J. Home Econ. Jpn. Vol. 46 No. 8 739•`744 ( 1995)

Antigenic Cross-Reactions among Major Buckwheat Proteins

Kenji TAKUMI, Makoto KANO, *1 Junko UDAKA,

Sachinobu MANABE*2 and Tetsuro KOGA*

Laboratory of Food and Nutrition, Faculty of Education, *1 Department of Bacteriology,

School of Medicine, Ehime University, Matsuyama 790, Japan*2 Department of Nutrition, School of Medicine, University of

Tokushima, Tokushima 770, Japan

The antigenic cross-reactivity among albumins, globulins and glutelins from buckwheat flour

was studied by using immunodiffusion and immunoblot analyses with antisera raised against

globulins or glutelins. Completely fused precipitin lines appeared between the albumins and globulins against anti-globulin serum, and this serum also bound to several polypeptide

bands separated by SDS-PAGE from both proteins, indicating antigenic homology between the

albumin and globulin. Little or no antigenic cross-reaction was detected between the globulins

and glutelins when using the anti-globulin serum absorbed by the glutelin or vice versa, dem-

onstrating antigenic heterogeneity between both the protein fractions.

(Received June 20, 1994)

Keywords: buckwheat, albumin, globulin, glutelin, immunoblotting.

INTRODUCTION

Most cereal proteins have generally been classified by the Osborne solvent method1) 2) into four major classes according to their solubility in water (albumins), in dilute salt solution (globulins), in 70% ethanol

(prolamins), and in dilute alkali or acid solution (glutelins). The solubility of cereal proteins, howev-er, is greatly affected by many factors, including the fineness of milling, mechanical impact from shaking or stirring, the ratio of solvent to meal, and the frequency of extraction.3) 4) Accordingly, application of the solvent method requires an adequate evaluation of the solvent efficiency toward cereal proteins.4)

Buckwheat (Fagopyrum esculentum) is not a true cereal, although usually being regarded as a cereal

grain, 5) and a number of studies on the physico-chemical properties of the grain have been performed by Pomeranz et al.6)-8) In these works, however, the individual protein fractions have been isolated by the solvent method, which inevitably resulted in mutual overlapping among the protein frac-tions.9)10) Thus, it is worth-while to monitor for cross-contamination in the protein fractions.

In the present study, the antigenic cross-reactivi-

ty between the soluble proteins (albumins plus

globulins) and glutelins from defatted buckwheat flour was compared by immunodiffusion and immunoblot analyses, using rabbit antisera raised against the globulin or glutelin proteins.

MATERIALS AND METHODS

Buckwheat flour Buckwheat flour (Shinano No.1) was obtained

commercially from Asahi Seifun (Sakurai-shi, Nara, Japan) and then defatted with petroleum ether.11)

Extraction of the albumins and globulins The proteins were extracted from the defatted

flour with distilled water and salt solutions of various concentration (3, 5 and 10%) by stirring for 2 h at room temperature. The water- and salt-soluble extracts obtained by centrifugation were dialyzed against tap water for 48 h at room temperature(20•Ž) to isolate the albumins and globulins.12)

Extraction of the glutelins The glutelins were extracted from the insoluble

residue that had been sequentially pre-extracted by 5% salt solution and 50% propanol. The dried residue (1 g each) was suspended in 20 ml of 0.1, 0.2 or 0.5% NaOH or of 0.05 or 0.1% KOH, and stirred for 1 h at room temperature before

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J. Home Econ. Jpn. Vol. 46 No. 8 ( 1995)

centrifuging at 21, 000 •~ g for 20 min. The glutelins

were recovered from each supernatant by isoelectric-

point precipitation (at pH 4. 2) and freeze-dried af-ter being washed twice with water.

Preparation of the antisera The globulins and glutelins isolated from the 5

% salt and 0.2% NaOH extracts, respectively, as just described were used for the immunogen. Rabbit antisera were raised by subcutaneously inject-ing 1 mg of the antigen protein in 0.05 M phosphate-buffered saline at pH 7. 2 that was supplemented with 0.5 M urea (U-PBS) diluted with 1 volume of Freund's complete adjuvant according to the

procedure described previously. 13) Two other injec-

tions, each of about 2 mg of the antigen, were

administrated at two-week intervals, and two weeks after the last injection, the rabbits were bled. The sera were mixed with 0.05% sodium azideand stored at - 20•Ž until needed for use.

Immunodiffusion test Immunodiffusion tests were carried out with 0.8

% Agar Noble (Difco Lab. , U. S. A.) in a barbital buffer (ionic strength of 0. 05, pH 8. 6) as reported

previously. 14) Each antigen sample was dissolvedin U-PBS by incubating at 60•Ž for 30 min while

shaking and then centrifuged in an Eppendorf tube

to remove the supernatant, which was used as

antigen solution. The gel plates were set in a wet

A B

c

The proteins were suspended in PBS (5 mg/ml) , incubated at 60•Ž for 30 min, and centri-

fuged. Aliquots (30 ,ƒÊl) of the supernatant containing about 600 ƒÊg of proteins were applied

to the wells. Panel A : peripheral well 1, W-alb ; 2, 3S-alb ; 3, 5S-alb ; 4 and 5, 10S-alb.

Panel B: peripheral well 1, W-glo ; 2, 3S-glo; 3, 5S-glo; 4, 10S-glo ; 5, 10S-alb. Panel C: well

1, W-alb ; 2, 5S-alb ; 3, 5S-glo. The center wells contain the antiserum.

Fig . 1. Immunodiffusion tests on the buckwheat albumin and globulin fracitons against

the anti-globulin serum

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Antigenic Cross-Reactions among Major Buckwheat Proteins

chamber and kept for 18-20 h at room temperature, the results being directly photographed by light scattering apparatus.

Immunoblot analysis Immunoblotting was performed according to the

method of Towbin et al.15) The proteins were first-ly separated by SDS-PAGE (sodium dodecylsulfate

polyacrylamide gel electrophoresis), which was carried out by the method of Laemmli16) with 15 % acrylamide gels. The separated proteins were then transferred to a nitrocellulose membrane in a blotting buffer solution by mini-cell apparatus (BioCraft BE-300, Tokyo, Japan). The membrane was washed twice for 20 min in T-PBS (0.5% Tween 20 in PBS, pH 7.2) , incubated with T-PBS containing 5% skim milk for 30 min while gently agitating, and then soaked in the same solution containing the rabbit antiserum (diluted 1:100 with

1 2 3 4

The proteins (100 ƒÊ g) were separated by SDS-PAGE

and transferred to a nitrocellulose membrane. The

blots were probed with anti-globulin serum diluted at

1:100. Lane 1, 5S-glo; 2, W-glo; 3, 5S-alb; 4, W-alb.

T-PBS) for 60 min. The membrane was extensive-ly washed with T-PBS and incubated with horserad-ish peroxidase-labeled anti-rabbit IgG goat serum (Bio-Rad, diluted at 1:1, 000 with T-PBS) for 60 min. Color development was carried out according to the manual for the apparatus.

RESULTS AND DISCUSSION

Antigenic homology between the albumins and globulins The results of immunodiffusion tests for the solu-

ble proteins when using the anti-globulin serum are shown in Fig. 1. Several recognizable differ-ences in the precipitation patterns were observed between the water-soluble (W-alb) and salt-soluble albumins, which had been prepared from the 3, 5 and 10% salt-soluble fractions (referred to as 3S-, 5S- and 10S-alb, respectively; panel A). Whereas W-alb displayed only one precipitin line, all the salt-albs (wells 2-5) had two or three lines, one of which growing near the center well was fused completely with one of those of W-alb. The fused precipitin lines were found to have almost identical intensity. The other lines emerging near the antigen wells, however, gradually increased their intensity with increasing salt concentration in the solvent by which the antigens were extracted.

These findings suggest that the albumins contained at least three different antigenic determinants, one

Well 1, 0.5% NaOH; 2, 0.1% NaOH; 3, 0.2% NaOH; 4, 0.05 M acetic acid; 5, 0.1% KOH; 6, H20. The center well contains the anti-glutelin (from the 0.2% NaOH extract) serum.

Fig . 2. Immunoblot analysis of the buckwheat

albumin and globulin fractions isolated from the water- and 5% salt-soluble extracts

Fig . 3. Results of immunodiffusion tests on the

alkali- and acid-soluble glutelins against

the anti-glutelin serum

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J. Home Econ. Jpn. Vol. 46 No. 8 (1995)

A BC

The glutelins used were prepared from the 0. 2% NaOH extract. Panel A: wells 1 and 3, glutelins ; 2

and 4, 5S-glo. The right and left wells contain the anti-glutelin serum, and the center well contains the

anti-globulin serum. Panel B: well 1, glutelins; 2, globulins. The center well contains the anti-glutelin

serum absorbed by glutelins. Panel C: well 1, glutelins; 2, globulins. The center well contains the anti-

globulin serum absorbed by globulins.

of them, the major one, being soluble in either water or salt, while the others were soluble only in salt solution.

In contrast, the globulin fractions (panel B, wells 1-4) demonstrated four completely fused

precipitin lines, two of them, which raised the central area, being fused with those of salt albumins 10S-alb (well 5), indicating that buckwheat albumins and globulins share at least two common antigenic determinants. A comparison of the precipitation

patterns for W-alb (well 1), 5S-alb (well 2) and 5S-glo (well 3) is shown in Fig. 1, panel C, clearly indicating antigenic ralationships among them.

The immunoblot analysis was carried out to determine the antigenic similarity or dissimilarity among the polypeptides separated from the water-and 5% salt-soluble proteins (Fig. 2). The anti-

globulins serum bound to so many polypeptide bands that it was difficult to determine which polypeptides were responsible for the common precipitin lines between the two protein fractions. However, four significantly stained bands, which

possessed respective apparent molecular weights of 16, 18, 32 and 47 kDa, were observed common-ly among the four fractions (W-alb and -glo, and 5S-alb and -glo), implying that these 4 polypeptide bands may have corresponded to the 4 precipitin lines raised in the immunodiffusion test. A polypeptide band with a relatively high molecular weight (shown by the arrow) was absorbed only for the albumin

proteins. Consequently, it seems likely that buckwheat albumins and globulins are primarily homogeneous to one another, although they share some specific antigenic properties.

Antigenic heterogeneity between the globulins and glutelins The results of immunodiffusion tests show that

various glutelins contained a common antigenic determinant to the homologous antiserum (Fig. 3). Since the strongest line was observed for the 0.2 % NaOH-glutelins (well 3), this glutelin fraction. was used for the present study. Antigenic relation- ships between the glutelins and globulins (5S-glo) were first assessed by immunodiffusion tests with the respective anti-sera (Fig. 4, panel A). The

glutelins (wells 1 and 3) displayed strong precipitin lines to the equivalent antiserum (large wells at both sides), although only a faint precipitin line was raised to the anti-globulin serum (center well), suggesting some contamination by globulins in the glutelin immunogen. Therefore, cross-absorbed antisera; an anti-globulin serum absorbed by

glutelins and vice versa, were prepared and tested. The anti-glutelin serum absorbed by globulins (panel B) revealed clear precipitin lines for the glutelins (well 1), but no precipitin lines were raised for the globulins (well 2). The inverse reac-tions (panel C) show completely opposite results to those of the former reactions, in which the glutelins (well 1) gave no precipitin line, while

Fig . 4. Results of immunodiffusion tests on the globulin and glutelin fractions from defatted

buckwheat flour

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Antigenic Cross-Reactions among Major Buckwheat Proteins

1 2

Lane 1, glutelins; 2, globulins. The proteins were

blotted as shown in Fig. 2 and probed with the anti-

glutelin serum absorbed by globulins.

the globulins (well 2) showed strong lines. The antigenic specificity of the glutelins was also evi-dent from the immunoblotting results (Fig. 5), in which some polypeptides with high molecular weight exclusively reacted with the absorbed glutelin serum. These results demonstrate no antigenic cross-reactivi-ty between buckwheat globulin and glutelin proteins.

No prolamin fractions were examined in the pres-ent work, because we were, unfortunately, unsuccessful in preparing antisera against these proteins. Further studies on this are in progress.

REFERENCES

1 ) Osborne, T. B.: Vegetable Proteins, Longmans

Green & Co. , London, 154 (1924)

2 ) Shukla, T. P.: Crit. Rev. Food Sci. Nutr., 6,

1•`75 (1975)

3 ) Miflin, B. J. , Field, J. M. and Shewry, P. R. :

Seed Proteins (ed. by Daussant, J. , Mosse, J.

and Vaughan, J.) , Academic Press, London,

255•`319 (1983)

4 ) Shewry, P. R. and Miflin, B. J.: Adv. Cereal Sci.

Technol. , 7, 1•`84 (1985)

5 ) Pomeranz, Y.: Crit. Rev. Food Sci. Nutr. , 19,

213•`258 (1983)

6 ) Pomeranz, Y. and Robbins, G. S. : J. Agric. Food

Chem., 20, 270•`274 (1972)

7 ) Biely, J. and Pomeranz, Y. : Poult. Sci. , 54,

761•`766 (1974)

8 ) Pomeranz, Y. , Marsgakk, H. G. , Robbins, G. S.

and Gilbertson, J. T. : Cereal Chem., 52, 479•`

492 (1975)

9 ) Dausant, J. and Skakoun, A.: Seed Proteins (ed.

by Daussan, J. , Mosse, J. and Vaughan, J.) ,

Academic Press, London, 101•`133 (1983)

10 ) Ewart, J. A. D.: Immunological Aspect of Foods

(ed. by Catsimpoolas, N.) , AVI Publ. Co. ,

Westport, CT, 87•`116 (1977)

11 ) Skerritt, J. H. : Adv. Cereal Sci. Technol., 9,

263•`338 (1989)

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Faulks, A. J. and Miflin, B. J.: J. Exp. Bot.,

31, 393•`407 (1980)

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Genetika, 13, 115•`121 (1981)

14 ) Takumi, K. and Takeoka, A.: Infect. Immun. ,

39, 1346•`1353 (1983)

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そば主要蛋白質問の抗原交差反応

宅見 賢 二,加 納 誠*1,宇 高順 子,真 鍋 裕之*2,古 賀 哲郎*2

(愛 媛大 学教 育学 部,*1医 学部,*2徳 島大 学 医学部)

平成6年6月20日 受理

そ ば の主要 な蛋 白質 ア ルブ ミン,グ ロブ リ ンお よび グル テ リンを オズ ボー ン法 で分 画 し,各

画分 の抗 原交 差性 を抗 うさ ぎグロ ブ リン とグルテ リン抗体 を用 いて,ゲ ル内拡散 法 とイ ムノ ブ

ロ ッ ト法 で調 べ た.そ の結 果,ア ルブ ミンで は水可 溶性 画分 か らの アル ブ ミン と食塩 水(3,5,

Fig . 5. Results of an immunoblot analysis of the

buckwheat glutelin and globulin fractions

from defatted flour

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J. Home Econ. Jpn. Vol. 46 No. 8 (1995)

10%)可 溶性 画分 か らの アル ブ ミンは見 かけ の抗原性 を異 にす るが,両 画分 か らの グ ロブ リ ン

抗 原 は 同質で あ るこ と,ア ル ブ ミン画 分 は抗原 的 にグ ロブ リ ン画分 の 部分 であ る こ と,な どが

示 され た.一 方,グ ルテ リ ン蛋 白質 は相 同抗体 とのみ反 応 し,水 また は食塩 水可 溶性 蛋 白質 と

は抗 原的 に異 質で あ る ことが判 明 した.

キ ー ワ ー ド:そ ば,ア ル ブ ミ ン,グ ロ ブ リ ン,グ ル テ リ ン,イ ム ノ ブ ロ ッ ト.

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