Heterogeneity of broad bean legumin

179

Biochimica et Biophysica Acta, 621 (1980) 179--189 © Elsevier/North-Holland Biomedical Press

BBA 38346

HETEROGENEITY OF BROAD BEAN LEGUMIN

SHIGERU UTSUMI and TOMOHIKO MORI

Research Institute for Food Science, Kyoto University, Uji, Kyoto 611 (Japan) (Received June 25th, 1979)

Key words: Legumin heterogeneity; Subunit structure; 11 S Globulin; (Broad bean)

Summary Legumins from various cultivars were fractionated on a DEAE-Sephadex A-50 column and their subunit structures were investigated. The results obtained indicated the heterogeneity of legumin molecular species. The nature of the heterogeneity was common to all cultivars examined from the standpoint of the molecular sizes of the subunits. Four groups of molecular species with molecular weights of 320 000, 350 000, 380 000 and 400 000 were detected by gradient gel electrophoresis. The smallest molecular species was composed of only three kinds of subunit, with molecular weights of 20 500, 23 000 and 36 000, and the largest one was composed of five kinds of subunit with molecular weights of 19 000, 23 000, 36 000, 49 000 and 51 000. All molecular species were composed of intermediary subunits which consisted of acidic and basic subunits. The intermediary subunits with molecular weights of 61 700, 59 800 and 48 000 are composed of the acidic subunits with molecular weights of 51 000, 49 000 and 36 000 and the b~sic subunits with molecular weights of 19 000, 19 000 and 23 000 or 20 500, respectively. The possible molecular species constructed from the subunit groups classified according to their sizes were presented. Introduction

Legumin, one of the most predominant components of seed storage proteins in various legumes [1], has been purified from broad bean by Wright and Boulter [2] and by Scholz et al. [3]. The subunit structure of legumin from broad bean has been described by the former authors [2]. They identified five distinct subunits, of which two were acidic (a) with a molecular weight of 37 000, and three basic (~) with molecular weights of 23 800, 20 900 and 20 100 in var. Triple White. They proposed that legumin of Triple White was composed of a6~6, which has also been reported in legumin-like protein of

180

Glycine max [4--6], Avena sativa [7], Sesamun indicum [8] and others [9]. However, in the preceding paper we reported observing another two acidic subunits with the molecular weights of 49 000 and 51 000 in var. Sanuki.Nagasaya [10]. The ratio of the amounts of three acidic subunits with molecular weights of 36 000, 49 000 and 51 000 was approx. 10 : 1 : 1 [10]. This ratio suggests that legumin may not exist as an unique molecular species having the subunit structure of a~6. In view of the evidence described above, legumins from various cultivars were fractionated and their subunit structures were investigated in order to elucidate the heterogeneity of legumin from broad bean in the present work. The results obtained indicate the heterogeneity of legumin molecular species. The possible molecular species of legumin which are common to all cultivars are presented. Materials and Methods .DEAE-Sephadex A-50 and Slab gradient gel (4--30%) were purchased from Pharmacia Co. Ltd. Ampholine (pH 3.5--10) was purchased from LKB. 2-mercaptoethanol and urea, specially prepared reagents, were obtained from Nakarai Chemicals. Sodium dodecyl sulfate (SDS), specially prepared reagent, was obtained from Wako Pure Chemicals. Other chemicals were guaranteed reagent grade. Seeds of Vicia faba vats. Sanuki-Nagasay, Kumamoto-Churyu and Otafuku were purchased from Takii Seed Co. Ltd.

Preparation of crude extract Crude extract of each cultivar of broad bean seeds was fractionated into with 50 mM sodium phosphate buffer (pH 7.0) containing 1 M NaC1 as described in the preceding paper [10]. Sucrose density gradient centrifugation Crude extract of each cultivar of broad bean seeds were fractionated into four fractions, called 2 S, 7 S, 11 S and 15 S fractions, by sucrose density gradient centrifugation as described in the preceding paper [10]. DEAE-Sephadex A-50 column chromatography DEAE-Sephadex A-50 column chromatography was performed using 1.5 X 20 cm column equilibrated with 50 mM potassium phosphate buffer (pH 7.6) containing 0.2 M NaC1 and 0.02% NaN3 at 4°C. Legumins from three cultivars prepared by sucrose density gradient centrifugation were equilibrated with the buffer and then applied on the columns. The column was washed with the buffer. Elution was performed by 500 ml of the buffer containing NaC1 in linear gradient concentration of 0.2 M to 0.35 M. Electrophoreses Polyacrylamide gel electrophoreses in the absence or presence of SDS were carried out according to the procedures described in the preceding paper [10]. Gel electrofocusing in the presence of 7 M urea was performed in 6% polyaczylamide gels according to the method of Wrigley [11] with a slight modifica-

181 tion. Anode and cathode solutions were 0.02 M phosphoric acid and 1 M NaOH, respectively. Prior to electrophoresis, 30/~g of legumin was equilibrated with 8 M urea and 0.2 M 2-mercaptoethanol. After the sample containing 2% Ampholine was layered on a gel and 50/~1 of 54 mM glutamic acid containing 2%Ampholine and 15% glycerol was layered on the sample, electrophoresis was carried out for 5.5 h at 4°C with a constant voltage of 180 V. After electrophoresis, gels were fixed and washed with two changes of 0.35% perchloric acid and then stained in 0.04% Coomassie brilliant blue G-250 in 0.35% perchloric acid. Slab gradient gel elect~ophoresis was performed by using 8 × 7 cm slab gradient gel, 4--30% (w/v) polyacrylamide in concentration, with Pharmacia Electrophoresis Apparatus GE-4. Electrode buffer was Tris-glycine buffer (16.5 mM Tris, 128 mM glycine, pH 8.3). Equilibrium run was carried out for 1 h at a constant voltage of 70 V. After the samples were applied on the gel, electrophoresis was can'ied out for 30 rain at a constant voltage of 70 V and for 72 h at a constant voltage of 100 V at 4°C. The molecular weights of legumin species were estimated by comparing their mobilities with those of markers of

known molecular weights. Molecular weight markers used were thyroglobulin (669 000), ferritin(460 000) and catalase(240 000). Results

DEAE-Sephadex A-50 column chromatography 11 S component fractions (legumin) from three cultivars of broad bean seeds were prepared by sucrose density gradient centrifugation and then were applied on ~the DEAE-Sephadex A-50 column. The chromatograms were shown in Fig. 1. The flow through fraction of each cultivar may be derived from the 7 S component which contaminated the 11 S component fraction of the sucrose density gradient centrifugation (Figs. 3-8, 4-8 and 5-8). Legumin of each cultivar was eluted as a single peak. However, it was a broad beak in the range of fractions 40 to 100 (approx.). The tk~actions from the columns of three cultivars were analyzed by various gel electrophoreses.

Polyacrylamide gel electrophoresis of legumin fractions from three cultivars The legumin fractions eluted from the DEAE-Sephadex A-50 column of three cultivars were analyzed by polyacrylamide gel electrophoresis. The electrophoretic patterns of Otafuku were shown in Fig. 2. Similar patterns were also obtained in the cases of Sanuki-Nagasaya and Kumamoto-Churyu. Fractions 8 and 11 of t h e flow through fraction gave very pale bands. The more quickly eluting fractions of legumin, e.g. fractions 44 and 47, gave a single band~ while those eluting more slowly gave the more bands.

Gradient gel electrophoresis of legumin fractions from three cultivars The legumin fractions eluted from the DEAE.Sephadex A-50 column of three cultivars were analyzed by-gradient gel electrophoresis in order to elucidate whether molecular species of legumin which were different in molecular weights were present in broad bean. The electrophoretic patterns of Otafuku were shown in Fig. 3. Similar patterns were also obtained in the cases of

182

O21 A

~-@ ~

0.1I-

0.4

z

0

20

02r

C

0

.

0

40

80

100

120 c o

1

20

(~0

~

40

.

~

60 80 Froction no.

100

1L:~O

Fig. 1. D E A E - S e p h a d e x A - 5 0 c o l u m n c h r o m a t o g r a p h y o f l e g u m i n s f r o m t h r e e cultiva~s. 55, 55 a n d 4 5 m g protel/ts o f 11 S f r a c t i o n s p r e p s x e d b y s u c r o s e d e n s i t y g r a d i e n t c e n t t d f u g a t i o n w e r e a p p l i e d o n t h e c o l u m n f o r S a n u k i - N a g a ~ a y a (A), K u m a m o t o - C h u r y u (B) a n d O ~ f u k u (C), r e s p e c t i v e l y , a n d t h e n c h r o m a t o ~ a p h e d as de~eribed i n Materials a n d M e t h o d s . T h e f l o w r a t e w a s 18 m l / h . F r a c t i o n v o l u m e w a s 6 ml. o o: a b s o r b a n c e a t 2 8 0 n m , - - - --: c o n c e n ~ r a t l o n o f NaCl.

Sanuki-Nagasaya and Kumamoto-Churyu. Unfractionated sample consisted of three components with molecular weightsof 320 000, 350 000 and 380 000. The fraction No. 8 gave a very pale band with molecular weight of about 200 000. The faster eluting fraction, 47, gave one band with molecular weight of 320 000. Fraction 59 gave two bands with molecular weights of 350 000 and 380 000. The fraction 89 gave three bands with molecular weights of 350 000, 380 000 and 400 000. These resultsdemonstrate the presence of four molecular species of legumins.

3DS-polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol of legumin fractions The legumin fractions eluted from the DEAE-Sephadex A-50 column of three cultivars were analyzed by SDS-polyacrylamide gel electrophoresis. The elect~ophoretic patterns of Otafuku were shown in Fig. 4. Similar results were also obtained in the cases of Sanuki-Nagasaya and Kumamoto-Churyu. Unfractionated sample consisted of six subunits with molecular weights of 19 000, 20 500, 23 000, 36 000, 49 000 and 51 000, of which the smaller three bands and the larger three bands were assigned to the basic subunits and the acidic subunits, respectively [10]. The faster eluting fractions of legumin, fractions 44

183

F i g . 2. P o l y a c r y l a m i d e gel e l e c t ~ o p h o r e s l s o f l e g u m i n f r a c t i o n s o f O t a f u k u . 2 0 ~ug p r o t e i n o f e a c h f r a c t i o n w a s d i a l y z e d a g a i n s t 5 0 rnM p o t a s s i u m p h o s p h a t e b u f f e r (PH 7 . 6 ) c o n t a i n i n g 0 . 2 M NaCI a t 4 ° C a n d t h e n e l e c t r o p h o r e s e d as d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s . U r e f e r s t o a n u n f r a c t i o n a t e d s a m p l e . T h e n u m b e r s u n d e r the gels a~e t h e f r a c t i o n n u m b e r s o f D E A E - S e p h a d e x A - 5 0 c o l u m n c h r o m a t o g r a p h y . M i g r a t i o n is f r o m t o p t o b o t t o m .

and 47, consisted of only three components with molecular weights of 20 500, 23 000 and 36 000. On the other hand, the more slowly eluting fractions contained all of the six bands. Thus the more slowly the legumin eluted, the more plentiful were the subunits with molecular weights of 19 000, 49 000 and 51 000, while the ratio of the amount of the subunit with molecular weight of 20 500 decreased. These results are summarized in Table I by estimating densitometrically the ratio of the amounts of six bands.

SDS-polyacrylamide gel electrophoresis in the absence of 2-mercaptoethanol of legurnin fractions The legumin fractions eluted from the DEAE-Sephadex A-50 column of three cultivars were analyzed by SDS-polyacrylamide gel electrophoresis in the absence of 2-mercaptoethanol in order to clarify the composition of intermedi-

184

Fig. 3. Slab gradient gel electrophoresis of legumin fractions of Otafuku. 10 ~ug I~rotein of each fraction equilibrated with 50 mM potassium phosphate buffer (pH 7.6) containing 0.2 M NaC1 was electrophoresed as described in Materiais and Methods. S.P. and U refer to s t a n d s z d proteins a n d an u n f r a c t i o n a t e d sample, respectively. The numbers under the gels ere the fraction n u m b e r s o f DEAE-Sephadex A-50 c o l u m n chro mato grap hy. Migration is from t o p t o b o t t o m .

ary subunits [10]. The electrophoretic patterns of Otafuku were shown in Fig. 5. Similar results were obtained also in the cases of Sanuki-Nagasaya and Kumamoto-Churyu. Unfractionated sample consisted of three intermediary subunits with molecular weights of 48 000, 59 800 and 61 700. The more quickly eluting fraction of legumin, fraction 47, consisted of only one kind of intermediary subunit with molecular weight of 48 000. O n the other hand, all of three bands were observed in the slower eluting fractions. Thus the more slowly the legumin eluted, the more plentiful were the intermediary subunits with molecular weights of 59 800 and 61 700. These results were summarized in Table II by estimating densitometrically the ratio of the amounts of three bands. These results are consistent with those obtained in SDS-polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol.

Subunit compositions of the intermediary subunits The intermediary subunits were elect~ophoresed on SDS-polyacrylamide gel in the presence of 2-mercaptoethanol. As shown in Fig. 6, the larger intermediary subunits, I and II, each gave two bands with molecular weights of 51 000 and 19 000, and 49 000 .and 19 000, respectively. On the other hand, since the band of intermediary subunit with a molecular weight of 48 000 is broad, the band was cut into two pieces, the upper (III) and the lower (IV) portions. They gave two bands with molecular weights of 36 000 and 20 500, and 36 000 and 23 000, respectively. It is of interest that the intermediary subunit composed of 36 000 and 20 500 migrated slower than that composed of 36 000 and 23 000. This may be due to the differences in compactness of the structure between these intermediary subunits.

185

F i g . 4. S D S - p o l y a c r y l a m i d e gel e l e c t r o p h o r e s t s o f l e g u m i n f r a c t i o n s o f O t a f u k u . 3 0 /~g p r o t e i n o f e a c h f r a c t i o n w a s d i a l y z e d ~_=inlt 6 2 . 5 m M T r i s - H C l b u f f e r Coil 6 . 8 ) c o n t s i n i n g 1 % S D S a n d 0 . 2 M 2 - m e r c a p t o e t h a n o l f o r 2 0 h a t r o o m t e m p e r a t u r e a n d t h e n e l e c t x o p h o r e s e d as d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s . U r e f e r s t o a n u n f r a c t i o n a t e d s a m p l e . T h e n u m b e r s u n d e r t h e geis e r e t h e f r a c t i o n n u m b e r s o f D E A E S e p h a d e x A - 5 0 c o l u m n c h r o m a t o g r a p h y . M i g r a t i o n is f r o m t o p t o b o t t o m . TABLE I R A T I O O F A M O U N T S O F S U B U N I T S IN E A C H F R A C T I O N E L U T E D F R O M D E A E - S E P H A D E X COLUMN

A-50

T h e r a t i o s w e r e e s t i m a t e d d e n s i t o m e t r i e a l l y , t a k i n g t h e b a s / c s u b u n i t w i t h m o l e e u l e r w e i g h t o f 2 3 0 0 0 as unity. Fraction numbers

apply 47 59

64 72 83 89

Acidic subunits with molecular weights of

Basic s u b t m i t s w i t h m o l e c u l a r weights of

51 0 0 0

49 000

36 000

23 000

20 500

19 000

0.29 -0.17 0.27 0.51 0.67 0.76

0.24 -0.19 0.23 0.27 0.22 0.26

1.96 2.80 2.38 2.27 2.04 1.72 2.00

1 1 1 1 1 1 1

0.73 1.10 0.78 0.73 0.67 0.52 0.32

0.49 --

0.31 0.41 0.57 0.83 0.86

186

Fig. 5. S D S - p o l y a c r y l a m i d e gel e l e c t x o p h o r e s i s of' l e g u m i n f r a c t i o n s o f O t a f u k u in t h e a b s e n c e o f 2 - m e r c a p t o e t h a n o L 3 0 ~g p r o t e i n o f e a c h f r a c t i o n w a s d i a l y z e d against 6 2 . 5 m M Tris-HCl b u f f e r ( p H 6 . 8 ) c o n talning 1% SDS f o r 20 h a t r o o m t e m p e r a t u r e and t h e n e l e c t r o p h o r e s e d as d e s c r i b e d in Materials and M e t h o d s . U r e f e r s to an u n f r a c t i o n a t e d s a m p l e . T h e n u m b e r s under t h e gels are t h e f r a c t i o n n u m b e r s o f D E A E - S e p h a d e x A-50 c o l u m n c h r o m a t o g r a p h y . Migration is f r o m t o p t o b o t t o m .

Discussion Heterogeneities have been reported for arachin of Arachis hypogaea [12--14], 7 S globulins of Glycine max [15--18], Pisum sativum [19] and Vicia faba [20] and legumins of Pisum sativum [19] and Lupinus angustifolius [21,22]. Only in arachin [14] and 7 S globulin of soybean [18] their subunit structures have been proposed. However, heterogeneity of legumin from broad bean has not yet been reported. This may be due to separation of each molecular species of legumin on ion exchange chromatography being difficult compared with that in another seeds (Fig. 1). The heterogeneity of legumin from each cultivar observed in this paper may not be as a result of artifacts such as deamidation [23] or proteolysis, on the basis of the following observa-

T A B L E II R A T I O OF A M O U N T S OF I N T E R M E D I A R Y D E A E - S E P H A D E X A-50 C O L U M N

SUBUNITS IN EACH F R A C T I O N E L U T E D F R O M

T h e ratios for t h r e e i n t e r m e d i a r y s u b u n i t s w e r e e s t i m a t e d d e n s i t o m e t r i c a l l y , t a k i n g t h e t o t a l i n t e r m e d i a r y s u b u n i t s as u n i t y . Fraction numbers

apply 47 59 64 72 83 89

Intermediary subunits with molecula~ weights of 61 7 0 0

59 8 0 0

48 000

0.10 -0.05 0.09 0.14 0.22 0.17

0.04 -0.05 0.04 0.06 0.06 0.09

0.86 1 0.91. 0.86 0.80 0.72 0.74

187

Fig. 6. S u b u n i t c o m p o s i t i o n s o f t h e i n t e r m e d i a r y s u b u n i t s . A t first, l e g u m i n ( 3 0 0 ~ g ) o f O t a f u k u w a s e l e c t r o p h o r e s e d o n S D S - p o l y a c r y l a m i d e gel i n t h e a b s e n c e o f 2 - m e r c a p t o e t h a n o l rating 1 X 1 0 c m g l a ~ t u b e . A f t e r c i e c t r o p h o r e s i s , t h e gel w a s s t a i n e d w i t h 0 . 5 % a m i d o b l a c k 1 0 B i n 2095 a c e t i c a c i d f o r 5 r a i n a n d d e s t e i n e d w i t h 7% a c e t i c a c i d . T h e b a n d s c o r r e s p o n d i n g t o t h e i n t e r m e d i a r y s u b u n i t s w e r e c u t o f f a n d washed with 63.5 mM Tris-HCl buffer (pH 6.8) containing 1% SDS, 0.2 M 2-mercaptoethanol and 30% sucrose for 30 rain at room temperatttre and another 30 rain at 50°C with change of buffer, and then put o n t h e S D S - p o l y a c r y l a m i d e gel in a 0 . 6 X 1 0 c m glass t u b e a n d e l e c t r o p h o r e s e d as d e s c r i b e d i n Fig. 4. M i g r a t i o n is f r o m t o p t o b o t t o m .

tions; (1) there was no change in the electrophoretic patterns of the subunits of legumin even if extracts prepared using 50 m M potassium phosphate buffer (pH 7.0) or 50 mM Tris-HC1 buffer (pH 7.6) containing 1 M NaC1 were incubated for 5 days at 20°C; (2) the pattern of heterogeneity was constant and reproducible for each cultivars. The subunit patterns of legumins from three cultivars were similar from the standpoint of the molecular sizes of the subunits (Figs. 4 and 5). Therefore, we attempted to list the possible molecular species of legumin which are common to the three cultivars, constructed from the subunit groups classified according to their sizes. The possible molecular species were shown in Table III. Each subunit composition was decided on the basis of the assumption that all the molecular species of legumin are composed of a6~6, which had been found in glycinin of Glycine max [4--6], 12 S globulin of A r e n a sativa [7], a-globulin of S es a mu m indicum [8] and others [9], and the following experimental evidence: (1) four kinds of molecular species which were different in molecular weights were present as legumin in broad bean (Fig. 3); (2) the intermediary subunits with molecular weights of 61 700, 59 800 and 48 000 are composed of the acidic subunits with molecular weights of 51 000, 49 000 and 36 000 and the basic subunits with molecular weights of :19 000, 19 000 and 23 000 or 20 500, respectively (Fig. 6); (3) the ratio of the amounts of intermediary subunits of each legumin species could be estimated from the ratio of the amount of each subunit and intermediary subunit of the legumin fractions as shown in Tables I and II. The molecular weight of each legurnin species was calculated from each mo-

188 TABLE III THE POSSIBLE MOLECULAR SPECIES OF LEGUMIN FROM BROAD BEAN Molecular species

I-1 I-2 II-1 II-2 III-1 III-2 IV

Subunit c o m p o s i t i o n *

3(A3B 1 ) 2(A3B1) 2(A3B 1) 2(A3BI) 2(A3B 1 ) 2(A3BI) 2(A3B 1 )

3(A3B 2) 4(A3B 2) 3(A3B2) 3(A3B2) 2(A3B 2 ) 2(A3B2)

Molecular weights

I ( A 2 B3 ) I ( A 2 B3 ) I ( A 2 B3 )

I(AIB3) I ( A I B 3) 2(AIB3) 3 ( A I B 3)

Calculated **

Observed ***

345 000

320 000

357 000

350 000

370 000

380 000

396 000

400 000

* The subunits w i t h molecular weights of 51 000, 49 0000 36 000, 23 0000 20 500 and 19 000 s how n in Fig. 4 were t e r m e d A I , A2, A3, BI, B 2 and B3, respectively. ** Calculated from each molecular weight of subunits. * ** E stimated from the results of the gradient gel electrophoresls (Fig. 3).

lecular weight of subunits, because there is correlation between molecular weights of intermediary subunit and its constituent subunits except either intermediary subunits III or IV (Fig. 6). As shown in Table III,the molecular weight of each legumin species calculated from the molecular weight of each subunit was in good agreement with that estimated from the results of the gradient gel electrophoresis.However, the molecular weight calculated from the

Fig. 7. Gel electzofocusing of legumins f~om three cultivars in the presence of 7 M urea. Electrofocusing was carried o u t as described in Materials and Methods; The electrophoretic pa t t e rns of Otafuku, SanukiNagasaya and K u m a m o t o - C h u r y u were shown in A, B and C, respectively. The u n f r a c t i o n a t e d samples were applied. Migration is from top to b o t t o m .

189 molecular weight of each intermediary subunit was not in good agreement with that observed. Thus, this agreement may be incidental and may not necessarily justify the validity of the deduction of subunit compositions of legumin species. We have no evidence to explain this discrepancy at present; however, it may be presumed that compactness and shape of the legumin molecules and those of its subunits are similar but not those of legumin and its intermediary subunits. As shown in Fig. 7B and C, legumin of Sanuki-Nagasaya and KumamotoChuryu gave four main bands as basic subunits, more than observed by SDSpolyacrylamide gel electrophoresis. Moreover, the kinds of main band of basic subunits were different among the three cultivars (Fig. 7). Therefore, when the subunit compositions of legumin species are represented by the subunits classified according to their charge, the number of possible molecular species of legumin must be much more than that presented in Table III. This suggests that each of seven molecular species presented in Table III may have further submolecular species. However, the subunit compositions of them cannot be deduced at present. Moreover, we have recently found similar heterogeneity of glycinin of soybean seeds (var. Tsuru-no-ko) from similar analyses to those described above. This evidence, together with the results of others [12--22] suggests that a major component of storage proteins of legume seeds exhibits the heterogeneity in general. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

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