Bovine Secretory Component* ISOLATION, MOLECULAR SIZE AND SHAPE, COMPOSITION, AND NH,-TERMINAL AMINO ACID SEQUENCE

Bovine free secretory component was purified from whey by salt precipitation, gel filtration, DEAE-cellulose and phosphocellulose chromatography, and immunoadsorption. It was obtained in immunologically pure form and in 56% yield. The Stokes radius of pure free secretory component was found to be 4.3 nm by gel filtration, and an .& of 4.1 S was determined by the ultracentrifuge. The molecular weight was 79,000 by sodium dodecyl sulfate gel electrophoresis and by sedimentation equilibrium in the ultracentrifuge, using a d of 0.73 determined by ultracentrifugation in D,O and H,O. A minimal axial ratio of approximately 5 was calculated. Amino acid analysis of bovine free secretory component showed remarkable similarity to that of human, dog, and rabbit but carbohydrate analysis showed significant differences. In contrast to the human, bovine free secretory component has 2 methionine residues/mol. The NH&rminal sequence was found to be Lys-Ser-Pro-Ile-Phe-Gly-Pro-Glu-Glu-Val-Asp-Ser-Val. This sequence is identical with that of the human and dog. However, the poor immunological cross-reactivity between the dog, human, and bovine proteins suggests that significant structural differences will be found in other regions of the molecule.

man (g-11), rabbit (12), and cow (13,14), as well as studies on the binding of human and bovine free secretory component to IgA (15), suggest similarities of secretory component in different species. Although the bovine free secretory component is easily available in relatively large amounts, no detailed studies have been made on its structure, and data on its molecular weight vary considerably (2). In the present paper we report the isolation of highly purified free secretory component, its molecular weight and conformational properties, amino acid and carbohydrate composition, and the NH,-terminal amino acid sequence. Interestingly, the amino acid sequences of the human and canine secretory component (16,17) show almost complete identity to that of bovine secretory component in the NH,-terminal 13 residues.

MATERIALS AND METHODS
Antisem-All antisera were prepared in rabbits by the subcutaneous injection of antigens into the foot pads in Freund's adjuvant. The specificity of the antisera was tested by double diffusion in agar and by immunoelectrophoresis.
Preparation of Bovine Free Secretory Component-Fresh adult cow milk was collected, and 0.2% sodium azide was added immediately. Fat was removed by centrifugation, and casein was precipitated at pH 4.5. The resulting whey was dialyzed overnight against cold running water and made 33% saturated with ammonium sulfate. After centrifugation the supernatant was made 50% saturated, and the precipitate was dialyzed against 0.1 M ammonium bicarbonate and lyophilized.
A solution of 1.0 g of the lyophilized ammonium sulfate fraction was dialyzed against 10 mM phosphate buffer at pH 7.4, applied to a DEAE-cellulose (DE52, Whatman) column (22 x 130 mm), and eluted with the same buffer. The unbound material was pooled, and its pH was reduced to 5.7 with 0.1 M phosphoric acid. This solution was then applied to a column of cellulose phosphate (Cellex-P, Bio-Bad, 66 x 22 mm) equilibrated with 0.05 M phosphate buffer, pH 5.7 (starting buffer). The absorbed proteins were eluted by a linear gradient produced from 250 ml of starting buffer (in a mixing chamber) and 250 ml of 0.1 M phosphate buffer, pH 7.5, containing 0.1 M NaCl in the reservoir of a gradient mixer GM-l (Pharmacia).
After elution with 306 ml of this gradient, 11 ml of 5.0 M NaCl in 0.1 M phosphate buffer, pH 7.5, were added to the reservoir to raise the final concentration of NaCl to 0.6 M, and the gradient was continued.
Eluted fractions were pooled in five pools, as shown in Fig. 1, and concentrated by ultrafiltration through a PM-10 Diaflo membrane. Pool 2 ( Fig. 1) was applied to a Sephadex G-200 column (110 x 2.5 cm) and eluted with 0.1 M ammonium bicarbonate solution. Two major peaks were obtained as shown in Fig. 2 by guest on March 24, 2020 http://www.jbc.org/ Downloaded from 72 hours. For amino acids showing loss by hydrolysis the data were extrapolated to zero time. whereas for slowI>-liberated amino acids the Xhour data were used. Methionine was determined as methionine sulfone. and cysteine (or cystine) as cysteic acid after performic acid oxidation (28). Tryptophan and tyrosine were determined spectrophotometrically in 6 M guanidine HCI (29) after correcting for cgstine absorption. Hydrolysis with mercaptoethanesulfonic acid for 22 hours (30) was also used for determination of tryptophan.
For amino-sugar determination hydrolysis by 4 N HCl for 6 hours at 100" and by 6 N HCl for 2 hours at 110' in sealed, evacuated tubes was used. Analysis was on the same amino acid analyzer. using a 20.cm column equilibrated at 49" with pH 5.26 citrate buffer (0.35 M in sodium).
Neutral sugar content was determined by the orcinol-sulfuric acid method as used by Francois et al. (31). Sialic acid was determined by the thiobarbituric acid method as described by Spiro (32).
Amino Acid Sequence Studies-The KHZ-terminal amino acid of performic acid-oxidized free secretory component was determined by polyamide thin layer chromatography after dansylation' in the presence of sodium dodecyl sulfate and hydrolysis with 6.0 N HCl as described by Gray (33). The NH,-terminal amino acid sequence analysis was performed with a Beckman model 890 C 'sequencer (34) using Quadrol buffer and a "fast" protein program (Beckman 072172C). For identification of each residue, gas-liquid chromatography of both PTH-derivatives as such, and after "on column" silylation, was performed.

RESULTS
Preparation and F'urity of Free Secretory Component-Results of typical experiments and yields at different steps in the preparation of free secretory component are shown in Table  I. It was important to accomplish these steps in as little time as possible to minimize proteolysis. Freezing of the purified free secretory component solution was preferred for storage.
Immunoelectrophoretic examination (Fig. 3, well 6) of the final free secretory component preparation showed only one precipitation line with polyvalent antiserum against whole whey that reacted with at least eight components in whole whey (Fig. 3, well I). Antiserum prepared by immunizing with these preparations showed only one line with whole whey, having the same electrophoretic mobility as the purified free secretory component. No reaction occurred with this antiserum against several whole bovine sera. Immunoelectrophoresis (Fig. 3), immunodiffusion, and acid urea gel electrophoresis were used to analyze the products of the different purification steps. The 33 to 50% ammonium sulfate fraction was markedly enriched in free secretory component, and almost free of serum albumin, but contained cY-lactalbumin and &lactoglobulin (Fig. 3, well 2). The DEAEcellulose effluent showed mainly IgG and free secretory component (Fig. 3, well 3), as well as several other minor components which were subsequently shown to be primarily lactoperoxidase and lactoferrin on antigenic analysis. The phosphocellulose column effectively removed lactoferrin and lactoperoxidase. Pool 2 of the phosphocellulose column showed only free secretory component and IgG on immunoelectrophoresis (Fig.  3, well 4), and these were partially resolved by the gel filration step (Fig. 2). The remaining IgG was completely removed by immunoadsorption ( Fig. 2A and Fig. 3, well 6).
Gel Electrophoresis Studies and Effects of Reduction-Disc gel electrophoresis at alkaline pH (Fig. 4, gel I) revealed only one band even on loading with relatively large amounts of free secretory component. However, this band occupied the top 1 cm of the gel, and this is the same position as for IgG and ' The abbreviations used are: dansyl, 5-dimethylaminonaphthalene-1-sulfonyl; PTH, 3-phenyl-2-thiohydantoin; FSC, free secretory component.  lactoferrin. Gel electrophoresis in acid urea produced better resolution ( Fig. 2A). It showed one heavy band in pure free secretory component preparations, whereas additional bands were seen with crude free secretory component preparations. Two faint, slightly faster bands were observed in some immunologically pure free secretory component preparations (Fig. 4,  gel 2). The proportion of these bands in the late fractions obtained from the free secretory component peak on gel by guest on March 24, 2020 http://www.jbc.org/ Downloaded from filtration was markedly higher than in the earlier fractions as quantitated by gel scanning. Reduced samples showed a marked decrease in mobility of the main band (Fig. 4, gel 3) and the appearance of more protein in the fast moving bands.
Gel electrophoresis in the presence of sodium dodecyl sulfate also showed one band in pure free secretory component preparations (Fig. 4, gel 4). Some faint fast bands were observed especially in gels of the last fractions of the free secretory component peak in gel filtration effluent. Reduced samples of the same preparations (Fig. 4, gel 5) showed some reduction in mobility as well as an increased amount of the fast bands. When samples of pure free secretory component were incubated at room temperature for 1 to 5 hours, the fast moving bands were detected in much larger amounts, representing up to 50% of the total protein (Fig. 4, gels 6 and 79, suggesting proteolysis. FIG. 4. Gel electrophoresis of free secretory component preparations. Gel 1 is a disc electrophoresis of pure free secretory component at alkaline pH. Gels 2 and 3 are acid urea gels of unreduced and reduced pure free secretory component, respectively. Gel 4 is a sodium dodecyl sulfate gel of an unreduced free secretory component preparation. Gels 5, 6, and 7 are sodium dodecyl sulfate gels of the same free secretory component preparation after preincubation for different times (0, 1, and 5 hours, respectively) at room temperature followed by reduction.

Size and Shape of Free Secretory
Component-Some physicochemical parameters of the free secretory component molecule were determined by the methods described above, and their results are summarized in Table II, together with similar results from the literature on bovine and human free secretory component.

Amino
Acid and Sugar Composition of Free Secretory Component-Amino acid analyses of three different pure free secretory component preparations did not differ by more than 5%. The average is shown in Table III together with literature data on the composition of free secretory component of different species. Results of carbohydrate analyses are also shown in Table III. Neutral sugar determination by the orcinolsulfuric method using galactose as a standard gave a value of 2.9 f 0.1% (w/w). The molar ratio of tryptophan to orcinol was less than 0.003 and interference would therefore be negligible (38). Sialic acid determinations in different preparations were in the range of 0.3 to 0.5% (w/w). Using a molecular weight of '79,000 for bovine free secretory component, the results of neutral sugar and sialic acid determinations were expressed as moles/100 moles of amino acids, and all the analytical results were expressed as residues/m01 of free secretory component.

Amino
Acid Sequence of NH,-terminal End-The NH,terminal amino acid residue could not be identified by gas chromatography after automatic sequencing, but with dansylation and polyamide thin layer chromatography it could be identified as lysine. The sequence of the first 13 residues was established after six runs on four different preparations as: Lys -Ser -Pro -Ile -Phe -Gly -Pro -Glu -Glu -Val -Asp -Ser-Val. Recoveries varied from 10 to 50% in these residues, and no other residues were observed other than those given in the sequence. Some ambiguities remain to be resolved in the sequence of residues 14 to 20, so only a tentative sequence is given as follows: A!!p (or Glu)-Gyy-Ser (or Cys)-Ser (or Cys)-Val-(Ser or Cys)-Be. DISCUSSION Several methods have been used to purify bovine free secretory component (13,15,39) but, in our hands, the method Physicochemical. parameters *Assuming a linear relation between elution volume and log molecular weight. This is not strictly true due to differences in f/f* c Literature values were obtained by other sedimentation equilibrium methods, and 6 was calculated from composition.  b Amino acids of bovine free secretory component were calculated from the molar ratio to glycine in Ref. 13, and amino sugars of rabbit free secretory component were calculated from the residues/m01 in Ref. 12. c Determined by mercaptoethanesulfonic acid hydrolysis; spectrophotometry gave slightly higher values. d From Ref. 14.
presented here was the only one suitable for preparing highly pure and intact free secretory component. Lactoferrin and lactoperoxidase were the most difficult contaminants to separate from free secretory component. Repeated precipitation with ammonium sulfate was used by Mach (15) to remove most of the lactoferrin, but from theoretical considerations (40) we did not expect this to be sufficient. On the other hand, chromatography on a phosphocellulose column was found to give good separation of these proteins, and no contamination of free secretory component with lactoperoxidase or lactoferrin could be detected immunologically.
By using an immunoadsorbent column to remove IgG we avoided repeated gel filtrations. Also, the presence of fragments of IgG in whey (39) makes it impossible to recover IgG-free free secretory component by gel filtrations alone. However, the gel filtration step was still necessary to first eliminate most of the IgG and allow resolution of partially degraded free secretory component.
Purity of the free secretory component preparations was shown by immunoelectrophoresis and immunodiffusion (Figs.  3 and 4). In sodium dodecyl sulfate-polyacrylamide gel electrophoresis about 96% of the protein migrated as one band. The appearance of faster bands in sodium dodecyl sulfate gels in some preparations having no immunologically detectable heterogeneity suggests that these bands are due to the presence of partially degraded molecules in the preparation. Their further explanation and suggests the presence of a protease in the preparation. The presence of protease activity in milk is well known (41,42). The increase of the faster bands on reduction suggests that some of the partially degraded molecules were apparently held intact by disulfide bonds. The decrease of the mobility of free secretory component on reduction either in sodium dodecyl sulfate gel or acid urea gel electrophoresis (Fig.  4) is interpreted as showing that disulfide bridges help to maintain the compact structure of the free secretory component molecule, complete unfolding being achieved only after reduction. This is in agreement with the finding of larger Stokes radii for reduced protein molecules in denaturing solvents compared to the unreduced molecules with intact intramolecular disulfide links (43). However, the faster bands observed in acid urea gel electrophoresis of pure free secretory component preparations may not be solely a result of proteolysis, as their proportion is much higher in acid urea than in sodium dodecyl sulfate gels. This may be due to variations in charge as a result of differences in carbohydrate composition, which is common with many glycoproteins.
Human free secretory component was found (37) to show marked microheterogeneity in isoelectric focusing, although other criteria showed 98% purity of the same preparation.
Much of the variation in the data on molecular weight of bovine free secretory component (Table II) (36) for human free secretory component (1.57 to 1.6) explain their retardation on gel filtration compared to more globular proteins and consequent high estimates of molecular weights by this method if no correction is made for differences in frictional ratios. From the frictional ratio of the bovine free secretory component, a minimum axial ratio can be estimated as 4 to 6 assuming a hydration value of 1 to 0.4 g of water/g of protein (44).
The amino acid composition of the bovine free secretory component shows a close similarity to the human, rabbit, and dog (Table III). The variation of the different analyses of the human free secretory component in different laboratories (9)(10)(11) appear to be larger than the variation between the different species, with a few exceptions. Taking the range of the human free secretory component data as a basis for comparison, the only significant differences are the higher valine and lower leucine in the bovine, higher aspartic and and lower alanine in the dog, and higher histidine and proline in the rabbit free secretory component. However, the similarities are more remarkable, especially if we allow for substitution of some valine for leucine residues in the bovine free secretory component, both residues probably playing a similar role in protein structure. Importantly, methionine seems to be completely absent in the human free secretory component, whereas it ranges from 0.24 to 1.0 mol/lOO mol of amino acids in cow, rabbit, and dog free secretory component. Because of its value in protein sequence studies, it was important to be sure that this small amount of methionine is not from a contaminant. Bovine lactoperoxidase and lactoferrm, two possible contaminants, are comparatively rich in methionine (45), but in the present studies both of these proteins were carefully excluded.
The similarity of the secretory component from various species is striking when their NH,-terminal sequences are compared. Bovine, human, and canine secretory component show complete identity of sequence up to residue 11, and identity to residue 13 is present in the bovine and human species. However, immunological cross-reactivity of human and bovine free secretory component with rabbit antisera could not be detected by us, or by other investigators (46), and it therefore seems likely that significant differences will be found in the amino acid sequence of the remainder of the polypeptide chain of these species. The elucidation of the full sequence of free secretory component will be of interest in connection with its affinity for certain polymeric immunoglobulins and its biological role, and to determine if any significant homologies occur between secretory component and immunoglobulins.
AcknoLuledgments-We gratefully acknowledge the participation of Dr. J. D. Capra in the early phase of this study. The first amino acid sequence data was obtained on a sample sent to him and subsequently this sequence was confirmed by us on three different preparations of free secretory component. We also acknowledge the excellent technical assistance of Mr. Karl Oles.