structural studies on bovine lactoferrin*

THE JOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 245, No. 1, Issue of September 10, pp. 4269-4275, 1970

Printed in U.S.A.

Structural Studies on Bovine Lactoferrin*

(Received for publication, March 19, 1970)

FRANCIS J. CASTELLINO,$ WAYNE TV. FISH,§ AND KENNETH G. MANNA

From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27706

SUMMARY

Bovine lactoferrin has a molecular weight of 77,100 f 1500 in dilute aqueous solution as determined by the method of sedimentation equilibrium in the ultracentrifuge. In 6 M guanidine hydrochloride in the presence or absence of mercaptoethanol, bovine lactoferrin has a molecular weight of 72,500 to 77,200 k1300 as measured by sedimentation equilibrium in the ultracentrifuge or by gel filtration on agarose columns. The molecular weight obtained by gel electrophoresis in the presence of sodium dodecyl sulfate is 76,000 f 2400. Tryptic peptide maps reveal a number of peptides approximately equal to the content of lysine plus arginine in the molecule. Quantitative end group analysis shows 1 residue of alanine at the amino terminus of the molecule. These data demonstrate that bovine lactoferrin is composed of a single polypeptide chain. Analysis of the carbohydrate content revealed 1 residue of terminal sialic acid, 10 to 11 residues of N-acetyl glucosamine, 5 to 6 residues of galactose, and 15 to 16 residues of mannose per molecule of lactoferrin. Another protein, isolated as a major contaminant in commercial preparations of bovine cY-lactalbumin showed identical properties to bovine lactoferrin except that it appeared, based upon molecular weight measurements, to be an aggregating system.

The transferrins are a group of nonheme iron-binding proteins found in various fluids of vertebrate animals. These proteins are believed to function by transporting iron from storage areas to red blood cells (1). Transferrins have been found in the blood

* This research was supported in part by Grant HE 06400 from the National Institutes of Health to Dr. Robert L. Hill and by Grant 5 lWl-AM-045709 from the National Institutes of Health to Dr. Charles Tanford.

1: Sunported in part, by Postdoctoral Research Grant 5 F02 AM 34,008 from the National Institutes of Health. Present address, Dcnartment, of Chemistrv. Universitv of Notre Dame, Notre Dame, Indiana 4655G. Tdkhom ins&es should be addressed.

5 Supported in part by Post,doctoral Research Grant 1 F02 AXI 33.249 from the National Institutes of Health. Present address, Department of Biochemistry, Medical University of South Carolina, Charleston, South Carolina 29401.

7 Supported in part by Postdoctoral Research Grant 5 F02 GM 34,119 from the Nat,ional Institutes of Health. Present address, Department of Biochemistry, University of Minnesota, St. Paul, Minnesota 55101.

serum of all vertebrates investigated, the milk of all mammals studied, and the egg white of all avians studied (2).

Human serum transferrin has been well characterized. Al- though this protein contains two equivalent iron-binding sites (3) and two identical carbohydrate side chains (4), evidence is quite clear that it is composed of a single polypeptide chain (5). Bovine lactoferrinl has also been studied and several of its properties have been reported. This protein also contains two equivalent iron-binding sites but the status of the carbohydrate moiety is uncertain. Molecular weights between 80,000 (6) and 88,000 (7) have been reported for this protein and amino acid compositions which are considerably different have also been reported (6,7). No studies have been made on the subunit structure of lactoferrin, although, as in the case of human serum transferrin, two polypeptide chains are implied based on the observa- tions that two equivalent iron-binding sites exist in this protein.

We have undertaken a study of the characterization of lactoferrin in order to establish many of its chemical and physical properties with emphasis on its subunit structure. This informa- tion is essential as a prerequisite to understanding the function of this protein in the iron transport system.

EXPERIMENTAL PROCEDURE

Jfateriuls

The lactoferrin used in these studies was either isolated from commercial preparations of bovine cu-lactalbumin (Pentex, Inc., Kankakee, Illinois) or generously donated by Dr. Philip Aisen. Both preparations were homogeneous as judged by disc gel electrophoresis at pH 8.5 (8) and pH 3.2 in the presence of 6.25 M

urea (9). Trypsin (treated with tosylphenylalanylchloromethyl ketone)

was obtained from Worthington. Guanidine hydrochloride was purchased from Heico, Inc. (ultra high purity) and used without further purification. Iodoacetic acid was purchased from Mathe- son, Coleman, and Bell and recrystallized from hexane. N- Ethylmorpholine was redistilled (b.p. 136-139”) and stored over sodium hydroxide pellets at 5’.

All other reagents were the best available commercial grade and used without further purification.

Tlethods

Isolation of Bovine Lactojerrin-Approximately 3 g of Pentex bovine Lu-lactalbumin were dissolved in 40 ml of 0.05 M ammo-

nium bicarbonate and the solution was adjusted to pH 8.6 with

1 Hereafter referred to as lactoferrin. When lartofttrrins from other species are mentioned their sources will bc spwifictd.

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Bovine Lactoferrin Vol. 245, No. 17

1 M sodium hydroxide. Insoluble material was centrifuged and discarded. The sample was placed on a column (5 x 140 cm) of Sephades G-100 equilibrated with 0.05 M ammonium bicarbonate. The column was developed with the same solvent at a flow rate of 36 ml per hour and 12-ml fractions were collected. A red protein appeared in the void volume of the column which was collected and pooled. Approximately 250 mg of this protein was obtained. This red protein was subsequently shown to be identical in most respects to authentic lactoferrin as supplied by Dr. Philip Aisen.

Sedimentation Equilibrium Studies-All sedimentation equilibrium studies were performed using the short column, high speed technique of Yphantis (10).

The samples used in these studies were dissolved in the appropriate solvent and dialyzed for at least 24 hours prior to sedimentation analysis. For determinations of native molecular weights the solvent used was 0.1 M sodium chloride, pH 7.0 and for subunit studies the solvent used was 6 M guanidine hydrochloride, 0.14 M mercaptoethanol, pH 8.6. In cases in which carboxymethylation was desired, a slight molar excess of iodo- acetate over mercaptoethanol in 6 M guanidine hydrochloride, pH 8.6 was added to the reduced protein.

All sedimentation equilibrium studies were performed with a Spinco model E analytical ultracentrifuge equipped with Ray- leigh interference optics and a temperature control unit. Ap- proximately 3-mm column heights and a temperature of 25” were used. Fluorocarbon FC-43 (0.02 ml, Beckman) was used to provide a flat, transparent cell bottom. Rotor speeds were chosen so that at equilibrium the meniscus concentration would be essentially 0 and the effective, reduced molecular weight, u, of the protein would be approximately 5 cm-* (10). Equilibrium times were measured by allowing all runs to proceed until there was no further increase in fringe displacement with time at a given radial distance (T) from the center of rotation. Following each experiment, a water blank was run without disassembling the cell to correct for minor effects of cell window distortions.

Molecular weights (.U) were calculated as described by Yphan- tis (10) using the following equation

d In c 2RlQ-

M= w2(1 - 4/d

0)

where 4’ is the effective partial specific volume of the protein in the solvent used, p is the solvent density, and w2 is the angular velocity of the rotor. The values of d In c/dr2 were obtained by linear least squares analysis of the plot of the natural logarithm of the blank corrected fringe displacements, In c, against the radial positions in the cell, r2.

The partial specific volume (0) of lactoferrin calculated from the amino acid composition is 0.722 ml per g and this value was used for 4’ in calculation of native molecular weights. The value of 4 in concentrated aqueous solutions of guanidine hydrochloride is somewhat ambiguous. Hade and Tanford (11) have demonstrated that 4’ for a number of proteins can be decreased by as much as 0.01 ml per g in 6 M guanidine hydrochloride. However Reithel and Sakura (12) found little change in fi for several proteins in 6 M guanidine hydrochloride. We have, therefore, based our molecular weights of lactoferrin in 6 M

guanidine hydrochloride on a c$’ value identical to the native value (0.722 ml per g) or decreased by 0.01 ml per g from the native value (0.712 ml per g).

Sedimentation Velocity Studies-Sedimentation velocities were measured in a Spinco model E analytical ultracentrifuge, using schlieren optics. All experiments were run at 25” and corrected to the density and viscosity of HZ0 at 20” (13).

Gel Filtration-The molecular weight of reduced, carboky- methylated lactoferrin was also determined in 6 M guanidine hydrochloride according to the gel filtration technique of Fish, Mann, and Tanford (14) using a column of 4%~ agarose equilibrated and developed with 6 M guanidine hydrochloride. The molecular weight of lactoferrin was estimated by comparison of its observed distribution coefficient with a calibration curve of log molecular weight versus distribution coefficient prepared for the column using proteins of known molecular weight.

Electrophoretic Analysis-The molecular weight of reduced lactoferrin was also obtained by the recently developed esten- sion (15) of the sodium dodecyl sulfate gel electrophoresis method (16). The molecular weight of lactoferrin was estimated by comparison of its electrophoretic mobility with a series of standard proteins run under the same conditions.

E&action CoeficientsThe extinction coefficient of native lactoferrin was determined by the ultracentrifugal method of Babul and Stellwagen (17).

The extinction coefficient of lactoferrin in 6 M guanidine hydrochloride was obtained by dilution of a known amount of protein with 7 M guanidine hydrochloride, final concentration of 6 M, and known volume and measuring the resulting absorbance.

Amino Acid Composition-Amino acid analyses were drter- mined by hydrolyzing l.O-mg samples of protein in 6 s HCl under reduced pressure for 24, 48, and 72 hours at I 10” (18) and performing the analysis on a Spinco model 120 13 automatic amino acid analyzer (19). Duplicate samples were analyzed for each time interval.

The total half-cystine content of lactoferrin was determined as cysteic acid by analyzing acid hydrolysates of performic acitl- treated protein (20). The sulfhydryl content was determined by the technique of Ellman (21) in 6 M guanidinc hydrochloride.

The tryptophan content of lactoferrin was estimated by the method of Edelhoch (22).

The total amide content (glutamine and asparagine) of l&o- ferrin was determined by amino acid analysis by measuring t,he amount of glycine incorporated into the free carboxyl groups of the protein after activation of the free carbosyl groups with l- ethyl-3-(3.dimethylaminopropyl)carbodiimide followed by reaction with glycine methyl ester (23). The number of glutamine and asparagine residues in the protein is equal to the total of the aspartic and glutamic acid residues obtained by amino acid analysis minus the glycine incorporated into the modified samples.

NHz-terminal Analysis-Quantitative amino-terminal amino acid studies were performed according to the method of Stark and Smyth (24). Lactoferrin (0.2 to 0.5 I.tmole) was carbamylated for 20 hours at 50” in 5 ml of 6 M guanidine hydrochloride containing 0.2 M N-ethylmorpholine acetate, pH 8.0, and 0.6 M

potassium cyanate. Excess cyanate was destroyed by addition of glacial acetic acid and the carbamylated protein was

dialyzed and lyophilized. The remaining procedures were as described earlier (24).

Peptide MapsPeptide maps were prepared from trgptic digests of S-carboxymethyl lactoferrin according to the method of Steers, Craven, and Anfinsen (25). Protein samples were reduced and carboxymethylated as described above. Tryptic


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hydrolysis of the reduced, carboxymethylated protein was car- ried out by suspending the protein in 0.2 M NH*HCOs, pH 8.2, at 37” and adding a sufficient amount of trypsin to give a final concentration of 2% of the weight of S-carboxymethyl lactoferrin. The reaction was terminated after 6 hours of digestion by acidification and lyophilization of the solution.

Peptide mapping was accomplished by descending chromatography in 1-butanol, glacial acetic acid, water (4: 1:5), followed by high voltage electrophoresis in pyridine, acetic acid, water (1:10:289) at pH 3.6. The peptides produced were located by a ninhydrin spray. Specific staining for histidine, tryptophan, and tyrosine was performed as described by Smith (26) after first bleaching the ninhydrin sprayed map with 1% HCl in acetone.

Carbohydrate Analysis-The total carbohydrate content (except amino sugars) of lactoferrin was determined by the phenol sulfuric acid method (27).

The amino sugar content of lactoferrin was determined by digesting the protein for 4 to 7 hours in 4 N HCI (28) and analyzing of the digest on the short column of the amino acid analyzer. The elution positions of glucosamine and galactosamine as well as their recovery values were determined by analyzing standard solutions of these amino sugars under the same conditions as used for the protein.

Sialic acid was determined by first incubating lactoferrin with neuraminidase (partially purified from Clostridium perjringens) to release the bound sialic acid and determining the amount of sialic acid released by the thiobarbituric acid assay (29).

Analysis of the neutral carbohydrates present in lactoferrin was kindly performed by Dr. Robert Barker, University of Iowa, in the following manner. A small quantity of lactoferrin was hydrolyzed in 1 N HzS04 for 16 hours at 90”. The sample was

then neutralized by addition of 1 N NaOH and reduced with sodium borohydride. Cations were removed by passing the solution through a column of Dowex 50 (H+), 8 x 200 mesh. Sulfate was removed by precipitation with Ba(OH)z and a batch- wise addition of Dowex 50 (H+), 8 X 200 mesh, was again added to remove excess Ba++. The solution was evaporated to dry- ness and dried down from methanol several times to remove borate. The residue was dissolved in pyridine and acetylated in the usual manner with acetic anhydride in order to render the carbohydrates volatile. The neutral carbohydrates present in the sample were identified by gas-liquid chromatography on a column (5 foot X i; inch) of 10% neopentyl glycol sebacate on Chromosorb W at 210’ (Analytical Engineering Laboratories, Hamden, Connecticut). The carrier gas was helium at 30 ml per min and detection was accomplished with a flame-ionization detector.

RESULTS

Comparison of Lactoferrins Used in This Study-The lactoferrin isolated as the major impurity in Pentex preparations of bovine cr-lactalbumin appeared to be identical to authentic lactoferrin in most of its properties. Each protein had an O.D.280:0.D.465 = 27, which indicates that two Fe+3 binding sites are present (3) per 76,000 molecular weight. Each protein migrated as a single band in the urea-acetate disc gel electrophoresis system and when equal quantities of the two proteins were mixed and characterized by this method, a single band was observed. Both proteins had almost identical amino acid and carbohydrate compositions and, as shown in Fig. 1, the tryptic peptide maps of these proteins were indistinguishable from each other. On the other hand, one property which these proteins did not have in common dealt with their association properties

j

FIG. 1. Comparison of the tryptic peptide maps of the two bovine lactoferrins used in this study. A, authenic bovine lacteferrin as supplied by Dr. Philip Aisen; R, the red protein component present in commercial preparations of bovine &&albumin. The conditions are as described under “Methods”. I, Histidine-positive peptides; ,%‘, tyrosine-positive peptides; 8, tryptophan-positive peptides.


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4272 Bovine Lactoferrin Vol. 245, No. 17

‘;; 6.0 5 t

s 5.5 0 c 5.0 -

/ / A B I I I I I I

50.8 51.0 51.2 51.4 50.8 51.0 51.2 51.4

rz(cmz 1

FIG. 2. Sedimentation equilibrium of bovine lactoferrin. A, lactoferrin in 0.1 M sodium chloride, pH 7.0, at 25” and 16,000 rpm; B, red component from bovine or-la&albumin in 0.1 M sodium chloride, pH 7.0, at 25” and 15,000 rpm.

6.5

‘;i

560 5 .- E

- 5.5 ”

I /

I I I I

504 506 50.8 51.0 51.2 51.4

r2 (cm*)

FIG. 3. Sedimentation equilibrium of bovine lactoferrin. 0, lactoferrin in 6 M guanidine hydrochloride, 0.14 M mercaptoethanol, pH 8.6, at 25” and 26,000 rpm. 0, lact,oferrin in 6 M guanidine hydrochloride at 25” and 30,000 rpm. The ordinate represents the natural logarithms of the blank corrected fringe displacements in microns and the abscissa represents the squares of the radial distances in centimeters. rb’, radial distance squared from the center of rotation to the cell bottom.

in the native state. As indicated from the plots of In c versus ~2, obtained by sedimentation equilibrium studies, and shown in Fig. 2, lactoferrin, conventionally purified, appeared to be a nonassociating system, whereas lactoferrin purified from preparations of bovine a-lactalbumin exhibited concentration- dependent association properties.

On studies in the denatured state, both proteins eluted at identical positions in the gel filtration experiments and both proteins when mixed migrated as a single band in the sodium dodecyl sulfate gels. Therefore, since these proteins appeared to be identical in most of their properties, they will be differentiated in this study only when describing experiments on the native protein.

Molecular Weights-The molecular weight of lactoferrin conventionally purified, in dilute aqueous salt solutions, obtained by sedimentation equilibrium in the analytical ultracentrifuge was 77,100 f 1500. This material was homogeneous with respect to molecular weight as evidenced from the linearity of the plots of In c versus r2 shown in Fig. 2A. On the other hand,

TABLE I

Summary of molecular weight data obtained for lactoferrin

Molecular weight

77,100 zt 15000

72,500 to 77,200 f 13006

77,000 f 2oooc

76,000 f 2400d

-

-

Solvent

0.1 M sodium chloride, pH 7.0

6 M guanidine hydrochloride with or without, 0.1 M mercaptoethanol, pH 8.6

6 M guanidine hydrochloride

Sodium dodecyl sulfate

Method

Sedimentation equilibrium

Sedimentation equilibrium

Gel filtration

Polyacrylamide electrophoresis

0 Average of three determinations b Average of three determinations in each solvent syst,em. The

range of values reported for the molecular weight reflects 4’ values of 0.722 to 0.712 used in the calculation.

c Protein present as S-carboxymethyllactoferrin, average of three determinations.

d Average of three determinations.

the lactoferrin purified from commercial preparations of bovine cu-lactalbumin exhibited heterogeneity with respect to molecular weight in dilute aqueous salt solutions as evidenced from the curvature of the plots of In c uersus r2 shown in Fig. 2B. Although a unique molecular weight could not be obtained for this protein under these conditions, the molecular weight estimated from Fig. 2B by the value of the limiting slope of In c versus r2 at the top of the cell was 76,000 and at the cell bottom molecular weights greater than 200,000 were estimated. Clearly, this protein associates in the native state and at the concentrations used in these experiments aggregates as high as trimers were obtained.

Studies performed on lactoferrin in 6 M guanidine hydrochloride in the presence or absence of reducing conditions indicate that the molecular weight of lactoferrin is not lowered in this solvent. Fig. 3 presents typical data obtained by sedimentation equilibrium analysis in 6 M guanidine hydrochloride in the presence or absence of mercaptoethanol. Clearly, a monodisperse system is obtained and mercaptoethanol is without influence on the molecular weight. The value of the molecular weight of lactoferrin in 6 M guanidine hydrochloride in the presence or absence of mercaptoethanol ranges from 72,500 to 77,200 f 1,300 de- pending upon the value of 4’ used in Equation 1.

Experiments conducted on the molecular weight of S-carboxymethyl-lactoferrin in 6 M guanidine hydrochloride by gel filtration according to Fish, Mann, and Tanford (14) gave a molecular weight of 77,000 =t 2,000, in excellent agreement with the results obtained by sedimentation equilibrium. Further, the molecular weight of the maximum subunit was determined by gel electrophoresis in sodium dodecyl sulfate and a value of 76,000 f 2,400 was obtained. These molecular weights are summarized in Table I and the data strongly suggest that lactoferrin, as human serum transferrin (5), is composed of a single polypeptide chain.

Sedimentation Velocity-The sedimentation coefficient (SZOJ of lactoferrin in 0.1 M NaCl, pH 7.0, conventionally purified, was found to be 5.3 S at a protein concentration of 5 mg per ml, in reasonable agreement with the value reported for human serum transferrin (5). A single apparently symmetrical peak was ob-


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TABLE II TABLE III

Amino acid composition of lactoferrin Summary of analysis of tryptic peptide patterns of S-carboxymethyllactoferrin Duplicate analyses were

Amino acid

Lysine. .................. Histidine ................ Arginine .................. Aspartic acid. ............ Threonine ................ Serine ................... Glutamic acid, ........... Proline ................... Glycine. .................. Alnnine. ................. Valine. ................... Methionine ............... Isoleucine ................ Leucine ................. Tyrosine ................. Phenylalanine Cysteic acid. ............. Glutamine + asparagine Tryptophan. .............

Total residues. ...........

-

--

:rformed on each hydrolysate.

Amount formed after hydrolysis

24 hr

I I

48 hr 72 hr

42.6 41.7 42.1 9.6 10.0 10.4

32.7 32.9 31.3 69.6 70.5 71.2 38.8 36.3 35.2 45.0 42.1 41.1 73.0 71.4 77.5 31.2 30.0 33.3 43.7 41.5 45.4 59.7 57.1 59.9 36.3 37.4 42.9

4.7 3.9 4.0 13.6 15.1 17.4 57.5 58.6 61.1 18.9 18.8 20.4 23.6 25.9 25.1

-

C

.-

&&ted number of residues/molecule

residues/molecule

42 10 32 71 39* 45” 73 31 43 59 43c

4 17c 61c 19 25 28 61d

9d

651

a The carbohydrate content is subtracted from the molecular weight used for actual calculation of the composition data (71,000).

* These values were extrapolated to zero time hydrolysis. c These values were taken from the 72 hours of hydrolysis time. d Measured separately as described in the text.

tained. On the other hand the sZO,W obtained for lactoferrin purified from commercial bovine ar-lactalbumin was 8.6 S at 5 mg per ml. There was a slight appearance of asymmetry in the peak but this is not unexpected in an associating-dissociating system.

&tinction Coeficient-A solution of Fe+3-saturated lactoferrin with a measured absorbance at 280 rnp was analyzed in a syn- thetic boundary cell in the ultracentrifuge with the aid of a solvent blank and the fringe displacement was measured. Babul and Stellwagen (17) have demonstrated that several proteins, at concentrations of 1 mg per ml, give fringe displacements of 1160 p across a boundary formed between the solvent and protein solution. The measured absorbance together with the measured fringe displacement for a given lactoferrin solution allowed us to calculate an t g0 = 14.5. Concentrated aqueous solutions of lactoferrin of known concentrations were diluted with guanidine hydrochloride and gave an cZsO - ‘% - 11.3 in 6 M

guanidine hydrochloride. There was no residual absorbance at 465 rnp of lactoferrin treated with 6 M guanidine hydrochloride in this manner. Therefore, the value of the ~f$ does not include contributions from Fe+3.

Amino Acid Composition-The amino acid composition of bovine lactoferrin is presented in Table II. All values reported represent the average of 24-, 4%, and 72-hour hydrolysis times except serine and threonine which were extrapolated to zero time

[ Spray reagent for detection of various peptides

Nad$gt;2des Content in lactoferrin

Total peptides.. 69-73 420 32*

Histidine peptides.. 7 10 Tryptophan peptides. 6-7 9 Tyrosine peptides.. 10 19

(L Lysine. * Arginine.

TABLE IV

Summary of carbohydrate analysis of lactoferrin

Carbohydrate Amount of lactoferrin

Method

residues/molecule

(N-acetyl)glucosamine 10-11 Amino acid analysis Mannose 15-16 Gas-liquid chromatog-

raphy Galactosea 5-6 Gas-liquid chromatog-

raphy Sialic acid 1 Thiobarbituric acid

after treatment with neuraminidase

a Identified as hexa-0-acetyl mannitol and hexa-O-acetyl galactitol.

and valine, isoleucine, and leucine which are given by the 72- hour hydrolysis time. Tryptophan values were determined in 6 M guanidine hydrochloride by the method of Edelhoch (22) and an average value of 9.2 residues per molecule was obtained. The total free carboxyl residues of lactoferrin including the carboxy-terminal amino acid, determined by incorporation of glycine, was found to be 84.1, which indicates that 61 of the total glutamic acid and aspartic acid residues are present as amides. The total for half-cystine residues, determined after performic acid oxidation, was 28.3. Titrations of lactoferrin in 6 M guanidine hydrochloride with Ellman’s reagent indicated that no free sulfhydryl groups were present. Therefore, lactoferrin contains 14 disulfide bonds.

NH2-terminal Amino Acid-Quantitative amino-terminal determinations according to the method of Stark and Smyth (24) indicated 0.9 + 0.2 residues of alanine, using a molecular weight of 76,000, present at the amino terminus of lactoferrin. An experiment performed with noncarbamylated lactoferrin was used as a control and appropriate corrections were made.

Tryptic Peptide Alaps-The tryptic peptide maps of S- carboxymethyl lactoferrin are shown in Fig. 1 and the data obtained are summarized in Table III. Clearly, the number of ninhydrin-positive spots obtained are approximately equal to the amount of lysine and arginine present in the molecule. Also, when the tryptic peptide maps of the two preparations of lactoferrin used in this study and shown in Fig. 1, A and B, are compared, it becomes quite clear that the proteins are indeed identical, within experimental limits, in their chemical structure.

Carbohydrate Analysis--The total neutral carbohydrate de-


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termined for each of the two lactoferrin preparations was 5.6 f 0.2% in three separate determinations. This value does not include amino sugars (27). Amino sugar analysis was performed after acid hydrolysis by amino acid analysis and 10 to 11 residues of (N-acetyl)glucosamine were found to be present per 76,000 daltons in three separate determinations. Sialic acid was determined by the thiobarbituric acid assay (29) after release of bound sialic acid with neuraminidase. A value of 0.95 moles of sialic acid per molecule of lactoferrin was obtained in one de- termination. Subtracting the sialic acid content from the total neutral carbohydrate leads to 21 to 22 residues of hexose (or pentose) present per molecule of lactoferrin. Identification of the hexose and pentose present by gas-liquid chromatography as described under “Methods” indicated that only mannose and galactose were present in a 2.6:1 ratio. The data obtained for the carbohydrate analysis on lactoferrin are summarized in Table IV.

DISCUSSION

The studies reported here indicate that lactoferrin has a molecular weight of 76,000, a value somewhat in disagreement with values reported earlier (6, 7). Further, this value of the molecular weight determined by sedimentation equilibrium is not lowered in 6 M guanidine hydrochloride-O.1 N mercaptoethanol, a solvent which has previously been shown to c ompletely disrupt the covalent and noncovalent bonds normally associated with subunit aggregation, yielding the molecular weight of individual subunits (30, 31). However, since molecular weight determina- tion by sedimentation equilibrium is a measure only of the mass of the particle, this technique cannot distinguish between a single polypeptide chain with a given mass and an incompletely dissociated or covalently linked set of polypeptide chains of the same cumulative mass. Therefore, we have calculated the molecular weight of S-carboxymethyl lactoferrin in 6 M guanidine hydrochloride by the gel filtration technique of Fish, Mann, and Tanford (14). The calculation of the molecular weight by this technique depends upon the molecule assuming the behavior of a linear random coil. An incompletely dissociated or z-linked molecule would yield a molecular weight by this technique lower than the molecular weight determined by sedimentation equilibrium since this method depends upon the hydrodynamic volume as a function of chain length. Since the molecular weight of lactoferrin determined by gel filtration was found to be 77,000, a value virtually identical to the molecular weight determined by sedimentation equilibrium, we conclude that reduced lactoferrin exists as a linear end-to-end random coil in 6 M guanidine hydrochloride and the molecule must exist as a single polypeptide chain. Of course, an incompletely dissociated molecule linked at the termini would also give the same results, but this circumstance is highly unlikely. Further confirmation of the molecular weight of the lactoferrin subunit was obtained by sodium dodecyl sulfate gel electrophoresis. This technique has been shown to yield the molecular weight of the maximum subunit for 40 different proteins (15) and a molecular weight of 76,000 has been obtained for lactoferrin. Thus, as in the case of human serum transferrin (5), although lactoferrin consists of two equivalent Fe+a-binding sites, it is apparently composed of a single polypeptide chain.

Analysis of the tryptic peptide maps of S-carboxymethyl lactoferrin, prepared under conditions which would be least expected to lead to artifacts, indicate that the structure is a

single polypeptide chain of nonrepeating sequence. In numerous trypt,ic-mapping experiments we have never observed fewer than 69 or greater than 73 ninhydrin-positive peptides. The theoretical number of peptides expected based on a nonrepeating sequence would be equal to the sum of lysine and nrginine content plus 1, i.e. 75. The observed number of histidine and tryptophan-containing tryptic peptides was 70c0 of the theoretical and the number of tyrosinc-containing tryptic peptides was approximately 50% of the theoretical. These numbers would be expected to be low since one tryptic peptide may con- tain more than one of the above residues. It had previously been reported that analysis of the tryptic peptide maps of human serum (5) and chicken egg white transferrin (32) led to a number of peptides which were considerably lower than expected based ou the lysine-arginine content of these proteins and it was pro- posed that portions of the transferrin polypeptidc chain in these species occurred in duplicate. Apparently, bovine lnctoferrin differs from human serum and chicken egg white transferrins in that it is either not homologous to these proteins, which ia nn- likely, or that greater differentiation of bovine lactoferrin during evolution has occurred.

There is nothing striking about the amino acid composition of lactoferrin except that the molecule is highly cross linked, (‘on- taining 14 disulfide bonds. One residue of alanine per molecule of lactoferrin is present at the amino terminus. If lactoferrin binds Fe+3 in the same manner as human serum transferrin, then 4 of its 10 histidine residues and 6 of its 19 tgrosine residues are functional in this respect. The amino acid composition of bovine lactoferrin differs in many respects from bovine serum transferrin (7) and more closely agrees with the composition of bovine lactoferrin reported by Groves and Basch (i) than with the composition reported by IUanc, Isujard, and 1Iauron (6).

The total carbohydrate content (sialic acid, neutral and amino sugars) of bovine lactoferrin was previously reported to be 7.2yC (7). Our data is in agreement with this value. In adtli- tion m-e have found the carbohydrate moiety to rollsist of 15 to 16 residues of mannose, 5 to 6 residues of galactosc, 1 residue of sialic acid, and 10 to 11 residues of glucosamine. The gluco.sa- mine is probably present as N-acetyl glucosamine as it nsunllg is in glycoproteins (28), although we could not establish this by the techniques used in this study. The terminal carbohydrate residue is I mole per mole of sialic acid. Jamieson (4) has reported that the carbohydrate of human serum transferrin was present on 2 glycopeptide chains, each of molecular weight 2350 daltons. Since we have found only 1 terminal residue of sinlic acid per molecule of lactoferrin, either all the carbohydrate is present on 1 glycopeptide chain in this protein or 2 nonidentical carbohydrate chains exist in bovine lactoferrin.

Finally, another protein, apparently identical to bovine lactoferrin, was purified from commercial preparations of bovine a-lactalbumin. This protein differed from authentic lnctoferrin only in the fact that it appeared to aggregate at increasing protein concentrations. At present, no esplanation for this observation can be forwarded. It, of course, may be an artifact due to the manner in which cY-lactalbumin is prepared but it is not impossible that it may structurally differ from authentic lactoferrin in a manner undetected by the techniques used in this study and these structural differences may be responsible for the observed aggregation of this protein.

Acknowledgments-We wish to thank Dr. Robert L. Hill and


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Dr. Charles Tanford for their helpful comments during the course 15. WEBER, K., AND OSBORN, M., J. Biol. Chem., 244,4406 (1969).

of this investigation. We also wish to thank Dr. Patrick Mat- 16. SHAPIRO, A. L., VINUEL.4, E., AND MAIZEL, J. V., Biochem.

tack for performing the molecular weights by the sodium dodecyl Biophys. Res. Commun., 28, 815 (1967).

sulfate electrophoresis method. 17. BABUL, J., AND STELLWBGEN, E., Anal. Biochem., 28, 216

(1968).

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Francis J. Castellino, Wayne W. Fish and Kenneth G. MannStructural Studies on Bovine Lactoferrin

1970, 245:4269-4275.J. Biol. Chem.

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