Immunochemical properties of the 11S component of soybean proteins

ARCHIVES

0)’

BIOCHEMISTRY

AND

BIOPHYSICS

Immunochemical

NICHOLAS Research

126, 742-750

Properties

of the

of Soybean

Proteins

CATSIMPOOLAS

Laboratory,

Chemurgy

Received

(1968)

August

AND

Division,

Central

28, 1967;

accepted

11s Component

EDWIN Soya

Co.,

November

W. JIEYER Chicago,

Illinois

60639

14, 1967

Immunization of rabbits with soybean proteins in Freund’s adjuvant results in the formation of specific antisera that can be used for the detection of these proteins by several immunochemical methods. The isolated 11s soybean protein was immunochemically homogeneous by double gel immunodiffusion, disc immunoelectrophoresis, and immunoelectrophoresis in agar. This protein retained its immunological properties after heating up to 80”. Its diffusion coefficient was calculated by the double gel gradient diffusion technique. A water-insoluble polymer of the 11s soybean protein obtained by the reaction of the protein with ethyl chloroformate was used as an immunoadsorbent for the isolation of the specific anti-11s soybean protein antibody.

6 One of the major components of the soybean globulins is the 11s protein, which accounts for about 30% of the total soybean proteins. In an earlier study (I), we reported the purification of the 11s component by ammonium sulfate fractionation followed by DEAE-Sephadex ionic strength gradient chromatography. Quantitative N-terminal amino acid analysis of the purified 11s protein indicated the presence of at least twelve polypeptide chains, eight of which end in glycine, two in phenylalanine, and two in either leucine or isoleucine. The purified 11s component was dissociated by 6 M guanidine hydrochloride containing 0.2 3~ mercaptoethanol into at least twelve subunits as shown by disc electrophoresis. In the present investigation, we present evidence that the purified 11s component is homogeneous as shown by several immunochemical methods, and that these methods can be effectively used in the detection of this protein and in the study of its physicochemical properties. The isolation of the anti-11s soybean protein antibody is also described. EXPERIMENTAL Protein grown in

samples. Harosoy 1964 and stored

PROCEDURE 63 variety soybeans, a.t 2.5O, were cracked, 742

dehulled, and flaked. The soybean flakes were defatted with pentane for 5 hours in a Soxhlet apparatus. The extracted flakes were then desolventized at room temperature. Whole soybean extract (WSE) was obtained by extraction of the defatted flakes with water (flake:water ratio, 1:lO) at 25” for 1 hour and cenbrifugation at 10,000 X g for 30 minutes to clarify the supernatant liquor. The isoelectric soybean globulins (ISG) were prepared from the whole soybean extract by isolelectric precipitation at pH 4.5 with 57, HCl, washed free of whey constituents with water, and adjusted to pH 7.6 with 1 N NaOH. The purified 11s component was prepared as described previously (1). All of the above-described fractions were dialyzed against pH 7.6 phosphate buffer (0.0325 M KzHPOa , 0.0026 M KH~POI) made 0.4 M in sodium chloride and 0.01 M in mercaptoethanol. This is designated standard buffer (2). Preparation of antisera. Young adult white rabbits were immunized by three intraperitoneal injections at 7-day intervals of 2% antigen solution (whole soybean extract or 11s component) in standard buffer mixed and homogenized with an equal volume of Freund’s complete adjuvant (Difco). The antigen dosage was increased weekly from 1 to 2 to a 5-ml volume. After a rest period of 4 weeks, the animals were given a 5-ml booster injection of antigen by the same route and bled after 2 weeks. The sera were collected and stored at 4” after filtration sterilization and addition of 1: 10,000 merthiolate.

11s SOYBEAN Quantitative precipitin test. Increasing amounts of 11s soybean protein in 0.5-ml volumes of standard buffer were added to test tubes containing 0.2 ml of the antiserum. The reaction mixtures were incubated for 30 minutes at 37” and then stored overnight at 4”. The resultant precipitates were centrifuged, washed twice with pH 7 buffered saline, and dissolved in 5 ml of 0.1 N NaOH. The absorbance of the solutions was measured at 280 rnp in a Beckman DU spectrophotometer. The molecular ratio of antigen to antibody present in the immunoprecipitate at equivalence was calculated from the extinction coefficients at 280 m and molecular weights of the 11s protein and rabbit r-globulin. E :TE, values of 9.2 for the 11s protein and 14.0 for the rabbit r-globulin (3) were used for approximate calculations. The molecular weights of the 11s protein (2) and rabbit r-globulin (4) were taken as 350,000 and 160,000, respectively. Double gel immunodiffusion. Double gel diffusion in agar was carried out in plates according to the method of Ouchterlony (5). The gel medium consisted of a 0.857, Ionagar No. 2 (Oxoid) solution in pH 6.8 phosphate buffer prepared as follows: 0.77 gm NapHPOa ,0.38 gm KHPOa , and 10.02 gm NaCl were dissolved in Hz0 and made up to 1 liter after the addition of 1 ml of lGj, merthiolate. The reactants were allowed to diffuse at room temperature, i.e., at 24-26”, for 2-3 days in a moist chamber. Results were recorded photographically. Disc immunoelectrophoresis. Polyacrylamide gel columns (7%) were prepared as described by Ornstein (6) and L)avis (7). Electrophoresis with a 0.2-0.4 mg protein sample was usually carried out for 30 minutes in Tris-glycine buffer (ionic strength 0.01; pH 8.3) with a current of 5 mA per gel column. Preliminary detection of the separated protein components was achieved by staining the columns for 1 hour with Amido-Schwartz dye followed by electrical destaining, as described by Davis (7). For immunodiffusion, the unstained columns were entirely embedded in the agar medium described above. After solidification of the agar, trenches were cut parallel to the line of columns and filled with the anti-serum. The reactants were allowed to diffuse at room temperature (24-26”) for 3-5 days in a moist chamber. Precipitin lines appeared usually in l-2 days, but in general, became more intense on longer standing. Results were recorded photographically. Immunoelectrophoresis in agar gel. These analyses were carried out according to the method of Grabar and Williams (8) as modified by Scheidegger (9). Characterization reactions were performed as described by Uriel (10). Single diffusion of the 1lS protein. A standard curve for determination of the 11s soybean protein was developed by using a single-diffusion

PROTEIN

743

technique. Glass tubes of 2-mm i.d. and approximately 100 mm long were coated with 0.1% Ionagar (Oxoid) and dried at 70”. For the single-diffusion procedure, these tubes were half-filled with 0.6% Ionagar in pH 7 buffer (0.87 gm Na2HPOI. HzO, 0.38 gm KHzHPOd , and 10.02 gm NaCl, made to 1 liter with H20) containing merthiolate (l:lO,OOO) and anti-11s soybean antiserum at a final dilution of 1:20. The 11s soybean protein (0.3 ml containing from 1Opg to 5 mg) in the above described standard gel diffusion bufler was layered above the gel, and the tubes were capped with Vaspar (white petrolatum:parafEn, .50:50). Migration of the antigen-antibody precipitate was measured with a measuring magnifier after incllbation of the tubes at 20” for several days. Isolation of the anti-ifs soybean protein antibody. This isolation was carried out according to the method of Avrameas and Ternynck (11) by using ethyl chloroformate to convert the antigen into an immunoadsorbent. The 11s soybean protein, 100 mg, was dissolved in 3ml of standard buffer, and while the solution was stirred, 0.2 ml of ethyl chloroformate (Baker) was added dropwise. After 30 minutes of stirring, a precipitate appeared, and the mixture was left without stirring for 1 hour. At the end of this period, the mixture was centrifuged for 10 minutes at 10,OOOg. The precipitate was suspended and washed several times with a large volume of phosphate-buffered 0.9c/ NaCl (pH 7.0) until the optical density of the eluates was 0 at 280 rnp. The washing procedure was repeated with 0.2 M glycine-HCI (pH 2.2) buffer. The precipitate was then washed with phosphate-buffered 0.970 NaCl unt,il the pII of the eluates was neutral. The suspension was then centrifuged at 10,OOOg for 10 minutes. The polymerized 11s protein was suspeneded in 36 ml of phosphate-buffered saline, and 4 ml of t.he anti-11s soybean protein antiserum was added. The mixture was stirred gently at 37” for 2 hours and centrifuged at 10,OOOg for 20 minutes. The precipitate was washed several times with phosphate-buffered saline until the optical density of bhe eluates was 0 at 280 rng. To elute the adsorbed anti-11s soybean protein antibody, the precipitat,e was stirred for 15 minutes with IO-ml portions of 0.2 M glycineHCl (pH 2.2) buffer and centrifuged at 10,OOOg for 20 minutes after each elution. Most of the antibody was eluted with the first lo-ml portion of the pH 2.2 buffer as determined by the 280 rnk absorbance. The combined eluates were neutralized carefully with 0.1 N NaOH and dialyzed against, phosphatebuffered saline at 4”. When necessary, t,he antibody preparation was concentrated by ammonium sulfate precipitation and resolubilization in the desired amount of phosphate-buffered saline. The purity of the antibody preparation was determined

744

CATSIMPOOLAS

by polyacrylamide gel disc electrophoresis, immunoelectrophoresis against goat anti-rabbit serum (Hyland), and quantitative precipitation (95% precipitable by 11s soybean protein). By using this procedure and the above quantities of antigen and antiserum, 15 mg of purified antibody was obtained per 100 mg of the 11s soybean protein. RESULTS

Double-diffusion plates in which soybean extract (WSE) was analyzed with rabbit extract antiserum anti-whole soybean (A-WSE) developed at least five precipitin

AND

MEYER

bands (Fig. 1A). The isoelectrically precipitated soybean globulin fraction (ISG) exhibited three bands, and the isolated 11s soybean protein (llS), one band. When these soybean protein fractions were allowed to diffuse against the anti-11s soybean protein antiserum (A-1X3), only one precipitin band appeared in each fraction (Fig. 1B). These results indicate that the isolated 11s soybean protein is immunochemically homogeneous if it is assumed that antibodies of all the soybean protein components that are antigenically distinct from each

[FIG. 1. Double gel immunodiffusion of soybean protein fractions. WSE, whole soybean extract; ISG, isoelectrically precipitated soybean globulins; llS, isolated 11s soybean protein. Upper figure (A), diffusion against anti-whole soybean extract antiserum (A-WSE). Lower figure (B), diffusion against anti-IIS soybean protein antiserum (A-11s).

11s SOYBEAN

other are present. The anti-11s soybean protein antiserum appears to be highly specific for the 11s component since the precipitin bands obtained with the whole soybean extract and isoelectric globulins are immunochemically identical to that of the 11s protein. No other bands developed in these two fractions. Although double gel diffusion is a very useful analytical tool, the possibility exists that two or more precipitin bands may coincide. To eliminate this possibility, soybean protein fractions were analyzed by both polyacrylamide gel disc immunoelectrophoresis and immunoelectrophoresis in agar. Figure 2 shows typical patterns of polyacrylamide gel disc electrophoresis of the whole soybean extract, isoelectric soybean globulins, and 11s component. When the unstained columns were immersed in agar, and

PROTEIN

745

the separated proteins were allowed to diffuse against anti-whole soybean extract and anti-11s soybean protein antisera, the results illustrated in Fig. 3A were obtained. Both the whole soybean extract and isoelectric soybean globulins were, as expected, immunochemically heterogeneous, and developed a minimum five and three (possibly four) precipitin arcs, respectively. The 11s soybean protein developed only one precipitin arc with the anti-whole soybean extract antiserum. Also, the heterogeneous fraction of isoelectric globulins exhibited only one band with the anti-l@ soybean protein antiserum. Similar results were obtained when the isoelectric soybean globulins and 11s component were electrophoresed in agar and diffused against anti-whole soybean extract and anti-11s soybean protein antisera (Fig. 3B). Again, the 11s component was found to be immunochemically homogeneous and its antiserum highly monospecific. The 11s soybean protein immunoprecipitate in agar developed a positive glycoprotein staining reaction (10). Staining reactions for lipoprotein were negative (10). The precipitin curve shown in Fig. 4 demonstrates results obtained in yuantitative precipitation of the 11s sovbean protein with anti-11s soybean protem antiserum. Ko precipitation was observed with sera before immunization. The precipitin curve shows a typical antigen-antibody reaction. At the point of maximum precipitation, it was found that approximately 1 mole of the 11s protein combines with 2 moles of rabbit antibody (see METHODS). The curve also demonstrates the sensitivity of t’he reaction since only a few micrograms of the 11s protein are required for detection. The effect of heat on the immunological properties of the isolated 11s protein was studied by heating 1% solutions of the protein in standard buffer at different temperatures (30-50”) for 1 hour. Figure 5A shows that the 11s component retained its ability to react with antibody after heating up to 80”. ,4t higher temperatures t#he protein becomes increasingly insoluble. After being heated at 110” for 1 hour the protein cannot be det,ected immunochemically. The reasons for this may be lack of diffusion of the pro-

746

CATSIMPOOLAS

FIG. 3. Immunoelectrophoresis of soybean tract; ISG, isoelectrically precipitated soybean A-WSE, anti-whole soybean extract antiserum; Upper figure (A), disc immunoelectrophoresis. agar.

tein because of loss of solubility or modification of the antigenic sites by excessive heat. The reason for the loss of antigenicity at higher temperatures is under investigation. Since the isolated 11s protein is in an immunologically pure state, its diffusion coefficient was determined by the immunochemical method of Allison and Humphrey (12). Briefly, antigen and antibody are placed in troughs cut at a 90” angle in a uniform layer of agar. It has been shown that, when the amounts of antigen and

AND MEYER

protein fractions. WSE, whole soybean exglobulins; llS, isolated 11s soybean protein; A-11& anti-118 soybean protein antiserum. Lower figure (B), immunoelectrophoresis in

antibody in the troughs are such as to give optimum proportions, precipitation occurs along a straight line inclined at an angle 8 to the antigen trough, such that tan 0 = where D, and Db are diffusion (WDdl’z, coefficients of antigen and antibody, respectively. The diffusion coefficient of rabbit antibody (Db) was taken as 3.8 X lo-’ cm2/second (13). The precipitin line obtained at 20” with the 11s component double gel gradient diffusion against whole soybean extract similar

antiserum is shown in Fig. 5B. A line was obtained by using anti-11s

11s

0.1

SOYBEAN

1.0 IIS

100

IO PROTEIN

ADDED(pg/O.Zml

FIG. 4. Quantitative precipitin curve. Increasing added to a standard volume of anti-whole soybean PROCEDURE). Absorbance at 280 mp indicates amount

antiserum. The diffusion coefficient of the 11s component with water as solvent was calculated to be DZO,=. = 1.47 -f 0.02 X 1O-7 cm2/second at 20”. Wolf and Briggs (14) reported that the diffusion constant of an impure 11s protein preparabion measured with a Neurath-type diffusion cell was 1.7 f 0.06 X 1O-7 cm2/second at 3.S”. This value corrected t,o 20” gave a value of 2.91 f 0.10 X 1O-7 cm2/second. While this correct,ion is widely employed, it results in a relatively large correction over a temperature difference as wide as 16”. Oft,en, the corrected values do not agree with values obtained directly at 20” (15). In view of these considerations, it would be desirable to reevaluate the diffusion constant of the purified 11s component. This may lead to an assignment of a higher molecular weight than the present’ly accepted value of 350,000. Immunodiffusion methods that can be used to determine diffusion coefficients of antigens can also be employed for quantitation. Single-diffusion tests have been used to quantitate a number of proteins (16). The possibility that an immunoassay can be developed for the quantitative det,ermination of the 11s soybean protein is demonstrated by the results shown in Fig. 6. The data were obtained from single diffusion

747

PROTEIX

serum)

amounts of the extract antiserum of antigen-antibody

11s soybean protein (see EXPERIMENTAL complex.

experiments of the 11s soybean protein in agar gel containing anti-11s soybean protein antiserum. The log of the 11s protein concentrations of 10 pg/O.3 ml to 5 mg/0.3 ml approximated straight lines when plotted against the distances of migration of the precipitin bands (in millimeters at 20’) for three different time intervals of incubation. The development of specific immunoassays for the determination of the 11s and other soybean proteins, in different fractions and food products, based on single diffusion techniques is under investigation. The immunoadsorbent properties of ethyl chloroformate-polymerized 11s soybean protein were investigated. It was found to be efficient and specific for the isolation of the anti-11s antibody. Polymerization of the protein at pH 7.6 occurred readily, and the water-insoluble polymer retained its immunological properties. The anti-11s antibody was adsorbed efficiently and was eluted with pH 2.2 glycine-HCI buffer. The purity of the antibody preparation was determined by disc electrophoresis and immunoelectrophoresis. Figure 7A shows disc electrophoresis patterns of the anti-11s antiserum and purified anti-11s antibody. The purified antibody migrates in the -yG-globulin region. No other contaminants were found to be

748

CATSIMPOOLAS

AND

MEYER

FIG. 5. Upper figure (A), double gel immunodiffusion of the at different temperatures (3040”) for 1 hour against anti-whole Lower figure (B), double gel gradient diffusion of the 11s soybean soybean extract antiserum.

present either from the antiserum proteins or the 11s soybean protein. When the electrophoresed anti-11s antiserum and purified antibody were developed with goat antirabbit serum, the anti-11s antibody exhibited only one precipitin line in the region of yG-globulin (Fig. 7B). The purified antibody was immunologically active as determined by quantitative precipitation and double gel diffusion against the 11s soybean protein. The quantitative precipitation test showed that the purified antibody was 95 % precipitable by the 11s soybean protein. This indicates that the purified antibody is only slightly contaminated by other nonspecific r-globulins.

11s soybean protein heated soybean extract antiserum. protein against anti-whole

DISCUSSION

The specificity and sensitivity of immunochemical reactions can be utilized effectively to define a protein in a mixture and to demonstrate its purity or homogeneity. This report presents evidence that immunochemical methods can be used to characterize soybean protein fractions. In the past, ultracentrifugal analysis, chromatography, and electrophoretic migration were the main analytical tools used to detect the presence of soybean protein components in isolation procedures. However, the production of antisera specific to soybean proteins makes it possible to analyze fractions by a simple and rapid technique. By this method, we have shown that the

11s

SOYBEAN

FIG. 6. Single diffusion of the 11s soybean protein against anti-11s soybean protein antiserum. Migration (in millimeters)of different amounts of the 11s soybean diffusion at 20”.

protein

at 18, 42, and

66 hours

of

PROTEIN

749

isolated 11s soybean protein (1) that was found to be homogeneous by disc electrophoresis and ion-exchange chromatography is also immunochemically homogeneous. The production of a monospecific ant,i-11s soybean protein antibody facilitates the detection of this protein in microgram quantities. The standard curve obtained by the single-diffusion method offers a, possibility for the development of a quantitative procedure to follow the fate of the llSprotein during maturity and germination of t’he soybean seed, and its estimation in food products and isolated soybean fractions. The main disadvantage of the immunochemical method is that the number of antigenic components detected is to be considered as a minimum. Several immune sera have to be tested and pooled to obtain a maximum number of identifiable components. Although ultracentrifugal data on the isoelectric soybean globulins (14,17) that indicate the presence of mainly four components (2S, 7S, llS, and 1.55) are not in gross disagreement with our immunochem-

FIG. 7. Upper figure (A), disc electrophoresis on polyacrylamide gel of anti-11s soybean protein antiserum (A-11s) and isolated anti-11s soybean protein antibody (A-11s Ab). Lower figure (B), immunoelectrophoresis in agar of anti-11s soybean protein antiserum (A-11s) and isolated anti-11s soybean protein antibody (A-1lSAb) against goat anti-rabbit serum.

750

CATSIMPOOLAS

ical results, disc electrophoresis analysis does show a difference. A larger number of stained protein bands separated by disc electrophoresis is present as compared with the precipitin arcs obtained by diffusion of these proteins against the antiserum. A possible explanation is that soybean protein components with a minor difference in electrophoretic mobility appear as separate bands, but immunochemically behave as a single component because of structural similarities. However, the possibility exists that the antisera obtained may not contain antibodies to all soybean protein components. Testing of antisera from a large number of immunized rabbits is being continued. However, experiments in progress with 40 rabbits immunized with soybean proteins are in agreement with the abovereported results. The isolation of the specific anti-11s soybean protein antibody by the recently described ethyl chloroformate method of Avrameas and Ternynck (11) demonstrates the usefulness of this technique in obtaining soybean protein polymers that are still biologically active. The method offers an opportunity for the isolation of other specific antibodies against immunochemically active and homogeneous soybean proteins. Since to the authors’ knowledge the anti-11s soybean protein antibody is the only isolated antibody to a seed or cereal protein, it would be interesting to study its physicochemical properties. ACKNOWLEDGMENTS The authors acknowledge the technical assistance of Mr. E. Leuthner, Miss D. A. Rogers, and Mrs. C. R. Ekenstam.

AND

MEYER REFERENCES

1. CATSIMPOOLAS, N., ROGERS, D. A., CIRCLE, S. J., AND MEYER, E. W., Cereal Chem. 44, 631 (1967). 2. WOLF, W. J., B~BCOCI~, G. E., AND SMITH, A. K., Arch. Biochem. Biophys. 99, 265 (1962). 3. PORTER, R. R., Biochem. J. 66,677 (1957). 4. PHELPS, R. A., AND PUTNAM, F. W., in “The Plasma Proteins” (F. W. Putnam, ed.), Vol. 1, p. 143. Academic Press, New York (1960). 5. OUCHTERLONY, ii., Acta Pathol. Microbial. Stand. 26,185 (1948). 6. ORNSTEIN, (1964). 7. DAVIS, B. 8. 9. 10.

11.

L.,

Ann.

J., Ann.

N.Y.

Acad.

Sci.

121, 321

A7.Y.

Acad.

Sci.

121,

404

(1964). GRUIAR, P., AND WILLIAMS, C. A., Biochim. Biophys. Acta 10, 193 (1953). SCHEIDEGGER, J. J., Intern. Arch. Allergy Appl. Immunol. 7,103 (1955). URIEL, J., in “Immuno-electrophoretic Analysis” (P. Grabar, and P. Burtin, eds.), p. 30. Elsevier, New York (1964). AvRAMEBS, S., AND TERNYNCK, T., J. Biol. Chem. 242,1651 (1967).

12. ALLISON, munology 13. KORNGOLD, Immunol.

A. C., AND HUMPHREY, J. H., Im3,95 (1960). L., AND T’.~N LEEUWEN, G., J. 78, 172 (1957).

14. WOLF, W. J., AND BRIGGS, D. R., Arch. Biothem. Biophys. 86, 186 (1959). E. A., AND MAYER, M. M., “Experi15. KABAT, mental Immunochemistry,” 2nd edition, p. 680. Thomas, Springfield, Illinois (1931). 16. CROWLE, A. J., “Immunodiffusion,” p. 161. Academic Press, New York (1961). W. E. F., Biochim. Biophys. Acia 17. NAISMITH, 16, 203 (1955).