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Plant Foods fo r Hu ma n Nutr i tion 50:211-222, 1997.(~) 1997 Klu we r Aca dem ic Publishers. Printed in the Netherlands.

C h a r a c t e r is t ic s o f t w o m a j o r l ec t in s fr o m m u n g b e a n( V i g n a r a d i a ta ) s e e d sK . N . S U S E E L A N 1, C .R . B H A T I A 2 an d R . M I T R A t*1Nuclear Agricul ture Divis ion, Bha bha Atom ic Research Centre , Mu mb ai-400 085, India217, Ro hini Cooperative Ho usin g Society, Sector 9A, Vashi, Na vi Mum bai, In dia(* autho r fo r correspondence)Received 10 A pril 1996; accepted in revised form 16 Decem ber 1996

Abstract. Two major lectins, MBL -I and M BL-II, were purified from Vigna radiata L. seedsusing ion-exchan ge and gel filtration chromatography techniques. M BL -I was fo und to be atetram er w ith native M .W. of 132 kDa and su bunit M .W. of 33 kD a havin g c~-galactosidaseactivity. M BL -II consisted of two mo nom eric lectins w ith M.W. of 94 kDa and 89 kD awhich w ere associated mainly w ith fl-galactosidase activity. Bo th M BL -I and M BL -II areD-galactose-spec ific lectins.Key words: c~-Galactosidase, fl-Galactosidase, Greengram, L ectins, M onom eric, Mung bean

I n t r o d u c t i o n

L e g u m e s e e d s c o n s t it u t e a n i m p o r t a n t p a r t o f t h e h u m a n d i e t e s p e c i a ll y a s as o u r c e p r o t e i n . H o w e v e r , if c o n s u m e d i n t h e ra w s t at e , t h e y a r e to x i c d u e t ot h e p r e s e n c e o f s u b s t a n c e s l ik e t ry p s i n i n h ib i to r s, c o m p o u n d s c a u s i n g f a v i s m ,l a t h y r o g e n s , p h y t a t e , s a p o n i n s , a l k a lo i d s , c y a n o g e n i c a n d g o i t r o g e n i c f a c to r sa n d p h y t o h e m a g g l u t i n i n s o r l e c ti n s [ 1 - 2 ] . L e c t i n s a r e a n t in u t r it io n a l b e c a u s et h e y e i t h e r i n t e r f e r e w i t h t h e d i g e s t i v e e n z y m e s a n d r e d u c e t h e a v a i l a b i l i t yo f n u t r ie n t s o r t h e y d i s r u p t t h e e p i th e l i a l c e ll s h a m p e r i n g t h e tr a n s p o r t o fnu t r i e n t s i n to t he c e l l [ 3 - 5 ] . Though the r e a r e s t r uc tu r a l s imi l a r i t i e s be twe e ns e v e r a l l e g u m e l e c t in s , t h e y d i f f e r w i t h r e s p e c t t o c a r b o h y d r a t e b i n d i n g s p e c i -f ic it y . A n o t h e r i n t e re s t in g a s p e c t o f l ec t in s i s t h at t h e y c o n t a i n a s e c o n d t y p e o fb i n d i n g s i te th a t i n t er a c ts w i t h a n o n - c a r b o h y d r a t e l i g a n d a n d t h is b i f u n c t i o n a ln a t u r e o f l e c ti n s h a s b e e n s h o w n t o b e b i o l o g i c a l l y s i g n i fi c a n t [ 6] .

V i g n a r a d i a t a L . W i l c z e k ( m u n g b e a n o f g r e e n g ra m ) i s a n i m p o r t an t p u l s ec r o p o f I n d ia . E x t e n s i v e b r e e d i n g w o r k h a s b e e n c a r r i e d o u t o n th i s c r o p f o rt h e i m p r o v e m e n t o f y i e l d a n d p r o t e i n [ 7 - 8 ] . C h a r a c t e ri z a t io n o f s e e d s t o r a g ep r o t e i n s a n d e v o l u t i o n a r y a s p e c t s h a v e b e e n r e p o r t e d [ 9] .

E a r l i e r m u n g b e a n l e c ti n h a s b e e n r e p o r t e d a s a g a l a c t o s e - s p e c i fi c , te t r a m e r -i c p r o t e in w i th i n t r i n s i c o~- ga l a c to s ida se a c t iv i t y [ 10 ] . The p r e se n t r e po r t

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2 1 2d e s c r i b e s t h e p r e s e n c e o f t w o g a l a c t o s e - s p e c i fi c , h e a t - l a b il e l e c t in s o f m u n g -bean , a s s oc i a t ed w i th e i t he r o~- o r / 3 -g a l ac tos ida se ac t i v it i e s .

M a t er i a l s a n d m e t h o d s

Material . D r y s e e d s o f m u n g b e a n (Vigna radiata L . W i l c z e k ) c v. T A P - 7 w e r eu s e d a s t h e s o u r c e o f l e ct in . S e e d s w e r e o b t a i n e d f r o m N u c l e a r A g r i c u l t u r eD i v i s io n o f B h a b h a A t o m i c R e s e a r c h C e n t re , M u m b a i , I n d ia .Extract ion a nd precipi ta t ion of lec t in . T h e l e c ti n s w e r e p u r i f ie d u s i n g a m o d -i f ic a t i o n o f t h e m e t h o d o f H a n k i n s e t a l. [ 1 1 ]. S e e d s w e r e g r o u n d i n a U d yC y c l o n e m i l l a n d p a s s e d t h r o u g h a 4 0 m e s h s i e v e. S e e d m e a l ( 5 0 g) , w a s s t ir r e do v e r n i g h t i n 2 0 0 m l o f e x t r a c ti o n b u f fe r (5 0 m M N a - p h o s p h a t e b u f fe r , p H 5 .2c o n t a i n i n g 0 .4 M N a C 1 , 1 m M D - g a la c t o s e , 1 0 m M / 3 - m e r c a p t o e t h a n o l a n d1 m M P M S F ) a t 4 ~ T h e s lu r r y w a s c e n t r if u g e d a t 1 2, 00 0 g f o r 2 0 m i n in ar e f r i g e r a t e d c e n t r i f u g e (K u b o t a ) . T h e s u p e r n a t a n t w a s s a v e d .

T h e l e c t i n in t h e s u p e r n a t a n t w a s p r e c i p i t a t e d a t 7 0 % s a t u r a ti o n o f ( N H 4)2S O 4 . T h e p r e c i p i t a t e d p r o t e i n w a s d i a l y z e d a g a i n s t a d ia l y s i s b u f f e r ( 1 0 m MN a - p h o s p h a t e b u f fe r , p H 5 .2 c o n t a in i n g 1 m M D - g a l a c t o s e a n d 1 0 m M / 3 -m e r c a p t o e t h a n o l ) .Ion-exch ange chromatography. Th e d i a l y z e d s a m p l e w a s l o a d e d o n a C M -S e p h a r o s e c o l u m n ( 6 0 x 1 5 m m ) , p r e - e q u i l ib r a t e d w i t h th e d i a l y s is b u ff e r. T h er e s i n - b o u n d p r o t e i n w a s e l u t e d w i t h a s a l t g r a d i e n t p r e p a r e d i n b u f f e r s w i t hl o w e r l im i t o f 1 0 m M N a - p h o s p h a t e b u f f e r ( P B ) a n d u p p e r l i m i t o f 5 0 m MP B c o n t a i n i n g 0. 4 M N aC 1. B o t h t h e b u f f e rs c o n t a i n e d 1 m M D - g a l a c t o s ea n d 1 0 m M / 3 - m e r c a p t o e t h a n o l . E l u t io n o f t h e c o l u m n w a s a t a f lo w ra t e o f6 0 m l / h a n d 8 m l f r a c t io n s w e r e c o l l e c t e d u s i n g a f r a c t io n c o l l e c t o r F R A C -1 0 0 ( P h a r m a c i a ) . A l l f r a c t i o n s w e r e c h e c k e d f o r a g g l u t i n a t io n a s w e l l a s o~-a n d / 3 - g a l a c t o s i d a s e a c t i v i t i e s . T h e f r a c t i o n s s h o w i n g a g g l u t i n a t i o n a c t i v i t yw e r e p o o l e d a n d t h e p r o t e in w a s p r e c i p i ta t e d at 7 0 % s a t u r a ti o n o f (NH4)2SO4.G el fi l tration chromatography. L e c t i n s a m p l e s p r e c i p i t a t e d f r o m t h e f r a c -t io n s c o l l e c t e d a ft e r i o n - e x c h a n g e c h r o m a t o g r a p h y w e r e d i s s o lv e d i n e x t r a c -t io n b u f fe r a nd c h r o m a t o g r a p h e d u si n g a U l tr o g e l A c A 4 4 c o l u m n ( 5 6 0 x1 6 r a m ) , p r e - e q u i l i b r a t e d w i th t h e e x t r a c t i o n b u ff e r . T h e c o l u m n w a s e l u t e dw i t h t h e s a m e b u f f e r a t a f l o w ra t e o f 1 2 m l / h a n d 1 m l f r a c t i o n s w e r e c o l l e c t e d .Native molecular weight es t imation. M o l e c u l a r w e i g h t s o f th e t w o m a j o rl e c ti n s ( M B L - I a n d M B L - I I ) w e r e e s t im a t e d u s in g a U l tr o g e l A c A 4 4 c o l u m n

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213( 5 6 0 1 6 m m ) . T h e c a l i b r a t io n c u r v e w a s p l o tt e d u s in g a r a n g e o f p r o t e i n sw i th k n o w n m o l e c u l a r w e ig h t.SDS -polyacrylamide gel electrophoresis. Th e p u r it y o f l e c ti n s w a s c h e c k e du s i n g 1 2 % S D S - g e l b y t h e m e t h o d o f L a e m m l i [1 2 ]. T h e s u b u n it m o l e c u l a rw e i g h t s o f t h e l e c ti n s w e r e e s t i m a t e d f r o m t h e s t a n d a r d c u r v e p l o t te d f o r t h em a r k e r p r o te i n s ( P h a r m a c i a S D S - g e l m a r k e r s ) lo a d e d o n t h e s a m e g e l.Agglutination assay. A g g l u t i n a t i o n a s s a y w a s c a r r i e d o u t u s i n g 2 % t r y p s i nt r e a t e d r a b b i t e r y t h r o c y t e s u s p e n s i o n i n P B S [ 11 ]. T w o - f o l d s e r ia l d il u t io n so f 5 0 # 1 o f l e ct in s o l u ti o n ( 1 0 - 5 0 # g ) u s i n g 0.3 M N a C I w e r e i n c u b a t e d w i t h5 0 # 1 o f e r y t h r o c y t e s u s p e n s i o n i n a m i c r o t it r e p l a te f o r 3 0 m i n , a t r o o mt e m p e r a t u r e ( 25 ~ a n d e x a m i n e d u n d e r a m i c r o s c o p e . A g g l u t i n a t io n u n i t( A U ) o f l e c t i n w a s c a l c u l a t e d a s th e r e c i p r o c a l o f t h e l a s t d i l u ti o n w h i c h g a v et h e d e t e c t a b l e a g g l u t in a t i o n .Protein e stimation. P r o t e in w a s e s t i m a t e d u s i n g t h e p r o c e d u r e o f L o w r y e t a l.[ 1 3]. L i p i d - f r e e b o v i n e s e r u m a l b u m i n ( B S A ) w a s u s e d a s t h e s t a n d a r d p r o t e in .Assay fo r galactosidases. Th e a c ti v it ie s o f ~ - a n d / 3 - g a l a c t o s i d a s e e n z y m e sw e r e a s s a y e d f o ll o w i n g t h e m e t h o d o f M u r r a y [ 14 ], u s i n g p - N i t r o p h e n y l -o ~ - / /% D - g a la c t o p y ra n o s id e s a s s u b s t r a te s . T h e a s s a y m i x t u r e c o n t a i n e d 2 0 -1 0 0 # 1 o f l e c t i n s o l u t i o n a n d 1 m l o f 3 m M s u b s t r a t e s o l u t i o n p r e p a r e d i n0 .1 M s o d i u m a c e t a t e b u f f e r, p H 4 .7 . T h e m i x t u r e w a s i n c u b a t e d a t 3 0 ~f o r 4 5 m i n . T h e r e a c t i o n w a s s t o p p e d b y a d d i n g 2 m l o f 0 .2 M s o d i u m c a r -b o n a t e . T h e e n z y m e a c ti v it y w a s d e t e r m i n e d b y m e a s u r i n g t h e a b s o rp t io n a t4 0 0 n m o n a P e r k i n - E l m e r L a m b d a 1 U V / V I S S p e c t ro p h o to m e t e r. T h e u n i to f e n z y m e a c ti vi ty w a s d e f i n e d as t h e a m o u n t o f e n z y m e w h i c h h y d r o l y z e s1 # m o l e o f s u b s t ra t e p e r m l , p e r m i n u t e a t 3 0 ~pH stability. T h e e f f e c t o f p H o n M B L w a s e x a m i n e d us in g v a r io u s b u ff e rso f p H 3 - 1 0 . L e c t i n s o l u ti o n w a s in c u b a t e d w i th th e b u f f e r a t 4 ~ o v e r n ig h t .A g g l u t i n a t i o n a s s a y s w e r e c a r r i e d o u t a s d e s c r i b e d e a rl ie r .Therm ostability and thermal inactivation. L e c t i n s a m p l e s w e r e i n c u b a t e d a t4 0 ~ f o r u p t o 9 0 m i n . A l i q u o ts w e r e d r a w n a t 5 m i n i n t e rv a l s u p t o 2 0 m i n ,f o l l o w e d b y 1 0 m i n i n t e rv a l s f o r t h e r e s t o f th e i n c u b a t i o n ti m e . T h e e x p e r i -m e n t w a s c a r r i e d o u t i n th e p r e s e n c e o f 2 m M D - g a l a c t o s e f o r b o t h t h e l e ct in s .I n a c ti v a ti o n te m p e r a t u r e o f M B L w a s d e t e r m i n e d b y i n c u b a ti n g l e c ti n s o lu -t io n in 1 0 m M P B , p H 5 . 2, f o r 1 0 m i n u p t o 9 0 ~ a t i n t e rv a l s o f 1 0 ~I n b o t h c a s e s , a l i q u o ts w e r e d r a w n a n d a s s a y e d f o r a g g l u t in a t i o n a s w e l l a s

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2 1 4

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