UBC Theses and Dissertations

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UBC Theses and Dissertations

Fibrinogen adsorption to human erythrocytes Janzen, Johan 1985

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F I B R I N O G E N ADSORPTION TO HUMAN ERYTHROCYTES by J o h a n J a n z e n B . S c , U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1 9 7 0 A T H E S I S SUBMITTED I N P A R T I A L F U L F I L L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE S T U D I E S ( THE DEPARTMENT OF PATHOLOGY ) We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE U N I V E R S I T Y OF B R I T I S H COLUMBIA A P R I L , 1 9 8 5 © J o h a n J a n z e n , 1 9 8 5 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of Pathology The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date A p r i l 15th 1985 (3/81) - i i -ABSTRACT F i b r i n o g e n a d s o r p t i o n t o human e r y t h r o c y t e s h a s b e e n i m p l i c a t e d a s t h e m e c h a n i s m r e s p o n s i b l e f o r t h e r e v e r s i b l e a g g r e g a t i o n o f r e d c e l l s . The o b j e c t o f t h i s t h e s i s was t o m e a s u r e t h e b i n d i n g o f f i b r i n o g e n t o human e r y t h r o c y t e s a n d t o c o m p a r e t h e b i n d i n g w i t h 1 25 a g g r e g a t i o n b e h a v i o u r . The b i n d i n g o f i s o l a t e d I - f i b r i n o g e n t o r e d c e l l s was e s t i m a t e d f r o m t h e amount o f r a d i o l a b e l r e m o v e d f r o m s o l u t i o n u p o n a d d i t i o n o f c e l l s , f r o m t h e amount o f r a d i o l a b e l r e t a i n e d i n c e l l p e l l e t s p r o d u c e d b y u l t r a c e n t r i f u g a t i o n a n d c o r r e c t e d f o r i n t r a c e l l u l a r r a d i o l a b e l , a n d f r o m r a d i o l a b e l r e t a i n e d b y c e l l s a f t e r e x h a u s t i v e w a s h i n g i n p r o t e i n - f r e e b u f f e r . B i n d i n g o f human a l b u m i n t o r e d c e l l s was d e t e r m i n e d b y t h e same m e t h o d s . The f r e e e n e r g y o f f o r m a t i o n o f r e d c e l l / r e d c e l l c o n t a c t s d u e t o i s o l a t e d f i b r i n o g e n was d e t e r m i n e d b y t h e m e t h o d o f E v a n s a n d Buxbaum ( B i o p h y s . J. 3 4 , 1 - 1 2 , 1 9 8 1 ) . The s o l u t i o n d e p l e t i o n m e t h o d r e q u i r e d c o r r e c t i o n s f o r d i l u t i o n e f f e c t s a n d was n o t s e n s i t i v e e n o u g h f o r d e t a i l e d d e t e r m i n a t i o n o f b i n d i n g o f t h e s e p r o t e i n s t o r e d c e l l s . B i n d i n g e s t i m a t e d f r o m c e l l p e l l e t s a n d c o r r e c t e d f o r i n t r a c e l l u l a r r a d i o l a b e l i n c r e a s e d l i n e a r i l y w i t h p r o t e i n c o n c e n t r a t i o n t o 12 mg/mL f o r f i b r i n o g e n a n d 5 0 mg/mL f o r a l b u m i n . The b i n d i n g a t 1 mg/mL was b e t w e e n 5 0 a n d 2 5 0 m o l e c u l e s p e r c e l l f o r f i b r i n o g e n a n d b e t w e e n 1 0 0 0 a n d 1 4 0 0 m o l e c u l e s p e r c e l l f o r a l b u m i n . B i n d i n g d e t e r m i n e d f r o m wash - i i i -a n a l y s e s was s i m i l a r t o p e l l e t b i n d i n g c o r r e c t e d f o r f r e e r a d i o l a b e l . T h e s u r f a c e a f f i n i t y o f r e d c e l l s f o r r e d c e l l b e a d s was -3 2 d e t e r m i n e d t o be 1.8 +_ 0.5 x 10 e r g / c m (mean + SEM, n = 4 ) a n d 2.8 + 0.8 x 1 0 "3 e r g / c m2 (mean + SEM, n = 2 8 ) a t 4.4 a n d 8.8 mg/ml f i b r i n o g e n r e s p e c t i v e l y . A d h e s i o n b e t w e e n r e d c e l l s a n d r e d c e l l b e a d s o c c u r r e d a t 2.2 mg/mL f i b r i n o g e n , b u t was t o o weak t o a l l o w e s t i m a t i o n o f t h e s u r f a c e a f f i n i t y . T he s u r f a c e a f f i n i t i e s a n d b i n d i n g e s t i m a t e s a l l o w c a l c u l a t i o n o f t h e mean b i n d i n g e n e r g y f o r f i b r i n o g e n t o t h e r e d c e l l s u r f a c e . The s u r f a c e a f f i n i t y p e r m o l e c u l e , a s s u m i n g 2 5 0 m o l e c u l e s p e r c e l l a t 1 mg/mL, was 30 +_ 10 a n d 24 + 7 k i l o c a l o r i e s / m o l e a t 4.4 a n d 8.8 mg/mL r e s p e c t i v e l y . The d i f f e r e n c e was n o t s i g n i f i c a n t . T h i s r e s u l t p o s e s a p r o b l e m a s b i n d i n g e n e r g i e s o f t h i s o r d e r s u g g e s t t h a t t h e a d h e s i o n w o u l d b e i r r e v e r s i b l e . I f f i b r i n o g e n b i n d i n g w e r e 1 5 0 0 m o l e c u l e s p e r c e l l a t 1 mg/mL a b i n d i n g e n e r g y o f 4 + 1 k i l o c a l o r i e s / m o l e w o u l d be i m p l i e d . T h e s e r e s u l t s a r e i n t e r p r e t e d a s i n d i c a t i n g t h a t f i b r i n o g e n b o u n d t o r e d c e l l s may be d i s p l a c e d d u r i n g u l t r a c e n t r i f u g a t i o n . - i v -TABL E OF CONTENTS P a g e A b s t r a c t i i L i s t o f T a b l e s v i i L i s t o f F i g u r e s x L i s t o f A p p e n d i c e s x i i i A c k n o w l e d g e m e n t s x i v C h a p t e r 1 INTRODUCTION 1 1.1 THE C L I N I C A L RELEVANCE OF ERYTHROCYTE AGGREGATION 1 1.2 MECHANISMS OF CELL-ADHESION 5 1.2.1 F r o h l i c h i n t e r a c t i o n : a l o n g r a n g e m e c h a n i s m . . 6 1.2.2 P h a s e s e p a r a t i o n 8 1.2.3 M o l e c u l a r b r i d g i n g 13 1.3 P R O T E I N ADSORPTION AND AGGREGATION OF RED C E L L S . 2 2 1.3.1 M e a s u r e m e n t o f p r o t e i n a d s o r p t i o n t o c e l l s . . . . 2 2 1.3.2 M e a s u r e s o f c e l l a g g r e g a t i o n 2 7 1.4 O B J E C T I V E S AND METHODS 3 0 C h a p t e r 2 M A T E R I A L S AND METHODS 31 2.1 MATERIALS 31 2.1.1 R e a g e n t s 3 1 2.1. 2 S o l u t i o n s a n d b u f f e r s 3 2 2.2 PROTEINS 3 7 2.2.1 F i b r i n o g e n i s o l a t i o n 3 7 2.2.2 PAGE a n a l y s i s 3 9 2.2.3 P r o t e i n c o n c e n t r a t i o n 41 2.2.4 R a d i o l a b e l l i n g 4 2 2.2.5 G e l f i l t r a t i o n o f r a d i o l a b e l e d p r o t e i n s . . . . 4 3 2.3 ERYTHROCYTES 4 5 2.3.1 I s o l a t i o n 4 5 2.3.2 Q u a n t i t a t i o n 4 5 2.3.3 M o d i f i c a t i o n o f Red C e l l s 4 9 - V -P a g e 2.4 GAMMA COUNTING 5 0 2.5 B I N D I N G EXPERIMENTS 5 5 2.5.1 A d s o r b a t e s o l u t i o n s 5 5 2.5.2 C e l l s u s p e n s i o n s 5 5 2.5 . 3 I n c u b a t i o n 56 2.5.4 S e p a r a t i o n o f c e l l s f r o m s u s p e n s i o n s 5 6 2.6 B I N D I N G A N A L Y S I S 6 0 2.6.1 E s t i m a t i o n o f b i n d i n g i n c e l l p e l l e t s 6 0 2. 6 . 2 E s t i m a t i o n o f b i n d i n g f r o m s o l u t i o n r a d i o l a b e l c o n c e n t r a t i o n 6 3 2.6 . 3 E s t i m a t i o n o f f r e e r a d i o l a b e l 6 4 2.6.4 B i n d i n g i s o t h e r m 6 5 2.7 WASH A N A L Y S I S 68 2.7.1 E s t i m a t i o n o f c e l l - a s s o c i a t e d r a d i o l a b e l . . . . 6 8 2.7 . 2 A n a l y s i s o f w a s h c u r v e s 6 9 C h a p t e r 3 RESULTS AND D I S C U S S I O N 7 3 3.1 PREPARATION OF PROTEINS 7 3 3.1.1 SDS-PAGE a n a l y s i s 7 3 3.1.2 F i b r i n o g e n i s o l a t i o n 79 3.1. 3 A l b u m i n 9 3 3.1.4 J -2 5I o d i n e r a d i o l a b e l e d p r o t e i n s 9 3 3.1.5 P r o t e i n c o n c e n t r a t i o n 1 0 0 3.1.6 Summary 1 0 3 3.2 APPARENT BINDING 1 0 5 3.2.1 C e n t r i f u g a t i o n r a t e a n d t i m e 1 0 5 3.2.2 C e l l a n d f r e e l a b e l c o n c e n t r a t i o n 1 1 4 3.2. 3 I o d i d e p a r t i t i o n b e t w e e n t h e c e l l a n d f l u i d c o m p a r t m e n t s 1 2 0 3.2.4 E s t i m a t i o n o f f r e e l a b e l e s t i m a t e s f r o m a p p a r e n t b i n d i n g 1 2 2 3.2.5 Summary 1 2 7 3.3 WASH A N A L Y S I S 1 2 9 3.3.1 I n t e r c e l l u l a r r a d i o l a b e l 1 3 0 3. 3 . 2 I n t r a c e l l u l a r r a d i o l a b e l 1 3 3 3.3. 3 R a d i o i o d i d e w a s h o u t c u r v e s 1 3 4 3.3.4 Wash c u r v e s i n p r o t e i n b i n d i n g e x p e r i m e n t s . . . 1 4 2 3.3 . 5 B o u n d r a d i o l a b e l 1 51 3.3.6 Summary 1 6 9 - v i -P a g e 3.4 SOLUTION A N A L Y S I S AND TUBE ADSORPTION 1 7 0 3.4.1 A d s o r p t i o n t o p o l y a l l o m e r t u b e s 171 3 . 4 . 2 S o l u t i o n a n a l y s i s 1 7 5 3.5 B I N D I N G E S T I M A T E S 181 3.5.1 E s t i m a t e s o f Q a n d 181 3 . 5 . 2 E s t i m a t e s o f Sj_ - T 1 9 2 3. 5 . 3 B i n d i n g i s o t h e r m s 1 9 5 3.5.4 I n t e r c e l l u l a r v o l u m e i n c e l l p e l l e t s 2 0 4 3. 5 . 5 B i n d i n g e s t i m a t e d f r o m w a s h i n t e r c e p t s 2 0 6 3 . 5 . 6 Summary 2 1 2 3.6 C E L L - C E L L ADHESION 2 1 5 3.6.1 C o m p a r i s o n o f s u r f a c e a f f i n i t y a n d a d s o r p t i o n . . 2 1 7 3. 6 . 2 M i n i m u m a d s o r p t i o n e s t i m a t e d f r o m a c r o s s -b r i d g i n g m o d e l 2 2 5 C h a p t e r 4 SUMMARIZING D I S C U S S I O N 2 2 9 4.1 UNDERESTIMATION OF B I N D I N G 2 2 9 4.2 CAUSES OF B I N D I N G UNDERESTIMATION 2 3 3 4.3 RELEVANCE OF THE OBSERVED B I N D I N G 2 3 6 C h a p t e r 5 CONCLUSIONS 2 3 9 B i b l i o g r a g h y 2 4 2 A p p e n d i c e s 2 6 1 - v i i -L I S T OF T A B L E S T a b l e P a g e 2.1 O s m o l a l i t y a n d c o n c e n t r a t i o n o f b u f f e r c o m p o n e n t s . . 3 6 2.2 C o m p o s i t i o n o f p h o s p h a t e b u f f e r s 3 6 2.3 C o n t a m i n a t i o n o f P B S w a s h e d r e d c e l l p r e p a r a t i o n s . . 4 6 2.4 P a r a m e t e r s f o r e q u a t i o n 3.9 47 2 . 5 A t t e n u a t i o n o f 1 2 5: gamma p h o t o n s 52 2.6 G a m m a - c o u n t i n g e f f i c i e n c y 54 3.1 M o l e c u l a r w e i g h t e s t i m a t e s f o r p l a s m a p r o t e i n s . . . 78 3.2 F i b r i n o g e n r e c o v e r y i n t h e PEG p r e c i p i t a t i o n m e t h o d . 8 6 3.3 P u r i t y o f F i b r i n o g e n i s o l a t i o n 3 0 . . 9 0 3.4 R a d i o l a b e l p u r i t y o f i s o l a t i o n s 3 4 AND 3 5 9 2 3.5 I o d i n e s u b s t i t u t i o n i n r a d i o l a b e l e d p r o t e i n s . . . . 9 6 3.6 F r e e r a d i o l a b e l i n s t o r e d 1 2 5I - A l b u m i n 1 0 0 3 . 7 OD28O a n d L o w r y p r o t e i n c a l i b r a t i o n c u r v e s 1 0 2 3.8 P e l l e t d i s t r i b u t i o n r a t i o (Q) a s a f u n c t i o n o f c e n t r i f u g a t i o n a n d t h e s a m p l e d i s t a n c e ( L ) 1 0 8 3.9 The o s m o l a l i t y o f w a s h b u f f e r s a n d c e l l s u s p e n s i o n s u p e r n a t a n t s a f t e r c e n t r i f u g a t i o n I l l 3.10 A l b u m i n d i s t r i b u t i o n r a t i o (Q) a s a f u n c t i o n o f c e l l a n d f r e e r a d i o l a b e l c o n c e n t r a t i o n s 116 3.11 F i b r i n o g e n d i s t r i b u t i o n r a t i o (Q) a s a f u n c t i o n o f c e l l a n d p r o t e i n c o n c e n t r a t i o n s 1 1 9 3 . 1 2 D i s t r i b u t i o n o f 1 2 5I o d i d e b e t w e e n t h e c e l l p e l l e t a n d s u p e r n a t a n t 121 3 . 1 3 F r e e l a b e l d e t e r m i n e d by a n a l y s i s o f a p p a r e n t b i n d i n g a n d g e l f i l t r a t i o n 1 2 5 - v i i i -T a b l e P a g e 3.1A A p p a r e n t b i n d i n g i n p e l l e t s p r o d u c e d by u l t r a a n d l o w - s p e e d c e n t r i f u g a t i o n 131 3 . 1 5 l2 5I - A l b u m i n w a s h a n a l y s e s a n d t h e a m o u n t o f f r e e r a d i o l a b e l d e t e r m i n e d b y g e l f i l t r a t i o n 1 3 6 3.16 l2 5I o d i d e w a s h o u t f r o m r e d c e l l p e l l e t s 1 3 9 3.17 F r e e l a b e l d e t e r m i n e d f r o m wash a n a l y s e s a n d g e l f i l t r a t i o n 1 4 3 3.18 I n t e r c e l l u l a r v o l u m e e s t i m a t e d f r o m w a s h a n a l y s e s . . 1 4 8 3.19 R a d i o l a b e l c a r r y - o v e r i n RBCM p r e p a r a t i o n s 1 5 4 3.20 R a d i o l a b e l d i s t r i b u t i o n i n 3 % SDS-PAGE a n a l y s e s o f 1 2 5I - F i b r i n o g e n a n d RBCM 158 3.21 Wash a n a l y s i s o f f i b r i n o g e n a g g r e g a t e a n d monomer b i n d i n g 1 6 3 3.22 E f f e c t o f w a s h m e d i a o n c e l l r e t a i n e d a c t i v i t i e s i n wash a n a l y s e s 1 6 7 3.23 F i b r i n o g e n b i n d i n g t o p r e - t r e a t e d p o l y a l l o m e r t u b e s . 1 7 3 3.24 S o l u t i o n d e p l e t i o n e s t i m a t e s o f b i n d i n g 1 7 6 3.25 F r e e r a d i o l a b e l a n d e s t i m a t e s o f t h e p a r a m e t e r s Q a n d Qj[ i n A l b u m i n e x p e r i m e n t s 1 8 3 3.26 F r e e r a d i o l a b e l a n d e s t i m a t e s o f t h e p a r a m e t e r s Q a n d Qi i n F i b r i n o g e n e x p e r i m e n t s 1 84 3.27 Mean v a l u e s f o r Q, Qj_, Q j / , a n d Si - T 187 3. 2 8 S i V a l u e s i n A l b u m i n a n d F i b r i n o g e n e x p e r i m e n t s . . 1 9 3 3.29 F i b r i n o g e n b i n d i n g a s a f u n c t i o n o f c o n c e n t r a t i o n . . 1 9 6 3.30 A l b u m i n b i n d i n g a s a f u n c t i o n o f c o n c e n t r a t i o n . . . . 197 3.31 S u r f a c e b i n d i n g e s t i m a t e s i n m o l e c u l e s p e r c e l l . . . . 2 0 4 - i x -T a b l e P a g e 3 . 3 2 I n t e r c e l l u l a r v o l u m e e s t i m a t e s 2 0 5 3 . 3 3 Wash a n a l y s i s e s t i m a t e s o f a d s o r b e d A l b u m i n 2 0 7 3.34 Wash a n a l y s i s e s t i m a t e s o f a d s o r b e d F i b r i n o g e n . . . 2 0 8 3.35 E n c a p s u l a t i o n a n d n o r m a l i z e d s u r f a c e a f f i n i t y 2 2 1 3.36 C o m p a r i s o n o f f i b r i n o g e n s u r f a c e c o n c e n t r a t i o n a n d a d h e s i o n s u r f a c e a f f i n i t y 2 2 3 A l E s t i m a t i o n o f t h e f r e e r a d i o l a b e l f r a c t i o n f r o m c h r o m a t o g r a p h i c d a t a 2 7 4 A2 D e p e n d e n c e o f T±2 e s t i m a t e s o n c h a n g e s i n y^-, when u s i n g e q u a t i o n 1 1 . 1 5 2 9 3 - X -L I S T OF F I G U R E S F i g u r e P a g e 2 . 1 a pH a s a f u n c t i o n o f ^ H P O z ^ / N a h ^ P O ^ m o l e r a t i o . . . . 3 4 2.1b O s m o l a r i t y o f t h e b u f f e r c o m p o n e n t s 3 4 2.2 H e m a t o c r i t ( H ) o f r e d c e l l s r e s u s p e n d e d i n a l b u m i n b u f f e r a s a f u n c t i o n o f t h e w e i g h t r a t i o fb 4 8 2.3 R e l a t i v e e f f i c i e n c y o f 1 2 5 j d e t e c t i o n a s a f u n c t i o n o f s a m p l e v o l u m e . . 51 2.4 R e l a t i v e e f f i c i e n c y ( R E ) v e r s u s a p p a r e n t c o u n t i n g r a t e (CPM) f o r 1 2 5I 5 3 2.5 S e p a r a t i o n o f c e l l p e l l e t s f r o m s u p e r n a t a n t s 58 2.6 F l o w c h a r t o f a d s o r p t i o n m e a s u r e m e n t s 6 7 2.7 Summary o f n o t a t i o n f o r t h e q u a n t i t i e s u s e d i n t h e b i n d i n g a n d w a s h a n a l y s e s 7 0 3.1 O p t i c a l d e n s i t y p r o f i l e s o f 3 % SDS-PAGE a n a l y s e s o f human p l a s m a 7 5 3.2 M o l e c u l a r w e i g h t v e r s u s r e l a t i v e m o b i l i t y i n 3 % SDS-PAGE 7 7 3.3 T h r e e p e r c e n t SDS-PAGE a n a l y s i s o f i s o l a t i o n 2 3 . . . . 81 3.4 a F i b r i n o g e n OD280 p r o f i l e o f e l u a t e f r o m a f f i n i t y c h r o m a t o g r a p h y 8 2 3.4b F i b r i n o g e n OD28O p r o f i l e o f e l u a t e f r o m d e - s a l t i n g o n S e p h a d e x G150 8 2 3.5 a T h r e e p e r c e n t SDS-PAGE a n a l y s i s o f f i b r i n o g e n i s o l a t i o n 2 3 , p o o l s C a n d D 84 3.5b C o m p a r i s o n o f l y o p h i l i z e d a n d g l u t a r a l d e h y d e c r o s s - l i n k e d f i b r i n o g e n 8 4 3.6 T h r e e p e r c e n t SDS-PAGE a n a l y s i s o f i s o l a t i o n 3 0 . . . . 8 9 3.7 O p t i c a l d e n s i t y p r o f i l e o f -*-25I r a d i o l a b e l e d S i g m a human a l b u m i n a n a l y s e d b y 3 % SDS-PAGE 94 3.8 S e p h a d e x G150 c h r o m a t o g r a p h y o f r e a c t i o n p r o d u c t s f r o m r a d i o i o d i n a t i o n 9 7 - x i -F i g u r e P a g e 3.9 R e c h r o m a t o g r a p h y o f l2 5I - p r o t e i n p r e p a r a t i o n s 9 9 3.10 P r o t e i n c o n c e n t r a t i o n c a l i b r a t i o n c u r v e s 101 3.11 P e l l e t d i s t r i b u t i o n r a t i o f o r l2 5I - a l b u m i n i n c e n t r i f u g e d r e d c e l l p e l l e t s 1 0 6 3.1 2 D i s t r i b u t i o n r a t i o (Q) a s a f u n c t i o n o f r e d c e l l c o n c e n t r a t i o n (/2>e) 1 1 5 3.13 A n a l y s i s o f t h e a p p a r e n t b i n d i n g t o d e t e r m i n e t h e f r e e r a d i o l a b e l r a t i o T^2 I2 4 3.14 C o m p a r i s o n o f w a s h c u r v e s f o r c e l l p e l l e t s p r o d u c e d b y u l t r a a n d l o w s p e e d c e n t r i f u g a t i o n 1 3 2 3.1 5 Wash a n a l y s e s o f e x p e r i m e n t s 3 8 , 4 5 , a n d 4 7 135 3.16 l2 5I o d i d e w a s h o u t f r o m human r e d c e l l s 1 3 8 3.1 7 l2 5I - a l b u m i n w a s h a n a l y s i s , e x p e r i m e n t 4 7 . P l o t o f Ac 2( i ) v e r s u s K ( i ) 1 4 5 3.18 Wash a n a l y s i s t o r e c o v e r b o u n d r a d i o l a b e l f o r 3 % SDS-PAGE a n a l y s i s 1 5 3 3.19 N a t i v e a n d r e d u c e d l2 5I - f i b r i n o g e n a d s o r b a t e a n d s u p e r n a t a n t d i s t r i b u t i o n s o n 3 % SDS-PAGE 1 5 5 3.2 0 RBCM a n a l y s e d o n 3 % SDS-PAGE 156 3 . 2 1 a S e p a r a t i o n o f a g g r e g a t e a n d monomer f i b r i n o g e n o n S e p h a c r y l S 3 0 0 1 6 0 3.2 1 b A g g r e g a t e a n d monomer f i b r i n o g e n o n 3 % SDS-PAGE . . . 1 6 0 3 . 2 2 a C o m p a r i s o n o f a g g r e g a t e a n d monomer f i b r i n o g e n b i n d i n g t o human e r y t h r o c y t e s . R a d i o l a b e l d i s t r i b u t i o n o f a d s o r b a t e s o n 3 % SDS-PAGE 161 3 . 2 2 b C o m p a r i s o n o f a g g r e g a t e a n d monomer f i b r i n o g e n b i n d i n g t o human e r y t h r o c y t e s . Wash a n a l y s e s o f c e l l p e l l e t s a f t e r u l t r a c e n t r i f u g a t i o n 161 3.23 I n f l u e n c e o f p r o t e i n o n f i b r i n o g e n w a s h c u r v e s . . . . 166 - x i i -F i g u r e P a g e 3.24 F r e q u e n c y h i s t o g r a m s o f t h e b i n d i n g p a r a m e t e r s Q a n d Q i 1 8 5 3.25 F r e q u e n c y h i s t o g r a m s o f t h e b i n d i n g p a r a m e t e r s Q l ( A ) , Q i ' ( A ) , a n d Q i ( B ) 1 8 6 3.2 6 F r e q u e n c y h i s t o g r a m s o f t h e b i n d i n g p a r a m e t e r S i . . . 194 3.27 F i b r i n o g e n b i n d i n g a s a f u n c t i o n o f p r o t e i n c o n c e n t r a t i o n 1 9 9 3. 2 8 P r o t e i n a d s o r p t i o n i s o t h e r m s b a s e d o n Q] 2 0 0 3. 2 9 P r o t e i n a d s o r p t i o n i s o t h e r m s b a s e d o n 2 0 2 3.30 B i n d i n g e s t i m a t e s d e t e r m i n e d f r o m w a s h a n a l y s e s . . . . 2 0 9 3.31 Human e r y t h r o c y t e r o u l e a u x i n i s o l a t e d f i b r i n o g e n a t a c o n c e n t r a t i o n o f 4 mg/mL 2 1 6 3.3 2 Human e r y t h r o c y t e e n c a p s u l a t i o n o f a r e d c e l l p a r t i c l e i n i s o l a t e d human f i b r i n o g e n 2 1 8 3 . 3 3 a M e a s u r e m e n t o f t h e e n c a p s u l a t i o n p a r a m e t e r s Zc a n d Rs 2 1 9 3.33b S u r f a c e a f f i n i t y o f t h e r e d c e l l s u r f a c e f o r a r e d c e l l b e a d a s a f u n c t i o n o f f i b r i n o g e n c o n c e n t r a t i o n . . 2 1 9 3.34 S u r f a c e c o n c e n t r a t i o n i n a c r o s s - b r i d g e d c o n t a c t . . . 2 2 7 A l R e l a t i o n b e t w e e n 5 m i n u t e m i c r o h e m a t o c r i t ( H ) a n d t h e w e i g h t r a t i o (/2>) f o r r e d c e l l s r e s u s p e n d e d i n P B S / a l b u m i n 2 7 0 A2 S e p h a d e x G75 c h r o m a t o g r a p h y o f 1 2 5I - a l b u m i n s h o w i n g t h e s e p a r a t i o n d a t a a f t e r c o r r e c t i o n f o r c o u n t i n g - r a t e , b a c k g r o u n d , a n d t a i l i n g 2 7 3 A3 D e t e r m i n a t i o n o f p a r a m e t e r s f o r e q u a t i o n 5.7 2 7 7 A4 D e p e n d e n c e o f u ) ( o ( i ) o n t h e d i f f e r e n c e (/V-/V 2 9 1 A5 S y s t e m a t i c e r r o r i n e s t i m a t i o n o f r f 2 f r o m r - j ^ d u e t o c h a n g e s i n t h e v a l u e o f P i 2 9 6 - x i i i -L I S T OF A P P E N D I C E S A P P E N D I X P a g e 1 L i s t o f s y m b o l s a n d a b b r e v i a t i o n s 2 6 1 2 D e f i n i t i o n o f c e l l a n d s o l u t i o n c o m p a r t m e n t s . . . . 2 6 6 3 R e l a t i o n b e t w e e n a n d H 2 6 8 4 F r e e v e r s e s p r o t e i n - b o u n d r a d i o l a b e l 2 7 1 5 R e l a t i v e e f f i c i e n c y a s a f u n c t i o n o f a p p a r e n t CPM . . 2 7 5 6 D e f i n i t i o n o f d i s t r i b u t i o n r a t i o s 2 7 9 7 R a d i o l a b e l i n t h e i n t e r c e l l u l a r c o m p a r t m e n t 2 8 1 8 R a d i o l a b e l i n t h e i n t r a c e l l u l a r c o m p a r t m e n t 2 8 2 9 a n d e s t i m a t e s 2 8 4 1 0 B i n d i n g e s t i m a t e d f r o m s o l u t i o n m e a s u r e m e n t s . . . . 2 8 5 11 E s t i m a t e o f i n t r a c e l l u l a r r a d i o l a b e l 2 8 8 1 2 R e l a t i o n b e t w e e n ri 2 a n d rf 2 2 9 4 1 3 B i n d i n g i s o t h e r m a n d s u r f a c e c o n c e n t r a t i o n 2 9 7 14 I n t e r c e l l u l a r v o l u m e a n d s e p a r a t i o n 2 9 9 15 Wash p r o c e d u r e 3 0 1 16 E x p o n e n t i a l m o d e l f o r w a s h c u r v e s 3 0 4 1 7 A n a l y s i s o f w a s h c u r v e s 3 0 6 - x i v -ACKNOWLEDGEMENTS I w i s h t o e x p r e s s my s i n c e r e t h a n k s t o Don B r o o k s , my s u p e r v i s o r , f o r h i s s u p p o r t a n d e n c o u r a g e m e n t , b o t h a c a d e m i c a n d o t h e r w i s e , a n d t o t h e B r i t i s h C o l u m b i a n a n d C a n a d i a n H e a r t F o u n d a t i o n s f o r f i n a n c i a l s u p p o r t . I e x t e n d s p e c i a l t h a n k s t o D r . E v a n E v a n s f o r m a k i n g h i s f a c i l i t i e s f o r c e l l - c e l l a d h e s i o n s t u d i e s a v a i l a b l e , f o r h i s d i s c u s s i o n o f r e s u l t s , a n d t o B a r b a r a K u k a n f o r h e r e x p e r t i s e i n c a r r y i n g o u t t h e r e l a t e d e x p e r i m e n t s . I w i s h t o t h a n k D r . P h i l R e i d , C h a r l e s Ramey, a n d K i m S h a r p f o r t h e i r d i s c u s s i o n s a n d i d e a s , D r . B a s i l C h i u f o r c e l l e l e c t r o p h e t i c m e a s u r e m e n t s , W a i - P a n C h a n a n d J o h n C a v a n a g h f o r d e v e l o p m e n t o f a n d a s s i s t a n c e w i t h t h e i s o l a t i o n o f f i b r i n o g e n , a n d Anne B i s h o p f o r h e l p a n d g u i d a n c e i n t h e u s e o f t h e d e p a r t m e n t a l w o r d p r o c e s s o r . I n a d d i t i o n t o t h e s e s p e c i f i c o b l i g a t i o n s , I f e e l t h a t I owe a c o n s i d e r a b l e t h a n k s t o t h e s t a f f a n d s t u d e n t s f o r c o n t r i b u t i n g t o t h e g o o d a t m o s p h e r e o f t h e d e p a r t m e n t a n d t o f r i e n d s a n d f a m i l y f o r p u t t i n g up w i t h me d u r i n g t h i s t i m e . - XV -' t h e m o s t u n i v e r s a l q u a l i t y i s d i v e r s i t y ' MONTAIGNE - 1 -CHAPTER 1 INTRODUCTION 1.1 THE CLINICAL RELEVANCE OF ERYTHROCYTE AGGREGATION The sedimentation of freshly drawn blood before normal coagulation occurred was observed in antiquity and was associated with disease. The humoral theory of pathology stated that the observation of the phlegma , later the "inflammatory crust" of coagulated blood, was evidence of imbalance of the humors - the cause of disease (Fahraeus 1929). In anticoagulated blood, the mechanism responsible for blood sedimentation was shown by Fahraeus (1921) to be aggregation and sedimentation of the erythrocytes. The measurement of erythrocyte sedimentation rate (ESR) followed from his observations and, because increased ESR was associated with a wide range of diseases, was widely adopted as a non-specific indicator of disease and disease progression (Thorsen and Hint 1950, Hardwicke and Squire 1952). Changes in ESR were shown to reflect the composition of the plasma rather than the properties of the c e l l s . Red c e l l aggregation was observed in plasma and the fibrinogen fraction of plasma, but was not seen in normal serum (Fahraeus 1921). The reversible, largely linear red c e l l aggregates were called rouleaux due to their - 2 -c h a r a c t e r i s t i c a p p e a r a n c e . The g l o b u l i n f r a c t i o n o f b l o o d was shown t o a g g r e g a t e r e d c e l l s a s w e l l , b u t l e s s s t r o n g l y t h a n f i b r i n o g e n ( F a h r a e u s 1 9 2 1 , H a r d w i c k e a n d S q u i r e 1 9 5 2 ) . I n t e r e s t i n p l a s m a s u b s t i t u t e s l e d t o t h e i n v e s t i g a t i o n o f t h e e f f e c t s o f d e x t r a n s u p o n e r y t h r o c y t e s e d i m e n t a t i o n . T h o r s e n a n d H i n t ( 1 9 5 0 ) s h o w e d t h a t ESR a n d r e d c e l l a g g r e g a t i o n i n c r e a s e d w i t h d e x t r a n c o n c e n t r a t i o n a n d m o l e c u l a r w e i g h t . R e v e r s i b l e a g g r e g a t i o n o f r e d c e l l s was t h e r e f o r e i n t e r p r e t e d a s b e i n g m e d i a t e d b y i n t e r a c t i o n o f m a c r o m o l e c u l e s w i t h t h e c e l l s u r f a c e . A c o u n t e r p a r t o f e r y t h r o c y t e s e d i m e n t a t i o n in v i t r o was o b s e r v e d i n v i v o a n d was r e f e r r e d t o a s ' b l o o d s l u d g i n g ' ( P l o m a n 1 9 2 0 , K n i s e l y e t a l . 1 9 4 7 ) . L i k e e r y t h r o c y t e s e d i m e n t a t i o n , i n v i v o r e d c e l l a g g r e g a t i o n was f o u n d t o be n o n - s p e c i f i c a l l y a s s o c i a t e d w i t h a w i d e v a r i e t y o f p a t h o l o g i c a l c o n d i t i o n s ( K n i s e l y e t a l . 1 9 4 7 ) . I n many d i s e a s e s t h e r e i s s e c o n d a r y e l e v a t i o n o f t h e p l a s m a f i b r i n o g e n c o n c e n t r a t i o n , s o t h a t t h e same m e c h a n i s m t h a t c a u s e s r e d c e l l a g g r e g a t i o n i n v i t r o may be e f f e c t i v e i n v i v o . I n c r e a s e d ESR a n d i n t r a v a s c u l a r b l o o d s l u d g e h a v e a l s o b e e n o b s e r v e d i n a s s o c i a t i o n w i t h m u l t i p l e m y e l o m a , w h e r e t h e g l o b u l i n f r a c t i o n o f t h e p l a s m a i s e l e v a t e d ( M a r m o n t e t a l . 1 9 5 7 ) , a n d a s a r e s u l t o f i n t r a v e n o u s i n f u s i o n o f h i g h m o l e c u l a r w e i g h t d e x t r a n ( T h o r s e n a n d H i n t 1 9 5 0 ) . N o r m a l r e d c e l l s a r e e a s i l y d e f o r m e d . T h i s a n d t h e i r f a v o u r a b l e s u r f a c e a r e a t o v o l u m e r a t i o e n a b l e s r e d c e l l s t o p a s s t h r o u g h v e s s e l s i n t h e m i c r o c i r c u l a t i o n w h e r e t h e f r e e s p a c e i s l e s s t h a n t h e c e l l ' s maximum d i a m e t e r (Canham a n d B u r t o n 1 9 6 8 , B r a n e m a r k 1 9 7 1 ) . A g g r e g a t i o n o f r e d c e l l s i m p e d e s n o r m a l b l o o d f l o w ( B r a n e m a r k - 3 -1 9 7 1 , B i c h e r 1 9 7 2 ) . F a h r a e u s ( 1 9 2 1 ) f e l t t h a t l o w l e v e l a g g r e g a t i o n m i g h t h a v e a p o s i t i v e p h y s i o l o g i c a l e f f e c t w i t h r e s p e c t t o t i s s u e p e r f u s i o n , w h i l e a g g r e g a t e s w h i c h w e r e l a r g e r a n d m o r e r e s i s t a n t t o d i s a g g r e g a t i o n , c o u l d a g g r a v a t e c l i n i c a l c o n d i t i o n s o r e v e n r e s u l t i n m o r t a l i t y . K n i s e l y e t a l . ( 1 9 4 7 ) o b s e r v e d v a r i a t i o n s i n t h e s i z e a n d c h a r a c t e r o f b l o o d s l u d g e a g g r e g a t e s . T hey d e s c r i b e d a g g r e g a t e s a s s o c i a t e d w i t h d i s e a s e a s a g g l u t i n a t e d ( " n o t l i k e r o u l e a u x " ) a n d f e l t t h a t c l i n i c a l c o n s e q u e n c e s s u c h a s f o c a l n e c r o s i s , e d e m a , c o m a , a n d a n e m i a c o u l d r e s u l t . B i c h e r ( 1 9 7 2 ) h a s r e v i e w e d t h e c l i n i c a l c a u s e s a n d c o n s e q u e n c e s o f i n t r a v a s c u l a r a g g r e g a t i o n . I n v i t r o v i s c o m e t r i c s t u d i e s o f a n t i c o a g u l a t e d w h o l e b l o o d h a v e f u r t h e r e d u n d e r s t a n d i n g o f i n v i v o r e d c e l l a g g r e g a t i o n . S i n c e t h e i n t r o d u c t i o n o f l o w s h e a r r a t e v i s c o m e t e r s , i t h a s b e e n shown t h a t t h e a p p a r e n t v i s c o s i t y o f b l o o d , o r o f s u s p e n s i o n s o f e r y t h r o c y t e s i n p l a s m a , i n c r e a s e s s t r o n g l y w i t h d e c r e a s i n g s h e a r r a t e b e l o w 10 sec-"'' ( W e l l s a n d M e r r i l l 1 9 6 1 , M e r r i l l e t a l . 1 9 6 3 ) . I t was s u g g e s t e d t h a t t h e l o w s h e a r r a t e i n c r e a s e i n t h e v i s c o s i t y . was d u e t o i n t e r a c t i o n o f p l a s m a p r o t e i n s w i t h t h e r e d c e l l membrane ( W e l l s e t a l . 1 9 6 2 ) . A g g r e g a t i o n o f r e d c e l l s was o b s e r v e d t o i n c r e a s e a t l o w e r s h e a r r a t e s , s o t h a t i n c r e a s e d s u s p e n s i o n v i s c o s i t y was r e l a t e d t o r e d c e l l a g g r e g a t i o n ( G o l d s t o n e e t a l . 1 9 7 0 ) . The f o r m a t i o n o f e x t e n s i v e r e d c e l l a g g r e g a t e s was a l s o i m p l i e d b y t h e o b s e r v a t i o n o f a y i e l d s t r e s s f o r b l o o d , i . e . a d i s t i n c t s h e a r s t r e s s w o u l d be r e q u i r e d t o make b l o o d f l o w ( C o k e l e t e t a l . 1 9 6 3 , M e r r i l l e t a l . 1 9 6 3 , W e l l s e t a l . 1 9 6 4 ) . T h e s e p r o p e r t i e s o f n o r m a l b l o o d w e r e shown t o be r e l a t e d t o t h e f i b r i n o g e n c o n c e n t r a t i o n o f t h e p l a s m a ( M e r r i l l e t a l . 1 9 6 3 , W e l l s - 4 -e t a l . 1 9 6 4 ) . V i s c o m e t r i c s t u d i e s i n d i c a t e t h a t b u l k s h e a r s t r e s s e s g r e a t e r 2 t h a n 2.6 d y n e s / c m w i l l d i s a g g r e g a t e n o r m a l r e d c e l l a g g r e g a t e s ( G o l d s t o n e e t a l . 1 9 7 0 , S c h m i d - S c h o n b e i n e t a l . 1 9 7 6 ) . When c o m p a r e d t o e s t i m a t e s o f i n v i v o s h e a r s t r e s s , r e d c e l l a g g r e g a t e s due t o n o r m a l p l a s m a a r e e x p e c t e d t o be o b s e r v e d p r e d o m i n a n t l y i n t h e p o s t - c a p i l l a r y v e n u l e s , a s s h e a r s t r e s s e s i n t h e r e s t o f t h e c i r c u l a t i o n a r e l i k e l y s u f f i c i e n t t o c a u s e d i s a g g r e g a t i o n ( G o l d s t o n e e t a l . 1 9 7 0 , B e r m a n 1 9 6 4 ) . H o w e v e r , r e d c e l l a g g r e g a t e s f o r m e d i n d i a b e t e s a n d m y e loma p l a s m a a r e more r e s i s t a n t t o s h e a r d i s a g g r e g a t i o n ( S c h m i d - S c h o n b e i n e t a l . 1 9 7 6 ) a n d t h u s a g g r e g a t e s a s s o c i a t e d w i t h d i s e a s e c a n be e x p e c t e d t o be more w i d e l y d i s t r i b u t e d i n t h e c i r c u l a t i o n . The c u r r e n t v i e w o f i n v i v o e r y t h r o c y t e a g g r e g a t i o n i s t h a t r e d c e l l a g g r e g a t e s a s s o c i a t e d w i t h d i s e a s e a n d t r a u m a a r e s e c o n d a r y e f f e c t s , b u t c a n h a v e c l i n i c a l c o n s e q u e n c e s ( B i c h e r 1 9 7 2 , B e r g e n t z e t a l . 1 9 7 7 ) . I n a d d i t i o n , a l t h o u g h a g g r e g a t e s i n d i s e a s e a r e m o r e r e s i s t a n t t o s h e a r d i s a g g r e g a t i o n a n d a r e c o r r e l a t e d w i t h i n c r e a s e s i n g l o b u l i n p r o t e i n s a s w e l l a s f i b r i n o g e n , t h e r o u l e a u x o b s e r v e d i n p h y s i o l o g i c a l p l a s m a r e p r e s e n t t h e same p h e n o m e n a , w i t h r e s p e c t t o t h e m e c h a n i s m s o f r e d c e l l - r e d c e l l a d h e r e n c e , a s a g g r e g a t e s f o r m e d s e c o n d a r y t o d i s e a s e ( B e r g e n t z e t a l . 1 9 7 7 ) . T h u s , t h e s t u d y o f f i b r i n o g e n ' s i n t e r a c t i o n w i t h t h e r e d c e l l membrane i s a t t r a c t i v e a s f i b r i n o g e n i s i m p l i c a t e d i n a m o d e l o f c l i n i c a l l y r e l e v e n t i n t r a v a s c u l a r a g g r e g a t i o n . - 5 -1.2 MECHANISMS OF C E L L - A D H E S I O N The a g g r e g a t i o n a n d a d h e s i o n o f c e l l s h a s b e e n s t u d i e d i n a w i d e r a n g e o f p h y s i o l o g i c a l a n d m o d e l s y s t e m ( C u r t i s 1 9 7 3 ) . The m e c h a n i s m s p r o p o s e d t o e x p l a i n c e l l - c e l l a n d c e l l - s u r f a c e a d h e s i o n s may be d i v i d e d i n t o t h o s e w h i c h a r e b a s e d o n p h y s i c a l i n t e r a c t i o n s a n d t h o s e b a s e d o n m o l e c u l a r i n t e r a c t i o n s ( C u r t i s 1 9 7 3 ) . The m o l e c u l a r m e c h a n i s m s i n v o l v e s h o r t r a n g e i n t e r a c t i o n s b e t w e e n s u r f a c e m o l e c u l e s o f a d j a c e n t c e l l s ( T y l e r 1 9 4 0 , B e l l 1 9 7 8 ) , b e t w e e n c e l l s u r f a c e m o l e c u l e s a n d i n t e r p o s e d n o n - c e l l u l a r m o l e c u l e s ( T h o r s e n a n d H i n t 1 9 5 0 , R u h e n s t r o t h - B a u e r 1 9 6 1 , C h i e n a n d J a n 1 9 7 3 ) , o r due t o i n t e r a c t i o n s b e t w e e n c e l l s u r f a c e a n d s o l u t i o n m o l e c u l e s ( S i l b e r b e r g 1 9 8 3 ) a n a l o g o u s t o t h o s e w h i c h r e s u l t i n t h e p h a s e s e p a r a t i o n o f m i x e d p o l y m e r s o l u t i o n s ( F l o r y 1 9 5 3 , L a u r e n t 1 9 6 3 , A l b e r t s s o n 1 9 7 0 ) . A p h y s i c a l m e c h a n i s m b a s e d u p o n a b a l a n c e b e t w e e n t h e a t t r a c t i v e , e l e c t r o d y n a m i c L o n d o n - V a n d e r W a a l ' s f o r c e a n d r e p u l s i v e e l e c t r o s t a t i c ( C u r t i s 1 9 6 0 , P e t h i c a 1 9 6 1 ) a n d s t e r i c f o r c e s ( L e r c h e 1 9 8 3 ) i s d e r i v e d f r o m t h e DLVO t h e o r y o f c o l l o i d s t a b i l i t y ( D e r j a g i n a n d L a n d a u 1 9 4 1 , V e r w e y a n d O v e r b e e k 1 9 4 8 ) . R e c e n t l y i t h a s b e e n p r o p o s e d t h a t a n i n t e r a c t i o n due t o p o l a r i z e d c e l l membrane m o l e c u l e s ( F r o h l i c h 1 9 6 8 ) may b e r e l e v a n t t o a g g r e g a t i o n o f e r y t h r o c y t e s ( R o w l a n d s e t a l . 1 9 8 2 ) . Some e v i d e n c e f o r a d h e s i o n o f p a r t i c l e s ( o r c e l l s ) b y v a r i o u s m e c h a n i s m s m e n t i o n e d h e r e h a s b e e n p u t f o r w a r d . A s o n e m i g h t e x p e c t , d i f f e r e n t f a c t o r s c o n t r i b u t e t o a n e f f e c t t o d i f f e r e n t d e g r e e s i n d i f f e r e n t s y s t e m s . The a g g r e g a t i o n o f c e l l s i n a g i v e n s i t u a t i o n may be d e p e n d e n t u p o n s e v e r a l m e c h a n i s m s , h o w e v e r , f u r t h e r d i s c u s s i o n h e r e - 6 -w i l l b e r e s t r i c t e d t o t h o s e w h i c h r e q u i r e t h e p r e s e n c e o f m a c r o m o l e c u l e s i n s o l u t i o n w i t h t h e a g g r e g a t i n g c e l l s . 1.2.1 F r o h l i c h i n t e r a c t i o n : a l o n g r a n g e m e c h a n i s m An i n t e r a c t i o n b e t w e e n c e l l s b o u n d e d b y membranes p o l a r i z e d b y a t r a n s - m e m b r a n e e l e c t r o s t a t i c p o t e n t i a l d i f f e r e n c e a t d i s t a n c e s o n t h e o r d e r o f m i c r o n s , d u e t o e n e r g y e x c h a n g e s a t f r e q u e n c e s o n t h e o r d e r o f I O1 1 H e r t z ( p h o n o n s ) , h a s b e e n p r o p o s e d b y F r o h l i c h ( 1 9 6 8 ) . T h i s m e c h a n i s m o f c e l l a g g r e g a t i o n h a s b e e n u s e d t o e x p l a i n i n c r e a s e s i n r e d c e l l d i f f u s i o n r a t e s ( R o w l a n d s e t a l . 1 9 8 2 , F r i t z 1 9 8 4 ) f o r c e l l s w i t h n o r m a l membrane p o t e n t i a l s r e l a t i v e t o t h o s e f o r m e t a b o l i c a l l y d e p l e t e d , membrane d e p o l a r i z e d , o r g l u t a r a l d e h y d e - f i x e d r e d c e l l s . I n a d d i t i o n t o a n o r m a l membrane p o t e n t i a l , t h e i n t e r a c t i o n a p p e a r s t o r e q u i r e m a c r o m o l e c u l e s l i k e p o l y v i n y l p y r r o l i d o n e ( P V P ) ( F r i t z 1 9 8 4 ) , d e x t r a n , o r f i b r i n o g e n ( S e w c h a n d e t a l . 1 9 8 2 ) i n s o l u t i o n . A s p o i n t e d o u t by S e w c h a n d e t a l . ( 1 9 8 2 ) , t h e t h e o r y p u t f o r w a r d a s a n e x p l a n a t i o n o f t h e s e o b s e r v a t i o n s d o e s n o t i n c l u d e t h e i n f l u e n c e o f m a c r o m o l e c u l e s . T h u s , i f t h e F r o h l i c h i n t e r a c t i o n i s t o be c o n s i d e r e d , t h e r e q u i r e m e n t f o r m a c r o m o l e c u l e s m u s t be i n c o r p o r a t e d i n t o - t h e t h e o r y . H o w e v e r , i t i s c l a i m e d t h a t m i t o t i c a l l y a c t i v e c e l l s d e m o n s t r a t e a F r o h l i c h - t y p e i n t e r a c t i o n w i t h i n o r g a n i c p a r t i c l e s ( P o h l 1 9 8 1 ) , w i t h o u t m a c r o m o l e c u l e s p r e s e n t . I f t h e s e o b s e r v a t i o n s a r e a c c e p t e d , t h e m o d i f i c a t i o n s t o t h e t h e o r y w i l l h a v e t o d i s t i n g u i s h t h i s c a s e f r o m t h a t o f c e l l - c e l l a g g r e g a t i o n . S e w c h a n d e t a l . ( 1 9 8 2 ) p r o p o s e d t h a t t h e s e c o n d a r y o r t e r t i a r y s t r u c t u r e o f t h e m a c r o m o l e c u l e d e t e r m i n e s w h e t h e r o r n o t t h e i n t e r a c t i o n w i l l be t r a n s m i t t e d , b u t d o e s n o t d i s c u s s t h e c o n s i d e r a b l e - 7 -c h e m i c a l a n d c o n f o r m a t i o n a l d i f f e r e n c e s b e t w e e n P V P , d e x t r a n , f i b r i n o g e n , a n d a l b u m i n . The f i r s t t h r e e o f t h e s e do ' t r a n s m i t ' t h e i n t e r a c t i o n w h i l e a l b u m i n , e v e n a t p h y s i o l o g i c a l c o n c e n t r a t i o n s , d o e s n o t . The c o n f o r m a t i o n a l e x p l a n a t i o n i s f u r t h e r c o m p l i c a t e d b y t h e f a c t t h a t a l b u m i n a g g r e g a t e d b y h e a t i n g s e r u m c a u s e s r e d c e l l s t o f o r m r o u l e a u x ( F o r s d y k e e t a l . 1 9 8 2 ) . The c o n f o r m a t i o n o f h e a t a g g r e g a t e d a l b u m i n i s l i k e l y t o be l e s s p r e d i c a b l e t h a n t h a t o f PVP o r d e x t r a n . The d i f f u s i o n r a t e d e t e r m i n a t i o n s r e q u i r e d e s t i m a t e s o f p a r t i c l e n u m b e r s w i t h t i m e ( R o w l a n d s e t a l . 1 9 8 2 ) o r l i g h t s c a t t e r i n g a u t o c o r r e l a t i o n w i t h t i m e ( F r i t z 1 9 8 4 ) b o t h o f w h i c h c h a n g e b e c a u s e o f c e l l a g g r e g a t i o n . N e i t h e r m e t h o d i s s p e c i f i c f o r t h e d e t e c t i o n o f F r o h l i c h i n t e r a c t i o n s , a s a n y c e l l - c e l l a t t r a c t i o n o f l o n g o r s h o r t r a n g e w o u l d a f f e c t t h e m e a s u r e m e n t s . F r i t z ( 1 9 8 4 ) n o t e d t h a t t h e d i f f u s i o n r a t e f o r r e d c e l l s was s l i g h t l y e l e v a t e d i n t h e p r e s e n c e o f h i g h m o l e c u l a r w e i g h t P V P e v e n when t h e c e l l s w e r e m e t a b o l i c a l l y i n a c t i v e . He s u g g e s t e d t h a t some a d d i t i o n a l i n t e r a c t i o n b e t w e e n t h e c e l l s a n d m a c r o m o l e c u l e s was o c c u r r i n g . T h u s t h e e x p e r i m e n t a l d e t e r m i n a t i o n o f t h e F r o h l i c h i n t e r a c t i o n , i f p r e s e n t , c o u l d h a v e s h o r t e r r a n g e i n t e r a c t i o n s s u p e r i m p o s e d u p o n t h e m . P r e s u m a b l y , c e l l - c e l l c o n t a c t s f o r m e d d u e t o s i m u l t a n e o u s a c t i o n o f t w o a t t r a c t i v e m e c h a n i s m s w o u l d b e more e n e r g e t i c t h a n t h o s e d u e t o t h e s e p a r a t e m e c h a n i s m s . A s a r e s u l t , c o m p a r i s o n o f t h e f o r m a t i o n r a t e a n d s t r e n g t h o f c o n t a c t s b e t w e e n r e d c e l l s w i t h o r w i t h o u t membrane p o t e n t i a l s ( m e t a b o l i c a l l y a c t i v e o r d e p l e t e d ) w o u l d p u t t h e F r o h l i c h m e c h a n i s m i n p e r s p e c t i v e . S u c h m e a s u r e m e n t s h a v e n o t b e e n r e p o r t e d , h o w e v e r . - 8 -W i t h o u t d e t e c t i o n o f t h e p h o n o n r a d i a t i o n , t h e F r o h l i c h m e c h a n i s m m u s t be c o n s i d e r e d a s u n l i k e l y . C h a n g e s i n t h e ' d i f f u s i o n r a t e s ' o f t h e r e d c e l l s may w e l l h a v e b e e n d u e t o c h a n g e s i n r e d c e l l s h a p e o r f l e x i b i l i t y ( R a m p l i n g a n d S i r s 1 9 7 2 , D u p o n t a n d S i r s 1 9 7 7 ) . I n a d d i t i o n , t h e f a c t t h a t a ) m e t a b o l i c a l l y a c t i v e r e d c e l l s do n o t v i s i b l y a g g r e g a t e i n t h e a b s e n c e o f m a c r o m o l e c u l e s , a n d b ) t h a t m e t a b o l i c a l l y i n a c t i v e ( f i x e d ) r e d c e l l s do a g g r e g a t e i n t h e p r e s e n c e o f , f o r e x a m p l e , d e x t r a n ( K n o x ejt a l . 1 9 7 5 ) a r g u e a g a i n s t t h e F r o h l i c h m e c h a n i s m . 1.2.2 P h a s e s e p a r a t i o n I t h a s b e e n p r o p o s e d t h a t w e a k , n o n - s p e c i f i c c e l l u l a r a g g r e g a t i o n i s d u e t o t h e e x c l u s i o n o f c e l l s u r f a c e g l y c o p r o t e i n s by m a c r o m o l e c u l e s i n s o l u t i o n ( B y c h k o v a n d K u z ' m i n a 1 9 7 7 , E d w a r d s 1 9 7 8 ) . C e l l a g g r e g a t i o n i s t h u s s e e n a s d u e t o t h e m e c h a n i s m r e s p o n s i b l e f o r t h e p h a s e s e p a r a t i o n o f m i x t u r e s o f p o l y m e r s ( F l o r y 1 9 5 3 , A l b e r t s s o n 1 9 7 0 ) a n d t h e p r e c i p i t a t i o n o f p r o t e i n s b y p o l y m e r s ( L a u r e n t 1 9 6 3 , P o i s o n e t a l . 1 9 6 4 , M c P h e r s o n 1 9 7 6 ) . F o r p h a s e s e p a r a t i o n t o o c c u r t h e e n t r o p y d e c r e a s e a s s o c i a t e d w i t h t h e s e p a r a t i o n m u s t be m o r e t h a n o f f s e t by a d e c r e a s e i n t h e i n t e r n a l e n e r g y o f t h e s y s t e m . The c h a n g e i n t h e f r e e e n e r g y o f m i x i n g a s s o c i a t e d w i t h t h e s e p a r a t i o n w i l l b e a s s o c i a t e d w i t h a d e c r e a s e i n t h e number o f i n t e r a c t i o n s b e t w e e n u n l i k e m o l e c u l e s w h i l e t h e number o f i n t e r a c t i o n s b e t w e e n l i k e m o l e c u l e s i n c r e a s e s . When t h e a f f i n i t y o f t h e l i k e - f o r - l i k e e x c e e d s t h a t o f l i k e - f o r - u n l i k e b y m o r e t h a n a c e r t a i n a m o u n t , p h a s e s e p a r a t i o n r e s u l t s . I n many i n s t a n c e s t h e f r e e e n e r g y o f m i x i n g o f p o l y m e r s y s t e m s a r e r a i s e d b y m i x i n g s o t h a t - 9 -c o n d i t i o n s , o f s a y t e m p e r a t u r e a n d c o n c e n t r a t i o n , may be f o u n d w h e r e p h a s e s e p a r a t i o n o c c u r s ( S i l b e r b e r g 1 9 8 3 ) . P h a s e s e p a r a t i o n i s s o m e t i m e s d e s c r i b e d a s b e i n g d u e t o ' s t e r i c e x c l u s i o n ' . U s e o f t h i s t e r m e m p h a s i s e s a m e c h a n i c a l p o i n t o f v i e w o f t h e a b o v e m e c h a n i s m . E x c l u s i o n o f o n e p o l y m e r m o l e c u l e f r o m t h e d o m a i n o f a m o l e c u l e o f t h e s e c o n d p o l y m e r s p e c i e s r e s u l t s f r o m more f a v o u r a b l e i n t e r a c t i o n s b e t w e e n l i k e p o l y m e r s e g m e n t s t h a n b e t w e e n u n l i k e s e g m e n t s . S t u d i e s o f r e d c e l l ( B y c h k o v a n d K u z ' m i n a 1 9 7 7 , F o r s d y k e e t a l . 1 9 8 2 ) a n d r e t i n a l c e l l ( M o r r i s 1 9 7 9 ) a g g r e g a t i o n h a v e b e e n r e p o r t e d a n d i n t e r p r e t e d a s s u p p o r t i v e o f t h e p h a s e s e p a r a t i o n m e c h a n i s m . B y c h k o v a n d K u z ' m i n a ( 1 9 7 7 ) d e m o n s t r a t e d t h a t t h e g l y c o s a m i n o g l y c a n s (GAG) p o t a s s i u m h y a l u r o n a t e a n d c h o n d r o i t i n - A -s u l p h a t e i n d u c e a g g r e g a t i o n o f r a b b i t r e d c e l l s . The a g g r e g a t i o n was s h o w n t o i n c r e a s e i n a d o s e d e p e n d e n t m a n n e r a n d was i n h i b i t e d b y a d d i t i o n o f h e p a r i n . The i n h i b i t i o n o f a g g r e g a t i o n by h e p a r i n was e x p l a i n e d a s d u e t o t h e d i s r u p t i o n o f t h e c e l l e x c l u s i o n e f f e c t . No m e a s u r e m e n t s o f t h e b i n d i n g o f t h e s e s u b s t a n c e s t o r e d c e l l s w e r e r e p o r t e d o r r e f e r r e d t o . W i t h o u t a d e m o n s t r a t i o n t h a t b i n d i n g o f GAG d i d n o t o c c u r i t i s n o t p o s s i b l e t o c l e a r l y e x c l u d e t h e p o s s i b i l i t y t h a t a g g r e g a t i o n was d u e t o t h e c r o s s - b r i d g i n g m e c h a n i s m . I n a d d i t i o n , t h e a c t i o n o f h e p a r i n i n a g g r e g a t i o n e x p e r i m e n t s may be m o r e c o m p l e x t h a n s u g g e s t e d a s t h i s GAG h a s b e e n r e p o r t e d t o c a u s e human r e d c e l l s t o a g g r e g a t e ( J a n a n d C h i e n 1 9 7 6 ) a t c o n c e n t r a t i o n s j u s t a b o v e t h o s e u s e d t o i n h i b i t r a b b i t r e d c e l l a g g r e g a t i o n . S p e c i f i c b i n d i n g o f h e p a r i n t o t h e human r e d c e l l s u r f a c e was i m p l i e d by t h e o b s e r v a t i o n s - 10 -t h a t a ) h e p a r i n - i n d u c e d a g g r e g a t i o n was r e d u c e d b y r e m o v a l o f r e d c e l l s i a l i c a c i d , a n d t h a t b ) t h e r e d c e l l z e t a p o t e n t i a l i n c r e a s e d w i t h i n c r e a s i n g h e p a r i n c o n c e n t r a t i o n ( J a n a n d C h i e n 1 9 7 6 ) . F o r s d y k e e t a l . ( 1 9 8 2 ) s h o w e d t h a t r e d c e l l a g g r e g a t i o n c o u l d b e c a u s e d by p o l y m e r i z e d a l b u m i n a n d a r g u e d t h a t t h i s e f f e c t was d u e t o a s t e r i c e x c l u s i o n m e c h a n i s m . They a c c e p t e d a l a c k o f c h a n g e i n c e l l e l e c t r o p h o r e t i c m o b i l i t y a s e v i d e n c e f o r n e g l i g i b l e b i n d i n g o f a l b u m i n t o human e r y t h r o c y t e s ( M o n a g h a n a n d W h i t e 1 9 3 5 ) . H o w e v e r , t h e e l e c t r o p h o r e t i c m o b i l i t y o f c e l l s d e p e n d s o n a n u m b er o f p a r a m e t e r s . T h e s e i n c l u d e s u r f a c e c h a r g e d i s t r i b u t i o n , t h i c k n e s s o f t h e g l y c o c a l y x a n d t h e c o n c e n t r a t i o n a n d d i s t r i b u t i o n o f m o l e c u l a r e l e m e n t s t h a t p r o v i d e f l o w r e s i s t a n c e a s s o c i a t e d w i t h t h e c e l l s u r f a c e ( L e v i n e e t a l . 1 9 8 3 ) . L a c k o f c h a n g e i n m o b i l i t y c o u l d be d u e t o o f f s e t t i n g c h a n g e s i n t w o o r m o r e o f t h e s e f a c t o r s . I n a d d i t i o n , s i n c e t h e s e n s i t i v i t y o f t h i s m e t h o d i s a t b e s t 1 % a c h a n g e i n e f f e c t i v e s u r f a c e c h a r g e o f l e s s t h a n 1 x 1 0 ^ e s u w o u l d n o t be r e s o l v e d , s o t h a t b i n d i n g w h i c h i s s i g n i f i c a n t f r o m t h e p o i n t o f v i e w o f c e l l a g g r e g a t i o n may n o t be d e t e c t e d . M o r r i s ( 1 9 7 9 ) s h o w e d t h a t r e t i n a l c e l l s c a n be a g g r e g a t e d b y c h o n d r o i t i n s u l p h a t e a n d p o l y m e r s s u c h a s d e x t r a n , p o l y v i n y l -p y r r o l i d o n e , a n d p o l y e t h y l e n e g l y c o l ( P E G ) . He n o t e d f i v e c r i t e r i a t o t e s t t h e s t e r i c e x c l u s i o n h y p o t h e s i s . E x c l u s i o n s h o u l d be i n d e p e n d e n t o f c h a r g e o n t h e p o l y m e r a n d e x c l u d e d c e l l s i f c o m p l e x e s do n o t f o r m , s h o u l d i n c r e a s e w i t h i n c r e a s i n g p o l y m e r c o n c e n t r a t i o n , s h o u l d i n c r e a s e w i t h m o l e c u l a r w e i g h t , s h o u l d be g r e a t e r f o r l i n e a r t h a n f o r b r a n c h e d p o l y m e r s , a n d t h e m a c r o m o l e c u l e s s h o u l d n o t b i n d o r be p r e c i p i t a t e d - 11 -w i t h t h e c e l l s . E x c e p t f o r t h e f i r s t a n d l a s t p o i n t s , s u b s t a n t i a l l y t h e same p r e d i c t i o n s c a n b e made f o r a g g r e g a t i o n d u e t o a c r o s s - b r i d g i n g m e c h a n i s m . C o m p a r i s o n o f t h e a g g r e g a t i o n i n d u c e d b y v a r i o u s p o l y m e r s a n d p o l y m e r m o l e c u l a r w e i g h t f r a c t i o n s g e n e r a l l y s u p p o r t e d t h e f i r s t f o u r ' c r i t e r i a ' . H o w e v e r , t h e o b s e r v a t i o n t h a t c e l l a g g r e g a t i o n f i r s t i n c r e a s e d a n d t h e n d e c r e a s e d w i t h i n c r e a s i n g PEG c o n c e n t r a t i o n , i s more r e a d i l y e x p l a i n e d b y a c r o s s - b r i d g i n g t h a n a p h a s e s e p a r a t i o n m e c h a n i s m . M o r r i s s u g g e s t e d t h a t t h e e f f e c t o f i n c r e a s e d s o l u t i o n v i s c o s i t y r e d u c e d a g g r e g a t i o n b y i m m o b i l i z i n g c e l l s . H o w e v e r , he d i d n o t n o t e t h a t t h e c e l l s s h o u l d h a v e n o n e t h e l e s s e v e n t u a l l y a g g r e g a t e d . I n a d d i t i o n , t h e o p t i m u m a g g r e g a t i o n c o n c e n t r a t i o n s f o r t h e 2 0 , 0 0 0 a n d 6 , 0 0 0 m o l e c u l a r w e i g h t PEG f r a c t i o n s w e r e r e s p e c t i v e l y , 1 2 5 a n d 6 2 mg/mL . T h e s e r e s u l t s a r e i n f a c t c o n t r a r y t o t h e v i s c o s i t y h y p o t h e s i s a s t h e i m m o b i l i z i n g v i s c o s i t i e s i n t h e s e c a s e s w o u l d be r a d i c a l l y d i f f e r e n t . I n a c r o s s - b r i d g i n g m e c h a n i s m , d i s a g g r e g a t i o n may b e e x p l a i n e d a s d u e t o s a t u r a t i o n o f b i n d i n g s i t e s b y n o n - b r i d g i n g l i g a n d s . T h e b i n d i n g m e a s u r e m e n t s r e p o r t e d f o r r e t i n a l p r o t e o g l y c a n s a n d g l y c o s a m i n o g l y c a n s s h o w e d t h a t o n l y a b o u t 2 % o f t h e r a d i o a c t i v i t y p r e s e n t o n t h e a g g r e g a t i n g a g e n t s was p r e s e n t i n c e l l p e l l e t s . H o w e v e r , p e l l e t a c t i v i t y was s i g n i f i c a n t l y r e d u c e d by a d d i t i o n o f c a r r i e r c h o n d r o i t i n s u l p h a t e , s u g g e s t i n g t h a t s a t u r a b l e b i n d i n g was i n v o l v e d ( H o l l e n b e r g a n d C u a t r e c a s a s 1 9 7 9 ) . No d a t a was p r o v i d e d t o a l l o w a d e t e r m i n a t i o n o f t h e a mount o f GAG b o u n d . No b i n d i n g d a t a was s hown f o r t h e o t h e r p o l y m e r s i n v e s t i g a t e d , h o w e v e r , d e x t r a n ( B r o o k s 1 9 7 3 b ) a n d PEG ( S h a r p 1 9 8 5 ) b i n d i n g t o r e d c e l l s h a s b e e n m e a s u r e d . - 1 2 -D e x t r a n b i n d i n g t o r e d c e l l s h a s b e e n r e l a t e d t o r e d c e l l a g g r e g a t i o n b y B r o o k s ( 1 9 7 3 c ) . D e x t r a n - i n d u c e d a g g r e g a t i o n , l i k e P E G - i n d u c e d a g g r e g a t i o n , f i r s t i n c r e a s e s a n d t h e n d e c r e a s e s w i t h i n c r e a s i n g p o l y m e r c o n c e n t r a t i o n . The e f f e c t o f v i s c o s i t y o n r e d c e l l a g g r e g a t i o n was r u l e d o u t d i r e c t l y i n e x p e r i m e n t s w h e r e r e d c e l l s w e r e b r o u g h t i n t o c l o s e o p p o s i t i o n b y m i c r o m a n i p u l a t i o n (Buxbaum e t a l . 1 9 8 2 ) . T h e s e r e s u l t s w i l l b e d e s c r i b e d m o r e f u l l y i n S e c t i o n 1.2.3 . B i n d i n g o f 8 0 0 0 m o l e c u l a r w e i g h t PEG t o r e d c e l l s a t 6 0 mg/mL i s more -3 -3 t h a n 0.4 x 10 g a n d l e s s t h a n 1 x 1 0 g p e r g o f c e l l s ( S h a r p 1 9 8 5 ) . The l o w e r l i m i t i n g v a l u e r e p r e s e n t s PEG r e t a i n e d by c e l l s a f t e r w a s h i n g w i t h b u f f e r e d s a l i n e . T h i s a m ount d i d n o t s u p p o r t a g g r e g a t i o n a n d was e l u t e d when c e l l s w e r e w a s h e d w i t h P E G . The u p p e r l i m i t i n g v a l u e c o r r e s p o n d s t o a p p a r e n t PEG b i n d i n g i n c e l l p e l l e t s c o r r e c t e d f o r i n t e r c e l l u l a r t r a p p i n g . T h u s t h e p r o p o s a l t h a t w e a k , n o n - s p e c i f i c a g g r e g a t i o n o f c e l l s i s d u e t o a p h a s e s e p a r a t i o n m e c h a n i s m i s n o t s u p p o r t e d i n m o s t c a s e s . I n h y a l u r o n a t e - i n d u c e d r e d c e l l a g g r e g a t i o n i t may be t h a t t h i s m e c h a n i s m was e f f e c t i v e . H y a l u r o n a t e o f 2 x 1 06 d a l t o n m o l e c u l a r w e i g h t i s e s t i m a t e d t o e f f e c t i v e l y o c c u p y 1 0 0 % o f t h e a v a i l a b l e s o l u t i o n v o l u m e ( T o o l e 1 9 7 5 ) a t t h e same c o n c e n t r a t i o n a t w h i c h r e d c e l l a g g r e g a t i o n r e a c h e d p l a t e a u v a l u e s ( B y c h k o v a n d K u z ' m i n a 1 9 7 7 ) . H o w e v e r , i n t h e c a s e s o f PEG a n d d e x t r a n - i n d u c e d a g g r e g a t i o n a c r o s s - b r i d g i n g m e c h a n i s m i s m o r e r e a d i l y s u p p o r t e d . The g r e a t e s t d r a w - b a c k o f t h e p h a s e s e p a r a t i o n t h e o r y i s t h a t i t r e l i e s u p o n t h e d e m o n s t r a t i o n o f i n s i g n i f i c a n t b i n d i n g a n d t h u s t h e r e p u d i a t i o n o f t h e c r o s s - b r i d g i n g m e c h a n i s m f o r s u p p o r t . No t h e o r e t i c a l p r e d i c t i o n s h a v e - 1 3 -b e e n made a s t o t h e c o n c e n t r a t i o n a n d m o l e c u l a r w e i g h t r a n g e s o f p o l y m e r s o r p r o t e i n s w h i c h m i g h t i n d u c e c e l l s t o a g g r e g a t e by p h a s e s e p a r a t i o n a n d n o e x p e r i m e n t s h a v e b e e n c a r r i e d o u t t o s e e i f c e l l s u r f a c e p r o t e i n s ( g l y c o c a l y x ) u n d e r g o p h a s e s e p a r a t i o n f r o m c e l l a g g r e g a t i n g a g e n t s . 1.2.3 M o l e c u l a r b r i d g i n g The m o l e c u l a r b r i d g i n g m o d e l o f a g g r e g a t i o n i s i n t i m a t e l y a s s o c i a t e d w i t h t h e a d s o r p t i o n o f b r i d g i n g m o l e c u l e s t o t h e c e l l s u r f a c e s i n v o l v e d . The i d e a t h a t m o l e c u l e s o f a t o x i c m a t e r i a l o r d r u g m i g h t i n t e r a c t w i t h s p e c i f i c m o l e c u l e s o f a c e l l o r i g i n a t e d w i t h t h e w o r k o f L a n g l e y a n d E h r l i c h i n t h e f i r s t d e c a d e o f t h i s c e n t u r y . The c o n c e p t o f a c e l l r e c e p t o r a n d t h e m e t h o d s u s e d t o d i s t i n g u i s h r e c e p t o r f r o m n o n - r e c e p t o r i n t e r a c t i o n s a r e r e v i e w e d by H o l l e n b e r g a n d C u a t r e c a s a s ( 1 9 7 9 ) . I n b i n d i n g s t u d i e s t h e a m o unt o f a m a t e r i a l , o r l i g a n d , b o u n d t o a c e l l ( o r t i s s u e ) i s t o be r e l a t e d t o a c e l l u l a r r e s p o n s e . I n p r a c t i c e , a l i g a n d may b i n d t o more t h a n t h e r e c e p t o r o f i n t e r e s t , s o t h a t i t i s n e c e s s a r y t o d i s t i n g u i s h r e s p o n s e - s p e c i f i c b i n d i n g f r o m n o n - s p e c i f i c b i n d i n g . R e s p o n s e - s p e c i f i c b i n d i n g m u s t o c c u r w i t h a n a p p r o p r i a t e t i s s u e a n d m u s t c o r r e l a t e w i t h q u a n t i t a t i v e m e a s u r e s o f t h e r e s p o n s e a t p h y s i o l o g i c a l c o n c e n t r a t i o n s o f t h e l i g a n d . I n a d d i t i o n , r e s p o n s e - s p e c i f i c b i n d i n g i s u s u a l l y a s s o c i a t e d w i t h l i m i t e d a m o u n t s o f b o u n d l i g a n d p e r c e l l a n d i n h i b i t i o n o f t h e b i n d i n g a n d a l t e r a t i o n o f t h e r e s p o n s e by l i g a n d s w i t h s i m i l a r m o l e c u l a r s t r u c t u r e s . P h a r m a c o l o g i c a l s t u d i e s h a v e l e d t o t h e i d e a t h a t d r u g s a n d h o r m o n e s i n t e r a c t w i t h s p e c i f i c c e l l u l a r m o l e c u l e s p r i m a r i l y b e c a u s e o f - 14 -c o m p l e m e n t a r y m o l e c u l a r s t r u c t u r e s . The a b o v e c o n c e p t s , d e v e l o p e d i n p h a r m a c o l o g i c a l b i n d i n g s t u d i e s , h a v e b e e n a d a p t e d t o t h e s t u d y o f c e l l u l a r a g g r e g a t i o n . T h u s , c r o s s - b r i d g i n g m o l e c u l e s w o u l d b e e x p e c t e d t o i n t e r a c t w i t h s t r u c t u r e s l o c a t e d a t t h e c e l l s u r f a c e . A g g r e g a t i o n w o u l d b e e x p e c t e d t o c o r r e l a t e w i t h b i n d i n g o f t h e c r o s s - b r i d g i n g a g e n t . B i n d i n g o f l o w e r m o l e c u l a r w e i g h t a n a l o g s o r s a t u r a t i o n o f t h e s u r f a c e b i n d i n g w o u l d r e s u l t i n r e d u c e d a g g r e g a t i o n . I n a d d i t i o n , t h e b i n d i n g a f f i n i t y o f t h e c r o s s - b r i d g i n g a g e n t a n d t h e s u r f a c e c o n c e n t r a t i o n o f t h e b r i d g i n g e l e m e n t s d e f i n e a s u r f a c e a f f i n i t y w h i c h may be r e l a t e d t o t h e w o r k d o n e t o f o r m a c e l l - c e l l c o n t a c t ( E v a n s 1 9 8 0 , S k a l a k e t a l . 1 9 8 1 ) . S t u d i e s o f b o t h m o d e l a n d p h y s i o l o g i c a l s y s t e m s show t h a t c r o s s - b r i d g i n g i s a n i m p o r t a n t m e c h a n i s m i n c e l l u l a r a g g r e g a t i o n . I n i n s t a n c e s w h e r e t h e m o l e c u l a r i n t e r a c t i o n s a r e r e l a t i v e l y s p e c i f i c o r o f h i g h a f f i n i t y , r e l a t i o n s h i p s b e t w e e n b i n d i n g a n d c r o s s - b r i d g i n g h a v e b e e n d e m o n s t r a t e d . Where b i n d i n g a f f i n i t i e s a r e l o w e r , d e m o n s t a t i o n o f a l i n k w i t h a g g r e g a t i o n i s more d i f f i c u l t . Some e x a m p l e s o f c e l l s y s t e m s l i k e l y t o i n t e r a c t v i a b r i d g i n g f o l l o w . Human p l a t e l e t s i n t e r a c t s p e c i f i c a l l y w i t h f i b r i n o g e n v i a s u r f a c e r e c e p t o r s ( M u s t a r d e t a l . 1 9 7 8 ) . The s u r f a c e r e c e p t o r s a r e e x p o s e d when t h e p l a t e l e t s a r e a c t i v a t e d b y a n y o f a number o f a g o n i s t s , i n c l u d i n g t h r o m b i n a n d a d e n o s i n e d i p h o s p h a t e ( M a r g u e r i e e t a l . 1 9 7 9 , B e n n e t t a n d V i l a i r e 1 9 7 9 ) . The r e c e p t o r p o p u l a t i o n c o n s i s t s o f b o t h h i g h a n d l o w a f f i n i t y s i t e s a n d may b e a s s o c i a t e d w i t h t h e p l a t e l e t membrane g l y c o p r o t e i n s l i b a n d I l i a ( N i e w i a r o w s k i e t a l . 1 9 8 3 ) . The c o m p l e m e n t a r y b i n d i n g d o m a i n s o f f i b r i n o g e n a r e a s s o c i a t e d - 15 -w i t h c a r b o x y t e r m i n a l s e g m e n t s o f t h e Aot a n d Y c h a i n s ( N i e w i a r o w s k i e t a l . 1 9 8 3 , H a w i g e r e t a l . 1 9 8 3 ) . P l a t e l e t a g g r e g a t i o n b y f i b r i n o g e n i s i n i t i a l l y r e v e r s i b l e , b u t p r o g r e s s e s t o i r r e v e r s i b i l i t y ( Z u c h e r a n d P e t e r s o n 1 9 6 8 , M a r g u e r i e e t a l . 1 9 8 0 ) . The f i b r i n o g e n b i n d i n g r e q u i r e s C a+ + a n d i t i s p r o p o s e d t h a t a g g r e g a t i o n o f p l a t e l e t s i n v o l v e s a f i b r i n o g e n b r i d g i n g m e c h a n i s m ( N i e w i a r o w s k i e t a l . 1 9 8 3 ) . T h e f i b r o n e c t i n s a r e a c l a s s o f m a c r o m o l e c u l e w h i c h may a c t a s b r i d g i n g e l e m e n t s ; t h e i r f u n c t i o n s h a v e b e e n r e v i e w e d by H y n e s a n d Yamada ( 1 9 8 2 ) . The c e l l s u r f a c e - a t t a c h e d a n d s o l u b l e f i b r o n e c t i n s a r e b o t h i n v o l v e d i n c e l l - c e l l a n d c e l l - s u b s t r a t e a d h e s i o n . The p o s s i b l e r a n g e o f i n v o l v e m e n t i s v e r y w i d e b e c a u s e f i b r o n e c t i n h a s b e e n s h o w n t o i n t e r a c t s p e c i f i c a l l y w i t h c o l l a g e n , f i b r i n o g e n , f i b r i n , f a c t o r X H I a , a n d p r o t e o g l y c a n s a s w e l l a s w i t h c e l l s a n d b a c t e r i a . I n v i t r o a d h e s i o n a n d / o r s p r e a d i n g o f a v a r i e t y o f c e l l t y p e s i s p r o m o t e d b y f i b r o n e c t i n a d s o r b e d t o c u l t u r e d i s h e s ( G r i n n e l l 1 9 7 8 , H y n e s 1 9 8 1 ) . I t h a s b e e n e s t i m a t e d t h a t w e l l - s p r e a d h a m s t e r f i b r o b l a s t s may make 4 5 , 0 0 0 s u r f a c e c o n t a c t s w i t h f i b r o n e c t i n c o a t e d d i s h e s u n d e r o p t i m a l c o n d i t i o n s ( H u g h e s e t a l . 1 9 7 9 ) . S p e c i f i c i n t e r a c t i o n o f f i b r o n e c t i n w i t h f i b r o b l a s t s h a s r e c e n t l y b e e n d e m o n s t r a t e d t o b e m e d i a t e d b y a t e t r a p e p t i d e s e q u e n c e o f t h e m o l e c u l e ( P i e r s c h b a c k e r a n d R u o s l a h t i 1 9 8 4 ) a n d s u g g e s t s t h a t f i b r o n e c t i n ' s a d h e s i v e f u n c t i o n s i n v o l v e m o l e c u l a r b r i d g i n g . C e l l s i n c u l t u r e o f t e n r e q u i r e t r e a t m e n t w i t h e n z y m e s o r c h e l a t i n g a g e n t s t o p e r m i t r e s u s p e n s i o n ( G r i n n e l l 1 9 7 8 ) . Immune a g g l u t i n a t i o n o f r e d c e l l s i n h e m a g g l u t i n a t i o n r e a c t i o n s h a s b e e n s h o w n t o i n v o l v e s p e c i f i c c e l l s u r f a c e g l y c o p r o t e i n s a n d g l y c o l i p i d s ( G i b l e t t 1 9 7 9 ) . The s p e c i f i c i t y o f many b l o o d g r o u p - 16 -determinants i s associated with the terminal sugars of the oligosaccharides, however, amino acids may be involved in some cases (Springer and Desai 1975). Agglutination of red c e l l s occurs by intercellular cross-bridging of the surface antigens by soluble antibody and may be achieved by binding at as few as one hundred sites per c e l l (Greenbury et a l . 1963). Agglutinated red cells adhere tenaciously and when forcibly separated, result in myelin figures apparently produced from red c e l l membrane materials (Bessis 1973). In these physiologically important c e l l - c e l l interactions, high a f f i n i t y , specific adsorption and cross-bridging play important roles. The number of cross-linking bonds may be quite small, but the binding a f f i n i t i e s are frequently such that irreversible or relatively irreversible aggregation occurs. Determination of the binding relevant to aggregation has been achieved using radiotracers in conjunction with the analysis of binding inhibition. The binding analysis i s simplified in instances of high a f f i n i t y binding because the dissociation rates are frequently low enough to permit separation of the cells from the bulk medium without perturbing the amount bound (O'Brien 1975). However, not a l l of these techniques are applicable to the study of red c e l l aggregation associated with a r t i f i c i a l and natural polymers and proteins like fibrinogen. Studies have demonstrated that aggregation of red cells by positively charged macromolecules (Katchalsky et a l . 1959, Lalezari 1968, Jan and Chien 1973) is due to a cross-bridging mechanism. Adsorption of polylysine to red blood cells i s reversible and the amounts adsorbed may be determined from the c e l l concentration - 17 -dependence of electrophoretic mobility changes i n the presence of a constant amount of added polymer (Nevo et a l . 1955). However, aggregation induced by polylysine does not appear to be due simply to reduction of net c e l l charge as aggregation occurred without a large decrease i n c e l l zeta potential (Katchalsky et a l . 1959). Electron microscopic study of the contacts between red c e l l s due to different molecular weight polylysine species strongly suggested that t h i s agent bridges the gap between adjacent c e l l s . The morphological data was supported by the observation that although polylysine binding was reversed by washing with buffered s a l i n e , c e l l aggregation was not, the binding being s u f f i c i e n t l y strong to prevent e f f i c i e n t removal of the polylysine from the aggregation seam. In addition, disaggregation by addition of polyacids was most effective when these were of molecular weight comparable to that of the aggregating polylysine species. Polylysine has been shown to interact with the s i a l i c acid groups of red c e l l surface glycoproteins (Danon et a l . 1965) and the red c e l l aggregation that results i s most l i k e that observed with immunagglutination. Unlike red c e l l rouleaux induced by dextran, such agglutinated c e l l s do not s l i d e into a configuration which maximizes the surface contact between adjacent c e l l s . The difference i n behaviour i s f e l t to r e f l e c t the difference i n the binding a f f i n i t i e s of these agents for the c e l l surface (Chien 1975). Reversible, rouleaux-like aggregation of red c e l l s by dextrans was f i r s t studied by Thorsen and Hint (1950). Dextran induced rouleaux, l i k e those observed i n plasma, appear to be due to weak interactions as they are dispersed when washed with saline or subjected - 18 -t o m i l d f l u i d s h e a r f o r c e s ( B r o o k s 1 9 7 6 ) . N e v e r t h e l e s s , g o o d e v i d e n c e f o r t h e b r i d g i n g m e c h a n i s m o f c e l l a g g r e g a t i o n h a s b e e n p r o v i d e d . Red c e l l a g g r e g a t i o n a n d c e l l - c e l l s e p a r a t i o n d i s t a n c e h a s b e e n c o r r e l a t e d w i t h d e x t r a n c o n c e n t r a t i o n a n d m o l e c u l a r w e i g h t ( C h i e n a n d J a n 1 9 7 3 ) . The d e g r e e o f a g g r e g a t i o n was e s t i m a t e d f r o m t h e mean number o f c e l l s p e r a g g r e g a t e , E S R , a n d l o w s h e a r r a t e v i s c o m e t r y , w h i l e c e l l s e p a r a t i o n was d e t e r m i n e d b y t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y . D e x t r a n b i n d i n g t o r e d c e l l s a s a f u n c t i o n o f d e x t r a n c o n c e n t r a t i o n , m o l e c u l a r w e i g h t , a n d t h e i o n i c s t r e n g t h o f t h e medium h a s a l s o b e e n r e l a t e d t o r e d c e l l z e t a p o t e n t i a l s a n d a g g r e g a t i o n ( B r o o k s a n d Seaman 1 9 7 3 , B r o o k s 1 9 7 3 a , b , c ) . R e d c e l l a g g r e g a t i o n o c c u r s w i t h d e x t r a n s o f w e i g h t a v e r a g e m o l e c u l a r w e i g h t s g r e a t e r t h a n a b o u t 4 5 , 0 0 0 d a l t o n s ( B r o o k s 1 9 7 3 c , C h i e n a n d J a n 1 9 7 3 ) . A g g r e g a t i o n i n c r e a s e s t o a maximum a n d t h e n d e c r e a s e s t o d i s a g g r e g a t i o n w i t h i n c r e a s i n g f r e e d e x t r a n c o n c e n t r a t i o n . D e x t r a n b i n d i n g i n c r e a s e d more r a p i d l y a b o v e t h e d i s a g g r e g a t i o n c o n c e n t r a t i o n t h a n b e l o w i t . The o n s e t o f a g g r e g a t i o n i n d u c e d b y 7 7 , 0 0 0 d a l t o n d e x t r a n o c c u r r e d a t a c o n c e n t r a t i o n o f 5 mg/mL a n d was a s s o c i a t e d w i t h b i n d i n g o f a b o u t 4 0 , 0 0 0 m o l e c u l e s d e x t r a n p e r c e l l ( B r o o k s 1 9 7 3 b ) . I t was a r g u e d t h a t t h e a d s o r p t i o n i n c r e a s e a b o v e t h e d i s a g g r e g a t i o n c o n c e n t r a t i o n was d u e t o t h e i n c r e a s e d a r e a made a v a i l a b l e f o r a d s o r p t i o n when b r i d g i n g d e x t r a n m o l e c u l e s d e s o r b f r o m o n e o r t h e o t h e r o f t h e i n i t i a l l y a d j a c e n t c e l l s u r f a c e s ' . D i s a g g r e g a t i o n a t h i g h p o l y m e r c o n c e n t r a t i o n s was r e l a t e d t o e l e c t r o s t a t i c r e p u l s i o n w h i c h i n c r e a s e d w i t h i n c r e a s i n g p o l y m e r c o n c e n t r a t i o n a n d d e c r e a s i n g i o n i c s t r e n g t h ( B r o o k s 1 9 7 3 c , B r o o k s - 19 -1 9 7 6 ) , a l t h o u g h t h i s e x p l a i n a t i o n s e e m s l e s s l i k e l y now ( B r o o k s 1 9 8 5 ) . I n a d d i t i o n , r e d c e l l s t r e a t e d w i t h n e u r a m i n i d a s e t o r e m o v e c e l l s u r f a c e s i a l i c a c i d a r e a g g r e g a t e d b y l o w e r m o l e c u l a r w e i g h t d e x t r a n s a n d do n o t d i s a g g r e g a t e a t h i g h e r d e x t r a n c o n c e n t r a t i o n s . The s a t u r a t i o n b i n d i n g p e r c e l l was l e s s f o r a g g r e g a t e d , n e u r a m i n i d a s e t r e a t e d c e l l s t h a n f o r d i s a g g r e g a t e d , n o r m a l c e l l s a n d t h i s was a l s o i n t e r p r e t e d i n t e r m s o f t h e g r e a t e r c e l l s u r f a c e a r e a a v a i l a b l e f o r a d s o r p t i o n w i t h d i s a g g r e g a t e d c e l l s ( C h i e n e t a l . 1 9 7 7 ) . F i n a l l y , e x p e r i m e n t s i n w h i c h p a i r s o f r e d c e l l s a r e o b s e r v e d a n d c a n be t r a n s f e r r e d t o d i f f e r e n t m e d i a b y m i c r o m a n i p u l a t i o n show t h a t c e l l s a g g r e g a t e d b y d e x t r a n a n d t r a n s f e r r e d t o a d i s a g g r e g a t i n g ( h i g h e r ) d e x t r a n c o n c e n t r a t i o n do n o t d i s a g g r e g a t e s p o n t a n e o u s l y . I f t h e c e l l s a r e t h e n s e p a r a t e d by m i c r o m a n i p u l a t i o n , t h e y do n o t a g g r e g a t e , i n d i c a t i n g t h a t t h e c o n d i t i o n s i n t h e g a p b e t w e e n t h e c e l l s , n o t t h e e x t e r n a l medium g o v e r n s t h e a g g r e g a t i o n o f t h e c e l l s ( Buxbaum e t a l . 1 9 8 2 ) . I t h a s b e e n n o t e d t h a t d e x t r a n b i n d i n g t o r e d c e l l s i s r e a d i l y r e v e r s e d b y r e d u c t i o n o f t h e f r e e d e x t r a n c o n c e n t r a t i o n i n t h e s u s p e n d i n g s o l u t i o n ( B r o o k s 1 9 7 3 b ) . T h i s i s i n a c c o r d w i t h t h e r e a d y d i s a g g r e g a t i o n o f d e x t r a n - i n d u c e d r o u l e a u x b y s a l i n e . A s a r e s u l t t h e m e t h o d o l o g y u s e d t o d e t e r m i n i n g b i n d i n g m u s t a v o i d w a s h i n g t h e c e l l s . B r o o k s ( 1 9 7 3 b ) d e t e r m i n e d ^ H - d e x t r a n i n u l t r a c e n t r i f u g e d c e l l p e l l e t s a n d C h i e n e t a l . ( 1 9 7 7 ) d e t e r m i n e d d e x t r a n e l u t e d f r o m u l t r a c e n t r i f u g e d c e l l p e l l e t s c h e m i c a l l y . E v e n w i t h u l t r a c e n t r i f u g a t i o n , i t may be a r g u e d t h a t w i t h o u t a w ash p r o c e d u r e t h e s e m e t h o d s may r e s u l t i n a n o v e r e s t i m a t e o f b i n d i n g d u e t o s u p e r n a t a n t t r a p p e d w i t h t h e c e l l s . The - 2 0 -t e c h n i q u e o f c o m p a r i n g b i n d i n g i n t h e p r e s e n c e a n d a b s e n c e o f a s p e c i f i c i n h i b i t o r i s u n a v a i l a b l e t o d e x t r a n b i n d i n g s t u d i e s , s o t h a t l e s s d i r e c t a r g u e m e n t s w e r e r e q u i r e d t o e s t a b l i s h t h e v a l i d i t y o f t h e b i n d i n g e s t i m a t e s . H o w e v e r , t h e d e x t r a n b i n d i n g i s o t h e r m s ( b i n d i n g a s a f u n c t i o n o f c o n c e n t r a t i o n ) when c o m p a r e d w i t h t h e a g g r e g a t i o n b e h a v i o u r a r g u e f o r a c c e p t i n g t h e b i n d i n g e s t i m a t e s ( B r o o k s 1 9 7 3 c , C h i e n e t a l . 1 9 7 7 ) . I n a d d i t i o n , t h e o b s e r v a t i o n t h a t d e x t r a n b i n d i n g s h o w e d i n d i c a t i o n s o f s a t u r a t i o n ( C h i e n e t a l . 1 9 7 7 ) i m p l i e s t h a t t h e n o n - s p e c i f i c e f f e c t o f t r a p p i n g p l a y s o n l y a m i n o r r o l e . O f t h e m e c h a n i s m s by w h i c h n o n - c e l l u l a r m a c r o m o l e c u l e s may c a u s e c e l l u l a r a g g r e g a t i o n , o n l y t h e c r o s s - b r i d g i n g m e c h a n i s m i s s u p p o r t e d b y c o n v i n c i n g e v i d e n c e . I n t h e c a s e o f s p e c i f i c c r o s s - b r i d g i n g i n t e r a c t i o n s b i n d i n g h a s b e e n c o r r e l a t e d w i t h a g g r e g a t i o n , m o d i f i e d b y i n h i b i t o r s , a n d i n some c a s e s a s s o c i a t e d w i t h s u b s t r u c t u r e s o f t h e i n t e r a c t i n g m o l e c u l e s . T h e s e b r i d g i n g r e a c t i o n s may l e a d t o r e l a t i v e l y i r r e v e r s i b l e a g g r e g a t i o n o f c e l l s , s o t h a t , a l t h o u g h t h e y a r e p h y s i o l o g i c a l l y i m p o r t a n t , i t i s c l e a r t h a t i n t e r a c t i o n s o f t h i s t y p e a r e n o t r e s p o n s i b l e f o r t h e r o u l e a u x f o r m e d b y r e d c e l l s i n t h e p r e s e n c e o f f i b r i n o g e n . D e x t r a n - i n d u c e d a g g r e g a t i o n o f r e d c e l l s i s a g o o d m o d e l f o r f i b r i n o g e n - i n d u c e d r o u l e a u x f o r m a t i o n , b e c a u s e t h e a g g r e g a t e s p r o d u c e d h a v e s i m i l a r m o r p h o l o g y a n d a r e i n b o t h c a s e s r e a d i l y r e v e r s e d . The b r i d g i n g m e c h a n i s m i n d e x t r a n - i n d u c e d a g g r e g a t i o n i s s u p p o r t e d b y t h e r e l a t i o n b e t w e e n t h e a g g r e g a t i o n - d i s a g g r e g a t i o n b e h a v i o u r a n d t h e d e x t r a n b i n d i n g i s o t h e r m s . The r e l a t i o n s h i p b e t w e e n d e x t r a n m o l e c u l a r w e i g h t , d e x t r a n ' s e f f e c t s o n c e l l u l a r z e t a p o t e n t i a l s a n d - 21 -disaggregation i s further support for the cross-bridging mechanism. Thus, i t is certainly a strong possibility that rouleaux associated with fibrinogen are caused by a cross-bridging mechanism. In order to demonstrate this mechanism, i t i s necessary to relate c e l l surface binding of fibrinogen to a measure of cellular aggregation. - 2 2 -1.3 P R O T E I N ADSORPTION AND AGGREGATION OF RED C E L L S I n o r d e r t o d e t e r m i n e w h e t h e r o r n o t t h e c r o s s - b r i d g i n g m e c h a n i s m i s i m p o r t a n t i n f i b r i n o g e n - i n d u c e d a g g r e g a t i o n , f i b r i n o g e n b i n d i n g t o r e d c e l l s m u s t be r e l a t e d t o t h e a g g r e g a t i o n b e h a v i o u r o f t h e c e l l s . B e c a u s e o f t h e s i m i l a r i t y b e t w e e n d e x t r a n a n d f i b r i n o g e n - i n d u c e d r e d c e l l a g g r e g a t i o n , s i m i l a r t e c h n i c a l l i m i t a t i o n s i n d e t e r m i n i n g ' b i n d i n g m u s t be a n t i c i p a t e d . I n a d d i t i o n , t h e c o r r e l a t i o n b e t w e e n a g g r e g a t i o n b e h a v i o u r a n d b i n d i n g may n o t be a s c l e a r a s i t was f o r d e x t r a n - i n d u c e d a g g r e g a t i o n , b e c a u s e d i s a g g r e g a t i o n d o e s n o t o c c u r a t h i g h e r f i b r i n o g e n c o n c e n t r a t i o n s ( B r o o k s 1 9 7 6 ) . 1.3.1 M e a s u r e m e n t o f p r o t e i n a d s o r p t i o n t o c e l l s A b r a m s o n ( 1 9 2 9 ) a n d l a t e r M o n a g h a n a n d W h i t e ( 1 9 3 5 ) d e m o n s t r a t e d b i n d i n g o f p l a s m a p r o t e i n s t o p a r t i c l e s o f q u a r t z a n d g l a s s b y m e a s u r i n g t h e e l e c t r o p h o r e t i c m o b i l i t y o f t h e p a r t i c l e s . U s i n g t h i s t e c h n i q u e , t h e y c o n c l u d e d t h a t r e d c e l l s w e r e n o t c o a t e d w i t h a d s o r b e d p l a s m a p r o t e i n ( s ) i n e i t h e r s a l i n e o r p r o t e i n s o l u t i o n s . I t was a p p r e c i a t e d t h a t e l e c t r o p h o r e t i c m o b i l i t i e s m i g h t n o t b e p a r t i c u l a r l y s e n s i t i v e t o p a r t i a l c o a t i n g s o f p r o t e i n ( A b r a m s o n 1 9 2 9 ) . A m o d e l w h i c h a l l o w s q u a n t i t a t i v e e s t i m a t e s o f c e l l s u r f a c e c h a r g e h a s r e c e n t l y b e e n r e p o r t e d ( L e v i n e e t a l . 1 9 8 3 ) , h o w e v e r , t h i s a d v a n c e d o e s n o t o v e r c o m e t h e s e n s i t i v i t y l i m i t a t i o n m e n t i o n e d e a r l i e r . M e a s u r e m e n t o f r e v e r s i b l e p r o t e i n b i n d i n g t o r e d c e l l s b y d e t e c t i o n o f t h e a m o u n t o f p r o t e i n a d s o r b e d f r o m s o l u t i o n b y t h e c e l l s ( t h e s o l u t i o n d e p l e t i o n m e t h o d ) h a s b e e n a t t e m p t e d . F u r c h g o t t a n d P o n d e r ( 1 9 4 0 ) s t u d i e d a l b u m i n b i n d i n g b e c a u s e o f i t s e f f e c t s u p o n t h e s h a p e o f m a m m a l i a n r e d c e l l s ( F u r c h g o t t 1 9 4 0 ) . They e s t i m a t e d a l b u m i n - 23 -binding to be about 6 x 106 molecules per cell and recognized that this result was probably at odds with electrophoretic mobility data as the mean spacing between albumin molecules (~50A) suggested a monolayer of adsorbed protein. Subsequent work using similar methodology suggested that the binding could not be distinguished from zero; 5 5 estimates ranged from +4 x 10 to -8 x 10 molecules per cell (Brown 1956). Protein in these experiments was determined by the Kjeldahl method and the difference was attributed to technical limitation in the earlier experiments. Fibrinogen binding by solution depletion has been reported to be 3.5 x 104 molecules per red cell at a protein concentration of 1 mg/mL and to increase linearly to a concentration of at least 5 mg/mL (Traber and Kolmen 1965). However, this method requires that allowance be made for the amount of fluid added to the protein solution with the cells. These authors assumed that cell pellets prepared by centrifugation at 3500 x g for five minutes would not have significant amounts of trapped fluid, an assumption which is clearly incorrect, based on the present work. The binding they reported was equivalent to the dilution effect produced by using cell pellets with a cell volume fraction of approximately 0.9 . In an experiment where the cell volume fractions before and after combining the protein solution and cell suspension are 0.9 and 0.5 respectively, the dilution effect will reduce the protein concentration in solution by about 11 %. Binding of 2 x 10^ molecules fibrinogen per cell would reduce the protein concentration by an additional 8 % i f the protein concentration were 1 mg/mL and by 1 % i f i t were 10 mg/mL. However, an error of 0.01 in the - 2 4 -c e l l v o l u m e f r a c t i o n s w o u l d r e s u l t i n a n e r r o r o f 1.5 % i n t h e e s t i m a t e o f t h e d i l u t i o n e f f e c t o n p r o t e i n c o n c e n t r a t i o n . R e c e n t l y , b i n d i n g 1 2 5 1 2 5 e s t i m a t e s b y t h i s m e t h o d u s i n g I - f i b r i n o g e n a n d I - a l b u m i n w e r e r e p o r t e d t o be l e s s t h a n t h e e r r o r s a s s o c i a t e d w i t h t h e d i l u t i o n e f f e c t s ( B r a s h a n d U n i y a l 1 9 7 6 ) . E s t i m a t e s o f p r o t e i n b i n d i n g t o human e r y t h r o c y t e s u s i n g a t i t r a t i o n c a l o r i m e t r y m e t h o d h a s b e e n r e p o r t e d ( R e h f e l d e t a l . 1 9 7 5 ) . A l b u m i n b i n d i n g was e s t i m a t e d t o be 7 x 10 m o l e c u l e s p e r c e l l a t a p r o t e i n c o n c e n t r a t i o n o f a b o u t 0 . 5 mg/mL . An e q u i l i b r i u m a s s o c i a t i o n c o n s t a n t o f 5 x 1 0 " ^ L / m o l e a n d a n a d s o r p t i o n e n t h a l p y o f - 2 8 0 +_ 9 0 k c a l / m o l e w e r e a l s o r e p o r t e d . I t was s u g g e s t e d t h a t t h e e n t h a l p y c h a n g e m i g h t be d u e t o t h e f o r m a t i o n o f a p p r o x i m a t e l y 8 0 h y d r o g e n b o n d s b e t w e e n e a c h a l b u m i n m o l e c u l e a n d c e l l s u r f a c e p r o t e i n s . C e r t a i n l y , e n t h a l p y c h a n g e s o f t h i s o r d e r a s s o c i a t e d w i t h o n l y a few b o n d s w o u l d b e s u g g e s t i v e o f c o v a l e n t b o n d i n g e n e r g i e s a n d t h u s b i n d i n g w h i c h w o u l d b e i r r e v e r s i b l e . I f t h e i n f l u e n c e o f a l b u m i n o n r e d c e l l m o r p h o l o g y i s d u e t o a n i n t e r a c t i o n o f a l b u m i n w i t h t h e c e l l s u r f a c e ( F u r c h g o t t 1 9 4 0 , J a y 1 9 7 4 ) , t h i s c o n c l u s i o n c o u l d b e r e j e c t e d o n t h e b a s i s o f t h e r e a d y c h a n g e i n m o r p h o l o g y when c e l l s a r e w a s h e d w i t h b u f f e r . I n f a c t , e v e n w i t h t h e b i n d i n g e n e r g y d i s t r i b u t e d o v e r a l a r g e number o f weak i n t e r a c t i o n s , s o t h a t b i n d i n g i s i n p r i n c i p l e r e v e r s i b l e , t h e s m a l l p r o b a b i l i t y o f s i m u l t a n e o u s r e l e a s e o f a l a r g e n u m ber o f b o n d s ( B e l l 1 9 7 8 ) w o u l d r e s u l t i n d e s o r p t i o n r a t e s i n c o m p a t i b l e w i t h t h e r a p i d r e v e r s i b i l i t y o f c e l l m o r p h o l o g y . A l t h o u g h R e h f e l d e t a l . ( 1 9 7 5 ) f e l t t h a t t h i s m e t h o d was a d i r e c t m e a s u r e o f b i n d i n g , i t i s n o t c l e a r t h a t t h e e n t h a l p y c h a n g e s o b s e r v e d c a n be e x c l u s i v e l y a t t r i b u t e d t o - 2 5 -a d s o r p t i o n , e s p e c i a l l y s i n c e B r o w n ( 1 9 5 6 ) a n d T r a b e r a n d K o l m a n ( 1 9 6 5 ) c l a i m e d t h a t f e w e r t h a n 1 0 ^ m o l e c u l e s o f a l b u m i n w e r e b o u n d p e r c e l l a t h i g h e r a l b u m i n c o n c e n t r a t i o n s . R a d i o l a b e l t e c h n i q u e s h a v e b e e n u s e d t o d e t e r m i n e d p r o t e i n b i n d i n g t o r e d c e l l s . H u g h e s - J o n e s a n d G a r d n e r ( 1 9 6 2 ) d e t e r m i n e d r e t e n t i o n o f " ^ I l a b e l l e d s e r u m a n d " ^ I - p r o t e i n s b y r e d c e l l s a f t e r e x t e n s i v e w a s h i n g . T h e y f o u n d t h a t n o m o r e t h a n 0.1 % o f t h e b u l k r a d i o l a b e l r e m a i n e d a s s o c i a t e d w i t h t h e c e l l s e v e n t h o u g h t h e i n i t i a l c e l l v o l u m e e x c e e d e d t h e s o l u t i o n v o l u m e . F i b r i n o g e n a n d a l b u m i n r e t e n t i o n was a t m o s t 5 ug/mL o f p a c k e d c e l l s ( a b o u t 10"5 m o l e c u l e s f i b r i n o g e n p e r c e l l s ) . M u l l e r a n d G r a m l i c h ( 1 9 6 5 ) m e a s u r e d r a d i o l a b e l e d f i b r i n o g e n e l u t i o n f r o m r e d c e l l s u s i n g a q u a n t i t a t i v e , s e r i a l w a sh a n a l y s i s . Two r a t e s o f e l u t i o n w e r e o b s e r v e d a n d t h e b i n d i n g a s s o c i a t e d w i t h t h e more s l o w l y e l u t i n g c o m p o n e n t , when e x t r a p o l a t e d b a c k t o t h e i n i t i a l p e l l e t , was t a k e n t o r e p r e s e n t b o u n d p r o t e i n . T h e i r b i n d i n g e s t i m a t e s r a n g e d f r o m 1,000 t o 1 5 , 0 0 0 m o l e c u l e s p e r c e l l f o r f i b r i n o g e n a n d f r o m 1 3 , 0 0 0 t o 5 3 , 0 0 0 m o l e c u l e s p e r c e l l f o r a l b u m i n . F i b r i n o g e n b i n d i n g o f 4 + 2 x 1 0 ^ m o l e c u l e s p e r c e l l f o r r e d c e l l s w a s h e d o n c e a t a c e l l t o w a s h v o l u m e r a t i o o f 1:200 h a s b e e n r e p o r t e d ( G e o r g e 1 9 7 2 ) . The b i n d i n g r e p o r t e d a f t e r e x t e n s i v e w a s h i n g i s u n l i k e l y t o r e p r e s e n t m a t e r i a l s i n v o l v e d i n r e v e r s i b l e c e l l a g g r e g a t i o n b e c a u s e r o u l e a u x a r e d i s a g g r e g a t e d b y w a s h i n g . I n a d d i t i o n , s i n c e t h e b o u n d q u a n t i t i e s a r e s o s m a l l , i t i s n o t o b v i o u s t h a t t h e r e t a i n e d r a d i o l a b e l s h o u l d b e i d e n t i f i e d w i t h t h e m a j o r r a d i o l a b e l e d s p e c i e s i n t h e b u l k s o l u t i o n , r a t h e r t h a n a r a d i o l a b e l e d c o n t a m i n a n t . T h u s , t h e d a t a - 2 6 -a v a i l a b l e o n f i b r i n o g e n b i n d i n g t o r e d c e l l s i s e i t h e r i n d o u b t d u e t o m i s i n t e r p r e t a t i o n o r i s i n s u f f i c i e n t t o e s t a b l i s h a b i n d i n g i s o t h e r m t h a t m i g h t be r e l a t e d t o f i b r i n o g e n - i n d u c e d r o u l e a u x f o r m a t i o n . The a v a i l a b l e d a t a s u g g e s t s t h a t t h e m e t h o d s u s e d t o d e t e r m i n e b i n d i n g s h o u l d be a b l e t o d e t e c t w e a k , n o n - s p e c i f i c a d s o r p t i o n o f a p p r o x i m a t e l y 3 5 10 t o 10 m o l e c u l e s p e r c e l l . W i t h r e s p e c t t o n o n - s p e c i f i c b i n d i n g o f p r o t e i n s a t s u r f a c e s , a d s o r p t i o n o f p l a s m a p r o t e i n s t o n o n - b i o l o g i c a l m a t e r i a l s h a s b e e n r e v i e w e d i n r e l a t i o n t o t h e u s e o f p r o s t h e t i c d e v i c e s i n t h e c i r c u l a t o r y s y s t e m ( B r a s h 1 9 7 7 , V r oman a n d L e o n a r d 1 9 7 7 , S a l z m a n 1 9 8 1 ) . P r o t e i n a d s o r p t i o n t o v a r i o u s p l a s t i c s , g l a s s e s , a n d m e t a l s o c c u r s r a p i d l y ( B a i e r a n d D u t t o n 1 9 6 9 , M a s o n e t a l . 1 9 7 4 ) a n d p r e c e d e s a d h e r e n c e o f p l a t e l e t s a t t h e s u r f a c e . The a m o u n t s o f p r o t e i n a d s o r b e d f r o m s i n g l e p r o t e i n s o l u t i o n s v a r i e s f r o m 0.01 t o a p p r o x i m a t e l y 2 1.0 ug/cm d e p e n d i n g u p o n t h e p r o t e i n a n d t h e n a t u r e o f t h e s u r f a c e ( B r a s h a n d U n i y a l 1 9 7 9 ) . P r o t e i n b i n d i n g i s o t h e r m s a p p e a r t o b e o f t h e L a n g m u i r t y p e ( D i l l m a n a n d M i l l e r 1 9 7 2 , L e e a n d K i m 1 9 7 4 ) , h o w e v e r c o m p l e t e r e v e r s i b i l i t y o f t h e b i n d i n g i s n o t n e c e s s a r i l y s e e n ( M o r r i s s e y 1 9 7 7 , v a n E n c k e v o r t e t a l . 1 9 8 4 ) . On h y d r o p h o b i c s u r f a c e s p l a t e a u b i n d i n g f r e q u e n t l y c o r r e s p o n d s t o a m o n o l a y e r o f a d s o r b e d p r o t e i n a n d d e s o r p t i o n d o e s n o t o c c u r ( M a c r i t c h i e 1 9 7 2 ) , a l t h o u g h e x c h a n g e h a s b e e n o b s e r v e d ( B r a s h a n d Samak 1 9 7 8 , C h a n a n d B r a s h 1 9 8 0 ) . On h y d r o p h i l i c s u r f a c e s b i n d i n g a p p e a r s t o be l e s s , b u t C o o p e r e t a l . ( 1 9 8 1 ) h a v e n o t e d t h a t t h i s m i g h t b e d u e t o p r o t e i n d e s o r p t i o n d u r i n g w a s h i n g p r o c e d u r e s . C o m p e t i t i o n e f f e c t s a r e o b s e r v e d when a d s o r p t i o n o c c u r s f r o m m i x t u r e s ( K i m a n d Lyman 1 9 7 3 , L e e - 2 7 -e t a l . 1 9 7 4 , M a c r l t c h i e 1 9 7 8 ) a n d r a p i d t i m e - d e p e n d e n t c h a n g e s i n t h e a m o u n t s o f a s p e c i e s b o u n d a r e o b s e r v e d i n a d s o r p t i o n f r o m b l o o d p l a s m a ( V r o m a n a n d Adams 1 9 6 7 , V r o m a n e t a l . 1 9 8 0 ) . A l t h o u g h p r o t e i n a d s o r p t i o n t o i m p l a n t e d d e v i c e s h a s b e e n s h o w n t o b e r e l e v a n t t o p l a t e l e t a d h e r e n c e ( Z u c k e r a n d V r o m a n 1 9 6 9 , L e e e t a l . 1 9 7 4 , B a i e r 1 9 7 7 ) a n d t h e t e n d e n c y t o t h r o m b o g e n e s i s , i t a p p e a r s u n l i k e l y t h a t p r o t e i n a d s o r p t i o n t o h y d r o p h o b i c s u r f a c e s w i l l b e o f g r e a t r e l e v a n c e t o t h e m e c h a n i s m o f f i b r i n o g e n i n t e r a c t i o n w i t h r e d c e l l s . C l o s e l y - p a c k e d m o n o l a y e r a d s o r p t i o n o f f i b r i n o g e n c o r r e s p o n d s 2 t o s u r f a c e c o n c e n t r a t i o n o f b e t w e e n 0.2 a n d 1.6 ug/cm d e p e n d i n g u p o n m o l e c u l a r o r i e n t a t i o n ( S c h m i t t e t a l . 1 9 8 3 ) . T h e s e s u r f a c e 5 6 c o n c e n t r a t i o n s w o u l d c o r r e s p o n d t o b e t w e e n 5 x 1 0 a n d 4 x 10 m o l e c u l e s p e r r e d c e l l . F i b r i n o g e n a d s o r p t i o n t o h y d r o p h i l i c m a t e r i a l s may n o t b e i r r e l e v a n t a s t h e r e a r e i n d i c a t i o n s t h a t some o f t h e b i n d i n g i s r e v e r s i b l e . F i b r i n o g e n r e t a i n e d b y h y d r o p h i l i c p o l y u r e t h a n e s h a s 2 b e e n r e p o r t e d t o be i n t h e r a n g e o f 0 . 0 3 t o 0 . 0 8 ug/cm ( B r a s h a n d U n i y a l 1 9 7 9 ) . 1.3.2 M e a s u r e s o f c e l l a g g r e g a t i o n A v a r i e t y o f m e t h o d s h a v e b e e n u s e d t o a s s e s s c e l l u l a r a g g r e g a t i o n , b u t f e w r e f l e c t s i m p l y t h e f o r c e s w h i c h g o v e r n a g g r e g a t i o n a n d d i s a g g r e g a t i o n ( B r o o k s 1 9 7 6 ) . M e t h o d s i n v o l v i n g f l u i d f l o w t e c h n i q u e s i n c l u d e e r y t h r o c y t e s e d i m e n t a t i o n ( F a h r a e u s 1 9 2 1 , T h o r s e n a n d H i n t 1 9 5 0 , C h i e n a n d J a n 1 9 7 3 ) , l o w s h e a r r a t e v i s c o m e t r y ( C h i e n e t a l . 1 9 7 0 , B r o o k s 1 9 7 6 , C h i e n e t a l . 1 9 7 7 , J a n 1 9 7 9 ) , y i e l d s t r e s s m e a s u r e m e n t s ( C o k e l e t e t a l . 1 9 6 3 ) , a n d d i s a g g r e g a t i o n i n f l o w c h a m b e r s ( C h i e n e t a l . 1 9 7 7 ) . E s t i m a t e s o f l o c a l s t r e s s e s o n t h e c e l l s w o u l d - 2 8 -h a v e t o be made f r o m b u l k s h e a r s t r e s s e s a n d s h e a r r a t e s ( B r o o k s 1 9 7 6 ) . H o w e v e r , e v e n when t h e g e o m e t r y i s r e l a t i v e l y s i m p l e ( C h i e n e t a l . 1 9 7 7 ) e s t i m a t e s o f t h e a g g r e g a t i n g o r d i s a g g r e g a t i n g f o r c e s h a v e n o t b e e n made. R a n k i n g o f t h e m i c r o s c o p i c a p p e a r a n c e o f a g g r e g a t e s ( B r o o k s 1 9 7 3 c , M o r r i s 1 9 7 9 ) a n d i n d i c e s b a s e d o n t h e n u m b e r s o f s i n g l e c e l l s a n d a g g r e g a t e s ( C h i e n a n d J a n 1 9 7 3 , B y c h k o v a n d K u z ' m i n a 1 9 7 7 ) p r o v i d e r e l a t i v e m e a s u r e s o f a g g r e g a t i o n . S a m s e l a n d P e r e l s o n ( 1 9 8 2 ) a n d P e r e l s o n a n d W i e g e l ( 1 9 8 2 ) h a v e e x t e n d e d e a r l i e r m o d e l s o f r o u l e a u x f o r m a t i o n . A p p l i c a t i o n o f t h e s e t h e o r i e s t o e s t i m a t e r a t e c o n s t a n t s f o r ' r e a c t i o n s ' b e t w e e n c e l l s ( S a m s e l a n d P e r e l s o n 1 9 8 2 ) a n d t h e s u r f a c e a f f i n i t i e s f o r r o u l e a u x e l o n g a t i o n a n d b r a n c h p o i n t f o r m a t i o n ( P e r e l s o n a n d W i e g e l 1 9 8 2 ) r e q u i r e r e l a t i v e l y d e t a i l e d k n o w l e d g e o f t h e d i s t r i b u t i o n s o f r o u l e a u x s i z e a n d b r a n c h i n g f r e q u e n c i e s . C e r t a i n l y , d e t e r m i n a t i o n o f a g g r e g a t i o n r a t e c o n s t a n t s r e q u i r e s c a r e f u l c o n t r o l o f t h e e x p e r i m e n t a l m i x i n g c o n d i t i o n s ( L i n n e m a n s e t a l . 1 9 7 6 a , L i n n e m a n s e t a l . 1 9 7 6 b , J o n e s a n d P e r r y 1 9 7 9 ) . R e c e n t l y , a n a l y s e s o f t h e d e f o r m a t i o n o f r e d c e l l s c a u s e d b y a g g r e g a t i o n h a v e b e e n d e v e l o p e d t o e s t i m a t e t h e w o r k o f f o r m a t i o n o f c o n t a c t s b e t w e e n r e d c e l l s ( S k a l a k e t a l . 1 9 7 7 , E v a n s 1 9 8 0 , S k a l a k e t a l . 1 9 8 1 ) . The m e t h o d s u s e t h e e s t a b l i s h e d m a t e r i a l p r o p e r t i e s o f t h e r e d c e l l membrane ( S k a l a k e t a l . 1 9 7 3 , E v a n s a n d H o c h m u t h 1 9 7 8 , E v a n s a n d S k a l a k 1 9 8 0 ) t o e s t i m a t e t h e s u r f a c e f r e e e n e r g y p e r u n i t a r e a a s s o c i a t e d w i t h t h e f o r m a t i o n o f a n a d h e s i v e c o n t a c t . T h i s g e n e r a l m e t h o d h a s b e e n m o d i f i e d b y u s i n g a m o r e f a v o u r a b l e c o n t a c t g e o m e t r y t o i m p r o v e s e n s i t i v i t y . I n a d d i t i o n , t h e a n a l y s i s h a s b e e n - 2 9 -s i m p l i f i e d b y t h e f a c t t h a t , a s t h e c o n t a c t b e t w e e n t h e t w o c e l l s u r f a c e s r e a c h e s e q u i l i b r i u m , t h e c h a n g e i n t h e e l a s t i c f r e e e n e r g y o f t h e r e d c e l l d e f o r m a t i o n i s e q u a l t o t h e c h a n g e i n t h e f r e e e n e r g y o f f o r m a t i o n o f t h e c o n t a c t ( E v a n s a n d Buxbaum 1 9 8 1 ) . S u r f a c e a f f i n i t i e s - 3 -2 2 r a n g i n g f r o m a b o u t 1 x 10 t o 7 x 1 0 e r g / c m may be e s t i m a t e d u s i n g t h i s m o r e s e n s i t i v e m e t h o d . D e x t r a n - i n d u c e d a g g r e g a t i o n i s a s s o c i a t e d w i t h p e a k r e d c e l l s u r f a c e a f f i n i t i e s o f 5 x I O-"5 a n d -2 2 2 x 10 e r g / c m f o r d e x t r a n s o f 7 0 , 0 0 0 a n d 1 5 0 , 0 0 0 w e i g h t a v e r a g e m o l e c u l a r w e i g h t s r e s p e c t i v e l y , w h i l e p l a s m a i s a s s o c i a t e d w i t h a -3 2 s u r f a c e a f f i n i t y o f a b o u t 2 x 1 0 e r g / c m (Buxbaum e t a l . 1 9 8 2 ) . C h a r a c t e r i z a t i o n o f r e d c e l l a g g r e g a t i o n b y t h i s m e t h o d o l o g y i s c l e a r l y d e s i r a b l e b e c a u s e t h e m e a s u r e t h a t r e s u l t s i s d i r e c t l y r e l a t e d t o t h e f o r m a t i o n o f a c e l l - c e l l c o n t a c t . I n a d d i t i o n , i t i s p o s s i b l e i n p r i n c i p l e t o e s t i m a t e t h e b i n d i n g e n e r g y a s s o c i a t e d w i t h t h e a d s o r p t i o n o f a c r o s s - b r i d g i n g s p e c i e s t o t h e c e l l s u r f a c e b y c o m p a r i n g t h e m e a s u r e d b i n d i n g a n d s u r f a c e a f f i n i t i e s . - 30 -1.4 O B J E C T I V E S AND METHODS The o b j e c t i v e s o f t h e p r e s e n t w o r k a r e t o c h a r a c t e r i z e t h e a d s o r p t i o n o f human f i b r i n o g e n t o human e r y t h r o c y t e s a n d t o c o m p a r e t h i s b e h a v i o u r w i t h r e d c e l l s u r f a c e a f f i n i t i e s d e t e r m i n e d u n d e r s i m i l a r c o n d i t i o n s . I f t h e s u r f a c e a f f i n i t i e s c a n be i n t e r p r e t e d i n t e r m s o f t h e n u m b er o f a d s o r b e d f i b r i n o g e n m o l e c u l e s , new i n f o r m a t i o n c o n c e r n i n g t h e w e a k , a p p a r e n t l y n o n - s p e c i f i c i n t e r a c t i o n o f a p l a s m a m a c r o m o l e c u l e w i t h c e l l s u r f a c e c o m p o n e n t s w i l l be p r o v i d e d . I n a d d i t i o n , some s u p p o r t f o r t h e r e l e v a n c e o f f i b r i n o g e n c r o s s - b r i d g i n g i n t h e m e c h a n i s m o f r o u l e a u x f o r m a t i o n w i l l b e g a i n e d . 1 2 5 The b i n d i n g s t u d i e s t o be d e s c r i b e d e m p l o y i o d i n e r a d i o l a b e l e d f i b r i n o g e n s o t h a t l o w l e v e l s o f b i n d i n g may be d e t e r m i n e d . U l t r a c e n t r i f u g a t i o n c o m p a r a b l e t o t h a t u s e d t o s t u d y d e x t r a n a d s o r p t i o n t o r e d c e l l s ( B r o o k s 1 9 7 3 b ) i s u s e d t o r e d u c e e x t r a c e l l u l a r t r a p p i n g i n c e l l p e l l e t s . C e l l p e l l e t s a r e t h e n s u b j e c t e d t o q u a n t i t a t i v e w a s h a n a l y s e s ( M u l l e r a n d G r a m l i c h 1 9 6 5 ) t o a s s e s s t h e p o s s i b l e c o n t r i b u t i o n o f t r a p p e d r a d i o l a b e l t o a p p a r e n t b i n d i n g . A l s o , a l t h o u g h t h e s o l u t i o n d e p l e t i o n m e t h o d a p p e a r s f r o m t h e l i t e r a t u r e t o b e i n s e n s i t i v e t o t h e l e v e l s o f b i n d i n g a n t i c i p a t e d , e s t i m a t e s o f b i n d i n g u s i n g t h i s m e t h o d a r e made s o t h a t a n u p p e r l e v e l f o r a d s o r p t i o n , i n d e p e n d e n t o f t h e u l t r a c e n t r i f u g a t i o n p r o c e d u r e , may b e e s t a b l i s h e d . F i n a l l y , s u r f a c e a f f i n i t i e s f o r r e d c e l l s i n s o l u t i o n s o f i s o l a t e d f i b r i n o g e n a r e d e t e r m i n e d by t h e e n c a p s u l a t i o n t e c h n i q u e d e s c r i b e d b y E v a n s a n d Buxbaum ( 1 9 8 1 ) , t h e r e s u l t s c o m b i n e d w i t h t h e a d s o r p t i o n m e a s u r e m e n t s , a n d t h e r e l a t i o n s h i p b e t w e e n t h e t w o d i s c u s s e d . - 31 -CHAPTER 2 M A T E R I A L S AND METHODS 2.1 MATERIALS 2.1.1 R e a g e n t s B e n z a m i d i n e h y d r o c h l o r i d e h y d r a t e f r o m A l d r i c h , Box 3 5 5 , M i l w a u k e e , W i s c o n s i n , 5 3 2 0 1 USA; h y d r o g e n p e r o x i d e 3 0 % a n d r e a g e n t g r a d e s u c r o s e f r o m A m e r i c a n S c i e n t i f i c a n d C h e m i c a l , S e a t t l e , WA, 9 8 1 9 9 USA; s o d i u m 1 2 5 i o d i d e I M S - 3 0 f r o m Amersham C o r p . , O a k v i l l e , O n t a r i o , C a n a d a L 6 H 2 R 3 ; a c r y l a m i d e , B I S N , N ' - m e t h y l e n e - b i s - a c r y l a m i d e ( B I S ) , s o d i u m d o d e c y l s u l p h a t e ( S D S ) a l l o f e l e c t r o p h o r e t i c p u r i t y , a n d E n z y m o b e a d L a c t o p e r o x i d a s e - G l u c o s e o x i d a s e # 1 7 0 - 6 0 0 1 f r o m B i o Rad L a b s . , R i c h m o n d , C A , 9 4 8 0 4 USA; a c e t i c a c i d a n a l y t i c a l , D e c o n 7 5R m e t h a n o l A C S - 5 3 1 , 3 - ( N - M o r p h o l i n o ) p r o p a n e s u l p h o n i c a c i d (MOPS) a n d SDS-PAGE M o l e c u l a r W e i g h t M a r k e r #44230 f r o m B r i t i s h D r u g H o u s e C h e m i c a l s ( B D H ) , T o r o n t o , O n t a r i o , C a n a d a M8Z 1 K 5 ; n e u r a m i n i d a s e ( V i b r o c h o l e r a ) # 4 8 0 7 1 7 f r o m C a l b i o c h e m - B e h r i n g C o r p . , B o x 1 2 0 8 7 S a n D i e g o , CA 9 2 1 1 2 USA; C o o m a s s i e B l u e G 2 5 0 , N , N , N ' , N ' , - T e t r a - m e t h y l - e t h y l e n e d i a m i n e ( T E M E D ) , a n d t r i c h l o r o a c e t i c a c i d f r o m E a s t m a n K o d a k C o . , R o c h e s t e r , NY 1 4 6 5 0 USA; b e n z y l b e n z o a t e ( p u r i f i e d ) B 2 7 6 , e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d d i s o d i u m s a l t ( E D T A ) , a n d p e r c h l o r i c a c i d ( A CS r e a g e n t ) A 2 2 9 f r o m - 32 -Fisher Scientific Co., Nepean, Ontario, Canada, K2E 7L6; Protein Standard Plasma OTFI, Standard Human Serum ORDT, and radial immunodiffusion (RID) plates: fibrinogen OTBN and albumin OTBG from Hoechst Canada Ltd., Montreal, Quebec, Canada H4R 1R6; poly(ethyleneglycol) 6000 (PEG) and pyronin Y from J.T.Baker Chemical Co., Weston, Ontario, Canada, M9L 1R9; cottonseed o i l (refined) CX-1960 from North American Scientific Chemical Ltd. (Matheson Coleman and Bell) Norwood, Ohio, 45212 USA; Trasylol R 10,000 KIU/mL from Miles Laboratories, Ontario, Canada M9W 1G6; Blue Dextran 2000R, R R R Sepharose-4B , Lysine-Sepharose-4B , Sephadex G75 , Sephadex G150R, and Sephacryl S300R from Pharmacia, Uppsala, S-751 04 Sweden; Albumin (human) crystallized and lyophillized A-9511, collagen Type 1 from swine skin G-2500, N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) H-3375, hirudin Grade IV H-4256, lactoperoxidase from bovine milk L-2005, 2-mercaptoethanol Type 1 M-6250, phenylmethylsulfonylchloride (PMSF) P-7626, anhydrous sodium phosphate dibasic S-0876 and monobasic S-0751, thrombin T-6759, and Tris(hydroxymethyl)-aminomethane hydrochloride (TRIS) T-3253 from Sigma, Box 14508, St.Louis, MO 63178 USA. Hemoglobin was isolated from freshly isolated red c e l l s lysed with 5 mM phosphate pH 8. 2.1.2 Solutions and buffers a) Fibrinogen Isolation IM barium chloride : 1.0 M barium chloride, 50 U/mL Trasylol, 25 mM benzamidine-HCl; IM sodium citrate : 1.0 M sodium citrate, 50 U/mL Trasylol, 25 mM benzamidine-HCl; 40 % poly(ethylene glycol) 6000 : 40 % w/v with d i s t i l l e d water; CSTB : 20 mM sodium citrate, - 3 3 -15 0 mM s o d i u m c h l o r i d e , 5 0 U/mL T r a s y l o l , 2 5 mM b e n z a m i d i n e - H C l , 0 . 0 3 % s o d i u m a z i d e , a d j u s t t o pH 5.6 o r 7.6 w i t h N a O H / H C l . b ) G e l E l e c t r o p h o r e s i s I P X b u f f e r : 1.0 M T R I S , 2.0 M s o d i u m a c e t a t e , 0 .2 M EDTA, pH 7.4; 3 0 % A c r y l a m i d e / B I S : 3 0 % w/v a c r y l a m i d e , 1.2 % w/v B I S ; C o o m a s s i e B l u e s t a i n / f i x : 0.2 g C o o m a s s i e B l u e G 2 5 0 , 2 7 . 5 mL m e t h a n o l , 5.5 mL a c e t i c a c i d , 2 5 mL p e r c l o r i c a c i d , t o 5 0 0 mL w i t h w a t e r ; m e t h a n o l d e s t a i n : 3 0 0 mL m e t h a n o l , 5 0 mL a c e t i c a c i d , t o 1 L w i t h w a t e r ; r e s e r v o i r b u f f e r : 1 0 0 mL 10X b u f f e r , 5 0 mL 4 % S D S , t o 1 L w i t h w a t e r ; s a m p l e r e a g e n t : 2 0 mM T r i s - H C l , 2 mM EDTA, 2 % w/v S D S , 14 % w/v s u c r o s e , 2 0 g/mL p y r o n i n Y , t o pH 8 w i t h a c e t i c a c i d . F o r r e d u c i n g c o n d i t i o n s 5 0 mM 2 - m e r c a p t o e t h a n o l was a d d e d . H e m o g l o b i n was a d d e d when a s t a i n i n g r e f e r e n c e was d e s i r e d . c ) A d s o r p t i o n I n m o s t e x p e r i m e n t s 2 0 mM p h o s p h a t e was u s e d t o e s t a b l i s h Jt h e pH a n d s o d i u m c h l o r i d e c o n t r i b u t e d t h e m a j o r p o r t i o n o f t h e s o l u t i o n o s m o l a l i t y . B u f f e r s o f t h i s g e n e r a l c o m p o s i t i o n w i l l b e r e f e r r e d t o a s P B S b u f f e r s . B u f f e r pH was d e t e r m i n e d f o r d i b a s i c t o m o n o b a s i c s o d i u m p h o s p h a t e m o l e r a t i o s b e t w e e n 0 . 0 9 a n d 10 a t o s m o l a l i t i e s o f 39 a n d 2 8 6 mosm, F i g u r e 2 . 1 a . The r e l a t i o n s w h i c h d e s c r i b e t h e pH v e r s u s m o l e r a t i o d a t a a r e , OSMOLALITY a R E L A T I O N STANDARD D E V I A T I O N 39+1 pH = 6 . 9 8 3 + 0 . 9 8 4 l o g ( D / M ) 0 . 0 0 8 ( n = 9 ) 286+3 pH = 6 . 7 0 7 + 1.019 l o g ( D / M ) 0 . 0 0 9 ( n = 1 4 ) a ) mean + maximum d e v i a t i o n Figure 2.1 a) pH as a function of the mole ratio of Na2HP04 to Nar^PO^ at 39 mosm O O and 289 mosm ++ . b) Osmolarity of the buffer components determined by freezing point depression as a function of the solution concentration : 1. CaCl2 2. Na2HP04 3, NaCl 4. NaH2P04 5. sucrose. - 3 5 -w h e r e D/M i s t h e m o l e r a t i o o f d i b a s i c t o m o n o b a s i c s o d i u m p h o s p h a t e . A t 2 8 9 mosm, a d i b a s i c t o m o n o b a s i c s o d i u m p h o s p h a t e m o l e r a t i o o f A.79 r e s u l t e d i n pH 7.A b u f f e r s . The r e l a t i o n s w e r e d e t e r m i n e d by f i t t i n g t h e d a t a u s i n g l e a s t s q u a r e s l i n e a r r e g r e s s i o n a n a l y s i s ( L R A ) . The s t a n d a r d d e v i a t i o n i s t h e e s t i m a t e f o r t h e s t a n d a r d e r r o r o f t h e e r r o r t e r m i n t h e r e g r e s s i o n a n a l y s i s . I n a l l s u b s e q u e n t l i n e a r r e g r e s s i o n a n a l y s e s , e s t i m a t e s o f t h e s t a n d a r d d e v i a t i o n s o f t h e i n t e r c e p t a n d s l o p e a r e r e p o r t e d . T h e s e w e r e c a l c u l a t e d f r o m t h e e s t i m a t e o f t h e s t a n d a r d e r r o r ( M e n d e n h a l l e t a l . 1 9 8 1 , C h a p t e r 1 1 ) . The c o n t r i b u t i o n s o f t h e v a r i o u s b u f f e r c o m p o n e n t s t o t h e s o l u t i o n o s m o l a l i t y w e r e c a l c u l a t e d f r o m t h e c o n c e n t r a t i o n i n m i l l i m o l e s a n d t h e m i l l i o s m o l e s / m i l l i m o l e f o r e a c h c o m p o n e n t . F i g u r e 2.1b s h o w s t h e r e l a t i o n s b e t w e e n o s m o l a l i t y , d e t e r m i n e d by f r e e z i n g p o i n t d e p r e s s i o n , a n d t h e s o l u t i o n c o n c e n t r a t i o n f o r s e v e r a l b u f f e r c o m p o n e n t s . T h e s e r e l a t i o n s a r e s u m m a r i z e d i n T a b l e 2.1 a n d w e r e u s e d r o u t i n e l y t o c o n v e r t b e t w e e n m i l l i m o l e s a n d m i l l i o s m o l e s . The o s m o l a l i t y o f t h e b u f f e r s was g e n e r a l l y d e s i g n e d t o e q u a l t h a t o f human s e r u m ( H e n d r y 1 9 6 1 ) . The b u f f e r s w e r e d e s i g n e d by c h o o s i n g t h e m o l a r i t y , a n d t h u s o s m o l a l i t y , o f a l l c o m p o n e n t s b u t s o d i u m c h l o r i d e . The m o l a r i t y o f s o d i u m c h l o r i d e r e q u i r e d t o b r i n g t h e t o t a l o s m o l a l i t y t o t h e d e s i r e d v a l u e was t h e n c a l c u l a t e d . The o s m o l a l i t y o f t h e s e b u f f e r s was f o u n d t o be w i t h i n 2 % o f t h e d e s i g n e d v a l u e s . T h r e e e x a m p l e P B S b u f f e r s a r e d e s c r i b e d i n T a b l e 2.2 . - 36 -TABLE 2.1 R E L A T I O N S BETWEEN OSMOLALITY AND THE CONCENTRATION OF BUFFER COMPONENTS COMPONENT SLOPE a I N T E R C E P T a nb DATA RANGE (mosm/mM) (mM) (mM) N a H2P 04 1 . 76 + 0 . 0 2 2.9 + 1 . 7 6 2 0 0 N a2H P 04 2 . 18 0 . 0 2 7.6 1 . 7 6 1 2 5 N a C l 1 . 8 4 0 . 0 1 0.7 2 . 7 1 5 4 0 0 N a N3 1 . 8 7 0.04 0 . 5 0 . 8 7 3 0 b e n z a m i d i n e - H C l 1 . 6 2 0 . 0 2 - 0 . 8 1 . 2 10 1 0 0 N a l 1 . 5 3 0.08 1 0 . 4 5 . 9 1 0 1 0 0 C a C l2 c 2 . 5 7 • • 3.7 • • 3 1 4 0 N a2E D T A 2 . 8 8 0 . 1 0 - 1 . 0 0 . 7 8 10 s u c r o s e c 1 . 0 7 0.01 - 2 . 6 1 . 1 7 1 9 0 H e p e s 1 . 0 0 H h 0 . 0 2 1 2 . 3 + 1 . 6 6 1 0 0 a ) mean + o n e s t a n d a r d d e v i a t i o n ( L R A ) b ) number o f d a t a p o i n t s c ) W e a s t a n d A s t l e ( 1 9 7 9 , p p . D 2 6 5 - D 3 1 4 ) TABLE 2.2 COMPOSITION OF THREE PHOSPHATE pH 7 . 4 , 2 8 9 mosm BUFF E R S BUFFER P B S P B S / N a N3 P B S / N a N3/ b e n z a m i d i n e - H C l (mM) (mosm) (mM) (mosm) (mM) (mosm N a H2P 04 3.4 6.0 3.4 6.0 3.4 6 . 0 N a2H P 04 1 6 . 6 3 6 . 2 1 6 . 6 3 6 . 2 1 6 . 6 3 6 . 2 N a C l 1 3 4 . 2 2 4 6 . 8 131.1 2 4 1 . 2 1 2 2 . 3 2 2 5 . 0 NaN3 3.0 5.6 3.0 5.6 b e n z a m i d i n e - H C l ... • • * * • • • • • 1 0 . 0 1 6 . 2 - 3 7 -2.2 PROTEINS 2.2.1 F i b r i n o g e n i s o l a t i o n U n i t s o f c i t r a t e - p h o s p h a t e - d e x t r o s e ( C P D ) a n t i c o a g u l a t e d b l o o d w e r e g i f t s f r o m t h e V a n c o u v e r Red C r o s s T r a n s f u s i o n S e r v i c e . The b l o o d was d r a w n o n t h e same d a y a s t h e i s o l a t i o n p r o c e d u r e was b e g u n . F i b r i n o g e n was i s o l a t e d by a m o d i f i c a t i o n o f t h e p o l y ( e t h y l e n e g l y c o l ) ( P E G ) p r e c i p i t a t i o n m e t h o d ( P o i s o n e t a l . 1 9 6 4 , P o i s o n a n d R u i z - B r a v o 1 9 7 2 ) a s u s e d b y J o h n s o n ( 1 9 7 6 ) . A l l o p e r a t i o n s , e x c e p t f o r t h e c o l l e c t i o n o f c h r o m a t o g r a p h i c f r a c t i o n s , w e r e c a r r i e d o u t a t room t e m p e r a t u r e . W h e r e v e r p o s s i b l e , p o l y p r o p y l e n e p l a s t i c - w a r e was u s e d i n p r e f e r e n c e t o g l a s s - w a r e . C e l l s w e r e f i r s t r e m o v e d b y c e n t r i f l i g a t i o n . T r a s y l o l a n d b e n z a m i d i n e - H C l w e r e a d d e d t o t h e p l a s m a t o f i n a l c o n c e n t r a t i o n s o f 5 0 KIU/mL a n d 2 5 mM r e s p e c t i v e l y . The p l a s m a was t h e n t r e a t e d w i t h b a r i u m t o r e m o v e p r o t h r o m b i n b y a d s o r p t i o n ( B a j a j a n d Mann 1 9 7 3 ) . The b a r i u m was p r e c i p i t a t e d a s b a r i u m c i t r a t e . S o d i u m c i t r a t e a n d b a r i u m c h l o r i d e w e r e a d d e d a s IM s o l u t i o n s p e r w e i g h t o f i n i t i a l p l a s m a . F i r s t , 6 mL s o d i u m c i t r a t e s o l u t i o n was a d d e d p e r 100 g o f p l a s m a , t h e n b a r i u m c h l o r i d e was a d d e d w i t h s l o w c o n t i n u o u s m i x i n g t o 12 mL p e r 1 0 0 g o f i n i t i a l p l a s m a . M i x i n g was c o n t i n u e d f o r 1 0 m i n u t e s . The s u p e r n a t a n t was d e c a n t e d a f t e r c e n t r i f u g a t i o n ( 5 0 0 0 x g f o r 10 m i n u t e s ) a n d r e t a i n e d . The b a r i u m a d s o r p t i o n was r e p e a t e d w i t h a d d i t i o n o f 8 mL b a r i u m c h l o r i d e p e r 1 0 0 g o f i n i t i a l p l a s m a , f o l l o w e d by 10 m i n u t e s m i x i n g , a d d i t i o n o f 9.5 mL s o d i u m c i t r a t e s o l u t i o n p e r 1 0 0 g i n i t i a l p l a s m a , a n d c e n t r i f u g a t i o n . The s u p e r n a t a n t was d e c a n t e d a n d a d j u s t e d - 3 8 -t o pH 7.6 w i t h 0 . 5 M NaOH a n d d i l u t e d t o t w i c e t h e i n i t i a l p l a s m a v o l u m e w i t h CSTB pH 7.6 . F i b r i n o g e n was t h e n p r e c i p i t a t e d b y a d d i t i o n o f PEG 6 0 0 0 t o A.8 % ( 1 4 . 1 g o f AO % w/v PEG p e r 1 0 0 g p r o t e i n s o l u t i o n ) . The p r e c i p i t a t e was c o l l e c t e d b y c e n t r i f u g a t i o n a t 2 0 0 0 x g f o r 3 m i n u t e s . The p r e c i p i t a t e was d i s s o l v e d i n a m i n i m a l v o l u m e o f CSTB pH 7.6 a n d t h e n d i l u t e d w i t h CSTB pH 7.6 t o t h e i n i t i a l p l a s m a v o l u m e . M o r e c e n t r i f u g a t i o n c a n make t h e d i s s o l u t i o n d i f f i c u l t . P r o t e i n was a g a i n p r e c i p i t a t e d by a d d i t i o n o f PEG 6 0 0 0 t o A.8 % a n d t h e p r e c i p i t a t e was r e d i s s o l v e d i n CSTB pH 7.6 t o a b o u t 1 0 % o f t h e i n i t i a l p l a s m a v o l u m e . The p r o t e i n c o n c e n t r a t e was c h r o m a t o g r a p h e d o v e r n i g h t i n s e r i e s o n R R L y s i n e - S e p h a r o s e AB a n d g e l a t i n - S e p h a r o s e AB t o r e m o v e p l a s m i n o g e n ( M a t s u d a e t a l . 1 9 7 2 , C e d e r h o l m - W i l l i a m s a n d F e n n e l l 1 9 8 1 ) a n d f i b r o n e c t i n ( V u e n t o a n d V a h e r i 1 9 7 9 ) r e s p e c t i v e l y . G e l a t i n -S e p h a r o s e AB was p r e p a r e d u s i n g T y p e 1 c o l l a g e n ( g e l a t i n f r o m s w i n e s k i n ) a n d c y a n o g e n b r o m i d e a c t i v a t e d S e p h a r o s e AB ( M a r c h e t a l . 1 9 7 A ) . The a f f i n i t y c o l u m n s w e r e e q u i l i b r a t e d a n d e l u t e d w i t h CSTB pH 7.6 a n d t h e e l u a t e was c o l l e c t e d i n a A°C c o l d - c a b i n e t . The e l u a t e was d i v i d e d i n t o p o o l s o n t h e b a s i s o f a b s o r p t i o n a t 2 8 0 nm, F i g u r e 3.Aa . A d u a l beam d e t e c t o r was r e q u i r e d a s b e n z a m i d i n e a b s o r b s s t r o n g l y i n t h i s w a v e l e n g t h r e g i o n . The e a r l y p e a k s w e r e l i p i d - r i c h f r a c t i o n s d e t e c t e d d u e t o l i g h t s c a t t e r i n g r a t h e r t h a n a b s o r b a n c e . The p r o t e i n p e a k was c o l l e c t e d , a d j u s t e d t o pH 5.6 w i t h c i t r i c a c i d a n d d i l u t e d t o t h e o r i g i n a l p l a s m a v o l u m e w i t h CSTB pH 5.6 . T h i s p o o l was p r e c i p i t a t e d b y a d d i t i o n o f PEG 6 0 0 0 t o 3.0 % ( 8 . 3 6 g o f AO % w/v PEG p e r 1 0 0 g p r o t e i n s o l u t i o n ) . The p r e c i p i t a t e was r e d i s s o l v e d i n - 3 9 -p P B S / b e n z a m i d i n e a n d c h r o m a t o g r a p h e d o n S e p h a d e x G150 o r S e p h a c r y l p S - 3 0 0 t o e x c h a n g e s a l t s a n d r e m o v e P E G . The p r o d u c t was e i t h e r l y o p h i l i z e d a n d s t o r e d a t - 2 0°C , o r was u s e d d i r e c t l y f o r e x p e r i m e n t s . 2.2.2 PAGE a n a l y s i 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 p h o r e s i s i n s o d i u m d o d e c y l s u l p h a t e ( S D S - PAGE) was u s e d t o a s s a y t h e c o m p o s i t i o n o f i s o l a t e d p r o t e i n s a n d r a d i o l a b e l e d m a t e r i a l s f r o m v a r i o u s s t a g e s o f t h e b i n d i n g e x p e r i m e n t s . The a c r y l a m i d e s y s t e m u s e d was a m o d i f i c a t i o n o f t h a t u s e d by F a i r b a n k s e t a l . ( 1 9 7 1 ) . The f o l l o w i n g r e a g e n t s w e r e c o m b i n e d i n t h e o r d e r g i v e n a n d c a s t a s r o d g e l s i n 1 2 5 x 5 mm i n s i d e d i a m e t e r ( I D ) g l a s s t u b e s . The f i n a l a c r y l a m i d e c o n c e n t r a t i o n was 3 % . 3 0 % A c r y l a m i d e / B I S 3 mL l O x b u f f e r 3 mL 4 % w/v SDS 1.5 mL 0 . 5 % v / v TEMED 1.5 mL w a t e r 18 mL 1.5 % w/v ammonium p e r s u l p h a t e 3 mL Two m i l l i l i t e r s o f t h e g e l s o l u t i o n was u s e d p e r t u b e a n d a n o v e r l a y o f t h e same s o l u t i o n , e x c e p t t h a t w a t e r r e p l a c e d t h e a c r y l a m i d e c o m p o n e n t , was a p p l i e d t o p r o d u c e a f l a t u p p e r s u r f a c e o n t h e g e l . The s o l u t i o n g e l l e d i n 30 t o 4 0 m i n u t e s a t r o o m t e m p e r a t u r e . The r o d s w e r e m o u n t e d i n a B i o - R a d m o d e l 15 0 A e l e c t r o p h o r e s i s c h a m b e r a n d t h e o v e r l a y s o l u t i o n was f l u s h e d away w i t h f r e s h r e s e r v o i r b u f f e r . S a m p l e s w e r e l a y e r e d o n t h e g e l s u r f a c e w i t h a p o s i t i v e d i s p l a c e m e n t m i c r o p i p e t t o r i n v o l u m e s r a n g i n g f r o m 5 t o 4 0 u L . The d e n s i t y o f t h e s a m p l e s was s u c h a s t o a v o i d m i x i n g w i t h t h e r e s e r v o i r b u f f e r . - 4 0 -The e l e c t r o p h o r e s i s was c a r r i e d o u t a t a f i x e d c u r r e n t , w h i c h was s e t a t 0 . 5 m i l l i a m p e r e s p e r t u b e u n t i l t h e s a m p l e h a d e n t e r e d t h e g e l a n d w h i c h was t h e n i n c r e a s e d t o b e t w e e n 4 a n d 8 m i l l i a m p e r e s p e r t u b e u n t i l t h e e n d o f t h e r u n . The r u n n i n g t i m e was b e t w e e n 1.5 a n d 3 h o u r s . The a p p a r a t u s was k e p t a t a b o u t 4°C i n a c o l d c a b i n e t . The g e l s w e r e r e m o v e d b y s l i p p i n g a l o n g f l e x i b i l e h y p o d e r m i c n e e d l e b e t w e e n t h e g e l a n d t h e g l a s s t u b e w a l l w h i l e e x p r e s s i n g a 2 % v / v s o l u t i o n o f g l y c e r o l a n d w a t e r . The p y r o n i n Y t r a c k i n g d y e was m a r k e d w i t h I n d i a i n k a n d t h e g e l was p l a c e d i n a p a r t i t i o n e d t r a y f o r f i x i n g , s t a i n i n g ( C o o m a s s i e B l u e G 2 5 0 ) , a n d c l e a r i n g . F i x a t i o n a n d s t a i n i n g was m o d i f i e d f r o m R e i s n e r e t a l . ( 1 9 7 5 ) . I n i t i a l f i x a t i o n a n d s t a i n i n g was f o r o n e h o u r i n C o o m a s s i e B l u e s t a i n / f i x , f o l l o w e d by f u r t h e r f i x a t i o n a n d c l e a r i n g i n m e t h a n o l d e s t a i n f o r o n e h o u r . F i n a l c l e a r i n g i n 7 % a c e t i c a c i d r e q u i r e d 24 t o 3 6 h o u r s a t r o o m t e m p e r a t u r e . G e l s a n a l y s e d f o r p r o t e i n w e r e p h o t o g r a p h e d a n d / o r s c a n n e d f o r d y e b i n d i n g a t a w a v e l e n g t h o f 5 9 5 nm i n a B e c k m a n M o d e l 2 5 s p e c t r o p h o t o m e t e r f i t t e d w i t h a g e l s c a n n i n g a t t a c h m e n t . G e l s w e r e p h o t o g r a p h e d i n t h e 1 5 0 x 10 mm I D g l a s s t u b e s i n w h i c h t h e y w e r e s t o r e d a f t e r c l e a r i n g . G e l s s c a n n e d i n t h e s p e c t r o p h o t o m e t e r w e r e h e l d i n a r e c t a n g u l a r q u a r t z c u v e t t e e i t h e r 5 o r 7 mm i n w i d t h a n d 9 5 mm l o n g . The s l i t w i d t h was a d j u s t e d t o 1.0 mm a n d t h e e x i t a p e r t u r e was s e t f o r m i c r o c u v e t t e s . The g e l d r i v e m e c h a n i s m was r u n a t t h e minimum r a t e o f 0 . 5 cm/min t o a c h i e v e o p t i m u m r e s o l u t i o n . The s c a n s w e r e d i g i t i z e d u s i n g a H e w l e t t - P a c k a r d 9 8 7 2 A p l o t t e r . G e l s a n a l y s e d f o r r a d i o l a b e l w e r e r u n a s d u p l i c a t e s , o n e o f w h i c h was s t a i n e d . The o t h e r g e l was i m m e d i a t e l y s l i c e d o n a B i o - R a d - A l -M o d e l 1 9 5 e l e c t r i c g e l s l i c e r w i t h 1 mm b l a d e - t o - b l a d e s e p a r a t i o n . S l i c e s w e r e t r a n s f e r r e d t o gamma c o u n t i n g t u b e s t o w h i c h a b o u t 0 . 5 mL p o f 2 % D e c o n 7 5 was a d d e d t o p r e v e n t t h e s l i c e s f r o m a d h e r i n g t o t h e t u b e w a l l s . 2 . 2 . 3 P r o t e i n c o n c e n t r a t i o n A l b u m i n a n d f i b r i n o g e n c o n c e n t r a t i o n s w e r e d e t e r m i n e d by o n e o f t h r e e m e t h o d s . R a d i a l i m m u n o d i f f u s i o n ( R I D ) was u s e d w h e r e i n t e r f e r r i n g p r o t e i n s w e r e p r e s e n t o r s a m p l e s w e r e s m a l l . P r o t e i n o p t i c a l d e n s i t y a t a w a v e l e n g t h o f 2 8 0 nm ( O D ^ g ) ( S c h u l t z e a n d H e r e m a n s 1 9 6 6 ) was u s e d t o d e t e r m i n e t h e c o n c e n t r a t i o n o f p r o t e i n s t o c k s o l u t i o n s when t h e s e w e r e f r e e f r o m i n t e r f e r e n c e d ue t o b e n z a m i d i n e -H C 1 . The L o w r y a s s a y ( L o w r y e t a l . 1 9 5 1 ) was u s e d when b e n z a m i d i n e was p r e s e n t . B o t h o f t h e o p t i c a l m e t h o d s w e r e c a l i b r a t e d a g a i n s t t h e R I D m e t h o d . The R I D m e t h o d was c a l i b r a t e d w i t h B e h r i n g S t a n d a r d P l a s m a o r S e r u m . I n t h e R I D m e t h o d 1 t o 18 u g f i b r i n o g e n o r 2 t o 2 9 u g a l b u m i n p a r e a p p l i e d t o a n a p p r o p r i a t e B e h r i n g M P a r t i g e n p l a t e i n a 5 u L v o l u m e . The p r o t e i n d i f f u s e s i n t o t h e a n t i b o d y - c o n t a i n i n g g e l t o f o r m a p r e c i p i t a t i o n r i n g . The r i n g s d e v e l o p o v e r a p e r i o d o f t w o t o t h r e e d a y s , d e p e n d i n g u p o n t h e p r o t e i n a n d i t s c o n c e n t r a t i o n , a n d a r e t h e n a p p a r e n t l y i m m o b i l e ( M a n c i n i e t a^L. 1 9 6 5 ) . A Z e i s s b i n o c u l a r m i c r o s c o p e w i t h 1 2 . 5 X e y e p i e c e a n d 2.5X o b j e c t i v e was u s e d t o m e a s u r e r i n g d i a m e t e r s . E a c h r i n g was m e a s u r e d a c r o s s t w o d i a m e t e r s t o a p r e c i s i o n o f +0.05 mm u s i n g t h e m i c r o s c o p e ' s v e r n i e r d r i v e n s t a g e a n d a n a d j u s t a b l e e y e p i e c e p o i n t e r . I n t h e R I D m e t h o d , t h e r i n g d i a m e t e r s q u a r e d i s l i n e a r i l y r e l a t e d t o t h e p r o t e i n - 4 2 -c o n c e n t r a t i o n . The c a l i b r a t i o n d a t a was f i t t e d u s i n g l i n e a r r e g r e s s i o n a n a l y s i s a n d u n k n o w n s w e r e i n t e r p o l a t e d u s i n g t h e r e l a t i o n t h u s d e t e r m i n e d . 2.2.4 R a d i o l a b e l l i n g I o d i n a t i o n o f p r o t e i n s was c a r r i e d o u t u s i n g a m o d i f i e d l a c t o p e r o x i d a s e m e t h o d ( K r o h n a n d W e l c h 1 9 7 4 , D a v i d 1 9 7 2 ) . The p r o t e i n t o b e i o d i n a t e d was d i s s o l v e d i n a n d d i a l y s e d a g a i n s t o r c h r o m a t o g r a p h e d i n 7 0 mM p h o s p h a t e pH 8 . The p r o t e i n c o n c e n t r a t i o n was b e t w e e n 1 a n d 10 mg/mL . I n f i b r i n o g e n i o d i n a t i o n s t h e b u f f e r i n c l u d e d 1 0 mM b e n z a m i d i n e . F r e e l a c t o p e r o x i d a s e was p r e p a r e d i n t h e same b u f f e r a t a c o n c e n t r a t i o n b e t w e e n 0 . 2 a n d 0 . 5 mg/mL . I m m o b i l i z e d l a c t o p e r o x i d a s e ( E n z y m o b e a d s ) was r e s u s p e n d e d a s d i r e c t e d by t h e m a n u f a c t u r e r , d i v i d e d i n t o a l i q u o t s , a n d s t o r e d a t -20°C u n t i l r e q u i r e d . H y d r o g e n p e r o x i d e was f r e s h l y p r e p a r e d f r o m 3 0 % p e r o x i d e s t o c k b y d i l u t i o n t o 0 . 0 4 uM w i t h d i s t i l l e d w a t e r . The r e a c t i o n was q u e n c h e d w i t h p o t a s s i u m i o d i d e a n d s o d i u m a z i d e made up a t 6 a n d 3 mM r e s p e c t i v e l y a s a s i n g l e s o l u t i o n . T h e r e a c t i o n s w e r e c a r r i e d o u t i n 1.5 mL E p p e n d o r f m i c r o t e s t t u b e s ( B r i n k m a n ) w i t h i n t e r m i t t a n t m i x i n g b y i n v e r s i o n . R e a g e n t s i - i v w e r e a d d e d a s f o l l o w s : i ) p r o t e i n 2 0 0 - 5 0 0 u L i i ) l a c t o p e r o x i d a s e ( f r e e ) 2 0 u L o r ( b e a d s ) 1 0 u L i i i ) Na I 1 - 20 u L i v ) H202 ( l - 3 ) x 2 0 u L v ) K l / a z i d e 2 0 u L - 4 3 -The p r o g r e s s o f t h e r e a c t i o n was m o n i t o r e d by a p r o t e i n p r e c i p i t a t i o n a s s a y . H e r e , a p p r o x i m a t e l y 1 uL o f t h e r e a c t i o n m i x t u r e was t r a n s f e r r e d t o 1 mL o f a 2 mg/mL a l b u m i n s o l u t i o n . The p r o t e i n was p r e c i p i t a t e d b y a d d i t i o n o f 1 mL 0 . 5 M t r i c h l o r o a c e t i c a c i d s o l u t i o n a n d c e n t r i f u g a t i o n a t 6 5 0 x g f o r 3 m i n u t e s . One m i l l i l i t e r o f t h e s u p e r n a t a n t was t r a n s f e r r e d t o a s e c o n d t u b e a n d b o t h s a m p l e s w e r e c o u n t e d f o r gamma a c t i v i t y . The f r a c t i o n o f t h e r a d i o l a b e l b o u n d t o p r o t e i n was e s t i m a t e d by t h e r a t i o o f t h e d i f f e r e n c e , t o t h e sum o f t h e s a m p l e a c t i v i t i e s . The r e a c t i o n was t e r m i n a t e d b y t h e a d d i t i o n o f t h e K l / a z i d e s o l u t i o n . 2 . 2 . 5 G e l f i l t r a t i o n o f r a d i o l a b e l e d p r o t e i n s I m m e d i a t e l y a f t e r i o d i n a t i o n , r a d i o l a b e l e d p r o t e i n s w e r e s e p a r a t e d f r o m f r e e r a d i o l a b e l by g e l f i l t r a t i o n . T h i s k e p t w a s t e v o l u m e s t o a m inimum a n d a l l o w e d d e t e r m i n a t i o n o f t h e l a b e l l i n g e f f i c i e n c y . I f t h e p r o d u c t was t o be u s e d i n t h e n e x t two d a y s , a l i q u o t s o f t h e p r o t e i n f r a c t i o n s w e r e d i a l y s e d a t 4°C a g a i n s t t h e r e q u i r e d b u f f e r . I f t h e p r o d u c t was t o be u s e d l a t e r , t h e p r o t e i n f r a c t i o n s w e r e p o o l e d , s p l i t i n t o c o n v e n i e n t a l i q u o t s , a n d s t o r e d a t -20°C . B e f o r e u s i n g s t o r e d t r a c e r s i n b i n d i n g e x p e r i m e n t s , t h e 1 2 5 I - p r o t e i n s w e r e e i t h e r d i a l y s e d o r c h r o m a t o g r a p h e d t o r e m o v e f r e e r a d i o l a b e l . F i b r i n o g e n was c h r o m a t o g r a p h e d o n S e p h a d e x G150 w h i l e a l b u m i n was r u n o n S e p h a d e x G75 . T h e s e m e d i a w e r e c h o s e n b e c a u s e t h e p r o t e i n s e l u t e d a t o r n e a r t h e v o i d v o l u m e . The a p p r o p r i a t e g e l was p a c k e d i n t o a 1 x 20 cm B i o - R a d E c o n o - c o l u m n a n d e q u i l i b r a t e d w i t h t h e r e q u i r e d b u f f e r . G e l c h r o m a t o g r a p h y was u s e d b o t h t o p r e p a r e r a d i o l a b e l e d p r o t e i n s f o r b i n d i n g e x p e r i m e n t s a n d t o a s s a y f o r f r e e - 4 4 -r a d i o l a b e l . When p r e p a r i n g r a d i o l a b e l e d p r o t e i n s f o r d i r e c t u s e i n a d s o r p t i o n e x p e r i m e n t s , t h e b u f f e r was t h a t u s e d i n t h e a d s o r p t i o n . When a s s a y i n g a d s o r b a t e s o r s u p e r n a t a n t s f r o m a d s o r p t i o n e x p e r i m e n t s , t h e b u f f e r i n c l u d e d lOmM N a l . N y l o n mesh d i s c s c u t t o s e t t l e f r e e l y t o t h e g e l / b u f f e r i n t e r f a c e r e d u c e d d i s t u r b a n c e o f t h e g e l o n a d d i t i o n o f s a m p l e s . C h r o m a t o g r a p h y o f r a d i o l a b e l e d p r o t e i n s was p r e c e d e d a n d f o l l o w e d b y a d d i t i o n o f r a d i o l a b e l - f r e e p r o t e i n . When m a t e r i a l was b e i n g p r e p a r e d f o r a n a d s o r p t i o n e x p e r i m e n t , t h i s p r o t e i n was f r o m t h e same s t o c k a s t h a t u s e d i n t h e i o d i n a t i o n r e a c t i o n . R a d i o l a b e l e d p r o t e i n s w e r e a p p l i e d i n a v o l u m e o f b e t w e e n 5 0 a n d 8 0 0 u L , a n d c o l l e c t e d i n 1 0 , 2 0 , o r 30 d r o p f r a c t i o n s ( 0 . 0 1 8 g / d r o p ) . The f l o w r a t e was r e g u l a t e d b y a P h a r a m a c i a P 3 p e r i s t a l t i c pump o n t h e i n p u t s i d e o f t h e c o l u m n . A l i q u o t s o f i o d i n a t i o n p r o d u c t s w e r e t a k e n f o r gamma c o u n t i n g w i t h 1 u L d i s p o s a b l e m i c r o - c a p i l l a r i e s (Drummond M i c r o c a p s ) . When a s s a y i n g f o r f r e e l a b e l t h e t o t a l a c t i v i t y was s e l e c t e d s o t h a t w h o l e f r a c t i o n s c o u l d b e c o u n t e d . The f r a c t i o n s w e r e c o l l e c t e d i n m o d i f i e d 1.9 mL c e n t r i f u g e t u b e s ( E v e r g r e e n ) w h i c h c o u l d f i t d i r e c t l y i n t o t h e c a r r i e r t u b e s u s e d f o r gamma c o u n t i n g . F r a c t i o n s w e r e c o l l e c t e d w i t h a G i l s o n M i c r o - f r a c t i o n a t o r M o d e l FC 8 0 K . D a t a f r o m c h r o m a t o g r a p h i c a s s a y s w e r e a n a l y s e d a s d e s c r i b e d i n A p p e n d i x 4 . - 1x5 -2.3 ERYTHROCYTES 2.3.1 I s o l a t i o n B l o o d was e i t h e r f r e s h l y d r a w n o r h a d b e e n s t o r e d a s w a s h e d c e l l s a t 4°C i n P B S / a z i d e . F r e s h l y d r a w n b l o o d was o c c a s i o n a l l y a v a i l a b l e f r o m t h e V a n c o u v e r R e d C r o s s T r a n s f u s i o n S e r v i c e , b u t was u s u a l l y o b t a i n e d f r o m c o - w o r k e r s o r m y s e l f . L o c a l l y o b t a i n e d b l o o d was d r a w n f r o m t h e a n t e r i o r c u b i t a l v e i n b y s y r i n g e a n d a n t i c o a g u l a t e d w i t h e i t h e r EDTA ( d r y ) o r a c i d - c i t r a t e - d e x t r o s e ( A C D , N a t i o n a l I n s t i t u t e s o f H e a l t h , f o r m u l a A ) . A p p r o x i m a t e l y t h r e e m i l l i l i t r e s o f b l o o d was d r a w n f o r e a c h m i l l i l i t r e o f p a c k e d c e l l s r e q u i r e d . The b l o o d was f i r s t c e n t r i f u g e d a t 1 5 0 0 x g f o r 3 m i n u t e s t o s e p a r a t e r e d c e l l s f r o m p l a s m a a n d w h i t e c e l l s . A d d i t i o n o f b u f f e r o r h i g h e r c e n t r i f u g a t i o n r a t e s p r o d u c e d a n a d h e r e n t l e u c o c y t e l a y e r ( b u f f y c o a t ) a n d r e d u c e d t h e t h e r e c o v e r y o f e r y t h r o c y t e s . The r e d c e l l p e l l e t was t h e n w a s h e d r e p e a t e d l y w i t h p r o t e i n f r e e b u f f e r . I f c a r e was t a k e n t o a s p i r a t e r e s i d u a l w h i t e c e l l s i n e a c h c y c l e , a c e l l p e l l e t w i t h no a p p a r e n t t r a c e s o f w h i t e c e l l c o n t a m i n a t i o n was u s u a l l y p r o d u c e d a f t e r f o u r w a s h c y c l e s w i t h a b u f f e r t o c e l l v o l u m e o f 4:1 . A b l o o d c e l l c o u n t o f a s e r i e s o f p e l l e t s w a s h e d i n t h i s m a n n e r was p e r f o r m e d w i t h a C o u l t e r C o u n t e r t h r o u g h t h e c o u r t e s y o f t h e C l i n i c a l L a b o r a t o r y o f t h e A c u t e C a r e H o s p i t a l a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , t o d e t e r m i n e l e u c o c y t e a n d p l a t e l e t c o n t a m i n a t i o n o f r e d c e l l p r e p a r a t i o n s , T a b l e 2.3 . 2.3.2 Q u a n t i t a t i o n M e a s u r e m e n t o f h e m a t o c r i t (H) g i v e s a n e s t i m a t e o f t h e v o l u m e c o n c e n t r a t i o n o f c e l l s i n s u s p e n s i o n . I n t h i s w o r k t h e c e l l q u a n t i t i e s - 4 6 -TABL E 2.3 CONTAMINATION OF P B S WASHED RED C E L L PREPARATIONS BY WHITE C E L L S AND P L A T E L E T S P B S WASHES a RBC/uL WBC/uL P L A T E L E T S / u L H % b F r e s h B l o o d 0 w a s h 1.63 X 1 06 2.4 x 1 03 8 5 x 1 03 1 4 1 w a s h 1.84 X 1 06 1.3 x 1 03 1 X 1 03 1 6 4 w a s h e s 3 . 1 0 X 1 06 0.8 x 1 03 1 X 1 03 2 7 S t o r e d R ed C e l l s c 0 w a s h 2 . 1 4 X 1 06 0.1 x 1 03 17 X 1 03 1 8 3 w a s h e s 3.37 X 1 06 0.0 x 1 03 2 X 1 03 2 9 % CONTAMINATION f r e s h 0 . 0 2 6 0 . 0 3 2 s t o r e d 0 . 0 0 2 0 . 0 5 9 a ) 1:4 c e l l t o w a s h v o l u m e r a t i o b ) h e m a t o c r i t d e t e r m i n e d b y C o u l t e r C o u n t e r c ) R e d b l o o d c e l l s ( R B C ) w e r e s t o r e d 8 d a y s i n p l a s m a a t 4°C a f t e r r e m o v a l o f w h i t e b l o o d c e l l s ( W B C ) . w e r e d e t e r m i n e d b y w e i g h t . The r a t i o o f t h e c e l l t o s u s p e n s i o n w e i g h t , y i , was u s e d t o e x p r e s s c e l l c o n c e n t r a t i o n s a n d i s a n a l o g o u s t o h e m a t o c r i t , A p p e n d i x 2 . A r e l a t i o n b e t w e e n H a n d fb may be d e t e r m i n e d i f c e l l p e l l e t s f r e e o f e x t r a c e l l u l a r f l u i d c a n be p r e p a r e d . E x p e r i m e n t s w i t h 125 I - a l b u m i n show t h a t c e n t r i f u g a t i o n a t 2 0 0 , 0 0 0 x g f o r 30 m i n u t e s p r o d u c e s c e l l p e l l e t s w i t h a t r a p p e d s p a c e o f l e s s t h a n 0 . 5 % , s e c t i o n 3.5.1 . T h u s , p e l l e t s p r e p a r e d by u l t r a c e n t r i f u g a t i o n w e r e - 4 7 -r e s u s p e n d e d t o g i v e n v a l u e s o f fb w h i l e t h e m i c r o h e m a t o c r i t (H) was d e t e r m i n e d u s i n g a s t a n d a r d 5 m i n u t e c e n t r i f u g a t i o n a t 1 3 , 0 0 0 x g i n a n I n t e r n a t i o n a l M i c r o C a p i l l a r y C e n t r i f u g e M o d e l MB. F i g u r e 2.2 s h o w s t h e o b s e r v e d r e l a t i o n . E q u a t i o n 3.9 d e s c r i b e s a f u n c t i o n H"(/S) u s e d t o r e l a t e m e a s u r e d h e m a t o c r i t s t o t h e c o r r e s p o n d i n g w e i g h t r a t i o s . H" = m/b + l o t s - i o g /5 - °) 3.9 V a l u e s f o r t h e c o n s t a n t s m, s , a n d b a r e l i s t e d i n T a b l e 2.4 . The c o m b i n e d means w e r e u s e d i n c a l c u l a t i o n s t o c o n v e r t v o l u m e f r a c t i o n s t o m a s s f r a c t i o n s . The i n v e r s e f u n c t i o n /3(H) , was e v a l u a t e d by a n i t e r a t i v e a p p r o x i m a t i o n m e t h o d . F i g u r e 2.2b s h o w s t h a t e q u a t i o n 3.9 d e s c r i b e s t h e o b s e r v e d r e l a t i o n b e t w e e n H a n d y 6 t o w i t h i n 0 . 0 0 5 u n i t s i n H o v e r t h e f u l l r a n g e o f fb . A r e l a t i o n b e t w e e n H a n d fb b a s e d o n r e d c e l l d e n s i t y , e q u a t i o n 3 . 6 , r e s u l t e d i n h i g h e r e s t i m a t e s o f /2> a t g i v e n v a l u e s o f H , A p p e n d i x 3 . T h i s may h a v e TABLE 2.4 PARAMETERS FOR EQUATION 3 . 9 PARAMETERS m a s a b a E X P . NO. 1 2 3 1,2,3 0 . 9 5 1 0 . 9 4 9 0 . 9 4 7 + 0 . 0 0 5 ( 1 8 )b 0 . 9 5 0 + 0 . 0 0 3 ( 2 6 ) 2.68 + 0.11 2.90 0 . 1 5 2.36 0 . 0 9 2 . 5 0 + 0 . 0 7 1.20 + 0 . 0 2 ( 2 2 )b 1.15 0 .04 ( 1 2 ) 1.19 0 . 0 2 ( 3 8 ) 1.20 + 0 . 0 2 ( 7 2 ) a ) mean .+ o n e s t a n d a r d d e v i a t i o n ( L R A ) b ) number o f d a t a - 48 --.0.02 * ' • • • 1 0 ILS 1 F i g u r e 2.2 a ) H e m a t o c r i t (H) o f r e d c e l l s r e suspended i n albumin b u f f e r as a f u n c t i o n o f t h e weight r a t i o y6 . b) D i f f e r e n c e s between e q u a t i o n s 3.9 and 3.6 • • • • and e q u a t i o n 3.9 and t h e H(yS ) d a t a ++ . - 4 9 -b e e n d u e t o i n c r e a s i n g i n t e r c e l l u l a r v o l u m e a l o n g t h e m i c r o h e m a t o c r i t t u b e s d u e t o t h e c h a n g e i n a c c e l e r a t i o n w i t h r a d i u s . 2 . 3 . 3 M o d i f i c a t i o n o f R e d C e l l s R e d c e l l m e m b ranes (RBCM) w e r e p r e p a r e d f o l l o w i n g t h e m e t h o d d e s c r i b e d b y F a i r b a n k s e t a l . ( 1 9 7 1 ) . L y s i s was c a r r i e d o u t i n 50 mL p o l y c a r b o n a t e c e n t r i f u g e t u b e s . C e n t r i f u g a t i o n was a t 4°C a n d 2 x 1 04 x g a t t h e b o t t o m o f t h e t u b e f o r 20 m i n u t e s u s i n g a S o r v a l HB-4 r o t o r . Two r a t h e r t h a n t h r e e w a s h e s w i t h 5 mM p h o s p h a t e pH 8 w e r e u s e d t o p r o d u c e RBCM. R e m o v a l o f s i a l i c a c i d f r o m r e d c e l l s ( C o o k e t a l . 1 9 6 1 ) was c a r r i e d o u t b y i n c u b a t i o n o f w a s h e d RBCs i n T r i s / C a+ + b u f f e r ( 5 0 mM T r i s , 5 mM C a C l2, 1 2 0 mM N a C l ) pH 7.0 w i t h 0.04 IU/mL n e u r a m i n i d a s e ( V i b r i o c h o l e r a e ) . The i n c u b a t i o n s w e r e f o r o n e h o u r a t 37°C a n d a c e l l c o n c e n t r a t i o n o f b e t w e e n 3 5 a n d 5 0 % h e m a t o c r i t . The e l e c t r o p h o r e t i c m o b i l i t i e s ( C o o k e t a l . 1 9 6 1 , C h i u 1 9 8 4 ) o f s a m p l e s f r o m t h e c e l l p r e p a r a t i o n s w e r e d e t e r m i n e d t o c h e c k t h e r e a c t i o n s . E x p e r i m e n t s t o d e t e r m i n e t h e i n t e r c e l l u l a r a d h e s i o n e n e r g i e s e m p l o y e d r e d c e l l s m o d i f i e d b y h e a t i n g . H e a t i n g r e d c e l l s t o b e t w e e n 4 8 a n d 52°C c a u s e s t h e c e l l s t o v e s i c u l a t e (Ham e t a l . 1 9 6 8 , Rakow a n d H o c h m u t h 1 9 7 5 , ) t o f o r m r e d c e l l b e a d s w i t h d i a m e t e r s i n t h e r a n g e o f 2-5 urn. The b e a d s w e r e u s e d a s r i g i d t e s t s u r f a c e s i n t h e a d h e s i o n e n e r g y m e a s u r e m e n t s ( E v a n s a n d Buxbaum 1 9 8 1 ) . F r e s h l y d r a w n b l o o d o r P B S w a s h e d r e d c e l l s w e r e d i l u t e d t o a h e m a t o c r i t o f 0.1 % i n P B S b u f f e r w i t h pH 7.4 a n d t o n i c i t y 2 0 mosm h i g h e r t h a n t h a t o f t h e b u f f e r u s e d i n t h e a d h e s i o n m e a s u r e m e n t s . The c e l l s u s p e n s i o n was h e a t e d f o r 6 0 s e c o n d s a t b e t w e e n 4 8 a n d 52°c - 5 0 -2.4 GAMMA COUNTING 125 The e n e r g y s p e c t r u m o f I was d e t e r m i n e d a n d a c o u n t i n g w i n d o w was s e t t o a v o i d c h a n g e s i n c o u n t i n g e f f i c i e n c y d u e t o c h a n g e s 129 i n d e t e c t o r v o l t a g e . I n a d d i t i o n , a I r e f e r e n c e s o u r c e , New E n g l a n d N u c l e a r N ES-227 l o t 2 8 5 - 1 7 7 , was r o u t i n e l y c o u n t e d t o v e r i f y s t a b i l i t y . The s a m p l e v o l u m e d e p e n d e n c e o f t h e c o u n t i n g e f f i c i e n c y was 1 2 5 d e t e r m i n e d u s i n g I i n a 10 mM N a l b u f f e r . A v o l u m e o f 1 mL was t a k e n a s t h e r e f e r e n c e v o l u m e , a n d s a m p l e s w e r e e i t h e r made up t o 1 mL b e f o r e c o u n t i n g , o r c o u n t s p e r m i n u t e (CPM) d a t a was c o r r e c t e d u s i n g t h e c a l i b r a t i o n c u r v e s s h o w n i n F i g u r e 2.3 . I n a d d i t i o n , q u e n c h i n g due t o v a r i o u s t u b e m a t e r i a l s , r e d c e l l s u s p e n s i o n s , a n d t r i c h l o r o a c e t i c a c i d w e r e d e t e r m i n e d , T a b l e 2 .5 . F i n a l l y , t h e d e p e n d e n c e o f t h e c o u n t i n g e f f i c i e n c y u p o n a p p a r e n t CPM was d e t e r m i n e d i n s e r i a l d i l u t i o n e x p e r i m e n t s d e s c r i b e d i n A p p e n d i x 5 . A r e l a t i v e 1 2 9 e f f i c i e n c y ( R E ) o f 1 0 0 % was c h o s e n t o c o r r e s p o n d w i t h t h e I s o u r c e c o u n t i n g r a t e a n d t h e p l a t e a u o f t h e r e l a t i v e e f f i c i e n c y v e r s u s CPM c u r v e , F i g u r e 2 . 4 a . The d e p e n d e n c e o f RE o n CPM i s d e s c r i b e d b y : RE = ( 1 + d-CPM + h - C P M V ) -1 5.7 N o n - l i n e a r i t y a t h i g h c o u n t r a t e s was c o r r e c t e d u s i n g t h i s r e l a t i o n . A p l o t o f t h e d i f f e r e n c e b e t w e e n t h e d a t a a n d e q u a t i o n 5.7 a s a f u n c t i o n o f t h e a p p a r e n t CPM i s g i v e n i n F i g u r e 2.4b . The p a r a m e t e r s f o r e q u a t i o n 5.7 a r e l i s t e d i n T a b l e 2.6 . Two gamma c o u n t e r s w e r e u s e d , a N u c l e a r C h i c a g o M o d e l 1 0 8 5 a n d a n L K B Compugamma M o d e l 1 2 8 2 . The s t a n d a r d o p e r a t i n g s e t t i n g s f o r t h e N u c l e a r C h i c a g o c o u n t e r w e r e : a t t e n u a t i o n ( c o a r s e ) 1 ( f i n e ) 2 5 , h i g h 0 VOLUME (mL) VOLUME (mL) Figure 2.3 Relative efficiency of ^ 2 5 I detection as a function of sample volume. The buffer i s PBS + lOmM Nal and the reference volume i s 1.0 ml. a)Nuclear Chicago Counter: 12 x 125mm carrier tubes , 12 x 75mm centrifuge tubes OO . b)LKB Compugamma: 17 x 100mm carrier tubes ++ . - 5 2 -v o l t a g e 1 0 6 6 , b a s e 1 . 5 , w i n d o w 8.5 ( w i d e ) . The L KB Compugamma was s e t w i t h a w i n d o w f r o m c h a n n e l 3 5 t o 1 1 0 i n c l u s i v e . The a c t i v i t y o f a g i v e n s a m p l e was c a l c u l a t e d b y f i r s t c o r r e c t i n g t h e a p p a r e n t CPM t o a c o r r e c t e d CPM u s i n g e q u a t i o n 5 . 7 , t h e n T A B L E 2.5 ATTENUATION OF GAMMA PHOTONS MATERIAL CONCENTRATION R E L A T I V E % SAMPLE VOLUME E F F I C I E N C Y a (mL) t r i c h l o r o a c e t i c a c i d 0 . 0 m o l e s / L 1 0 0 . 0 1.0 r e d c e l l s 0 . 0 % h c t 1 0 0 . 0 1.1 0 . 0 1.5 8 8 . 0 3.0 7 7 . 0 0 . 0 1 2 1 0 0 . 5 16 1 0 0 . 2 5 9 1 0 0 . 8 6 1 1 0 0 . 3 7 6 1 0 0 . 3 8 4 1 0 0 . 5 0 1 0 0 . 0 77 9 8 . 8 7 9 9 9 . 7 8 2 9 8 . 8 8 2 9 8 . 8 + 0.2 + 0.4 p o l y a l l o m e r . . 9 8 . 9 + 0.4 p o l y s t y r e n e . . 9 8 . 9 a ) mean +. o n e s t a n d a r d d e v i a t i o n e s t i m a t e d a s s u m i n g P o i s s o n s t a t i s t i c s - 5 3 -O o LU < 3 4 LOG (CPM) F i g u r e 2.4 a ) R e l a t i v e e f f i c i e n c y ( R E ) v e r s u s a p p a r e n t c o u n t i n g r a t e (CPM) f o r ^2 5I b ) D i f f e r e n c e s b e t w e e n ( R E ) a n d e q u a t i o n 5 . 5 . N u c l e a r C h i c a g o C o u n t e r <>o, L K B Compugamma ++ . S a m p l e v o l u m e was 1.0 mL. - 54 -s u b t r a c t i n g t h e b a c k g r o u n d a c t i v i t y , a n d f i n a l l y c o r r e c t i n g t o a 1 mL e q u i v a l e n t r a t e . T he a b s o l u t e e f f i c i e n c i e s a n d t h e p a r a m e t e r s e s t i m a t e d t o c o r r e c t f o r n o n - l i n e a r i t y a t h i g h c o u n t r a t e s a r e : TABLE 2.6 COUNTING E F F I C I E N C Y AND PARAMETERS FOR EQUATIONS 5.5 AND 5.7 N u c l e a r C h i c a g o L K B Compugamma E F F I C I E N C Y ( a t 1 5 , 0 0 0 CPM) 6 3 . 2 % 77.1 % PARAMETER a d x l O9 e c x l O2 l n ( h ) x l O "2 V 7.43 - 0 . 2 3 - 1 . 0 6 6.89 + 0 . 3 2 + 0 . 2 2 + 0 . 0 3 + 0 . 2 0 ( l l )b ( 1 1 ) ( 1 3 ) ( 1 3 ) 4 . 9 5 + 0 . 2 2 0 . 1 9 + 0.11 ( 1 8 ) b ( 1 8 ) a ) mean _+ o n e s t a n d a r d d e v i a t i o n ( L R A ) b ) number o f d a t a c ) d e f i n e d i n e q u a t i o n 5.5 - 5 5 -2.5 B I N D I N G EXPERIMENTS 2.5.1 A d s o r b a t e s o l u t i o n s A d s o r b a t e s o l u t i o n s w e r e p r e p a r e d f r o m c o n c e n t r a t e d s t o c k s o f l a b e l l e d a n d l a b e l - f r e e p r o t e i n s . I n e x p e r i m e n t s a s s e s s i n g a d s o r p t i o n d e p e n d e n c e o n p r o t e i n c o n c e n t r a t i o n , b o t h s t o c k s o r i g i n a t e d f r o m t h e same p r o t e i n p r e p a r a t i o n . S o l u t i o n s w e r e m e a s u r e d by w e i g h t i n t o c l e a n , d r y p o l y a l l o m e r c e n t r i f u g e t u b e s o r p o l y c a r b o n a t e b o t t l e s . A l i q u o t s o f a d s o r b a t e s o l u t i o n w e r e r e t a i n e d f o r a s s a y o f t h e p r o t e i n c o n c e n t r a t i o n ( C ^ ) , t h e a c t i v i t y p e r g r a m ( A ^ ) , a n d , i n some c a s e s , t h e f r e e l a b e l r a t i o ( r ^2) . 2 . 5 . 2 C e l l s u s p e n s i o n s A f t e r t h e i n i t i a l p r e p a r a t i o n o f t h e c e l l s , t h e c e l l s w e r e w a s h e d a f i n a l t i m e a n d c e n t r i f u g e d a t 1 5 , 0 0 0 x g f o r 10 m i n u t e s . The s u p e r n a t a n t was a s p i r a t e d o f f t o p r o d u c e a c e l l p e l l e t w i t h a h e m a t o c r i t i n e x c e s s o f 9 0 % . T h i s c e l l p e l l e t was m i x e d b y g e n t l e r o t a t i o n a n d a l i q u o t s w e r e p r o m p t l y d r a w n f o r a d d i t i o n t o t h e a d s o r b a t e s o l u t i o n . C e l l s u s p e n s i o n s i n t h i s h e m a t o c r i t r a n g e a r e o f a s y r u p y c o n s i s t e n c y a n d w e r e t r a n s f e r r e d w i t h d i s p o s a b l e p i p e t t e s , t h e t i p s o f w h i c h h a d b e e n c u t b a c k s o t h a t a n e x i t b o r e o f 5 t o 7 mm was a v a i l a b l e . C e l l s u s p e n s i o n s b e f o r e a d d i t i o n o f a d s o r b a t e w e r e c h a r a c t e r i z e d by t h e c e l l t o s u s p e n s i o n w e i g h t r a t i o ( / ^ ) . T h i s r a t i o c a n be d e t e r m i n e d f r o m t h e c e l l s u s p e n s i o n h e m a t o c r i t ( H ^ ) a s i s d e s c r i b e d i n A p p e n d i x 3 . A f t e r r e s u s p e n s i o n w i t h t h e a d s o r b a t e s o l u t i o n , t h e c e l l c o n c e n t r a t i o n i s c h a r a c t e r i z e d b y fte • I n e x p e r i m e n t s w h e r e A - was r e q u i r e d f o r t h e b i n d i n g e s t i m a t e , i t was c a l c u l a t e d f r o m /2> u s i n g e q u a t i o n 2 . 1 2 . - 5 6 -2 . 5 . 3 I n c u b a t i o n The a d s o r b a t e / c e l l s u s p e n s i o n s w e r e m i x e d by i n c l i n i n g t h e t u b e a n d r e v o l v i n g i t p a r a l l e l t o i t s l o n g a x i s u s i n g a F i s h e r M o d e l 96 R o t o - R a c k . The i n c u b a t i o n h e m a t o c r i t s (H ) w e r e d e t e r m i n e d f o r d u p l i c a t e s a m p l e s a n d v a r i a t i o n s t h a t w o u l d i n d i c a t e i n h o m o g e n e i t y w e r e n e v e r e v i d e n t . I n c a s e s w h e r e t h e i n c u b a t i o n was n o t c a r r i e d o u t i n c e n t r i f u g e t u b e s , a l i q u o t s o f t h e a d s o r b a t e / c e l l s u s p e n s i o n s w e r e t r a n s f e r r e d t o c e n t r i f u g e t u b e s a f t e r He h a d b e e n d e t e r m i n e d . The i n c u b a t i o n s u s p e n s i o n s w e r e t h e n c e n t r i f u g e d i n a n I n t e r n a t i o n a l C l i n i c a l C e n t r i f u g e M o d e l C L , f o r 5 m i n u t e s a t 6 5 0 x g . T h i s c e n t r i f u g a t i o n a l l o w e d s a m p l i n g o f t h e a d s o r p t i o n s u p e r n a t a n t ( A ^ ) p r i o r t o u l t r a c e n t r i f u g a t i o n . The i n c u b a t i o n t i m e was t a k e n t o be t h e i n t e r v a l f r o m f i r s t m i x i n g u n t i l t h e b e g i n n i n g o f t h e l o w s p e e d c e n t r i f u g a t i o n . I n c u b a t i o n s w e r e a t r o o m t e m p e r a t u r e , 21+_1°C u n l e s s o t h e r w i s e n o t e d . 2.5.A S e p a r a t i o n o f c e l l s f r o m s u s p e n s i o n s The o b j e c t i v e i n t h i s p r o c e d u r e was t o r e d u c e t h e t r a p p i n g a r t i f a c t t o a l o w l e v e l w i t h o u t p e r t u r b i n g t h e a d s o r p t i o n e s t a b l i s h e d i n a n i n c u b a t i o n . To t h i s e n d , u l t r a c e n t r i f u g a t i o n a t 3 5 , 0 0 0 rpm f o r 3 0 m i n u t e s u s i n g a B e c k m a n L 5 - 6 5 H u l t r a c e n t r i f u g e ( S W A l - T i r o t o r ) was e s t a b l i s h e d a s t h e s t a n d a r d p e l l e t i n g c o n d i t i o n . B e c k m a n 9/16 x 3-1/2 i n c h p o l y a l l o m e r t u b e s w e r e u s e d . The a c c e l e r a t i o n a t t h e e n d o f t h e t u b e s , 1A.7 c e n t i m e t e r s f r o m t h e a x i s o f r o t a t i o n , was 2 0 2 , 0 0 0 x g . Low s p e e d p e l l e t i n g was c a r r i e d o u t i n a S o r v a l RC5 c e n t r i f u g e u s i n g a n HB-A h e a d f i t t e d w i t h a d a p t o r s t o c a r r y t h e i n t h e p o l y a l l o m e r t u b e s . H e r e t h e e n d s o f t h e t u b e s w e r e e s t i m a t e d t o be 1 3 . 6 cm f r o m t h e a x i s - 5 7 -o f r o t a t i o n . When s a m p l e s w e r e n o t l a r g e e n o u g h t o s u f f i c i e n t l y f i l l t h e p o l y a l l o m e r u l t r a c e n t r i f u g e t u b e s , a n i m m i s c i b l e o i l was u s e d t o p r e v e n t c o l l a p s e o f t h e t u b e s d u r i n g u l t r a c e n t r i f u g a t i o n . I n e a r l i e r e x p e r i m e n t s t h e o i l was c o m p o s e d t o be o f a d e n s i t y i n t e r m e d i a t e t o t h a t o f t h e e r y t h r o c y t e s a n d t h e a d s o r b a t e s o l u t i o n . T h i s o i l was a m i x t u r e o f c o t t o n s e e d o i l ( 1 p a r t b y w e i g h t ) a n d b e n z y l b e n z o a t e ( 2 . 6 p a r t s b y w e i g h t ) o f f i n a l d e n s i t y 1.056 g/mL . O i l o f d e n s i t y i n t e r m e d i a t e t o t h e c e l l s a n d f l u i d was o f m e c h a n i c a l a s s i s t a n c e i n p r e v e n t i n g t h e r e - i n t r o d u c t i o n o f s u p e r n a t a n t i n t o c e l l p e l l e t s . H o w e v e r , c o m p l e t e r e m o v a l o f t h e o i l f r o m t h e r e s u l t i n g p e l l e t s c o u l d n o t b e v e r i f i e d i n a r o u t i n e m a n n e r , s o t h a t t h e f o l l o w i n g a d d i t i o n a l s t e p s w e r e t a k e n . When c e l l s w e r e n o t r e q u i r e d f o r a wash a n a l y s i s , t h e p e l l e t s f r o m t h e u l t r a c e n t r i f u g a t i o n w e r e f r o z e n (-70°C) a f t e r r e m o v a l o f t h e s u p e r n a t a n t a n d some o f t h e o i l . S e g m e n t s o f t h e f r o z e n t u b e c o u l d t h e n b e s l i c e d o f f , s t a r t i n g f r o m t h e b o t t o m a n d w o r k i n g t o w a r d s t h e o i l - p e l l e t i n t e r f a c e . The t u b e a n d c o n t e n t s w e r e s l i c e d w i t h a L e w i s u t i l i t y k n i f e a t p o s i t i o n s i n d i c a t e d i n F i g u r e 2.5 . The e x p o s e d f a c e s o f t h e s e s l i c e s w e r e s e a l e d o f f f r o m a i r w i t h p a r a f i l m t o p r e v e n t c o n d e n s a t i o n a r t i f a c t s w h i l e t h e s l i c e s w e r e a l l o w e d t o t h a w . S a m p l e s o f t h e s l i c e s w e r e t h e n t r a n s f e r r e d t o t u b e s f o r gamma c o u n t i n g . When c e l l s w e r e r e q u i r e d f o r a w a s h a n a l y s i s , a g u i l l o t i n e d e v i s e d t o s l i c e t h e v e r t i c a l l y h e l d t u b e was u s e d . The s u p e r n a t a n t a n d o i l w e r e f i r s t r e m o v e d a n d t h e g u i l l o t i n e was t h e n u s e d t o s l i c e t h e t u b e a b o u t 5 mm b e l o w t h e p e l l e t s u r f a c e . The b l a d e r e m a i n e d i n - 58 -oil oil intermediate overlay + density • frozen oil guillotine slicing supernatant F i g u r e 2.5 S e p a r a t i o n o f c e l l p e l l e t s f r o m s u p e r n a t a n t s . The c o m p o s i t i o n o f t h e o i l s a n d t h e d e t a i l s o f t h e t e c h n i q u e s a r e g i v e n i n s e c t i o n 2 . 5 . A . - 5 9 -p l a c e u n t i l t h e s a c r i f i c e d p o r t i o n o f t h e p e l l e t was c l e a n e d a w a y . P a r a f i l m was u s e d t o c o v e r t h e e x p o s e d f a c e o f t h e p e l l e t t o p r e v e n t d r y i n g a r t i f a c t s . T h e s e t u b e s l i c i n g s t e c h n i q u e s made i t p o s s i b l e t o e l i m i n a t e t h e u s e o f t h e o i l l a y e r w h e r e c o l l a p s e o f t h e c e n t r i f u g e t u b e s was n o t a p r o b l e m . The u s e o f i m m i s c i b l e o i l m i x t u r e s i n t h e d e n s i t y s e p a r a t i o n o f c e l l s f r o m s u s p e n d i n g m e d i a h a s b e e n d e s c r i b e d ( B a l l e n t i n e a n d B u r f o r d 1 9 6 0 , D a n o n a n d M a r i k o v s k y 1 9 6 4 ) . C o m p a r i s o n o f f r o z e n , s l i c e d c e l l p e l l e t s a n d p e l l e t s r e c o v e r e d f o r w a s h a n a l y s i s s h o w e d t h a t no s i g n i f i c a n t d i f f e r e n c e s i n t h e a c t i v i t y p e r g r a m o f p e l l e t o c c u r r e d w i t h t h e s e d i f f e r e n t p r o c e d u r e s . The a c t i v i t y p e r g r a m o f t h e o i l was u s u a l l y n o t s i g n i f i c a n t l y a b o v e b a c k g r o u n d a c t i v i t y . - 6 0 -2.6 B I N D I N G A N A L Y S I S 2.6.1 E s t i m a t i o n o f b i n d i n g i n c e l l p e l l e t s A n umber o f " c o m p a r t m e n t s " i n w h i c h i s o t o p e may r e s i d e a r e p r e s e n t i n e x p e r i m e n t s i n v o l v i n g c e l l s . T h e s e i n c l u d e t h e f l u i d i n w h i c h t h e c e l l s a r e s u s p e n d e d , t h e c e l l m e m b r a n e , a n d t h e i n t r a c e l l u l a r m a t r i x . The p r e s e n c e o f r a d i o l a b e l i n a c e l l p e l l e t i s t h e r e f o r e n o t a s p e c i f i c e s t i m a t e o f b i n d i n g s o t h a t s u i t a b l e c o n t r o l s o r c o r r e c t i o n s m u s t be a p p l i e d . As m e t h o d s t o s p e c i f i c a l l y i n h i b i t f i b r i n o g e n b i n d i n g a r e n o t a v a i l a b l e , p h y s i c a l e s t i m a t e s o f n o n - s p e c i f i c b i n d i n g m u s t be m a de. The f o l l o w i n g s e c t i o n s d e v e l o p t h e m o d e l t o c o r r e c t a p p a r e n t b i n d i n g e s t i m a t e d f r o n m c e l l p e l l e t s . The m e a s u r a b l e c o m p a r t m e n t s a r e d e f i n e d i n A p p e n d i x 2 . a ) I n t e r c e l l u l a r c o m p a r t m e n t The a c t i v i t y o b s e r v e d i n a c e l l p e l l e t i s i n p a r t d u e t o t r a p p e d s u p e r n a t a n t f l u i d . T h i s c o n t r i b u t i o n m u s t be s u b t r a c t e d f r o m t h e a p p a r e n t b i n d i n g . The r e l a t i o n s h i p b e t w e e n t h e c e l l a s s o c i a t e d a c t i v i t y p e r g r a m o f c e l l s ( Ac) , t h e s u p e r n a t a n t a c t i v i t y p e r g r a m o f f l u i d ( A ^ ) , a n d t h e a c t i v i t y p e r g r a m o f c e l l p e l l e t ( Ap) , i . e . c e l l s p l u s t r a p p e d s o l u t i o n , i s g i v e n b y Ac = v/^P - v ^ / y / ^ p 7-3 w h e r e fl i s t h e r a t i o o f t h e c e l l t o p e l l e t w e i g h t . E q u a t i o n 7.3 i s d e v e l o p e d i n A p p e n d i x 7 a n d a s s u m e s t h a t t h e a c t i v i t y o f t h e f l u i d t r a p p e d i n t h e p e l l e t a n d e x t e r n a l t o t h e c e l l s i s g i v e n b y A^ . The b i n d i n g c a n a l s o be d e s c r i b e d i n t e r m s o f t h e r a t i o s A /A-- 6 1 -a n d A p / ^ f • T h e s e r a t i o s d e s c r i b e t h e d i v i s i o n o f r a d i o l a b e l b e t w e e n t h e c o m p a r t m e n t s o f t h e s y s t e m a n d a r e r e f e r r e d t o r e s p e c t i v e l y a s t h e c e l l ( P ) a n d p e l l e t (Q) d i s t r i b u t i o n r a t i o s . The d i s t r i b u t i o n r a t i o s a r e d e f i n e d i n A p p e n d i x 6 a n d a l l o w e q u a t i o n 7.3 t o b e r e - w r i t t e n a s P = Q / /3 p - ( 1 -/4p)//3p 7.4 b ) I n t r a c e l l u l a r c o m p a r t m e n t When d e n s e c e l l p e l l e t s a r e p r o d u c e d t h e i n t r a c e l l u l a r c o m p a r t m e n t r e p r e s e n t s a l m o s t a l l o f t h e v o l u m e o f a c e l l p e l l e t . T h u s , r a d i o l a b e l w h i c h h a s a c c e s s t o t h e c e l l i n t e r i o r c a n h a v e a n e f f e c t o n t h e a p p a r e n t b i n d i n g w h i c h i s l a r g e i n c o m p a r i s o n t o i t s 12 5 c o n c e n t r a t i o n i n t h e a d s o r b a t e . I o d i d e h a s a c c e s s t o t h e human e r y t h r o c y t e i n t e r i o r v i a a n a n i o n c h a n n e l ( C a b a n t c h i k a n d R o t h s t e i n 1 9 7 4 , Z a k i e t a l . 1 9 7 5 ) . The u p - t a k e a n d e l u t i o n o f i o d i d e b y r e d c e l l s ( D a l m a r k a n d W i e t h 1 9 7 2 , D a l m a r k 1 9 7 6 ) s u g g e s t s t h a t t h e i n t r a c e l l u l a r c o m p a r t m e n t i s s i m p l e r a t h e r t h a n c o m p o u n d . E q u a t i o n 7.3 was e x t e n d e d t o a c c o m m o d a t e t h e e x i s t e n c e o f r a d i o l a b e l w h i c h h a s a c c e s s t o t h e i n t r a c e l l u l a r c o m p a r t m e n t . Two s p e c i e s o f r a d i o l a b e l w e r e r e c o g n i z e d a n d d i s t i n g u i s h e d b y d i f f e r e n t c e l l d i s t r i b u t i o n r a t i o s . N u m e r i c s u b s c r i p t s w e r e a d d e d t o t h e c u r r e n t 1 25 n o t a t i o n t o i n d i c a t e q u a n t i t i e s r e l a t e d t o t h e s e s p e c i e s ; 1 = I-12 5 p r o t e i n , 2 = i o d i d e . The e x t e n s i o n i s d e v e l o p e d i n A p p e n d i x 8 a n d t h e r e s u l t i s a n a l o g o u s t o e q u a t i o n 7.3 : A d = Af[Q / / 3 p - (l - / 3 p ) / / 3 p - rf 2P2] 8.3 - 6 2 -The l a s t t e r m a c c o u n t e d f o r r a d i o l a b e l i n t h e i n t r a c e l l u l a r c o m p a r t m e n t o f a c e l l p e l l e t . I t was n e c e s s a r y t o know r ^2 » t h e r a t i o o f t h e f r e e t o t o t a l r a d i o l a b e l i n t h e s u p e r n a t a n t f l u i d , a n d P2 , t h e c e l l d i s t r i b u t i o n r a t i o f o r i o d i d e . The v a l u e o f P2 was d e t e r m i n e d i n s e p a r a t e e x p e r i m e n t s . F o r p r o t e i n s l a r g e r t h a n h e m o g l o b i n , i t was a s s u m e d t h a t p r o t e i n - b o u n d r a d i o l a b e l d i d n o t h a v e a c c e s s t o t h e i n t r a c e l l u l a r c o m p a r t m e n t . c ) Membrane c o m p a r t m e n t D e m o n s t r a t i o n o f r a d i o l a b e l a s s o c i a t e d w i t h c e l l m embranes i s r e q u i r e d f o r d i r e c t e v i d e n c e o f t h e b i n d i n g o f e x o g e n o u s p r o t e i n . When t h e a m o u n t o f m e m b r a n e - a s s o c i a t e d a c t i v i t y i s s m a l l i t i s n e c e s s a r y t h a t t h e r a d i o l a b e l r e t a i n e d d e m o n s t r a t e some b e h a v i o u r c h a r a c t e r i s t i c o f t h e r a d i o l a b e l e d e x o g e n o u s p r o t e i n . T h i s i s n e c e s s a r y s o t h a t b i n d i n g r e l a t e d t o c o n t a m i n a t i n g p r o t e i n s a n d / o r c h e m i c a l t r a n s f e r o f r a d i o l a b e l t o i n t e g r a l membrane p r o t e i n s may be e x c l u d e d . T h u s SDS-PAGE a n a l y s i s was u s e d t o c o m p a r e t h e r a d i o l a b e l e d s p e c i e s r e t a i n e d b y r e d c e l l m e m b ranes a n d t h o s e p r e s e n t i n a d s o r b a t e p r e p a r a t i o n s . The d i s t r i b u t i o n r a t i o P^ was d e f i n e d a s t h e r a t i o o f t h e p r o t e i n - b o u n d r a d i o l a b e l p e r g r a m o f c e l l s t o t h e p r o t e i n - b o u n d r a d i o l a b e l p e r g r a m o f s u p e r n a t a n t . T h u s , i n p r i n c i p l e , P-^  d i d n o t d i s t i n g u i s h b e t w e e n t h e membrane a n d i n t r a c e l l u l a r c o m p a r t m e n t s . H o w e v e r , s i n c e p r o t e i n i s e x c l u d e d f r o m t h e i n t r a c e l l u l a r c o m p a r t m e n t ( P o n d e r a n d S a s l o w 1 9 3 0 ) , P^ d i d r e p r e s e n t t h e membrane c o m p a r t m e n t . E q u a t i o n 8.3 c a n be r e - w r i t t e n t o e x p r e s s t h e b i n d i n g - 6 3 -e s t i m a t e i n t e r m s o f : P i = [Q//&p - (l-/3 p)//3p - r f 2 P 2 ] / r f i 8.A 2.6 . 2 E s t i m a t i o n o f b i n d i n g f r o m s o l u t i o n r a d i o l a b e l c o n c e n t r a t i o n A l t h o u g h t h e p e l l e t e s t i m a t i o n m e t h o d d e s c r i b e d i s d i r e c t a n d s e n s i t i v e i t r e q u i r e s a l l o w a n c e s t o be made f o r t r a p p e d m a t e r i a l . B i n d i n g e s t i m a t e s b a s e d o n c h a n g e s i n t h e c o n c e n t r a t i o n o f t h e a d s o r b a t e h a v e d i f f e r e n t l i m i t a t i o n s . The m e t h o d n e e d n o t r e q u i r e p r e p a r a t i o n o f a d e n s e c e l l p e l l e t , n o r e s t i m a t i o n o f e x t r a c e l l u l a r p r o t e i n . H o w e v e r , t h e c o n c e n t r a t i o n c h a n g e s m e a s u r e d c a n be due i n p a r t t o d i l u t i o n e f f e c t s a n d a d s o r p t i o n t o s u r f a c e s o t h e r t h a n c e l l p l a s m a m e m b r a n e s . A r e l a t i o n s h i p w h i c h t a k e s i n t o a c c o u n t t h e a d s o r p t i o n a n d d i l u t i o n e f f e c t s i s d e r i v e d i n A p p e n d i x 10 f r o m t h e s i m u l t a n e o u s c o n s e r v a t i o n o f p r o t e i n - b o u n d a n d f r e e r a d i o l a b e l s . Ac l = A i fc " A ,/3i/3e The b i n d i n g i n t e r m s o f Pj_ i s - Af 1 fle + rf 2P2 -C P Mt 1 0 . 5 P i = *f\fiifiel fle / rf l - T 10 . 6 w h e r e A^ a n d a r e r e s p e c t i v e l y t h e i n i t i a l a d s o r b a t e r a d i o l a b e l a n d c e l l c o n c e n t r a t i o n s a n d Af a n d fb& a r e t h e c o r r e s p o n d i n g f i n a l c o n c e n t r a t i o n s . The t o t a l t u b e b o u n d r a d i o l a b e l i s r e p r e s e n t e d by T . - 6 4 -2 . 6 . 3 E s t i m a t i o n o f f r e e r a d i o l a b e l 1 2 5 F r e e I was r e m o v e d f r o m p r e p a r a t i o n s o f r a d i o l a b e l e d p r o t e i n s b y g e l f i l t r a t i o n a n d / o r d i a l y s i s m e t h o d s . A s s a y s o f a d s o r b a t e a n d s u p e r n a t a n t s o l u t i o n s w e r e c a r r i e d o u t b y t h e g e l f i l t r a t i o n m e t h o d s d e s c r i b e d i n A p p e n d i x 4 . A l t h o u g h t h e s e a s s a y s c o u l d be d o n e o n a r o u t i n e b a s i s , t h e a p p a r e n t r e l e a s e o f r a d i o l a b e l f r o m p r o t e i n s d u r i n g t h e c o u r s e o f t h e e x p e r i m e n t s , t h e i n t e r p r e t a t i o n o f c h r o m a t o g r a m s c o m p l i c a t e d b y p r o t e o l y t i c d e g r a d a t i o n o f f i b r i n o g e n , a n d t h e o c c a s i o n a l o c c u r r e n c e o f a p p a r e n t l y n e g a t i v e b i n d i n g when c o r r e c t i o n s f o r " f r e e r a d i o l a b e l " w e r e a t t e m p t e d , r e d u c e d t h e r e l i a b i l i t y o f f r e e l a b e l e s t i m a t e s f r o m g e l f i l t r a t i o n d a t a . An a l t e r n a t e m e t h o d was d e v e l o p e d t o e v a l u a t e t h e a m o unt o f i n t r a c e l l u l a r r a d i o l a b e l i n b i n d i n g e x p e r i m e n t s . The e s t i m a t e i s b a s e d u p o n t h e d e p e n d e n c e o f a p p a r e n t b i n d i n g o n c e l l c o n c e n t r a t i o n . The d e p e n d e n c e o c c u r s b e c a u s e f r e e r a d i o l a b e l h a s a c c e s s t o t h e r e d c e l l v o l u m e w h e r e a s p r o t e i n - b o u n d r a d i o l a b e l d o e s n o t . T h u s , when a c e l l s u s p e n s i o n i s a d d e d t o a n a d s o r b a t e s o l u t i o n , t h e c o n c e n t r a t i o n o f t h e f r e e r a d i o l a b e l i s d i l u t e d t o a g r e a t e r e x t e n t t h a n t h a t o f t h e p r o t e i n - b o u n d r a d i o l a b e l . T h i s d i f f e r e n c e i n d i l u t i o n b e c o m e s g r e a t e r a s t h e c e l l c o n c e n t r a t i o n i s i n c r e a s e d . M e a s u r i n g t h e a p p a r e n t b i n d i n g ( A p ) o f a g i v e n a d s o r b a t e p r e p a r a t i o n a t t w o o r more c e l l c o n c e n t r a t i o n s (/2>e), a l l o w s e s t i m a t i o n o f t h e f r e e l a b e l f r a c t i o n o f t h e a d s o r b a t e ( r . _ ) . E q u a t i o n 1 1 . 1 5 i s d e v e l o p e d i n A p p e n d i x 11 . CO = Ac l/ A i + «i r i 2 1 1 . 1 5 - 6 5 -Where i s d e f i n e d b y u j = ( A p / A i - < X3) / < X2 1 1 - 1 6 a n d A "/3e " 1 = P 2 / 3 i ( l - / 3 e + P 2 / 3 e ) cx2 = (/2>p -/%)/a-/%) 11.12 <*3 = C/a± -fie)a-fo)/fii(l-fie) 1 1 - 1 3 A p l o t o f a> a g a i n s t (X^ h a s a s l o p e o f v a l u e ri 2, w h i c h i s c o n s t a n t i f ft^ e q u a l s o n e , A p p e n d i x 11 . The v a l u e o f /3 i n u l t r a c e n t r i f u g e d c e l l p e l l e t s was n o t d e t e r m i n e d i n d e p e n d e n t l y , h o w e v e r a l o w e r l i m i t o f 0 . 9 9 5 f o r t h i s p a r a m e t e r i s e s t i m a t e d i n s e c t i o n 3.5.1 . E s t i m a t e s o f f r e e l a b e l b y t h i s a n a l y s i s w e r e made f o r e q u a l t o 0 . 9 9 5 a n d t h e u p p e r l i m i t i n g v a l u e o f 1 . A m e t h o d t o d e t e r m i n e t h e s u p e r n a t a n t f r e e l a b e l ( i y2) f r o m wash a n a l y s i s d a t a i s d e s c r i b e d i n s e c t i o n 2.7.2 . 2.6.A B i n d i n g i s o t h e r m The s u r f a c e c o n c e n t r a t i o n ( f ) o f p r o t e i n b o u n d t o r e d c e l l s , i n m o l e c u l e s / c e l l i s g i v e n b y ' = M-Cc H e r e , JSf i s A v a g a d r o ' s n u m b e r , J^c i s t h e r e d c e l l s u r f a c e a r e a i n 2 _ 2 m , L,c i s t h e r e d c e l l s p e c i f i c s u r f a c e a r e a i n m / g , M i s t h e m o l e c u l a r w e i g h t o f t h e b o u n d p r o t e i n i n g / m o l e , a n d t h e s p e c i f i c b i n d i n g (SB-^) i s g i v e n b y - 6 6 -S B l = A c i C f i / ( Af l^ f ) = P1C F L - 1 0 -3/ / ' f 1 3 . 3 The s u p e r n a t a n t d e n s i t y ( ^ y ) was a s s u m e d t o be 1 . 0 g/mL a n d t h e p r o t e i n c o n c e n t r a t i o n i s g i v e n i n mg/mL . The p r o t e i n c o n c e n t r a t i o n o f t h e a d s o r b a t e s u p e r n a t a n t ( C ^ ) was e i t h e r m e a s u r e d d i r e c t l y o r c a l c u l a t e d f r o m t h e p r o t e i n c o n c e n t r a t i o n o f t h e a d s o r b a t e b e f o r e a d d i t i o n o f t h e c e l l s ( C . Q ) , e q u a t i o n 1 3 . 5 . The c a l c u l a t i o n a s s u m e s t h a t t h e c o n c e n t r a t i o n d i f f e r e n c e i s d u e p r i m a r i l y t o t h e d i l u t i o n e f f e c t o f t h e a d d e d c e l l s u s p e n s i o n . T h e d i s t r i b u t i o n r a t i o was e v a l u a t e d f r o m t h e p e l l e t a n a l y s i s u s i n g e q u a t i o n 8 . A o r f r o m t h e s o l u t i o n a n a l y s i s u s i n g e q u a t i o n 1 0 . 6 . The b i n d i n g i s o t h e r m s a r e p l o t t e d a s g r a m s p r o t e i n b o u n d p e r g r a m o f c e l l (SB-^) v e r s u s t h e f r e e p r o t e i n c o n c e n t r a t i o n ( C ^ ) i n mg/mL . T h u s , i f a b i n d i n g i s o t h e r m i s l i n e a r t h e s l o p e o f t h e i s o t h e r m w i l l b e g i v e n b y P ^ I O-"5/ f f, e q u a t i o n 1 3 . 3 . F i g u r e 2 . 6 s u m m a r i z e s t h e m e a s u r e d m a t e r i a l w e i g h t s , a c t i v i t i e s , a n d c e l l c o n c e n t r a t i o n s i n t h e f o r m o f f l o w c h a r t . - 6 7 -ADSORBATE ( f i b r i n o g e n ) w e i g h t A i a c t i v i t y / g r a m ADSORBENT ( R e d C e l l s ) H i h e m a t o c r i t ( y 3 i w e i g h t r a t i o ) INCUBATION SUSPENSION We w e i g h t He h e m a t o c r i t ( / oe w e i g h t r a t i o ) c e n t r i f u g e SUPERNATANT C E L L P E L L E T A f a c t i v i t y / g r a m Ap a c t i v i t y / g r a m w a s h a n a l y s i s F i g u r e 2.6 F l o w c h a r t o f m e a s u r e d w e i g h t s , a c t i v i t i e s a n d c e l l c o n c e n t r a t i o n s i n v o l v e d i n t h e p e l l e t a n d s o l u t i o n d e p l e t i o n b i n d i n g e s t i m a t e s . - 6 8 -2.7 WASH A N A L Y S I S Wash a n a l y s e s c o n s i s t e d o f a s e r i e s o f w a s h c y c l e s w h e r e known q u a n t i t i e s o f c e l l s w e r e r e s u s p e n d e d i n r a d i o l a b e l - f r e e m e d i u m , c e n t r i f u g e d , a n d s e p a r a t e d f r o m t h e s u p e r n a t a n t . B o t h t h e c e l l p e l l e t a n d t h e s u p e r n a t a n t w e r e a s s a y e d f o r r a d i o l a b e l . The o b j e c t i v e o f t h e a n a l y s i s was t o d e t e r m i n e i f t h e a p p a r e n t b i n d i n g ( A p ) was h o m o g e n e o u s o r n o t a n d t o e s t i m a t e t r a p p i n g i n c e l l p e l l e t s . 2.7.1 E s t i m a t i o n o f c e l l - a s s o c i a t e d r a d i o l a b e l The c e l l - a s s o c i a t e d a c t i v i t y ( Ap) r e t a i n e d b y c e l l s i n t h e w a s h p r o c e s s was c a l c u l a t e d u s i n g e q u a t i o n 7.3 . Ac = A p / / bp - Af( l - / b p ) / / 3 p 7.3 The e v a l u a t i o n o f Ac was n o t p o s s i b l e i n c e l l p e l l e t s p r o d u c e d f r o m a d s o r p t i o n s u s p e n s i o n s d u e t o t h e v e r y l o w l e v e l s o f b i n d i n g . H o w e v e r , w a s h i n g i n c r e a s e d t h e r a t i o o f t h e c e l l - a s s o c i a t e d t o s o l u t i o n -a s s o c i a t e d r a d i o l a b e l a n d made t h e u s e o f e q u a t i o n 7.3 p o s s i b l e . U l t r a c e n t r i f u g a t i o n was u s e d t o m i n i m i z e t h e f l u i d c a r r y - o v e r f r o m t h e a d s o r p t i o n c y c l e . I n t h e s u b s e q u e n t w a s h c y c l e s 4 m i n u t e s c e n t r i f u g a t i o n a t 6 5 0 x g i n a n I n t e r n a t i o n a l M o d e l HN c e n t r i f u g e was s u f f i c i e n t . The c e l l q u a n t i t y (/3p) c a n be e s t i m a t e d f r o m h e m a t o c r i t m e a s u r e m e n t s , h o w e v e r , t h i s w o u l d h a v e b e e n c o s t l y w i t h r e s p e c t t o t i m e a n d m a t e r i a l , s o t h e f o l l o w i n g m e t h o d was u s e d . A l i q u o t s o f t h e c e l l p e l l e t s p r o d u c e d b y u l t r a c e n t r i f u g a t i o n w e r e w e i g h e d i n t o c l e a n g a m m a - c o u n t i n g t u b e s . Wash b u f f e r was a d d e d a n d s u p e r n a t a n t f l u i d was r e m o v e d q u a n t i t a t i v e l y . I f c e l l s w e r e t r a n s f e r r e d , t h e c e l l s u s p e n s i o n was made h o m o g e n e o u s a n d t h e amount o f - 6 9 -s u s p e n s i o n t r a n s f e r r e d was d e t e r m i n e d b y w e i g h t . T h u s i t was p o s s i b l e t o c a l c u l a t e b o t h t h e c e l l t o p e l l e t (fb ) a n d t h e c e l l t o s u s p e n s i o n (fb ) r a t i o s f o r e a c h c y c l e . The m e t h o d u s e d t o d e t e r m i n e t h e q u a n t i t y o f c e l l s i n t h e w a s h a n a l y s i s a s s u m e d t h a t t h e c e l l p e l l e t s u p p l i e d t o t h e wash a n a l y s i s h a d a known q u a n t i t y o f t r a p p e d f l u i d a s s o c i a t e d w i t h i t . I n t h e c a s e o f u l t r a c e n t r i f u g e d c e l l p e l l e t s , t h e q u a n t i t y o f t r a p p e d f l u i d was l e s s t h a n 0 . 5 % o f t h e p e l l e t v o l u m e , s e c t i o n 3.5.1 . T h i s was n e g l i g i b l e c o m p a r e d t o t h e q u a n t i t i e s o f w a s h f l u i d u s e d i n t h e a n a l y s e s . T h u s , e a c h w a s h a n a l y s i s was d e s c r i b e d by t h e r a t i o s fbQS a n d a n d e s t i m a t e s o f A^, , A , a n d A£ a s f u n c t i o n s o f t h e n umber o f w a s h c y c l e s . The w a s h a n a l y s i s n o t a t i o n i s g i v e n i n F i g u r e 2.6 a n d t h e c a l c u l a t i o n s a r e o u t l i n e d i n A p p e n d i x 15 . 2 . 7 . 2 A n a l y s i s o f w a s h c u r v e s A w a s h c u r v e was d e f i n e d b y A ( i ) f o r l ^ i ^ n . T h i s d a t a was d e s c r i b e d m a t h e m a t i c a l l y a s t h e sum o f t w o e x p o n e n t i a l l y d e c a y i n g t e r m s . The Ac( i ) d a t a f o r a p a r t i c u l a r e x p e r i m e n t was f i r s t e x p r e s s e d a s t h e r a t i o A ( i ) / A ( 0 ) , a n d was t h e n f i t t e d t o t h e r c p e x p o n e n t i a l m o d e l A c ( i ) / A p ( 0 ) = a j e x p ( A ] _ i ) + a 2 e x p ( / \ > 2 i ) 16.1 The p a r a m e t e r s , A ^ , a2 , a n d A2 w e r e d e t e r m i n e d b y a n o n - l i n e a r , l e a s t s q u a r e s r e g r e s s i o n m e t h o d o u t l i n e d i n A p p e n d i x 16 . A m o d e l f o r t h e b e h a v i o u r o f a s i n g l e , e q u i l i b r a t i n g r a d i o l a b e l d u r i n g a w a s h e x p e r i m e n t was d e v e l o p e d , A p p e n d i x 17 . The m o d e l r e l a t e s t h e e x p o n e n t i a l f a c t o r X a n d t h e w a s h p a r a m e t e r s ( n t h C Y C L E ) INPUT ADSORPTION ( i s t C Y C L E ) WASH a d s o r b a t e a d s o r b e n t ( c e l l s ) C Y C L E STAGES c e l l a l i q u o t s u s p e n s i o n o f CS c e l l p e l l e t a l i q u o t o f CP V t r a n s f e r c e n t r i f u g e t r a n s f e r c e l l s u s p e n s i o n GRAMS o f c e l l s f l u i d t o t a l C E L L/TOTAL TRANSFER Wi CO WC w Wp wc w f We wc' Wf' Wp' WC' wc o ' wp' wc " , wc o ' Wn" A fit A t l WC Wf w, CS CS CPM/GRAM o f f l u i d p e l l e t c e l l Af Af' A p ' A c ' Af" Ap" An" A f F i g u r e 2.7 N o t a t i o n f o r q u a n t i t i e s r e q u i r e d i n b i n d i n g a n d w a s h a n a l y s e s , o m i t t i n g t h e c y c l e n u m b e r s . The n o t a t i o n f o r c y c l e s 0 t h r o u g h n a r e i d e n t i c a l e x c e p t f o r t h e c y c l e n u m b e r a n d t h e c h a n g i n g o f s u b s c r i p t e t o c s . - 71 -/ oc s a n d fSp t o t h e w a s h d i s t r i b u t i o n r a t i o ( p ) . a . m ;p - v1 - »i ( P + / ^ c s "1 - D i + l A n d r e a r r a n g i n g 1 - / 3p- l ( i ) + e x p ( A ) ( / 3c s- l ( i+l ) - 1 ) p = c i 1 7 . 1 6 1 - e x p ( A ) U s i n g e q u a t i o n 1 7 . 1 6 , v a l u e s f o r t h e f r e e l a b e l d i s t r i b u t i o n r a t i o ( p2) w e r e c a l c u l a t e d . The e x p o n e n t i a l f a c t o r A2 was d e t e r m i n e d a s d e s c r i b e d i n A p p e n d i x 1 6 , w h i l e / ^ p ^; a n c l / ^ c s ^+^ w e r e a v e r a g e d o v e r w a s h e s l ^ i ^ n - 1 . T h i s m o d e l was a l s o u s e d t o e s t i m a t e t h e v a l u e o f t h e i n t e r c e p t ftc(°)- ! t was n o t p o s s i b l e t o s i m p l y e v a l u a t e e q u a t i o n 1 6 . 1 a t i = 0 , b e c a u s e t h e v a l u e o f fl) c h a n g e d s i g n i f i c a n t l y b e t w e e n i = 0 a n d i = 1 . T h u s i t was n e c e s s a r y t o c a l c u l a t e v a l u e s f o r A ^ a n d A2 i n t h e i n t e r v a l i = 0 t o i = 1 , s o t h a t v a l u e s f o r Aci ( 0 ) a n d Ac 2( ° ) c o u l d b e c a l c u l a t e d f r o m Ac l( D a n d Ac 2( l ) . T h i s r e q u i r e d t h a t p-^  a n d p2 be i n d e p e n d e n t o f t h e w a s h m e c h a n i c s , a s s h o w n i n s e c t i o n 3.3.3 . The v a l u e o f Ac( 0 ) was t a k e n a s t h e sum o f Ac^ ( 0 ) a n d Ac 2( 0 ) • An e s t i m a t e o f t h e s u p e r n a t a n t f r e e r a d i o l a b e l r a t i o ( r ^2) may be made f r o m w a s h a n a l y s i s r e s u l t s . The q u a n t i t y Ac 2( i ) w a s d e t e r m i n e d f o r 1 - i ^ n u s i n g A c 2 ( i ) = A r j C i ) - A p ( 0 ) a ] e x p ( A ] i ) 1 7 . 1 7 -12 -The s e c o n d t e r m i s t h e s l o w l y e l u t i n g c o m p o n e n t d e t e r m i n e d by t h e n o n - l i n e a r f i t t i n g a n a l y s i s . The q u a n t i t y Ac 2( i ) w a s P l o t t e d a g a i n s t t h e q u a n t i t y K ( i ) g i v e n by i - 1 K ( i ) = P2A f ( 0 ) TT k=0 (p + /2>p-l - l )k ( P + /3cs- 1 " Dk+l 1 7 . 2 0 E q u a t i o n 1 7 . 1 9 i m p l i e s t h a t t h e p l o t w i l l h a v e a s l o p e o f r ^2 a n d a z e r o i n t e r c e p t . - 7 3 -CHAPTER 3 R E S ULTS AND D I S C U S S I O N 3.1 PREPARATION AND CHARACTERIZATION OF R A D I O L A B E L L E D P R O T E I N S The f i b r i n o g e n i s o l a t i o n p r o c e d u r e c o n s i s t e d o f t h r e e b a r i u m c i t r a t e p r e c i p i t a t i o n t o r e m o v e p r o t h r o m b i n , f o l l o w e d b y t w o A.8 % PEG p r e c i p i t a t i o n s a t pH 7 . 6 , a f f i n i t y c h r o m a t o g r a p h y t o r e m o v e p l a s m i n o g e n a n d f i b r o n e c t i n , a 3 % PEG p r e c i p i t a t i o n a t pH 5 . 6 , a n d c h r o m a t o g r a p h y t o r e m o v e PEG a n d e x c h a n g e s a l t s . Human a l b u m i n was p u r c h a s e d f r o m S i g m a a n d u s e d w i t h o u t f u r t h e r p u r i f i c a t i o n . B o t h p r o t e i n s w e r e r a d i o i o d i n a t e d u s i n g l a c t o p e r o x i d a s e . The c o m p o s i t i o n o f t h e i s o l a t e d p r o t e i n s was a s s e s s e d u s i n g SDS-PAGE. P r o t e i n c o n c e n t r a t i o n s w e r e d e t e r m i n e d v i a o p t i c a l d e n s i t y a t 2 8 0 nm, t h e L o w r y p r o t e i n a s s a y , o r b y r a d i a l i m m u n o d i f f u s i o n . 3.1.1 SDS-PAGE a n a l y s i s The m o b i l i t i e s o f p r o t e i n s i n a g i v e n p o r e - s i z e SDS-PAGE s y s t e m a r e g o v e r n e d p r i m a r i l y b y t h e p r o t e i n m o l e c u l a r w e i g h t (Weber a n d O s b o r n 1 9 6 9 , T h o r u n 1 9 7 1 ) a n d t h e a m ount o f SDS c o m p l e x e d w i t h t h e p r o t e i n ( P i t t - R i v e r s a n d I m p i o m o b a t o 1 9 6 8 , R e y n o l d s a n d T a n f o r d 1 9 7 0 a ) . SDS b i n d i n g a n d p r o t e i n m o b i l i t i e s a r e r e d u c e d when p o l y p e p t i d e s a r e c r o s s - l i n k e d a r t i f i c i a l l y ( D a v i e s a n d S t a r k 1 9 7 0 ) o r - 7 4 -n a t u r a l l y ( F u r l a n a n d B e c k 1 9 7 5 ) . G l y c o p r o t e i n s , l i p o p r o t e i n s , membrane p r o t e i n s ( P i t t - R i v e r s a n d I m p i o m o b a t o 1 9 6 8 ) , a n d m o s t p r o t e i n s w h i c h r e t a i n t h e i r n a t i v e c o n f o r m a t i o n s s i m i l a r l y b i n d r e d u c e d a m o u n t s o d SDS ( R e y n o l d s a n d T a n f o r d 1 9 7 0 a , R e y n o l d s a n d T a n f o r d 1 9 7 0 b ) . T h u s t h e r e l a t i o n b e t w e e n m o b i l i t y a n d p r o t e i n m o l e c u l a r w e i g h t i s n o t i d e n t i c a l f o r a l l p r o t e i n s . P r o t e i n s i n SDS-PAGE a n a l y s e s w e r e d e t e c t e d u s i n g C o o m a s s i e B l u e s t a i n i n g o r r a d i o l a b e l . C o o m a s s i e B l u e b i n d i n g t o p r o t e i n s i s d u e t o e l e c t r o s t a t i c i n t e r a c t i o n o f d y e s u l p h a t e a n d p r o t e i n NH* g r o u p s ( F a z e k a s de S t . G r o t h e t a l . 1 9 6 3 ) . H o w e v e r , b i n d i n g p e r g r a m p r o t e i n v a r i e s w i t h d i f f e r e n t p r o t e i n s ( K a h n a n d R u b i n 1 9 7 5 ) a n d t h e e f f i c i e n c y o f d y e u p t a k e i s r e d u c e d a t h i g h e r p r o t e i n c o n c e n t r a t i o n s ( F i s h b e i n 1 9 7 2 ) . T h u s t h e d y e d i s t r i b u t i o n s o b s e r v e d i n SDS-PAGE a n a l y s e s w i l l b e o f r e s t r i c t e d u s e i n d e t e r m i n i n g t h e c o m p o s i t i o n o f p r o t e i n p r e p a r a t i o n s . I d e n t i f i c a t i o n o f p r o t e i n s p e c i e s o t h e r t h a n f i b r i n o g e n , a l b u m i n , a n d h e m o g l o b i n was t h e r e f o r e t e n t a t i v e . E l e c t r o p h o r e s i s o f p l a s m a on 3 % p o l y a c r y l a m i d e g e l i n s o d i u m d o d e c y l s u l p h a t e d e m o n s t r a t e d t w e n t y o r m o r e p r o t e i n b a n d s . The b a n d s a r e r e f e r r e d t o b y t h e n u m b e r s a s s i g n e d i n F i g u r e 3.1 . B a n d s 9 a n d 10 w e r e i d e n t i f i e d a s f i b r i n o g e n b y c o m p a r i s o n o f t h e p r o f i l e s o f p l a s m a a n d t h r o m b i n - c l o t t e d p l a s m a . On t h e b a s i s o f r e l a t i v e a b u n d a n c e a n d m o l e c u l a r w e i g h t s ( F e l g e n h a u e r 1 9 7 0 , P u t n a m 1 9 7 5 , A n d e r s o n a n d A n d e r s o n 1 9 7 7 , L o n b e r g - H o l m e t a l . 1 9 8 2 ) , b a n d s 8 , 1 4 , 1 5 , 1 8 , a n d 19 w e r e i d e n t i f i e d a s h a p t o g l o b i n 2 - 2 , h a p t o g l o b i n 2 - 1 , I g G , t r a n s f e r r i n , a n d a l b u m i n r e s p e c t i v e l y . R e l a t i v e a b u n d a n c e s w e r e c a l c u l a t e d f r o m p e a k a r e a s e s t i m a t e d b y t r a p a z o i d s u m m a t i o n o f t h e g e l s t a i n d i s t r i b u t i o n s . OPTICAL DENSITY (595 nm) - -- 7 6 -On t h e b a s i s o f m o b i l i t y , b a n d o n e w o u l d n o r m a l l y b e i d e n t i f i e d a s p ^ - r o a c r o g l o b u l i n , h o w e v e r , t h i s p l a s m a s a m p l e was u n u s u a l i n t h a t m o r e p r o t e i n was a p p a r e n t a t t h i s m o b i l i t y t h a n n o r m a l . S i n c e t h e d o n o r was r e c o v e r i n g f r o m m i l d s e p t i c e m i a , t h e p o s s i b i l i t y t h a t b a n d o n e r e p r e s e n t e d IgM was c o n s i d e r e d . H e m o g l o b i n ( b a n d 2 1 ) was a d d e d t o s a m p l e s a n d u s e d a s t h e m o b i l i t y r e f e r e n c e . A r e l a t i o n b e t w e e n t h e a s s i g n e d m o l e c u l a r w e i g h t s (M) a n d t h e o b s e r v e d r e l a t i v e m o b i l i t i e s (R^,) f o r t h e a b o v e p r o t e i n s a n d p o l y m e r i z e d a l b u m i n i s s h o w n i n F i g u r e 3.2 . The d a t a i n t h i s f i g u r e was d r a w n f r o m f o u r d i f f e r e n t SDS-PAGE a n a l y s e s t o i n d i c a t e t h e s c a t t e r o b s e r v e d i n r e l a t i v e m o b i l i t y d e t e r m i n a t i o n s . A c u r v e was f i t t e d t o t h e d a t a by e y e a n d u s e d t o i n t e r p o l a t e m o l e c u l a r w e i g h t s . T a b l e 3.1 l i s t s t h e r e l a t i v e m o b i l i t i e s a n d e s t i m a t e d m o l e c u l a r w e i g h t s f o r t h e p e a k s n o t e d i n F i g u r e 3.1 . The mean a n d s t a n d a r d d e v i a t i o n o f t h e p e r c e n t d i f f e r e n c e b e t w e e n t h e m o l e c u l a r w e i g h t s e s t i m a t e d f r o m t h e f i t t e d c u r v e a n d t h e r e f e r e n c e m o l e c u l a r w e i g h t s was 0.01 +_ 3.8 % , n = 21 . The p r e c i s i o n o f m o l e c u l a r w e i g h t e s t i m a t e s was t h e r e f o r e b e t t e r t h a n 4 % . H o w e v e r , i t was c l e a r f r o m t h e m o b i l i t i e s o f t h e f i b r i n o g e n p o l y p e p t i d e c h a i n s t h a t l a r g e r d e v i a t i o n s , p r e s u m a b l y d u e t o d i f f e r e n c e s i n S D S - b i n d i n g , o c c u r . The r e l a t i v e m o b i l i t i e s o f t h e f i b r i n o g e n p o l y p e p t i d e c h a i n s p r o d u c e d b y 2 - m e r c a p t o e t h a n o l r e d u c t i o n o f i s o l a t e d f i b r i n o g e n , a r e p l o t t e d a t m o l e c u l a r w e i g h t s d e t e r m i n e d b y e q u i l i b r i u m s e d i m e n t a t i o n ( M c K e e e t a l . 1 9 6 6 ) a n d SDS-PAGE (McD o n a g h e t a l . 1 9 7 2 ) . F o r t h e a n a l y s e s o f f i b r i n o g e n i s o l a t i o n s a n d i s o l a t e d a l b u m i n , i t was a s s u m e d t h a t t h e m o l e c u l a r w e i g h t s o f t h e p r o t e i n s -11 -1000 10 -I • 1 1 1 1 1 1 1 1 1 1 0 RELATIVE MOBILITY 1 F i g u r e 3.2 M o l e c u l a r w e i g h t v e r s u s r e l a t i v e m o b i l i t y i n 3 % SDS-PAGE. The b a n d ( m o b i l i t y ) n u m b e r i n g i s t h a t o f F i g u r e 3.1 . The m o l e c u l a r w e i g h t a s s i g n m e n t s a r e t e n t a t i v e a n d g i v e n i n T a b l e 3.1 . A l b u m i n monomer " t h r o u g h p e n t a m e r a r e i n d i c a t e d by • . The d a t a was d r a w n f r o m f o u r s e p a r a t e SDS-PAGE a n a l y s e s . - 78 -T A B L E 3.1 R E L A T I V E M O B I L I T Y AND MOLECULAR WEIGHT E S T I M A T E S FOR PLASMA P R O T E I N S BY 3 % SDS-PAGE A N A L Y S I S PLASMA PEAK PEAK MOLECULAR WEIGHT x l O "3 P R O T E I N N o . R f a M b M c 1 0 . 0 4 7 - 0 . 0 5 1 7 2 5 / 9 5 0 ... o i ^ M / i g M 2 0 . 0 8 2 6 1 0 - 6 6 0 3 0 . 0 9 0 59 0 - 6 3 0 4 0 . 1 2 4 - 0 . 1 2 9 51 0 - 5 3 5 5 0 . 1 3 2 4 9 5 - 5 1 0 6 0.151 4 6 0 - 4 7 0 7 0 . 1 6 1 44 0 - 4 5 0 8 0 . 2 0 0 - 0 . 2 0 6 4 0 0 3 7 5 H a p t o g l o b i n 2-2 9 0 . 2 1 9 - 0 . 2 3 7 3 4 0 3 5 0 F i b r i n o g e n 10 0 . 2 4 5 - 0 . 2 6 3 3 2 0 3 2 0 F i b r i n o g e n 11 0 . 3 0 3 266 1 2 0 . 3 3 6 2 3 8 13 0 . 3 5 7 2 2 0 14 0 . 3 7 7 - 0 . 3 8 2 2 0 0 2 0 4 H a p t o g l o b i n 2-1 15 0 . 4 6 0 - 0 . 4 6 1 1 6 0 1 5 5 I g G 16 0 . 5 2 4 1 2 5 17 0 . 6 1 4 9 2 18 0 . 6 5 4 - 0 . 6 5 6 76 8 1 T r a n s f e r r i n 19 0 . 6 9 4 - 0 . 7 1 8 66 6 7 A l b u m i n 2 0 0 . 9 1 2 2 9 21 1.000 1 6 . 6 • • • H e m o g l o b i n A * 0 . 6 4 5 - 0 . 6 5 3 6 4 81 F i b r i n o g e n Ac< 0 . 6 8 6 - 0 . 6 8 7 5 6 71 F i b r i n o g e n B^> 0 . 7 3 0 - 0 . 7 3 4 4 8 6 0 F i b r i n o g e n y 0 . 2 4 8 3 3 0 3 2 5 A l b u m i n ( p e n t a m e r ) 0 . 3 0 2 - 0 . 3 1 1 2 6 4 2 6 5 " ( t e t r a m e r ) 0 . 3 8 1 - 0 . 3 9 5 1 9 8 2 0 0 " ( t r i m e r ) 0 . 5 0 8 - 0 . 5 2 7 1 3 2 1 2 8 " ( d i m e r ) 0 . 6 9 4 - 0 . 7 1 8 6 6 67 " (monomer) a ) The maximum r a n g e o f R f v a l u e s o b s e r v e d i s g i v e n f o r t h e s p e c i e s u s e d t o e s t a b l i s h M v e r s u s R f , F i g u r e 3.2 . b ) P u t n a m 1 9 7 5 c ) I n t e r p o l a t e d f r o m M v e r s u s R f , F i g u r e 3.2 . - 79 -resolved on SDS-PAGE were given by the relation shown in Figure 3.2 and that the peak areas determined from the Coomassie Blue stain distributions were proportional to protein quantity. Comparison of 125 peak areas and radiolabel for I-fibrinogen showed that these were proportional to radiolabel for optical densities to 1 OD unit. Above 2 OD units peak areas underestimated radiolabel quantities. 3.1.2 Fibrinogen isolation Precipitation of proteins by PEG (Poison and Ruiz-Bravo 1972, McPherson 1976), when the pH i s held constant and the molecular weight of the PEG i s above 6000 daltons, depends primarily on PEG concentration, protein molecular weight, and isoelectric point (Poison et a l . 1964, Juckes 1971). The suggested mechanism i s exclusion of the protein from the polymer phase (Laurent 1963, Juckes 1971). As such, specificity for fibrinogen precipitation was primarily due to i t s large size relative to most other plasma proteins. Therefore, precipitation of higher molecular weight proteins with fibrinogen was expected and was observed. However, other protein characteristics, presumably differences in isoelectric point, play a role as was seen by the fact that a protein band with mobility just less than fibrinogen did not precipitate. Except where noted, SDS-PAGE analyses of isolated proteins were carried out on samples of equal volume. In Figure 3.3a, lanes 1 and 2 show fresh and thrombin clotted plasma from one source. Lane 3 shows plasma from a second source. Lanes 4 and on show subsequent steps in the isolation of fibrinogen from the second source. Supernatants from the series of three barium citrate precipitations are - 80 -shown i n lanes A, 5, and 6 . Supernatants from the two A.8 % PEG precipitations are shown i n lanes 7 and 8, while lanes 9 and 10 show the redissolved second PEG precipitate. I t appears that there were no substantial changes i n the plasma band pattern due to the barium c i t r a t e p r e c i p i t a t i o n . In contrast, A.8 % PEG precipitates bands 9 and 10 e f f i c i e n t l y as well as bands A and 5 . Trace contaminants were detected i n the region of bands 1,6 and 7 and between 16 and 17. The second A.8 % PEG p r e c i p i t a t i o n was shown to be of value as the second supernatant, lane 8, contained protein with the mobility of band 19 (albumin). The samples shown i n lanes 11 and 12 are the same as those of lanes 5 and 7 of Figure 3.3b . A f f i n i t y chromatography of the redissolved second PEG precipitate was followed by fractionation of the protein concentrate into four pools designated A, B, C, and D, Figure 3.Aa . I t was observed that variable amounts of l i p i d were present i n pools A and B. Lipoproteins are complexes with dimensions on the order of nanometers (Gustafson 1976) and thus would be expected to be precipitated by PEG (Juckes 1971). Pools A and B contained proteins with mobilities less than those of band 1 and greater than those of band 21, Figure 3.3b lanes 3 and A . Low density lipoprotein and very low density lipoprotein have molecular weights i n excess of 2 x 10^ daltons and high density lipoproteins range from 1.7 to 3.6 x 10 5 daltons (Gustafson 1976). The bands which barely entered the 3 % gels (Figure 3.3b, lanes 3 and A) may represent lipoprotein complexes. The polypeptide chains of lipoproteins range i n molecular weight from 7,000 to 27,000 daltons (Gustafson 1976), and thus may be responsible for the - 81 -a 1 — 4 , 5 — 9 — 1 0 — mmm fe] - -mmwm ft - • i i i i i 1 18 — 19 — * l i t 5 1 S 1 121 — t 1 f $ * I Lane 1 2 3 A 5 6 7 8 9 10 11 12 21 Lane Pool * * * ? IJlil f l I II I I I 1 2 3 A 5 6 7 8 9 10 11 12 A B C D D D D D C D Figure 3.3 Three percent SDS-PAGE analysis of fibrinogen isolation 23. The lanes show progressive stages in the isolation procedure and are described in the text, section 3.1.1 . - 82 -0 0 BED VOLUME FRACTION 1.2 F i g u r e 3.A F i b r i n o g e n OD280 p r o f i l e s a ) E l u a t e f r o m S e p h a r o s e AB c h r o m a t o g r a p h y s h o w i n g t h e p o o l s s e l e c t e d f o r 3 % PEG p r e c i p i t a t i o n , b ) D e - s a l t i n g o n S e p h a d e x G150 o f p o o l D. B e n z a m i n i n e - H C l a t o n e c o l u m n v o l u m e . - 8 3 -o b s e r v e d l o w m o l e c u l a r w e i g h t s p e c i e s . C h r o m a t o g r a p h y p a r t i a l l y c o n c e n t r a t e d b a n d s 4 a n d 5 i n p o o l B. C o n t a m i n a n t s w i t h m o b i l i t i e s s i m i l a r t o b a n d s 1 a n d 19 w e r e p a r t i a l l y c o n c e n t r a t e d i n p o o l C , l a n e 5 . The f r a c t i o n s c o m p r i s i n g p o o l D show c o n t a m i n a t i n g b a n d s o f m o b i l i t i e s b o t h f a s t e r a n d s l o w e r t h a n t h o s e o f b a n d s 9 a n d 10 . Some o f t h e s e b a n d s may be c o n t a m i n a n t s n o t p r e v i o u s l y d e t e c t e d a t t h e l o w e r s a m p l e c o n c e n t r a t i o n s , h o w e v e r , i t i s p o s s i b l e t h a t some o f t h e b a n d s w e r e d u e t o e i t h e r p r o t e o l y s i s . o r a g g r e g a t i o n . The s e r i e s o f b a n d s i n t h e r e g i o n o f b a n d 1 a n d t h o s e b e t w e e n b a n d s 5 a n d 9 a r e b e l i e v e d t o be a g g r e g a t i o n e f f e c t s , w h e r e a s t h e b a n d b e t w e e n 16 a n d 17 was p r o b a b l y a s i m p l e c o n t a m i n a n t . L a n e 10 s h o w s a t w o - f o l d d i l u t i o n o f t h e s a m p l e r u n i n l a n e 7 . P o o l s C a n d D w e r e c o l l e c t e d a n d p r e c i p i t a t e d o n c e w i t h 3 % P E G . L a n e s 11 a n d 1 2 , F i g u r e 3 . 3 b , show t h e r e d i s s o l v e d p r e c i p i t a t e s o f p o o l s C a n d D r e s p e c t i v e l y . A l t h o u g h d i s t o r t i o n o f b a n d s a n d m o b i l i t i e s o c c u r r e d d u e t o o v e r l o a d i n g , i t i s c l e a r t h a t c o n t a m i n a t i n g p r o t e i n s a r e p r e s e n t . The p r o t e i n c o n c e n t r a t e s w e r e a s s a y e d u n d e r b o t h n a t i v e a n d r e d u c i n g SDS-PAGE c o n d i t i o n s a n d a r e s h o w n i n l a n e s 1 t h r o u g h 4 o f F i g u r e 3 . 5 a a t o n e - f i f t h t h e s a m p l e l o a d o f l a n e s 11 a n d 1 2 , F i g u r e 3.3b . P o o l C was n o t p r o c e s s e d a n y f u r t h e r a n d was n o t u s e d i n a d s o r p t i o n e x p e r i m e n t s . The D p o o l c h r o m a t o g r a p h e d o n S e p h a d e x G150 a s a s i n g l e p e a k , F i g u r e 3.4b . L a n e s 5 a n d 6 o f F i g u r e 3 . 5 a show t h e c h r o m a t o g r a p h e d p r o d u c t , w h i c h was t h e n l y o p h i l i z e d . A r e c o n s t i t u t i o n o f t h e l y o p h i l i z e d m a t e r i a l i s s h own i n l a n e s 7 a n d 8 . L a n e s 9 a n d 10 show an a l b u m i n m o l e c u l a r w e i g h t s t a n d a r d ( B D H ) . I n c o m p a r i s o n t o human s e r u m a l b u m i n , t h e monomer - 84 -Lane 1 2 3 A 5 6 7 8 9 10 11 12 Pool C C D D D D D D D D 2 0 • b A E C '[ 51 E m ** * I I m V > - A . R E L A T I V E M O B I L I T Y Figure 3.5 Three percent SDS-PAGE analysis of fibrinogen isolation 23. a) Pools C and D, the samples are described in the text. b)Comparison of lyophilized and glutaraldehyde cross-linked .... fibrinogen. - 8 5 -m o l e c u l a r w e i g h t was e s t i m a t e d t o be 5 8 , 0 0 0 d a l t o n s . L a n e s 11 a n d 1 2 show r e s p e c t i v e l y t h e n a t i v e a n d r e d u c e d D p o o l a t h a l f t h e s a m p l e l o a d o f l a n e s 3 a n d 4 . The r e c o v e r y o f f i b r i n o g e n p r o t e i n , when u s i n g t h e PEG i s o l a t i o n m e t h o d , was d e t e r m i n e d by R I D a n a l y s e s o f t h r e e i s o l a t i o n s , T a b l e 3.2 . A n a l y s i s o f t h e 4.8 % PEG p r e c i p i t a t e o n 3 % SDS-PAGE, F i g u r e 3 . 3 a , s h o w e d t h a t t h e m a j o r c o m p o n e n t was t h e t h r o m b i n -c l o t t a b l e p r o t e i n o f b a n d s 9 a n d 10 . The o c c u r r e n c e o f t w o b a n d s w i t h m o l e c u l a r w e i g h t s i n t h e same r a n g e a s o b s e r v e d h e r e , h a s b e e n a s s o c i a t e d w i t h l i m i t e d p r o t e o l y s i s o f t h e f i b r i n o g e n A c h a i n b y p l a s m i n - l i k e p r o t e a s e s ( M o s e s s o n e t a l . 1 9 7 3 , L i p i n s k a e t a l . 1 9 7 4 , S e m e r a r o e t a l . 1 9 7 7 , G a l a n a k i s e t a l . 1 9 7 8 , W e i n s t e i n a n d D e y k i n 1 9 7 8 ) . On r e d u c t i o n , t h e p r o d u c t s h o w e d t h e c h a r a c t e r i s t i c f i b r i n o g e n t r i p l e t , F i g u r e 3 . 5 a . The m o b i l i t i e s o f t h e t h r e e b a n d s i m p l i e d m o l e c u l a r w e i g h t s o f 8 1 , 0 0 0 , 7 1 , 0 0 0 , a n d 6 0 , 0 0 0 d a l t o n s f o r t h e A d , Eyo , a n d "Jf c h a i n s r e s p e c t i v e l y , F i g u r e 3.2 . The s t r u c t u r a l m o l e c u l a r w e i g h t s o f t h e c h a i n s h a v e b e e n r e p o r t e d t o b e r e s p e c t i v e l y 6 6 , 0 6 2 , 5 4 , 3 5 8 , a n d 4 9 , 5 2 9 ( H e n s c h e n e t a l . 1 9 8 3 ) . T h u s t h e g r a m / g r a m b i n d i n g o f SDS t o t h e f i b r i n o g e n c h a i n s a p p e a r s t o b e r e d u c e d r e l a t i v e t o p r o t e i n s s u c h a s a l b u m i n . The f r a c t i o n a l p e a k a r e a s f o r t h e f i b r i n o g e n c h a i n s w e r e 3 6 , 3 5 , a n d 2 9 % , s i m i l a r t o t h o s e p r e d i c t e d f r o m t h e s t r u c t u r a l m o l e c u l a r w e i g h t s . O f t h e c o n t a m i n a n t s i n t h e l y o p h i l i z e d p r o d u c t , t h e b a n d s w i t h m o b i l i t i e s b e t w e e n t h o s e o f b a n d s 1 a n d 4 a p p e a r e d t o b e i n t e n s i f i e d b y l y o p h i l i z a t i o n a n d s u s c e p t i b l e t o r e d u c t i o n . The b a n d s b e t w e e n 5 a n d 9 w e r e a l s o s u s c e p t i b l e t o r e d u c t i o n , a n d i t a p p e a r e d t h a t some o r a l l o f - 8 6 -T A B L E 3.2 FI B R I N O G E N RECOVERY FROM FRESH PLASMA USING THE PEG P R E C I P I T A T I O N METHOD I S O L A T I O N 28 3 2 3 5 s t a r t i n g v o l u m e (mL) 2 6 8 2 5 6 2 5 0 c o n c e n t r a t i o n ' ( m g / m L )a 2 . 6 0 2.78 2.98 PERCENT OF STARTING P R O T E I N ° I S O L A T I O N STAGE B a+ + s u p e r n a t a n t 8 0 + 11 - 9 2 + 5 r e s u s p e n s i o n o f 1 s t p p t . 8 0 + 11 - 6 1 + 3 r e s u s p e n s i o n o f 2 n d p p t . - 5 1 3 a f f i n i t y c h r o m a t o g r a p h y <> p o o l A-C 5 + 1 - -p o o l D 3 5 + 3 5 1 + 4 5 7 + 3 r e s u s p e n s i o n o f 3 r d p p t - - -s a l t c h a n g e S e p h a d e x G 1 5 0R 26 + 5 — — S e p h a c r y l S 3 0 0R - 3 3 + 3 -d i a l y s i s — — 4 3 + 3 f i n a l v o l u m e (mL) 78 1 1 0 2 2 c o n c e n t r a t i o n ( m g / m L )a 2.34 2 . 1 3 1 4 . 6 e f f i c i e n c y 2 6 + 5 3 3 + 3 4 3 + 3 a ) f i b r i n o g e n d e t e r m i n e d by R I D b ) mean + maximum d e v i a t i o n t h e s e b a n d s c o l l a p s e d t o m o b i l i t i e s c o i n c i d e n t w i t h t h e f i b r i n o g e n f\o( , Bfb , a n d / o r "ft b a n d s . F i g u r e 3.5b s h o w s t h e C o o m a s s i e B l u e s t a i n i n g p r o f i l e s o f t h e l y o p h i l z e d p r o d u c t a n d g l u t a r a l d e h y d e c r o s s - l i n k e d p r o d u c t r u n o n 3 % SDS-PAGE. M i l d g l u t a r a l d e h y d e c r o s s - l i n k i n g ( F u r l a n - 8 7 -a n d B e c k 1 9 7 5 ) c a u s e d i n c r e a s e s i n s t a i n i n g c o i n c i d e n t w i t h t h e b a n d s p r e s e n t i n t h e u n t r e a t e d f i b r i n o g e n . Z o n e s A t h r o u g h E w e r e d e f i n e d b y t h e b a n d s o b s e r v e d i n F i g u r e 3.5b t o d i s t i n g u i s h s p e c i e s w i t h m o b i l i t i e s l e s s t h a n d i m e r f i b r i n o g e n ( A ) , d i m e r f i b r i n o g e n ( B ) , l e s s t h a n monomer f i b r i n o g e n a n d g r e a t e r t h a n d i m e r f i b r i n o g e n ( C ) , monomer f i b r i n o g e n ( D ) , a n d g r e a t e r t h a n monomer f i b r i n o g e n ( E ) . The t h i r d b a n d o f z o n e A i n c r e a s e d f r o m 1.7 t o 8.7 % , a n d t h e b a n d s o f z o n e B i n c r e a s e d f r o m 6.1 t o 1 8 . 7 % , w h i l e t h e monomer f i b r i n o g e n b a n d ( z o n e D) d e c r e a s e d f r o m 74 t o 4 4 . 5 % o f t h e t o t a l s t a i n i n g . A b a n d w i t h a n a p p a r e n t m o l e c u l a r w e i g h t o f a b o u t 1 0 0 , 0 0 0 d a l t o n s r u n n i n g i n z o n e E b e t w e e n b a n d s 16 a n d 1 7 o f t h e p l a s m a p r o f i l e was s u b s t a n t i a l a n d may r e p r e s e n t p l a s m i n o g e n . T h i s b a n d d e c r e a s e d f r o m 3.4 % t o l e s s t h a n 0 . 3 % u p o n c r o s s - l i n k i n g w i t h f i b r i n o g e n . T h i s i s o l a t i o n was s e r i o u s l y d e g r a d e d b y s t o r a g e o f t h e l y o p h i l i z e d m a t e r i a l a t r o o m t e m p e r a t u r e . S u b s e q u e n t i s o l a t i o n s w e r e s t o r e d a t -20°C . P l a s m i n o g e n i s a p r o t e i n o f m o l e c u l a r w e i g h t 8 7 , 0 0 0 d a l t o n s a n d i s f o u n d a t a c o n c e n t r a t i o n o f a b o u t 0.4 mg/mL i n p l a s m a ( P u t n a m 1 9 7 5 ) . I t i s t h e p r e c u r s o r o f p l a s m i n a n d i s a n i m p o r t a n t c o n t a m i n a n t i n - f i b r i n o g e n p r e p a r a t i o n s , a s p l a s m i n i s t h e p r o t e a s e p r i m a r i l y r e s p o n s i b l e f o r f i b r i n ( o g e n ) o l y s i s . The b a n d b e t w e e n b a n d s 16 a n d 17 i n l a n e 12 o f F i g u r e 3 . 3 a a n d i n l a n e s 6 t h r o u g h 8 , 1 0 , a n d 1 2 o f F i g u r e '3.3b, a t a p p r o x i m a t e l y t h e same m o b i l i t y , may r e p r e s e n t p l a s m i n o g e n . L y s i n e - S e p h a r o s e a f f i n i t y c h r o m a t o g r a p h y was u s e d t o s p e c i f i c a l l y r e m o v e p l a s m i n o g e n ( M a t s u d a e t a l . 1 9 7 2 ) . The p r e s e n c e o f p l a s m i n o g e n i n t h e a b o v e f r a c t i o n s a p p e a r s t o i n d i c a t e t h a t t h e l y s i n e - S e p h a r o s e a f f i n i t y medium h a d b e e n o v e r l o a d e d . - 8 8 -F i b r o n e c t i n i s a p r o t e i n o f m o l e c u l a r w e i g h t 4 5 0 , 0 0 0 d a l t o n s a n d i s f o u n d i n p l a s m a a t a c o n c e n t r a t i o n o f 0 . 3 +_ 0.1 mg/mL ( M o s e s s o n 1 9 7 7 ) . T h i s p r o t e i n p r o m o t e s c e l l a d h e s i o n i n v i t r o ( H y n e s 1 9 8 1 ) , p o s s i b l y v i a a s p e c i f i c b i n d i n g s i t e ( P i e r s c h b a c h e r a n d R u o s l a h t i 1 9 8 4 ) a n d t h e r e f o r e g e l a t i n - a f f i n i t y c h r o m a t o g r a p h y was u s e d t o r e m o v e f i b r o n e c t i n f r o m f i b r i n o g e n p r e p a r a t i o n s . E i t h e r o f b a n d s 4 o r 5 a r e o f a p p r o x i m a t e l y t h e c o r r e c t p l a s m a a b u n d a n c e , b u t t h e m o b i l i t i e s a p p e a r e d t o b e l o w b y a b o u t 1 5 % . B a n d s 6 a n d 7 a r e o f t h e c o r r e c t a p p a r e n t m o b i l i t y , b u t a r e o f l o w e r t h a n e x p e c t e d a b u n d a n c e . B a n d s 6 a n d 7 a r e n o t r e a d i l y e v i d e n t a f t e r p r e c i p i t a t i o n w i t h 4 . 8 % PEG ( l a n e s 9 a n d 1 0 , F i g u r e 3 . 3 a ) a n d a r e n o t d i s t i n g u i s h a b l e i n h e a v i l y l o a d e d g e l s o f t h e p r o t e i n c o n c e n t r a t e a f t e r g e l a t i n - a f f i n i t y c h r o m a t o g r a p h y . B a n d s 4 a n d 5 w e r e e v i d e n t a f t e r 4 . 8 % PEG p r e c i p i t a t i o n , b u t w e r e p r i m a r i l y i n p o o l B a f t e r g e l a t i n - a f f i n i t y c h r o m a t o g r a p h y . T h u s t h e s e b a n d s w e r e a s s o c i a t e d w i t h t h e l i p i d c o n t a i n i n g f r a c t i o n s o f c h r o m a t o g r a p h e d p r o t e i n c o n c e n t r a t e s , w e r e n o t b o u n d by g e l a t i n , a n d w e r e t h e r e f o r e u n l i k e l y t o r e p r e s e n t f i b r o n e c t i n . F i b r o n e c t i n was n o t i d e n t i f i e d i n i s o l a t i o n 2 3 . E s t i m a t e s o f t h e a m o u n t s o f p o s s i b l e c o n t a m i n a n t s i n i s o l a t e d f i b r i n o g e n w e r e d e t e r m i n e d f r o m t h e d i s t r i b u t i o n s o f C o o m a s s i e B l u e s t a i n a n d r a d i o l a b e l i n 3 % SDS-PAGE g e l s . F i g u r e s 3 . 6 a a n d b show t h e d y e a n d r a d i o l a b e l d i s t r i b u t i o n s f o r i s o l a t i o n 3 0 . T h e s e a n a l y s e s e m p l o y e d p y r o n i n Y a s t h e r e f e r e n c e m a t e r i a l , r a t h e r t h a n h e m o g l o b i n , s o t h a t s c a l e f a c t o r s ' g i v e n i n T a b l e 3.3 w e r e r e q u i r e d t o c o n v e r t o b s e r v e d m o b i l i t y v a l u e s t o t h o s e o f t h e M v e r s u s R^. p l o t , F i g u r e 3.2 . The f a c t o r s a r e o f t h e f o r m K = Rf/ Rf' , w h e r e Rf - 8 9 -E c LO (TI ID . a o o 1 23 A! 7 J89 13! I 10 11 12 15 16 17 0 — i - i - i 0 Q_ o RELATIVE MOBILITY F i g u r e 3.6 T h r e e p e r c e n t SDS-PAGE a n a l y s i s o f f i b r i n o g e n i s o l a t i o n 3 0 , n a t i v e ^ r e d u c e d . a ) S t a i n i n g p r o f i l e , b ) ^2 5I r a d i o l a b e l . T h e s t a i n a n d r a d i o l a b e l d i s t r i b u t i o n s a r e l i s t e d i n T a b l e 3.3 . - 9 0 -TA B L E 3.3 P U R I T Y OF F I B R I N O G E N I S O L A T I O N 3 0 PERCENT OF TOTAL a PLASMA ZONE R f ' M S T A I N 1 2 5I S T A I N 1 2 5I BANDS x l O- 3 ( n a t i v e ) ( r e d u c e d ) A B D F G 0 . 0 i 1 0 . 0 5 7 8 3 3 1.3 1.5 0.1 0.0 l 2 0 . 1 1 0 5 7 0 4 . 5 3.3 0.2 0.1 2,3 3 0 . 1 3 3 5 0 9 1.8 1.8 4 0 . 1 8 1 4 1 1 2 . 0 0.8 4 - 7 5 0 . 1 9 8 3 8 3 2.3 1.5 0.4 0 . 4 6 0 . 2 2 4 3 4 8 0 . 2 0 . 2 R i n 7 0 . 2 5 8 3 1 0 8 4 . 9 8 4 . 6 • • • O— J.U 8 0 . 2 6 9 3 0 0 1.8 1.3 9 0 . 3 0 0 2 6 7 1.3 3.8 10 0 . 4 1 1 18 3 0 . 3 0.1 1.5 11 1 £L 11 0 . 4 8 0 146 1.2 2.8 11-16 1 2 0 . 5 3 5 121 1.2 2.0 13 0 . 5 9 0 1 0 0 0.1 . 0.2 1.6 3.1 14 0 . 7 1 5 6 5 0 . 3 0 . 3 9 0 . 2 8 4 . 5 1 7 , 1 8 15 0 . 7 9 8 4 8 0.1 0.6 2.3 2.5 16 0 . 8 9 5 31 0 . 2 0.5 0.5 1.0 1 9 - 2 1 17 1.04 10 1.2 0.8 0.2 0.6 PAGE EXPERIMENT # 6 0 / 6 7 1 / 2 6 0 / 9 7 1 / 8 S C A L E FACTOR K 1.047 1.058 1.108 1.084 a ) The b l a n k s d o n o t i n d i c a t e z e r o v a l u e s . The p e r c e n t s t a i n f o r t h e n a t i v e p r o f i l e f r o m p o s i t i o n 5 t h r o u g h 7 was 8 4 . 9 % . - 9 1 - . was m o b i l i t y r e l a t i v e t o h e m o g l o b i n a n d R^1 was m o b i l i t y r e l a t i v e t o p y r o n i n Y . The p r o f i l e s i n F i g u r e 3 . 6 a w e r e t h e n d i v i d e d i n t o z o n e s o n t h e b a s i s o f t h e a p p a r e n t b a n d s . The f r a c t i o n a l a r e a s u n d e r t h e p r o f i l e s f o r t h e s e z o n e s w e r e d e t e r m i n e d u s i n g t r a p a z o i d s u m m a t i o n . The same z o n e s w e r e u s e d i n t h e a n a l y s i s o f t h e r a d i o l a b e l d i s t r i b u t i o n s , F i g u r e 3.6b . The r e l a t i o n s h i p b e t w e e n t h e z o n e s d e f i n e d i n F i g u r e 3.5b a n d t h o s e d e f i n e d i n F i g u r e 3 . 6 a i s n o t e d i n F i g u r e 3.6b . The same s u m m a t i o n p o i n t s w e r e u s e d i n t h e a n a l y s i s o f r a d i o l a b e l e d i s o l a t i o n 3 0 , 3 4 , a n d 3 5 . The a n a l y s e s o f t h e s t a i n a n d r a d i o l a b e l d i s t r i b u t i o n s a r e s u m m a r i z e d i n T a b l e s 3.3 a n d 3.4 . The d y e - b i n d i n g a n d r a d i o l a b e l d i s t r i b u t i o n s f o r i s o l a t e d f i b r i n o g e n w e r e g e n e r a l l y s i m i l a r , b u t d i f f e r e n c e s w e r e o b s e r v e d s u g g e s t i n g t h a t c o n t a m i n a t i n g s p e c i e s w e r e p r e s e n c e . I s o l a t i o n 3 0 , T a b l e 3 . 3 , s u g g e s t s t h a t r e l a t i v e t o r a d i o l a b e l , t h e amount o f d y e a s s o c i a t e d w i t h t h e f i b r i n o g e n c h a i n s , z o n e F , was o n a v e r a g e a b o u t 6 % g r e a t e r t h a n t h a t a s s o c i a t e d w i t h o t h e r s p e c i e s . A s i m i l a r d i s p r o p o r t i o n was n o t o b s e r v e d f o r monomer f i b r i n o g e n ( z o n e D ) , h o w e v e r , t h i s may b e d u e t o u n d e r e s t i m a t i o n o f f i b r i n o g e n b y d y e s t a i n i n g a s t h e s t a i n i n g was v e r y i n t e n s e i n t h i s c a s e , F i g u r e 3.6 . A t r e l a t i v e m o b i l i t i e s b e t w e e n 0.41 a n d 0 . 5 9 ( z o n e E ) , t h e p e r c e n t r a d i o l a b e l was 5.3 % g r e a t e r t h a n t h a t o f d y e a n d s u g g e s t s t h a t n o n - f i b r i n o g e n s p e c i e s w e r e p r e s e n t . I n z o n e C t w o n o n - f i b r i n o g e n s p e c i e s w i t h r e l a t i v e m o b i l i t i e s o f 0 . 1 2 2 a n d 0 . 1 9 0 ( a p p a r e n t m o l e c u l a r w e i g h t s o f 5 4 0 , 0 0 0 a n d 3 9 5 , 0 0 0 d a l t o n s ) r e p r e s e n t e d r e s p e c t i v e l y 1.8 a n d 2.3 % o f t h e t o t a l s t a i n i n g , a n d s i m i l a r p e r c e n t a g e s o f t h e r a d i o l a b e l . The p r o f i l e o f t h e r e d u c e d - 9 2 -TAB L E 3.4 RADIOLABEL P U R I T Y OF I S O L A T I O N S 34 AND 3 5 I S O L A T I O N 3 4 I S O L A T I O N 3 5 ZONE PERCENT OF TOTAL A C T I V I T Y PERCENT OF TOTAL A C T I V I T Y n a t i v e r e d u c e d n a t i v e r e d u c e d A 1.9 0.8 2.1 0. 3 1 2 . 7 0 . 3 B 2.4 1.7 4.1 0.5 1 1 . 5 0.4 C 7.1 5.9 5.6 2.0 5.2 0 . 3 D 8 3 . 6 8 5 . 3 7 6 . 3 0.4 6 4 1 0. 3 E 1.6 1.5 4.1 8 . 8 2 2.7 6.2 F 0 . 6 0 . 9 0.7 7 5 . 5 0.8 8 5 . 7 G 2.8 3.9 7.1 1 2 . 5 3.0 6.8 EXPER # 7 8 / 3 7 8 / 5 7 9 / 5 7 9 / 7 8 1 / 5 8 1 / 1 1 p r o d u c t s h o w s o n e s p e c i e s o f r e l a t i v e m o b i l i t y 0 . 2 4 6 ( a p p a r e n t m o l e c u l a r w e i g h t 3 2 5 , 0 0 0 d a l t o n s ) . a n d t w o more a t 0 . 4 4 5 a n d 0 . 5 0 7 ( m o l e c u l a r w e i g h t s 1 2 0 , 0 0 0 a n d 1 8 0 , 0 0 0 d a l t o n s ) c o m p r i s i n g 1 . 8 , 1.2 a n d 1.2 % o f t h e t o t a l s t a i n i n g r e s p e c t i v e l y . The n a t i v e b a n d w i t h r e l a t i v e m o b i l i t y 0 . 1 2 2 a n d t h e r e d u c e d b a n d w i t h r e l a t i v e m o b i l i t y 0 . 2 4 6 w e r e o f e q u a l a d u n d a n c e . The m o b i l i t i e s a n d q u a n t i t a t i v e r e l a t i o n b e t w e e n t h e s e b a n d s s u g g e s t s t h a t t h e s e may r e p r e s e n t f i b r o n e c t i n ( V u e n t o a n d V a h e r i 1 9 7 8 ) . The b a n d - 9 3 -w i t h r e l a t i v e m o b i l i t y 0 . 1 9 0 a p p e a r s t o r e p r e s e n t h a p t o g b o b i n 2-2 . S p e c i e s w i t h r e l a t i v e m o b i l i t i e s l e s s t h a n 0 . 7 1 5 ( a p p a r e n t m o l e c u l a r w e i g h t l e s s t h a t 6 5 , 0 0 0 d a l t o n s ) r e p r e s e n t e d l e s s t h a n 2 % i n n a t i v e a n d A % i n r e d u c e d g e l s a n d h a v e n o t b e e n i d e n t i f i e d . The d i s t r i b u t i o n s f o r i s o l a t i o n s 3 0 , 3 A , a n d 3 5 s h o w e d t h a t a b o u t 8 5 % o f t h e d e t e c t e d r a d i o l a b e l r a n c o i n c i d e n t w i t h t h e Ac< , Bfl , a n d "tf c h a i n s o f f i b r i n o g e n . I n i s o l a t i o n 3A t h e a m o u n t o f r a d i o l a b e l a s s o c i a t e d w i t h monomer f i b r i n o g e n , z o n e D, was l o w e r i n s u b s e q u e n t a n a l y s e s , b u t t h e f r a c t i o n s o f t h e t o t a l r a d i o l a b e l d e t e c t e d i n z o n e s D a n d F w e r e e q u a l , a s o b s e r v e d i n i s o l a t i o n 3 0 . I n c r e a s e i n t h e r a d i o l a b e l p r e s e n t i n z o n e G s h o w s t h a t some d e g r a d a t i o n o f t h e p r o d u c t o c c u r r e d . I n i s o l a t i o n 3 5 , t h e f r a c t i o n s o f t h e t o t a l r a d i o l a b e l d e t e c t e d i n z o n e s D a n d F w e r e n o t e q u a l . H o w e v e r , t h e sum o f t h e p e r c e n t r e d u c a b l e r a d i o l a b e l i n z o n e s A , B, a n d D was o n l y 1.5 % more t h a n t h e r e d u c e d r a d i o l a b e l i n z o n e F . T h i s s u g g e s t e d t h a t 9 0 % o f t h e r a d i o l a b e l i n z o n e s A. a n d B may r e p r e s e n t a g g r e g a t e d f i b r i n o g e n . 3 . 1 . 3 A l b u m i n C r y s t a l l i z e d a n d l y o p h i l i z e d human a l b u m i n was p u r c h a s e d f r o m S i g m a a n d u s e d w i t h o u t f u r t h e r p u r i f i c a t i o n . T h r e e p e r c e n t SDS-PAGE a n a l y s i s d e t e c t e d a g g r e g a t e s up t o h e x a m e r s , F i g u r e 3.7 . The d e g r e e o f a g g r e g a t i o n was n o t n o t i c e a b l y a l t e r e d o n r e d u c t i o n w i t h 2 - m e r c a p t o e t h a n o l . 1 2 5 3.1.A I o d i n e r a d i o l a b e l e d p r o t e i n s 1 2 5 I o d i n e was s e l e c t e d a s t h e l a b e l f o r t h e a d s o r p t i o n s t u d y a s t h e i n f l u e n c e o f i o d i n a t i o n o n t h e b e h a v i o u r o f f i b r i n o g e n h a d b e e n d e s c r i b e d e x t e n s i v e l y . B i o l o g i c a l c l e a r a n c e o f r a d i o l a b e l e d F i g u r e 3.7 O p t i c a l d e n s i t y p r o f i l e o f 12 5I r a d i o l a b e l e d S i g m a human a l b u m i n a n a l y s e d by 3 % SDS-PAGE. The s t a i n u s e d was C o o m a s s i e B l u e G 2 5 0 . The 12 5I a c t i v i t y o f a s e c o n d g e l was d e t e r m i n e d f o r 1mm s l i c e s a n d p l o t t e d a s a s u p e r i m p o s e d h i s t o g r a m . P y r o n i n Y was t h e m o b i l i t y r e f e r e n c e . - 9 5 -f i b r i n o g e n v a r i e s w i t h t h e m e t h o d o f p r e p a r a t i o n ( M e t z g e r e t a l . 1 9 7 3 ) a n d t h i s may r e f l e c t t h e d i f f e r e n t p r o t e i n r e s i d u e s l a b e l l e d b y t h e s e m e t h o d s ( K r o h n a n d W e l c h 1 9 7 4 , K r o h n e t a l . 1 9 7 7 ) . F o r m a t i o n o f p r o t e i n a g g r e g a t e s a n d d e c r e a s e i n t h r o m b i n c l o t t i n g t i m e h a s b e e n o b s e r v e d when m o r e t h a n 3 a t o m s o f i o d i n e w e r e i n c o r p o r a t e d p e r m o l e c u l e o f f i b r i n o g e n ( L y a n d K i e r u l f 1 9 7 5 ) . G e n e r a l l y , i n c o r p o r a t i o n o f l e s s t h a n o n e a t o m i o d i n e p e r m o l e c u l e f i b r i n o g e n h a s n o t b e e n f o u n d t o a l t e r i n v i v o o r i n v i t r o b e h a v i o u r . E n z y m a t i c i o d i n a t i o n u s i n g l a c t o p e r o x i d a s e ( K r o h n a n d W e l c h 1 9 7 4 ) was c h o s e n f o r t h e s e s t u d i e s b e c a u s e o f i t s s i m p l i c i t y , g r e a t e r l a b e l l i n g e f f i c i e n c y ( A r d a i l l o u a n d L a r r i e u 1 9 7 4 , K r o h n e t a l . 1 9 7 7 ) , a n d h i g h s p e c i f i c i t y f o r t y r o s i n e s u b s t i t u t i o n ( K r o h n e_t a l . 1 9 7 7 ) . F i b r i n o g e n c l o t t a b i l i t y a n d m o b i l i t y i n SDS-PAGE h a v e b e e n f o u n d t o be r e l a t i v e l y i n s e n s i t i v e t o i o d i n a t i o n 1 2 5 ( R e g o e c z i 1 9 7 1 , L y a n d K i e r u l f 1 9 7 5 ) . I n a d d i t i o n , i o d i n e e m i t s m o s t l y 2 7 - 3 5 KeV gamma p h o t o n s a n d t h e s e a r e n o t s i g n i f i c a n t l y q u e n c h e d b y b i o l o g i c a l m a t e r i a l s . T h u s i t was p o s s i b l e t o r a p i d l y a n d r e p e a t e d l y a s s a y w h o l e c e l l p e l l e t s f o r r a d i o l a b e l i n p r o c e d u r e s s u c h a s w a s h a n a l y s e s . U n d e r t h e c o n d i t i o n s d e s c r i b e d i n s e c t i o n 2.2.4 b o t h a l b u m i n a n d f i b r i n o g e n w e r e r a d i o l a b e l e d t o l e s s t h a n 0.5 m o l e s i o d i n e p e r m o l e o f p r o t e i n , T a b l e 3.5 . The r e a c t i o n p r o d u c t s , f i b r i n o g e n o r a l b u m i n , w e r e c h r o m a t o g r a p h e d o n S e p h a d e x G150 o r G75 r e s p e c t i v e l y , t o r e m o v e t h e m a j o r p o r t i o n o f t h e u n r e a c t e d r a d i o l a b e l a n d t o a s s e s s t h e r e a c t i o n e f f i c i e n c y . The e l u t i o n o f p r o t e i n s a n d i o d i d e was e x p r e s s e d a s t h e r a t i o o f t h e e l u t i o n v o l u m e o f a s p e c i e s t o t h e v o l u m e o f t h e c o l u m n b e d . - 96 -T A B L E 3.5 1 2 5I 0 D I N E S U B S T I T U T I O N I N R A D I O L A B E L L E D PROTEINS P R O T E I N REACTION . BOUND A C T I V I T Y m o l e s I s p e c i e s q u a n t i t y ( m g ) E F F I C I E N C Y (CPM) m o l e p r o t e i n A l b u m i n 2 . 0 3 2 % 1.4 X 1 0 8 0 . 0 0 1 9.6 7 5 % 7.0 x i r j 8 0 . 0 0 1 5.2 7 6 % 1.5 X 1 0 * 0 . 0 0 4 2.1 9 2 % 1.2 X 1 0 * 0 . 0 0 8 F i b r i n o g e n 3.0 81 % 2.8 X I O 9 0 . 0 6 6 5 . 0 9 3 % 1.6 X 1 0 * 0 . 0 2 3 1.5 9 0 % 2.1 X I O 9 0 . 1 0 T h i s r a t i o was r e f e r r e d t o a s t h e b e d v o l u m e f r a c t i o n . F i b r i n o g e n , 1 2 5 R I - f i b r i n o g e n , a n d t h e v o i d v o l u m e m a r k e r B l u e D e x t r a n 2 0 0 0 , e l u t e d a t b e d v o l u m e f r a c t i o n s b e t w e e n 0 . 3 4 a n d 0 . 3 6 o n S e p h a d e x G 1 5 0 . B l u e D e x t r a n r e c o v e r i e s w e r e b e t w e e n 9 0 a n d 1 0 0 % . F i b r i n o g e n r e c o v e r i e s i n n e w l y p r e p a r e d c o l u m n s w e r e a b o u t 7 0 % . F i b r i n o g e n r e c o v e r i e s i n u s e d c o l u m n s w e r e b e t w e e n 8 5 a n d 9 0 % . R e c o v e r y o f r a d i o l a b e l e d p r o t e i n p r e p a r a t i o n s was i m p r o v e d b y r u n n i n g t h e s e w i t h a p r o t e i n - c a r r i e r . A l b u m i n c h r o m a t o g r a p h e d w i t h a b e d v o l u m e f r a c t i o n o f a b o u t 0.4 o n S e p h a d e x G75 a n d was r e c o v e r e d w i t h e f f i c i e n c i e s s i m i l a r 1 2 5 t o t h o s e f o r f i b r i n o g e n . I o d i d e e l u t e d a t a b e d v o l u m e f r a c t i o n b e t w e e n 0 . 9 8 a n d 1.0 . B o v i n e l a c t o p e r o x i d a s e ( L P ) e l u t e d i n p a r t i a l o v e r l a p w i t h 1 25 f i b r i n o g e n , F i g u r e 3.8 . I n I - f i b r i n o g e n p r e p a r a t i o n s , t h e p e r c e n t o f t h e t o t a l L P r e t a i n e d w i t h t h e p r e p a r a t i o n was d e c r e a s e d b y - 97 -1 f . a X X nm) (280 OD + Y 0 BED VOLUME FRACTION F i g u r e 3.8 S e p h a d e x G150 c h r o m a t o g r a p h y o f r e a c t i o n p r o d u c t s f r o m r a d i o i o d i n a t i o n . a ) F i b r i n o g e n + + a n d b o v i n e l a c t o p e r o x i d a s e . b ) * 25I - f i b r i n o g e n 0 D2 8 0 + +, CPM , a n d 1 2 5 i0d i d e . - 9 8 -s a c r i f i c i n g t h e t r a i l i n g f r a c t i o n s o f t h e f i b r i n o g e n p e a k ( f r o m 1 2 t o 3 % i n F i g u r e 3 . 8 a when f r a c t i o n 1 0 was d i s c a r d e d ) . The u s e o f i m m o b i l i z e d l a c t o p e r o x i d a s e made no n o t i c a b l e d i f f e r e n c e t o b i n d i n g e s t i m a t e s . On S e p h a c r y l S 3 0 0 , B l u e D e x t r a n 2 0 0 0 c h r o m a t o g r a p h e d a t a b e d v o l u m e f r a c t i o n b e t w e e n 0 . 3 7 a n d 0 . 3 8 a n d r e c o v e r y was 9 5 t o 1 0 0 % . F i b r i n o g e n e l u t e d a t a b e d v o l u m e f r a c t i o n b e t w e e n 0 . 4 4 a n d 0 . 4 7 , w h i l e a g g r e g a t e d f i b r i n o g e n e l u t e d b e t w e e n 0 . 3 8 a n d 0 . 4 2 . R e c o v e r y was s i m i l a r t o t h a t i n S e p h a d e x G 1 5 0 . On s t o r a g e , p r e p a r a t i o n s o f I - f i b r i n o g e n a n d I-a l b u m i n w e r e f o u n d t o c o n t a i n i n c r e a s i n g a m o u n t s o f c h r o m a t o g r a p h i c a l l y d e t e c t a b l e r a d i o l a b e l r u n n i n g a t a b e d v o l u m e f r a c t i o n c o m p a r a b l e t o I O C I rye i o d i d e ( ' f r e e * r a d i o l a b e l ) . I n t h e c a s e o f I - a l b u m i n p r e p a r a t i o n s , r e l e a s e r a t e s up t o 0.2 % p e r d a y w e r e o b s e r v e d f o r m a t e r i a l s t o r e d a t - 2 0°C , T a b l e 3.6 . The m a j o r d e i o d i n a t i o n p r o d u c t h a s b e e n shown t o be I" a n d i t s r e l e a s e was f o u n d t o be m i n i m i z e d when t y r o s i n e was t h e p r e d o m i n a n t r e s i d u e i o d i n a t e d ( K r o h n e t a l . 1 9 7 7 ) . C h r o m a t o g r a p h i c s e p a r a t i o n o f p r o t e i n - b o u n d a n d f r e e r a d i o l a b e l was a t t i m e s o n l y p a r t i a l . F i g u r e s 3 . 9 a a n d b show a n a l y s e s 1 2 5 1 2 5 o f I - a l b u m i n o n S e p h a d e x G75 a n d I - f i b r i n o g e n o n S e p h a d e x G150 r e s p e c t i v e l y . I n b o t h c a s e s t h e p r o f i l e s c o m p a r e t h e p r o t e i n - b o u n d a n d ' f r e e ' r a d i o l a b e l p o o l s f r o m j u s t p r e v i o u s l y c h r o m a t o g r a p h e d p r e p a r a t i o n s . The e s t i m a t e d f r e e r a d i o l a b e l c o n t a m i n a n t s w e r e 0.2 a n d 0 . 4 % f o r t h e a l b u m i n a n d f i b r i n o g e n e x p e r i m e n t s r e s p e c t i v e l y , w h i l e t h e b o u n d - l a b e l c o n t a m i n a n t s w e r e 1.2 a n d 1.8 % . I n t h e c a s e o f t h e f i b r i n o g e n e x p e r i m e n t , 1.8 % e s t i m a t e s F i g u r e 3.9 R e c h r o m a t o g r a p h y o f l2 5I - p r o t e i n p o o l , f r e e r a d i o l a b e l a s s o c i a t e d p o o l — b y s h a d i n g , f r e e , p r o t e i n - a s s o c i a t e d p r e p a r a t i o n s . P r o t e i n - a s s o c i a t e d r a d i o l a b e l C r o s s c o n t a m i n a t i n g s p e c i e s a r e i n d i c a t e d . a ) 1 2 5I - a l b u m i n b ) 1 25 l - f i b r i n o g e n . - 1 0 0 -T A B L E 3 . 6 F R E E R A D I O L A B E L I N STORED 1 2 5I - A L B U M I N ASSAYED BY GEL F I L T R A T I O N ON SEPHADEX G 7 5 I O D I N A T I O N STORAGE FREE R A D I O L A B E L a d a y s t e m p e r a t u r e % # 2 1 , a l b u m i n ( i o d i n a t i o n 2 3 - 2 0 ° C 0 . 8 e f f i c i e n c y 7 5 %) 3 6 - 2 0 ° C 1 . 6 6 5 - 2 0 ° C A.6 # 2 2 , a l b u m i n ( i o d i n a t i o n 0 1 . 6 e f f i c i e n c y 7 6 %) 6 - 2 0 ° C 2 . 8 2 8 - 2 0 ° C A.8 6 5 - 2 0 ° C 6 . 1 a ) s e c o n d p e a k o f t h e c h r o m a t o g r a p h i c s e r p a r a t i o n s c o r r e c t e d f o r t a i l i n g e f f e c t s , A p p e n d i x A t h e c o n t a m i n a t i o n b y n a t i v e p r o t e i n . The c h r o m a t o g r a m s h o w e d t h a t c o n s i d e r a b l e d e g r a d a t i o n o f t h i s f i b r i n o g e n p r e p a r a t i o n h a d o c c u r r e d . 3 . 1 . 5 P r o t e i n c o n c e n t r a t i o n P r o t e i n c o n c e n t r a t i o n was d e t e r m i n e d b y m e a s u r e m e n t o f o p t i c a l d e n s i t y a t 2 8 0 nm ( O D ^ Q ) o r a t 7 0 0 nm when t h e L o w r y m e t h o d ( L o w r y e t a l . 1 9 5 1 ) was u s e d . T h e s e m e t h o d s w e r e c a l i b r a t e d a g a i n s t a r a d i a l i m m u n o d i f f u s i o n ( R I D ) m e t h o d ( M a n c i n i e t a l . 1 9 6 5 ) f o r b o t h human f i b r i n o g e n a n d a l b u m i n . The o p t i c a l d e n s i t i e s b y t h e s e t w o m e t h o d s a s f u n c t i o n s o f p r o t e i n c o n c e n t r a t i o n a r e shown i n F i g u r e 3 . 1 0 . The c a l i b r a t i o n r e s u l t s a r e s u m m a r i z e d i n T a b l e 3 . 7 . The N D 2 8 0 c a l i b r a t i o n c u r v e s a r e c h a r a c t e r i z e d b y i n s i g n i f i c a n t i n t e r c e p t s a n d F i g u r e 3 . 10 P r o t e i n c o n c e n t r a t i o n c a l i b r a t i o n c u r v e s . S t o c k c o n c e n t r a t i o n s d e t e r m i n e d b y RID o r a s q u o t e d by K A B I a n d d i l u t e d by w e i g h t . The l i n e a r r e g r e s s i o n a n a l y s i s i s s u m m a r i z e d i n T a b l e 3.7 . a ) 0 D28 0 F i b r i n o g e n A p r i l / 7 8 , F e b / 8 2 O O , A l b u m i n N o v / 8 1 , J a n / 8 2 D D , b ) L o w r y : F i b r i n o g e n F e b / 8 2 O O , A l b u m i n J a n / 8 2 n a . - 1 0 2 -c o n s t a n t s l o p e s . The s l o p e s a r e s i m i l a r t o r e p o r t e d a d s o r p t i o n c o e f f i c i e n t s . The a b s o r b a n c e c o e f f i c i e n t ( a b s o r b a n c e a t 0 . 1 % p r o t e i n c o n c e n t r a t i o n f o r a l i g h t p a t h o f 1 cm) a t 2 8 0 nm f o r human f i b r i n o g e n a t pH 7.1 i n 5 mM s o d i u m c i t r a t e h a s b e e n r e p o r t e d t o be 1.39 ( K a z a l e t a l . 1 9 6 3 ) a n d f o r human a l b u m i n t o b e 0 . 5 2 6 ( E d w a r d s e t a l . 1 9 6 9 ) . T h e s e a r e c l o s e t o t h e v a l u e s o f 1.29 a n d 0 . 5 7 f o u n d h e r e . T A B L E 3.7 SUMMARY OF 0 D28 0 AND LOWRY P R O T E I N C A L I B R A T I O N CURVES 0 D28 0 S T0 C K CONC. SLOPE a I N T E R C E P T a (mg/mL) (OD-mL/mg) (OD) F I B R I N O G E N A p r i l 7 8 b 2 0 . 3 1.300 + 0 . 0 0 9 - 0 . 0 3 7 + 0 . 0 1 2 F e b 8 2 c 2 2 . 1 + 0 . 6 1.287 0 . 0 0 3 - 0 . 0 1 8 0 . 0 0 2 mean 1.294 + 0 . 0 3 5 ALBUMIN Nov 81 c 4 . 6 6 + 0 . 0 7 0 . 5 3 8 + 0 . 0 0 1 0 . 0 0 6 + 0 . 0 0 5 J a n 8 2 c 1 0 . 2 6 0.21 0 . 6 3 4 0 . 0 0 2 0 . 0 0 6 0 . 0 0 3 mean d 0 . 5 6 5 + 0 . 0 0 7 LOWRY MAX SLOPE F I B R I N O G E N 2 2 . 1 + 0.6 2.68 + 0 . 0 2 ALBUMIN 1 0 . 3 + 0.2 2 . 6 4 + 0 . 0 2 a ) mean +_ o n e s t a n d a r d d e v i a t i o n ( L R A ) b ) K a b i l y o p h i l i z e d human f i b r i n o g e n c ) D e t e r m i n e d b y R I D d ) w e i g h t e d m e a n , +_ o n e s t a n d a r d d e v i a t i o n , S q u i r e s ( 1 9 6 8 ) - 1 0 3 -3.1.6 Summary A p p r o x i m a t e l y 8 5 % o f t h e p r o t e i n i n t h e f i b r i n o g e n p r e p a r a t i o n s was i d e n t i f i e d a s f i b r i n o g e n b y SDS-PAGE a n a l y s e s . T h i s e s t i m a t e c o r r e s p o n d s t o t h e f r a c t i o n o f d y e o r r a d i o l a b e l a s s o c i a t e d w i t h t h e r e d u c e d f i b r i n o g e n c h a i n s a n d was f e l t t o be a minimum e s t i m a t e . C l o t t a b i l i t y d e t e r m i n a t i o n s made o n p r e p a r a t i o n s u s i n g t h e same i s o l a t i o n p r o t o c o l c o n s i s t e n t l y i n d i c a t e d a p u r i t y i n t h e 9 5 % t o 9 8 % r a n g e a n d a p p e a r e d o p t i m i s t i c . P r e l i m i n a r y e x p e r i m e n t s h a d shown t h a t r a d i o l a b e l a s s o c i a t e d w i t h r e d c e l l s i n u l t r a c e n t r i f u g e d c e l l p e l l e t s n o r m a l l y a c c o u n t e d f o r l e s s t h a n t w o o r t h r e e p e r c e n t o f t h e t o t a l r a d i o l a b e l . T h u s t h e i d e n t i t y o f t h e a d s o r b e d p r o t e i n s p e c i e s c o u l d n o t b e i n f e r r e d f r o m t h e c o m p o s i t i o n o f t h e f i b r i n o g e n p r e p a r a t i o n s . I n s t e a d , t h e a d s o r b e d s p e c i e s w e r e i d e n t i f i e d by SDS-PAGE a n a l y s i s o f r e d c e l l m e m b r a n e s . The c o n t a m i n a n t s o f c o n c e r n w e r e p l a s m i n ( o g e n ) , f i b r o n e c t i n , a n d f i b r i n o g e n a g g r e g a t e s . P r o t e o l y t i c d e g r a d a t i o n o f f i b r i n o g e n p r e p a r a t i o n s i n d i c a t e d t h a t t h e l y s i n e - S e p h a r o s e a f f i n i t y c h r o m a t o g r a p h y t e c h n i q u e , a s u s e d h e r e , d i d n o t r e m o v e a l l o f t h e p l a s m i n ( o g e n ) p r e s e n t i n a l l i s o l a t i o n s . T h u s t h e i n h i b i t o r s b e n z a m i d i n e a n d T r a s y l o l w e r e a d d e d t o f i b r i n o g e n i s o l a t i o n s t o r e t a r d t h e p r o t e o l y t i c a c t i o n o f p l a s m i n . C o l l a g e n - S e p h a r o s e a f f i n i t y c h r o m a t o g r a p h y was u s e d t o r e m o v e f i b r o n e c t i n f r o m f i b r i n o g e n p r e p a r a t i o n s , h o w e v e r , SDS-PAGE a n a l y s i s o f i s o l a t i o n 3 0 s u g g e s t e d t h a t t h e b i n d i n g c a p a c i t y o f t h e a f f i n i t y l i g a n d may h a v e b e e n o v e r l o a d e d i n some c a s e s . A n a l y s i s o f r a d i o l a b e l r e t a i n e d b y r e d c e l l m e m b r a n e s , s e c t i o n 3 . 3 . 5 , i n d i c a t e d h o w e v e r , t h a t t h e m a j o r p a r t o f t h e b o u n d - 1 04 -r a d i o l a b e l was a s s o c i a t e d w i t h f i b r i n o g e n . The p o s s i b l i t y t h a t t h e b i n d i n g o f f i b r i n o g e n a g g r e g a t e s t o r e d c e l l s m i g h t be d i f f e r e n t f r o m t h a t o f monomer f i b r i n o g e n was i n v e s t i g a t e d by c o m p a r i s o n o f t h e b i n d i n g o f i s o l a t e d monomer a n d a g g r e g a t e d f i b r i n o g e n , s e c t i o n 3 . 3 . 5 . F i b r i n o g e n a n d a l b u m i n w e r e l a b e l l e d t o l e v e l s o f l e s s t h a n o n e m o l e i o d i n e p e r m o l e p r o t e i n . No a l t e r a t i o n o f t h e p r o t e i n s was d e t e c t e d b y SDS-PAGE o r g e l f i l t r a t i o n a n d t h e l e v e l o f l a b e l l i n g was b e l o w t h a t r e p o r t e d t o a l t e r f i b r i n o g e n ' s c l o t t i n g b e h a v i o u r . The i o d i n a t i o n c o n d i t i o n s f o r a l b u m i n w e r e s u c h a s t o a v o i d i o d i n a t i o n o f s u l p h y d r a l g r o u p s ( C r a n d a l l e t a l . 1 9 8 1 ) . 1 2 5 T h e amount o f r a d i o l a b e l a s i o d i d e , r e f e r r e d t o a s f r e e i o d i d e , was e s t i m a t e d b y g e l f i l t r a t i o n . H o w e v e r , p r o t e o l y s i s a n d t h e c o n t i n u e d r e l e a s e o f l a b e l f r o m r a d i o l a b e l e d p r o t e i n s made t h e e s t i m a t i o n o f t h e a p p r o p r i a t e a m o u n t s o f f r e e r a d i o l a b e l c o m p l e x . A l t e r n a t e m e t h o d s f o r e s t i m a t i n g t h e a m o u n t s o f f r e e r a d i o l a b e l a r e d e s c r i b e d i n s e c t i o n s 3 .2.4 a n d 3 . 3 . 4 . I n t h e f o l l o w i n g s e c t i o n , 1 2 5 i o d i d e was shown t o b e a f a c t o r c o n t r i b u t i n g t o t h e a p p a r e n t b i n d i n g t o r e d c e l l s e s t i m a t e d f o r r a d i o i o d i n a t e d p r o t e i n s . - 1 0 5 -3.2 APPARENT BINDING The b i n d i n g d e t e r m i n e d f r o m t h e a c t i v i t y p e r g r a m o f c e l l p e l l e t , Ap , was a n a p p a r e n t m e a s u r e o f b i n d i n g b e c a u s e t h e a c t i v i t y was c o m p o s e d o f i n t e r - a n d i n t r a c e l l u l a r a c t i v i t i e s , a s w e l l a s c e l l - b o u n d a c t i v i t y . M e t h o d s t o r e d u c e a n d / o r e s t i m a t e t h e s e c o m p o n e n t s o f a p p a r e n t b i n d i n g w e r e i n v e s t i g a t e d . The i n t e r c e l l u l a r a c t i v i t y o f c e l l p e l l e t s was r e d u c e d b y u l t r a c e n t r i f u g a t i o n . The e l u t i o n o f a p p a r e n t b i n d i n g i n w a s h a n a l y s e s , s e c t i o n 3 . 3 , was i n t e r p r e t e d t o e s t i m a t e i n t e r c e l l u l a r r a d i o l a b e l . I n t r a c e l l u l a r 1 2 5 a c t i v i t y was r e l a t e d t o i o d i d e i n p r o t e i n p r e p a r a t i o n s . I t was s h o w n t h a t t h e i n t r a c e l l u l a r a c t i v i t y c o u l d b e e s t i m a t e d f r o m c h a n g e s i n a p p a r e n t b i n d i n g w i t h c e l l c o n c e n t r a t i o n , s e c t i o n 3 . 2 , a n d f r o m wash a n a l y s e s , s e c t i o n 3.3 . 3.2.1 C e n t r i f u g a t i o n r a t e a n d t i m e E x p e r i m e n t s t o c o m p a r e t h e a p p a r e n t b i n d i n g o f r a d i o l a b e l e d p r o t e i n s t o r e d c e l l s i n c e n t r i f u g e d c e l l p e l l e t s w e r e c a r r i e d o u t w i t h " I O C 1 Q £ I - a l b u m i n a n d I - f i b r i n o g e n . The d e p e n d e n c e o f t h e a p p a r e n t 1 2 5 b i n d i n g o f I - a l b u m i n , e x p r e s s e d a s t h e p e l l e t d i s t r i b u t i o n r a t i o ( Q ) , u p o n t h e r a t e a n d d u r a t i o n o f c e n t r i f u g a t i o n a n d t h e s a m p l e l o c a t i o n i n t h e c e l l p e l l e t i s s u m m a r i z e d i n F i g u r e 3.11 a n d T a b l e 3.8 . The c e l l p e l l e t s w e r e p r o d u c e d b y c e n t r i f u g a t i o n a t 1 8 7 0 x g , 1 5 , 2 0 0 x g , a n d 2 0 2 , 0 0 0 x g f o r 3 0 0 a n d / o r 3 0 m i n u t e s . A s e r i e s o f s a m p l e s w e r e o b t a i n e d f r o m e a c h p e l l e t by f r e e z i n g t h e p e l l e t t o -70°C a n d f r a c t u r i n g t h e t u b e a n d c o n t e n t s a t m e a s u r e d p o s i t i o n s a l o n g t h e t u b e . The c u t s w e r e made i n o r d e r f r o m t h e b o t t o m o f t h e t u b e t o w a r d s t h e c e l l / s u p e r n a t a n t i n t e r f a c e a s d e s c r i b e d i n - 1 0 6 -F i g u r e 3.11 P e l l e t d i s t r i b u t i o n r a t i o f o r 1 2 ^ 1 - a l b u m i n i n c e n t r i f u g e d r e d c e l l p e l l e t s a s a f u n c t i o n o f t h e a v e r a g e d i s t a n c e o f t h e s a m p l e f r o m t h e b o t t o m o f t h e c e n t r i f u g e t u b e . C e n t r i f u g a t i o n c o n d i t i o n s : 1 8 7 0 x g f o r 3 0 m i n ++ ; I 5 , 2 0 0 x g f o r 3 0 m i n o o , f o r 3 0 0 m i n • • ; 2 0 2 , 0 0 0 x g f o r 3 0 m i n OO, f o r 3 0 0 m i n . The e x p e r i m e n t s a r e r e f e r r e d t o i n T a b l e 3.8 b y t h e n u m b e r s g i v e n i n t h i s F i g u r e . TABLE 3 . 8 P E L L E T D I S T R I B U T I O N RATIO (Q) AS A FUNCTION OF CENTRIFUGATION RATE AND TIME AND THE SAMPLE DISTANCE ( L ) FROM THE BOTTOM OF THE CENTRI F U G E TUBE EXPERIMENT CENTRIFUGATION L I N E A R REGRESSION : Q a s a f u n c t i o n o f L s l o p e a i n t e r c e p t a r D n x g m i n ( Q / c m ) x l 02 Q x l O2 27 1 8 7 0 3 0 0 . 9 4 + 0 . 0 5 3 . 0 8 + 0 . 0 9 0 . 9 9 6 5 2 8 1 5 , 2 0 0 3 0 . 0 . 1 2 5 + 0 . 0 0 9 2 . 4 2 + 0 . 0 2 0 . 9 8 3 9 1 5 , 2 0 0 3 0 0 0 . 1 1 0 + 0 . 0 1 8 2.36 + 0 . 0 3 0 . 9 1 0 1 0 2 8 2 0 2 , 0 0 0 3 0 0 . 0 2 9 + 0 . 0 0 9 2.19 + 0 . 0 2 0 . 7 5 3 1 0 2 0 2 , 0 0 0 3 0 0 0 . 0 1 2 + 0 . 0 0 4 2 . 2 5 + 0.01 0 . 7 2 3 1 2 3 0 2 0 2 , 0 0 0 3 0 0 . 0 1 8 + 0 . 0 0 2 0 . 8 0 5 + 0 . 0 0 3 0 . 9 8 8 5 0 . 0 2 4 + 0 . 0 0 5 0 . 9 8 6 + 0 . 0 0 8 0 . 9 5 5 4 3 5 2 0 2 , 0 0 0 3 0 0.011 + 0 . 0 0 2 0 . 2 4 7 + 0 . 0 0 3 0 . 9 6 8 5 0 . 0 1 4 + 0 . 0 0 3 0 . 2 6 2 + 0 . 0 0 5 0 . 9 4 5 5 5 1 2 0 2 , 0 0 0 3 0 0 . 0 1 9 + 0.001 0 . 3 6 5 + 0 . 0 0 1 0 . 9 9 9 4 a ) mean + o n e s t a n d a r d d e v i a t i o n ( L R A ) b) s a m p l e c o r r e l a t i o n c o e f f i c i e n t - 1 0 8 -s e c t i o n 2 . 5 . 4 . The l e n g t h ( L ) was m e a s u r e d f r o m t h e b o t t o m o f t h e t u b e t o t h e m i d d l e o f t h e s a m p l e . E x p e r i m e n t s 2 7 a n d 28 s h o w e d t h a t t h e p e l l e t d i s t r i b u t i o n r a t i o i n c r e a s e d l i n e a r i l y w i t h t h e d i s t a n c e f r o m t h e b o t t o m o f t h e p e l l e t . The s l o p e o f Q v e r s u s L d e c r e a s e d w i t h i n c r e a s i n g c e n t r i f u g a t i o n r a t e . I n c r e a s i n g t h e c e n t r i f u g a t i o n t i m e by a f a c t o r o f t e n d i d n o t r e d u c e t h e s l o p e s i g n i f i c a n t l y a t r a t e s b e l o w 1 5 , 2 0 0 x g . A t 2 0 2 , 0 0 0 x g t h e s l o p e d e c r e a s e d o n l y s l i g h t y w i t h i n c r e a s e d c e n t r i f u g a t i o n t i m e , t h u s a s t a n d a r d p e l l e t i n g c o n d i t i o n o f 2 0 2 , 0 0 0 x g f o r 3 0 m i n u t e s was a d o p t e d . The v a r i a t i o n s s e e n i n t h e s l o p e o f Q v e r s u s L i n s u b s e q u e n t e x p e r i m e n t s i n d i c a t e d t h a t l o n g e r c e n t r i f u g a t i o n t i m e s d i d n o t r e s u l t i n m o r e r e l i a b l e d a t a . Th e i n c r e a s e i n a p p a r e n t b i n d i n g w i t h s a m p l e p o s i t i o n was i n t e r p r e t e d a s d u e t o i n t e r c e l l u l a r t r a p p i n g . I n t e r c e l l u l a r t r a p p i n g w o u l d be e x p e c t e d t o i n c r e a s e w i t h t h e d e c r e a s e d c e n t r i p e t a l a c c e l e r a t i o n a t p o s i t i o n s n e a r e r t h e a x i s o f r o t a t i o n . The s l o p e o f Q v e r s u s L i s n o t d u e t o s e p a r a t i o n o f y o u n g e r a n d o l d e r r e d c e l l s ( R i g a s a n d K o l e r 1 9 6 1 , P i o m e l l i e t a l . 1 9 6 7 ) . I f t h i s w e r e t h e c a s e , a l o n g e r c e n t r i f u g a t i o n i n t e r v a l w o u l d h a v e i n c r e a s e d t h e s l o p e o f Q v e r s u s L . The d i f f e r e n c e s i n t h e i n t e r c e p t v a l u e s o f Q v e r s u s L , u n d e r c o n s t a n t c e n t r i f u g a t i o n c o n d i t i o n s , w e r e i n t e r p r e t e d a s d u e t o d i f f e r e n c e s i n t h e a m o u n t o f f r e e r a d i o l a b e l p r e s e n t i n t h e c e l l s . T h i s i n t e r p r e t a t i o n was s u p p o r t e d by e s t i m a t e s o f t h e a m o u n t s o f f r e e r a d i o l a b e l p r e s e n t i n p r o t e i n p r e p a r a t i o n s . The a m o unt o f r a d i o l a b e l p r e s e n t i n r e d c e l l s was s h o w n t o be p r o p o r t i o n a l t o t h e c o n c e n t r a t i o n - 109 -o f f r e e r a d i o l a b e l i n c e l l s u s p e n s i o n s u p e r n a t a n t s , s e c t i o n s 3 . 2 . 4 a n d 3.3.4 . I t f o l l o w e d t h a t t h e b i n d i n g p e r g r a m o f c e l l s was r e p r e s e n t e d b y d i s t r i b u t i o n r a t i o s o n t h e o r d e r o f t h o s e o f e x p e r i m e n t 3 5 r a t h e r t h a n t h o s e o f e x p e r i m e n t 2 8 . The u l t r a c e n t r i f u g a t i o n c o n d i t i o n s u s e d h e r e s u b j e c t e d r e d c e l l s t o l a r g e h y d r o s t a t i c p r e s s u r e s . F o r a c e l l p e l l e t f o u r c e n t i m e t e r s d e e p , p r e s s u r e s o f a b o u t 5 5 0 a n d 1 3 0 0 a t m o s p h e r e s w e r e c a l c u l a t e d f o r r e s p e c t i v e l y t h e b u f f e r - c e l l i n t e r f a c e a n d t h e b o t t o m o f t h e p e l l e t . H o w e v e r , a f t e r u l t r a c e n t r i f u g a t i o n r e d c e l l s a p p e a r n o r m a l u p o n m i c r o s c o p i c e x a m i n a t i o n ( B r o o k s 1 9 7 3 b ) . I n a d d i t i o n , r e d c e l l m e m b r anes a p p e a r e d n o r m a l when a n a l y s e d b y SDS-PAGE, s e c t i o n 3 . 3 . 5 . I t h a s b e e n s u g g e s t e d t h a t t h e v o l u m e o f r e d c e l l s may be r e d u c e d b y t h e p r e s s u r e s p r o d u c e d b y c e n t r i f u g a t i o n ( P o n d e r 1 9 4 8 , p . 5 7 ) . E v i d e n c e f o r c o m p r e s s i o n o f s w o l l e n r e d c e l l s b y c e n t r i f u g a t i o n f o r 3 0 m i n a t 2 0 0 0 x g ( a p p r o x i m a t e maximum p r e s s u r e o f 14 a t m o s p h e r e s ) h a s b e e n p r e s e n t e d b y H e n d r y ( 1 9 5 4 ) . The v o l u m e o f s w o l l e n r e d c e l l s c a l c u l a t e d f r o m p r o t e i n d i l u t i o n m e a s u r e m e n t s was i n a g r e e m e n t w i t h t h e o s m o t i c t h e o r y o f r e d c e l l v o l u m e c h a n g e s ( P o n d e r 1 9 4 8 , p . 8 3 ) , w h e r e a s v o l u m e s d e t e r m i n e d b y c e n t r i f u g a t i o n w e r e l o w e r . I n c o n t r a s t , s i g n i f i c a n t c o m p r e s s i o n o f n o r m a l c e l l s u n d e r t h e s e c e n t r i f u g a t i o n c o n d i t i o n s was r u l e d o u t ( H e n d r y 1 9 5 4 ) . I t h a s b e e n r e p o r t e d t h a t c e n t r i f u g a t i o n o f n o r m a l r e d c e l l s f o r 5 t o 1 0 m i n u t e s a t 1 2 , 0 0 0 x g ( a p p r o x i m a t e maximum p r e s s u r e o f 9 0 a t m o s p h e r e s ) r e s u l t e d i n c e l l v o l u m e e s t i m a t e s w h i c h w e r e s i g n i f i c a n t l y 1 2 5 l o w e r t h a n t h o s e d e t e r m i n e d f r o m I - a l b u m i n d i l u t i o n ( S i r s 1 9 6 8 ) . T h e s e r e s u l t s w e r e i n t e r p r e t e d i n t e r m s o f d i s p l a c e m e n t o f w a t e r f r o m - 1 1 0 -t h e i n t r a c e l l u l a r v o l u m e . I n t h i s i n t e r p r e t a t i o n , t h e c h a n g e i n o s m o t i c p r e s s u r e was s e t e q u a l t o a n e f f e c t i v e h y d r o s t a t i c p r e s s u r e c a l c u l a t e d f r o m t h e d e n s i t y d i f f e r e n c e b e t w e e n t h e c e l l s a n d s u s p e n d i n g m e d i u m . H o w e v e r , D e l l i m o r e a n d G o s l i n g ( 1 9 7 3 ) r e p o r t e d t h a t no d e t e c t a b l e d i f f e r e n c e s w e r e o b s e r v e d i n t h e i o n i c c o n c e n t r a t i o n s o f s u p e r n a t a n t s c o l l e c t e d e i t h e r w i t h o u t c e n t r i f u g a t i o n o r b y c e n t r i f u g a t i o n a t r a t e s r a n g i n g f r o m AO t o 6 0 0 0 x g . T h o u g h t h e c e n t r i f u g a t i o n r a t e was o n l y h a l f t h a t u s e d b y S i r s ( 1 9 6 8 ) , t h e d i l u t i o n e f f e c t s p r e d i c t e d f r o m c o n s i d e r a t i o n s a n a l o g o u s t o t h o s e e m p l o y e d b y S i r s s h o u l d h a v e b e e n r e a d i l y d e t e c t e d . I n t h e p r e s e n t w o r k , t h e o s m o l a l i t i e s o f s u p e r n a t a n t s f r o m c e l l s u s p e n s i o n s c e n t r i f u g e d a t e i t h e r A , 1 0 0 o r 2 0 2 , 0 0 0 x g f o r 3 0 m i n u t e s w e r e m e a s u r e d . I r r e v e r s i b l e d i l u t i o n d u e t o a s h i f t o f w a t e r o u t o f t h e c e l l s d i d n o t o c c u r . F o r t h e s e e x p e r i m e n t s , f r e s h r e d c e l l s w e r e f i r s t w a s h e d i n i s o t o n i c P B S / a z i d e b u f f e r w i t h 2 mg/ml a l b u m i n a n d w e r e s u s p e n d e d i n t h e same b u f f e r a t c e l l c o n c e n t r a t i o n s o f a p p r o x i m a t e l y AO o r 8 0 % w/w. C e n t r i f u g a t i o n was c a r r i e d o u t i n c a p p e d p o l y a l l o m e r t u b e s a n d t h e o s m o l a l i t i e s o f t h e s u p e r n a t a n t s w e r e d e t e r m i n e d u s i n g a W e s c o r M o d e l 5 1 0 0 c v a p o u r p r e s s u r e o s m o m e t e r . Red c e l l h e m o l y s i s was d e t e r m i n e d f r o m h e m o g l o b i n a b s o r b a n c e a t A 1 5 nm A / N - l u s i n g a n e x t i n c t i o n c o e f f i c i e n t o f 1 2 . 9 x 1 0 L ' ( m o l e s - c m ) ( K i r s c h e n b a u m 1 9 7 3 ) . The r e s u l t s a r e s u m m a r i z e d i n T a b l e 3.9 . The o s m o l a l i t i e s w e r e d e t e r m i n e d t o a p r e c i s i o n o f a p p r o x i m a t e l y o n e p e r c e n t . The s u p e r n a t a n t o s m o l a l i t i e s f o l l o w i n g c e n t r i f u g a t i o n w e r e b e t w e e n 2 a n d A % g r e a t e r t h a n t h e w a s h b u f f e r o s m o l a l i t y . The c o n c e n t r a t i o n o f h e m o g l o b i n d u e t o h e m o l y s i s was l e s s TABLE 3.9 THE OSMOLALITY OF WASH BUFFERS AND C E L L S U S PENSION SUPERNATANTS AFTER CENTRIFUGATION CENTRIFUGATION CONDITIONS SUPERNATANT r e c o v e r y o s m o l a l i t y3 (SB) (mosm) h e m o g l o b i n (mM) C E L L fbe f r a c t i o n a l v o l u m e PRESSURE o s m o t i c i n t e r f a c e b o t t o m ( a t m . ) ( a t m . ) ( a t m . ) PBS/ALBUMIN 28 3 + 3 SUPERNATANT 2 0 2 , 0 0 0 x g SUPERNATANT 2 0 2 , 0 0 0 x g SUPERNATANT 4 , 1 0 0 x g SUPERNATANT 4 , 1 0 0 x g 29 7 + 1 0 . 0 6 4 9 8 . 0 2 9 4 . 5 + 1.5 0 . 0 0 9 9 0 . 0 2 9 3 . 5 + 0.5 0 . 0 6 8 97. 4 2 9 1 . 5 + 0.5 0 . 0 1 3 0.779 0 . 4 0 7 0 . 7 7 9 0 . 4 0 7 - 0 . 0 1 1 - 0 . 0 4 6 - 0 . 0 0 8 - 0 . 0 3 0 - 0 . 3 7 - 0 . 6 5 - 0 . 2 7 - 0 . 4 3 1 7 5 1 3 1 0 5 9 0 1 2 7 0 11 24 2 3 a ) mean + maximum d e v i a t i o n - 1 1 2 -t h a n 0 . 0 7 mM . The h e m o l y s i s was e s t i m a t e d t o r e p r e s e n t l y s i s o f 0.4 % o f t h e c e l l s a n d was i n d e p e n d e n t o f t h e c e l l c o n c e n t r a t i o n a n d c e n t r i f u g a t i o n c o n d i t i o n s i n t h e v a r i o u s e x p e r i m e n t s . The v o l u m e c h a n g e s a s s o c i a t e d w i t h w a t e r s h i f t s i m p l i e d b y o s m o l a l i t y c h a n g e s r a n g e d f r o m - 0 . 8 t o - 4 . 6 % , i . e . w a t e r a p p a r e n t l y moved i n t o t h e c e l l s . The o s m o t i c p r e s s u r e c h a n g e s a s s o c i a t e d w i t h t h e s e v o l u m e c h a n g e s w e r e c a l c u l a t e d u s i n g t h e v a n ' t H o f f - B o y l e r e l a t i o n ( D e l l i m o r e a n d G o s l i n g 1 9 7 3 ) a n d r a n g e d f r o m - 0 . 3 t o -0.7 a t m o s p h e r e s . H y d r o s t a t i c p r e s s u r e s a t t h e b u f f e r - c e l l i n t e r f a c e s a n d t h e b o t t o m s o f 3 t h e p e l l e t s w e r e c a l c u l a t e d u s i n g d e n s i t i e s o f 1.0 a n d 1.09 g/cm f o r t h e s u p e r n a t a n t a n d c e l l s r e s p e c t i v e l y a n d c e n t r a l a c c e l e r a t i o n p r o p o r t i o n a l t o t h e r a d i a l d i s t a n c e f r o m t h e a x i s o f r o t a t i o n . C o m p a r i s o n o f t h e p r e s s u r e a n d v o l u m e c h a n g e s i n d i c a t e d t h a t t h e a b s o l u t e h y d r o s t a t i c p r e s s u r e c o u l d n o t be r e l a t e d t o t h e o b s e r v e d v o l u m e c h a n g e s u s i n g a m o d e l w h i c h summed t h e o s m o t i c a n d h y d r o s t a t i c p r e s s u r e s . I f t h e a b s o l u t e h y d r o s t a t i c p r e s s u r e s w e r e p e r t i n e n t t o t h e o s m o t i c b a l a n c e o f w a t e r a c r o s s t h e r e d c e l l m e m b r a n e , c h a n g e s i n t h e c e l l w a t e r v o l u m e , a n d t h u s t h e s u p e r n a t a n t o s m o l a l i t y , v e r y much l a r g e r t h a n o b s e r v e d h e r e w o u l d h a v e o c c u r r e d . I f i t i s a s s u m e d t h a t t h e e f f e c t i v e p r e s s u r e i s d u e t o t h e d i f f e r e n t i a l d e n s i t y o f t h e c e l l s a n d s u s p e n d i n g m e d i u m , p r e s s u r e s i n e x c e s s o f 5 5 a t m o s p h e r e s a r e c a l c u l a t e d f o r t h e u l t r a c e n t r i f u g e d p e l l e t s w h i l e p r e s s u r e s o f 1 t o 2 a t m o s p h e r e s a r e e s t i m a t e d f o r t h e l o w s p e e d c e l l p e l l e t s . A l t h o u g h t h e p r e s s u r e s i n t h e l a t t e r c a s e s w e r e o f t h e c o r r e c t m a g n i t u d e t o e x p l a i n v o l u m e c h a n g e s o n t h e o r d e r o f 1 0 % , t h e p r e s s u r e s i n t h e f o r m e r c a s e s w o u l d h a v e p r o d u c e d s u b t a n t i a l l y l a r g e r v o l u m e c h a n g e s . H o w e v e r , l a r g e - 1 1 3 -d i f f e r e n c e s i n c e l l v o l u m e s w e r e n o t o b s e r v e d a t t h e d i f f e r e n t c e n t r i f u g a t i o n r a t e s . T h u s i t i s c l e a r t h a t h y d r o s t a t i c p r e s s u r e s do n o t i r r e v e r s i b l y d e c r e a s e r e d c e l l v o l u m e s d u r i n g c e n t r i f u g a t i o n . T h e o s m o l a l i t y m e a s u r e m e n t s i m p l i e d t h a t e i t h e r w a t e r was r e m o v e d f r o m , o r s o l u t e s a d d e d t o , t h e s u p e r n a t a n t s . L o s s o f w a t e r t h r o u g h e v a p o r a t i o n was a v o i d e d b y u s i n g c a p p e d c e n t r i f u g e t u b e s . I n t h e c a s e s o f t h e e x p e r i m e n t s a t l o w e r c e l l c o n c e n t r a t i o n s , t h e s u p e r n a t a n t r e c o v e r i e s s u p p o r t e d t h i s v i e w . L i k e w i s e , h e m o l y s i s o f c e l l s c o u l d n o t e x p l a i n t h e o b s e r v e d o s m o l a l i t y c h a n g e s . The s u p e r n a t a n t h e m o g l o b i n c o n c e n t r a t i o n s c o u l d a c c o u n t f o r no more t h a n 1 % o f t h e c h a n g e s i n o s m o l a l i t y , t h u s r u l i n g o u t t h e r e l e v a n c e o f h e m o l y s i s i n t h i s e f f e c t . T h e r e r e m a i n s t h e p o s s i b i l i t y t h a t w a t e r s h i f t e d i n t o t h e c e l l s . A l t h o u g h s p h e r i n g o f r e d c e l l s b y p r e s s u r e s i n e x c e s s o f 1 0 0 0 a t m o s p h e r e s h a s b e e n r e p o r t e d ( H a u b r i c h 1 9 3 8 ) , t h e same m e c h a n i s m p r o b a b l y d o e s n o t a p p l y h e r e s i n c e t h e r e was no c o n s i s t e n t d i f f e r e n c e b e t w e e n t h e a p p a r e n t v o l u m e s h i f t s u n d e r t h e d i f f e r e n t c e n t r i f u g a t i o n c o n d i t i o n s . N o n e t h e l e s s , a w a t e r s h i f t i n t o t h e c e l l s d u e t o some a s p e c t o f t h e i r h a n d l i n g , o t h e r t h a n c e n t r i f u g a t i o n , a p p e a r s t o be t h e m o s t r e a s o n a b l e e x p l a n a t i o n f o r t h e s m a l l i n c r e a s e s i n s u p e r n a t a n t o s m o l a l i t y o b s e r v e d . The b i n d i n g e x p r e s s e d a s m o l e c u l e s p e r c e l l a s s u m e d v a l u e s f o r t h e v o l u m e ( E v a n s a n d F u n g 1 9 7 2 ) a n d d e n s i t y ( L i e f a n d V i n o g r a d 1 9 6 4 ) o f t h e c e l l s . The v a l u e s w e r e r e p r e s e n t a t i v e o f c e l l s i n n o r m a l i s o t o n i c m e d i a . T h u s , i f t h e s e c e l l c h a r a c t e r i s t i c s w e r e c h a n g e d by u l t r a c e n t r i f u g a t i o n , t h e r e p o r t e d b i n d i n g w o u l d n e e d t o b e c o r r e c t e d f o r t h e s e c h a n g e s . I f w a t e r w e r e d r i v e n o u t o f t h e c e l l s by - 114 -u l t r a c e n t r i f u g a t i o n , t h e mass p e r c e l l w o u l d be r e d u c e d a n d t h e s u p e r n a t a n t a c t i v i t y w o u l d d e c r e a s e d u e t o d i l u t i o n . H o w e v e r , s i n c e t h e s u p e r n a t a n t s a m p l e s w e r e t a k e n p r i o r t o u l t r a c e n t r i f u g a t i o n , o n l y t h e c h a n g e i n t h e c e l l m a s s n e e d be c o n s i d e r e d . A f r a c t i o n a l d e c r e a s e i n c e l l m ass o f o w o u l d r e s u l t i n more c e l l s p e r g r a m o f p e l l e t a n d l o w e r b i n d i n g p e r c e l l . The a p p a r e n t s u r f a c e c o n c e n t r a t i o n , T , e q u a t i o n 1 3 . 7 , w o u l d t h e r e f o r e be r e l a t e d t o t h e c o r r e c t e d s u r f a c e c o n c e n t r a t i o n P ' b y r ' = ra - &) The e x p e r i m e n t s h e r e s h o w e d t h a t c h a n g e s i n c e l l v o l u m e w e r e l e s s t h a n 5 % . S i n c e t h e v o l u m e o f t h e c e l l s a p p e a r e d t o i n c r e a s e r a t h e r t h a n d e c r e a s e w i t h c e n t r i f u g a t i o n , t h e c o r r e c t e d s u r f a c e c o n c e n t r a t i o n w o u l d b e g r e a t e r t h a n t h e a p p a r e n t s u r f a c e c o n c e n t r a t i o n s b y no more t h a n 5 % . The a p p a r e n t s u r f a c e c o n c e n t r a t i o n s w i t h o u t c o r r e c t i o n s a r e r e p o r t e d i n s e c t i o n 3.5 . 3 . 2 . 2 C e l l a n d f r e e l a b e l c o n c e n t r a t i o n 1 2 5 E x p e r i m e n t s w i t h I - a l b u m i n s h o w e d t h a t a p p a r e n t b i n d i n g d e p e n d e d u p o n c e l l a n d f r e e r a d i o l a b e l c o n c e n t r a t i o n s , F i g u r e 3 . 1 2 a a n d T a b l e 3.10 . A p p a r e n t b i n d i n g was r e l a t i v e l y i n d e p e n d e n t o f i n c u b a t i o n t i m e a n d n o t a f f e c t e d b y s t o r a g e o f t h e r e d c e l l s t o 14 d a y s o r b y c h a n g e s i n a l b u m i n p r o t e i n c o n c e n t r a t i o n b e t w e e n 2 a n d 1 5 mg/mL . C e l l c o n c e n t r a t i o n s w e r e d e t e r m i n e d a s h e m a t o c r i t a n d w e r e c o n v e r t e d t o w e i g h t f r a c t i o n s a s d e s c r i b e d i n A p p e n d i x 3 . S a m p l e s o f t h e 1 2 5 I - a l b u m i n p r e p a r a t i o n s w e r e a s s a y e d f o r f r e e r a d i o l a b e l b y c h r o m a t o g r a p h y o n S e p h a d e x G75 . The i n c u b a t i o n t i m e s w e r e t h e C O N C E N T R A T I O N if>e) C O N C E N T R A T I O N (y&e) F i g u r e 3 .12 D i s t r i b u t i o n r a t i o (Q) a s a f u n c t i o n o f r e d c e l l c o n c e n t r a t i o n (/3 e) d u r i n g t h e a d s o r p t i o n i n t e r v a l . a ) 1 2 5I - a l b u m i n p r e p a r a t i o n #20, i n c u b a t i o n t i m e : 4 5 m i n O O , 1 4 5 m i n • • ; p r e p a r a t i o n # 2 1 , i n c u b a t i o n t i m e : 4 5 m i n a a , 1 1 0 m i n • • , 3 0 m i n a t 2 , 7 . 7 , a n d 1 5 . 4 mg/mL © . b ) l2 5I - f i b r i n o g e n e x p e r i m e n t 53 ++ a n d 5 4 OO a t t h e i n d i c a t e d m i l l i g r a m p e r m i l l i l i t e r p r o t e i n c o n c e n t r a t i o n s . TABLE 3.10 ALBUMIN D I S T R I B U T I O N RATIO (Q) A S A FUNCTION C E L L AND FREE RADIOLABEL CONCENTRATIONS OF ALBUMIN PREPARATION #20 a #21 b #21 b RED C E L L S R e d C r o s s 0 Rh+ f r e s h P B S w a s h e d R e d C r o s s 0 Rh+, 1 4 d a y s (4°C) PBS w a s h e d ( J J ) A Rh+ f r e s h P B S w a s h e d INCUBATION TIME ( m i n ) 4 5 1 4 5 4 5 1 1 0 30 •fle Q x l O2 fie Q x l O2 fie Q x l O2 [ p r o t e i n ] 0 . 2 1 1 0 . 4 1 4 3.02 3.15 2.34 2.53 0 . 2 1 2 0 . 3 6 0 1.01 1.22 0 . 8 3 0.97 0 . 4 5 0 0 . 9 7 0.96 1 5 . 4 0 . 6 1 5 0 . 8 0 5 2.00 2.02 1.65 1.55 0 . 4 1 0 0 . 5 6 4 0 . 7 5 0 . 9 4 0 . 6 3 0 . 7 6 0 . 4 5 0 0 . 9 6 0 . 9 5 7.7 0 . 8 0 4 0 . 3 6 0 0 . 4 0 0 . 4 8 0.81 1.00 c 0 . 4 5 0 0.98 0 . 9 8 2.1 A l b u m i n (mg/ml) 1.7 1.7 v a r i e d a ) G75 e s t i m a t e b ) G75 e s t i m a t e o f f r e e o f f r e e r a d i o l a b e l : 4 - 5 % r a d i o l a b e l : 1 % c ) No m i x i n g a f t e r i n i t i a l r e s u s p e n s i o n o f c e l l s i n a d s o r b a t e . - 117 -i n t e r v a l s d u r i n g w h i c h t h e c e l l s u s p e n s i o n s w e r e u n i f o r m . C e l l s u s p e n s i o n s w e r e p e l l e t e d b y s t a n d a r d u l t r a c e n t r i f u g a t i o n . P r o t e i n c o n c e n t r a t i o n s w e r e d e t e r m i n e d by 0 D2 8 Q o r R I D a n d d i l u t i o n d a t a . A t e q u a l c e l l c o n c e n t r a t i o n s , a n i n c r e a s e i n f r e e r a d i o l a b e l i n t h e a d s o r b a t e f r o m 1.0 % f o r p r e p a r a t i o n #21 t o 4.2 % f o r p r e p a r a t i o n #20 was a s s o c i a t e d w i t h a t h r e e f o l d i n c r e a s e i n a p p a r e n t b i n d i n g . I n v e s t i g a t i o n o f i o d i d e d i s t r i b u t i o n b e t w e e n t h e c e l l u l a r a n d e x t r a c e l l u l a r c o m p a r t m e n t s o f r e d c e l l s u s p e n s i o n s , s e c t i o n 3 . 2 . 3 , s h o w e d t h a t a t e q u i l i b r i u m t h e i n t r a c e l l u l a r r a d i o i o d i d e p e r g ram o f c e l l s was a b o u t 8 0 % o f t h a t f o u n d p e r g r a m o f s u p e r n a t a n t . The t h r e e -f o l d c h a n g e i n a p p a r e n t b i n d i n g w i t h t h e a p p r o x i m a t e l y f o u r - f o l d c h a n g e i n f r e e i o d i d e c o n c e n t r a t i o n r e f l e c t e d t h e o b s e r v e d i o d i d e d i s t r i b u t i o n r a t i o . The a p p a r e n t b i n d i n g d e c r e a s e d u n i f o r m l y w i t h i n c r e a s i n g c e l l c o n c e n t r a t i o n . The d e p e n d e n c e o f t h e a p p a r e n t b i n d i n g u p o n f r e e i o d i d e a n d t h u s t h e c e l l u l a r c o m p a r t m e n t o f t h e c e l l s u s p e n s i o n s , r e p r e s e n t e d b y /^>e , o f f e r e d a n e x p l a n a t i o n f o r t h i s d e p e n d e n c e . A m o d e l i n t e r p r e t i n g t h e a p p a r e n t b i n d i n g a s a f u n c t i o n o f c e l l c o n c e n t r a t i o n was d e v e l o p e d , s e c t i o n 3 . 2 . 4 , w h i c h p e r m i t t e d e s t i m a t i o n o f t h e amount o f f r e e i o d i d e p r e s e n t i n t h e c e l l s . L o n g e r i n c u b a t i o n i n t e r v a l s r e s u l t e d i n s l i g h t i n c r e a s e s i n a p p a r e n t b i n d i n g . T h e s e i n c r e a s e s c a n be e x p l a i n e d b y i n c r e a s e s i n t h e a m o u n t o f f r e e i o d i d e w i t h t i m e . The f r e e i o d i d e i n r a d i o i o d i n a t e d p r o t e i n p r e p a r a t i o n s was r e d u c e d by c h r o m a t o g r a p h y a n d / o r d i a l y s i s , h o w e v e r s u b s e q u e n t i n c r e a s e s w e r e u s u a l l y d e t e c t e d . F o r e x a m p l e , t h e a l b u m i n p r e p a r a t i o n s u s e d w e r e s t o r e d a t - 2 0 0 Q a n cj s no w e d i n c r e a s e s - 1 1 8 -i n c h r o m a t o g r a p h i c a l l y d e t e c t a b l e f r e e i o d i d e o f a b o u t 0.1 t o 0.2 % p e r d a y . C r a n d a l l e t a l . ( 1 9 8 1 ) r e p o r t e d r e l e a s e f r o m b o v i n e s e r u m a l b u m i n o f i o d i d e a t r a t e s o f a b o u t 9 % p e r d a y a t 3 7°C . The i n c r e a s e i n a p p a r e n t b i n d i n g w i t h i n c u b a t i o n t i m e i n e x p e r i m e n t 3 0 i m p l i e d t h a t t h e f r e e l a b e l f r a c t i o n i n c r e a s e d a t a r a t e o f 8 % p e r d a y a t r o o m t e m p e r a t u r e i n t h e p r e s e n c e o f r e d c e l l s . A s i m i l a r t h o u g h s m a l l e r i n c r e a s e a p p e a r s t o h a v e o c c u r r e d i n e x p e r i m e n t 2 8 . The m e c h a n i s m s r e s p o n s i b l e f o r p r o t e i n d e i o d i n a t i o n a r e s e v e r a l a n d may be c o m p l e x . C r a n d a l l e t a l . ( 1 9 8 1 ) n o t e d t h a t t h e s u l f h y d r y l c o n t e n t o f m e r c a p t a l b u m i n was r e d u c e d w i t h i n c r e a s i n g i o d i n a t i o n . A t l o w I~ c o n c e n t r a t i o n s s u l f e n y l i o d i d e d e r i v a t i v e s may d e c o m p o s e t o y i e l d I- a n d a n o x i d i z e d d e r i v a t i v e (Thomas a n d A u n e 1 9 7 7 ) . D e i o d i n a t i o n o f f i b r i n o g e n w i t h t h e f o r m a t i o n o f I~ h a s b e e n o b s e r v e d u n d e r c o n d i t i o n s o f a c i d i c a n d b a s i c p r o t e o l y s i s ( K r o h n e t a l . 1 9 7 7 ) . F r e e r a d i c a l s f o r m e d b y r a d i o l y s i s o f w a t e r r e a c t w i t h p r o t e i n s c a u s i n g p r o t e i n d e g r a d a t i o n a n d may a l s o r e s u l t i n f r e e i o d i d e f o r m a t i o n ( A n b a r a n d N e t a 1 9 6 7 ) . F r e e r a d i o l a b e l i n p r o t e i n b i n d i n g e x p e r i m e n t s e n t e r e d r e d c e l l s , s e c t i o n 3 . 2 . A , a n d e l u t e d f r o m c e l l s , s e c t i o n 3 . 3 . A , l i k e i o d i d e ; b e y o n d t h i s t h e i d e n t i t i e s o f t h e f r e e r a d i o l a b e l s p e c i e s w e r e n o t d e t e r m i n e d i n t h e s e e x p e r i m e n t s . The a p p a r e n t b i n d i n g o f f i b r i n o g e n , l i k e t h a t o f a l b u m i n , was d e p e n d e n t u p o n c e l l c o n c e n t r a t i o n . H o w e v e r , t h e a p p a r e n t f i b r i n o g e n b i n d i n g i n e x p e r i m e n t s 5 3 a n d 5A was a l s o d e p e n d e n t u p o n p r o t e i n c o n c e n t r a t i o n , F i g u r e s 3 . 1 2 b a n d T a b l e 3.11 . I n t h e s e e x p e r i m e n t s t h e a p p a r e n t b i n d i n g d e c r e a s e d w i t h i n c r e a s i n g p r o t e i n c o n c e n t r a t i o n . The f r e e l a b e l c a l c u l a t e d f r o m t h e d e p e n d e n c e o f a p p a r e n t b i n d i n g o n c e l l - 1 1 9 -TABLE 3.11 APPARENT F I B R I N O G E N B I N D I N G , EXPRESSED AS Q , AS A FUNCTION OF C E L L AND PR O T E I N CONCENTRATIONS EXPERIMENT 5 3 EXPERIMENT 5A f i b r i n o g e n h Q x l O2 f i b r i n o g e n Q x l O2 (mg/mL) (mg/mL) 1 2 . 3 0.A95 0.A82 1 1 . 3 0 .A79 0 . 2 A 8 0 . 6 0 7 0.391 0 . 5 6 9 0 . 2 1 2 0 . 7 6 5 0 . 2 6 3 0 . 7 1 0 0 . 1 6 2 6.2 0 . 5 0 2 0.53A 5.5 0.A71 0 . 2 8 3 0 . 6 1 8 0.A1A 0 . 5 6 0 0 . 2 A 7 0 . 7 6 7 0 . 2 7 6 0 . 7 0 0 0.19A 3.1 0 . 5 0 5 0 . 5 9 3 2.8 0 . A 8 0 0.30A 0 . 6 1 8 0.A67 0 . 5 6 9 0 . 2 6 9 0 . 7 6 2 0 . 3 0 7 0 . 7 0 9 0 . 2 0 2 1.5 0 . A 6 9 0 . 3 6 2 0 . 5 6 8 0 . 3 1 6 0 . 7 0 8 0 . 2 A 3 c o n c e n t r a t i o n d e c r e a s e d f r o m a b o u t 1.3 % a t 3 mg/mL t o 1.0 % a t 12 mg/mL i n e x p e r i m e n t 5 3 a n d f r o m a b o u t 0 . 6 5 % a t 1 mg/mL t o 0.A8 % a t 11 mg/mL i n e x p e r i m e n t 5 A , T a b l e 3.26 The r e s u l t was u n u s u a l s i n c e f o r e a c h e x p e r i m e n t t h e f i b r i n o g e n s o l u t i o n s w e r e p r e p a r e d f r o m o n e 125 I - f i b r i n o g e n s t o c k a n d t h e p e r c e n t f r e e l a b e l w o u l d o r d i n a r i l y b e e x p e c t e d t o be i n d e p e n d e n t o f p r o t e i n c o n c e n t r a t i o n . T he p r o t e i n c o n c e n t r a t i o n i n t h e s e e x p e r i m e n t s was v a r i e d by a d d i n g c o n c e n t r a t e d n o n - r a d i o l a b e l l e d f i b r i n o g e n a n d b u f f e r t o r a d i o l a b e l e d f i b r i n o g e n . T h u s i t was p o s s i b l e t h a t f r e e i o d i d e was - 120 -r e d u c e d b y t h e a d d i t i o n o f f i b r i n o g e n . A l t h o u g h t h e r e i s e v i d e n c e f o r b i n d i n g o f i o d i d e t o p r o t e i n s w i t h o u t i o d i n a t i o n ( S c a t c h a r d a n d Yap 1 9 6 4 , C r a n d a l l e t a l . 1 9 8 1 ) , o n l y a s m a l l o r n e g l i g i b l e c h a n g e i n a p p a r e n t b i n d i n g , a n d t h e r e f o r e f r e e r a d i o l a b e l , o c c u r r e d w i t h c h a n g e s i n a l b u m i n c o n c e n t r a t i o n s . T h u s i t m u s t be a s s u m e d t h a t t h i s b e h a v i o u r i s c h a r a c t e r i s t i c o f f i b r i n o g e n . On t h e o t h e r h a n d , s i n c e i t t o o k s e v e r a l h o u r s t o c o m p l e t e a s e r i e s o f e x p e r i m e n t s a t d i f f e r e n t c e l l a n d p r o t e i n c o n c e n t r a t i o n s , i t i s p o s s i b l e t h a t t h e r e l a t i o n b e t w e e n f r e e r a d i o l a b e l a n d p r o t e i n c o n c e n t r a t i o n was a n a r t i f a c t . I n e x p e r i m e n t 5 3 , a r a d i o i o d i d e r e l e a s e r a t e o f a b o u t 2 % p e r d a y w o u l d a c c o u n t f o r t h i s r e s u l t . 3 . 2 . 3 I o d i d e p a r t i t i o n b e t w e e n t h e c e l l a n d f l u i d c o m p a r t m e n t s 1 2 5 The d i s t r i b u t i o n r a t i o f o r i o d i d e b e t w e e n t h e c e l l a n d f l u i d c o m p a r t m e n t s , P2 , was d e t e r m i n e d u n d e r c o n d i t i o n s s i m i l a r t o 1 2 5 t h o s e u s e d t o d e t e r m i n e I - p r o t e i n b i n d i n g . The d i s t r i b u t i o n r a t i o 1 2 5 f o r i o d i d e b e t w e e n a c e l l p e l l e t a n d t h e p e l l e t s u p e r n a t a n t , Q2 , was m e a s u r e d , b u t d e p e n d s u p o n t h e c o n c e n t r a t i o n o f c e l l s i n t h e p e l l e t . H o w e v e r , f o r c e l l p e l l e t s p r e p a r e d ' by u l t r a c e n t r i f u g a t i o n t h e i n t e r c e l l u l a r t r a p p i n g was i n s i g n i f i c a n t a n d P2 was n o t s i g n i f i c a n t l y d i f f e r e n t f r o m Q2 , A p p e n d i x 9 . 1 2 5 The r e s u l t s o f e x p e r i m e n t s w i t h e i t h e r Na I o r t h e ' f r e e ' r a d i o l a b e l p e a k c o l l e c t e d f r o m c h r o m a t o g r a p h e d , r a d i o l a b e l e d p r o t e i n s a r e s u m m a r i z e d i n T a b l e 3 . 1 2 . The d i s t r i b u t i o n r a t i o Q2 was a p p r o x i m a t e l y t w o o r d e r s o f m a g n i t u d e g r e a t e r t h a n t h e a p p a r e n t d i s t r i b u t i o n r a t i o s o b s e r v e d f o r p r o t e i n - b o u n d r a d i o l a b e l . T h u s c o n t a m i n a t i o n o f a n a d s o r b a t e b y a s l i t t l e a s 1 % 1 2 5 iO Cj ic|e r e s ui t e d - 121 -TABLE 3 . 1 2 P A R T I T I O N OF 1 2 5I 0 D I D E BETWEEN THE C E L L P E L L E T AND SUPERNATANT COMPARTMENTS OF RED C E L L SUSPENSIONS E X P E R . RADIOLABEL C E L L S (CPM/g) Q2 3 3 N a1 2 5I f r e s h 3 . 2 3 x l 06 0 . 8 0 1 3 7 f r e e p e a k f r o m f i b r i n o g e n i o d i n a t i o n 2 4 f r e s h +1 d a y 9 . 0 0 x l 05 9 . 1 0 x l 05 8 . 6 7 x l 05 8 . 7 1 x l 05 0 . 8 1 4 0 . 8 2 3 0 . 7 7 5 0 . 7 7 9 3 8 f r e e p e a k f r o m a l b u m i n i o d i n a t i o n 2 2 f r e s h 7 . 7 5 x l 05 7 . 9 3 x l 05 0 . 8 0 5 0 . 8 2 4 mean +_ 1 SD (n=5) (n=2) (n=7) f r e s h +1 d a y c o m b i n e d 0 . 8 1 3 + 0 . 0 1 0 0 . 7 7 7 0 . 0 0 3 0 . 8 0 3 + 0 . 0 2 0 i n s i g n i f i c a n t i n c r e a s e s i n t h e a p p a r e n t b i n d i n g . I o d i d e h a s a c c e s s t o t h e i n t r a c e l l u l a r c o m p a r t m e n t o f r e d c e l l p e l l e t s v i a t h e a n i o n c h a n n e l a s s o c i a t e d w i t h b a n d I I I , a membrane-s p a n n i n g p r o t e i n o f r e d c e l l s ( C a b a n t c h i k a n d R o t h s t e i n 1 9 7 4 , Z a k i e t a l . 1 9 7 5 ) . D a l m a r k a n d W i e t h ( 1 9 7 2 ) a n d D a l m a r k ( 1 9 7 6 ) r e p o r t e d a n i o d i d e v o l u m e d i s t r i b u t i o n r a t i o o f 0 . 8 2 a t 38°C a n d pH 7.0 w h i c h i n c r e a s e d t o 1.27 a t 0°C. The w e i g h t d i s t r i b u t i o n r a t i o r e p o r t e d h e r e c o r r e s p o n d s t o a v o l u m e d i s t r i b u t i o n r a t i o o f 0 . 8 7 . - 1 2 2 -3 . 2 . 4 E s t i m a t i o n o f f r e e l a b e l f r o m a p p a r e n t b i n d i n g S e c t i o n 3 . 2 . 2 s h o w e d t h a t a p p a r e n t b i n d i n g was d e p e n d e n t o n c e l l c o n c e n t r a t i o n . I f t h e a p p a r e n t b i n d i n g d i d r e p r e s e n t s u r f a c e a d s o r p t i o n , t h e e q u i l i b r i u m b i n d i n g w o u l d h a v e t o h a v e b e e n d e p e n d e n t u p o n c e l l c o n c e n t r a t i o n . T h i s w o u l d i m p l y i n t e r a c t i o n b e t w e e n t h e c e l l s s u c h a s t o r e d u c e b i n d i n g . D e c r e a s e d b i n d i n g m i g h t be e x p e c t e d w h e r e t h e s u r f a c e a d s o r b e d s p e c i e s o c c u p i e d a s u b s t a n t i a l p o r t i o n o f t h e c e l l s u r f a c e , o r s u r f a c e b i n d i n g s i t e s , a n d c e l l c o n c e n t r a t i o n s w e r e i n c r e a s e d s o t h a t some a d s o r b e d s p e c i e s o c c u p i e d s i t e s o n a d j a c e n t c e l l s . D e c r e a s e d d e x t r a n b i n d i n g i n a s s o c i a t i o n w i t h a g g r e g a t i o n o f r e d c e l l s h a s b e e n r e p o r t e d ( B r o o k s 1 9 7 3 b ) . H o w e v e r , a m o d e l d e r i v e d i n A p p e n d i x 11 s h o w s t h a t p e l l e t a c t i v i t i e s c a n d e c r e a s e s i g n i f i c a n t l y w i t h i n c r e a s i n g c e l l c o n c e n t r a t i o n i f , i n a d d i t i o n t o p r o t e i n - b o u n d r a d i o l a b e l , a r a d i o l a b e l s p e c i e s w i t h a l a r g e r c e l l - t o - m e d i u m d i s t r i b u t i o n r a t i o i s p r e s e n t . The m o d e l d e r i v e d i n A p p e n d i x 11 was b a s e d o n t h e i n d e p e n d e n t c o n s e r v a t i o n o f t h e t w o s p e c i e s o f r a d i o l a b e l b e f o r e a n d a f t e r a d d i t i o n o f t h e c e l l s u s p e n s i o n t o t h e a d s o r b a t e m e d i u m . An e x p r e s s i o n r e l a t i n g t h e a p p a r e n t b i n d i n g ( Ap) , t o t h e i n i t i a l a d s o r b a t e a c t i v i t y ( A ^ ) , t h e i n i t i a l a d s o r b a t e f r e e r a d i o l a b e l f r a c t i o n (rj_2^» ^e c e l l - a s s o c i a t e d p r o t e i n - b o u n d r a d i o l a b e l ( A c i ) > t h e r a d i o i o d i d e d i s t r i b u t i o n r a t i o (P^), a n d t h e c e l l w e i g h t r a t i o s fb^ , fb^ , a n d y i p was d e r i v e d , e q u a t i o n 1 1 . 1 1 . U n f o r t u n a t e l y , h o w e v e r , t h e v a l u e o f t h e mass f r a c t i o n o f t h e u l t r a c e n t r i f u g e d c e l l p e l l e t (/$p) i s n o t known w i t h s u f f i c e n t a c c u r a c y t o a p p l y t h i s m o d e l i n i t s e n t i r e t y . - 123 -I f t h e v a l u e o f fl was a c c u r a t e l y k n o w n , t h e v a l u e o f t h e b i n d i n g e s t i m a t e A ^ c o u l d be e s t i m a t e d d i r e c t l y . The w e i g h t r a t i o was g r e a t e r t h a n 0 . 9 9 5 i n u l t r a c e n t r i f u g e d c e l l p e l l e t s , s e c t i o n 3.5.1 . H o w e v e r , e v e n w i t h jb^ r e s t r i c t e d t o t h e r a n g e 0 . 9 9 5 t o 1 . 0 , a w i d e r a n g e o f e s t i m a t e s f o r Ac^ r e s u l t e d . A m o d e l c a l c u l a t i o n , A p p e n d i x 1 1 , s h o w e d t h a t c h a n g e s i n fl^ r e s u l t e d i n l a r g e c h a n g e s i n t h e e s t i m a t e s f o r A£ l a n d r e l a t i v e l y s m a l l c h a n g e s i n t h e f r e e l a b e l e s t i m a t e , r ^2 , a t c e l l - t o - s u s p e n s i o n w e i g h t r a t i o s ( l e s s t h a n 0.8 . T h i s r e s u l t e d f r o m t h e n o n - l i n e a r d e p e n d e n c e o f t h e b i n d i n g e s t i m a t e u) , e q u a t i o n 1 1 . 1 6 , u p o n t h e p a r a m e t e r o(^ , e q u a t i o n 1 1 . 6 , when ^ was l e s s t h a n o n e . T h u s e x t r a p o l a t i o n o f d a t a p l o t t e d a s CJ v e r s u s 0( ^  t o e s t i m a t e t h e i n t e r c e p t Acj /Ai w a s n°t r e l i a b l e . V a r i a t i o n s i n fl^ b e t w e e n 0 . 9 9 5 a n d 1.0 r e s u l t e d i n e s t i m a t e s o f A ^ t h a t v a r i e d by a s much a s A 0 0 % , w h i l e e s t i m a t e s o f r ^2 v a r i e d by a b o u t 30 % . T h u s t h e d e p e n d e n c e o f t h e a p p a r e n t b i n d i n g o n t h e c e l l c o n c e n t r a t i o n ( f l & ) c o u l d be u s e d t o e s t i m a t e t h e a d s o r b a t e f r e e r a d i o l a b e l f r a c t i o n ( r ^2) e v e n t h o u g h t h e v a l u e o f y 3 p was n o t a c c u r a t e l y k n o w n , A p p e n d i x 11 . U s i n g t h e v a l u e d e t e r m i n e d i n s e c t i o n 3 . 2 . 3 f o r t h e c e l l d i s t r i b u t i o n r a t i o o f i o d i d e ( P2) , e s t i m a t e s w e r e made o f t h e a m o unt 125 o f f r e e r a d i o l a b e l p r e s e n t i n I - a l b u m i n a d s o r b a t e s w h i c h h a d a l s o b e e n a s s a y e d by g e l f i l t r a t i o n . F i g u r e 3.13 s h o w s d a t a p l o t t e d t o e v a l u a t e t h e r a t i o s Acj /Ai a r |d ri 2 ' r e s p e c t i v e l y t h e i n t e r c e p t a n d s l o p e o f e q u a t i o n 1 1 . 1 5 . I n t h i s F i g u r e , t h e p a r a m e t e r y3p was e q u a l t o o n e . T a b l e 3 . 1 3 l i s t s t h e i n t e r c e p t s a n d s l o p e s - 124 -0 . 0 4 0 . 0 2 -0 . 0 0 A-S - - - B t 2 8 V 4 7 3 0 3 2 0 . 5 l -A(1-/VP 2£e) 1 . 0 F i g u r e 3 . 1 3 A n a l y s i s o f t h e d e p e n d e n c e o f a p p a r e n t b i n d i n g o n c e l l c o n c e n t r a t i o n t o d e t e r m i n e t h e f r e e r a d i o l a b e l r a t i o r ^ . The m o d e l d e v e l o p e d i n A p p e n d i x 11 p r e d i c t s a l i n e a r r e l a t i o n b e t w e e n u) a n d o< ^ w i t h s l o p e r ^2 a n d i n t e r c e p t Ac] / A j when A p a p p r o a c h e s 1 . The a n a l y s e s a r e s u m m a r i z e d i n T a b l e 3 . 1 3 . TABLE 3.13 FREE L A B E L FRACTIONS ( ri 2) DETERMINED BY A N A L Y S I S OF APPARENT B I N D I N G AND GEL F I L T R A T I O N OF ADSORBATES. INCUBATION L I N E A R REGRESSION : u ) ( y 5 p ) VERSUS #1, EQUATION 1 1 . 1 5 GEL F I L T R A T I O N INTERCEPT a : AC 1/ A J X 1 02 1.0 0 . 9 9 5 SLOPE 3 : r1 2x l 02 1.0 0 . 9 9 5 r i2x l 02 a EXPERIMENT 2 8 ( n = 4 , 4 5 m i n ) ( n = 4 , 1 4 5 m i n ) 0.12 + 0 . 0 6 - 0 . 3 2 + 0 . 0 6 0.19 + 0 . 0 4 - 0 . 2 5 + 0.04 4 . 3 + 0.1 4 . 4 + 0.1 4.2 + 0.1 4 . 3 + 0.1 3 0 ( n = 4 , 4 5 m i n ) 0 . 0 7 5 + 0 . 0 0 5 - 0 . 3 7 5 + 0 . 0 0 9 1.30 + 0.01 1.22 + 0 . 0 2 ( n = 4 , 1 1 0 m i n ) 0 . 0 7 3 + 0 . 0 0 2 - 0.376" + 0 . 0 0 5 1.58 + 0.01 1.50 + 0.01 3 2 ( n = 6 , 3 5 m i n ) 4 7 ( n = 5 , 3 0 m i n ) 0.11 + 0 . 0 4 - 0.37 + 0 . 0 4 0.09 + 0 . 0 3 - 0.41 + 0 . 0 3 2 . 2 4 + 0 . 0 9 2 . 2 7 + 0 . 1 0 4 . 2 + 0.1 ( n = 3 ) 1.0 0 . 5 5 + 0 . 0 7 0 . 5 3 + 0 . 0 7 0.7 + 0.2 ( n = 2 ) 2 . 2 a ) means + o n e s t a n d a r d d e v i a t i o n - 126 -f o r c a l c u l a t i o n s w i t h fl>^ e q u a l t o 0 . 9 9 5 a n d 1.0 . The a g r e e m e n t b e t w e e n t h e e s t i m a t e s f o r f r o m t h i s a n a l y s i s a n d g e l f i l t r a t i o n , a n d t h e l i n e a r i t y o f t h e r e l a t i o n s b e t w e e n u J a n d cK-^, a r e t h e b a s e s o n w h i c h t h e c h a n g e i n a p p a r e n t b i n d i n g w i t h c e l l c o n c e n t r a t i o n i s a t t r i b u t e d t o i n t r a c e l l u l a r r a d i o i o d i d e . T h u s i t was c o n s i d e r e d u n l i k e l y t h a t t h e d e p e n d e n c e o f a p p a r e n t b i n d i n g u p o n c e l l c o n c e n t r a t i o n was d u e t o r e d u c e d b i n d i n g a s s o c i a t e d w i t h c e l l u l a r i n t e r a c t i o n . The p r e s e n c e o f i n t r a c e l l u l a r r a d i o i o d i d e h a s b e e n n o t e d w i t h r e s p e c t t o t h e e s t i m a t i o n o f t h e i n t e r c e l l u l a r v o l u m e i n r e d c e l l p e l l e t s ( V a z q u e z e t a l . 1 9 5 2 , M a i z e l s a n d R e m i n g t o n 1 9 5 9 , F u n d e r a n d W i e t h 1 9 6 7 ) , h o w e v e r t h e u s e o f c h a n g e s i n t h e a p p a r e n t b i n d i n g w i t h c e l l c o n c e n t r a t i o n t o d e t e r m i n e s u p e r n a t a n t r a d i o i o d i d e c o n c e n t r a t i o n d o e s n o t a p p e a r t o h a v e b e e n r e p o r t e d p r e v i o u s l y . The a n a l y s i s o f A p p e n d i x 11 c o r r e c t s f o r t h e r a d i o l a b e l i n b o t h t h e i n t r a - a n d i n t e r c e l l u l a r c o m p a r t m e n t s a t o n c e . The u n r e l i a b i l i t y o f t h e b i n d i n g e s t i m a t e A ^ , when u s i n g t h e a n a l y s i s o f A p p e n d i x 1 1 , was d u e m o r e t o t h e c o r r e c t i o n f o r i n t e r c e l l u l a r r a d i o l a b e l t h a n f o r i n t r a c e l l u l a r r a d i o l a b e l . The s e n s i t i v i t y o f t o t h e u n c e r t a i n t y i n t h e p e l l e t m ass f r a c t i o n was l e s s s e v e r e t h a n t h a t o f t h e b i n d i n g e s t i m a t e Ac i . T h u s b i n d i n g e s t i m a t e s w e r e made by c o r r e c t i n g t h e a p p a r e n t b i n d i n g f o r i n t r a c e l l u l a r r a d i o l a b e l d e t e r m i n e d f r o m t h e v a l u e o f r ^ • T n e c a l c u l a t i o n u s e d e q u a t i o n 8.3 a n d a l l o w e d s e p a r a t i o n o f t h e i n t r a - a n d i n t e r c e l l u l a r c o r r e c t i o n s , t h u s s e p a r a t i n g t h e m a j o r s o u r c e o f u n c e r t a i n t y f r o m t h i s b i n d i n g e s t i m a t e . The b i n d i n g c a l c u l a t i o n s a r e s u m m a r i z e d i n s e c t i o n 3.5 . - 127 -The v a l u e s o f t h e i n t e r c e p t s d e t e r m i n e d when y3 was a s s u m e d t o e q u a l o n e w e r e o f t h e o r d e r o f 1 x 1 0 ~3 . The b i n d i n g p a r a m e t e r r e p r e s e n t i n g t h e a p p a r e n t b i n d i n g c o r r e c t e d f o r i n t r a c e l l u l a r r a d i o i o d i d e , , was s i m i l a r , s e c t i o n 3.5.1 . T h i s s u g g e s t s t h a t t h e i n t e r c e l l u l a r m a s s f r a c t i o n was c l o s e r t o z e r o ( f b ^ = 1 ) , t h a n t o t h e a s s u m e d maximum v a l u e o f 0 . 0 0 5 . 3 . 2 . 5 Summary U l t r a c e n t r i f u g a t i o n was e f f e c t i v e i n r e d u c i n g i n t e r c e l l u l a r v o l u m e a n d t h e r e f o r e o v e r e s t i m a t i o n o f s u r f a c e a d s o r p t i o n . No s i g n i f i c a n t a l t e r a t i o n s i n t h e c h a r a c t e r i s t i c s o f t h e r e d c e l l s c o u l d b e a s s o c i a t e d w i t h t h e c e n t r i f u g a t i o n c o n d i t i o n s . H e m o l y s i s o f c e l l s was o c c a s i o n a l l y n o t e d , b u t was a f u n c t i o n o f t h e p r i o r t r e a t m e n t o f t h e c e l l s r a t h e r t h a n t h e c e n t r i f u g a t i o n c o n d i t i o n s . The p r e s e n c e o f i o d i d e , b e c a u s e i t h a s a c c e s s t o t h e i n t r a c e l l u l a r c o m p a r t m e n t o f r e d c e l l s , may r e s u l t i n s i g n i f i c a n t o v e r e s t i m a t i o n o f t h e b i n d i n g o f r a d i o l a b e l e d p r o t e i n s t o r e d c e l l s . " I O C "I o r I n a d d i t i o n , i o d i d e i n I - p r o t e i n p r e p a r a t i o n s c a n c a u s e a p p a r e n t b i n d i n g t o be d e p e n d e n t u p o n c e l l c o n c e n t r a t i o n , w h i l e 1 2 5 ' d e i o d i n a t i o n ' o f I - p r o t e i n s c a n c a u s e a p p a r e n t b i n d i n g t o be d e p e n d e n t u p o n i n c u b a t i o n t i m e . 1 2 5 C o m p a r i s o n o f t h e a m ount o f f r e e r a d i o l a b e l i n I - a l b u m i n p r e p a r a t i o n s d e t e r m i n e d b y g e l c h r o m a t o g r a p h y a n d a n a l y s i s o f a p p a r e n t b i n d i n g s h o w e d t h a t t h e i n t r a c e l l u l a r f r e e r a d i o l a b e l c o u l d be i d e n t i f i e d w i t h r a d i o i o d i d e . T h u s , t h e d e p e n d e n c e o f a p p a r e n t b i n d i n g u p o n c e l l c o n c e n t r a t i o n c o u l d be u s e d t o e s t i m a t e t h e a m ount o f r a d i o i o d i d e p r e s e n t i n t h e i n t r a c e l l u l a r c o m p a r t m e n t a n d t o c o r r e c t f o r - 128 -t h i s c o m p o n e n t i n b i n d i n g c a l c u l a t i o n s . T h i s m e t h o d o f c o r r e c t i n g e s t i m a t e s o f a p p a r e n t b i n d i n g h a d t h e a d v a n t a g e o f a v o i d i n g u n c e r t a i n t i e s a s s o c i a t e d w i t h t h e e s t i m a t i o n o f f r e e r a d i o l a b e l f r o m c h r o m a t o g r a p h i c a n a l y s e s , s p e c i f i c a l l y t h e p r o b l e m s o f d i s t i n g u i s h i n g b e t w e e n i o d i d e a n d i o d i n a t e d p r o t e i n f r a g m e n t s a n d c o r r e c t i n g b i n d i n g e s t i m a t e s f o r t i m e d e p e n d e n t d e i o d i n a t i o n o f r a d i o i o d i n a t e d p r o t e i n s . - 129 -3.3 WASH A N A L Y S I S I n o r d e r t o d e t e r m i n e w h e t h e r o r n o t t h e a p p a r e n t b i n d i n g o b s e r v e d i n u l t r a c e n t r i f u g e d c e l l p e l l e t s r e p r e s e n t e d a s i n g l e b i n d i n g r e a c t i o n , c e l l p e l l e t s w e r e w a s h e d u s i n g a q u a n t i t a t i v e a n a l y s i s d e s c r i b e d i n A p p e n d i x 1 5 . I n t h i s a n a l y s i s , t h e r a d i o l a b e l r e t a i n e d i n a s s o c i a t i o n w i t h t h e c e l l s o f a c e l l p e l l e t ( Ac) was c a l c u l a t e d , u s i n g e q u a t i o n 7 . 3 , f r o m t h e m e a s u r e d p e l l e t a n d s u p e r n a t a n t a c t i v i t i e s a n d t h e amount o f s u p e r n a t a n t c a r r i e d o v e r w i t h t h e c e l l s f r o m w a s h t o w a s h . The q u a n t i t y Ac g e n e r a l l y d e c r e a s e d r a p i d l y f o r t h e f i r s t t h r e e o r f o u r w a s h e s a n d t h e n l e s s r a p i d l y f o r t h e s u b s e q u e n t w a s h e s . The same r e l a t i v e c e l l t o w a s h v o l u m e s a n d w a s h i n t e r v a l s w e r e m a i n t a i n e d , i m p l y i n g t h a t t h e a p p a r e n t b i n d i n g was c o m p o s e d o f a t l e a s t t w o c o m p o n e n t s . Wash c u r v e s w e r e c o n s t r u c t e d b y p l o t t i n g Ac a s a f r a c t i o n o f t h e a p p a r e n t b i n d i n g v e r s u s t h e w a s h n u mber o n s e m i - l o g c o o r d i n a t e s . The w a s h c u r v e s w e r e w e l l c h a r a c t e r i z e d a s t h e sum o f t w o e x p o n e n t i a l s w i t h t h e w a s h n u mber ( i ) a s a n i n t e g e r v a r i a b l e , And) = a - , e x p ( A i i ) + a0e x p ( A . o i ) 16.1 Ap( 0 ) 1 1 w h e r e A c ( i ) was t h e a c t i v i t y p e r g r a m o f c e l l s i n t h e i t h wash p e l l e t , Ap(0) was t h e a c t i v i t y p e r g r a m o f t h e c e l l p e l l e t p r o d u c e d f r o m t h e i n i t i a l a d s o r p t i o n s u s p e n s i o n . The p a r a m e t e r s a-^ , , , a n d A 2 w e r e d e t e r m i n e d b y a n o n - l i n e a r f i t t i n g a n a l y s i s w h i c h m i n i m i z e d t h e sum o f t h e s q u a r e d e r r o r s , A p p e n d i x 1 6 . - 1 3 0 -3.3.1 I n t e r c e l l u l a r r a d i o l a b e l The a p p a r e n t b i n d i n g d e t e r m i n e d f r o m c e l l p e l l e t s was s i g n i f i c a n t l y l o w e r a t h i g h e r r a t e s o f c e n t r i f u g a t i o n , s e c t i o n 3.2.1 . T h i s r e d u c t i o n was i n t e r p r e t e d a s due t o r e d u c t i o n o f i n t e r c e l l u l a r t r a p p i n g . C o m p a r i s o n o f w a s h a n a l y s e s o f c e l l p e l l e t s p r o d u c e d a t d i f f e r e n t r a t e s o f c e n t r i f u g a t i o n s u p p o r t e d t h i s i n t e r p r e t a t i o n . A s u s p e n s i o n o f P B S w a s h e d r e d c e l l s , e q u i l i b r a t e d w i t h 1 2 5 I - a l b u m i n , was s p l i t i n t o a l i q u o t s a n d c e l l p e l l e t s w e r e p r o d u c e d b y s t a n d a r d u l t r a c e n t r i f u g a t i o n o r b y l o w s p e e d c e n t r i f u g a t i o n ( 6 0 0 x g f o r 2 0 m i n u t e s ) . The c e l l q u a n t i t y i n t h e u l t r a c e n t r i f u g e d c e l l p e l l e t was d e t e r m i n e d by w e i g h t t o a n e r r o r o f l e s s t h a n 0.5 % . I n t h e c a s e o f t h e p e l l e t p r o d u c e d by l o w s p e e d c e n t r i f u g a t i o n , t h e c e l l q u a n t i t y was d e t e r m i n e d by a h e m a t o c r i t m e a s u r e m e n t o f t h e c e l l r e s u s p e n s i o n a f t e r t h e f i r s t w a s h , flcs = 0 . 2 0 8 + 0 . 0 1 0 . The c e l l - a s s o c i a t e d r a d i o l a b e l p e r g r a m o f c e l l s ( Ac) was c a l c u l a t e d f o r t h e c e l l p e l l e t s o f e a c h w a s h c y c l e a s d e s c r i b e d i n s e c t i o n 2.7.1 . The d e v i a t i o n s i n Ac v a l u e s a s s o c i a t e d w i t h 5 % v a r i a t i o n s i n t h e c e l l q u a n t i t y w e r e a l s o c a l c u l a t e d . T h e s e r e s u l t s a r e s u m m a r i z e d i n T a b l e 3.14 a n d F i g u r e 3 . 1 4 . The a p p a r e n t b i n d i n g i n t h e p e l l e t p r o d u c e d by l o w s p e e d c e n t i f u g a t i o n was a b o u t t w e n t y t i m e s t h a t o f t h e p e l l e t p r o d u c e d by u l t r a c e n t r i f u g a t i o n . H o w e v e r , a f t e r e i g h t P B S w a s h e s , t h e a c t i v i t i e s r e t a i n e d p e r g r a m o f c e l l s w e r e 6 1 0 0 a n d 5 7 0 0 CPM/g r e s p e c t i v e l y . The d i f f e r e n c e s b e t w e e n t h e h i g h a n d l o w s p e e d Ac v a l u e s w e r e n o t s i g n i f i c a n t f o r w a s h e s o n e t h r o u g h t h r e e , a n d w e r e l e s s t h a n 7 % f o r t h e s u b s e q u e n t w a s h e s . - 131 -TABLE 3.14 COMPARISON OF APPARENT B I N D I N G I N P E L L E T S PRODUCED BY ULTRACENTRIFUGATION AND LOW-SPEED CENTRIFUGATION WASH A N A L Y S I S Af Ap ^>p . Ac a A AC b ULTRACENTRIFUGATION 0 1 . 6 3 x l 07 9 . 0 7 x l 04 • • • • 1 1.79 1 04 2.05 1 04 0 . 7 8 7 2 . 1 2 x l 04 1 6 0 2 4 . 0 3 1 03 8.72 1 03 0 . 7 7 6 1.01 1 04 3 0 0 3 9 4 8 5 . 9 6 " 0 . 8 4 0 6.92 1 03 2 8 0 4 1 7 9 5.14 " 0 . 8 1 4 6.28 " 2 9 0 5 6 7 5 . 0 4 11 0 . 8 3 5 6.01 " 2 8 0 6 2 7 4 . 8 0 " 0 . 8 1 2 5.91 " 2 8 0 7 2 6 4 . 7 8 " 0 . 8 2 5 5.79 " 2 7 0 8 1 9 4 . 7 8 x l 03 0 . 8 3 7 5 . 7 l x l 03 2 8 0 LOW-SPEED CENTRIFUGATION w a s h 0 1 . 6 3 x l 07 2 . 0 7 x l 06 . . . 1 6 . 0 8 1 05 1.42 1 05 0 . 8 0 8 2 . 9 8 x l 04 2 . 7 8 x l 04 2 4 . 1 4 1 04 1.76 1 04 0 . 8 1 0 1.19 1 04 1 . 4 2 x l 03 3 7.56 1 03 6.85 1 03 0 . 8 2 2 7.56 1 03 1 9 0 4 4 1 0 5.50 " 0 . 8 1 8 6.59 " 2 8 0 5 7 0 5 . 4 0 " 0 . 8 3 7 6.39 " 2 9 0 6 2 9 5.02 " 0 . 7 9 3 6.28 " 2 9 0 7 14 5 . 1 7 " 0 . 8 2 3 6.23 " 2 9 0 8 12 5 . 1 1 x l 03 0 . 8 3 6 6 . 1 3 x l 03 2 9 0 a ) u s i n g e q u a t i o n 7.3 ; Ac = A p / y i p - A f ( l - / 3 p ) / / 5 p , w h e r e Ap i s t h e a c t i v i t y p e r g r a m o f p e l l e t , A f i s t h e a c t i v i t y p e r g r a m o f s u p e r n a t a n t , a n d fbp i s t h e c e l l t o p e l l e t w e i g h t r a t i o . b ) Mean c h a n g e i n Ac d u e t o c h a n g e s o f + 5 % i n t h e e s t i m a t e d c e l l q u a n t i t y . - 1 3 2 -W A S H NUMBER 10 F i g u r e 3 . 1 4 C o m p a r i s o n o f w a s h c u r v e s f o r c e l l p e l l e t s p r o d u c e d b y u l t r a c e n t r i f u g a t i o n • D a n d l o w s p e e d c e n t r i f u g a t i o n ( 6 0 0 x g f o r 2 0 m i n ) <>•••<> . The a d s o r b a t e was l2 5I - a l b u m i n . The w a s h a n a l y s i s i s s u m m a r i z e d i n T a b l e 1 3 . 1 4 . The e r r o r b a r s i n d i c a t e d e v i a t i o n s w h i c h r e s u l t f r o m c h a n g e s o f 5 % i n t h e c e l l w e i g h t . M o s t o f t h e d e v i a t i o n s a r e s m a l l e r t h a n t h e s y m b o l s u s e d . C l o s e d s y m b o l s i n d i c a t e a c t i v i t i e s p e r g r a m o f p e l l e t , o p e n s y m b o l s a c t i v i t i e s p e r g r a m o f c e l l . - 1 3 3 -I n p r i n c i p l e , t h e q u a n t i t y Ac d o e s n o t i n c l u d e i n t e r c e l l u l a r r a d i o l a b e l , t h u s w ash c u r v e s d e t e r m i n e d f o r p e l l e t s p r o d u c e d b y d i f f e r e n t r a t e s o f c e n t r i f u g a t i o n s h o u l d b e e q u i v a l e n t . The r e l a t i v e l y s m a l l d i f f e r e n c e s b e t w e e n t h e w a s h c u r v e s o f F i g u r e 3 . 1 4 , i n s p i t e o f t h e t w e n t y - f o l d d i f f e r e n c e i n A p ( 0 ) v a l u e s , s u p p o r t e d t h i s v i e w . The d i f f e r e n c e s i n t h e f i r s t t h r e e w a s h e s b e t w e e n t h e Ac q u a n t i t i e s a t d i f f e r e n t c e n t r i f u g a t i o n r a t e s w e r e s m a l l e n o u g h t o be a c c o u n t e d f o r b y t h e u n c e r t a i n t y i n t h e q u a n t i t y o f c e l l s . I t f o l l o w e d t h a t t h e d i f f e r e n c e b e t w e e n A p ( 0 ) a n d t h e i n t e r c e p t o f a w a s h c u r v e a t w a s h z e r o , s h o u l d r e p r e s e n t i n t e r c e l l u l a r r a d i o l a b e l . E s t i m a t i o n o f t h e wash c u r v e i n t e r c e p t s d e p e n d e d u p o n a m o d e l f o r t h e b e h a v i o u r o f t h e e l u t i n g c o m p o n e n t o f t h e w a s h c u r v e s d e v e l o p e d i n s e c t i o n s 3 . 3 . 3 a n d 3 . 3 . 4 . E s t i m a t e s o f i n t e r c e l l u l a r r a d i o l a b e l a r e made i n s e c t i o n 3 . 3 . 4 . The w a s h c u r v e s w e r e t h e r e f o r e i n t e r p r e t e d a s r e p r e s e n t i n g r a d i o l a b e l w h i c h was s t r o n g l y b o u n d t o t h e c e l l s a n d l a b e l w h i c h was e i t h e r i n t r a c e l l u l a r , o r r e v e r s i b l y - b o u n d , o r b o t h . T h e s e e x p e r i m e n t s w e r e n o t a b l e t o show a d i f f e r e n c e i n t h e amount o f r a d i o l a b e l t h a t c o u l d b e r a p i d l y e l u t e d f r o m t h e c e l l s d u e t o c h a n g e s i n c e n t r i f u g a t i o n r a t e . I n g e n e r a l , g r e a t e r p r e c i s i o n was a c h i e v e d i n t h e e s t i m a t i o n o f A v a l u e s a f t e r s e v e r a l w a s h e s b e c a u s e t h e c o n t r i b u t i o n o f t h e c s u p e r n a t a n t was t h e n i n s i g n i f i c a n t . U l t r a c e n t r i f u g a t i o n was u s e f u l i n t h i s r e g a r d , a s t h e s u p e r n a t a n t a c t i v i t y was r e d u c e d i n f e w e r w a s h e s , d u e t o r e d u c e d i n t e r c e l l u l a r t r a p p i n g . 3 . 3 . 2 I n t r a c e l l u l a r r a d i o l a b e l T he s i m i l a r i t y o f t h e i n i t i a l s l o p e s o f t h e wash c u r v e s f o r a - 134 -number o f e x p e r i m e n t s s u g g e s t e d t h a t a s i n g l e c e l l - a s s o c i a t e d c o m p a r t m e n t o r c o m p o n e n t m i g h t be i n v o l v e d , F i g u r e 3 . 1 5 . R a d i o i o d i d e e l u t i n g f r o m t h e i n t r a c e l l u l a r c o m p a r t m e n t o f t h e c e l l s was t h e m o s t l i k e l y e x p l a n a t i o n f o r t h i s b e h a v i o u r . I n e x p e r i m e n t s 3 8 , 4 5 , a n d 4 7 t h e r e l a t i v e a m o u n t s o f r a d i o l a b e l a s s o c i a t e d w i t h t h e t w o c o m p o n e n t s o f w a s h c u r v e s w e r e c o m p a r e d t o t h e r e l a t i v e a m o u n t s o f f r e e a n d p r o t e i n - b o u n d r a d i o l a b e l d e t e r m i n e d b y g e l f i l t r a t i o n . T a b l e 3 . 15 s u m m a r i z e s t h e w a s h a n a l y s e s f o r t h e s e e x p e r i m e n t s a n d s h o w s t h a t t h e p a r a m e t e r a-^ d e c r e a s e d a s t h e p e r c e n t f r e e r a d i o l a b e l i n c r e a s e d . T h u s t h e p o s s i b i l i t y t h a t f r e e r a d i o l a b e l a c c o u n t s q u a n t i t a t i v e l y f o r t h e e l u t i n g c o m p o n e n t i n w a s h a n a l y s e s was i n v e s t i g a t e d . T e s t i n g t h i s h y p o t h e s i s r e q u i r e d a m o d e l w h i c h l i n k e d t h e r a d i o l a b e l e l u t i o n r a t e s a n d q u a n t i t i e s w i t h t h e v o l u m e s o f t h e c o m p a r t m e n t s i n a wash a n a l y s i s a n d t h e d i s t r i b u t i o n r a t i o s o f t h e r a d i o l a b e l c o m p o n e n t s b e t w e e n t h e c o m p a r t m e n t s . A m o d e l was d e v e l o p e d , A p p e n d i x 1 7 , a n d was t e s t e d i n 1 2 5 w a s h e x p e r i m e n t s i n v o l v i n g t h e e l u t i o n o f i o d i d e f r o m c e l l s . 3 . 3 . 3 R a d i o i o d i d e w a s h o u t c u r v e s The m o d e l d e v e l o p e d i n A p p e n d i x 17 a s s u m e d t h a t t h e i n t r a c e l l u l a r v o l u m e o f a c e l l s u s p e n s i o n was a r e s e r v o i r f o r 1 2 5 i o d i d e . T h u s t h e s l o p e o f t h e r a p i d l y e l u t i n g c o m p o n e n t o f a wash c u r v e was e x p e c t e d t o v a r y w i t h t h e r a t i o o f t h e c e l l t o w a s h v o l u m e u s e d i n a w a s h a n a l y s i s . T h i s p r e d i c t i o n was t e s t e d i n t h e f o l l o w i n g e x p e r i m e n t . F r e s h P B S w a s h e d r e d c e l l s w e r e e q u i l i b r a t e d w i t h pH 7.4 1 2 5 P B S / i o d i d e b u f f e r f o r 3 0 m i n u t e s a t r o o m t e m p e r a t u r e a n d a h e m a t o c r i t o f 5 0 % w i t h a n d w i t h o u t 1 0 mM N a l . C a r r i e r i o d i d e was - 135 -F i g u r e 3 . 1 5 Wash a n a l y s e s o f e x p e r i m e n t s 3 8 , 4 5 , a n d 4 7 . B o t h t h e d a t a a n d t h e e x p o n e n t i a l m o d e l s l i s t e d i n T a b l e 3 . 1 5 a r e s h o w n . The q u i c k l y e l u t i n g c o m p o n e n t i s a s s o c i a t e d w i t h f r e e r a d i o l a b e l . E s t i m a t e s o f t h e f r e e l a b e l f r o m t h e w a s h a n a l y s e s a r e c o m p a r e d w i t h s i m i l a r e s t i m a t e s f r o m g e l f i l t r a t i o n i n T a b l e 3 . 1 7 . TABLE 3 . 1 5 1 25 l - A L B U M I N WASH ANALYSES AND THE AMOUNT OF F R E E RADIOLABEL DETERMINED BY GEL F I L T R A T I O N WASH A N A L Y S I S GEL F I L T R A T I O N WASH RATIOS a EXPONENTIAL MODEL PARAMETERS b EXPERIMENT / ic sx l O O / o p x l O O - A2 a 2 x l 0 0 a i x l O O r i 2x 1 0 0 3 8 / 1 17.7 + 0.3 7 0 . 6 + 0 . 1.477 0 . 0 7 2 0 3 1 . 6 0 2 6 . 6 4 0 . 1 8 / 2 1 6 . 8 + 0.2 6 9 . 3 + 0 . 1.347 0 . 0 7 6 3 1 9 . 4 9 2 9 . 1 7 / 3 c 16 . 8 + 0.1 6 9 . 0 + 0 . 1.556 • • • 6 2 . 6 3 • • • 9 9 . 3 4 5 / 1 17.2 + 0.1 76 . 9 + 0.7 1.629 0 . 0 1 7 6 5 4 . 7 1 6.05 4 . 8 d /2 18.2 + 0.1 8 0 . 8 + 0.5 1.557 0 . 0 3 1 6 5 4 . 7 6 6.24 2.8 d 4 7 / 1 2 0 . 0 + 0.1 7 8 . 2 + 0.7 1.449 0 . 0 2 2 2 6 4 . 5 0 3 . 5 0 /2 22.1 + 0.1 78 . 4 + 0.9 1.367 0 . 0 2 5 7 6 6 . 5 7 2.87 /3 2 0 . 7 + 0.1 7 6 . 8 + 1.1 1.396 0 . 0 2 7 9 6 3 . 4 2 2.54 2 . 2 4 /4 20.-2 + 0.1 78.1 + 1.1 1.414 0 . 0 2 8 8 5 9 . 5 9 2 . 1 9 /5 2 2 . 0 + 0.1 78.1 + 1.2 1.368 0 . 0 3 4 0 6 0 . 3 9 2 . 1 6 /6 c 24.1 + 0.1 79.1 + 1.1 1.322 0 . 1 2 0 1 6 7 . 0 5 0 . 3 3 9 5 a ) mean +_ o n e s t a n d a r d d e v i a t i o n b ) mean +_ e s t i m a t e d s t a n d a r d d e v i a t i o n , A p p e n d i x 16 c ) A d s o r b a t e i s p r i m a r i l y f r e e r a d i o l a b e l . d ) C a l c u l a t e d f r o m e s t i m a t e s o f r f2 . - 137 -a d d e d t o s e e w h e t h e r o r n o t s a t u r a t i o n o f t h e i o d i d e e x c h a n g e m i g h t n e e d t o be c o n s i d e r e d . Two c e l l p e l l e t s w e r e p r o d u c e d by u l t r a c e n t r i f u g a t i o n a n d a l i q u o t s o f t h e s e w e r e s u b j e c t e d t o w a s h a n a l y s e s . The w a s h c u r v e s w e r e d e t e r m i n e d i n P B S b u f f e r w i t h a n d w i t h o u t 1 0 mM N a l , c o r r e s p o n d i n g t o t h e p r e v i o u s i n c u b a t i o n c o n d i t i o n s . The w ash p r o c e d u r e was c a r r i e d o u t i n p o l y s t y r e n e c o u n t i n g t u b e s . R e s u s p e n s i o n o f t h e p e l l e t s a m p l e s was a c h i e v e d b y b r i e f v o r t e x i n g , f i r s t w i t h a 1:1 c e l l t o f l u i d r a t i o a n d t h e n a t t h e f i n a l c e l l t o f l u i d r a t i o o f t h e p a r t i c u l a r e x p e r i m e n t . The r e s u s p e n d e d c e l l s w e r e a l l o w e d t o s t a n d f o r f i v e m i n u t e s b e f o r e c e n t r i f u g a t i o n a t a b o u t 8 0 0 x g f o r 4 m i n u t e s . S u p e r n a t a n t s w e r e r e m o v e d a n d k e p t f o r l a t e r a n a l y s i s . C e l l p e l l e t s w e r e g a m m a - c o u n t e d d i r e c t l y , t h e n w e i g h e d , a n d t h e c y c l e r e p e a t e d . The p r o c e d u r e t o o k a b o u t 2 5 m i n u t e s p e r w a s h . A l l t h e w a s h q u a n t i t i e s w e r e d e t e r m i n e d by w e i g h t . 1 2 5 F i g u r e 3.16 s h o w s t h e i o d i d e w a s h c u r v e s . T a b l e 3 . 1 6 s u m m a r i z e s t h e m o d e l p a r a m e t e r s a i > A^ > a 2 > a n c ' ^ 2 ^ o r t h e w a sh c u r v e s a n d t h e r a t i o s /2>cs a n d jh^ w h i c h c h a r a c t e r i z e d t h e c e l l , e x c h a n g e , a n d c a r r y - o v e r v o l u m e s o f t h e w a s h a n a l y s e s . The s l o p e s o f t h e w a s h c u r v e s d e p e n d e d o n / 3 C S a n d /3 , b u t w e r e i n d i f f e r e n t t o t h e p r e s e n c e o f 1 0 mM i o d i d e . W i t h s u f f i c i e n t w a s h i n g , a s e c o n d , s l o w l y e l u t i n g c o m p o n e n t e q u a l t o a b o u t 0.1 % o f t h e i n i t i a l p e l l e t a c t i v i t y ( 6 x l 06 CPM/g) was d e t e c t e d . I n t h e c a r r i e r f r e e e x p e r i m e n t , . t h i s r e p r e s e n t e d a b o u t 3xl0~^ m o l e s i o d i d e p e r g r a m o f c e l l s ( o r a b o u t 0 . 2 m o l e c u l e s / c e l l ) . - 138 -0 W A S H N U M B E R F i g u r e 3 . 1 6 ^2 5I o d i d e w a s h o u t f r o m human r e d c e l l s . T he a c t i v i t y r e t a i n e d w i t h t h e c e l l s p e r g r a m o f c e l l s i s p l o t t e d a s a p e r c e n t o f t h e a c t i v i t y p e r g r a m o f u l t r a c e n t r i f u g e d c e l l p e l l e t . The w a s h m e d i a was P B S +++ o r P B S w i t h 10 mM N a l OO . The c e l l a n d c a r r y - o v e r v o l u m e s w e r e a p p r o x i m a t e l y c o n s t a n t a n d e q u a l . The w a s h e x c h a n g e v o l u m e i s g i v e n i n t h e F i g u r e . TABLE 3 . 16 A N A L Y S I S OF 125TQDIDE WASHOUT FROM ULTRACENTRIFUGED RED C E L L P E L L E T S WASH PARAMETERS / > c sa / 3 p a b * 2b a i b x l O O x l O O z 1 x l O O x l O O MEDIA PBS + N a l 2 mL 34 . 0 + 0.2 7 9 . 1 + 0.6 0 . 9 2 2 + 0 . 0 0 3 6 4 . 5 + 0. 2 3 2 3 . 8 + 0.1 7 6 . 8 + 0.9 1.296 + 0.002 0 . 0 0 + 0 . 0 4 7 0 . 1 + 0.1 0 . 0 7 + 0 . 0 2 5 1 6 . 3 + 0.2 7 6 . 5 + 1.5 1.691 + 0.001 0 . 1 3 + 0.01 7 0 . 2 + 0.1 0 . 1 0 + 0.01 PBS 2 3 0 . 9 + 0.1 7 7 . 6 + 0.8 0.991 + 0 . 0 0 3 6 8 . 9 + 0 . 3 3 2 5 . 5 + 0.1 7 8 . 0 + 0.5 1.215 + 0.009 0.04* + 0 . 3 4 6 6 . 8 + 0.4 0 . 0 6 + 0.11 5 1 7 . 2 + 0.1 7 7 . 6 + 0.6 1.641 + 0.001 0. 0 9 + 0.01 7 1 . 8 + 0.1 0 . 0 9 + 0 . 0 4 P 2 c P I c a2' c x l O O a i ' c x l O O 5+NaI 2 mL 0 . 8 4 3 + 0. 0 1 4 • * • 8 2 . 9 + 1.7 • • 4 3 0 . 7 9 0 0 . 0 1 6 1 0 4 0 + 4 0 9 4 . 8 3 . 5 0 . 0 7 + 0.0 2 5 0 . 7 8 6 0 . 0 2 3 3 5 1 9 5 . 9 7.4 0 . 1 0 0.01 PBS 2 0 . 8 5 9 0 . 0 1 7 • • • 9 0 . 7 2.4 3 0 . 8 3 3 0 . 0 1 9 6 5 2 2 8 8 . 2 3.1 0 . 0 6 0.11 5 0 . 7 9 7 + 0 . 0 1 0 5 0 + 1 9 7 . 1 + 3. 2 0 . 0 9 + 0 . 0 0 a ) mean +_ o n e s t a n d a r d d e v i a t i o n b ) mean e s t i m a t e d s t a n d a r d d e v i a t i o n , A p p e n d i x 16 c ) mean + rms p r o p a g a t e d e r r o r ( o n e s t a n d a r d d e v i a t i o n ) - 140 -a ) I n t r a c e l l u l a r r a d i o i o d i d e The m o d e l d e v e l o p e d i n A p p e n d i x 17 l e d t o a n e s t i m a t e o f t h e d i s t r i b u t i o n r a t i o b e t w e e n t h e c e l l a n d f l u i d c o m p a r t m e n t s ( p ) f o r c o m p o n e n t s d e f i n e d b y t h e w a s h a n a l y s i s . F o r a n y c o m p o n e n t t h e e x p o n e n t i a l p a r a m e t e r A a n d t h e mean wash r a t i o s / 3 c s a n d d e t e r m i n e p , e q u a t i o n 1 7 . 1 6 . The r e s u l t s f o r t h e s i x r a d i o i o d i d e e l u t i o n e x p e r i m e n t s a r e g i v e n i n T a b l e 3.16 . The mean e s t i m a t e f o r P2 was 0 . 8 2 +_ 0 . 0 3 ( n = 6 ) . T h i s was n o t s i g n i f i c a n t l y d i f f e r e n t ( t t e s t , p = 0 . 5 0 ) f r o m P2 e s t i m a t e d f r o m u l t r a c e n t r i f u g e d c e l l p e l l e t s , T a b l e 3.11 . D i s t r i b u t i o n r a t i o s c a l c u l a t e d f o r t h e b o u n d c o m p o n e n t o f t h e s e w a s h a n a l y s e s r a n g e d f r o m 35 t o 1 0 0 0 . The w i d e r a n g e r e f l e c t s i n p a r t t h e e x p o n e n t i a l d e p e n d e n c e o f p o n A . D i f f e r e n c e s i n t t e s t s w e r e c o n s i d e r s i g n i f i c a n t when p 0 . 0 5 ( M e n d e n h a l l e t a l . 1 9 8 1 , p p . 3 9 0 - 3 9 5 ) . D a l m a r k a n d W i e t h ( 1 9 7 2 ) s h o w e d t h a t t h e e q u i l i b r i u m e x c h a n g e o f i o d i d e f r o m r e d c e l l s f o l l o w e d f i r s t o r d e r k i n e t i c s ( t j y2 = 5^ s e c a t 2 0°C) u n t i l a t l e a s t 9 8 % o f t h e i n t r a c e l l u l a r i o d i d e h a d b e e n e x c h a n g e d . The e q u i v a l e n c e o f t h e d i s t r i b u t i o n r a t i o s , p2 a n d P2 s h o w e d t h a t i o d i d e was a b l e t o r e - e q u i l i b r a t e b e t w e e n t h e c e l l a n d s u p e r n a t a n t c o m p a r t m e n t s d u r i n g e a c h w a s h c y c l e . T h u s t h e w ash 12 5 a n a l y s e s c o u l d b e u s e d t o i d e n t i f y i n t r a c e l l u l a r i o d i d e . The b i n d i n g o f i o d i d e r e p o r t e d h e r e was o n l y e v i d e n t a f t e r e l u t i o n o f more t h a n 9 9 . 5 % o f t h e i n t r a c e l l u l a r r a d i o l a b e l . The a m o u n t s r e t a i n e d w e r e s m a l l e n o u g h n o t t o r e p r e s e n t s i g n i f i c a n t b i n d i n g i n t h e e x p e r i m e n t s 1 2 5 a n d may h a v e b e e n d u e t o i m p u r i t i e s i n t h e i o d i d e s t o c k s . - 141 -b) Intercellular radioiodide The wash curve parameters a-^  and a 2 represented the apparent intercepts of the wash components extrapolated to the zero*"*"1 wash. The intercepts were apparent because the wash curves depended upon the wash volumes of a particular experiment as well as the distribution ratio of the components. The calculation of corrected intercepts, Appendix 1 7 i i i , assumed that p was constant for a given component and took into account the change in the value of /2> from one for the zero^*"1 pellet to between 0.7 and 0.8 for the subsequent pellets. Estimates of p 2 made at successive washes in the iodide experiments varied by no more than 3 % in the linear portion of the wash curves. Table 3.16 l i s t s the corrected intercepts, a^' and a 2' . The correction to the a-^  value was always negligible. In the iodide experiments, the sum of the apparent intercept a l + a2 represented between 65 and 80 % of the i n i t i a l pellet activity, Ap(0). The range of a-^ 1 + a 2' was 0.82 to 1.06 . A value of one was expected in the case that a l l the radiolabel in the i n i t i a l c e l l pellets was accounted for in the wash analysis. The mean value for a-^ ' + a 2' was 0.92 _+ 0.05 (n=6). The mean value was significantly less than one (t test, p = 0.006). The difference between the apparent binding at the zero*''"1 wash, Ap(0), and the intercept extrapolated from a wash curve was expected to represent intercellular trapping, section 3.3.1 . However, 125 in I-albumin experiments, radiolabel in c e l l pellets produced by ultracentrifugation indicated that the intercellular volume was on the order of 0.5 % or less, section 3.5.1 . Thus the 8 % mean difference - 142 -between the intercepts and Ap(0) was greater than expected on the basis of an i n t e r c e l l u l a r volume of 0.5 % . Review of the experiment indicated that the difference may have been due i n part to the high counting rates, approximately 4 x 10 6 CPM, associated with the p e l l e t samples. At high counting rates, counting e f f i c i e n c y decreases, section 2.4 . The corrections applied to the apparent a c t i v i t i e s of the ultracentrifuged and f i r s t wash p e l l e t s were respectively +67 and +15 % . I f the correction i n the f i r s t case had been too great, the i n t e r c e l l u l a r volume would have been overestimated, accounting for the discrepancy. 3.3.4 Wash curves i n protein binding experiments The wash curves for the albumin experiments described i n Table 3.15 were analysed to determine the wash parameters p^ and P 2 , the free l a b e l r a t i o r ^ 2 , and the corrected intercepts a-^1 and a 2* . The estimation of the d i s t r i b u t i o n r a t i o s and intercept values was as described for the radioiodide experiments. The supernatant free radiolabel r a t i o , r ^ 2 , was estimated from the wash analysis data on the basis that the eluting component represented radioiodide. The difference between the t o t a l wash curve and the bound component therefore represented the radioiodide per gram of c e l l s , A c 2 . The model for t h i s analysis i s described i n Appendix 17iv and predicts that the r ^ 2 i s equal to the r a t i o of A c 2 ( i ) and a factor K ( i ) which i s dependent upon P 2 , p 2 , ^3C S , /2>p » and A^(0) , equation 17.20 . Table 3.17 summarizes these results. TABLE 3.17 FREE L A B E L ESTIMATES DETERMINED FROM WASH A N A L Y S E S AND GEL F I L T R A T I O N WASH A N A L Y S I S GEL F I L T R A T I O N I N T E R C E P T S D I S T R I B U T I O N RATIO a FREE L A B E L EXPERIMENT a 2 * x l 0 0 a j / x l O O P2 P I r f 2 X l 0 0 r i 2 x l 0 0 b r j ^ x l O O 3 8 / 1 46.7 2 6 . 8 0.84 + 2.2 5 6 + 2 0.16 0 . 2 7 n l o / 2 2 6 . 8 2 9 . 4 1.13 + 4.1 56 + 2 0.11 0 . 1 9 U. 10 / 3 c 100 . 0 • • • 0.75 + 0 . 0 2 • • > • 9 7 . 3 9 9 . 3 9 9 . 3 4 5 / 1 70.8 6.1 0.86 + 0.05 2 5 6 + 1 0. 3 5 1.47 4 . 8 b / 2 70 . 0 6 .3 0 . 8 9 + 0 . 0 3 1 3 3 + 1 0 . 4 3 1.75 2 . 8 ° 4 7 / 1 85 . 4 3.5 0.86 + 0 . 0 5 166 + 1 1.31 2.31 / 2 88 . 0 2.9 0.83 + 0 . 0 4 1 2 5 + 1 1.08 2 . 4 3 / 3 85 1 2.5 0.86 + 0 . 0 3 124 + 1 0.89 2 . 5 2 2 . 2 4 /4 9 2 . 6 2.2 0. 9 0 + 0 . 0 2 125 + 1 0.71 3 . 0 9 /5 8 0 . 0 2.2 0.84 + 0 . 0 3 9 4 + 1 0. 4 8 2 . 6 5 /6 C 8 8 . 3 0.3 0.78 + 0 . 0 2 2 2 + 0 . 5 1 0 5 . 2 1 0 2 . 7 9 5 a ) mean +_ rms p r o p a g a t e d e r r o r ( o n e s t a n d a r d d e v i a t i o n ) b ) C a l c u l a t e d f r o m e s t i m a t e s o f r f 2 , e q u a t i o n 1 2 . 9 . c ) A d s o r b a t e i s p r i m a r i l y f r e e r a d i o l a b e l . - 144 -a ) I n t r a c e l l u l a r r a d i o l a b e l The mean d i s t r i b u t i o n r a t i o o f t h e e l u t i n g c o m p o n e n t o f t h e w a s h c u r v e s ( p2) , when e x p e r i m e n t 3 8 / 2 was e x c l u d e d , was 0 . 8 4 _+ 0 . 0 5 ( n = 1 0 ) a n d was n o t s i g n i f i c a n t l y d i f f e r e n t ( t t e s t , p = 0 . 1 7 ) f r o m t h e e q u i l i b r i u m d i s t r i b u t i o n r a t i o o b s e r v e d f o r r a d i o i o d i d e , T a b l e 3 . 1 1 . T h u s i n t r a c e l l u l a r r a d i o l a b e l was i d e n t i f i e d w i t h r a d i o i o d i d e i n p r o t e i n b i n d i n g e x p e r i m e n t s . D i s t r i b u t i o n r a t i o s f o r t h e b o u n d c o m p o n e n t (p-^) w e r e g r e a t e r t h a n 5 0 . The m o d e l d e v e l o p e d i n A p p e n d i x 17 t o e s t i m a t e p v a l u e s a s s u m e d t h a t a c o m p o n e n t was p a r t i t i o n e d b e t w e e n t h e c e l l a n d e x t e r n a l v o l u m e s . H o w e v e r , e x p e r i m e n t s i n s e c t i o n 3 . 3 . 5 s h o w e d t h a t b o u n d ' r a d i o l a b e l was o n l y a s s o c i a t e d w i t h t h e membrane o f r e d c e l l s . I n a d d i t i o n , d e s o r p t i o n o f t h e b o u n d c o m p o n e n t d e p e n d e d u p o n t h e f r e e p r o t e i n c o n c e n t r a t i o n , s e c t i o n 3 . 3 . 5 . T h u s p-^  v a l u e s w e r e o n l y u s e d t o d e s c r i b e t h e e l u t i o n b e h a v i o u r o f t h e b o u n d c o m p o n e n t a n d w e r e n o t f u r t h e r i n t e r p r e t e d . D e t e r m i n a t i o n o f t h e amount o f s u p e r n a t a n t f r e e r a d i o l a b e l f r o m w a s h c u r v e s i s s h o w n i n F i g u r e 3.17 f o r e x p e r i m e n t 4 7 . F i g u r e 3 . 17 s h o w s t h e w a s h d a t a p l o t t e d a c c o r d i n g t o t h e a n a l y s i s o f A p p e n d i x 1 7 i v . The l i n e a r r e l a t i o n s b e t w e e n Ac 2 a n d K ( i ) r e f l e c t e d t h e c o n s t a n c y o f t h e d i s t r i b u t i o n r a t i o p2 . The s l o p e s i n t h i s F i g u r e e s t i m a t e d t h e v a l u e s o f r ^2 i n e a c h o f s i x e x p e r i m e n t s . E x c e p t f o r t h e f r e e r a d i o l a b e l e x p e r i m e n t , t h e g r e a t e s t s l o p e was a s s o c i a t e d w i t h t h e w a s h a n a l y s i s o f t h e e x p e r i m e n t w i t h t h e l o w e s t c e l l c o n c e n t r a t i o n , /2>e . The v a r i a t i o n o f r ^2 was i n a g r e e m e n t w i t h t h e d e p e n d e n c e o f a p p a r e n t b i n d i n g o n t h e c e l l - 1 4 5 -F i g u r e 3 . 1 7 1 2 5I - a l b u m i n w a s h a n a l y s i s , e x p e r i m e n t 4 7 . P l o t o f AC2 ( i ) e s t i m a t e d f r o m e q u a t i o n 1 7 . 1 7 v e r s u s K ( i ) d e f i n e d b y e q u a t i o n 1 7 . 1 9 . The s l o p e e s t i m a t e s t h e f r e e r a d i o l a b e l r a t i o o f t h e s u p e r n a t a n t . The v a l u e s o f fle f o r t h e c u r v e s 1-6 a r e r e s p e c t i v e l y 0 . 4 9 5 , 0 . 6 1 6 , 0 . 7 0 0 , 0 . 8 1 3 , 0 . 8 5 3 , a n d 0 . 5 3 5 . - 146 -c o n c e n t r a t i o n fle , s e c t i o n 3 . 2 . 3 . U s i n g t h e m o d e l f o r t h e d i l u t i o n o f r a d i o i o d i d e d u e t o a d d i t i o n o f c e l l s , e q u a t i o n 1 2 . 9 , t h e c o r r e s p o n d i n g a d s o r b a t e f r e e l a b e l r a t i o s ( ri 2) w e re c a l c u l a t e d . ' The mean v a l u e f o r r ^ f r o m t h e wash a n a l y s e s o f e x p e r i m e n t 4 7 was 2.6 +_ 0 . 3 % ( n = 5 ) a n d was i n g o o d a g r e e m e n t w i t h t h e f r e e r a d i o l a b e l d e t e r m i n e d b y g e l f i l t r a t i o n . W i t h t h e e x c e p t i o n o f e x p e r i m e n t s 4 5 / 1 a n d 4 5 / 2 , t h e w a s h e s t i m a t e s f o r i n o t h e r e x p e r i m e n t s w e r e a l s o i n a g r e e m e n t w i t h t h e e s t i m a t e s made by g e l f i l t r a t i o n , T a b l e 3 . 17 . I n t h e e x c l u d e d e x p e r i m e n t s i t a p p e a r e d t h a t t h e g e l f i l t r a t i o n r e s u l t s w e r e i n e r r o r . T h i s was e v i d e n t b e c a u s e t h e a p p a r e n t b i n d i n g c o r r e c t e d f o r f r e e r a d i o l a b e l was n e g a t i v e when t h e g e l f i l t r a t i o n v a l u e s f o r ri 2 w e r e u s e d» i n d i c a t i n g t h a t t h e v a l u e s d e t e r m i n e d b y g e l f i l t r a t i o n w e r e t o o h i g h . T h u s t h e e l u t i n g c o m p o n e n t o f t h e w a s h c u r v e s r e p r e s e n t e d p r i m a r i l y r a d i o i o d i d e . The e l u t i n g c o m p o n e n t may h a v e i n c l u d e d r e v e r s i b l y a d s o r b e d p r o t e i n . H o w e v e r , t h e a g r e e m e n t b e t w e e n t h e c h r o m a t o g r a p h i c a n d w a s h a n a l y s e s s u g g e s t e d t h a t l e s s t h a n 1 0 % o f t h e e l u t i n g c o m p o n e n t c o u l d b e a t t r i b u t e d t o o t h e r t h a n r a d i o i o d i d e , T a b l e 3 . 1 7 . M u l l e r a n d G r a m l i c h ( 1 9 6 3 , 1 9 6 5 ) made s i m i l a r a n a l y s e s o f p r o t e i n b i n d i n g t o r e d c e l l s a n d o b s e r v e d w a s h c u r v e s w h i c h i m p l i e d t h a t a p p a r e n t b i n d i n g c o n s i s t e d o f a t l e a s t t w o c o m p o n e n t s . They d i d n o t i n t e r p r e t t h e r a p i d l y e l u t i n g c o m p o n e n t o f t h e i r w a s h c u r v e s . H o w e v e r , t h e y n o t e d t h a t f r e e i o d i d e i n t h e i r p r o t e i n p r e p a r a t i o n s v a r i e d b e t w e e n 0.2 a n d 8 % ( M u l l e r a n d G r a m l i c h 1 9 6 5 ) . E l u t i o n o f v a r i a b l e a m o u n t s o f i n t r a c e l l u l a r r a d i o l a b e l i s s u g g e s t e d b y v a r i a t i o n s i n t h e w a s h c u r v e s f o r t h e i r p r o t e i n p r e p a r a t i o n s , s i m i l a r t o t h o s e - 1 4 7 -s h o w n i n F i g u r e 3 . 1 5 . b ) I n t e r c e l l u l a r r a d i o l a b e l When t h e d i f f e r e n c e s b e t w e e n t h e a p p a r e n t b i n d i n g a n d t h e sum o f t h e c o r r e c t e d i n t e r c e p t s w e r e i n t e r p r e t e d a s i n t e r c e l l u l a r r a d i o l a b e l , t h e i n t e r c e l l u l a r v o l u m e s i n p e l l e t s p r o d u c e d b y u l t r a c e n t r i f u g a t i o n w e r e e s t i m a t e d t o be l e s s t h a n 0 . 5 % o f t h e c e l l v o l u m e . T h e s e e s t i m a t e s o f t h e i n t e r c e l l u l a r v o l u m e , c a l c u l a t e d u s i n g e q u a t i o n 1 4 . 7 , a s s u m e d t h a t t h e c o n c e n t r a t i o n s o f t h e p r o t e i n s i n t h e s u p e r n a t a n t a n d i n t e r c e l l u l a r v o l u m e s w e r e e q u a l , a n d a r e s u m m a r i z e d i n T a b l e 3 . 1 8 . The v o l u m e f r a c t i o n f o r t h e a l b u m i n e x p e r i m e n t s 3 8 , 4 5 , 4 7 , a n d 4 9 was ( 1 . 0 0 + 0 . 3 2 ) x 1 0 ~3 (mean + s t a n d a r d d e v i a t i o n , n = l l ) a n d f o r t h e f i b r i n o g e n e x p e r i m e n t s 4 0 , 5 3 , a n d 5 4 was ( 0 . 2 4 + 1.20) x 1 0 "3 ( n = 9 ) . E x c e p t f o r e x p e r i m e n t s 4 5 , 4 7 , a n d 4 9 , t h e i n d i v i d u a l e s t i m a t e s o f t h e v o l u m e f r a c t i o n s w e r e s m a l l e r t h a n t h e e s t i m a t e d e r r o r s . T h u s i n d i v i d u a l w a s h a n a l y s e s w e r e f r e q u e n t l y u n a b l e t o r e s o l v e a d i f f e r e n c e b e t w e e n t o t a l p e l l e t a c t i v i t i e s a n d c o r r e c t e d w a s h i n t e r c e p t s . When t h e e s t i m a t e d e r r o r s w e r e c o n s i d e r e d , m o s t o f t h e v o l u m e f r a c t i o n s w e r e b e l o w 0 . 5 % . A t p r o t e i n c o n c e n t r a t i o n s o f 1 mg/mL t h e mean i n t e r c e l l u l a r v o l u m e f r a c t i o n s w e r e e q u i v a l e n t t o 8 6 0 ± 9 0 a n d 4 0 +_ 70 m o l e c u l e s p e r c e l l (mean + s t a n d a r d e r r o r o f t h e mean) f o r a l b u m i n a n d f i b r i n o g e n r e s p e c t i v e l y . The i n t e r c e l l u l a r v o l u m e s may a l s o b e e x p r e s s e d i n t e r m s o f a n a v e r a g e f i l m t h i c k n e s s ( D ) c a l c u l a t e d a s s u m i n g t h a t t h e f l u i d v o l u m e i n t h e p e l l e t i s u n i f o r m l y d i s t r i b u t e d o v e r t h e t o t a l c e l l s u r f a c e a r e a , e q u a t i o n 1 4 . 8 . The t h i c k n e s s r e p r e s e n t s h a l f t h e mean c e l l - t o - c e l l s e p a r a t i o n . The c e l l - t o - c e l l s e p a r a t i o n s c a l c u l a t e d f o r - 1 4 8 -T a b l e 3.18 I N T E R C E L L U L A R VOLUME ESTIMATED FROM WASH AN A L Y S E S EXPERIMENT Q x l O3 a2' a a i ' a V c c / V c : x l 03 b 38/1 2 . 56 0 . 4 7 + 2.06 0 . 2 7 + 0.04 0 . 7 2 + 5.7 2 2 . 4 5 0 . 2 7 1.44 0 . 2 9 0 . 0 5 1 . 21 4 . 0 4 5 / 1 3 . 77 0.71 0.07 0 . 0 6 0.001 0 . 9 4 0 . 2 8 2 4 . 51 0 . 7 0 0 . 0 4 0.06 0.001 1 . 17 0 . 1 9 4 7 / 1 1 1 . 5 0 . 8 5 0 . 0 7 0 . 0 3 5 0 . 0 0 1 1 . 4 3 0 . 8 7 2 9 . 4 9 0.88 0 . 0 6 0 . 0 2 9 0 . 0 0 1 0 . 9 3 0 . 6 2 3 7 . 9 4 0 . 8 5 0 . 0 5 0 . 0 2 5 0 . 0 0 1 1 . 0 7 0 . 4 3 4 5 . 51 0 . 9 3 0 . 0 5 0 . 0 2 2 0 . 0 0 1 0 . 2 9 0 . 3 0 5 4 . 6 2 0 . 8 0 0 . 0 5 0 . 0 2 2 0 . 0 0 1 0 . 8 9 0 . 2 5 4 9 / 1 5 . 5 5 0 . 7 0 0 . 1 0 0 . 0 6 8 0 . 0 0 2 1 . 3 9 0 . 6 0 2 2 . 6 6 0 . 6 2 0.08 0 . 0 5 9 0 . 0 0 2 0 . 9 2 0 . 2 3 4 0 / 1 7 . 7 2 0 . 9 4 0 . 9 8 0 . 0 8 8 0 . 0 0 3 - 0 . 2 3 8.2 2 8 . 0 4 0 . 7 6 0 . 3 3 0 . 1 2 2 0 . 0 0 2 1 . 0 2 2.9 3 7 . 6 8 0 . 6 5 0 . 2 4 0 . 1 2 7 0 . 0 0 4 1. 8 5 2.0 4 7 . 6 8 1.02 1.43 0 . 1 0 5 0 . 0 0 4 - 1 . 0 4 1 2 5 7 . 6 0 0 . 9 0 0 . 2 6 0 . 0 8 2 0 . 0 0 1 0 . 15 2.1 6 7 . 4 4 0 . 6 6 0 . 1 3 0 . 1 1 5 0 . 0 0 3 1 . 81 1.1 5 3 / 1 4 . 7 7 0 . 8 0 0 . 4 2 0 . 1 1 4 0 . 0 0 7 0 . ,44 2.2 3 5 . 9 1 0 . 8 3 0 . 3 2 0 . 1 0 8 0 . 0 0 4 0 . 4 0 2.0 5 4 / 1 3 . 6 0 1.20 + 0 . 5 5 0 . 2 6 7 + 0 . 0 0 4 - 1 . 8 2 + 2.1 a ) mean +_ e s t i m a t e d s t a n d a r d d e v i a t i o n , A p p e n d i x 16 b ) mean _+ p r o p a g a t e d s t a n d a r d d e v i a t i o n t h e i n t e r c e l l u l a r v o l u m e f r a c t i o n s a s s o c i a t e d w i t h a l b u m i n a n d o f i b r i n o g e n w e r e r e s p e c t i v e l y 14 +_ 1.4 a n d 4 + 6 A . L a r g e r v o l u m e s a n d s e p a r a t i o n s w o u l d o n l y b e i m p l i e d i f t h e p r o t e i n c o n c e n t r a t i o n i n t h e i n t e r c e l l u l a r s p a c e was r e d u c e d r e l a t i v e t o t h a t o f t h e - 1 4 9 -s u p e r n a t a n t . H o w e v e r , p r e f e r e n t i a l movement o f f i b r i n o g e n o r a l b u m i n o u t o f c e l l p e l l e t s d u r i n g c e n t r i f u g a t i o n r e l a t i v e t o w a t e r w o u l d t h e n h a v e t o o c c u r . I f t h e e s t i m a t e s o f t h e i n t e r c e l l u l a r v o l u m e a r e a c c e p t e d , t h e n i t w o u l d seem t h a t t h e v o l u m e s c o u l d n o t b e u n i f o r m l y d i s t r i b u t e d , a s t h e mean i n t e r c e l l u l a r s e p a r a t i o n s w e r e l e s s t h a n t h e m inimum d i m e n s i o n s o f e i t h e r a l b u m i n ( S q u i r e e t a l . 1 9 6 8 ) o r f i b r i n o g e n ( S h u l m a n 1 9 5 3 , C o h e n e t a l . 1 9 8 3 ) . T h i s s u g g e s t s t h e p r e s e n c e o f i n t e r c e l l u l a r s e a m s w h e r e m o r e t h a n t w o c e l l s a b u t . I n t e r c e l l u l a r v o l u m e s i n d e x t r a n b i n d i n g e x p e r i m e n t s h a v e b e e n e s t i m a t e d i n a n a n a l o g o u s m a n n e r a f t e r c e n t r i f u g a t i o n o f c e l l s a t 1 6 5 , 0 0 0 x g f o r 6 0 m i n u t e s ( B r o o k s e t a l . 1 9 8 0 ) . The a p p a r e n t i n t e r c e l l u l a r v o l u m e was d e p e n d e n t u p o n t h e a g g r e g a t i o n o f t h e c e l l s a n d t h e d e x t r a n c o n c e n t r a t i o n o f t h e s u p e r n a t a n t . The i n t e r c e l l u l a r v o l u m e f r a c t i o n f o r r e d c e l l s a g g r e g a t e d by D e x t r a n 150 ( a p p r o x i m a t e m o l e c u l a r w e i g h t 1 5 0 , 0 0 0 d a l t o n s ) was a p p r o x i m a t e l y 0.5 x 1 0-"5 a n d _2 i n c r e a s e d t o v a l u e s i n t h e r a n g e o f 1 t o 1.5 x 10 a b o v e t h e c r i t i c a l d i s a g g r e g a t i o n c o n c e n t r a t i o n f o r t h i s d e x t r a n f r a c t i o n . D e x t r a n 3 6 . 5 d o e s n o t a g g r e g a t e r e d c e l l s a n d was a s s o c i a t e d w i t h i n t e r c e l l u l a r v o l u m e s w h i c h i n c r e a s e d l i n e a r l y f r o m a b o u t 0.3 t o _2 1.0 x 10 w i t h i n c r e a s i n g d e x t r a n c o n c e n t r a t i o n . D i f f e r e n c e s i n i n t e r c e l l u l a r v o l u m e a s s o c i a t e d w i t h c h a n g e s i n f i b r i n o g e n c o n c e n t r a t i o n c o u l d n o t be d i s t i n g u i s h e d . The mean a p p a r e n t i n t e r c e l l u l a r v o l u m e f r a c t i o n i n e x p e r i m e n t 4 0 was ( 0 . 6 +_ 1.2) x 10""5 . The f i b r i n o g e n c o n c e n t r a t i o n was 0.6 mg/mL, n o t e n o u g h t o r e s u l t i n a g g r e g a t i o n o f r e d c e l l s . I n e x p e r i m e n t s 5 3 / 1 , 5 3 / 3 , a n d 5 4 / 1 , t h e s u p e r n a t a n t f i b r i n o g e n c o n c e n t r a t i o n s w e r e - 1 5 0 -r e s p e c t i v e l y 1 1 . 5 , 2 . 9 , a n d 2.6 mg/mL, e n o u g h i n e a c h c a s e t o c a u s e r e d c e l l a g g r e g a t i o n . T h o u g h t h e e s t i m a t e s o f i n t e r c e l l u l a r v o l u m e w e r e n o t w e l l d e f i n e d i n t h e f i b r i n o g e n e x p e r i m e n t s , t h e y w e r e c l e a r l y l e s s t h a n t h o s e a s s o c i a t e d w i t h r e d c e l l s d i s a g g r e g a t e d by D e x t r a n 1 5 0 , w e r e p r o b a b l y l e s s t h a n t h o s e m e a s u r e d b y D e x t r a n 3 6 . 5 , a n d w e r e e q u i v a l e n t t o t h o s e f o r c e l l s a g g r e g a t e d by D e x t r a n 1 5 0 . I n e x p e r i m e n t s w i t h f i b r i n o g e n , d i s a g g r e g a t i o n o f r e d c e l l s d u e t o h i g h p r o t e i n c o n c e n t r a t i o n s h a s n o t b e e n o b s e r v e d . I f t h e i n t e r p r e t a t i o n o f t h e w a s h a n a l y s e s i s c o r r e c t , i t f o l l o w s t h a t f i b r i n o g e n m e a s u r e d a s m a l l e r i n t e r c e l l u l a r v o l u m e t h a n D e x t r a n 3 6 . 5 a n d D e x t r a n 1 5 0 , when t h e l a t t e r was a b o v e i t s d i s a g g r e g a t i o n c o n c e n t r a t i o n . The i n t e r c e l l u l a r v o l u m e a s s o c i a t e d w i t h a l b u m i n was g r e a t e r t h a n t h a t o f f i b r i n o g e n a n d s m a l l e r t h a n t h a t o f D e x t r a n 3 6 . 5 . I n t e r c e l l u l a r v o l u m e s i n r e d c e l l p e l l e t s h a v e b e e n e s t i m a t e d i n c o n n e c t i o n w i t h t h e d e t e r m i n a t i o n o f r e d c e l l v o l u m e s b y t h e h e m a t o c r i t m e t h o d ( P o n d e r a n d S a s l o w 1 9 3 0 , V a z q u e z e t a l . 1 9 5 2 ) a n d r e d c e l l e l e c t r o l y t e c o n c e n t r a t i o n s ( M a i z e l s a n d R e m i n g t o n 1 9 5 9 , F u n d e r a n d W i e t h 1 9 6 7 ) . E x c e p t f o r t h e s t u d y r e p o r t e d b y F u n d e r a n d W i e t h ( 1 9 6 7 ) , t h e i n t e r c e l l u l a r v o l u m e e s t i m a t e s w e r e a s s o c i a t e d w i t h r e l a t i v e l y l o w s p e e d c e n t r i f u g a t i o n c o n d i t i o n s . I n t h e l a t t e r c a s e , a n i n t e r c e l l u l a r v o l u m e f r a c t i o n o f 4.7 x l O-"5 was r e p o r t e d f o r r e d c e l l p e l l e t s a f t e r 3 0 m i n u t e s c e n t r i f u g a t i o n a t 7 5 , 0 0 0 x g when ^ I - a l b u m i n was u s e d a s a v o l u m e m a r k e r . H o w e v e r , t h e s e a u t h o r s d i d n o t c o n s i d e r b i n d i n g o f a l b u m i n t o t h e r e d c e l l s u r f a c e n o r d i d t h e y c o r r e c t t h e i r e s t i m a t e s f o r f r e e r a d i o i o d i d e . T h u s t h e i r v a l u e o v e r e s t i m a t e d - 151 -i n t e r c e l l u l a r v o l u m e . H o w e v e r , t h e y w e r e a l s o i n t e r e s t e d i n c o m p a r i s o n o f i n t e r c e l l u l a r v o l u m e s d e t e r m i n e d b y ' ^ ' ' " I - a l b u m i n , " ^ C - s u c r o s e , 4 2 2 2 K, a n d Na . W i t h t h e l a s t t h r e e m a r k e r s , t h e y e s t i m a t e d a _2 v o l u m e f r a c t i o n o f 2.2 x 1 0 , s i g n i f i c a n t l y l a r g e r t h a n t h a t f o u n d w i t h l a r g e r m a r k e r s . T h u s t h e e s t i m a t e d i n t e r c e l l u l a r v o l u m e s o f u l t r a c e n t r i f u g e d r e d c e l l p e l l e t s d e p e n d u p o n m o l e c u l a r s i z e . P r e s u m a b l y , some r e g i o n s o f t h e i n t e r c e l l u l a r v o l u m e a r e i n a c c e s s i b l e t o l a r g e s p e c i e s . I t f o l l o w s t h a t a v o l u m e m a r k e r t o e s t i m a t e a c o r r e c t i o n t o a p p a r e n t f i b r i n o g e n b i n d i n g f o r i n t e r c e l l u l a r f i b r i n o g e n , was n o t l i k e l y t o b e f o u n d . S u c h a m a r k e r w o u l d h a v e t o be a m o l e c u l e o f c o m p a r a b l e s i z e a n d s h a p e , a n d w o u l d f i r s t h a v e t o d e m o n s t r a t e a s i g n i f i c a n t l y l o w e r a p p a r e n t b i n d i n g . 3 . 3 . 5 B o u n d r a d i o l a b e l a ) SDS-PAGE a n a l y s i s Wash a n a l y s e s o f u l t r a c e n t r i f u g e d r e d c e l l s u s p e n s i o n s h a v e s h o w n t h a t t h e a p p a r e n t b i n d i n g a s s o c i a t e d w i t h t h e c e l l p e l l e t s was d u e i n p a r t t o ' i r r e v e r s i b l y ' b o u n d r a d i o l a b e l . I n f e r r i n g t h e r a d i o l a b e l s p e c i e s r e s p o n s i b l e f o r t h i s b i n d i n g f r o m t h e c o m p o s i t i o n o f t h e p r o t e i n a d s o r b a t e s was n o t p o s s i b l e . The l e v e l o f i m p u r i t i e s i n t h e f i b r i n o g e n i s o l a t i o n s w e r e o f t h e o r d e r o f 1 0 % , w h e r e a s t h e f r a c t i o n o f t h e a d s o r b a t e r a d i o l a b e l r e t a i n e d i n c e l l p e l l e t s a f t e r w a s h a n a l y s i s was o f t h e o r d e r o f 0.1 % . I n a d d i t i o n , i t was o b s e r v e d 1 2 5 t h a t r a d i o l a b e l s p e c i e s p r e s e n t i n i o d i d e s t o c k s a l s o b o u n d t o c e l l s . The o b j e c t o f t h e f o l l o w i n g e x p e r i m e n t was t o d e t e r m i n e t h e m o l e c u l a r w e i g h t c h a r a c t e r i s t i c s o f t h e b o u n d c o m p o n e n t u s i n g SDS-PAGE. F i b r i n o g e n , i o d i n a t e d u s i n g e n z y m o b e a d s , was c h r o m a t o g r a p h e d - 1 5 2 -on S e p h a d e x G150 a n d t h e n d i a l y s e d t o r e m o v e f r e e i o d i d e . The b u f f e r s y s t e m was P B S / a z i d e pH 7.4 w i t h 1 0 mM b e n z a m i d i n e - H C l a n d 2 5 u n i t s / m L T r a s y l o l . The d i a l y s i s was c a r r i e d o u t a t 4°C a g a i n s t t w o 5 0 0 : 1 c h a n g e s o f b u f f e r . The d i a l y s e d a d s o r b a t e was i n c u b a t e d w i t h P B S w a s h e d r e d c e l l s f o r 1 h o u r a t ro o m t e m p e r a t u r e a n d a s u s p e n s i o n h e m a t o c r i t o f 8 0 % . R a d i o l a b e l b i n d i n g a n d w a s h o u t was t h e same f o r f r e s h a n d s t o r e d ( 8 d a y s a t 4°C) c e l l s . C e l l p e l l e t s w e r e i s o l a t e d b y c e n t r i f u g a t i o n f o r 10 m i n u t e s a t 1 5 , 0 0 0 x g . A s i x c y c l e w ash p r o c e d u r e w i t h y &c s ^ 0 . 2 0 , ^ ^ 0 . 8 7 a n d t r a n s f e r s t o c l e a n t u b e s a f t e r t h e f i r s t , s e c o n d , a n d s i x t h w a s h e s was u s e d t o r e m o v e a c t i v i t y t r a p p e d i n t h e p e l l e t s . R ed c e l l m embranes (RBCM) w e r e p r e p a r e d u s i n g a m e t h o d m o d i f i e d f r o m F a i r b a n k s e t a l . ( 1 9 7 1 ) . Two w a s h e s w i t h 5mM p h o s p h a t e pH 8 ( 5 P 8 ) a t c e l l t o w a s h v o l u m e s o f 1:25 w e r e s u f f i c i e n t t o r e m o v e m o s t o f t h e c e l l h e m o g l o b i n . S t a n d a r d 3 % SDS-PAGE a n a l y s e s w e r e c a r r i e d o u t u n d e r b o t h n a t i v e a n d r e d u c i n g c o n d i t i o n s . The w a s h o u t c u r v e s , F i g u r e 3 . 1 8 , show t h a t t r a p p e d r a d i o l a b e l 1 2 5 was r e m o v e d i n t h e f i r s t t h r e e w a s h e s . The I - f i b r i n o g e n a c t i v i t y P e q u i l i b r a t e d w i t h t h e c e l l s was 1.3 x 1 0 CPM/g o f c e l l s . T h i s was 5 r e d u c e d t o 1.6 x 10 CPM/g o f c e l l s a f t e r t h e s i x t h P B S w a s h . The 0.1 % r e t e n t i o n o f a d s o r b a t e was r e p r e s e n t a t i v e o f m o s t b i n d i n g e x p e r i m e n t s ; a p p a r e n t b i n d i n g u s u a l l y r e p r e s e n t e d a b o u t 1 % o f t h e a d s o r b a t e a c t i v i t y a n d t h e b o u n d c o m p o n e n t was a b o u t 1 0 % o f t h e a p p a r e n t b i n d i n g . The t w o l y s i n g w a s h e s r e d u c e d t h e a c t i v i t y p e r g r a m t o 1.3 x 1 04 CPM/g o f RBCM p e l l e t a n d t h e c a r r y - o v e r a c t i v i t y t o l e s s t h a n 1 6 0 CPM/g. The r a d i o l a b e l d i s t r i b u t i o n s f o r RBCM w e r e b a s e d o n t o t a l a c t i v i t i e s o f 1 1 0 0 CPM r u n o n 5 s e p a r a t e g e l s . T h e s e w e r e s l i c e d - 153 -10' H 1 1 1 1 H H r-3 . 10 7 Q_ < 3 10 5 D RBCM 1/2 • + 10' H r-RBCM 6/2 D 0 W A S H N U M B E R 10 F i g u r e 3 . 1 8 Wash a n a l y s i s t o r e c o v e r b o u n d r a d i o l a b e l f o r 3 % SDS-PAGE a n a l y s i s . C o u n t s p e r m i n u t e p e r g r a m o f p e l l e t • , f r e s h c e l l s + — + , s t o r e d c e l l s O O , a n d RBCM o a . - 154 -a n d c o r r e s p o n d i n g s l i c e s w e r e p o o l e d f o r a c t i v i t y d e t e r m i n a t i o n s . O f t h e 1 1 0 0 CPM, l e s s t h a n 1 5 CPM c a n b e a t t r i b u t e d t o c a r r y - o v e r i f t h e s a m p l e s t a k e n w e r e h o m o g e n e o u s . Some e v i d e n c e f o r i n h o m o g e n e i t y e x i s t e d a s t h e r a d i o l a b e l r e c o v e r y i n t h e c a s e o f t h e r e d u c e d a n a l y s i s was a b o u t 1 6 0 % . H o w e v e r , t h e maximum c a r r y - o v e r i n t h e w h o l e o f t h e membrane p r e p a r a t i o n was 1 4 0 CPM, s o t h a t c a r r y - o v e r c o u l d n o t a c c o u n t f o r more t h a n 1 6 % o f t h e a c t i v i t y i n t h e RBCM r a d i o l a b e l d i s t r i b u t i o n s . E s t i m a t e s f o r t h e a m o u n t s o f r a d i o l a b e l c a r r y - o v e r a r e s u m m a r i z e d i n T a b l e 3 . 1 9 . RBCM 6/2 d e s i g n a t e s r e d c e l l m embranes p r o d u c e d b y s i x i s o t o n i c P B S w a s h e s f o l l o w e d b y t w o 5 P 8 w a s h e s . TABLE 3.19 R A D I O L A B E L CARRY-OVER I N RBCM PREPARATIONS WASH P E L L E T /bp w e i g h t ( g ) p e l l e t TOTAL CPM c e l l s c a r r y - o v e r PERCENT c a r r y - o v e r 0 6 6/2 0 . 8 4 2.61 0 . 7 5 1.63 0.91 1 . 3 6 x l 08 1.73 1 05 1.18 1 04 1 . 7 i x l 05 1 8 8 0 1 4 0 i i i 1.2 I n F i g u r e s 3 . 1 9 t h e 3 % SDS-PAGE d i s t r i b u t i o n s o f 1 2 5 I - f i b r i n o g e n a d s o r b a t e a n d s u p e r n a t a n t a r e s h o w n f o r b o t h n a t i v e a n d r e d u c e d m a t e r i a l . I n F i g u r e 3 . 20 t h e n a t i v e a n d r e d u c e d d i s t r i b u t i o n s f o r t h e 6/2 RBCM r a d i o l a b e l a n d C o o m a s s i e B l u e s t a i n i n g a r e s h o w n . The i n i t i a l p u r i t y o f t h i s p r e p a r a t i o n was e s t i m a t e d t o - 1 5 5 -O CD Q_ O in O CU I n Q_ R E L A T I V E M O B I L I T Y F i g u r e 3 . 1 9 N a t i v e a n d r e d u c e d • • • • r a d i o l a b e l d i s t r i b u t i o n s o n 3 % SDS-PAGE. a ) 1 2 5I - f i b r i n o g e n a d s o r b a t e . b ) 1 2 5I - f i b r i n o g e n s u p e r n a t a n t . T a b l e 3 . 2 0 l i s t s t h e p e r c e n t a c t i v i t y p e r z o n e . E c LO CD ID Q O R E L A T I V E M O B I L I T Y F i g u r e 3 . 20 RBCM a n a l y s e d o n 3 % SDS-PAGE : n a t i v e a n d r e d u c i n g c o n d i t i o n s . a ) RBCM r a d i o l a b e l d i s t r i b u t i o n a f t e r s i x PBS w a s h e s a n d t w o 5 P 8 w a s h e s , b ) C o r r e s p o n d i n g C o o m a s s i e B l u e s t a i n i n g o f RBCM. - 157 -h a v e b e e n a b o u t 8 8 % f r o m t h e r e d u c e d r a d i o l a b e l d i s t r i b u t i o n , T a b l e 3 . 2 0 , z o n e s 4 a n d 5 . The s u p e r n a t a n t a n a l y s i s i m p l i e d a s i m i l a r l e v e l o f i m p u r i t y a n d t h a t p r o t e o l y s i s o f t h e f i b r i n o g e n o ( - c h a i n h a d o c c u r r e d d u r i n g t h e a d s o r p t i o n i n t e r v a l . A l p h a c h a i n d e g r a d a t i o n i s a n e a r l y f e a t u r e o f p l a s m i n p r o t e o l y s i s o f f i b r i n o g e n ( M o s e s s o n e t a l . 1 9 7 3 , L i p i n s k a e t a l . 1 9 7 4 ) , h a s b e e n d e m o n s t r a t e d i n f i b r i n o g e n i s o l a t e d f r o m f r e s h p l a s m a a n d i s mo r e e x t e n s i v e i n f i b r i n o g e n f r o m o u t o f d a t e b l o o d a n d i n s t o r e d p r e p a r a t i o n s ( S e m e r a r o e t a l . 1 9 7 7 ) . The r a d i o l a b e l e d f r a g m e n t s o f z o n e 7 a r e o f l e s s t h a n 3 0 , 0 0 0 m o l e c u l a r w e i g h t a n d t h u s f r a g m e n t s r e f e r r e d t o a s A c x 7 ^n a p p e a r t o be t h e l a r g e s t p r e s e n t ( G a l a n a k i s e t a l . 1 9 7 8 , W e i n s t e i n a n d D e y k i n 1 9 7 8 ) . W i t h t h e e x c e p t i o n o f t h e r a d i o l a b e l a t r e l a t i v e m o b i l i t i e s l e s s t h a n 0 . 0 2 a n d t h e r e f o r e a s s o c i a t e d w i t h p r o t e i n a g g r e g a t e s i n e x c e s s o f 1 06 d a l t o n s , F i g u r e 3 . 2 0 a s h o w e d t h a t t h e n a t i v e a n d r e d u c e d r a d i o l a b e l p e a k s a s s o c i a t e d w i t h RBCM i d e n t i f i e d t h e a s s o c i a t e d p r o t e i n a s f i b r i n o g e n . I n a d d i t i o n , t h e m o l e c u l a r w e i g h t s o f t h e p e a k s w e r e n o r m a l f o r f i b r i n o g e n i n d i c a t i n g t h a t t h e ' i r r e v e r s i b l e ' b i n d i n g o b s e r v e d w h i l e w a s h i n g w i t h P B S b u f f e r was r e v e r s e d o r d i d n o t i n v o l v e l a r g e p r o t e i n s p e c i e s o f t h e c e l l m e m b r a n e . F i g u r e 3 . 2 0 b s h o w e d t h a t t h e s h i f t i n t h e r a d i o l a b e l p e a k w i t h r e d u c t i o n c o u l d n o t be a t t r i b u t e d t o a g r o s s s h i f t o f t h e membrane p r o t e i n s u p o n r e d u c t i o n . The a n a l y s i s a l s o s u g g e s t e d t h a t cX - c h a i n f r a g m e n t s w e r e p r e s e n t i n t h e r e t a i n e d r a d i o l a b e l . The p e r c e n t r a d i o l a b e l o f z o n e 7 was s i m i l a r f o r t h e s u p e r n a t a n t a n d RBCM a n a l y s e s s u g g e s t i n g t h a t t h e r e was n o p r e f e r e n c e f o r b i n d i n g o f t h e s e f r a g m e n t s o v e r t h e r e m a i n d e r o f t h e f i b r i n o g e n m o l e c u l e . T h u s t h e r a d i o l a b e l r e t a i n e d b y c e l l s a f t e r e x t e n s i v e PBS TABLE 3 . 2 0 RADIOLABEL D I S T R I B U T I O N I N 3 % SDS-PAGE A N A L Y S I S OF 12 5I - F I B R I N 0 G E N AND RBCM PERCENT OF TOTAL COUNTS a SAMPLE a d s o r b a t e s u p e r n a t a n t RBCM 1/2 RBCM 6/2 n r n r n r n r ZONE 1 6.7 0.2 2.7 0.1 2 5 . 0 + 1.0 3.2 + 0 . 3 3 2 . 8 + 2 . 5 8 . 4 + 0 . 8 2 9 0 . 6 2.2 7 6 . 8 1.0 4 6 . 8 1.5 3.4 0 . 3 3 3 . 2 2 . 5 7.7 0 . 7 3 0.4 7.0 1.0 3.9 4.8 0.4 7.4 0.6 8.0 1.2 1 6 . 4 1.2 4 0.2 7 7 . 3 0.5 54.1 2.1 0.3 4 5 . 9 1.5 4.0 0 . 9 2 6 . 5 1.6 5 0.3 1 0 . 7 0.4 1 8 . 3 1.6 0.3 1 4 . 8 0 . 8 2 . 3 0.6 7.9 0 . 8 6 0.9 1.9 1.0 3.2 6.3 0.6 1 1 . 6 0.7 4.5 0 . 8 1 2 . 2 0.9 7 1.0 0 . 8 17.4 1 9 . 2 1 3 . 4 0.7 1 3 . 9 + 0 . 8 14.1 1.6 1 8 . 8 1.4 0.2 0.3 0.1 + 0.1 . . . . 1.6 + 0.5 2 . 3 + 0 . 4 TOTAL 5.87 6.20 6.12 5.85 x l O5 2 9 2 0 3 1 1 0 8 8 0 1 8 6 0 a ) mean + o n e s t a n d a r d d e v i a t i o n ( P o i s s o n s t a t i s t i c s ) n : n a l i v e ( S D S ) r : r e d u c i n g ( S D S ) - 1 5 9 -w a s h i n g r e p r e s e n t e d , i n l a r g e p a r t , f i b r i n o g e n r a t h e r t h a n a n u n i d e n t i f i e d i m p u r i t i e s i n f i b r i n o g e n p r e p a r a t i o n s . H o w e v e r , s i n c e b u f f e r w a s h e d r e d c e l l s do n o t f o r m r o u l e a u x , t h e f i b r i n o g e n b o u n d a f t e r w a s h i n g t h e c e l l s c a n n o t r e p r e s e n t t h e p r o t e i n r e s p o n s i b l e f o r a g g r e g a t i o n o f t h e c e l l s . b ) A g g r e g a t e d v e r s u s m o n o m e r i c f i b r i n o g e n b i n d i n g F i b r i n o g e n a g g r e g a t e s w e r e o n e o f t h e c o n t a m i n a n t s f o u n d i n f i b r i n o g e n p r e p a r a t i o n s , F i g u r e 3 . 5 a . The f o r m a t i o n o f t h e s e a g g r e g a t e s was e n h a n c e d b y l y o p h i l i z a t i o n a n d / o r s t o r a g e . An h y p o t h e s i s p r o p o s e d t o e x p l a i n t h e t w o c o m p o n e n t wash c u r v e s s t a t e d t h a t t h e b o u n d c o m p o n e n t was a s s o c i a t e d w i t h a g g r e g a t e d f i b r i n o g e n w h i l e t h e e l u t e d c o m p o n e n t was a s s o c i a t e d w i t h monomer f i b r i n o g e n ( B r o o k s e t a l . 1 9 8 0 ) . The f o l l o w i n g e x p e r i m e n t s h o w e d t h a t t h e d i f f e r e n c e i n t h e a m o u n t s o f a g g r e g a t e a n d monomer f i b r i n o g e n b o u n d p e r g r a m o f c e l l s w e r e t o o s m a l l f o r t h i s t o - b e t h e c a s e . F i b r i n o g e n i s o l a t i o n s w h i c h c o n t a i n e d h i g h p r o p o r t i o n s o f r e d u c i b l e , a g g r e g a t e d f i b r i n o g e n w e r e i d e n t i f i e d b y 3 % SDS-PAGE a n a l y s i s . The a g g r e g a t e d a n d monomer c o m p o n e n t s w e r e s e p a r a b l e b y c h r o m a t o g r a p h y o n S e p h a c r y l S 3 0 0 , F i g u r e 3 . 2 1 a . I s o l a t i o n 18 was r a d i o l a b e l e d a n d c h r o m a t o g r a p h e d o n S e p h a c r y l S 3 0 0 t o s e p a r a t e t h e a g g r e g a t e d a n d monomer f r a c t i o n s . F i g u r e 3 . 2 1b s h o w s t h e C o o m a s s i e B l u e s t a i n i n g p r o f i l e s o f f r a c t i o n s 5 1 t h r o u g h 56 o f t h e S e p h a c r y l s e p a r a t i o n ( l a n e s 1 - 6 ) , t h e s t a r t i n g m a t e r i a l ( l a n e 7 ) , a n d a BDH a l b u m i n m o l e c u l a r w e i g h t s t a n d a r d ( l a n e 8 ) . The 3 % SDS-PAGE r a d i o l a b e l d i s t r i b u t i o n s o f t h e t w o f r a c t i o n s a r e shown i n F i g u r e 3 . 2 2 a . The monomer p o o l was 8 0 % monomer a n d t h e a g g r e g a t e d - 160 -ELUTION V O L U M E (mL) 120 Figure 3.21 Separation of aggregate and monomer fibrinogen. a) Preparative separation on Sephacryl S300, protein by OD28O » Blue Dextran 2000 ••• b) Three percent SDS-PAGE analysis of selected fractions. - 161 -R E L A T I V E M O B I L I T Y 0 W A S H N U M B E R F i g u r e 3 . 2 2 C o m p a r i s o n o f a g g r e g a t e f i b r i n o g e n b i n d i n g t o human R B C . • a n d monomer a ) R a d i o l a b e l d i s t r i b u t i o n " o f a d s o r b a t e s o n 3 % SDS-PAGE. b ) a n a l y s e s o f c e l l p e l l e t s a f t e r u l t r a c e n t r i f u g a t i o n . Wash - 1 6 2 -p o o l was 7 5 % m u l t i m e r . T h e s e p o o l s w e r e t h e n a d s o r b e d w i t h r e d c e l l s , a n d c e l l p e l l e t s w e r e i s o l a t e d b y u l t r a c e n t r i f u g a t i o n . Wash a n a l y s e s w e r e c a r r i e d o u t t o s e e i f t h e wash c u r v e s w e r e i n f l u e n c e d by t h e d i f f e r e n t p r o p o r t i o n s o f a g g r e g a t e d a n d monomer f i b r i n o g e n s i n t h e c h r o m a t o g r a p h e d p r e p a r a t i o n s . The w a s h a n a l y s e s , F i g u r e 3 . 2 2 b , show t h e f i t t e d e x p o n e n t i a l m o d e l s a n d t h e a p p a r e n t b i n d i n g i n g r a m s p r o t e i n p e r g r a m o f r e d c e l l s . T he r e s u l t s a r e s u m m a r i z e d i n T a b l e 3.21 . The s p e c i f i c a c t i v i t i e s o f t h e a g g r e g a t e d a n d monomer f r a c t i o n s w e r e 8 8 2 . 0 9 x 1 0 a n d 1 . 8 6 x 1 0 CPM/mg r e s p e c t i v e l y . P r o t e i n was d e t e r m i n e d b y t h e L o w r y m e t h o d ( L o w r y e t a l . 1 9 5 1 ) . T h e w a s h a n a l y s e s i n d i c a t e d t h a t t h e a m o u n t o f t h e b o u n d c o m p o n e n t ( a - ^ ' ) , o f t h e a g g r e g a t e d f r a c t i o n , was l a r g e r ( 1 1 4 % , t t e s t , p = 0 . 0 0 2 ) t h a n t h e c o r r e s p o n d i n g c o m p o n e n t o f t h e monomer f r a c t i o n . The a m o u n t o f e l u t i n g c o m p o n e n t ( a2' ) , o f t h e a g g r e g a t e d f r a c t i o n was a l s o l a r g e r ( 1 2 8 % , t t e s t , p = 0 . 0 2 9 ) t h a n t h e c o r r e s p o n d i n g monomer c o m p o n e n t . The w a s h c u r v e s f o r b o t h o f t h e p r e p a r a t i o n s s h o w e d t h a t a t l e a s t t w o c o m p o n e n t s w e r e p r e s e n t . H o w e v e r , b e c a u s e o f t h e c o m p o s i t i o n o f t h e p r e p a r a t i o n s , t h e p r o p o s e d h y p o t h e s i s i m p l i e d t h a t o n e c o m p o n e n t s h o u l d p r e d o m i n a t e i n o n e c u r v e a n d t h e o t h e r s h o u l d p r e d o m i n a t e i n t h e s e c o n d c u r v e . T h u s , a s b o t h o f t h e i n t e r c e p t s f o r t h e a g g r e g a t e e n r i c h e d a d s o r b a t e w e r e h i g h e r t h a n t h o s e o f t h e monomer a d s o r b a t e , t h e w a s h a n a l y s e s d i d n o t d i s t i n g u i s h w h i c h c o m p o n e n t , a g g r e g a t e d o r monomer, s h o u l d b e a s s o c i a t e d w i t h e i t h e r t h e b o u n d o r t h e e l u t e d r a d i o l a b e l . T h u s a m o d e l w h e r e a g g r e g a t e d f i b r i n o g e n s p e c i e s w o u l d a c c o u n t f o r o n e c o m p o n e n t o f a wash c u r v e w h i l e monomer f i b r i n o g e n - 1 6 3 -T a b l e 3.21 COMPARISON OF AGGREGATE AND MONOMER B I N D I N G BY WASH A N A L Y S I S PERCENT R A D I O L A B E L D I S T R I B U T I O N ON 3 % SDS-PAGE ZONE 1 FRACTIONS 1-5 a g g r e g a t e 4 5 . 5 monomer 7.0 2 3 4 5-11 1 2 - 1 9 2 0 - 2 5 2 9 . 0 2 3 . 2 0 . 4 1 0 . 2 7 9 . 5 0.6 5 6 7 2 6 - 3 9 4 0 - 5 9 6 0 - 6 5 0.4 1.5 0.1 0.5 2.1 0.1 BI N D I N G A N A L Y S I S AGGREGATE MONOMER APPARENT B I N D I N G ; (Q) 0 . 0 1 3 1 0 . 0 1 0 9 WASH A N A L Y S I S ; b i n d i n g ( u g / g c e l l ) 0 2 . 3 2 1.90 1 0 . 6 6 5 0 . 5 9 7 2 0 . 3 1 8 0 . 3 0 3 3 0 . 2 1 6 0 . 2 0 6 4 0 . 1 7 3 0 . 1 7 3 5 0 . 1 5 9 0 . 1 6 2 6 0 . 1 4 8 0 . 1 5 3 7 0 . 1 3 7 0 . 1 4 4 WASH CURVE PARAMETERS / a c s x l O O a 1 9 . 6 + 1.0 1 8 . 8 + 1.1 /3>p x l O O 8 9 . 4 + 2.8 8 9 . 6 + 2.4 a2 b 1.64 + 0 . 0 5 0 1.35 + 0 . 0 2 6 al 0 . 2 1 2 +_ 0 . 0 1 2 0 . 1 8 7 + 0 . 0 0 7 - 1 . 2 6 2 + 0 . 0 4 5 - 1 . 1 7 5 + 0 . 0 3 0 * 1 - 0 . 0 6 2 + 0 . 0 1 0 - 0 . 0 3 6 + 0 . 0 0 7 a2' c 2 . 1 3 + 0.37 1.66 + 0 . 2 3 0 . 2 1 6 + 0 . 0 1 2 0 . 1 8 9 + 0 . 0 0 7 a ) mean _+ o n e s t a n d a r d d e v i a t i o n b ) mean + e s t i m a t e d s t a n d a r d d e v i a t i o n , A p p e n d i x 1 6 c ) mean +_ rms p r o p a g a t e d s t a n d a r d d e v i a t i o n - 1 64 -a c c o u n t e d f o r t h e o t h e r was r e j e c t e d . The o b s e r v e d r e s u l t s w e r e c o m p a t i b l e w i t h t h e i n t r a c e l l u l a r - r a d i o i o d i d e / b o u n d - r a d i o l a b e l m o d e l . The b i n d i n g r e p r e s e n t e d b y t h e i n t e r c e p t a ^ ' i n c r e a s e d a s a r e s u l t o f e n r i c h m e n t o f t h e a g g r e g a t e d s p e c i e s . The r e s u l t s o f s e c t i o n 3 . 5 . i n d i c a t e t h a t s u r f a c e a d s o r p t i o n u n d e r t h e s e c o n d i t i o n s was p r o p o r t i o n a l t o t h e s o l u t i o n c o n c e n t r a t i o n o f t h e p r o t e i n s p e c i e s . W i t h t h i s a s s u m e d a n d t h e a-^' i n t e r c e p t t a k e n t o r e p r e s e n t t h e sum o f t h e a d s o r b e d a g g r e g a t e d a n d monomer s p e c i e s , w h i l e t h e r e l a t i v e s o l u t i o n c o n c e n t r a t i o n s o f t h e s p e c i e s w e r e g i v e n by t h e PAGE a n a l y s i s , t h e a m o unt a d s o r b e d p e r u n i t c o n c e n t r a t i o n f o r o n e s p e c i e s r e l a t i v e t o t h e o t h e r , was c a l c u l a t e d . From t h i s e x p e r i m e n t , a d s o r p t i o n o f t h e a g g r e g a t e d s p e c i e s was 1 2 5 % o f t h a t o f t h e monomer s p e c i e s . A t h e o r e t i c a l b a s i s u p o n w h i c h t h e b i n d i n g o f a p o l y m e r t o a s u r f a c e w o u l d b e d e p e n d e n t u p o n t h e number o f p o l y m e r s e g m e n t s h a s b e e n p r o p o s e d ( S i l b e r b e r g 1 9 7 0 ) . E x p e r i m e n t a l e v i d e n c e f o r p r e f e r e n t i a l a d s o r p t i o n o f p o l y m e r i c a l b u m i n t o p o l y s t y r e n e h a s b e e n r e p o r t e d ( G r e i g a n d B r o o k s 1 9 8 1 ) . c ) D e s o r p t i o n i n p r o t e i n - c o n t a i n i n g w a s h m e d i a The b o u n d c o m p o n e n t o f t h e r a d i o l a b e l i n u l t r a c e n t r i f u g e d c e l l p e l l e t s was shown t o r e p r e s e n t t h e c o m p o s i t i o n o f t h e p r o t e i n p r e p a r a t i o n a d s o r b e d w i t h t h e c e l l s . I n P B S w a s h a n a l y s e s , t h i s r a d i o l a b e l was e s s e n t i a l l y i r r e v e r s i b l y a s s o c i a t e d w i t h t h e c e l l s . H o w e v e r , i n w a s h a n a l y s e s w h e r e p r o t e i n was p r e s e n t i n t h e w a s h m e d i a , t h e r e was a f u r t h e r r e d u c t i o n i n t h e r a d i o l a b e l r e t a i n e d b y t h e c e l l s . A s s h own i n F i g u r e 3 . 2 3 a , w a s h i n g w i t h p l a s m a , 3.2 _+ 0.1 mg/mL f i b r i n o g e n by R I D , r e d u c e d t h e b o u n d r a d i o l a b e l t o a b o u t o n e - t e n t h o f - 1 6 5 -t h a t f o u n d a f t e r w a s h i n g w i t h P B S . The e x p e r i m e n t s s h o w n i n F i g u r e 3 . 2 3b r e p e a t e d t h e c o m p a r i s o n o f P B S a n d p l a s m a w a s h i n g , 3.0 +_ 0.1 mg/mL f i b r i n o g e n b y R I D . I n a d d i t i o n , t h r o m b i n - c l o t t e d p l a s m a , 0 . 5 3 +_ 0 . 0 3 mg/mL f i b r i n o g e n b y R I D , r e s u l t e d i n a w a s h c u r v e v e r y s i m i l a r t o t h a t p r o d u c e d b y p l a s m a w a s h i n g . T h e s e o b s e r v a t i o n s i n d i c a t e t h a t 9 0 % o f t h e r a d i o l a b e l p r e s e n t i n r e d c e l l p e l l e t s a f t e r e x t e n s i v e P B S w a s h i n g was a s s o c i a t e d w i t h t h e c e l l s u r f a c e a n d t h a t t h e d e s o r p t i o n was p r o b a b l y n o t d e p e n d e n t u p o n t h e s p e c i e s i n s o l u t i o n . W a s h i n g w i t h 1 mg/mL a l b u m i n a l s o d e c r e a s e d b i n d i n g r e l a t i v e t o P B S , b u t t o a l e s s e r e x t e n t t h a n t h a t o b s e r v e d w i t h p l a s m a . The d i f f e r e n c e s b e t w e e n t h e c e l l - r e t a i n e d a c t i v i t y f o r P B S a n d p r o t e i n m e d i a w a s h e s a r e s u m m a r i z e d i n T a b l e 3 . 2 2 . E x c e p t f o r t h e e x p e r i m e n t w h e r e a l b u m i n was u s e d i n t h e w a s h m e d i a , t h e c o n s t a n c y o f t h e d i f f e r e n c e s b e t w e e n t h e P B S a n d p l a s m a w a s h c u r v e s i n d i c a t e d t h a t d e s o r p t i o n i n d u c e d b y p r o t e i n was c o m p l e t e i n t h e f i r s t w a s h . I n t h e c a s e o f t h e a l b u m i n e x p e r i m e n t , t h e i n i t i a l d e s o r p t i o n was a b o u t o n e - t h i r d o f t h a t o b s e r v e d f o r p l a s m a o r s e r u m , t h u s t h e d e s o r p t i o n was d e p e n d e n t o n t h e c o n c e n t r a t i o n o f t h e p r o t e i n i n t h e m e d i a . The a l b u m i n e x p e r i m e n t a l s o i n d i c a t e d t h a t when t h e p r o t e i n c o n c e n t r a t i o n i n t h e w a s h m e d i a was l o w , t h e d e s o r p t i o n p r o c e s s was t i m e d e p e n d e n t . The r a d i o l a b e l r e t a i n e d a f t e r p l a s m a w a s h i n g was n o t e n t i r e l y a c c o u n t e d f o r b y b i n d i n g a s s o c i a t e d w i t h r a d i o i o d i d e , s e c t i o n 3 . 3 . 3 . The a c t i v i t y r e t a i n e d i n e x p e r i m e n t 5 3 was 5 6 0 CPM/g a t w a s h s i x ( 9 9 0 CPM/g a t t h e i n t e r c e p t , w a s h z e r o ) , w h e r e a s t h e f r e e r a d i o l a b e l r e t e n t i o n was e s t i m a t e d a t b e t w e e n 6 0 a n d 3 0 0 CPM/g . T h i s r a n g e was F i g u r e 3 . 2 3 I n f l u e n c e o f p r o t e i n on f i b r i n o g e n w a s h c u r v e s , a ) A d s o r p t i o n 5 3 : T h e w a s h m e d i a w e r e P B S b u f f e r f o l l o w e d by p l a s m a ( 1 a n d 3 ) a n d p l a s m a ( 2 ) . b ) A d s o r p t i o n 5 4 : The w ash m e d i a w e r e PBS b u f f e r f o l l o w e d b y p l a s m a ( 1 ) , P B S + a l b u m i n ( 2 ) , p l a s m a ( 3 ) , a n d c l o t t e d - p l a s m a ( 4 ) . Wash c u r v e 5 s h o w s t h e e l u t i o n o f t h e f r e e l a b e l p e a k o f t h e G 1 5 0 S e p h a d e x a d s o r b a t e p r e p a r a t i o n by P B S b u f f e r . - 1 6 7 -TABLE 3 . 2 2 D I F F E R E N C E S BETWEEN RETAINED A C T I V I T I E S FOR D I F F E R E N T WASH MEDIA EXPERIMENT 5 3 54 54 54 S E R I E S a ( 1 - 2 ) ( 1 - 3 ) ( 1 - 4 ) ( 1 - 2 ) CPM/g WASH 1 4 0 1 0 4 8 5 0 4 7 2 0 1 5 5 0 2 4 3 8 0 4 7 1 0 4 7 9 0 1 5 7 0 3 4 2 9 0 4 8 0 0 4 8 3 0 1 9 7 0 4 4 1 5 0 4 7 2 0 4 7 1 0 2 1 9 0 5 4 1 8 0 4 5 8 0 4 5 6 0 2 2 8 0 a ) 5 3 ( 1 - 2 ) : P B S -. p l a s m a , 5 4 ( 1 - 3 ) : P B S - c l o t t e d - p l a s m a , 5 4 ( 1 - 4 ) : P B S - p l a s m a , 5 4 ( 1 - 2 ) : P B S - a l b u m i n ( 1 mg/mL). c a l c u l a t e d f r o m t h e s u p e r n a t a n t f r e e l a b e l a c t i v i t y a t wash z e r o a n d a n e s t i m a t e d f r e e l a b e l r e t e n t i o n o f b e t w e e n 0 . 0 0 1 a n d 0 . 0 0 5 . T h e s e f a c t o r s r e p r e s e n t e d t h e r e t e n t i o n o f r e s p e c t i v e l y r a d i o i o d i d e i n F i g u r e 3 . 1 6 a n d o f f r e e r a d i o l a b e l i n F i g u r e 3 . 2 3 b , e x p e r i m e n t 5 4 / 4 . I n e x p e r i m e n t 54 t h e f r e e r a d i o l a b e l r e t e n t i o n c a l c u l a t e d f o r t h e p l a s m a w a s h e x p e r i m e n t was 1 0 0 CPM/g, u s i n g t h e f a c t o r 0 . 0 0 5 , w h e r e a s a t w a s h s i x t h e r e t a i n e d a c t i v i t y was 6 2 0 CPM/g ( 1 0 0 0 CPM/g a t wash z e r o ) . T h u s t h e r e t a i n e d r a d i o l a b e l was b e t w e e n t w o a n d t e n t i m e s g r e a t e r t h a n t h e e s t i m a t e d a m o u n t s o f f r e e r a d i o l a b e l . The r a d i o l a b e l e d s p e c i e s r e s p o n s i b l e f o r t h e r e s i d u a l b i n d i n g h a v e n o t b e e n i d e n t i f i e d . - 1 6 8 -T h e s e o b s e r v a t i o n s i m p l i e d t h a t d e s o r p t i o n o f s u r f a c e a d s o r b e d f i b r i n o g e n d e p e n d e d u p o n t h e f r e e p r o t e i n i n s o l u t i o n . T h i s s u g g e s t s a d e s o r p t i o n m e c h a n i s m w h e r e a m o l e c u l e i n t h e s o l u t i o n d i s p l a c e s a s u r f a c e a d s o r b e d m o l e c u l e . A s i m i l a r m e c h a n i s m h a s b e e n p r o p o s e d t o e x p l a i n d e s o r p t i o n o f p r o t e i n s f r o m man-made m a t e r i a l s ( B r a s h a n d Samak 1 9 7 8 , C h a n a n d B r a s h 1 9 8 0 ) . B r a s h a n d Samak ( 1 9 7 8 ) s h o w e d t h a t a l b u m i n a d s o r b e d t o p o l y e t h y l e n e was q u a n t i t a t i v e l y e x c h a n g e d w i t h a l b u m i n i n s o l u t i o n . I n b o t h t h e s e s t u d i e s , t h e e x t e n t o f e x c h a n g e i n c r e a s e d w i t h p r o t e i n c o n c e n t r a t i o n . The t i m e r e q u i r e d t o e x c h a n g e 5 0 % o f t h e a d s o r b e d p r o t e i n was m e a s u r e d i n h o u r s s o t h a t t h e e x c h a n g e o b s e r v e d h e r e was much f a s t e r . The d i f f e r e n c e may h a v e b e e n d u e t o t h e h i g h e r p r o t e i n c o n c e n t r a t i o n s u s e d i n e x p e r i m e n t s r e p o r t e d h e r e . The d i s p l a c e m e n t m e c h a n i s m s u p p o s e s c l o s e i n t e r a c t i o n b e t w e e n s u r f a c e - a d s o r b e d a n d f r e e m o l e c u l e s . T h i s f o l l o w s f r o m t h e o b s e r v a t i o n t h a t s u r f a c e - a d s o r b e d p r o t e i n d e s o r b e d o n l y i n t h e p r e s e n c e o f f r e e p r o t e i n . P r e s u m a b l y a n a d s o r b e d m o l e c u l e i s p r o g r e s s i v e l y d i s p l a c e d b y a s e c o n d m o l e c u l e a t t h e s u r f a c e l o c a t i o n s r e s p o n s i b l e f o r i t s a d s o r p t i o n . When a c e r t a i n f r a c t i o n o f t h e s e ' c o n t a c t s ' a r e b r o k e n , d e s o r p t i o n i s c o m p l e t e . The m e c h a n i s m i m p l i e s t h a t a s u r f a c e a d s o r b e d m o l e c u l e i s i n c o n t a c t w i t h more t h a n one l o c a t i o n o f a s u r f a c e . I n t e r a c t i o n o f p r o t e i n m o l e c u l e s w i t h man-made m a t e r i a l s a t m o r e t h a n o n e r e s i d u e i s s u p p o r t e d b y e v i d e n c e o f c o n f o r m a t i o n a l c h a n g e s i n a d s o r b e d p r o t e i n s ( S o d e r q u i s t a n d W a l t o n 1 9 8 0 , C h a n a n d B r a s h 1 9 8 1 ) , o f s u r f a c e d i f f u s i o n o f a d s o r b e d p r o t e i n s ( M i c h a e l i e t a l . 1 9 8 0 ) , a n d by e s t i m a t e s o f t h e n u m b er o f c a r b o n y l m o i e t i e s p e r m o l e c u l e i n v o l v e d i n p r o t e i n a d s o r p t i o n ( M o r r i s s e y a n d S t r o m b e r g 1 9 7 3 ) . The s u r f a c e - 1 6 9 -a d s o r p t i o n o b s e r v e d b y B r a s h a n d Samak ( 1 9 7 8 ) a n d by C h a n a n d B r a s h ( 1 9 8 0 ) was e s t i m a t e d t o r e p r e s e n t a m o n o l a y e r o f p r o t e i n a t t h e a d s o r b i n g s u r f a c e . H o w e v e r , t h e a m o u n t s o f p r o t e i n b o u n d t o r e d c e l l s w e r e f a r s h o r t o f m o n o l a y e r c o v e r a g e , s e c t i o n 3 . 5 . 5 . T h u s , i f t h e e x c h a n g e m e c h a n i s m i s a c c e p t e d , i t a p p e a r s t h a t a l a r g e f r a c t i o n o f t h e r e d c e l l s u r f a c e i s n o t i n v o l v e d i n t h e b i n d i n g r e p r e s e n t e d by t h e r a d i o l a b e l r e t a i n e d a f t e r w a s h i n g c e l l s w i t h b u f f e r . 3 . 3 . 6 Summary Wash a n a l y s e s c o n f i r m e d t h a t a p p a r e n t b i n d i n g r e p r e s e n t e d i n t r a - a n d i n t e r c e l l u l a r c o m p o n e n t s a s w e l l a s s u r f a c e a d s o r p t i o n . The i n t r a c e l l u l a r c o m p o n e n t was i d e n t i f i e d a s r a d i o i o d i d e a n d i n m o s t e x p e r i m e n t s was t h e d o m i n a n t c o n t r i b u t i o n t o t h e a p p a r e n t b i n d i n g i n c e l l p e l l e t s p r o d u c e d b y u l t r a c e n t r i f u g a t i o n . U l t r a c e n t r i f u g a t i o n s u b s t a n t i a l l y r e d u c e d t h e i n t e r c e l l u l a r c o m p o n e n t , w h i l e t h e b o u n d c o m p o n e n t was r e d u c e d b y l e s s t h a n 10 % . The amount o f f r e e r a d i o i o d i d e a s s o c i a t e d w i t h c e l l s was d e t e r m i n e d f r o m w a s h c u r v e s a n d c o r r e s p o n d e d t o t h e a m o u n t s d e t e r m i n e d b y c h r o m a t o g r a p h i c a n a l y s e s o f a d s o r p t i o n s u p e r n a t a n t s . T h u s t h e e l u t i n g c o m p o n e n t o f t h e w a s h c u r v e s r e p r e s e n t e d p r i m a r i l y r a d i o i o d i d e . The c o m p o n e n t t h a t r e m a i n e d b o u n d w h i l e t h e c e l l s w e r e w a s h e d w i t h P B S b u f f e r was f o u n d t o r e p r e s e n t f i b r i n o g e n i n f i b r i n o g e n a d s o r p t i o n e x p e r i m e n t s . The c e l l s u r f a c e l o c a t i o n o f t h i s c o m p o n e n t was i m p l i e d b y t h e o b s e r v a t i o n t h a t more t h a n 9 0 % o f t h i s c o m p o n e n t was e l u t e d when c e l l s w e r e w a s h e d w i t h p l a s m a . Q u a n t i t a t i v e e s t i m a t e s o f t h e amount o f p r o t e i n r e t a i n e d by c e l l s d u r i n g P B S w a s h i n g a r e s u m m a r i z e d i n s e c t i o n 3 . 5 . - 1 7 0 -3.4 SOLUTION A N A L Y S I S AND TUBE ADSORPTION The d e t e r m i n a t i o n o f s u r f a c e a d s o r p t i o n i n c e l l p e l l e t s r e q u i r e d c o r r e c t i o n s f o r i n t e r c e l l u l a r t r a p p i n g o f s u p e r n a t a n t a n d i n t r a c e l l u l a r t r a p p i n g o f f r e e r a d i o l a b e l . As a n a l t e r n a t e m e t h o d , o n e may d e t e r m i n e b i n d i n g f r o m t h e d e c r e a s e i n a d s o r b a t e c o n c e n t r a t i o n u p o n a d d i t i o n o f a d s o r b e n t . I n t h i s s y s t e m , t h e a d s o r b e n t was i n t h e f o r m o f a c e l l s u s p e n s i o n , a n d t h e r e f o r e d i l u t i o n o f t h e a d s o r b a t e h a d t o be t a k e n i n t o a c c o u n t . T h i s d i l u t i o n was r e f l e c t e d i n t h e c o m p l e m e n t a r y d i l u t i o n o f t h e c e l l s u p e n s i o n a n d was e x p r e s s e d i n t e r m s o f A -t u b e s u s e d f o r t h e i n c u b a t i o n s h a d t o be c o n s i d e r e d . S i n c e t h e a d s o r b a t e a c t i v i t e s w e r e d e t e r m i n e d o n a l i q u o t s o f t h e a d s o r b a t e b e f o r e e x p o s u r e t o t h e t u b e s , t h e s u r f a c e a d s o r p t i o n r e p r e s e n t e d b y t h e t u b e t e r m ( T ) , was s u b t r a c t e d f r o m t h e b i n d i n g e s t i m a t e S-^  . The v a l u e o f T f o r a p a r t i c u l a r e x p e r i m e n t was c a l c u l a t e d u s i n g e q u a t i o n 1 0 . 1 0 . a n d / ie . F r e e r a d i o l a b e l a n d d i l u t i o n a r e b o t h t a k e n i n t o c o n s i d e r a t i o n i n t h e b i n d i n g e s t i m a t e S , , e q u a t i o n 1 0 . 1 1 . 10 . 1 1 I n a d d i t i o n t o t h e d i l u t i o n e f f e c t s , p r o t e i n a d s o r p t i o n t o t h e 1 0 . 1 0 H e r e J4 i s t h e t u b e a r e a , T i s t h e a d s o r b e d s u r f a c e c o n c e n t r a t i o n , i s t h e s u p e r n a t a n t d e n s i t y i n g/mL, Wc i s t h e c e l l w e i g h t , a n d Cf l i s t h e s u p e r n a t a n t p r o t e i n c o n c e n t r a t i o n i n mg/mL . T h e s e - 171 -b i n d i n g e s t i m a t e s w e r e made i n B e c k m a n p o l y a l l o m e r c e n t r i f u g e t u b e s . 3.4.1 A d s o r p t i o n t o p o l y a l l o m e r t u b e s I n p r e l i m i n a r y e x p e r i m e n t s a t t e m p t s w e r e made t o d e t e r m i n e p r o t e i n b i n d i n g i n c e l l p e l l e t s w i t h o u t t r a n s f e r r i n g t h e c e l l s f r o m t h e t u b e s i n w h i c h t h e a d s o r p t i o n was c a r r i e d o u t . W i t h t h i s m e t h o d , a d s o r p t i o n t o t h e t u b e s i n t h e p r e s e n c e o f c e l l s n e e d e d t o be 1 2 5 d e t e r m i n e d . A c o m p a r i s o n was made o f I - f i b r i n o g e n a d s o r p t i o n t o p o l y a l l o m e r c e n t r i f u g e t u b e s w i t h a n d w i t h o u t p r e v i o u s e x p o s u r e t o p r o t e i n o r c e l l s a n d s u b s e q u e n t t o w a s h a n a l y s i s w i t h a n d w i t h o u t c e l l s p r e s e n t . 1 2 5 B i n d i n g o f I - f i b r i n o g e n t o p o l y a l l o m e r c e n t r i f u g e t u b e s was c o m p a r e d a f t e r p r e - a d s o r p t i o n o f t u b e s w i t h e i t h e r f i b r i n o g e n o r r e d c e l l s i n 3 - ( N - m o r p h o l i n o ) p r o p a n e s u l p h o n i c a c i d (MOPS) b u f f e r ( 1 5 0 mM N a C l , 5 mM MOPS a d j u s t e d t o pH 7.4 w i t h N a O H ) . New, w a t e r w a s h e d t u b e s w e r e a d s o r b e d w i t h e i t h e r t w o mL o f a 5 mg/mL f i b r i n o g e n s o l u t i o n o r t w o mL o f a 5 4 % c e l l s u s p e n s i o n f o r 4 h o u r s a t ro o m t e m p e r a t u r e . C o n t r o l t u b e s w e r e new, w a t e r w a s h e d t u b e s w i t h n o p r e - a d s o r p t i o n t r e a t m e n t . The p r e - t r e a t m e n t m a t e r i a l s w e r e r e m o v e d a n d t h e t u b e s w a s h e d t w i c e w i t h t w o mL o f M O P S / s a l i n e b u f f e r . O n e - h a l f 1 2 5 m i l l i l i t e r o f a I - f i b r i n o g e n s o l u t i o n o f s p e c i f i c a c t i v i t y 3 . 0 5 x 1 0 ^ CPM/mg a n d a p r o t e i n c o n c e n t r a t i o n o f e i t h e r 4.6 o r 1. 5 mg/ml was a d s o r b e d f o r 4 0 m i n u t e s t o c o n t r o l a n d p r e - t r e a t e d t u b e s . The t u b e s w e r e t h e n w a s h e d b y r e p e a t e d a d d i t i o n a n d r e m o v a l o f t h r e e mL a l i q u o t s o f M O P S / s a l i n e b u f f e r . The t i m e i n t e r v a l b e t w e e n a d d i t i o n a n d r e m o v a l o f t h e w a s h b u f f e r was b e t w e e n 5 a n d 10 m i n u t e s . - 1 7 2 -The t u b e b o u n d a c t i v i t i e s w e r e d e t e r m i n e d b y s u b t r a c t i n g t h e a c t i v i t i e s o f t h e f l u i d c a r r y - o v e r f r o m t h e t u b e p l u s f l u i d a c t i v i t i e s . The t u b e b i n d i n g was e s s e n t i a l l y c o n s t a n t a f t e r t h e 2 n d w a s h . A s i m i l a r 1 2 5 a n a l y s i s was c a r r i e d o u t f o r i o d i d e b i n d i n g t o p o l y a l l o m e r t u b e s w i t h o u t p r e t r e a t m e n t . The t u b e b i n d i n g a f t e r 5 w a s h e s was l e s s t h a n t h e b a c k g r o u n d c o u n t i n g r a t e . The a d s o r p t i o n d e c r e a s e d b y 2 5 % a s a r e s u l t o f p r e t r e a t m e n t w i t h f i b r i n o g e n a n d 3 4 % a s a r e s u l t o f p r e t r e a t m e n t w i t h c e l l s . The 2 a d s o r p t i o n t o c l e a n e d t u b e s was 0 . 5 3 ug/cm a t 1.5 mg/mL f i b r i n o g e n 2 a n d 1.22 ug/cm a t 4.6 mg/mL, T a b l e 3 . 2 3 . Tne s u r f a c e c o n c e n t r a t i o n o b s e r v e d a t 1.5 mg/mL was l o w c o m p a r e d t o r e p o r t e d v a l u e s f o r f i b r i n o g e n b i n d i n g t o p o l y e t h y l e n e ( B r a s h a n d Lyman 1 9 6 9 , B r a s h a n d D a v i d s o n 1 9 7 6 ) . H o w e v e r , t h e s u r f a c e c o n c e n t r a t i o n a t 4.6 mg/mL was e q u i v a l e n t t o t h e s a t u r a t i o n b i n d i n g r e p o r t e d . S u r f a c e c o n c e n t r a t i o n s f o r c l o s e - p a c k e d , e n d - o n f i b r i n o g e n 2 m o n o l a y e r s h a v e b e e n c a l c u l a t e d t o be 1.57 ug/cm ( S c h m i t t e t a l . 1 9 8 3 ) . R e d u c t i o n o f f i b r i n o g e n s u r f a c e c o n c e n t r a t i o n s o n p o l y e t h y l e n e 2 t o 0 . 5 ug/cm , when a d s o r p t i o n o c c u r r e d i n t h e p r e s e n c e o f r e d c e l l s , 2 a n d t o 0.1 ug/cm , when a d s o r p t i o n o c c u r e d s u b s e q u e n t t o e x p o s i n g t h e s u r f a c e t o c e l l s , h a s b e e n r e p o r t e d ( B r a s h a n d U n i y a l 1 9 7 6 ) . B i n d i n g o f i n d i v i d u a l p r o t e i n s p e c i e s i s r e d u c e d when a d s o r p t i o n o c c u r s f r o m p r o t e i n m i x t u r e s ( L e e e t al. 1 9 7 4 , B r a s h a n d U n i y a l 1 9 7 9 ) a n d t h u s t h e r e d u c e d b i n d i n g i s a t t r i b u t e d t o c o m p e t i t i o n b e t w e e n a d s o r b i n g s p e c i e s f o r a l i m i t e d s u r f a c e a r e a . I t h a s b e e n s u g g e s t e d t h a t t h e e f f e c t a s s o c i a t e d w i t h r e d c e l l s i s d u e t o d e p o s i t i o n o f c e l l s u r f a c e m a t e r i a l s ( B r a s h a n d U n i y a l 1 9 7 6 ) . - 1 7 3 -TABL E 3 . 2 3 1 2 5I - F I B R I N 0 G E N B I N D I N G TO PRE-TREATED POLYALLOMER TUBES PRE-TREATMENT c o n t r o l f i b r i n o g e n c o n t r o l c e l l s p r o t e i n (mg/mL) 5.0 c e l l s (%) - 5 4 v o l u m e (mL) 2.0 2.0 INCUBATION p r o t e i n (mg/mL) 4.6 4.6 1.5 1.5 v o l u m e (mL) 0 . 5 0 0.51 0 . 5 0 0 . 5 0 a r e a ( c m2) a 2 . 4 3 2.49 2.46 2.46 WASH ( 3 m i s ) CPM BOUND D x 1 0 ~3 3 - 5 i ) 8.66 + 0 . 3 5 7.43 + 0 . 1 4 4 . 1 8 + 0. 0 4 2 . 1 8 + 0 . 0 4 i i ) 9 . 3 3 0.04 6.48 0.67 3.76 MEAN SURFACE CONCENTRATION 0 . 1 5 3.01 D ( u g / c m2) 0 . 0 2 3 - 5 1.22 + 0.06 0.91 + 0 . 0 9 0 . 5 3 + 0.03 0 . 3 5 + 0 . 0 6 a ) The a r e a was c a l c u l a t e f r o m t h e t u b e g e o m e t r y a n d s a m p l e v o l u m e . The e f f e c t s o f t h e p r e v i o u s w e t t i n g a n d t h e f o r m a t i o n o f a m e n i s c u s w e r e n o t c o n s i d e r e d . b ) mean + o n e s t a n d a r d d e v i a t i o n The s u r f a c e c o n c e n t r a t i o n s o b s e r v e d f o r p o l y a l l o m e r s h o w e d t h e same e f f e c t s a s t h o s e r e p o r t e d f o r p o l y e t h y l e n e . The p a r t i c u l a r s u r f a c e c o n c e n t r a t i o n s w e r e n o t a l w a y s e q u i v a l e n t t o t h e p r e v i o u s l y r e p o r t e d r e s u l t s , h o w e v e r t h e d i f f e r e n c e s a r e a t t r i b u t e d t o t e c h n i c a l s h o r t c o m i n g s o f t h e e x p e r i m e n t s r a t h e r t h a n t o d i f f e r e n c e s i n a d s o r p t i o n b e h a v i o u r . The c o n t r o l o f t h e s u r f a c e a r e a i n t h e s e e x p e r i m e n t s was n o t p r e c i s e a n d t h e g e o m e t r y o f t h e s a m p l e s may h a v e - 1 74 -a f f e c t e d t h e g a m m a - c o u n t i n g e f f i c i e n c y . B r a s h a n d U n i y a l ( 1 9 7 6 ) h a v e r e p o r t e d t h a t p r o t e i n a d s o r b e d t o p o l y e t h y l e n e t u b i n g was n o t r e m o v e d b y s u b s e q u e n t e x p o s u r e o f t h e t u b i n g t o f l o w i n g c e l l s u s p e n s i o n s . T h u s i t was a s s u m e d t h a t s i g n i f i c a n t t r a n s f e r o f p r e - a d s o r b e d a l b u m i n a n d f i b r i n o g e n f r o m t u b e s t o c e l l s o r t h e s u s p e n s i o n s u p e r