Biochimica et Biophysica Acta, 491 (1977) 82-92

© Elsevier/North-Holland Biomedical Press BBA 37606 ISOLATION AND C H A R A C T E R I Z A T I O N OF RAT a - L A C T A L B U M I N : A GLYCOPROTEIN

R. CLARK BROWN, WAYNE W. FISH*, BILLY G. HUDSON and KURT E. EBNER Department of Biochemistry, University of Kansas Medical Center, 39th and Rainbow Blvd., Kansas City, Kan. 66103 and the Department of Biochemistry, Oklahoma State University, Stillwater, Okla. 74074 (U.S.A.)

(Received September 14th, 1976)

SUMMARY a-Lactalbumin was purified to homogeneity from rat milk. Rat a-lactalbumin, in contrast to other a-lactalbumins, is a glycoprotein and exhibits an abnormally high molecular weight when obtained by gel filtration or electrophoresis in sodium dodecyl sulfate. The molecular weight by sedimentation equilibrium is 15 400 ~: 5 % and of the reduced and alkylated protein is 16 000 when determined by thin-layer chromatography in 6 M guanidine hydrochloride. At least, three major charge forms, all containing carbohydrate and active in the lactose synthetase reaction were demonstrated. The amino acid composition reveals a high proline content which is reflected in a low a-helical content.

INTRODUCTION a-Lactalbumin is a low molecular weight protein found in the milk of all species that produce lactose. The biological function of a-lactalbumin was described by Ebner et al. [1]. a-Lactalbumin is required for significant rates of lactose synthesis and in effect lowers the apparent Km of glucose a thousand fold of a galactosyltransferase which is responsible for the synthesis of lactose [2]. The amino acid sequence of bovine [3], guinea pig [4], human [5] and the partial sequence of kangaroo [6] alactalbumin's are completed, a-Lactalbumin has been purified from the milk of the goat, pig, and sheep [7]. a-Lactalbumins as generally isolated do not contain any significant amount of carbohydrate. Barman [8] has reported that bovine milk contains two forms of a-lactalbumin which may be separated by anion exchange chromatography. The major component had a molecular weight of 14 435 and a minor component represented about 15% of the total. This form had a molecular weight of 16 800 and contained 11-12 sugar residues per mol. Hopper and McKenzie [9] have isolated one major and three minor forms of bovine a-lactalbumin B. Two of the minor forms contained carbohydrate * Present address: Department of Biochemistry, Medical University of South Carolina, Charleston, S.C., U.S.A.

83 and both together represented about 3 ~ of the total a-lactalbumin. All of the minor forms of a-lactalbumin were active in the lactose synthesis reaction. a-Lactalbumin is a specific protein marker of secretory mammary tissue and hence can be used to identify tissues, tumors or cell cultures which may have secretory properties. The development of a sensitive radioimmune assay for rat a-lactalbumin required purification of a-lactalbumin to homogeneity for antibody production. The purified g-lactalbumin was unique from other a-lactalbumins as isolated to date in that it is a glycoprotein and exists in multiple forms which are probably due in part to variance in sialic acid. The present paper reports on the isolation and chemical and physical characterization of rat a-lactalbumin. EXPERIMENTAL PROCEDURE Materials. Fischer 344 pregnant females were from Charles River. DEAEcellulose 32 was from Whatman, Sephadex gels were from Pharmacia and Biogels were from Bio-Rad. Sodium pentobarbital was from Haver-Lockhart. Reagents for gel etectrophoresis were from Bio-Rad. Oxytocin, guanidine hydrochloride, neuraminidase and protein standards were from Sigma. Bovine a-lactalbumin [10] and fllactoglobulin [11 ] were isolated from milk. All other reagents were of reagent grade quality. Methods. Galactosyltransferase was assayed as previously described [12]. Polyacrylamide gel electrophoresis was run with 7.5 ~ [13] or 12 ~ gels [14] and the gels were stained with 0.007 ~ commassie blue in 40 ~ methanol and 7.5 ~ acetic acid. Gels were stained for carbohydrate [13] and charge forms were assessed [ 14]. Molecular weight determinations were made using sodium dodecyl sulfate gel electrophoresis [ 15]. a-Lactalbumin was extracted from one to 2-mm slices of 7.5 ~ acrylamide gel by freezing at --20 °C and thawing to room temperature in 100/A of 100 mM KC1, 20 mM Tris, pH 7.5. Circular dichroism spectra of a-lactalbumin were obtained at 24 °C on a Cary model 61 spectropolarimeter. Protein samples were in 100 mM KC1, 20 mM Tris, pH 7.5. Data are expressed as mean residue molar ellipticity and a mean residue weight of 118 was used to calculate the percentage of a-helix by the method of Chen and Yang [16]. Sedimentation equilibrium measurements [17, 18] were performed in a Beckman Model E analytical ultracentrifuge equipped with Schlieren and interference optics. The optics were aligned [19] and confirmed [20]. The apparent partial specific volume of the glycoprotein at 6 mg/ml (in 0.1 M NaC1, 0.1 ionic strength sodium phosphate buffer, pH 7.2) was estimated by density measurements on the protein solution and the solvent. Densities were measured with a Precision Density Meter DMA-02C (Anton Parr, Gratz) at 25.00 °, maintained constant to within ::[:0.008 °. Protein concentrations were estimated at 280 nm or by differential refractometry [21]. For amino analyses, samples were hydrolyzed with glass-distilled 6 N HCI under reduced pressure in sealed tubes at 105 °C for 24, 48, and 72 h. The amino acids in the hydrolysates were determined on a Beckman 121HP Analyzer. Tryptophan was determined spectrophotometrically [22] and half-cystine was determined as cysteic acid after performic acid oxidation [23]. Neutral monosaccharides were determined as alditol acetates by gas liquid

84 chromatography as described by Kim et al. [24] with modification. Sulfuric acid was removed with Dowex 1 and chromatography was performed on a single column (2 mm × 6 f t ) o f 3 ~ SP2340 (Supelco) on Supelcoport (80-120 mesh) with nitrogen as carrier gas at a flow rate of 20 ml/min in a Hewlett-Packard 5931A gas chromatograph. Temperature was programmed from 180 to 225 °C at a rate of 1 °C per min. Glucosamine and galactosamine were determined on the short column of the amino acid analyzer after hydrolysis of the protein with 4 N HC1 at 100 °C for 6 h [25]. Sialic acid was determined by the thiobarbituric method [26] after hydrolysis of protein with 0.1 N H2SO 4 at 80 °C for 1 h. The molecular weight of rat a-lactalbumin was also determined by thin-layer gel chromatography on Sephadex G-100 (super fine) in 6 M guanidine hydrochloride [27]. The standard proteins and a-lactalbumin were first reduced and alkylated [28]. At completion of the run, proteins were transferred to Whatman 3MM. The paper print was stained for protein with commassie brilliant blue by a modification of the method of Radola [29]. A straight line was obtained by plotting the logarithm of the molecular weight of the standard proteins against RM (the ratio of protein migration distance to that of bovine serum albumin). The molecular weights of standard proteins used were: bovine serum albumin, 68 000; ovalbumin, 43 000; chymotrypsinogen, 25 700; bovine a-lactalbumin, 14 000. RESULTS

Purification of rat a-lactalbumin Female Fischer 344 rats were milked at approximately 14 days after parturitition. Animals were injected intraperitoneally with sodium pentobarbital (0.05 mg/g body wt.) and after the rat became unconscious, 0.2 ml of oxytocin were injected intraperitoneally. After 5 min the teats were bathed with tepid water (22 °C) and the rats were milked by using very low suction. About 5 ml of milk were collected per rat and the milk was stored at --15 °C. After thawing to 4 °C, the rat milk was diluted with an equal volume of 0.9 ~ NaC1. a-Lactalbumin isolated from the milk of most species [7] ha s a molecular weight of about 14 500 and can be separated readily from the majority of the other whey proteins by chromatography on Bio-Gel P-30. However, rat a-lactalbumin did not separate from the majority of the whey proteins on Bio-Gel P-30 but was most effectively separated on Bio-Gel P-150. Rat milk contains a large amount of serum albumin which complicated chromatography on DEAE-cellulose and resolution on disc gels since both serum albumin and a-lactalbumin have similar charge characteristics. The purification scheme as evolved is as follows: 41 ml of rat milk were diluted with 41 ml of 0.9 ~ NaC1. After removal of the cream by centrifugation at 15 000 × g, the caseins were removed by centrifuging at 100 000 × g for 1 h. The supernatant solution was passed through a 0.45 and 0.22 #m millipore filter. The supernatant solution (56 ml) contained 740 mg of protein (A2s0, where 1 mg/ml equals an absorbance of 1.0). The proteins were precipitated with ammonium sulfate (56 g/100 ml) and after centrifugation for 20 min at 40 000 x g the pellet was dissolved in 9.9 ml of 20 mM Tris, 100 mM KCI, pH 7.6 at 4 °C and contained 590 mg of protein. In an alternate method casein was removed by lowering the pH of the rat skim

85 milk to pH 4.6 with 1 N HC1 followed by centrifugation at 20 000 x g for 20 min at 4 °C. The pH of the supernatant solution was raised to 7.4 with 0.5 N KOH, centrifuged, and then precipitated with ammonium sulfate as before. Both procedures removed the majority of the casein as evidenced by observing protein profiles on disc gel electrophoresis. The major whey protein (caseins removed) appeared to be serum albumin [31] since the molecular weight of this protein was about 65 000. A solution containing about 200 mg of protein was layered onto a 3.5 x 90 cm Bio Gel P-150 (100-200mesh) column equilibrated and eluted with 20 mM Tris. C1, 100 mM KC1, pH 7.76 at 4 °C. The elution profile is presented in Fig. 1 and fractions were assayed for galactosyltransferase and a-lactalbumin. Tubes from two separate columns containing a-lactalbumin were pooled and dialyzed against 4 1 of 20 mM Tris. C1, pH 7.25, changed every 6 h (4 changes). The dialyzed a-lactalbumin solution (120 mg in 139 mi) was placed on 1.0 x 24 cm DEAE 32 cellulose column equilibrated with 20 mM Tris. C1, pH 7.1. Proteins were eluted with a linear gradient from 0.0 M KC1, 2 0 m M Tris.C1, pH7.1 (300ml) to 3 0 0 m M KC1, 2 0 m M Tris.Cl, pH7.1 (300 ml). Two protein peaks contained a-lactalbumin. The fractions containing alactalbumin were pooled, lyophilized, and dissolved in 5 ml of deionized water. The pooled material, containing about 50 mg of protein, was layered on a 3.2 x 90 cm Bio Gel P-150 column equilibrated and eluted with 100 mM KCI, 2 0 m M Tris.Cl, pH 7.5. Fractions were collected and those containing c~-lactalbumin were pooled and dialyzed exhaustively against 6 liters of deionized water changed four times at 12 h intervals. The dialyzed a-lactalbumin was lyophilized and stored dry at --15 °C. About 0.9-1.0 mg of a-lactaibumin were isolated from 1 ml of rat milk.

0.03 2

0.01

0

40

60

BO

FRACTION

I00

I

120

0.00

NUMBER

Fig. 1. Chromatography of whey proteins from rat milk on a Bio Gel P-150 column. Whey proteins of skimmed rat milk were placed on a 3.15 x 90 cm Bio-Gel P-150 (100-200 mesh) column equilibrated and eluted with 20 mM Tris, and 100 mM KCI, pH 7.5. The sample contained 200 mg in 3.34 ml of 20 mM Tris, and 100 mM KC1, pH 7.5. Approximately 3.65 ml fractions were collected and measured for absorbance at 280 nm ( © - - O ) . a-Lactalbumin activity (O---O) and galactosyltransferase activity (11--11) were measured spectrophotometrically at 340 nm.

86

Properties of rat a-lactalbumin Rat a-lactalbumin was homogenous on sodium dodecyl sulfate acrylamide gels (Fig. 2) but did exhibit at least three major charge forms on 11 percent polyacrylamide disc gels (Fig. 2). Parallel lines were obtained when the relative mobility was plotted against percent gel (from 7 to 11 ~) indicating the presence of charge forms [14]. Each of the forms had a-lactalbumin activity in the lactose synthetase reaction and each form could be extracted from the disc gels. In addition, the three major charge forms were partially separated on DEAE-cellulose chromatography and each form was active in the lactose synthetase reaction. Rat a-lactalbumin is a glycoprotein as shown by subsequent analytical data and by the observation that all the protein bands which contained a-lactalbumin activity stained with the Schiffperiodate reagent [13]. Treatment of rat a-lactalbumin with neuraminidase altered the migration of all of the forms but did not alter the activity in the lactose synthetase reaction. The ultraviolet absorption spectrum was similar to a-lactalbumin isolated from other species [7] in that there was a pronounced shoulder at 290 nm which is

0.8

o 0.4

0.0 I 000

0.5 RELATIVE

I 1.0 MIGRATION

0.62

o 0.51

J 0.0 I 0,0

I 0,5 RELATIVE MIGRATION

l 1.0

Fig. 2. (a) Scan of purified rat a-lactalbumin migrated in 11 percent polyacrylamide sodium dodecyl gels. The sample size was 50 b~g. (b) Scans of purified rat a-lactalbumin migrated in 12 percent polyacrylamide gels. The sample size was 25/~g.

87

indicative of exposed tryptophan residues. The E2~~ of rat a-lactalbumin was determined to be 16.2 which is in the range of values reported for other a-lactalbumins The near and far ultraviolet circular dichroism spectra of a-lactalbumin are presented in Fig. 3. The percent a-helical content as calculated by the method of Chen and Yang [16] at 221 nm was 12.47o which is considerably lower than the value of 25-26 ~ for bovine a-lactalbumin [31 ]. Q

-i,o

o

-2.0 -3,0 -4.0

t~

-5.0 o m

-6,0

r-i

'~

-7.0 I

I

I

I

I

a

200

210

220

230

240

250

NANOMETERS 0.01- b

-4.0 'o

I m

x

-8.0

',1

-12.0

,4" =E tJ

O3 Ll.I

"n,-'

-16.0

o bJ o

-20.0

-24.0 250

270

290

310

:350

NANOMETERS

Fig. 3. (a) Far ultraviolet circular dichroism spectrum of rat a-lactalbumin. (b) Near ultraviolet circular dichroism spectrum of rat a-lactalbumin.

88 Data from both molecular sieve columns (Bio Gel 0.5 m agarose) and gel electrophoresis in sodium dodecyl sulfate gave an apparent molecular weight of 26 000 to 28 000 which is about double the value observed for most other a-lactalbumins. Accordingly, the molecular weight was determined on reduced and alkylated alactalbumin by chromatography on thin layers of Sephadex G-100 saturated and developed with 6 M guanidine hydrochloride. These results are presented in Fig. 4 and gave a molecular weight of 16 000 for rat a-lactalbumin. In this system, unreduced rat a-lactalbumin had a molecular weight slightly less than 16 000.

'O K

6'

4

J'

4, 3,

(J bJ 0

I ~ 0.5

I 0.6

I 0.7

i 0.8

I 0.9

I 1.0

RM Fig. 4. Molecular weight determination of rat a-lactalbumin by thin layer chromatography on Sephadex. The standard proteins are: 1, bovine a-lactalbumin; 3, chymotrypsinogen; 4, ovalbumin; and 5, bovine serum albumin. Rat a-lactalbumin was 2. RM is the ratio of the distance of protein migration to the distance of migration of serum albumin.

The apparent partial specific volume of rat a-lactalbumin at 6 mg/ml, as measured by densimetry, was 0.712 ml/g. This value was used for the partial specific volume, P2, since the apparent partial specific volume of native hydrophilic proteins exhibit little or no protein concentration dependence. A value for 92 of 0.708 ml/g, which was estimated for rat a-lactalbumin from its chemical composition (Tables I and I]), agrees well with the experimental measurement. Sedimentation equilibrium measurements on rat a-lactalbumin indicated a tendency for the protein to undergo reversible association-dissociation. Plots of In(fringe displacement) versus (radius) 2 exhibited upward curvature in two different buffer systems and at a number of beginning protein concentrations between 0.2 and 2 mg/ml. That this apparent sample heterogeneity resulted from association-dissociation and not from the presence of contaminating proteins is indicated by the fact that the apparent weight-average molecular weight of the sample increased with increasing protein concentration. Fig. 5 gives the combined sedimentation equilibrium data plotted as the apparent weight-average molecular weight as a function of radial protein concentration in the centrifuge cell. A linear extrapolation of these data to infinite dilution yields an estimated molecular weight for rat a-lactalbumin of 15 400 ± 5 ~ . Though a linear extrapolation of these limited data may not be the most correct description of the associating behavior of this protein, linearization is not without precedent in this concentration range [32].

89 TABLEI A M I N O ACID COMPOSITION OF RAT ct-LACTALBUMIN Amino acid

#mol/100mg ~

mg/100 mg b

Residues/mol c

Lysine Histidine Argine Aspartic acid g Threonine Serine Glutamic acid g Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Half-cystine Total

62.3 17.2 12.0 91.0 31.2 61.1 102 36.1 43.0 47.9 30.5 a 12.0 53.0 d 45.3 20.8 27.5 24.2 e 51.0 r 768

7.99 2.36 1.87 10.5 3.16 5.32 13.2 3.5 2.46 3.41 3.02 1.57 6.00 5.13 3.39 4.05 4.50 5.26 86.7

10 (10) 2.8 (3) 1.9 (2) 14.6 (15) 5.0 (5) 9.8 (10) 16.3 (16) 5.8 (6) 6.9 (7) 7.7 (8) 4.9 (5) 1.9 (2) 8.5 (8) 7.3 (7) 3.3 (3) 4.4 (4) 3.9 (4) 8.2 (8) 123 (123)

a Unless otherwise indicated, the values represent an average of the amounts obtained for 24, 48, and 72 h hydrolysis time. b Values calculated using residue molecular weight. c Values calculated on the basis of a molecular weight of 16 000. Numbers in parentheses are the residues per molecule rounded to the nearest integer. Values were taken from 72 h hydrolysis time. e Determined spectrophotometrically. r Determined as cysteic acid after performic acid oxidation. g Values include the amide.

TABLE I1 CARBOHYDRATE COMPOSITION OF RAT ct-LACTALBUMIN Monosaccharide Hexose Mannose Galactose Glucose Hexosamine Glucosamine Galactosamine Fucose Sialic acid Total

/~mol/100 mg

rag/100 mg a

16.2 16.1 1.2

2.62 2.61 0.19

16.1 1.7 5.0 10.9 69.6

3.27 c 0.35 ~ 0.73 3.17 13.4

Residues/mol b 2.6 (3) 2.6 (3) 0.2 (0) 2.6 0.3 0.8 1.7 11

(3) (0) (1) (2) (12)

a Values calculated using residue molecular weight. b Values calculated on the basis of a molecular weight of 16 000. The number in parenthesis is the residue per molecule rounded to the nearest integer. c Values expressed as N-acetyl derivative.

90 18

' • 17,5 x ¢L

17

]=

I=Z 16.5 16

15.5

r

O

0

•

0.4

0.8 1.2 1.6 [oc-LA] radial (mg/ml)

2.0

Fig. 5. Apparent weight-average molecular weights of rat a-lactalbumin in solution at pH 6.8-7.2. A, initial protein concentration -- 0.2 mg/ml and rotor speed 52 000 rev./min; O, initial protein concentration 0.47 mg/ml and rotor speed 34 000 rev./min, Q, initial protein concentration 1.4 mg/ml and rotor speed 28 000 rev./min; II, initial protein concentration 1.9 mg/ml and rotor speed 26 000 rev./min. [a-LA]rad~a,stands for the protein concentration at a given radial position in the ultracentrifuge cell; )f/w,,pp is the apparent weight average molecular weight of that radial position.

The amino acid composition of rat a-lactalbumin is presented in Table I and the carbohydrate composition is presented in Table II. DISCUSSION In general, a-lactalbumin is relatively easy to purify from most milks but in rat milk the problem was complicated by the high level of serum albumin which is the major whey protein (skim milk less the caseins). Both serum albumin and alactalbumin have similar charge properties and are difficult to separate on DEAEcellulose and thus it was necessary to separate these proteins by a molecular sieve column in the final step of purification. In addition rat a-lactalbumin is a glycoprotein and migrates on molecular sieves with an apparent molecular weight of 26 to 28 000 thus forcing the use of a Bio-Gel P-150 column to effect separation from the bulk of the serum albumin. The apparent molecular weight of rat a-lactalbumin from molecular sieve columns and from electrophoresis in sodium dodecyl sulfate was 26 000-28 000. However, the molecular weight obtained from the reduced and alkylated protein was 16 000 and 15,400 ± 5 ~ from sedimentation equilibrium determinations. The high molecular weights obtained by molecular sieve chromatography and electrophoresis in sodium dodecyl sulfate is probably reflective of the carbohydrate nature of rat a-lactalbumin and for the tendency for the protein to associate in dilute buffer• Rat a-lactalbumin is unique in that it is a glycoprotein and exists in multiple forms• Most a-lactalbumins do not contain carbohydrate though there is good evidence for a minor form of bovine a-lactalbumin which contains low levels of carbohydrate [8, 9]. All of the multiple forms of rat a-lactalbumin contain carbohydrate and are active in the lactose synthetase reaction and it appears that the multiple forms, in part, are due to differences in the content of sialic acid. Examination of the carbohy-

91 d r a t e c o m p o s i t i o n suggest t h a t the p r o m i n e n t c a r b o h y d r a t e unit w o u l d be o f the a s p a r a g i n e l i n k e d type since rat a - l a c t a l b u m i n contains a relatively high level o f m a n nose a n d glucosamine. The a m i n o acid c o m p o s i t i o n d a t a shows a relatively high level o f proline, 6 in r a t a - l a c t a l b u m i n as c o m p a r e d to 2 in bovine a - l a c t a l b u m i n a n d this high value is reflected in the circular d i c h r o i s m spectra since the calculated percent a-helix o f rat a - l a c t a l b u m i n is low c o m p a r e d to other a-lactalbumins. R a t a-lactalb u m i n has a higher level o f G l x t h a n Asx which is in c o n t r a s t to m o s t other a-lactalbumins. R a t a - l a c t a l b u m i n is a unique a - l a c t a l b u m i n in t h a t it is a g l y c o p r o t e i n t h o u g h still active in the lactose synthetase system. P r e l i m i n a r y experiments suggest that m o s t o f the c a r b o h y d r a t e is l o c a t e d in a single p e p t i d e unit a n d studies relating to the carb o h y d r a t e sequence a n d n a t u r e o f the multiple forms are in progress. ACKNOWLEDGEMENTS The a u t h o r s wish to t h a n k C h u n g - H o Hung, G. Schultz, a n d R. P r a s a d for assistance with some o f the experiments. This w o r k was s u p p o r t e d by grants f r o m the N a t i o n a l Science F o u n d a t i o n (GB 23809) a n d the N a t i o n a l Institutes o f H e a l t h ( A M 18257).

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92 27 28 29 30 31 32

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