weights of approximately 14,300, 4,100, and 9,900 Daltons. Corresponding S°20.w values of 1.22, 0.78, and 1.23 are reported. Ogston (23) reported sedimentation coefficients of 0.96 and 2.75 for two components observed in Aschaffenburg's sigma-proteose. Molecular weights of 4,900 and 23,900, respectively, were reported. Similar sedimentation coefficients (S°2o.~) were observed by Brunner and Thompson (6) for the two principal sedimentating species in proteosepeptone and its partial fractions. Because of the observation that Component 5 showed two zones in starch-urea, it is suggested

32

- I.l

:~

~

80

TIME (min)

H2

5 0

6.1

~) I

CONC (mg/ml)

FIG. 8. Representative ultracentrifuga] data characterizing the proteose-peptone fraetlons: A, Component 8-fast from unheated skimmilk; B, Component 8-slow from unheated skimmilk; and C, Component 5 from heated skimmilk. All determiuation~ were made in veronal buffer at pH 8.6, F/2 -- 0.1 and at 20 C. Sedlmentation-veloeity patterns represent trials at 59,780 rpm with protein concentrations of 7.0, A; %1, B; and 6.1 C mg/ml. Sedimentation-equillbrium patterns represent trials at about 23,000 rpm for ~he protein concentrations indicated. JOURNAL OF DAIRY SCIENCE ~*OL. 53, NO. 8

P R O T E O S E - P E P T O N E IN M I L K

that this component consists of two protein species, differing merely in simple amino acid substitutions (polymorphs) which sediment similarly, or that they represent two smaller proteins which sediment as a dimer. Obviously, more detailed studies are indicated before the complex nature of the proteose-peptone system can be described. DISTRIBUTION OF COMPONENTS 5 AND ~ IN T H E

(3)

(4)

(5)

M I L K PROTEIN SYSTEN[

Serum Components 5 and 8, together with Compoent 3, are unique members of the milk protein system. They constitute the principal constituents of the proteose-peptone fraction isolated from whey and, to a lesser degree, the casein complex. The distribution of Components 5 and 8 differs from that of Component 3, which does not constitute a part of the casein system (18,22). Therefore, should Components 5 and 8 be classified as caseins since they are recovered, in part, from skimmilk under the same conditions casein is obtained? Their relatively high concentration of phosphorus and, in Component 5 an equivalent concentration of proline suggest similarities with casein. And, like u-casein, they contain carbohydrate moieties. Furthermore, when skimmilk is saturated with NaC1, Component 5 is recovered as the principal proteose-peptone in the casein complex. Also, it has been noted that Components 5 and 8 arc gradually released from the casein complex by repeated isoeleetric precipitations of the casein or by a relatively mild heat treatment (95 C for 30 rain). Moreover, it is of interest that the yields of Components 5 and 8 are greater, as components of the proteose-peptone fraction, when isolated from heated than from unheated skimmilk. The functions, if any, of Components 5 and 8 as constituents of the casein complex are unknown. The observations that they occur in both the casein and whey fraction suggests they exist in a state of dynamic equilibrium between association with casein and in solution. Based upon the observations reported in this study and other general properties already mentioned, we tentatively designate milk serum Components 5 and 8 constituents of the classical proteose-peptone system as casein-associated glycoproteins.

(6)

(7)

(8) (9) (10)

(11)

(12) (13) (14) (15)

(16) (17)

References

(1) Aschaffenburg, R. 1946. Surface activity and proteins of milk. J. Dairy Res., 14: 316. (2) Aschaffenburg, 1~., and J. Drewry. 1959. New procedure for the routine determination of

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the various non-casein proteins of milk. Int. Dairy Congr., 15th, London, 3: 1631. Bezkorovainy, A. 1965. Comparative study of the aeld glyeoproteins isolated from bovine serum, colostrum and milk whey. Arch. Biochem. Biophys., 110: 558. Bezkorovainy, A. 1967. Physical and chemical properties of bovine milk and colostrum whey M-1 glycoproteins. J. Dairy Set., 50: 1368. Brunner, J. R., C. A. Ernstrom, R. A. Hollis, B. L. Larson, R. McL. Whitney, and C. A. Zittle. 1960. Nomenclature of the proteins of bovine milk--first revision. J. Dairy Set., 43 : 901. Brunner, J. R., and M. P. Thompson. 1961. Characteristics of several minor-protein fractions isolated from bovine miik. J. Dairy Sci., 44 : 1224. Dubois, M., K. A. Gilles, J. K. Hamilton, P. A. Reben, and F. Smith. 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem., 28: 350. Ellman, G.L. 1959. Tissue sulfhydryl groups. Arch. Biochem. Biophys., 82: 70. Fujita, H. 1962. Mathematical Theory of Sedimentation Analysis. Academic Press, New York. Ganguli, N. C., B. S. Gupta, O. N. Agarwala, and V. R. Bhalerao. 1965. Studies on the proteose-pcptone fraction of milk: Isolation and properties of proteose-peptone and proteose from cow and buffalo milk. Indian J. Biochem., 2: 189. Ganguli, N. C., S. K. Gupta, V. K. Joshi, and V. R. Bhalerao. 1967. Sialic acid and hexose contents of proteose-peptone of milk in relation to other milk proteins. Indian J. Dairy Set., 20 : 96. Hirs, G. H. W., W. It. Stein, and S. Moore. 1954. The amino acid composition of ribonuclease. J. Biol. Chem., 211: 941. Jenness, R. 1957. Characteristics of the milk protein fraction precipitated by salt but not by heat. J. Dairy Sci., 40: 598. Jenness, R. 1959. Characterization of milk serum Component 5. J. Dairy Set., 42: 895. Johansen, P. G., R. D. Marshall, and A. Ncuberger. 1960. Carbohydrates in proteins. 9. The hexose, hexosamine, acetyl and amidenitrogen contents of hen's-egg albumin. Biochem. J., 77: 239. ~:eyser, J. ~V. 1964. Staining of serum glycoproteins after eleetrophoretic separation in acrylamide gels. Anal. Biochem., 9: 249. Kolar, C. W., and J. R. Brunner. 1968. Characteristics of proteose-peptone Components Five and Eight. J. Dairy Sci., 51 : 945. Kolar, C. W., and J. R. Brunner. 1969. Proteose-peptone fraction of bovine milk: distribution in the protein system. J. Dairy Sci., 52 : 1541. JOUENAL OF DAIRI" SCIENCE VOL. 53, NO. S

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(19) Larson, B. L., and G. D. Rolleri. 1955. Heat denaturation of the specific serum proteins in milk. J. Dairy Sci., 38:351. (20) Marier, J. R., H. Tessier, and D. Rose. 1963. Sialic Acid as an index of the K-casein of bovine milk. J. Dairy Sei, 46: 373. (21) l~g, We Su, and J. R. Brunner. 1967. Whey protein Component "Three." J. Dairy Sci., 50 : 950. (22) Ng, W. S., J. R. Brunner, and K. C. Rhce. 1970. Proteose-peptone fraction of bovine milk: lacteal serum Component 3 - - a whey glycoprotein. J. Dairy Sei., 53: 987. (23) 0gston, A. G. 1946. Examination in the ultracentrifuge. J. Dairy Res., 14: 328. (24) Rowland, S. J. 1937. The soluble protein fraction of milk. J. Dairy Res., 8: 6.

JO~R.'qAL Or DAIRY Sc~g~cg VOL 53, NO. 8

(25) Spies, J. R. 1967. Determination of tryptophan in proteins. Anal. Chem., 39: 1412. (26) Sumner, J. B. 1944. A method for the colorimetric determination of phosphorus. Science, 100: 413. (27) Thompson, M, P., and J. R. Brunner. 1959. The carbohydrates of some glyeoproteins of bovine milk. J. Dairy S¢i., 42: 369. (28) Van Holde, K. E., and R. L. Baldwin. 1958. Rapid attainment of sedimentation equilibritun. J. Phys. Chem., 62:734. (29) Wake, R. G., and R. L. Baldwin. 1961. Analysis of casein fractions by zone electrophoresis in concentrated urea. Biochim. Biophys. Acta, 47: 225. (30) Warren, L. 1959. The thiobarbiturie acid assay of sialie acids. J . Biol. Chem., 234: 1971.