Chapter 10 Glycoproteins and Glycopeptides (Affinity Electrophoresis)

219

Chapter 10 GLYCOPROTEI N S AND GLYCOPEPTIDES (AFFINITY ELECTROPHORESIS) T.C.

BBG-HANSEN AND J. HAU

GENERAL ASPECTS A f f i n i t y e l e c t r o p h o r e s i s i s based on t h e r e a c t i o n between i n t e r a c t i n g compo-

.

nents d u r i n g e l e c t r o p h o r e s i s . An e a r l y s y s t e m a t i c s t u d y was conducted by Nakamura 1 L a t e r t h e t e r m a f f i n i t y e l e c t r o p h o r e s i s was i n t r o d u c e d by B6g-Hansen2 and H o r e j s i and Kocourek’.

Well known examples o f t h e p r i n c i p l e o f l e t t i n g i n t e r a c t i n g com-

ponents r e a c t d u r i n g e l e c t r o p h o r e s i s a r e c o u n t e r immunoelectrophoresis, r o c k e t immunoelectrophoresis and c r o s s e d immunoelectroohoresis, c o l l e c t i v e l y r e f e r r e d t o as electroimmunoassays o r g e l e l e c t r o i m m u n o p r e c i p i t a t i o n methods (see, f o r 4 i n s t a n c e , Axelsen ) . A f f i n i t y e l e c t r o p h o r e s i s o f o t h e r i n t e r a c t i n g systems has 5 been reviewed b r i e f l y by H o r e j s i e t a l .

.

0u r comb ina t ion o f qua n t it a t iv e immunoe 1e c t r oDho re s is and a f f i n ity e 1e c t r0p h o r e s i s w i t h l e c t i n s was developed f o r t h e i d e n t i f i c a t i o n , q u a n t i f i c a t i o n and c h a r a c t e r i z a t i o n o f g l y c o p r o t e i n s and t h e a n a l y t i c a l o r e d i c t i o n o f p r e p a r a t i v e s e p a r a t i o n s . I n t h i s c h a p t e r we s h a l l deal w i t h a n a l y t i c a l a f f i n i t y e l e c t r o p h o r e s i s w i t h l e c t i n s o f g l y c o p r o t e i n s . I n p r i n c i p l e t h e r e a r e t h r e e approaches which can be used f o r s t u d y i n g i n t e r a c t i n g comnonents by a n a l y t i c a l e l e c t r o p h o r e s i s : t h e i n t e r a c t i o n can t a k e p l a c e e i t h e r b e f o r e , d u r i n g o r a f t e r e l e c t r o p h o r e s i s . We have s t u d i e d t h e r e s u l t s o f i n t e r a c t i o n s d u r i n g e l e c t r o p h o r e s i s , because more i n f o r m a t i o n can be o b t a i n e d i n t h i s way. Moreover, t h e c h a r a c t e r i z a t i o n o f e l e c t r o p h o r e t i c a l l y s e p a r a t e d g l y c o p r o t e i n s and g l y c o n e p t i d e s by b i n d i n g o f l e c t i n s , such as f l u o r e s c e i n i s o t h i o c y a n a t e - l a b e l l e d l e c t i n , enzyme-labelled l e c t i n o r a n t i b o d y - l a b e l l e d l e c t i n , has been performed elsewhere (see, f o r i n s t a n c e , B i t t i g e r and S c h n e b l i 6 . A l s o r e a c t i o n s w i t h l e c t i n s a f t e r e l e c t r o D h o r e s i s , i n a system analogous t o t h e c l a s s i c a l i n n n u n o e l e c t r o p h o r e t i c a n a l y s i s a c c o r d i n g t o Grabar have been s t u d i e d b y o t h e r s (Murakawa and Nakamura7, Osunkoya and k l i l l i a m s

9 Spengler and Weber

.

a,

We p r e f e r t o use electroimmunoprecipitation as t h e a n a l y t i c a l r e f e r e n c e method. The main reason i s t h a t p r o t e i n s r e m a i n i n t h e i r n a t i v e s t a t e d u r i n g an e l e c t r o i m m u n o p r e c i p i t a t i o n e x p e r i m e n t and r e t a i n t h e i r b i o l o g i c a l a c t i v i t y . T h e r e f o r e , p r o t e i n - l i g a n d i n t e r a c t i o n s a r e n o t i m p a i r e d . G e n e r a l l y , complex f o r m a t i o n between p r o t e i n s and l i g a n d s t e n d t o change t h e i r e l e c t r o p h o r e t i c and a n t i g e n i c b e h a v i o u r .

220

Consequently, i n t e r a c t i n g p r o t e i n s may be i d e n t i f i e d and s t u d i e d by t h e changes induced by i n c l u s i o n o f l i g a n d s i n crossed immunoelectrophoresis. The changes a r e e v i d e n t w i t h macromolecular l i g a n d s , e x e m p l i f i e d h e r e by l e c t i n s and t h e i r a f f i n i t y t o the carbohydrate parts o f glycoproteins. Another reason why we p r e f e r crossed immunoelectrophoresis as t h e r e f e r e n c e e l e c t r o p h o r e t i c system i s t h a t we want t o t a k e advantage o f t h e e x t r a o r d i n a r y f e a t u r e s o f t h i s method f o r t h e a n a l y s i s and c h a r a c t e r i z a t i o n o f multicomponent p r o t e i n m i x t u r e s . The use o f a n t i b o d i e s o f f e r s t h e p o s s i b i l i t y o f t h e s p e c i f i c i d e n t i f i c a t i o n o f p r o t e i n s , b u t i t l i m i t s t h e a n a l y t i c a l uses o f l e c t i n s because of t h e g l y c o p r o t e i n n a t u r e o f t h e a n t i b o d i e s and t h e n e c e s s i t y f o r n o n - d e n a t u r i n g c o n d i t i o n s , The general usage and p r e p a r a t i o n o f a n t i b o d i e s f o r electroimmuno11 p r e c i p i t a t i o n has been d e s c r i b e d e l sewhere (Svendsen'', B$g-Hansen e t a1 ). The t e c h n i c a l d e t a i 1s o f t h e compound methods o f l e c t i n a f f i n i t y immunoelec2,12-20 t r o p h o r e s i s have been d e s c r i b e d p r e v i o u s l y

.

Many p r o t e i n s have been c h a r a c t e r i z e d and p u r i f i e d by a f f i n i t y chromatograohy

21

~ ( T u r k o v a ) and a l s o s p e c i f i c a l l y by i n t e r a c t i o n w i t h l e c t i n s as reviewed by 3ulaneyZ2. I t i s t h e o b j e c t o f t h i s c h a p t e r t o d e s c r i b e g e n e r a l a n a l y t i c a l t e c h nology t o f a c i l i t a t e : ( 1 ) i d e n t i f i c a t i o n o f l i g a n d - b i n d i n g p r o t e i n s ; ( 2 ) charact e r i z a t i o n o f the reactive s i t e ( s ) ; (3) q u a n t i f i c a t i o n o f ligand-binding proteins; and ( 4 ) p r e d i c t i o n o f p r e p a r a t i v e a f f i n i t y s e p a r a t i o n s . 3ESCRIPTION OF THE TECHNOLOGY

Method A: Reactions with Zectin before electrophoresis Reactions a f t e r i n c u b a t i o n w i t h l e c t i n a r e shown s c h e m a t i c a l l y i n F i g . l O . l a , b and c, which show t h e p a t t e r n s i n crossed immunoelectrophoresis. The c o n t r o l D a t t e r n i s shown i n F i g . 1 0 . l a . F i g . 1 0 . l b shows t h e p a t t e r n a f t e r r e a c t i o n w i t h i m m o b i l i z e d l e c t i n . The i n c u b a t i o n p r i o r t o e l e c t r o p h o r e s i s was oerformed by m i x i n g i m m o b i l i z e d l e c t i n w i t h t h e p r o t e i n sample. Only t h e s u p e r n a t a n t f l u i d was analysed by crossed immunoelectrophoresis. P r o t e i n 1 i s known n o t t o b i n d t o t h e l e c t i n , and i t s p r e c i p i t a t e i s unchanged and may b e used as an i n t e r n a l r e f e r e n c e . The t y p i c a l r e a c t i o n i s disappearance o f p r e c i p i t a t e s f r o m t h e p a t t e r n . E i t h e r t h e disappearance may be t o t a l , as seen f o r p r o t e i n 2, or p a r t i a l , as seen f o r p r o t e i n 3 ( o n l y a f r a c t i o n o f p r o t e i n 3 i s bound). T h i s p a t t e r n o c c u r s f r e q u e n t l y i n t h i s t y p e o f experiment. T h e o r e t i c a l l y o t h e r p a t t e r n s c o u l d occur, i n c l u d i n g s p l i t t i n g o f t h e p r e c i p i t a t e i n t o s e v e r a l l i n e s w i t h t h e same f i r s t - d i m e n s i o n m o b i l i t y (Fig. lO.la, p r o t e i n 4 mobilities (protein 5

-t

-+

4a t 4b) o r w i t h d i f f e r e n t f i r s t - d i m e n s i o n

5 t x ) accompanied by a change i n p r e c i p i t a t e morphology

( p r o t e i n 4) o r p r o f i l e ( p r o t e i n 5 ) .

221

7 7 1

Id

1

lI g

1

1

II

F i g . 10.1. L e c t i n i n t e r a c t i o n immunoelectroDhoresis. Schematic diagrams o f p r o t e i n p r e c i p i t a t e p a t t e r n s i n crossed immunoelectrophoresis w i t h and w i t h o u t l e c t i n . ( a ) The p r o t e i n p r e c i p i t a t e r e f e r e n c e p a t t e r n w i t h o u t l e c t i n ; c o n t r o l experiment. ( b ) The p a t t e r n a f t e r i n v i t r o i n c u b a t i o n w i t h i m m o b i l i z e d l e c t i n . ( c ) The p a t t e r n a f t e r i n v i t r o i n c u b a t i o n w i t h f r e e l e c t i n . ( d ) The r e f e r e n c e p a t t e r n w i t h o u t l e c t i n i n t h e i n t e r m e d i a t e g e l ( i . m . ) ; c o n t r o l exDeriment. ( e ) The o a t t e r n w i t h i m m o b i l i z e d l e c t i n ( l e c t i n - S e p h a r o s e , L-S) i n t h e i n t e r m e d i a t e g e l . ( f ) The o a t t e r n w i t h f r e e l e c t i n (L) i n t h e i n t e r m e d i a t e g e l . ( 9 ) The r e f e r e n c e p a t t e r n w i t h o u t l e c t i n i n t h e f i r s t - d i m e n s i o n g e l ; c o n t r o l experiment. ( h ) The p a t t e r n w i t h i m m o b i l i z e d l e c t i n ( l e c t i n - S e p h a r o s e , L-S) i n t h e f i r s t - d i m e n s i o n g e l . ( i ) The p a t t e r n w i t h f r e e l e c t i n (L) i n t h e f i r s t - d i m e n s i o n g e l . See t e x t f o r d e t a i l s . A b b r e v i a t i o n s : a = a f f i n i t y p r e c i p i t a t e ; i . m . = i n t e r m e d i a t e g e l ; L = l e c t i n ; L-S = l e c t i n - S e o h a r o s e ; 1+ = f i r s t - d i m e n s i o n e l e c t r o n h o r e s i s ; 2t = second-dimension e l e c t r o p h o r e s i s . F i g . 1 0 . 1 ~shows a s i m i l a r p a t t e r n a f t e r t h e r e a c t i o n w i t h f r e e l e c t i n . Some p r o t e i n s ( a r r o w s ) appear as l e c t i n complexes i n t h e s u p e r n a t a n t f l u i d and d u r i n g e l e c t r o p h o r e s i s , r e s u l t i n g i n a t y p i c a l p a t t e r n o f p a r t i a l i d e n t i t y (B#g-'Hansen

N

TABLE 10.1

LIST OF CHANGES IN THE IMMUNOPRECIPITATION PATTERN IN CROSSED IMMUNOELECTROPHORESIS WITH LECTINS Reactions seen f o r various glycoproteins w i t h conA. W i t h immobilized conA-Sepharose reactions 6 and 9 have not been seen. (Modified from T.C. BBg-Hansen e t a1 .20). No.

Reaction w i t h l e c t i n i n f i r s t dimension

Reaction w i t h l e c t i n in i n t e r mediate gel

Interpretation

1. 2.

No reaction. Disappearance of precipitate.

No reaction. Disappearance of precipitate.

3.

5.

S h i f t o f position in electrophoret i c pattern. Appearance of a multi-peak precip i t a t e ("camel" precipitate"). Decrease i n precipitate s i z e .

No molecules have a f f i n i t y f o r the l e c t i n . All molecules have a f f i n i t y f o r the ligand. The b i n d i n g t o immobilized l e c t i n may be observed w i t h glycoprotein enzymes. An aff i n i t y precipitate may appear, indicating that some or a l l molecules contain two o r more binding s i t e s f o r the ligand. Binding to ligand.

Decrease i n precipitate s i z e .

6.

Increase i n precipitate size.

Increase i n precipitate s i z e .

7.

Change i n precipitate profile. Change i n precipitate morphology. A . S p l i t t i n g of precipitate. B. Change t o diffuse precipitate. Reactions of "partial i d e n t i t.y".

Change i n precipitate profile. Change i n precipitate morphology. A. S p l i t t i n g of precipitate. B. Change t o diffuse precipitate. Reactions of "partial identity".

4.

8.

9.

Reveals various molecular forms w i t h d i f ferent a f f i n i t y t o the ligand. Only part of the molecules have binding s i t e s o r the binding i s weak. Binding o f ligand leads to s t e r i c hindrance of anti body-bi ndi ng . As 4. As 6 , o r interrupted precipitation.

As 4 and 6 , o r coupling of different prot e i n s through one molecule of ligand (the ligand mediates the "partial identity").

223 e t a1 .20) mediated by l e c t i n c r o s s - l i n k i n g o f d i f f e r e n t p r o t e i n species (compare below). With small amounts o f l e c t i n , r e l a t i v e t o g l y c o p r o t e i n , t h e p a t t e r n i s a f f e c t e d by t h e l e c t i n concentration, b u t above t h e " s a t u r a t i o n p o i n t " t h e p a t t e r n i s

l i t t l e changed by f u r t h e r a d d i t i o n s o f more l e c t i n . We i n t e r p r e t t h e l o s s o f a p r e c i p i t a t e as b i n d i n g o f t h e p r o t e i n t o t h e l e c t i n . The r e a c t i o n s described here as t h e y appear i n crossed immunoelectrophoresis have analogous c o u n t e r p a r t s i n o t h e r e l e c t r o p h o r e t i c systems.

Method B: Lectin in an intermediate gel The p r i n c i p l e o f i n c o r p o r a t i n g l e c t i n i n t o an i n t e r m e d i a t e gel i n crossed immunoelectrophoresis was i n t r o d u c e d by Bg5g-Hansen2 under t h e term "crossed

immunoaffinoelectrophoresis". Since i t s i n t r o d u c t i o n , t h e method has been used f o r t h e i d e n t i f i c a t i o n o f many g l y c o p r o t e i n s and f o r the p r e d i c t i o n o f s e p a r a t i o n experiments (see, e.g.,

r e f s . 2, 14, 17, 20, 23-32).

A l i m i t e d number o f r e a c t i o n s occur when immobilized l e c t i n i s i n c l u d e d i n an i n t e r m e d i a t e g e l i n crossed immunoelectrophoresis. C h a r a c t e r i s t i c r e a c t i o n s a r e

disappearance o r d i m i n u t i o n o f p r e c i p i t a t e s (see F i g . 1 0 . l d and e ) . T h e o r e t i c a l l y i t i s reasonable t o expect t h a t macromolecular complexes c o n t a i n i n g g l y c o p r o t e i n s

may g i v e more complex p a t t e r n s a f t e r r e a c t i o n w i t h l e c t i n . S p l i t t i n g o f p r e c i p i t a t e s , as shown i n F i g . 1 0 . l e ( p r o t e i n 4

-+

4a t 4b o f t h e same f i r s t - d i m e n s i o n

m o b i l i t y ) , may occur a f t e r r e a c t i o n i n t h e i n t e r m e d i a t e g e l . The change i n prec i p i t a t e p r o f i l e seen f o r p r o t e i n 5 i n F i g . 1 0 . l e i s c h a r a c t e r i s t i c f o r complex g l y c o p r o t e i n s and i s seen, f o r exmaple, w i t h human serum complement C3. Any o f these r e a c t i o n s may be accompanied by changes i n p r e c i p i t a t e morphology. Changes i n t h e p r e c i p i t a t e p a t t e r n s , i n c l u d i n g observations w i t h b o t h f r e e and immobilized l e c t i n s , a r e shown i n Table 10.1. F i g . 1 0 . l f shows t h e r e a c t i o n s w i t h f r e e l e c t i n i n t h e i n t e r m e d i a t e g e l . I n a d d i t i o n t o t h e p r e c i p i t a t e changes mentioned w i t h immobilized l e c t i n , o t h e r n o t a b l e f e a t u r e s appear w i t h f r e e l e c t i n s : f o r m a t i o n o f a f f i n i t y p r e c i p i t a t e s and c o p r e c i p i t a t i o n o f i n d i v i d u a l g l y c o p r o t e i n s (discussed below).

Method C. Lectin in the first-dimension g e l The p r i n c i p l e o f i n c o r p o r a t i n g l e c t i n i n t o t h e f i r s t - d i m e n s i o n g e l was i n t r o duced by B#g-Hansen e t a1.33 and i s analogous t o t h e use o f s p e c i f i c a n t i b o d i e s i n t h e f i r s t - d i m e n s i o n gel

-

a h i g h l y s p e c i f i c and s e n s i t i v e method f o r t h e

d e t e c t i o n and i d e n t i f i c a t i o n of i n t e r a c t i n g antigens and a n t i b o d i e s ( P l a t t e t a1 .34) Since i t s i n t r o d u c t i o n , t h e method w i t h l e c t i n s i n t h e f i r s t - d i m e n s i o n gel has been used f o r t h e i d e n t i f i c a t i o n , c h a r a c t e r i z a t i o n and q u a n t i f i c a t i o n o f g l y c o p r o t e i n s and p r e d i c t i o n o f s e p a r a t i o n experiments (see, e.g., r e f s . 16, 20, 23, 25, 27, 28, 35-61).

224

Fig. 10.2. Lectin i n t e r a c t i o n immunoelectrophoresis o f human serum protein. In each experiment 2 vl of human serum were analysed w i t h multivalent antibodies against human serum proteins (OAK0 Immunoglobulins, code lOOSF, 13 v1 / c d ) o r s o e c i f i c antibodies ( 1 . 3 pl/cm2). ( a ) The control experiment: the reference pat-

226

F i g . 10.3. L e c t i n i n t e r a c t i o n immunoelectrophoresis o f a 1 - a n t i t r y p s i n i n 2 u l o f human serum. The e f f e c t o f i n c r e a s i n g c o n c e n t r a t i o n o f f r e e conA i n t h e f i r s t 10-5 m o l / l conA. ( b ) 0.9 * 10-5 m o l / l conA. The p o s i dimension g e l . ( a ) 0.15 t i o n o f a l - a n t i t r y p s i n w i t h o u t conA i s i n d i c a t e d . The a n t i b o d y c o n c e n t r a t i o n was 1.3 p l / c m (DAKO Immunoglobulins, s p e c i f i c a n t i b o d y a g a i n s t a 1 - a n t i t r y p s i n ) . Otherw i s e as f o r F i g . 10.2.

-

F i g . 1 0 . l h and i shows t y p i c a l r e a c t i o n s when p r o t e i n s a r e e l e c t r o p h o r e s e d t h r o u g h a g e l c o n t a i n i n g i m m o b i l i z e d and f r e e l e c t i n , r e s p e c t i v e l y . The p a t t e r n i s h i g h l y dependent on t h e l e c t i n c o n c e n t r a t i o n . P r o t e i n 1 does n o t b i n d t o t h e l e c t i n and i s used as an i n t e r n a l r e f e r e n c e of unchanged o o s i t i o n ( T a b l e 10.1). The r e a c t i o n s a r e disappearance o f p r e c i p i t a t e s , f o r m a t i o n o f an a f f i n i t y p r e c i p i t a t e and changed p o s i t i o n and changed p r o f i l e s : p r o t e i n 2 i s l o s t f r o m t h e

-

t e r n w i t h o u t l e c t i n . ( b ) The p a t t e r n w i t h 0.9 10-5 m o l / l f r e e conA i n t h e f i r s t dimension g e l ( c ) As ( b ) b u t w i t h s p e c i f i c a n t i b o d i e s a g a i n s t G C - g l o b u l i n t o show t h e unchanged p a t t e r n o f G C - g l o b u l i n . ( d ) As ( b ) b u t w i t h s p e c i f i c a n t i b o d i e s a g a i n s t orosomucoid ( a 1 - a c i d g l y c o p r o t e i n ) t o show t h e t r i p l e peak o f orosomucoid w i t h conA. ( e ) As ( b ) b u t w i t h s p e c i f i c a n t i b o d i e s a g a i n s t h a p t o g l o b i n t o show t h e 10-5 double peak o f h a p t o g l o b i n w i t h conA. ( f ) The p a t t e r n o f orosomucoid w i t h 0 . 9 m o l / l conA-Sepharose i n t h e f i r s t dimension [compare w i t h (d)] t o show t h e d o u b l e peak o f orosomucoid w i t h conA-Sepharose. The b a r r e p r e s e n t s 1 cm. A b b r e v i a t i o n s : A = albumin; AT = a l - a n t i t r y p s i n ; GC = GC-globulin; HDL = h i g h - d e n s i t y l i p o p r o t e i n ; Hp = h a p t o g l o b i n ; I g = immunoglobulins; PA = prealbumin; 0 = orosomucoid ( a l - a c i d g l y c o p r o t e i n ) ; T = t r a n s f e r r i n . C o n d i t i o n s o f e l e c t r o p h o r e s i s : 1%agarose i n T r i s v e r o n a l (pH 8.6); f i r s t - d i m e n s i o n e l e c t r o p h o r e s i s 10 V/cm f o r 1 h; second-dimension e l e c t r o p h o r e s i s , 2 V/cm f o r 18 h.

226

p a t t e r n . P r o t e i n 3 i s s h i f t e d c a t h o d i c a l l y t o appear as a double-peak p r e c i p i t a t e , each peak w i t h a c h a r a c t e r i s t i c r e t a r d a t i o n t h a t i s dependent on t h e l e c t i n conc e n t r a t i o n . G l y c o p r o t e i n s t h a t appear r e p r o d u c i b l y as m u l t i - p e a k p r e c i p i t a t e s i n t h i s manner a r e f o r i n s t a n c e human serum a - 1 - a n t i t r y p s i n ,

orosomucoid ( a - 1 - a c i d

g l y c o p r o t e i n , AGP), a - f o e t o p r o t e i n (AFP) , h a p t o g l o b i n and h i g h - d e n s i t y l i p o p r o t e i n (HDL) ( s e e F i g s . 10.2 and 10.3). G e n e r a l l y l e c t i n s w i t h a l o w m o b i l i t y a t pH 8.6 (such as conA f r o m Pharmacia and P h a r m i n d u s t r i e ) i n d u c e a c a t h o d i c s h i f t o f t h e

b i n d i n g g l y c o p r o t e i ns , INTERPRETATION OF THE PATTERNS

VisuaZization of proteins bound t o i m o b i t i z e d t e e t i n s I m m o b i l i z e d l e c t i n s a r e p r o t e i n s themselves and s t a i n w i t h Coomassie B r i l l i a n t B l u e and o t h e r p r o t e i n s t a i n s , so t h a t bound p r o t e i n w i l l n o t b e d e t e c t e d u n l e s s a s p e c i f i c method i s a v a i l a b l e ; c f . , F i g . 10.2f i n w h i c h t h e human serum g l y c o -

F i g . 10.4. V i s u a l i z a t i o n o f b i n d i n g between g l y c o p r o t e i n s and l e c t i n i n l e c t i n i n t e r a c t i o n immunoelectrophoresis. ( a ) Crossed immunoelectrophoresis w i t h conASepharose i n t h e i n t e r m e d i a t e g e l . Two m i c r o l i t r e s o f human serum were analysed. A f t e r e l e c t r o p h o r e s i s t h e p l a t e was s t a i n e d f o r e s t e r a s e a c t i v i t i e s (Brogren and BBg-Hansen86). The arrows i n d i c a t e enzymes bound t o conA ? a r t i c l e s . ArE = HDLa s s o c i a t e d a r y l e s t e r a s e ; ChE = c h o l i n e s t e r a s e . ( b ) Crossed immunoelectrophoresis w i t h f r e e , r a d i o a c t i v e l y l a b e l l e d conA i n t h e i n t e r m e d i a t e g e l ( a u t o r a d i o g r a p h y ) ( c f . , B j e r r u m e t a l . 2 4 ) . Two m i c r o l i t r e s o f human serum were analysed. The m a j o r b i n d i n g p r o t e i n s a r e ( f r o m t h e r i g h t ) : a 2 - m a c r o g l o b u l i n Y h a p t o g l o b i n , LDL, complement C3 and IgM. Otherwise as f o r F i g . 10.2.

227

p r o t e i n s a r e p a r t i a l l y bound t o conA-Sepharose p a r t i c l e s . However, by a u t o r a d i o graphy o f r a d i o a c t i v e l y l a b e l l e d g l y c o p r o t e i n s o r b y h i s t o c h e m i c a l s t a i n i n g o f g l y c o p r o t e i n enzymes, t h e b i n d i n g t o l e c t i n p a r t i c l e s can b e v i s u a l i z e d . Thus, F i g . 10.4 shows I - l a b e l l e d human serum p r o t e i n s i n p r e c i p i t a t e s w i t h conA ( F i g . 10.4a) and human serum c h o l i n e s t e r a s e and h i g h - d e n s i t y l i p o p r o t e i n - a s s o c i a t e d a r y l e s t e r a s e ( a r r o w s ) bound t o conA p a r t i c l e s ( F i g . 10.4b). Appearance of affinity precipitate Most remarkable a f t e r e l e c t r o p h o r e s i s w i t h f r e e l e c t i n i s t h e appearance o f l e c t i n - p r e c i p i t a t e d g l y c o p r o t e i n i n one o r more d i s c r e t e " a f f i n i t y p r e c i p i t a t e s " . T h i s i s seen w i t h human serum p r o t e i n s when t h e y a r e e l e c t r o p h o r e s e d t h r o u g h a f i r s t - d i m e n s i o n g e l o r t h r o u g h an i n t e r m e d i a t e g e l c o n t a i n i n g f r e e conA ( F i g s . 1 0 . l f and i, 10.2, and 10.3). A p r e r e q u i s i t e f o r t h e f o r m a t i o n o f an a f f i n i t y p r e c i p i t a t e i s t h a t b o t h l e c t i n and g l y c o p r o t e i n have a t l e a s t two b i n d i n g s i t e s p e r m o l e c u l e . T h e r e f o r e , we may c h a r a c t e r i z e most o f t h e g l y c o p r o t e i n s i n t h e a f f i n i t y p r e c i p i t a t e as h a v i n g a t l e a s t two b i n d i n g s i t e s . On t h e o t h e r hand, i t cannot be excluded t h a t some g l y c o p r o t e i n s w i t h o n l y one b i n d i n g s i t e w i l l b i n d t o an a l r e a d y e x i s t i n g a f f i n i t y D r e c i p i t a t e . However, i t was p o s s i b l e t o d i s t i n g u i s h between t h e r e a c t i o n s o f two human g l y c o o r o t e i n enzymes [urine a c i d Dhosphatase ( p r o s t a t e ) and serum c h o l inesterase]

i n t h e intermediate g e l technique 14 ) . Both enzymes bound

w i t h conA-Sepharose and f r e e conA (Bdg-Hansen and Brogren

t o conA-Sepharose b u t o n l y c h o l i n e s t e r a s e gave an a f f i n i t y p r e c i p i t a t e w i t h f r e e conA, i n d i c a t i n g two o r more b i n d i n g s i t e s p e r m o l e c u l e f o r c h o l i n e s t e r a s e ( g l y c o p r o t e i n Type 2 ) and o n l y one b i n d i n g s i t e f o r u r i n e a c i d phosphatase ( g l y c o p r o t e i n Type l ) . Reactions of "partiaZ identity" The p r e c i p i t a t i o n p a t t e r n o f p a r t i a l l y i d e n t i c a l p r o t e i n s i n q u a n t i t a t i v e immunoelectrophoresis has been d e s c r i b e d i n d e t a i l by Bock and Axelsen62. When m i x t u r e s o f g l y c o p r o t e i n s a r e a n a l y s e d w i t h f r e e l e c t i n ( c f . , F i g . l O . l c , f and i ) , t h e common c a r b o h y d r a t e m o i e t i e s w i l l m e d i a t e r e a c t i o n s o f p a r t i a l i d e n t i t y and t h e l e c t i n w i l l c r o s s - r e a c t w i t h a l l g l y c o p r o t e i n s h a v i n g b i n d i n g c a p a c i t y . DETERMINATION OF AFFINITY The degree o f r e t a r d a t i o n d u r i n g t h e f i r s t - d i m e n s i o n e l e c t r o p h o r e s i s i n a g e l w i t h l e c t i n i s an e x p r e s s i o n o f t h e a f f i n i t y between t h e g l y c o p r o t e i n and t h e l e c t i n . The c o n d i t i o n s i n t h e e l e c t r o p h o r e s i s g e l a r e analogous t o c o n d i t i o n s i n an a f f i n i t y chromatographic column. H i g h e r a f f i n i t y means s t r o n g e r b i n d i n g and h i g h e r degree o f r e t a r d a t i o n .

228

The retardation coefficient

A s i m p l e way t o express t h e r e l a t i v e a f f i n i t y o f p r o t e i n s t o t h e same l i g a n d i s t o calculate the retardation coefficients, R =

ZdZr -

1, where Zr and Zo a r e

t h e m i g r a t i o n d i s t a n c e s i n t h e f i r s t - d i m e n s i o n e l e c t r o o h o r e s i s w i t h and w i t h o u t l i g a n d , r e s p e c t i v e l y (Bdg-Hansen e t a1 .20). The r e t a r d a t i o n c o e f f i c i e n t can be c a l c u l a t e d f r o m a s i n g l e experiment w i t h l i g a n d and p r o v i d e d t h a t an excess o f l i g a n d i s p r e s e n t , t h e r e t a r d a t i o n c o e f f i c i e n t i s independent o f l i g a n d conc e n t r a t i o n , d i s r e g a r d i n g t h e i n f l u e n c e o f d i f f e r e n c e s i n m o l e c u l a r w e i g h t , charge d e n s i t y , e t c . By d e t e r m i n a t i o n o f t h e r e t a r d a t i o n c o e f f i c i e n t w i t h conA o f human serum g l y c o p r o t e i n s i t was p o s s i b l e t o l i s t t h e s e p r o t e i n s a c c o r d i n g t o t h e i r a f f i n i t y t o conA (Bdg-Hansen e t a1.20). The advantage o f t h i s method i s t h a t o n l y one experiment needs t o be performed w i t h l e c t i n i n c o r p o r a t e d i n t h e g e l . The concentration-dependent retardation The c o n c e n t r a t i o n - d e p e n d e n t r e t a r d a t i o n i s examined u s i n g an i n c r e a s i n g conc e n t r a t i o n o f l e c t i n i n t h e f i r s t - d i m e n s i o n g e l . T h i s i s shown f o r human serum a - 1 - a n t i t r y p s i n i n F i g . 10.3. With f r e e conA i n t h e f i r s t - d i m e n s i o n g e l t h e

F i g . 10.5. Crossed affinoimmunoelectrophoresis o f mouse AFP ( a - f o e t o o r o t e i n ) . The a n t i g e n was a 3-day j u v e n i l e homogenate. The f i r s t - d i m e n s i o n g e l c o n t a i n e d 300 pg/cm2 o f conA (1.8 10-5 m o l / l ) . An i n t e r m e d i a t e g e l ( b l a n k ) was i n s e r t e d between t h e f i r s t - d i m e n s i o n ge1,and t h e a n t i b o d y - c o n t a i n i n g g e l ( g o a t a n t i s e r u m a g a i n s t murine AFP). Note t h e d i f f e r e n c e i n number of oeaks t o F i g . 10.7 (conA f r o m Pharmacia)

-

229

a - 1 - a n t i t r y p s i n shows a c h a r a c t e r i s t i c m u l t i - p e a k p r e c i p i t a t e and a l l components e x h i b i t b i n d i n a t o conA. F o r some c o n A - b i n d i n g g l y c o p r o t e i n s such as orosomucoid ( a - 1 - a c i d g l y c o p r o t e i n , AGP) o n l y some f r a c t i o n s o f t h e p r o t e i n b i n d ( F i g . 10.2d and f ) , whereas one f r a c t i o n does n o t b i n d ( a r r o w , F i g . 10.2d and f ) (Bplg-Hansen e t a1.33, Bplg-Hansen and Takeo16, W e l l s and co-workers60y61), as v e r i f i e d by a f 51 ). F i g . 10.5. shows t h e p a t t e r n s o f m u r i n e a - f o e t o p r o t e i n (rn-AFP) w i t h conA i n

f i n i t y chromatography ( N i l s s o n and B$g-Hansen

t h e f i r s t - d i m e n s i o n g e l s . Four m i c r o h e t e r o g e n e i t y forms can be d i s t i n g u i s h e d by t h e i r r e a c t i o n w i t h conA i n f o e t a l and j u v e n i l e t i s s u e . These were d e s i g n a t e d 0, 1, 2 and 3 a c c o r d i n g t o i n c r e a s i n g conA a f f i n i t y , f o r m 0 b e i n g t h e conA non-

reactive. The e x i s t e n c e o f a m o l e c u l a r f o r m o f f o e t a l m-AFP which c o u l d be c o m p l e t e l y p r e c i p i t a t e d b y f r e e conA was e x c l u d e d by comparison o f r o c k e t s i n affinoimmunoe l e c t r o p h o r e s i s w i t h r o c k e t s i n i m m u n o e l e c t r o p h o r e s i s w i t h o u t conA. The r o c k e t h e i g h t s were i d e n t i c a l i n t h e two t y p e s o f e l e c t r o o h o r e s i s , p r o v i n g t h a t a comp l e t e l y p r e c i p i t a b l e f o r m o f m-AFP was n o t p r e s e n t (Hau and co-workers 46,47 ) . The general T a k e o - N a h r a p l o t The t h e o r e t i c a l background of a f f i n i t y e l e c t r o p h o r e s i s was worked o u t by Takeo and N a k a m ~ r ai ~n ~t h e i r o r i g i n a l experiments w i t h enzymes and s u b s t r a t e s . U s i n g t h e i r t h e o r y , i t i s p o s s i b l e t o c a l c u l a t e t h e a f f i n i t y between a r e t a r d e d p r o t e i n and a l e c t i n as t h e d i s s o c i a t i o n c o n s t a n t f r o m a Takeo-Nakarnura p l o t , which i s a s i m p l e r e l a t i o n s h i p between t h e r e l a t i v e a i g r a t i o n v e l o c i t y and t h e l e c t i n concentration: 1

*mi

= L (1+41) Rmo K

(10.1

where K i s t h e d i s s o c i a t i o n c o n s t a n t o f t h e l e c t i n - g l y c o p r o t e i n complex, c i s t h e c o n c e n t r a t i o n o f l e c t i n expressed as t h e c o n c e n t r a t i o n o f b i n d i n g s i t e s , t h e " n o r m a l i t y " , Rmo i s t h e m o b i l i t y o f t h e g l y c o p r o t e i n w i t h o u t l e c t i n , Rmi i s t h e m o b i l i t y o f t h e g l y c o p r o t e i n i n t h e presence o f l e c t i n and Rmc i s t h e m o b i l i t y o f t h e l e c t i n - g l y c o p r o t e i n complex i n r e l a t i o n t o an i n t e r n a l s t a n d a r d such as bromophenol blue-marked a l b u m i n o r p r e a l b u m i n . U s i n g t h i s method, t h e d i s s o c i a t i o n c o n s t a n t f o r conA complexes o f s e v e r a l g l y c o D r o t e i n s f r o m hunfan serum have been e s t i m a t e d , c f . , T a b l e 10.2. The Takeo-Nakamura p l o t i s c o n f i n e d i m p l i c i t l y t o t h e s i t u a t i o n where t h e complex between t h e i n t e r a c t i n g components i s e l e c t r o D h o r e t i c a l l y immobile, a c o n d i t i o n which i s n o t a p r i o r i met w i t h f r e e macromolecular l i g a n d s such as l e c t i n s , b u t i s w i t h i m m o b i l i z e d l e c t i n s . However, i n many i n s t a n c e s i t would

230 TABLE 10.2 DISSOCIATION CONSTANTS OF COMPLEXES BETWEEN SERUM PROTEINS AND conA

Human orosomucoid component 2 component 3 Human a n t i t r y p s i n component 1 component 2 Human a l - l i p o p r o t e i n associated arylesterase Human a2-HS-glycoprotei n Human h a p t o g l o b i n Human t r a n s f e r r i n Human a - f o e t o p r o t e i n (AFP) component 1 Murine a - f o e t o p r o t e i n (AFP) component 1 component 2 Murine c a r b o x y l e s t e r a s e component 1 component 2 component 3

D i s s o c i a t i o n constant, mean ( m o l / l ) a

nb

1.5 1.1

10-5 10-5

16 12

3.1 1.3

10-5 10-5

7 15

1.1 4.0 3.2 3.9

10-5 10-5 10-5 10-5

2 11 4

5.0

10-6

C

4.5 3.3

10-6 10-6

d d

1.5 1.3 0.9

10-5 10-5 10-5

4

7

4 4

Ref, Bdg-Hansen and Take016

T h i s work

Bdg-Hansen e t al.38

: D i s s o c i a t i o n c o n s t a n t s c a l c u l a t e d a f t e r crossed i m u n o e l e c t r o p h o r e s i s w i t h conA. Number o f s e r a analysed. :Pool o f more t h a n 10 sera. Pool o f more t h a n 20 sera. be d e s i r a b l e t o work w i t h f r e e l e c t i n s because i t would save t h e e f f o r t s i n v o l v e d i n p r e p a r i n g t h e i m m o b i l i z e d l e c t i n s and e l i m i n a t e t h e d i f f i c u l t y o f e s t i m a t i n g t h e e x a c t amount o f bound l e c t i n . Complexes between g l y c o u r o t e i n s and f r e e l e c t i n s would be expected t o have an e i g e n m o b i l i t y . N o n - l i n e a r Takeo-Nakamura p l o t s f o r 63 k i d n e y phosphorylase i n complex w i t h g l y c o p r o t e i n (Takeo and Nakamura ) and human serum g l y c o p r o t e i n s i n complex w i t h conA have been d e s c r i b e d (664-Hansen 16 ). Therefore, t h e general equation f o r e l e c t r o p h o r e t i c determination

and Takeo

o f d i s s o c i a t i o n c o n s t a n t s was d e r i v e d : (10.2)

where Rmc i s t h e m o b i l i t y o f t h e c o n A - g l y c o p r o t e i n complex, i n r e l a t i o n t o t h e i n t e r n a l s t a n d a r ( o t h e r symbols as i n eqn. 10.1). T h i s e q u a t i o n r e p r e s e n t s a s t r a i g h t l i n e when ( l e c t i n c o n c e n t r a t i o n ) - ' i s p l o t t e d a g a i n s t (Rmo Rh) -1

-

.

231

- 3 - 2 - 1

0

1

2

3

4

5

F i g . 10.6. P l o t f o r d e t e r m i n a t i o n o f d i s s o c i a t i o n c o n s t a n t s f o r conA complexes w i t h human and m u r i n e AFP ( a - f o e t o p r o t e i n ) . 04, M u r i n e AFP, m o l e c u l a r f o r m 1; 0-0, m u r i n e AFP, m o l e c u l a r f o r m 2;0-n8, human AFP, m o l e c u l a r f o r m \1. The i n t e r c e p t w i t h t h e a b s c i s s a i s -K-1 b e e t e x t (ean. 10.1) f o r explanation].

t h e c-l a x i s i s -K-l.

- ~~1 -1

a x i s i s (Rmo - Rmc) The s l o p e o f t h e l i n e i s K/(RmO

The i n t e r c e p t on t h e (Rmo

-1 and t h e i n t e r c e p t on

- R,~).

The e q u a t i o n g i v e s t h e d i s s o c i a t i o n c o n s t a n t K as w e l l as t h e m o b i l i t y o f t h e c o n A - g l y c o p r o t e i n complex, Rmc ( c f . ,

F i g . 10.6). The values o f K and Rmc were

c a l c u l a t e d by t h e l e a s t - s q u a r e s method. The e q u a t i o n and i t s d e r i v a t i o n were subsequently v e r i f i e d and p u b l i s h e d b y H o r e j s i u s i n g o t h e r symbols ( H o r e j s i

64,65

I n t h e s i m p l e case where t h e m o b i l i t y o f t h e c o n A - g l y c o p r o t e i n complex i s z e r o (Rmc = 0 ) , eqn. 10.2 becomes eqn. 10.1). The c a l c u l a t i o n o f t h e d i s s o c i a t i o n c o n s t a n t deoends on t h e accuracy o f t h e d e t e r m i n a t i o n o f t h e l e c t i n c o n c e n t r a t i o n . T h i s a p p l i e s e s p e c i a l l y t o cases w i t h i m m o b i l i z e d l e c t i n , where t h e d e t e r m i n a t i o n o f bound l e c t i n p e r u n i t m a t r i x i s d i f f i c u l t . A c o m p l i c a t i n g f a c t o r i s t h e change i n b i n d i n g p r o p e r t i e s a f t e r i m m o b i l i z a t i o n . I n o u r p r e s e n t e x p e r i m e n t a l work t h e g r e a t e s t d i f f i c u l t y f o r s t a n d a r d i z a t i o n o f t h e s e methods f o r d i a g n o s t i c purposes seems t o b e t h e - i r r e p r o d u c i b i l i t y o f t h e l e c t i n preparation. T a b l e 10.2 l i s t s t h e d i s s o c i a t i o n c o n s t a n t s o f complexes o f conA and g l y c o o r o t e i n s i n human serum. Sets of d a t a were o b t a i n e d f r o m t h e same s e t s o f experiments. I n f a c t , f r o m one s e t o f experiments a s e t o f d a t a c o u l d be o b t a i n e d f o r each g l y c o p r o t e i n r e a c t i n g w i t h conA. When i n d i v i d u a l g l y c o p r o t e i n s f r o m i n d i v i d u a l human s e r a were compared, we found a c o n s i d e r a b l e and u n s y s t e m a t i c v a r i a t i o n i n t h e measured d i s s o c i a t i o n c o n s t a n t s f o r a l l b u t a few g l y c o p r o t e i n s . Among these, orosomucoid was surp r i s i n g l y c o n s t a n t , b o t h w i t h r e s p e c t t o i t s appearance as t h r e e components w i t h

).

232

F i g . 10.7. L e c t i n i n t e r a c t i o n immunoelectrophoresis o f human AFP ( l e f t ) and rnurine AFP ( r i g h t ) i n crossed affinoimmunoelectronhoresis w i t h d i f f e r e n t amounts o f conA added t o t h e f i r s t - d i m e n s i o n g e l . The amounts o f conA added were ( f r o m t h e t o p ) 0, 3.03 and 24.20 10-6 m o l / l . Note t h e number o f components d i f f e r e n t f r o m F i g . 10.5 (conA f r o m I B F ) .

-

233

a c o n s t a n t r a t i o ( W e l l s e t a1.60) and w i t h r e s p e c t t o d i s s o c i a t i o n c o n s t a n t s . When serum f r o m i n d i v i d u a l s l o w i n a - 1 - a n t i t r y p s i n was t e s t e d i n t h i s way, we observed t h e same p a t t e r n o f a m a j o r component and a m i n o r component w i t h t h e normal d i s s o c i a t i o n c o n s t a n t t o conA even though t h e a n t i t r y p s i n c o n c e n t r a t i o n 16 was as low as 1%o f t h e normal c o n c e n t r a t i o n (Bdg-Hansen and Takeo ) . U s i n g eqn. 10.2 we compared t h e d i s s o c i a t i o n c o n s t a n t s o f t h e two m o l e c u l a r forms of human AFP ( 0 and 1) and t h e t h r e e m o l e c u l a r forms o f m u r i n e AFP (0, 1 and 2). The r e a c t i o n o f t h e two m o l e c u l a r forms o f human AFP and t h e t h r e e mol e c u l a r forms o f m u r i n e AFP w i t h v a r i o u s amounts o f conA i s i l l u s t r a t e d i n F i g .

10.7. M i g r a t i o n d i s t a n c e s were determined as f i r s t - d i m e n s i o n m i g r a t i o n d i s t a n c e s f r o m t h e o r i g i n t o t h e p r o j e c t i o n o f t h e peak o f t h e p r e c i D i t a t e on t h e b a s e l i n e .

A l l o f t h e a n t i g e n samples were t e s t e d w i t h t h e same conA p r e p a r a t i o n u s i n g m o n o s p e c i f i c a n t i s e r u m t o t h e r e s p e c t i v e a - f o e t o p r o t e i n and p o l y v a l e n t a n t i s e r u n : which a l s o r e a c t e d w i t h albumin. The d i s s o c i a t i o n c o n s t a n t s were d e r i v e d f r o m t h e p l o t shown i n F i g . 10.6. The d i s s o c i a t i o n c o n s t a n t o f t h e conA-human AFP f o r m 1 complex was 3.9 The d i s s o c i a t i o n c o n s t a n t o f t h e conA-murine AFP f o r m 1 c o m l e x was 1.1

-

mol/l.

m o l / l and t h e d i s s o c i a t i o n c o n s t a n t o f conA-murine AFP f o r m 2 complex was 5.1

.

m o l / l . The c o r r e l a t i o n c o e f f i c i e n t s o f t h e s t r a i g h t l i n e s i n t h e p l o t were i n t h e range 0.97-1.00.

The electrophoretic conditions The e l e c t r o p h o r e t i c parameters were v a r i e d w i t h t h e c o n c l u s i o n t h a t t h e b e s t c o n d i t i o n s a r e t h o s e which a r e o p t i m a l f o r electroimmunoprecipitation.

I n a way

t h i s i s a l i m i t a t i o n t o t h e g e n e r a l use o f t h e method, as t h e v a r i a t i o n o f t h e d i s s o c i a t i o n c o n s t a n t as a f u n c t i o n o f pH and t e m p e r a t u r e can be s t u d i e d o n l y w i t h i n narrow l i m i t s . R o u t i n e l y , immunoelectroprecipitation i s performed i n t h e pH range 8.5-8.9 and i n t h e t e m p e r a t u r e range 16-18OC; e x c e D t i o n a l l y , t h e pH can be chosen as l o w as 5.5 and as h i g h as 9.2, and t h e t e m o e r a t u r e may be f r o m 10 t o 3OoC. Only s m a l l changes i n t h e d i s s o c i a t i o n c o n s t a n t s were observed i n t h e narrow i n t e r v a l s . I t was o u t s i d e t h e scope o f t h i s work t o t e s t extreme c o n d i t i o n s o r l e c t i n s w i t h o t h e r m o b i l i t y p r o p e r t i e s . I m m o b i l i z e d l e c t i n (Sepharosebound conA) was found t o i n f l u e n c e t h e e l e c t r o p h o r e t i c p r o p e r t i e s o f t h e f i r s t dimension g e l . I n a d d i t i o n , c o n t r o l o f t h e b i n d i n g s i t e c o n c e n t r a t i o n was d i f f i c u l t w i t h i m m o b i l i z e d l e c t i n . T h e r e f o r e , f r e e l e c t i n was used i n t h e s e e x p e r i ments as more c o n v e n i e n t and s a t i s f a c t o r y . P r o v i d e d t h a t t h e complex has a l o w e r m o b i l i t y t h a n t h e f r e e o r o t e i n , a h i g h e r a f f i n i t y means a h i g h e r degree o f r e t a r d a t i o n . Q u a l i t a t i v e l y , no d i f f e r e n c e s were found between a n a l y t i c a l a f f i n i t y e l e c t r o p h o r e s i s and a f f i n i t y chromatography (BCg-Hansen2 and K e r c k a e r t a t a l . 4 8 ) . We d i d n o t compare o u r p r e s e n t r e -

234

.2-

I. 1

2

3

PI se

F i g . 10.8. D e v i a t i o n o f t h e r e l a t i v e m o b i l i t y o f a p u r e p r o t e i n w i t h i n c r e a s i n g amounts o f c o n t a m i n a t i n g g l y c o p r o t e i n s : Ro-Rm o f p u r e m u r i n e serum c a r b o x y l i c e s t e r a s e component 1 (CxE-1, l e s s t h a n 1 ng p e r t e s t ) as a f u n c t i o n o f added serum (0-150 u g p e r t e s t ) a t a f i x e d conA c o n c e n t r a t i o n ( 6 10-4 m o l / l ) .

-

sul t s experimentally w i t h determinations o f d i s s o c i a t i o n constants by e i t h e r i n h i b i t i o n p r e c i p i t a t i o n , f r o n t a l a n a l y s i s , e l u t i o n a n a l y s i s o r any o t h e r method. The i n f l u e n c e o f o t h e r g l y c o p r o t e i n s was t e s t e d by comparison o f t h e o a t t e r n o f orosomucoid observed w i t h serum w i t h t h a t observed w i t h D u r i f i e d orosomucoid. The same three-peak p r e c i p i t a t e was observed i n b o t h i n s t a n c e s , which i n d i c a t e d t h a t t h e i n f l u e n c e o f competing g l y c o p r o t e i n s on t h e l e c t i n b i n d i n g i s n e g l i g i b under o u r s t a n d a r d c o n d i t i o n s w i t h a g l y c o p r o t e i n amount o f a b o u t 50 ng p e r ana 37 ) . However, we wondered whether o u r c o n d i t i o n s were i d e a l and

y s i s (Bdg-Hansen

c o u l d be compared w i t h c o n d i t i o n s w i t h e x t r e m e l y s m a l l amounts o f g l y c o p r o t e i n s T h e r e f o r e , we t e s t e d o u r systems w i t h a m i n o r c o n s t i t u e n t o f mouse serum, t h e a - c a r b o x y l e s t e r a s e , which s t a i n s w i t h a h i s t o c h e m i c a l s t a i n o n l y and i s presen i n an amount o f l e s s t h a n 1 ng i n t h e t e s t . W i t h t h i s o u r i f i e d enzyme ( F i g . 10.8) we c o u l d show o n l y small d e v i a t i o n s ( < l o % ) o f r e l a t i v e m o b i l i t i e s and d i s s o c i a t i o n c o n s t a n t s w i t h g l y c o p r o t e i n amounts f r o m l e s s t h a n 1 u g t o more t h a n 150 ug p e r t e s t . T h e r e f o r e , we concluded t h a t t h e i n f l u e n c e f r o m o t h e r g l y c o p r o t e i n s i s n e g l i g i b l e p r o v i d e d t h a t an excess o f l i g a n d (conA) i s p r e s e n t (Bdg-Hansen e t a l .

38,66

).

I t s h o u l d be n o t e d t h a t t h e c o n c e n t r a t i o n o f l i o a n d i s c a l c u l a t e d as t h e monomer

c o n c e n t r a t i o n and i t was n o t p o s s i b l e t o e s t i m a t e t h e e f f e c t o f t h e t e t r a m e r i c n a t u r e o f conA.

Evaluation of the dissociation Constants The method d e s c r i b e d h e r e f o r t h e d e t e r m i n a t i o n o f d i s s o c i a t i o n c o n s t a n t s i s a two-dimensional method t h a t a l l o w s t h e a n a l y s i s o f more complex systems because o f t h e s p e c i f i c i d e n t i f i c a t i o n o f components b y t h e i r immunological c h a r a c t e r i s t i c s . However, compared w i t h f r o n t a l o r e l u t i o n a n a l y s i s a f f i n i t y chromatography, i t i s a drawback t h a t t h e e l e c t r o p h o r e t i c method r e q u i r e s a number o f analyses t o be p e r -

235

formed a t w e l l determined l i g a n d c o n c e n t r a t i o n s . On t h e o t h e r hand, the e l e c trophoreses a r e s i m p l e t o perform and r e q u i r e o n l y a minimum o f m a t e r i a l f o r t h e d e t e r m i n a t i o n and t h e l i g a n d need n o t be immobilized. The values measured f o r d i s s o c i a t i o n constants o f g l y c o p r o t e i n s w i t h conA by o u r method a r e o f t h e o r d e r o f

m o l / l (Table 10.2). T h i s agrees w e l l w i t h

o t h e r measurements o f human serum g l y c o p r o t e i n s . The d i s s o c i a t i o n constants found i n a p r e c i p i t a t i o n i n h i b i t i o n t e s t were 0 . 4 0.7 Leon

67

-

mol/l f o r p u r i f i e d transferrin,

m o l / l f o r t r a n s f e r r i n glycopeDtide and

m o l / l f o r IgM (Young and

). The e a r l i e r s t u d i e s w i t h one-dimensional e l e c t r o p h o r e s i s o f enzymes and

immobile s u b s t r a t e s i n t h e medium y i e l d e d d i s s o c i a t i o n constants i n t h e f o l l o w i n g ranges: 6.1

*

-

27

and N a k a m ~ r a ~and ~ ) 2.5 p l e x (Takeo e t a1.68).

m o l / l f o r phosphorylase-glycogen complexes (Takeo

lo-‘ -

2.6

g/ml f o r amylase-soluble s t a r c h com-

S i m i l a r l y , B l u e Dextran has been used i n t h e medium and mol/l f o r im16

d i s s o c i a t i o n constants were obtained i n t h e range 0.4

-

-

-

m o b i l i z e d dye-complexes w i t h bovine h e a r t l a c t a t e dehydrogenase isoenzymes, 1.3

3

-

m o l / l f o r soybean l a c t a t e dehydrogenase isoenzymes, 2.2-7.9

f o r rape seed a l c o h o l dehydrogenase, 2.2 f o r pyruvate kinase and 8.6

m o l / l and 1.8

isoenzymes (Ticha e t a l . 6 9 ) . Analogously,

mol/l

m o l / l f o r a l d o l a s e , 2.6

mol/l

m o l / l f o r c r e a t i n e kinase

immobilized saccharides have been i n -

corporated i n t h e medium i n one-dimensional e l e c t r o p h o r e s i s t o g i v e d i s s o c i a t i o n constants f o r l e c t i n s i n t h e range 0.2-20 71 r e j s i e t al. ).

. loF4

m o l / l (Hauser e t a1.70 and Ho-

The development o f an easy method f o r d e t e r m i n a t i o n o f t h e a f f i n i t y o f a n t i bodies has h i g h p r i o r i t y . Thus Caron e t a l . 7 2 s t u d i e d D o s s i b i l i t i e s f o r assessing a n t i - a l b u m i n a f f i n i t y i n agarose a f f i n i t y e l e c t r o p h o r e s i s by d e t e r m i n a t i o n of t h e “ p a r t i t i o n i n g c o e f f i c i e n t “ , Kpy between immunosorbent and s o l u b l e a n t i g e n o r antibody. However, a more d i r e c t apDroach f o r a n t i b o d i e s was used by Takeo and Kabat73, i n analogy w i t h t h e o r i g i n a l work on enzymes: A s s o c i a t i o n constants were measured f o r mouse myeloma p r o t e i n s w i t h a n t i d e x t r a n o r a n t i - i s o m a l t o s e o l i g o saccharide a c t i v i t y by e l e c t r o D h o r e s i s i n polyacrylamide g e l s c o n t a i n i n g d e x t r a n 4 4 2 (K, = 3 10 - 6 10 ml/g) o r isomaltose oligosaccharides (K, = 8.3 10 m o l / l ) .

-

.

I t was concluded t h a t a f f i n i t y e l e c t r o o h o r e s i s i s u s e f u l f o r o b t a i n i n g b i n d i n g 2 6 constants r a n g i n g from 10 t o 10 l / m o l .

JUANTITATION OF MICROHETEROGENEITY FORMS The h e t e r o g e n e i t y o f g l y c o p r o t e i n s i n t h e i r r e a c t i o n w i t h conA, as observed w i t h b o t h these methods and o t h e r methods, may now be a s c r i b e d t o t h e e x i s t e n c e o f oopulations o f m i c r o h e t e r o g e n e i t y forms, each p o p u l a t i o n w i t h i t s c h a r a c t e r i s t i c carbohydrate s t r u c t u r e , p o s s i b l y r e f l e c t i n g d i f f e r e n t m e t a b o l i c stages r e l a t e d t o

236

237

238

Fig. 10.9. Q u a n t i f i c a t i o n of microheterogeneous forms o f individual glycoproteins. Crossed immunoelectrophoresis of concentrated human urine ( A ) , with insolubilized conA ( B ) and with f r e e conA i n t h e intermediate gel ( C ) . The r e a l t i v e amounts of glycoprotein with one, with twb o r more and without conA-binding s i t e s were determined by planimetry o f the immunoprecipitates ( s e e t e x t and Table 1 0 . 3 ) .

239 t h e f u n c t i o n o f t h e p r o t e i n . Therefore, i t seems i m p o r t a n t t o develop methods t o assess t h e q u a n t i t a t i v e changes t h a t take p l a c e i n v i v o f o r these microheterogenei t y forms.

&antitation b y t h e intermediate g el technique, Method B P r e c i p i t a t i o n w i t h f r e e l e c t i n versus b i n d i n g t o immobilized l e c t i n forms a b a s i s f o r a c l a s s i f i c a t i o n o f the m i c r o h e t e r o g e n e i t y forms o f a g l y c o p r o t e i n i n t o t h r e e classes: Type 0 (molecules w i t h o u t b i n d i n g s i t e s ) , Type 1 (molecules w i t h one b i n d i n g s i t e ) and Type 2 (molecules w i t h two o r more b i n d i n g s i t e s ) . F i g . 10.9 shows t h e experimental b a s i s f o r q u a n t i f i c a t i o n o f each o f t h e t h r e e types o f microheterogenei t y form mentioned above o f a number o f microheterogeneous g l y c o p r o t e i n s i n human u r i n e . Three d i f f e r e n t experiments were performed w i t h intermediate gels: A without l e c t i n i n the intermediate gel,

B with free lectin

i n t h e . i n t e r m e d i a t e g e l and C w i t h immobilized l e c t i n i n t h e i n t e r m e d i a t e g e l . By c a r e f u l p l a n i m e t r y o f t h e enclosed area o f each p r e c i p i t a t e , a r e l a t i v e e s t i m a t e

was obtained f o r t h e t o t a l amount o f each g l y c o p r o t e i n (A), t h e amount o f glycop r o t e i n o f Type 2 (B) and t h e amount o f g l y c o ? r o t e i n o f Type l p l u s Type 2 ( C ) . 'Table 10.3 shows t h e r e l a t i v e d i s t r i b u t i o n between t h e v a r i o u s m i c r o h e t e r o g e n e i t y classes o f 18 u r i n a r y g l y c o p r o t e i n s . TABLE 10.3 HETEROGENEITY OF URINARY GLYCOPROTEINS R e l a t i v e amount o f p r o t e i n i n each m i c r o h e t e r o g e n e i t y c l a s s f o r 13 human u r i n e p r o t e i n s . Note t h a t o n l y one p r o t e i n appears t o be homogeneous ( a d i c phosphatase); albumin appears t o be g l y c o s y l a t e d ( 1 0 % ) . Most u r i n a r y p r o t e i n s a r e p r e s e n t i n a t l e a s t t h r e e d i f f e r e n t g l y c o s y l a t i o n forms. P r o t e i n component

R e l a t i v e amount o f each h e t e r o g e n e i t y c l a s s (%) Without binding s i t e

u1 U2 = albumin u3 u4 u5 U6 u7 U8 u9 u 10 u11 u12 U13 = a c i d phosphatase

75 90 70 15 35

ao

35 75 50 30 50 55 0

With one binding s i t e

5 5 0 45 20 10 40 0 15 15 25 10

100

With two o r more binding s i t e s

20 5

30 40 45 10 25 25 35 55 25 35 0

240

Quantitation by the f i r s t dimension gel technique, Method C By t h e f i r s t - d i m e n s i o n t e c h n i q u e we d i s t i n g u i s h between m i c r o h e t e r o g e n e i t y forms d i f f e r i n g i n t h e i r a f f i n i t y t o t h e l e c t i n . the r a t i o o f f o u r d i s t i n c t m i c r o h e t e r o g e n e i t y components o f m-AFP i s s h i f t e d i n pregnancy and t h e percentage by which each o f t h e f o u r m o l e c u l a r forms o f m-AFP c o n t r i b u t e t o t h e t o t a l m-AFP l e v e l i n f o e t a l and j u v e n i l e homogenate i s shown i n F i g . 10.10 (Hau and co-workers 46,47 ) . I n 1'2-day-old f o e t u s e s t h e two forms w i t h weakest a f f i n i t y t o conA ( f o r m s 0 and 1) dominate, whereas i n homogenate o f j u v e n i ' l e s t h e two forms w i t h s t r o n g e s t a f f i n i t y t o conA ( f o r m s 2 and 3 ) dominate. A t t h e t i m e o f b i r t h a l l f o u r forms o f m-AFP a r e p r e s e n t and t h e y c o n t r i b u t e a p p r o x i m a t e l y 25% each t o t h e t o t a l AFP l e v e l . T h i s i s p a r a l l e l t o t h e t h r e e forms o f orosomucoid which a r e p r e s e n t n o r m a l l y i n a w e l l defined r a t i o , b u t s h i f t e d d u r i n g pregnancy and d u r i n g t r e a t m e n t w i t h hormone-like

50

12 13 14 15 16 17 18 19 0 1 2 3 4 5

----.

7

10

0%

F i g . 10.10. Q u a n t i f i c a t i o n o f microheterogeneous forms o f i n d i v i d u a l g l ! x o p r o t e i n s . ? l o t o f t h e r e l a t i v e amounts o f f o u r m o l e c u l a r forms o f murine AFP f r o m 12-day mof o e t u s e s t o 10-day j u v e n i l e s . 0---o M o l e c u l a r f o r m 0 ( n o n - b i n d i n g ) ; l ] --l e c u l a r form 1 ( s l i g h t binding); o - - - ~ molecular form 2 (intermediate binding); m o l e c u l a r f o r m 3 ( s t r o n g e s t b i n d i n g ) . Each p o i n t r e p r e s e n t s t h e mean v a l u e f o r 10 samples. I n s i d e t h e p o i n t s i s +1 s.e.m. ( c f . , F i g . 10.5). drugs. However, t h e i r b i n d i n g p r o p e r t i e s t o conA a r e unchanged ( W e l l s and co60,61

workers

).

As mentioned above, t h e number o f m i c r o h e t e r o g e n e i t y forms seems t o depend on t h e l e c t i n p r e p a r a t i o n . Here f o u r forms o f m-AFP were seen w i t h conA f r o m Pharmacia i n c o n t r a s t t o o n l y t h r e e forms w i t h conA f r o m IBF (compare F i g s . 10.7 and 10.10). The reason f o r t h i s i n c o n s i s t e n c y i s n o t c l e a r , b u t may l i e i n d i f f e r e n c e s i n t h e b i n d i n g s o e c i f i c i t i e s o f v a r i o u s commercial l e c t i n s orepared f r o m d i f f e r e n t

241 l i n e s o r c u l t i v a r s o f t h e same p l a n t , as such d i f f e r e n c e s a r e known t o o c c u r 13 (B&-Hansen ) . T h i s i n d i c a t e s t h a t c a r e s h o u l d be t a k e n t o s o e c i f y t h e s o u r c e o f l e c t i n when r e p o r t i n g q u a n t i t a t i v e s t u d i e s . OTHER ELECTROPHORETIC METHODS I n t h e i n t r o d u c t i o n we mentioned t h e h i s t o c h e m i s t r y - l i k e use o f l a b e l l e d l e c t i n s f o r t h e i d e n t i f i c a t i o n o f g l y c o p r o t e i n bands a f t e r e l e c t r o p h o r e s i s . T h i s appears t o be a v e r y s e n s i t i v e means f o r t h e i d e n t i f i c a t i o n o f g l y c o p r o t e i n bands a f t e r a c r y l a m i d e g e l e l e c t r o p h o r e s i s and i s a good supplement t o t h e mentioned t e c h n o l o g y o r f o r t h e p r e l i m i n a r y s c r e e n i n g f o r g l y c o p r o t e i n s . The methodology and r e f e r e n c e s were g i v e n by B i t t i g e r and S c h n e b l i

6

.

Se Zection e Zectrophoresis F o r c e r t a i n purposes i t i s n o t necessary o r d e s i r a b l e t o o e r f o r m t h e e l e c t r o p h o r e s i s as c r o s s e d i m m u n o e l e c t r o p h o r e s i s . S e l e c t i o n e l e c t r o p h o r e s i s i s a s i m p l e r method: a c o m b i n a t i o n o f one-dimensional e l e c t r o p h o r e s i s i n agarose w i t h subsequent d i f f u s i o n o f l e c t i n f r o m a t r o u g h , analogous t o t h e c l a s s i c a l immuno74 e l e c t r o p h o r e t i c a n a l y s i s a c c o r d i n g t o Grabar, c f . , F i g . 10.11 (Harboe e t a l . , Spengler and Weber9’75). The method has n o t been used e x t e n s i v e l y as t h e p a r e n t method w i t h a n t i b o d i e s . However, t h e g r e a t o o t e n t i a l o f g l y c o p r o t e i n a n a l y s i s w i t h l e c t i n s was shown b y Osunkoya and W i l l i a m s 8 i n t h e i r work w i t h human serum g l y c o p r o t e i n s f r o m p a t i e n t s w i t h v a r i o u s d i s e a s e s . F i g . 10.11 shows t h e p r e c i p i t a t i o n p a t t e r n s o f p u r i f i e d human 19s IgM and 7s kappa c h a i n s w i t h a n t i b o d i e s and four different lectins.

One-dimensionaZ a f f i n i t y eZectrophoresis Another example t h a t a r e f e r e n c e p a t t e r n o f c r o s s e d i m m u n o e l e c t r o p h o r e s i s i s n o t necessary i s p r o v i d e d by one-dimensional

a f f i n i t y e l e c t r o p h o r e s i s . Thus, f o r

the i d e n t i f i c a t i o n o f binding t o l e c t i n o r f o r the i d e n t i f i c a t i o n o f precipitat i o n w i t h l e c t i n , e l e c t r o p h o r e s i s may be p e r f o r m e d i n t o a g e l w i t h e i t h e r i m m o b i l i z e d l e c t i n o r f r e e l e c t i n , r e s p e c t i v e l y . T h i s was shown e a r l i e r f o r enzymes f r o m b a r l e y (Bdg-Hansen e t a 1 . l 7 ) . The method was a l s o used f o r t h e q u a n t i t a t i o n o f denatured g l y c o p r o t e i n s . We f o u n d t h a t d e n a t u r a t i o n d i d n o t i n t e r f e r e w i t h t h e s t r u c t u r e o f t h e b i n d i n g s i t e , and g l y c o p r o t e i n s denatured t o v a r i o u s e x t e n t s gave a f f i n i t y p r e c i p i t a t e s o f t h e same h e i g h t (Bfig-Hansen a t a1.l’).

F i g . 10.12a

shows g l y c o p r o t e i n enzymes f r o m a p l a n t e x t r a c t r e a c t i n g w i t h conA i n t h e g e l . F i g . 10.12b shows t h e r e v e r s e d t y p e one-dimensional a f f i n i t y e l e c t r o p h o r e s i s . The g e l c o n t a i n e d g l y c o p r o t e i n s (human serum) and e x t r a c t s o f v a r i o u s p l a n t sources were e l e c t r o p h o r e s e d i p t r t h e g l y c o p r o t e i n - c o n t a i n i n g g e l w i t h f o r m a t i o n

242

Fig. 10.11. Selection electrophoresis with lectins, an analogous method to the

243

Fic. 10.12. One-dimensional a f f i n i t y electrophoresis f o r q u a n t i f i c a t i o n and screening. ( a ) Glycoproteins electroDhoresea i n t o a conA-containing g e l : a plant enzyme e x t r a c t ( s t a i n e d f o r acid phosohatasej a n d p u r i f i e d human serum choline s t e r a s e ( s t a i n e d f o r e s t e r a s e ) . ( b ) Screening f o r l e c t i n a c t i v i t y : plant e x t r a c t s electrophoresed i n t o a gel containing human serum glycooroteins (from l e f t : ext r a c t of barley malted seeds, two d i f f e r e n t e x t r a c t s from UZex europaeus, t h r e e d i f f e r e n t e x t r a c t s of ooke weed, an e x t r a c t from soybeans and p u r i f i e d r i c e seed lectin). of a f f i n i t y p r e c i p i t a t e s as t h e r e s u l t . This tyoe of exoeriment seems t o be a f a s t screening method f o r g l y c o p r o t e i n - o r e c i p i t a t i n g l e c t i n s as well as f o r t h e i r 15 quanti t a t i o n (Bdg-Hansen and liord ) . Compound ge Z method: po Zyacry lamide and agarose

Fig. 10.12 shows how SDS-polyacrylamide gel electrophoresis of denatured glycoproteins can be combined with l e c t i n a f f i n i t y e l e c t r o p h o r e s i s . The protein sample was conA-binding glycoproteins from normal human serum purified by a f f i n i t y chromatography on conA-Sepharose. The samples were t r e a t e d with SDS before applicat i o n t o t h e SDS-polyacrylamide gel s l a b s . After separation i n t h e SDS g e l , lanes were c u t and soaked f o r 15 min in the agarose gel buffer o r stored frozen. The

c l a s s i c a l immunoelectrophoretic analysis according t o Grabar. The p r e c i p i t a t i o n pattern of purified human 19s IgM and 7 s kappa chains with antibodies (anti-IgM) and l e c t i n s (PHA, lens c u l i n a r i s l e c t i n , conA, a n d r i c i n ) i s shown. Reproduced with permission from G . A . Spengler, Bern.

h3

P P

TABLE 10.4 VARIOUS MODIFICATIONS OF AFFINITY ELECTROPHORESIS WTH INTERACTING CONPONENTS AND SOME APPLICATIONS

M o d i f i e d from Bdg-Hansen e t a1 .20. Conventional immunoelectrophoretic methods n o t included. No.

Modification

I n t e r a c t i n g cornponent which i s e l ectroDhoresed

Interacting component i n t h e medium

Purpose

Ref.

1.

Selection electrophoresis analogous = a f f i n i t y electrophoresis analogous t o immunoelectrophoret i c a n a l y s i s according t o Grabar

Glycoproteins

Lectinsa

Identification o f 1e c t in- b i n d i ng glycoproteins

Spengler and Weber75 Harboe e t a1 .74

2.

Crossing diagramsb

Enzyme

Substrate i n h i b i tor

Identification of i n t e r a c t i n g components

Nakamural

3.

One-dimensional a f f i n i t y electrophoresisb

4.

One-di mens ional a f f in it y electrophoresis

Glycoproteins

Lectin

Lectin

Carbohydrate

Identification of lectin

H o r e j s i and Kocourek3

Enzyme

Substrates

Takeo and N a k a ~ n u r a ~ ~

A n t i bodies

A n t i gens

Determination o f d i s s o c i a t i o n constants

Glycoprotein enzymes

Immobilized lectin

Identification o f g l y c o p r o t e i n enzymes

Takeo and Kabat73 Bdg-Hansen e t a1 .I7

5.

Rocket a f f i n i t y electrophoresis

Lectins

Glycoproteins

Polysaccharides

Lectins

G l y c o p r o t e i ns

Lectins

6.

Fused r o c k e t a f f in i ty electrophoresis

Glycoproteins

Lectins

7.

Crossed a f f i n i t y electrophoresis

Lectin

Glycoproteins

Polysaccharides

Lectins

Glycoproteins

Lectins

G l y c o p r o t e i ns

Lectins

Lectins

Glycoproteins

Immobilized a f f i n 1t i n s

Biologically active proteins

8. Crossed immunoelectroPhOreSiS w i t h j i g a n d i n an i n t e r m e d i a t e Ele 1

Screening f o r 1e c t ins Quantification o f polysaccharides Quantification o f g l y c o p r o t e i ns

Bdg-Hansen and Nordl5 Owen and Sal ton84 Bdg-Hansen e t al.20

Bdg-Hansen e t al.20 Analysis o f (1) f r a c t i o n a t i o n ( 2 ) p r o g r e s s i v e changes ( 3 ) treatments Identification o f

Bdg-Hansen and Nordl5

Identification o f i n t e r a c t i n g components I d e n t i f i c a t i o n and quantification o f l e c t i n-bi n d i n g g l y c o p r o t e i ns

Owen and Salton84 Owen e t a1.85

(1) I d e n t i f i c a t i o n o f interacting components ( 2 ) P a r t i a l characterization of number o f b i n d i n g sites (3) Prediction o f s e p a r a t i o n experiments

Bdg-Hansen*

1e c t i ns

Bdg-Hansen e t al.20

Bdg-Hansen and Brogrenl4

Bdg-Hansen2 Ramlau and Bock31 (Continued on p. 246)

TABLE 10.4

(continued)

No.

Modification

I n t e r a c t i n g cornponent which i s electrophoresed

Interacting component i n t h e medium

Purpose

9.

Crossed immunoelectrophoresis w i t h ligand i n the f i r s t dimension gel

Glycoprotei ns

Lectins

(1) I d e n t i f i c a BBg-Hansen e t a1 33 tion of intera c t i n g components

L e c t i n s and Glycoproteins

Ref.

( 2 ) Determination b i n d i ng speci f ic t y ( 3 ) Analysis o f BBg-Hansen e t a1 33 m i croheterogenei t y ( 4 ) Determination o f BBg-Hansen and Takeo16 dissociation constants N i l s s o n and Bdg-HansenSl (5) Prediction o f s e p a r a t i o n exper iment s

i L e c t i n a p p l i e d i n a trough b e f o r e d i f f u s i o n . Agarose gel was used as s u p p o r t i n g medium except i n 2' (paper) and 3 (polyacrylamide g e l ) .

247

F i g . 10.13. Combined S D S - p o l y a c r y l a m i d e g e l e l e c t r o o h o r e s i s and a f f i n i t y e l e c t r o p h o r e s i s o f 1 mg o f human serum g l y c o p r o t e i n . The l o w e r D a r t o f t h e p l a t e i s a T r i t o n X - 1 0 0 - c o n t a i n i n g a g a r o s e g e l ( 5 % ) . The w h i t e a r e a w i t h i n t h i s g e l shows where t h e SDS g e l was o v e r l a y e r e d w i t h t h e s e n a r a t e d p r o t e i n s , anode t o t h e r i g h t . The u p p e r g e l was a g a r o s e c o n t a i n i n g conA (1.6 10-6 m o l / i ) . F o r t u r r h e r d e t a i l s , see t e x t and B d g - H a n ~ e n ~ ~ .

-

s e c o n d - d i m e n s i o n e l e c t r o p h o r e s i s was p e r f o r m e d i n a g a r o s e w i t h n o n - i o n i c d e t e r G e n t ( s e e F i g . 10.13). As t h e p r e c i p i t a t e f o r m a t i o n i s d e p e n d e n t on t h e amount o f 7' g l y c o p r o t e i n and t h e amount o f l e c t i n a p o l i e d (Bfig-Hansen " ) t h i s method may a l s o b e u s e d q u a n t i t a t i v e l y f o r t h e d e t e r m i n a t i o n o f t h e amount o f S l y c o p r o t e i n app l i e d o r t h e amount o f l e c t i n - b i n d i n g g l y c o p r o t e i n i n each band. i t i s r e m a r k a b l e t h a t t h e SDS i n t h e a c r y l a m i d e g e l does n o t d i s t u r b t h e b i n d -

i n g t o t h e l e c t i n . T h i s i s p r e s u m a b l y due t o t h e n e u t r a l i z i n g e f f e c t o f T r i t o n b y i n c l u s i o n o f SDS i n t o m i c e l l e s , s o t h a t t h e l e c t i n i s n o t e x p o s e d t o f r e e SDS. 3n t h e o t h e r hand, l e c t i n s a r e known t o b e s t a b l e and r e a c t i v e i n t h e p r e s e n c e

o f d e t e r g e n t ( s e e , f o r i n s t a n c e , B j e r r u m e t a 1 . 7 7 ) . E a r l i e r we showed t h a t den a t u r a t i o n o f t h e g l y c o p r o t e i n has l i t t l e e f f e c t on t h e b i n d i n g t o l e c t i n ( c o n A ) , w h e t h e r d e n a t u r a t i o n was p e r f o r m e d w i t h d e t e r g e n t , h e a t o r a c i d t r e a t m e n t , and t h a t t h e p r e c i p i t a t i o n i n a f f i n i t y p r e c i p i t a t e s was q u a n t i t a t i v e i r r e s p e c t i v e o f t h e d e g r e e o f d e n a t u r a t i o n (Bdg-Hansen e t a l .

19

).

Preparative a j - f i n i t y electrophoresis One o f t h e m a i n d i f f i c u l t i e s i n o r e p a r a t i v e a f f i n i t y c h r o m a t o g r a p h y i s t h a t t h e a c t i v e component s t i c k s t o o w e l l t o t h e i m m o b i l i z e d l i g a n d . I n o r d e r t o o v e r -

248

come t h i s problem, Dean e t a1 .73 used electrophoreticdesorption o f t h e a f f i n i t y column a f t e r l o a d i n g and washing. E a r l i e r we performed a few e x p l o r a t o r y experiments on t h e p o s s i b l e p r e p a r a t i v e use o f t h e p r i n c i p l e o f a f f i n i t y e l e c t r o p h o r e s i s when we performed p r e p a r a t i v e i s o t a c h o p h o r e s i s i n a c r y l a m i d e g e l w i t h conA, b u t we d i d n o t c o n t i n u e t h i s approach because t h e a n a l y t i c a l aspects appeared more p r o m i s i n g (Bplg-Hansen e t a l .

18

BIOMEDICAL APPLICATIONS The a n a l y t i c a l e l e c t r o p h o r e t i c approach seems t o be an a l t e r n a t i v e t o preparat i v e methods and may be used when o n l y a s m a l l amount o f b i o l o g i c a l specimen i s a v a i l a b l e i n s t e a d o f t h e more m a t e r i a l - r e q u i r i n g column and b a t c h procedures. T h e r e f o r e , i t c o u l d be used r o u t i n e l y f o r d i a g n o s i s and c h e c k i n g o f human d i s eases.

Diagnosis of malignant diseases and neural tube defects As s e v e r a l i m p o r t a n t marker p r o t e i n s i n human diseases a r e g l y c o p r o t e i n s , t h e i r i n t e r a c t i o n w i t h l e c t i n s may g i v e i m p o r t a n t c l u e s about t h e s t a t e o f t h e disease. a - F o e t o p r o t e i n (AFP) i s one such p r o t e i n . A m n i o t i c f l u i d AFP f r o m normal pregnancies between 15 and 33.5 weeks o f pregnancy c o n s i s t s o f 12-45% o f t h e 79 conA n o n - r e a c t i v e f o r m ( S m i t h and K e l l e h e r ) , whereas f o e t a l serum has been r e p o r t e d t o c o n t a i n a s i g n i f i c a n t l y l o w e r o r o p o r t i o n o f conA n o n - r e a c t i v e v a r i a n t s 79 (2-10%) ( R u o s l a h t i e t al."), Smith and K e l l e h e r ) . T h i s l e d t o t h e d e m o n s t r a t i o n t h a t t h e p a t t e r n o f c o n A - a f f i n i t y v a r i a n t s i n a m n i o t i c f l u i d seems t o be v a l u a b l e i n d i a g n o s i n g f o e t a l a b n o r m a l i t i e s (Smith e t a1 .81).

I n f o e t a l a b n o r m a l i t i e s , c h a r a c t e r i z e d by a change i n AFP compartmentalizat i o n , such as t r a n s u d a t i o n o f f o e t a l serum across exposed f o e t a l membranes i n t h e presence o f n e u r a l t u b e d e f e c t s , a s h i f t i n t h e o a t t e r n o f a m n i o t i c f l u i d AFP c o n A - a f f i n i t y v a r i a n t s t o resemble t h a t o f f o e t a l serum, has been observed (Smith e t al.81, Smith and K e l l e h e r 79 ) . The measurement o f c o n k a f f i n i t y v a r i a n t s i n a m n i o t i c f l u i d has r e c e n t l y been performed u s i n g a f f i n i t y e l e c t r o p h o r e s i s w i t h conA i n c l u d e d i n t h e f i r s t - d i m e n s i o n g e l (Hinderson e t a1

.**, Ndrgaard-Pedersen

.

e t a1 52, T o f t a g e r - L a r s e n and NfJrgaard-

P e d e r ~ e n ~ ~T h) i. s method has been found t o b e more s i m p l e and r e l i a b l e t h a n t h e 82 ),

p r e v i o u s chromatographic t e c h n i q u e s (Hinderson e t a l .

The a f f i n i t y e l e c t r o p h o r e s i s system has a l s o been used i n t h e comparison o f 46 f o e t a l and hepatoma AFP l e c t i n - a f f i n i t y v a r i a n t p a t t e r n s ( K e r c k a e r t e t a l . , Breborowics and M a c k e w i c ~ ~ ~ The ) . p r o p o r t i o n o f t h e c o n A - a f f i n i t y v a r i a n t s seem t o be s i m i l a r i n hepatoma s e r a and s i m i l a r t o t h e p r o p o r t i o n i n f o e t a l sera.

).

249

However, t h e L C A - a f f i n i t y

p a t t e r n s v a r y among hepatoma sera, b u t g e n e r a l l y t h e

percentage o f t h e LCA-reactive v a r i a n t i s h i g h e r i n hepatoma s e r a t h a n i n f o e t a l 79 s e r a ( S m i t h and K e l l e h e r ).

Generalizations: o t h e r i n t e r a c t i n g systems By p e r f o r m i n g c r o s s e d immunoelectrophoresis w i t h h e p a r i n i n t h e f i r s t - d i m e n s i o n

g e l , B l e y l and P e i ~ h were l ~ ~a b l e t o s e p a r a t e a c t i v e a n t i t h r o m b i n f r o m t h e i n a c t i v e complex and t o measure i t q u a n t i t a t i v e l y i n p a t i e n t s under h e p a r i n t h e r a p y . T h i s example shows t h a t i t i s p o s s i b l e t o d i s t i n g u i s h between v a r i o u s m o l e c u l a r forms o f a c t i v e macromolecules a n a l o g o u s l y t o t h e m i c r o h e t e r o g e n e i t y forms o f g l y c o p r o t e i n s . A l s o , t h i s o f f e r s an o p p o r t u n i t y t o d i s t i n g u i s h between a c t i v a t o r c o n t r o l ( c o n t r o l by i n h i b i t o r s and a c t i v a t o r s and by a c t i v a t i o n o f p r e c u r s o r forms and i n a c t i v a t i o n by p a r t i a l d e g r a d a t i o n ) f r o m c o n t r o l by de novo s y n t h e s i s and t o t a l degradation. H o r e j s i6 4 y 6 5 p r e s e n t e d some t h e o r e t i c a l c o n s i d e r a t i o n s on t h e g e n e r a l q u a n t i t a t i v e use o f a f f i n i t y e l e c t r o p h o r e s i s , some g e n e r a l i z a t i o n s o f t h e p r e s e n t approach f o r t h e d e s c r i p t i o n o f r e c e p t o r f u n c t i o n s o f membrane p r o t e i n s were d i s cussed r e c e n t l y by B j e r r u m e t a1 .77 and Ramlau and Bock3’ d e s c r i b e d some o t h e r i n t e r a c t i n g systems.

ConcZuding remarks I n g e n e r a l , any macromolecular o r p a r t i c l e - b o u n d l i g a n d o r i n t e r a c t i n g subs t a n c e may be i n c l u d e d d u r i n g e l e c t r o p h o r e s i s t o S i v e a l i g a n d - i n d u c e d r e a c t i o n , such as a change o f t h e e l e c t r o o h o r e t i c m o b i l i t y o r a change o f t h e morDhology o r p r o f i l e o f t h e p r o t e i n . As seen f r o m some o f t h e examples mentioned above, t h e l i g a n d need n o t be charged. The main advantages o f t h e a n a l y t i c a l e l e c t r o p h o r e s i s approach appear t o be:

(1) I t can s e p a r a t e macromolecules t h a t i n t e r a c t w i t h a s p e c i f i c l i g a n d f r o m those t h a t do n o t . ( 2 ) I t can b e used f o r s t u d i e s o f i n t e r a c t i n g macromolecules. ( 3 ) I t i s n o t necessary t o p u r i f y i n t e r a c t i n g components. ( 4 ) A m u l t i t u d e o f p r o t e i n s r e a c t i n g w i t h t h e same l i g a n d may be s t u d i e d s i mu1taneous l y

.

( 5 ) I t can be g e n e r a l i z e d t o i n t e r a c t i o n s o t h e r t h a n t h o s e between l e c t i n s and g l y c o p r o t e i ns

.

( 6 ) I t can b e used f o r t h e p r e d i c t i o n o f p r e p a r a t i v e experiments. ACKNOWLEDGEMENTS !Is. P i a Jensen i s thanked f o r h e r s k i l f u l h e l p . The s t u d i e s were s u p p o r t e d by

t h e Danish Medical Research C o u n c i l .

250

REFERENCES

1 S. Nakamura, Cross Electrophoresis. Its PrincipZe and AppZications, I g a k u Shoin, Tokyo, and E l s e v i e r , Amsterdam, 1966. 2 T.C. Bdg-Hansen, Anal. Biochem., 56 (1973) 480. 3 V. H o r e j s i and J. Kocourek, Biochim. Biophys. Acta, 336 (1974) 338. 4 N.H. Axelsen ( E d i t o r ) , I m n o p r e c i p i t a t i o n Techniques i n Gel, U n i v e r s i t e t s f o r l a g e t , Oslo; Scand. J . InomtnoZ. Suppz., (1981) i n press. 5 V. H o r e j s i , M. T i c h a and J. Kocourek, TIBS, 4, 1 (1979) N6. 6 H. B i t t i g e r and H.P. S c h n e b l i , CmcanuvaZin A as a TooZ, R i l e y , London, 1976. 7 S. Murakawa and 5 . Nakamura, BUZZ. Yamaguchi MedicaZ SchooZ, 10 (1963) 11. 8 B.O. Osunkoya and A . I . O . W i l l i a m s , CZin. Exp. I m n o l . , 8 (1971) 205. 9 G.A. Spengler and R.M. Weber, i n T.C. Bdg-Hansen ( E d i t o r ) , Lectins, BioZogy, Biochemistry and CZinicaZ Biochemistry, V o l . 1, W. de G r u y t e r , B e r l i n , 1981, pp. 231-240. 10 P.J. Svendsen, i n Z. Deyl ( E d i t o r ) , ELectrophoresis. A Survey o f Techniques and 4ppZications. Part A: Techniques, E l s e v i e r , Amsterdam, 1979, np. 133-154, 307-326, 345-362. 11 T.C. Bdg-Hansen, I . Lorenc-Kubis and O.J. Bjerrum, i n B.J. Radola ( E d i t o r ) , EZectrophoresis '79. Advanced Methods. BiochsmicaZ and ClinicaZ AppZications, W. de G r u y t e r , B e r l i n , 1980, pp. 173-192. 12 T.C. Bdg-Hansen, i n J.M. E g l y ( E d i t o r ) , Proc. 3rd Int. Symp. A f f i n i t y Chromatography and MolecuZar Interactions, Inserm Symposia S e r i e s , Les c o l locrues de l ' I n s e r m , Inserm, P a r i s , Vol. 86, 1979, pp. 399-416. 13 T.C. Bdg-Hansen ( E d i t o r ) , Lectins, BioZogy, Biochemistry and CZinicaZ Biochemistry, Vol. 1, W. de G r u y t e r , B e r l i n , 1981. 14 T.C. Bdg-Hansen and C.H. Brogren, Scand. J . I m n o Z . , 4, Suppl. 2 (1975) 135. 15 T.C. Bdg-Hansen and M. Nord, J . B i o l . Educ., 8 (1974) 167. 16 T.C. Bdg-Hansen and K. Takeo, J . EZectrophoresis, 1 (1980) 67. 17 T.C. Bdg-Hansen, C.H. Brogren and I . Ycblurrough, J . Inst. Brming, 80 (1974) 443. 18 T.C. Bdg-Hansen, P.J. Svendsen and O.J. Bjerrum, i n P.G. R i g h e t t i ( E d i t o r ) , Progress i n IsoeZectric Focusing and Isotachophoresis, North-Hol l a n d , Amsterdam, 1975, p. 347. 19 T.C. Bdg-Hansen, O.J. B j e r r u m and C.H. Brogren, Anal. Biochem., 8 1 (1978) 78. 20 T.C. Bdg-Hansen, P. P r a h l and H. Ldwenstein, J . I m m Z . Methods, 22 (1978) 293. 21 J. Turkova, A f f i n i t y Chromatography, E l s e v i e r , Amsterdam, 1978. 22 J.T. Dulaney, MoZ. CeZZ Biochem., 21 (1979) 43. 23 S. B i s a t i , L. M i k k e l s e n and C.H. Brogren, i n T.C. Bdg-Hansen ( E d i t o r ) , Lectins, Biology, Biochemistry and CZinicaZ Biochemistry, V o l . 1, W. de G r u y t e r , B e r l i n , 1981, pp. 387-394. 24 O.J. Bjerrum, T.C. Bdg-Hansen, T. P l e s n e r and M. Wilken, i n T.C. Bdg-Hansen ( E d i t o r ) , Lectins, BioZogy, Biochemistry and CZinicaZ Biochemistry, Vol 1, W . de G r u y t e r , B e r l i n , 1981, pp. 259-268. 25 O.J. B j e r r u m and T.C. Bdg-Hansen, Biochim. Biophys. Actu, 455 (1976) 66. 26 C.H. Brogren and S . B i s a t i , i n T.C. Bplg-Hansen ( E d i t o r ) , Lectins, Biology, Biochemistry and CZinicaZ Biochemistry, V o l . 1, Lir. de G r u y t e r , B e r l i n , 1981, pp. 375-385. 27 J. Gerlach, O.J. Bjerrum, H.C. Rank and T.C. Bdg-Hansen, Protides BioZ. F ~ u i d s , 27 (1980) 479. 28 I . Hagen and 0.3. Bjerrum, Protides BioZ. FZuids, 27 (1979) 875. 29 H. Lfiwenstein, T.C. Bplg-Hansen and B. Weeke, Protides BioZ. FZuids, 27 (1980) 611. 30 M. R a f t e l l , Immunochemistry, 14 (1977) 787. 31 J . Ramlau and E. Bock, i n J.M. E g l y ( E d i t o r ) , Proc. 3rd I n t . Symp. A f f i n i t y chromatography and MoZecuZar Interactions, Inserm Symposia S e r i e s , Les Colloques de l ' I n s e r m , Inserm, P a r i s , Vol. 86, 1979, pp. 147-173.

.

251 32 B.F. Vestergaard and T.C. Bgig-Hansen, scand. J . I m n o Z . , 4, s u p p l . 2 (1975) 211. 33 T.C. BCg-Hansen, O.J. B j e r r u m and J. Ramlau, Scand. J . I m n o Z . , 4, Suppl 2 (1975) 141. 34 H.S. P l a t t , B.M. S e w e l l , T. Feldman and R.L. Souhami, Clin. Chim. Acta, 46 (19731 419. 35 6 . B a i a r d and J.P. K e r c k a e r t , Biochim. Biophys. Res. C o m n . , 95 (1980) 777. 36 O.J. B j e r r u m and T.C. BBg-Hansen, i n A.H. Maddy ( E d i t o r ) , BiochemicaZ Analysis o f Membranes, Chapman and H a l l , London, 1976, pp. 378-426. 37 T.C. Bdg-Hansen, Protides BioZ. FZuids, 27 (1980) 659. 38 T.C. Bdg-Hansen, P. Jensen, F. H i n n e r f e l d t and K. Takeo, i n T.C. BBg-Hansen ( E d i t o r ) , Lectins, Biology, Biochenristry and CZinicaZ Biochemistry, V O l 1, W. de G r u y t e r , B e r l i n , 1981, pp. 241-258. 39 J. Breborowicz and A. Mackiewicz, i n T.C. Bdg-Hansen ( E d i t o r ) , Lectins, Biology, Biochemistry and Clinical Biochemistry, V o l . 1, W. de G r u y t e r , B e r l i n , 1981, pp. 303-314. 40 C . F o u r n i e r , J.P. K e r c k a e r t , B. Bayard, M. C o l l y n and G. B i s e r t e , Protides Biol. Fluids, 27 (1980) 623. 41 M. F u r l a n , B.A. P e r r e t and E . A . Beck, Protides BioZ. Fluids, 27 (1980) 653. 42 P. Guldager and T.C. Bdg-Hansen, Protides Biol. Fluids, 27 (1979) 401. 43 I . Hagen and G. Gogstad, i n T.C. Bdg-Hansen ( E d i t o r ) , Lectins, Biology, Biochemistry and CZinicaZ Biochemistry, V o l . 1, W. de G r u y t e r , B e r l i n , 1981, pp. 347-354. 44 J. Hau, P. Svendsen, B. T e i s n e r and G.T. Pedersen, J . Reprod. Fert., 58 (1980) 389. 45 J . Hau, P. Svendsen, B. T e i s n e r and J. B r a n d t , Biol. Reprod., 24 (1981) 163. 46 J. Hau, P. Svendsen, B. T e i s n e r and G. Thomsen Pedersen, i n T.L. Bdg-Hansen ( E d i t o r ) , Lectins, BioZogy, Biochemistry and CZinicaZ Biochemistry, Vol 1, W. de G r u y t e r , B e r l i n , 1981, pp. 327-338. 47 J. Hau, P . Svendsen, B. T e i s n e r . G. Thomsen Pedersen and B.R. K r i s t i a n s e n , Bioi!. Reprod., 24 (1381) 683. 48 J.P. K e r c k a e r t , B. Bayard and G. B i s e r t e , Biochim. Biophys. Acta, 576 (1979) 99. 49 A. Mackiewicz and J. Breborowicz, i n T.C. Bdg-Hansen ( E d i t o r ) , Lectins, Biology, Biochemistry and CZinicaZ Biochemistry, V o l . 1, b!. de G r u y t e r , B e r l i n , 1981, pp. 315-326. 50 C.S. N i e l s e n and O.J. Bjerrurn, Biochim. Biophys. Acta, 466 (1977) 496. 51 M. N i l s s o n and T.C. Bdg-Hanse, Protides Biol. FZuids, 27 (1980) 599. 52 B. Nbrqaard-Pedersen, K. T o f t a q e r - L a r s e n , J. P h i l i D and P . Hindersson, CZin. . * Gen., 17 (1980) 1. 53 J.D. Oppenheim, P . Owen, M.S. Nachbar, K. C o l l e d g e and H.S. Kapian, Immnol. C o m n . . 6 (19771 167. 54 R.L. Or,; T:C. Big-Hansen and R.R. Mod, i n R.L. Ory ( E d i t o r ) , Antinutrients and NaturaZ Toxicants i n Foods, Food and N u t r i t i o n Press, Westport, CN, 1980. 55 R.L. Ory, R.R. Mod and T.C. Bdg-Hansen, Protides Bioi!. Fluids, 27 (1980) 387. 56 I . R . Pedersen and C.H. M o r d h o r s t , i n T.C. BBg-Hansen ( E d i t o r ) , Lectins, Biology, Biochemistry and CZinicaZ Biochemistry, Vol 1, W. de G r u y t e r , B e r l i n , 1981, pp. 395-399. 57 T. Plesner, O.J. B j e r r u m and M. Wilken, i n T.C. B6g-Hansen ( E d i t o r ) , Lectins, Biology and Biochemistry, V o l . 1, W . de G r u y t e r , 1981, P. 363-374. 58 K. T o f t a g e r - L a r s e n and B. Nfirgaard-Pedersen, i n T.C. BBg-Hansen ( E d i t o r ) , Lectins, Biology, Biochemistry and CZinicaZ Biochemistry, Vol 1, W. de G r u y t e r , B e r l i n , 1981, pp. 293-302. 59 K. T o f t a g e r - L a r s e n , P.L. P e t e r s e n and B. Ndrgaard-Pedersen, i n T.C. B$g-Hansen ( E d i t o r ) , Lectins, BioZogy, Biochemistry and CZinicaZ Biochemistry, V O l . 1, W. de G r u y t e r , B e r l i n , 1981, pp. 283-292. 60 C. Wells, E. Cooper and T.C. Bdg-Hansen, i n T.C. BBg-Hansen ( E d i t o r ) , Lectins, Biology, Biochemistry and CZinicaZ BiochemistPy, Vol 1, W. de G r u y t e r , B e r l i n , 1981, pp. 339-346.

.

.

.

.

.

.

252

6 1 C . Wells, E. Cooper, R.M. Glass and T.C. Bdg-Hansen, CZin. Chirn. Acta, (1981) i n press. 62 E. Bock and N.H. Axelsen, Scand. J . I m n o Z . , 2, sUpD1. 1, (1973) 95-99. 63 K. Takeo and S. Nakamura, Arch. Biochem. Biophys., 153 (1972) 45. 64 V. H o r e j s i , i n J.M. E g l y ( E d i t o r ) , Proc. 3rd I n t . Symp. A f f i n i t y chromatography and MoZecuZar Interactions, Inserm Symposia S e r i e s , Les Colloques de l ' I n s e r m , Inserm, P a r i s , V o l . 86, 1979, pp. 391-398. 65 V. H o r e j s i , J . Chromatogr., 178 (1979) 1. 66 T.C. Bdg-Hansen, Scand. J . I m o Z . , Suppl. 10 (1981) i n p r e s s . 67 N.M. Young and M.A. Leon, Biochim. Biophys. Acta, 365 (1974) 418. 68 K. Takeo, A. Kuwahara, H. Nakayama and S. Nakamura, Protides BioZ. FZuids, 23 (1976) 645. 69 M. Ticha, V. H o r e j s i and J. Barthova, Biochim. Biophys. Aeta, 534 (1978) 58. 70 K. Hauser, M. Ticha, V. H o r e j s i and K. Kocourek, Biochim. Biophys. Acta, 583 (1979) 103. 7 1 V. H o r e j s i , M. T i c h a and J. Kocourek, Biochim. Biophys. Acta, 499 (1977) 290. 72 M. Caron, A. Faure, R.G. Keros and P. C a m i l l o t , Biochim. Biophys. Acta, 491 (1977) 558. 73 K. Takeo and A. Kabat, J . I m n o Z . , 121 (1978) 2305. 74 Vl. Harboe, E. S a l t v e d t , 0. Closs and S. O l s n e i , Scand. J . ImmunoZ., 4, Suppl. 2 (19751 125. 75 G.A. S p i n g l e r and R.M. Weber, Protides BioZ. Fluids, 27 (1980) 615. 76 T.C. Bdg-Hansen, i n B.J. Radola ( E d i t o r ) , EZectrophoresis ' 7 9 . Advanced Methods. BiochemicaZ and CZinicaZ Applications, W. de G r u y t e r , B e r l i n , 1980, pp. 193-202. 77 O.J. Bjerrum, J. Ramlau, E. Bock and T.C. Blg-Hansen, i n S.J. Jacobs and P. Cuatrecasas ( E d i t o r s ) , Membrane Receptors: Methods f o r Characterization and Purification, Chapman and H a l l , London, 1980, pp. 117-156. 78 P.D.G. Dean, F. Q a d r i , W. Jessup, V . B o u r i o t i s , S. Angal , H. Potuzak, R.J. Leatherbarrow, T. Miron, E. George and M.R.A. Morgan, i n J.M. E g l y ( E d i t o r ) , Proc. 3rd rnt. Symp. A f f i n i t y Chromatography and MoZecuZar Interactions, Inserm Symposia Series, Les Colloques de l ' I n s e r m , Inserm, P a r i s , V o l . 86, 1979, pp. 321-344. 79 C.J. Smith and P.C. K e l l e h e r , Biochim. Biophys. Acta, 605 (1980) 1. 80 E. R u o s l a h t i , E. E n g v a l l , A. Pekkala and M. Seopala, Int. J . Cancer, 22 (1978) 515. 3 1 C.J. Smith, P.C. K e l l e h e r , L. Belanger and L. D a l l a i r e , B r i t . Med. J . , 1 (1979) 920. 82 P. Hinderson, K. ' T o f t a g e r - L a r s e n and B. Nfirgaard-Pedersen, Lancet, a c t . 27 1979) 906. 83 i.B l k y l and H. P e i c h l , i n B.J. Radola ( E d i t o r ) , Electrophoresis ' 7 9 . Advanced Methods. BiochemicaZ and CZinicaZ AppZications, W. de G r u y t e r , B e r l i n , 1980, pp. 733-742. 84 P. Owen and M.R.J. S a l t o n , Anal. Biochem., 73 (1976) 20. 85 P. Owen, J.D. Oppenheim, M.S. Nachbar and R.E. K e s s l e r , AnaZ. Biochem., 80 (1977) 446. 86 C.H. Brogen and T.C. Blg-Hansen, Scand. J . ~ m n o Z . , 4, Suppl. 2 (1975) 37.