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The role of α₂macroglobulin the pathogenesis of cystic fibrosis Bridges, Michael Anthony 1981

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THE ROLE OF a 2 M A C R 0 G L 0 B U L I N IN THE P A T H O G E N E S I S OF C Y S T I C F I B R O S I S by M I C H A E L ANTHONY B R I D G E S B . S c . , U n i v e r s i t y o f New M e x i c o , 1 9 6 9 . 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 , 1977 T H E S I S S U B M I T T E D IN 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 F A C U L T Y 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 AUGUST 1981 c) M i c h a e l A n t h o n y B r i d g e s , 1 981 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r a n a d v a n c e d d e g r e e 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 , I a g r e e t h a t t h e L i b r a r y s h a l l m a k e i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s m a y b e g r a n t e d b y t h e h e a d o f m y d e p a r t m e n t o r b y h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t b e a l l o w e d w i t h o u t m y w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f 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 2 0 7 5 W e s b r o o k P l a c e V a n c o u v e r , C a n a d a V 6 T 1W5 D a t e D E - 6 ( 2 / 7 9 ) 19 August 1981 TO WHOM IT MAY CONCERN: Permission is hereby granted to Michael Anthony Bridges to reproduce our papers, " I s o l a t i o n of Pure, Act ive o^Macroglobul in from s m a l l - s c a l e Plasma Samples" and "Protease Binding by o^Macroglobul in in C y s t i c F i b r o s i s " , ( in p ress , C l i n i c a Chimica A c t a , 1981) in his Ph.D. T h e s i s , "The Role of o^Macroglobul in in the Pathogenesis of C y s t i c F i b r o s i s " , Univers'rty of B r i t i s h Columbia, 1981, and to the National L ibrary of Canada to copy them on micro f i lm and to lend or s e l l copies of the f i l m . A l l other pub l i ca t ion r ights are reserved. Derek A. Applegarth A. George F. Davidson Lawrence T. K. Wong Joanne Johannson i A B S T R A C T F o l l o w i n g r e p o r t s b y S h a p i r a e t a]_. t h a t o ^ M a c r o -g l o b u l i n ( a ^ M ) i s a b n o r m a l i n c y s t i c f i b r o s i s ( C F ) , t h e a u t h o r s e t o u t t o e x a m i n e t h e p r o p e r t i e s o f c^M i s o l a t e d f r o m t h e p l a s m a o f c h i l d r e n w i t h CF a n d f r o m t h e p l a s m a o f a g e / s e x m a t c h e d c o n t r o l s . T o do s o , a t e c h n i q u e c a p a b l e o f i s o l a t i n g p u r e , p h y s i o l o g i c a l l y " a c t i v e " <*2M f r o m s m a l l p l a s m a s a m p l e s h a d t o be d e v e l o p e d . By a t w o - s t e p c h r o m a t o g r a p h i c t e c h n i q u e , i n v o l v i n g C i b a c r o n B l u e S e p h a r o s e c h r o m a t o g r a p h y a n d immuno-a d s o r p t i o n , t h e a u t h o r was a b l e t o i s o l a t e " a c t i v e " CF a n d c o n t r o l c^M o f a t l e a s t 98 p e r c e n t p u r i t y f r o m 5 ml o f p l a s m a , r e g a r d l e s s o f p l a s m a h a p t o g l o b i n t y p e . H a v i n g a c c o m p l i s h e d t h i s , c o m p a r a t i v e s t u d i e s o f CF a n d c o n t r o l o^M w e r e u n d e r t a k e n . F o u r p a r a m e t e r s w e r e i n v e s t i g a t e d : ( 1 ) t h e m o l a r p r o t e a s e b i n d i n g o f a , , M , ( 2 ) t h e i n t e r a c t i o n o f o ^ M - b o v i n e c a t i o n i c t r y p s i n ( B C T ) c o m p l e x e s w i t h t h e l o w m o l e c u l a r w e i g h t s u b s t r a t e B A E E , ( 3 ) t h e s t a b i l i t y o f f o r m e d a^M-BCT c o m p l e x e s , a n d ( 4 ) t h e s u b u n i t s t r u c t u r e o f o ^ M . C o n t r a r y t o t h e r e p o r t s o f S h a p i r a a n d h i s c o l l e a g u e s , t h i s a u t h o r f o u n d no d i f f e r e n c e s b e t w e e n t h e s u b u n i t s t r u c t u r e o f CF a n d c o n t r o l o^M n o r b e t w e e n t h e a b i l i t i e s o f CF a n d c o n t r o l o^M t o i n t e r a c t w i t h B CT. B a s e d u p o n t h e s e f i n d i n g s , t h e a u t h o r b e l i e v e s t h a t no f i r m e v i d e n c e e x i s t s t o i m p l i c a t e a n o^M d e f e c t i n t h e p a t h o g e n e s i s o f c y s t i c f i b r o s i s . i i T A B L E OF CONTENTS PAGE I . I NTRODUCTION 1 A. C y s t i c F i b r o s i s : C l i n i c a l a n d P a t h o l o g i c a l A s p e c t s 2 B. CF F a c t o r s : 5 ( 1 ) C i l i a r y D y s k i n e s i s ( C D ) F a c t o r s 6 ( 2 ) S o d i u m R e a b s o r p t i o n I n h i b i t o r y F a c t o r ( T h e M a n g o s F a c t o r ) 10 C. T h e P u t a t i v e P r o t e o l y t i c E n z y m e D e f i c i e n c y o f CF P l a s m a a n d S a l i v a 12 D. T h e R o l e o f a2M i n N o r m a l P h y s i o l o g y a n d E v i d e n c e f o r a n «2M D e f e c t i n CF 16 E . I s o l a t i o n o f P u r e , A c t i v e «2M f r o m S m a l l P l a s m a S a m p l e s 25 I I . M A T E R I A L S AND METHODS 28 A. M a t e r i a l s 28 B. E x p e r i m e n t a l S u b j e c t s 29 C. I s o l a t i o n o f ot2M f r o m CF a n d C o n t r o l B l o o d S a m p l e s 29 D. P u r i f i c a t i o n o f B o v i n e C a t i o n i c T r y p s i n 32 E . E s t i m a t i o n o f t h e P r o t e i n C o n t e n t s o f C h r o m a t o g r a p h y F r a c t i o n s a n d «2M I s o l a t e s 33 i i i T A B L E OF CONTENTS PAGE ( I I . M A T E R I A L S AND METHODS c o n t i n u e d ) F. A s s e s s m e n t o f P u r i t y o f C h r o m a t o g r a p h y F r a c t i o n s a n d a2M I s o l a t e s : 3 3 ( 1 ) I m m u n o e l e c t r o p h o r e s i s ( I E P ) 3 4 ( 2 ) G r a d i e n t PAGE 3 5 ( 3 ) SDS PAGE 3 7 G. A s s e s s m e n t o f P r o t e a s e B i n d i n g A b i l i t y o f c*2M I s o 1 a t e s : 3 8 ( 1 ) M o l a r P r o t e a s e B i n d i n g S t u d i e s 3 8 ( 2 ) G r a d i e n t PAGE 3 9 ( 3 ) SDS PAGE 3 8 H. E x p e r i m e n t s P e r f o r m e d t o C o m p a r e CF a n d C o n t r o l 012M: 3 9 ( 1 ) M o l a r P r o t e a s e B i n d i n g b y CF a n d C o n t r o l c«2M 3 9 ( 2 ) S u b s t r a t e K i n e t i c s o f CF a n d C o n t r o l a 2 M-BCT C o m p l e x e s 4 0 ( 3 ) S t a b i l i t y o f CF a n d C o n t r o l C12M-BCT C o m p l e x e s 4 1 ( 4 ) S u b u n i t S t r u c t u r e o f CF a n d C o n t r o l <*2M 4 4 I . S t a t i s t i c a l T r e a t m e n t o f D a t a 4 4 1 V - T A B L E OF CONTENTS PAGE I I I . R E S U L T S : 46 A. I s o l a t i o n o f 012M f r o m CF a n d C o n t r o l P l a s m a 46 B. D e t e r m i n a t i o n o f P r o t e i n C o n t e n t s o f P r e p a r a t i o n s 46 C. P r o o f o f P u r i t y o f a 2 M I s o l a t e s 51 D. P r o o f o f P r o t e a s e - B i n d i n g A b i l i t y o f « 2 M I s o l a t e s : 58 ( 1 ) M o l a r P r o t e a s e B i n d i n g b y 012M 58 ( 2 ) G r a d i e n t PAGE o f « 2 M I s o l a t e s 63 ( 3 ) SDS PAGE w i t h T h i o l R e d u c t i o n o f 012M I s o l a t e s a n d 012M-BCT C o m p l e x e s 64 E . E x p e r i m e n t s P e r f o r m e d t o C o m p a r e CF a n d C o n t r o l a 2 ^ ' 67 ( 1 ) M o l a r P r o t e a s e B i n d i n g b y U n t r e a t e d a 2 M a n d b y Ammonium I o n - T r e a t e d a 2 M 67 ( 2 ) I n t e r a c t i o n o f CF a n d C o n t r o l c » 2 M - B o v i n e C a t i o n i c T r y p s i n ( B C T ) C o m p l e x e s a n d N o n -c o m p l e x e d BCT w i t h B e n z o y l A r g i n i n e E t h y l E s t e r ( B A E E ) 67 ( 3 ) S t a b i l i t y o f CF a n d C o n t r o l « 2 M - B C T C o m p l e x e s t o H i g h M o l e c u l a r W e i g h t S u b s t r a t e s a n d l n h i b i t o r s 76 ( 4 ) S u b u n i t S t r u c t u r e o f CF a n d C o n t r o l 012M 81 V T A B L E OF CONTENTS PAGE I I V . D I S C U S S I O N 8 7 A. I s o l a t i o n o f P u r e , A c t i v e c*2M 8 7 B. T h e R o l e o f a 2 M i n t h e P a t h o g e n e s i s o f C F : 9 0 ( 1 ) M o l a r P r o t e a s e B i n d i n g b y CF a n d C o n t r o l c*2M 9 1 ( 2 ) I n t e r a c t i o n o f CF a n d C o n t r o l o ^ M - t r y p s i n C o m p l e x e s w i t h BAEE 9 7 ( 3 ) S t a b i l i t y o f CF a n d C o n t r o l c « 2 M - T r y p s i n C o m p l e x e s : 9 9 ( a ) S t a b i l i t y o f C o m p l e x e s t o S T I 1 0 1 ( b ) S t a b i l i t y o f C o m p l e x e s t o R B B - h i d e 1 0 2 ( 4 ) S u b u n i t S t r u c t u r e o f CF a n d C o n t r o l « 2 M 1 0 3 C. C o n c l u s i o n 1 0 7 V. B I B L I O G R A P H Y 1 0 9 v i L I S T O F T A B L E S N O . T I T L E P A G E M O L A R P R O T E A S E B I N D I N G R A T I O S F O R C F A N D C O N T R O L a 2 M N O T E X P O S E D T O A M M O N I U M S U L F A T E T R E A T M E N T 6 8 I I . M O L A R P R O T E A S E B I N D I N G R A T I O S - F O R C F A N D C O N T R O L c»2M T R E A T E D W I T H 2 0 0 mM A M M O N I U M S U L F A T E F O R 3 0 M I N U T E S A T 3 7 ° 7 0 I I I . I N T E R A C T I O N O F C F - A N D C O N T R O L a 2 M - B C T C O M P L E X E S A N D N O N - C O M P L E X E D B C T W I T H B A E E 7 7 I V . S T A B I L I T Y O F C F A N D C O N T R O L a 2 M - B C T C O M P L E X E S T O A H I G H M O L E C U L A R W E I G H T I N H I B I T O R ( S O Y B E A N T R Y P S I N I N H I B I T O R ) 7 9 V . S T A B I L I T Y O F C F A N D C O N T R O L a 2 M - B C T C O M P L E X E S T O A H I G H M O L E C U L A R W E I G H T S U B S T R A T E ( R E M A Z O L B R I L L I A N T B L U E - H I D E ) 8 2 VI 1 L I S T OF F I G U R E S NO. T I T L E 1 C i b a c r o n B l u e S e p h a r o s e ( C B S ) C h r o m a t o g r a p h y O f Human P l a s m a 2 I m m u n o a d s o r p t i o n C h r o m a t o g r a p h y O f CBS P o o l 3 A s s a y o f P r o t e i n C o n t e n t s o f « 2 M P r e p a r a t i o n s : C o r r e l a t i o n o f D e t e r m i n a t i o n s made b y 280 nm E x t i n c t i o n a n d b y t h e B r a d f o r d Dye B i n d i n g A s s a y 4 I m m u n o e l e c t r o p h o r e s i s o f a 2 M 5 G r a d i e n t PAGE o f a 2 M i n 4 t o 26 g / d l G e l s 6 SDS PAGE w i t h T h i o l R e d u c t i o n o f c^M I s o l a t e d f r o m H a p t o g l o b i n T y p e 2-2 P l a s m a 7 M o l a r P r o t e a s e B i n d i n g b y U n t r e a t e d <*2M a n d a 2 M P r e - t r e a t e d w i t h Ammonium I o n 8 SDS PAGE o f 012M S h o w i n g t h e S t o i c h i o m e t r i c C o n v e r s i o n b y BCT o f t h e c t 2 M M o n o m e r t o t h e a 2 M H a l f - M o n o m e r ( 8 5 K F r a g m e n t ) 9 S u b s t r a t e K i n e t i c s o f a 2 M - B C T C o m p l e x e s : V e l o c i t y vs S u b s t r a t e C o n c e n t r a t i o n P l o t 10 S u b s t r a t e K i n e t i c s o f o^M-BCT C o m p l e x e s : L i n e w e a v e r - B u r k P l o t 11 S u b - u n i t S t r u c t u r e o f CF a n d C o n t r o l « 2 M VI 1 1 L I S T OF A B B R E V I A T I O N S a 2 M B A E E BCT CBS CD CF cm C N B r C 3 a D E A E - S e p h a d e x d l E D T A E t O H 9 HC1 HOAc h r H 2 0 I E P 1 2 5 J K a 2 - M a - c r o g l o b u l i n b e n z o y l a r g i n i n e e t h y l e s t e r b o v i n e c a t i o n i c t r y p s i n C i b a c r o n B l u e S e p h a r o s e c i 1 i a r y d y s k i n e s i a c y s t i c f i b r o s i s c e n t i m e t e r c y a n o g e n b r o m i d e a n a p h y 1 o t o x i n d i e t h y l a m i n o e t h y l S e p h a d e x d e c i l i t e r 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 e t h a n o l g r a m h y d r o c h l o r i c a c i d a c e t i c a c i d h o u r w a t e r I m m u n o e l e c t r o p h o r e s i s i m m u n o g l o b u l i n i o d i n e - 1 2 5 t h o u s a n d i n h i b i t i o n c o n s t a n t a p p a r e n t a f f i n i t y m o l a r i x L I S T OF A B B R E V I A T I O N S ( c o n t i n u e d ) ma mi 1 1 i a m p e r e mg m i l l i g r a m ml m i l l i l i t e r mM m i l l i m o l a r u l m i c r o l i t e r y_M m i c r o m o l a r n n u m b e r i n s a m p l e Na ,K - A T P a s e s o d i u r n - , p o t a s s i u m - s t i m u l a t e d a d e n o s i n e 5 ' - t r i p h o s p h a t a s e N H ^ + ammoni urn i o n nm n a n o m e t e r p s t a t i s t i c a l p r o b a b i l i t y PAGE p o l y a c r y 1 a m i d e g e l e l e c t r o p h o r e s i s R c o r r e l a t i o n c o e f f i c i e n t R B B - h i d e R e m a z o l b r i l l i a n t b l u e - h i d e RID r a d i a l i m m u n o d i f f u s i o n S.D. s t a n d a r d d e v i a t i o n SDS s o d i u m d o d e c y l s u l p h a t e S E - S e p h a d e x s u l p h o e t h y l S e p h a d e x S T I s o y b e a n t r y p s i n i n h i b i t o r TAME a - N - t o s y l - L ^ - a r g i n i ne m e t h y l e s t e r T EMED N,N,N' , N ' - t e t r a m e t h y 1 e t h y 1 e n e d i a m i n e UV u l t r a v i o l e t V m a x i m a l v e l o c i t y ma x w/w w e i g h t p e r w e i g h t < 1 e s s t h a n 0 d e g r e e s C e n t i g r a d e X ACKNOWLEDGEMENTS I w i s h t o e x p r e s s my deep g r a t i t u d e t o D r . D e r e k A. A p p l e g a r t h f o r h i s many c o n t r i b u t i o n s t o my g r a d u a t e e d u a t i o n a n d p r o f e s s i o n a l d e v e l o p m e n t , a s w e l l a s f o r h i s f r i e n d s h i p a n d s upp o r t . I w i s h t o t h a n k D r . P h i l R e i d , D r . P e t e r C a n d i d o , D r . A. G e o r g e F. D a v i d s o n a n d D r . R.H. P e a r c e , members o f my R e s e a r c h A d v i s o r y C o m m i t t e e , f o r t h e i r p a r t i c i p a t i o n i n t h i s w o r k a s c o n s u l t a n t - s c i e n t i s t s . I a l s o g r a t e f u l l y a c k n o w l e d g e t h e i n v a l u a b l e a s s i s t a n c e o f D r . L a w r e n c e Wong, D r . A. G e o r g e F. D a v i d s o n a n d D r . J o h n T z e who p r o v i d e d t h e c l i n i c a l a n d c o n t r o l b l o o d s a m p l e s e x a m i n e d i n t h e s e s t u d i e s . I w o u l d a l s o l i k e t o t h a n k : Ms. J o a n n e J o h a n n s o n a n d M r s . M i l a n a F r o h l i c h f o r t h e i r e x p e r t t e c h n i c a l a s s i s t a n c e a n d t h o u g h t f u l s u g g e s t i o n s , Ms. J e n n i f e r T o o n e f o r h e r many i n t e l l e c t u a l c o n t r i b u t i o n s t o t h i s work a s w e l l a s f o r h e r a s s i s t a n c e i n p r e p a r i n g t h e d i a g r a m s f o r t h i s t h e s i s , M r . G o r d o n G e l l a n d t h e P h o t o g r a p h y D e p a r t m e n t o f C h i l d r e n ' s H o s p i t a l f o r t h e i r h e l p w i t h t h e p h o t o g r a p h y o f e 1 e c t r o p h o r e t i c g e l s . x i I a l s o g r a t e f u l l y a c k n o w l e d g e t h e g r e a t s k i l l a n d many h o u r s o f d i l i g e n c e b y Ms. G l o r i a M o r r i s t h a t w e n t i n t o t h e e x e c u t i o n o f t h i s m a n u s c r i p t . F i n a l l y , I w i s h t o t h a n k D r . L . T . K i r b y f o r h i s k i n d c o - o p e r a t i o n , a n d t h e f i n a n c i a l s u p p o r t o f T h e M e d i c a l R e s e a r c h C o u n c i l o f C a n a d a a n d T h e C a n a d i a n C y s t i c F i b r o s i s F o u n d a t i o n . x i i FOR ROSALYN AND J A C K I PAGE 1 I . I N T R O D UCTION T h i s t h e s i s a s k s t h e q u e s t i o n : I s t h e p l a s m a p r o t e a s e i n h i b i t o r , o ^ M a c r o g 1 o b u 1 i n ( a 2 M ) , a b n o r m a l i n p a t i e n t s w i t h c y s t i c f i b r o s i s ( C F ) ? Some i n v e s t i g a t o r s h a v e a n s w e r e d i n t h e a f f i r m a t i v e . On t h e b a s i s o f s e v e r a l s t u d i e s o f CF c» 2M, S h a p i r a et_ a j _ . [ 1 - 4 ] and W i l s o n and F u d e n b e r g [ 5 , 6 ] h a v e h y p o t h e s i z e d t h a t an a b n o r m a l i t y i n c t 2M may e v e n be t h e b a s i c d e f e c t i n C F . To t r a c e t h e h i s t o r i c a l d e v e l o p m e n t s w h i c h l e d i n v e s t i -g a t o r s t o s u s p e c t a r o l e f o r d e f e c t i v e c * 2 M i n t h e p a t h o g e n e s i s o f C F , we m u s t c o n s i d e r f o u r t o p i c s : ( 1 ) t h e c l i n i c a l a n d p a t h o -l o g i c a l a s p e c t s o f C F , ( 2 ) "CF f a c t o r s " , ( 3 ) a p u t a t i v e p r o -t e o l y t i c e n z y m e d e f i c i e n c y o f CF p l a s m a and s a l i v a , a n d ( 4 ) t h e r o l e o f a 2 M i n n o r m a l p h y s i o l o g y a n d t h e e v i d e n c e f o r an c * 2 M d e f e c t i n C F . In t h e i n t e r e s t o f c l a r i t y , t h e c o n n e c t i o n s r e p u t e d t o e x i s t b e t w e e n t h e s e f o u r e l e m e n t s w i l l be s u m m a r i z e d b e f o r e e a c h i s d i s c u s s e d a t l e n g t h : A m o l e c u l a r d e f e c t h a s b e e n s a i d t o be p r e s e n t i n CF a 2 M , w h i c h i m p a i r s i t s a b i l i t y t o r e g u l a t e p r o -t e o l y t i c e n z y m e a c t i v i t y i n a n o r m a l p h y s i o l o g i c a l way [ 1 - 6 ] ; PAGE 2 t h i s may l e a d t o a d e f i c i e n c y o f p r o t e o l y t i c e n z y m e a c t i v i t y w i t h a r g i n i n e e s t e r a s e s p e c i f i c i t y i n CF p l a s m a a n d e x o c r i n e g l a n d s and may p r e v e n t t h e n o r m a l m e t a b o l i s m o f c e r t a i n b a s i c a m i n o a c i d - r i c h p o l y p e p t i d e s , i n a p p r o p r i a t e l y c a l l e d "CF f a c t o r s " [ 7 - 1 1 ] . T h e c o n c e n t r a t i o n o f CF f a c t o r s may t h e n i n c r e a s e a n d c a u s e d e l e t e r i o u s e f f e c t s on t h e p l a s m a m e m b r a n e s o f c i l i a t e d c e l l s and g l a n d u l a r a c i n a r and d u c t a l c e l l s [ 6 ] . T h i s c o u l d r e s u l t i n d i s t u r b a n c e s o f c i l i a r y m o t i o n a nd a l t e r a t i o n s i n t h e c o m p o s i t i o n o f g l a n d u l a r s e c r e t i o n s , p r o d u c i n g t h e c l i n i c a l s y n d r o m e w h i c h we r e c o g n i z e as c y s t i c f i b r o s i s [ 6 , 1 0 ] . A. C y s t i c F i b r o s i s : C l i n i c a l a n d P a t h o l o g i c a l A s p e c t s : C y s t i c F i b r o s i s ( C F ) i s a c l i n i c a l d i s o r d e r c h a r a c t e r i z e d by c h r o n i c o b s t r u c t i v e l u n g d i s e a s e , e x o c r i n e p a n c r e a t i c i n -s u f f i c i e n c y a n d e l e v a t e d s w e a t e l e c t r o l y t e s [ 1 2 ] . T h e i n c i d e n c e o f CF among w h i t e p o p u l a t i o n s i s a p p r o x i m a t e l y 1 i n 2 , 0 0 0 l i v e b i r t h s , m a k i n g i t t h e m o s t common l e t h a l g e n e t i c d i s o r d e r o f C a u c a s i a n s [ 1 3 ] . T h e b e s t g e n e t i c e v i d e n c e i m p l i c a t e s an a u t o -s o m a l r e c e s s i v e mode o f t r a n s m i s s i o n o f t h e CF t r a i t ( s ) [ 1 1 ] . S i n c e t h e p r i m a r y l e s i o n ( s ) h a s ( h a v e ) n o t y e t b e e n i d e n t i f i e d , i t i s n o t c l e a r w h e t h e r CF i s a h o m o g e n e o u s o r a h e t e r o g e n e o u s d i s e a s e e n t i t y . PAGE 3 B i o c h e m i c a l and h i s t o l o g i c a l e v i d e n c e s h ows t h a t a l l e x o -c r i n e g l a n d s a r e a f f e c t e d i n C F . As a r u l e , t h e s e c r e t i o n s o f e x o c r i n e g l a n d s i n CF b e c o m e i n s p i s s a t e d [ 1 3 ] . T h e a b n o r m a l r h e o l o g i c a l p r o p e r t i e s o f s u c h s e c r e t i o n s c a u s e o b s t r u c t i o n o f t h e d u c t s o r p a s s a g e w a y s i n t o w h i c h t h e y a r e d i s c h a r g e d . S i t e s o f o b s t r u c t i o n i n c l u d e l u n g a i r w a y s , p a r a n a s a l s i n u s e s , m u c u s - s e c r e t i n g s a l i v a r y g l a n d s , s m a l l i n t e s t i n e , p a n c r e a s , b i l i a r y t r a c t , a n d m a l e a n d f e m a l e g e n i t a l t r a c t s [ 1 3 ] . T h e p a t h o g e n e s i s o f t h e i n s p i s s a t i o n o f e x o c r i n e s e c r e t i o n s i s u n c e r t a i n , b u t d e h y d r a t i o n , e l e c t r o l y t e a l t e r a t i o n s a n d c o m p o s i t i o n a l a b n o r m a l i t i e s i n m u c o u s g l y c o p r o t e i n s h a v e a l l b e e i m p l i c a t e d [ 1 3 ] . One c o n s e q u e n c e o f d u c t a l o b s t r u c t i o n i n c e r t a i n e x o c r i n e g l a n d s ( n o t a b l y t h e p a n c r e a s ) i s f i b r o s i s [ 1 1 ] . A n o t h e r r e s u l t o f t h i s o b s t r u c t i v e p r o c e s s i s t h e p r o d u c t i o n o f d e f i c i e n c y o f t h e e x o c r i n e s e c r e t i o n i n t h e p h y s i o l o g i c t a r g e t o r g a n [ 1 1 ] . F o r e x a m p l e , p a n c r e a t i c o b s t r u c t i o n r e s u l t s i n a d e f i c i e n c y o f d i g e s t i v e e n z y m e s i n t h e s m a l l i n t e s t i n e . T h i s , i t u r n , l e a d s t o ma 1 d i g e s t i o n and m a l a b s o r p t i o n . Two s e r o u s e x o c r i n e g l a n d s do n o t p r o d u c e i n s p i s s a t e d s e c r e t i o n s i n C F : e c c r i n e s w e a t g l a n d s a n d p a r o t i d s a l i v a r y g l a n d s [ 1 3 ] . H o w e v e r , t h e s e c r e t i o n s o f b o t h show c h e m i c a l a b n o r m a l i t i e s . CF e c c r i n e s w e a t i s h i g h i n s o d i u m a n d c h l o r i d e [ 1 4 ] , and CF p a r o t i d s a l i v a shows i n c r e a s e d c a l c i u m and p r o t e i n [ 1 1 ] . PAGE 4 M o s t m o r b i d i t y and m o r t a l i t y i n CF r e s u l t s f r o m c h r o n i c l u n g d i s e a s e [ 1 3 ] . T h e d i s e a s e p r o c e s s a p p e a r s t o be i n i t i a t e d by p l u g g i n g o f b r o n c h i a l g l a n d s w i t h m u c u s . O b s t r u c t i o n o f p e r i p h e r a l a i r w a y s s o o n f o l l o w s , r e s u l t i n g i n b a c t e r i a l c o l o n -i z a t i o n and t i s s u e d e s t r u c t i o n [ 1 3 ] . A d e f e c t i n m u c o c i l i a r y t r a n s p o r t o f p a r t i c u l a t e m a t t e r w i t h i n t h e l u n g i n CF h a s b e e n r e p o r t e d [ 1 5 ] and may c o n t r i b u t e t o c h r o n i c l u n g d i s e a s e . N o r m a l l y t h e m u c u s - c o a t e d c i l i a t e d e p i t h e l i u m , w h i c h l i n e s t h e r e s p i r a t o r y l u m e n , p e r f o r m s t h e t a s k o f p h y s i c a l l y r e m o v i n g n a t u r a l d e b r i s a n d i n h a l e d p a r t i c u l a t e s ( e . g . b a c t e r i a ) [ 1 3 ] . T h i s p h y s i o l o g i c a l d e f e n c e m e c h a n i s m i s c a l l e d " m u c o c i l i a r y t r a n s p o r t " . Wood ejt a J L [ 1 5 ] f o u n d t h a t t h e r a t e o f m u c o c i l i a r y t r a n s p o r t o f m a r k e r p a r t i c l e s a c r o s s t h e t r a c h e a l m u c o s a o f CF p a t i e n t s was 5 t o 1 0 - f o l d l o w e r t h a n t h e r a t e f o r n o r m a l a g e - m a t c h e d c o n t r o l s . R e s e a r c h i n t o t h e p a t h o g e n e s i s o f CF h a s f r e q u e n t l y c e n t e r e d u p o n two a s p e c t s o f CF p a t h o p h y s i o l o g y : ( 1 ) t h e c a u s e ( s ) o f t h e c o m p o s i t i o n a l a n d / o r r h e o l o g i c a l c h a n g e s i n e x o c r i n e s e c r e t i o n s a n d , ( 2 ) t h e c a u s e ( s ) o f t h e p u t a t i v e m u c o -c i l i a r y t r a n s p o r t d e f e c t o f l u n g . In 1 9 6 7 , two i n d e p e n d e n t g r o u p s o f i n v e s t i g a t o r s p u b l i s h e d s t u d i e s s u g g e s t i n g t h e p r e s e n c e o f p a t h o g e n i c p o l y p e p t i d e f a c t o r s i n CF s e r u m , s a l i v a a n d s w e a t [ 1 6 - 1 8 ] . T h e s e s o - c a l l e d 'CF f a c t o r s ' w e r e f o u n d t o i n d u c e PAGE 5 c h a n g e s i n i s o l a t e d p r e p a r a t i o n s o f n o r m a l a n i m a l t i s s u e s w h i c h m i m i c k e d some o f t h e p a t h o l o g i c a l c h a n g e s o c c u r r i n g i n t h e l u n g s and e x o c r i n e g l a n d s o f human s u b j e c t s w i t h C F . B. CF F a c t o r s : In t h e 1967 r e p o r t s , two d i f f e r e n t t y p e s o f CF f a c t o r a c t i v i t y w e r e d e s c r i b e d : S p o c k e_t a_l_. r e p o r t e d t h e p r e s e n c e o f a f a c t o r i n s e r u m f r o m p a t i e n t s w i t h CF and f r o m o b l i g a t e h e t e r o z y g o t e s w h i c h d i s -o r g a n i z e d c i l i a r y b e a t i n g i n r a b b i t t r a c h e a l e x p l a n t s [ 1 6 ] . T h i s s o - c a l l e d " c i l i a r y d y s k i n e s i a f a c t o r " may u n d e r l i e t h e p u l m o n a r y m u c o c i l i a r y t r a n s p o r t d e f e c t o b s e r v e d by Wood e_t a j _ . [ 1 5 ] . M a n g o s and c o - w o r k e r s d e s c r i b e d a s e c o n d t y p e o f f a c t o r c a p a b l e o f p r o d u c i n g i n h i b i t i o n o f r a t p a r o t i d d u c t s o d i u m r e a b s o r p t i o n f o l l o w i n g r e t r o g r a d e p e r f u s i o n o f CF s w e a t o r s a l i v a [ 1 7 , 1 8 ] . T h i s s o - c a l l e d " s o d i u m r e a b s o r p t i o n i n h i b i t o r y f a c t o r " may u n d e r l i e t h e h i g h s w e a t s o d i u m and c h l o r i d e e x c r e t i o n o b s e r v e d i n CF [ 1 4 ] . T h i s c o n c e p t i s s u p p o r t e d by m i c r o p u n c t u r e s t u d i e s o f t h e s e c r e t o r y c o i l o f CF s w e a t g l a n d s w h i c h show t h a t t h e f l u i d p r o d u c e d by t h e s e c r e t o r y p o r t i o n o f t h e g l a n d i s o f n o r m a l o s m o l a l i t y [ 1 9 ] . I t a p p e a r s t o be t h e r e a b s o r p t i o n o f s w e a t PAGE 6 e l e c t r o l y t e s w i t h i n t h e g l a n d ' s d u c t a l e l e m e n t s w h i c h i s d e f e c t i v e i n CF [ 1 2 ] . S i n c e 1 9 6 7 CF f a c t o r s h a v e b e e n t h e s u b j e c t o f i n t e n s e i n v e s t i g a t i o n b e c a u s e o f t h e i r p o s s i b l e i m p l i c a t i o n s t o t h e p a t h o g e n e s i s o f t h e d i s e a s e . What f o l l o w s i s a b r i e f o v e r v i e w o f w h a t i s known a b o u t CF f a c t o r s . ( 1 ) C i l i a r y D y s k i n e s i a ( CD) F a c t o r s : I n v e s t i g a t i o n o f t h e r o l e o f CD f a c t o r s i n CF p a t h o -p h y s i o l o g y h a s made s l o w p r o g r e s s b e c a u s e e s t i m a t i o n o f f a c t o r a c t i v i t y i n CF b i o l o g i c a l f l u i d s a n d i n c h r o m a -t o g r a p h y f r a c t i o n s h a s r e l i e d u p o n t h e u s e o f b i o a s s a y s e m p l o y i n g c i l i a t e d t i s s u e s i n o r g a n c u l t u r e . S u c h a s s a y s d e p e n d u p o n a c c u r a t e a n a l y s i s o f c i l i a r y m o t i o n a n d by t h e i r v e r y n a t u r e a l l o w t h e i n t r o d u c t i o n o f o b s e r v e r b i a s i n t o d a t a g a t h e r i n g [ 2 0 ] . F u r t h e r m o r e , i s o l a t e d c i l i a t e d t i s s u e s a r e s u b j e c t t o g r e a t v a r i a b i l i t y r e l a t i n g t o t h e s o u r c e a n d h e a l t h o f t h e d o n o r o r g a n i s m [ 2 0 ] . Two c i l i a t e d s y s t e m s h a v e b e e n u s e d w i d e l y t o d e m o n -s t r a t e CD f a c t o r a c t i v i t y [ 1 3 ] : r a b b i t t r a c h e a l e x p l a n t s [ 1 6 , 2 1 ] and A t l a n t i c o y s t e r g i l l p r e p a r a t i o n s [ 2 2 ] . T h e r e i s r e a s o n t o b e l i e v e t h a t t h e CD f a c t o r a c t i v i t i e s a s s e s s e d PAGE 7 by t h e s e two d i f f e r e n t b i o a s s a y s r e f l e c t d i f f e r e n t CD f a c t o r s , p o s s e s s i n g some common c h a r a c t e r i s t i c s . B o t h f a c t o r s h a v e b e e n s e m i - p u r i f i e d a nd shown t o be c a t i o n i c p o l y p e p t i d e s h a v i n g a m o l e c u l a r w e i g h t b e t w e e n 4 , 0 0 0 t o 1 0 , 0 0 0 , and b o t h a r e c a p a b l e o f n o n - c o v a l e n t , n o n - a n t i g e n i c b o n d i n g t o i m m u n o g l o b u l i n G [ 2 3 - 2 8 ] . B o t h h a v e b e e n c l a i m e d t o be p r o d u c e d by h o m o z y g o t e and o b l i g a t e h e t e r o -z y g o t e s f o r CF [ 1 6 , 2 1 , 2 2 ] and t o be s y n t h e s i z e d by f i b r o -b l a s t s , p e r i p h e r a l b l o o d l e u c o c y t e s a nd l y m p h o c y t e s i n c u l t u r e [ 2 9 - 3 1 ] . H o w e v e r , t h e CD f a c t o r a c t i v i t y o f CF f l u i d s a s s e s s e d by t h e r a b b i t t r a c h e a l a s s a y h a s b e e n shown ( 1 ) t o be i n a c t i v a t e d by t r e a t m e n t w i t h c a r b o x y p e p t i d a s e ; ( 2 ) t o r e q u i r e t h e p r e s e n c e o f I g G f o r e x p r e s s i o n o f i t s a n t i c i l i a r y a c t i v i t y [ 3 2 ] , a n d , ( 3 ) t o be i n h i b i t e d by e x p o s u r e t o a n t i - C 3 a s e r u m , i m p l y i n g i d e n t i t y t o , o r a t l e a s t c r o s s - r e a c t i v i t y o f t h i s f a c t o r w i t h , a n a p h y 1 o t o x i n ( C 3 a ) [ 3 2 , 3 3 ] . T h e CF f a c t o r a c t i v i t y o f CF f l u i d s a s s e s s e d by t h e o y s t e r g i l l b i o a s s a y d o e s n o t show t h e s e p r o p e r t i e s [ 2 0 , 2 5 , 2 8 ] . G i v e n t h e e x i s t e n c e o f CD f a c t o r s i n CF b i o l o g i c f l u i d s , t h e i n t r i g u i n g q u e s t i o n a r i s e s : What i s t h e r e -l a t i o n s h i p b e t w e e n CD f a c t o r s a n d t h e p r i m a r y g e n e t i c d e f e c t o f CF? S t a t e d a n o t h e r way: A r e CD f a c t o r s t h e d e f e c t i v e g e n e p r o d u c t o r a r e t h e y i t s c o n s e q u e n c e ? PAGE 8 A c c o r d i n g t o Wood et_ a]_. [ 1 3 ] , t h e e l a b o r a t i o n o f CD f a c t o r s b y c u l t u r e d c e l l s i s o l a t e d f r o m CF p a t i e n t s a n d o b l i g a t e h e t e r o z y g o t e s s u g g e s t s t h a t t h e s e a g e n t s b e a r a p r o x i m a l r e l a t i o n s h i p t o t h e p r o d u c t o f t h e d e f e c t i v e g e n e i n C F . H o w e v e r , a number o f l i n e s o f e v i d e n c e p r e c l u d e CD f a c t o r s f r o m b e i n g t h e a b n o r m a l g e n e p r o d u c t : CD f a c t o r s a r e n o t a s i n g l e p o l y p e p t i d e . As d i s c u s s e d a b o v e , f a c t o r a c t i v i t i e s m e a s u r e d by t h e r a b b i t and o y s t e r b i o a s s a y s a r e p r o b a b l y d i f f e r e n t i n t h e i r b i o c h e m i c a l p r o p e r t i e s a n d m e c h a n i s m s o f a c t i o n [ 2 0 , 2 5 , 2 8 , 3 1 - 3 3 ] . S c h m o y e r e_t a j _ . w e r e a b l e t o d i s c r i m i n a t e by i s o e l e c t r i c f o c u s i n g a t l e a s t t h r e e c a t i o n i c f r a c t i o n s p o s s e s s i n g CD a c t i v i t y , d e r i v e d f r o m CF h o m o z y g o t e and h e t e r o z y g o t e s e r a [ 3 4 , 3 5 ] . CD f a c t o r s a r e n o t u n i q u e t o C F . S e r u m f r o m p a t i e n t s w i t h c e r t a i n a u t o i m m u n e o r a l l e r g i c d i s e a s e s ( e . g . s y s t e m i c l u p u s e r y t h e m a t o s u s , c o e l i a c d i s e a s e , b r o n c h i a l a n d a s p i r i n - s e n s i t i v e a s t h m a s ) a l s o p o s s e s s CD f a c t o r a c t i v i t y as m e a s u r e d by t h e r a b b i t t r a c h e a l b i o a s s a y [ 3 6 , 3 7 ] . S e m i - p u r i f i e d f r a c t i o n s o f n o r m a l s e r u m [ 3 4 ] and c o n t r o l s e r u m t r e a t e d w i t h t h e c a r b o x y p e p t i d a s e i n h i b i t o r e - a m i n o -c a p r o i c a c i d [ 3 3 , 3 8 ] a l s o p r o d u c e t h i s r e a c t i o n . When c u l t u r e d CF s k i n f i b r o b l a s t s a r e i n g r o w t h p h a s e , an PAGE 9 I g G - d e p e n d e n t CD f a c t o r a c t i v i t y i s e l a b o r a t e d ( a s a s s e s s e d by t h e r a b b i t b i o a s s a y ) ; t h i s i s n o t t h e c a s e w i t h c u l t u r e d n o r m a l f i b r o b l a s t s [ 3 2 ] . H o w e v e r , b o t h n o r m a l and CF f i b r o b l a s t s i n c o n f l u e n t c u l t u r e s y n t h e s i z e I g G - i n d e p e n d e n t CD f a c t o r a c t i v i t i e s [ 3 2 ] . CD f a c t o r s may r e p r e s e n t a f a m i l y o f b i o l o g i c a l l y a c t i v e p o l y p e p t i d e s , n o r m a l l y p r e s e n t i n human b o d y f l u i d s b u t w h i c h a r e o v e r - p r o d u c e d o r u n d e r - d e g r a d e d i n C F , l e a d i n g t o a g r o s s e x c e s s o f t h e s e a g e n t s i n CF p l a s m a a n d e x o c r i n e s e c r e t i o n s . E v i d e n c e f o r an e n d o g e n o u s d e f i c i e n c y o f t h e a r g i n i n e e s t e r a s e - t y p e o f p r o t e o l y t i c a c t i v i t y i n CF p l a s m a a n d e x o c r i n e f l u i d s w i l l be r e v i e w e d i n t h e p a g e s t h a t f o l l o w . R e g a r d l e s s o f t h e p r e c i s e r e l a t i o n s h i p o f CD f a c t o r a c t i v i t y t o t h e CF g e n e t i c d e f e c t , t h e s e a g e n t s may p l a y a p r i m a r y r o l e i n t h e p a t h o g e n e s i s o f t i s s u e p a t h o l o g y i n C F . By i n h i b i t i n g c i l i a r y m o t i o n and t h e m u c o c i l i a r y t r a n -s p o r t o f d e b r i s f r o m t h e l u n g s o f CF p a t i e n t s , CD f a c t o r s may p r e d i s p o s e a f f e c t e d i n d i v i d u a l s t o c h r o n i c p u l m o n a r y b a c t e r i a l c o l o n i z a t i o n a n d i t s i n e v i t a b l e s e q u e l a e [ 1 3 ] . S o d i u m R e a b s o r p t i o n I n h i b i t o r y F a c t o r ( T h e M a n g o s PAGE 10 F a c t o r ) : I n 1967 M a n g o s e_t a j _ . r e p o r t e d t h a t m i x e d s a l i v a [ 1 8 ] and s w e a t [ 1 7 ] f r o m CF p a t i e n t s c o n t a i n e d a f a c t o r w h i c h , when p e r f u s e d i n t o t h e i s o l a t e d r a t p a r o t i d g l a n d , i s c a p a b l e o f i n h i b i t i n g s o d i u m r e a b s o r p t i o n by t h e g l a n d ' s e x c r e t o r y d u c t a l e l e m e n t s . T h e p r e s e n c e o f s o d i u m r e -a b s o r p t i o n i n h i b i t o r y f a c t o r ( M a n g o s f a c t o r ) i n CF s w e a t a n d s a l i v a h a s b e e n c o n f i r m e d by o t h e r l a b o r a t o r i e s [ 3 9 , 4 0 ] . Thje c o n f i r m a t i o n by K a i s e r e t a j _ . [ 3 9 ] u s i n g n o r m a l human d i g i t a l s w e a t g l a n d s j_n s i t u s t r o n g l y a r g u e s t h a t t h e i s o l a t e d r a t p a r o t i d g l a n d i s a r e a s o n a b l e m o d e l f o r i n v i t r o s t u d y o f t h e p u t a t i v e g l a n d u l a r e l e c t r o l y t e t r a n s p o r t d e f e c t i n d u c i b l e b y CF f a c t o r s J_n v i v o . I n 1 9 7 3 , M a n g o s r e p o r t e d on m i c r o p e r f u s i o n s t u d i e s c o n d u c t e d on i s o l a t e d human s w e a t g l a n d d u c t s o b t a i n e d f r o m b i o p s y and a u t o p s y s p e c i m e n s [ 4 1 ] : when n o r m a l s w e a t was p e r f u s e d i n t o CF s w e a t d u c t s , n o r m a l l e v e l s o f s o d i u m r e -a b s o r p t i o n w e r e r e c o r d e d , b u t when CF s w e a t was p e r f u s e d i n t o n o r m a l o r CF s w e a t d u c t s , d e c r e a s e d l e v e l s o f s o d i u m u p t a k e w e r e o b s e r v e d . T h e s e o b s e r v a t i o n s [ 4 1 ] a n d t h o s e o f K a i s e r e_t aV. [ 3 9 ] s u g g e s t t h a t t h e CF s w e a t e l e c t r o l y t e d e f e c t i s an i n d u c i b l e p h e n o m e n o n p r o d u c e d by h u m o r a l PAGE 11 f a c t o r s i n CF s w e a t and i s n o t an i n h e r e n t d e f e c t o f d u c t a l t i s s u e . T h e m e c h a n i s m o f a c t i o n o f t h e M a n g o s f a c t o r i s n o t c l e a r , b u t i t d o e s n o t a p p e a r t o i n v o l v e d i r e c t i n -h i b i t i o n o f m e m b r a n e - b o u n d N a + , K + - A T P a s e [ 1 8 ] . T h e r o l e o f t h e M a n g o s f a c t o r i n t h e p r o d u c t i o n o f m a l f u n c t i o n s i n CF t i s s u e s o t h e r t h a n s w e a t a n d s a l i v a r y g l a n d s i s u n c e r t a i n. T h e M a n g o s f a c t o r h a s n o t b e e n i s o l a t e d i n p u r e f o r m and c h a r a c t e r i z e d due t o i t s l a b i l e n a t u r e [ 1 7 ] and b e c a u s e b i o a s s a y o f t h e f a c t o r i s t e d i o u s a n d t i m e - c o n s u m i n g [ 2 0 ] . H o w e v e r , l i k e t h e CD f a c t o r s , t h e M a n g o s f a c t o r i s p r o b a b l y a p o s i t i v e l y c h a r g e d m a c r o m o l e c u l e , b e c a u s e , ( 1 ) i t i s a d s o r b e d f r o m CF s a l i v a a n d s w e a t by t r e a t m e n t w i t h n e g a -t i v e l y c h a r g e d m o l e c u l e s ( e . g . h e p a r i n ) a n d by e x p o s u r e t o n e g a t i v e l y c h a r g e d s u r f a c e s ( e . g . g l a s s ) [ 4 2 ] , a n d ( 2 ) i t s a c t i v i t y i n t h e b i o a s s a y i s m i m i c k e d by p o s i t i v e l y c h a r g e d m a c r o m o l e c u l e s ( e . g . p o l y l y s i n e a n d a r g i n i n e - r i c h h i s t o n e ) [ 4 2 ] . Some o f t h e s e p r o p e r t i e s h a v e a l s o b e e n o b s e r v e d i n CD f a c t o r s a s s e s s e d by t h e o y s t e r b i o a s s a y [ 2 2 , 4 3 ] . How-e v e r , i d e n t i t y o f t h e M a n g o s f a c t o r a n d t h e CD f a c t o r s i s u n l i k e l y [ 1 3 ] . I t seems more p r o b a b l e t h a t M a n g o s f a c t o r i s o n l y o n e o f a f a m i l y o f b i o l o g i c a l l y a c t i v e c a t i o n i c p o l y p e p t i d e s n o r m a l l y p r e s e n t i n human b o d y f l u i d s PAGE 12 b u t w h i c h a c c u m u l a t e i n CF t i s s u e s and may c o n t r i b u t e t o t h e p a t h o g e n e s i s o f t h i s d i s e a s e . T h e o r e t i c a l l y , CF f a c t o r a c c u m u l a t i o n c o u l d come a b o u t e i t h e r by o v e r - p r o d u c t i o n o r by d e f i c i e n t d e g r a d a t i o n . In t h e n e x t s e c t i o n , we r e v i e w t h e e v i d e n c e f o r t h e e x i s t e n c e o f an e n d o g e n o u s d e f i c i e n c y o f p r o t e o l y t i c e n z y m e a c t i v i t y i n CF p l a s m a a n d s a l i v a . S u c h a d e f e c t m i g h t u n d e r l i e t h e a c c u m u l a t i o n o f CF f a c t o r s i n t h e s e f l u i d s . C. T h e P u t a t i v e P r o t e o l y t i c E n z y m e D e f i c i e n c y o f C F P l a s m a a n d S a l i v a : C i l i a r y d y s k i n e s i a (CD) f a c t o r s a n d t h e s o d i u m r e a b s o r p t i o n i n h i b i t o r y f a c t o r h a v e b e e n c l a i m e d t o be c a t i o n i c p o l y p e p t i d e s , r i c h i n a r g i n i n e a n d l y s i n e a m i n o a c i d r e s i d u e s [ 2 2 , 4 2 , 4 3 ] . Rao and N a d l e r , s u s p e c t i n g t h a t i n c r e a s e d a m o u n t s o f s u c h f a c t o r s m i g h t r e f l e c t a d e f i c i e n c y o f " a r g i n i n e e s t e r a s e " - t y p e o f p r o t e o -l y t i c a c t i v i t y i n t h e b o d y f l u i d s o f CF p a t i e n t s , e x a m i n e d 1 t r y p s i n - 1 i k e 1 a c t i v i t y i n CF s a l i v a [ 7 ] . T h e s e i n v e s t i g a t o r s m e a s u r e d ' t r y p s i n - 1 i k e 1 a c t i v i t y as t h e d i f f e r e n c e b e t w e e n t h e BAEE ( a - N - b e n z o y 1 - L - a r g i n i n e e t h y l e s t e r ) e s t e r a s e a c t i v i t y o f CF a n d ' n o r m a l s a l i v a 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 s o y b e a n t r y p s i n i n h i b i t o r ( S T I ) . T h e S T I - s e n s i t i v e a r g i n i n e e s t e r a s e a c t i v i t y o f PAGE 13 CF s a l i v a was 1/10 o f t h a t f o u n d i n n o r m a l s a l i v a . When s p e c i f i c t r y p s i n s u b s t r a t e s ( e . g . a - N - b e n z o y 1 - D L - a r g i n i n e - p - n i t r o - a n i 1 i d e ) w e r e t e s t e d , t h e y w e r e n o t h y d r o l y s e d b y e i t h e r CF o r c o n t r o l s a l i v a ; t h i s s u g g e s t e d t o Rao a n d N a d l e r t h a t t h i s 1 t r y p s i n - 1 i k e 1 a c t i v i t y was n o t t r y p s i n a t a l l , b u t some o t h e r p r o t e a s e w i t h a r g i n i n e e s t e r a s e a c t i v i t y , s u c h as an S T I - s e n s i t i v e k a l l i k r e i n [ 8 ] . T h e f a c t t h a t k a l l i k r e i n s o c c u r i n e x o c r i n e g l a n d s ( e . g . p a n c r e a s a n d s a l i v a r y , l a c r i m a l a n d s w e a t g l a n d s ) , known t o be a f f e c t e d b y t h e d i s e a s e p r o c e s s e s i n CF [ 1 1 , 1 3 ] , made t h i s an a t t r a c t i v e p r o p o s i t i o n . S i n c e C o l e m a n e_t a j _ . [ 4 4 ] h a d e v i d e n c e t h a t a r g i n i n e e s t e r a s e a c t i v i t y a c t i v a t e d b y k a o l i n o r e l l a g i c a c i d t r e a t m e n t o f p l a s m a m i g h t be k a l l i k r e i n , Rao e_t a j _ . [ 8 ] m e a s u r e d t h i s a c t i v i t y , u s i n g TAME ( a - N - t o s y 1 - L - a r g i n i n e m e t h y l e s t e r ) as s u b s t r a t e , 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 S T I i n CF a n d c o n t r o l p l a s m a . T h e y a s c r i b e d t h e TAME e s t e r a s e a c t i v i t y t o k a l l i k r e i n a n d f o u n d t h a t t o t a l a n d S T I - s e n s i t i v e ' k a l l i k r e i n ' a c t i v i t i e s i n CF p l a s m a ( i . e . e l l a g i c a c i d - a c t i v a t e d a r g i n i n e e s t e r a s e s o f p l a s m a ) w e r e a p p r o x i m a t e l y h a l f n o r m a l l e v e l s . ( I t s h o u l d be n o t e d h e r e t h a t e l l a g i c a c i d - a c t i v a t e d a r g i n i n e e s t e r a s e s o f p l a s m a h a v e s u b s e q u e n t l y b e e n shown t o r e p r e s e n t n o t o n l y k a l l i -k r e i n b u t a number o f o t h e r d i f f e r e n t p r o t e a s e s p e c i e s as w e l l [ 4 5 ] ) . T h e f i n d i n g s o f Rao et. a j _ . [ 8 ] w e r e d i s p u t e d by L i e b e r m a n [ 4 6 ] , who u s e d BAEE t o m e a s u r e e l l a g i c a c i d - a c t i v a t e d a r g i n i n e PAGE 14 e s t e r a s e s o f CF and c o n t r o l p l a s m a , b u t c o n f i r m e d b y C h a n et_ a l . [ 4 7 ] , who f o l l o w e d t h e a n a l y t i c a l m e t h o d s o f Rao e_t a j _ . m o r e c l o s e l y a n d , i n p a r t i c u l a r , u s e d TAME as a s u b s t r a t e f o r e s t e r a s e a c t i v i t y . Rao a n d N a d l e r [ 9 ] u s e d i o n e x c h a n g e c h r o m a t o g r a p h y a n d i s o - e l e c t r i c f o c u s i n g o f e l l a g i c a c i d - t r e a t e d p l a s m a f r o m CF a n d n o r m a l s u b j e c t s t o show t h a t n o t o n l y q u a n t i t a t i v e b u t a l s o q u a l i t a t i v e d i f f e r e n c e s o c c u r i n t h e a r g i n i n e e s t e r a s e s o f CF p l a s m a . T h e s e w o r k e r s w e r e a b l e t o r e s o l v e a r g i n i n e e s t e r a s e a c t i v i t i e s i n t o two c o m p o n e n t s by D E A E - S e p h a d e x c h r o m a t o g r a p h y : P e a k I c o n t a i n e d a p p r o x i m a t e l y 60 p e r c e n t o f t o t a l a r g i n i n e e s t e r a s e a c t i v i t y a nd was e n t i r e l y S T I - s e n s i t i v e ; P e a k I I c o n t a i n e d t h e b a l a n c e o f a c t i v i t y a n d was a l m o s t e n t i r e l y ST I -r e s i s t a n t . Rao and N a d l e r [ 9 ] f o u n d t h a t i n c h r o m a t o g r a p h e d CF p l a s m a , P e a k I was s m a l l e r t h a n i n c o n t r o l s , w h i l e P e a k I I was s i m i l a r t o c o n t r o l s . I s o e l e c t r i c f o c u s i n g o f CF and n o r m a l p l a s m a , f o l l o w e d by s t a i n i n g f o r a r g i n i n e e s t e r a s e a c t i v i t y , s h o w e d s i x r e a c t i v e b a n d s i n n o r m a l p l a s m a , b u t o n l y f i v e i n CF [ 9 ] , F u r t h e r m o r e , t h e i d e n t i t y o f t h e a b s e n t a r g i n i n e e s t e r a s e b a n d i n CF p l a s m a v a r i e d f r o m i n d i v i d u a l t o i n d i v i d u a l . Rao and N a d l e r [ 9 ] i n t e r p r e t e d t h e s e r e s u l t s t o r e f l e c t e i t h e r t h e g e n e t i c h e t e r o g e n e i t y o f t h e d i s e a s e o r a b n o r m a l a g g r e g a t i o n o r d i s a g g r e g a t i o n o f a r g i n i n e e s t e r a s e e n z y m e m o l e c u l e s i n t h e p l a s m a o f a f f e c t e d i n d i v i d u a l s . PAGE 15 As an e x t e n s i o n o f t h i s w o r k , S h a p i r a e_t aj _ . [ 1 ] u s e d S T I -a f f i n i t y c h r o m a t o g r a p h y a nd p r e p a r a t i v e i s o - e l e c t r i c f o c u s i n g t o i s o l a t e , f r o m p o o l e d c o n t r o l p l a s m a , t h e s u b - f r a c t i o n o f t h e S T I - s e n s i t i v e a r g i n i n e e s t e r a s e s h a v i n g a p i o f 5 . 5 . T h i s m a t e r i a l was t h e n e m p l o y e d t o r a i s e a n t i - s e r u m i n r a b b i t s . T h e I g G f r a c t i o n o f t h i s a n t i - s e r u m was t h e n u s e d t o a n a l y z e p r e p -a r a t i o n s o f CF and c o n t r o l p l a s m a by i m m u n o e l e c t r o p h o r e t i c a nd c r o s s e d - i m m u n o e l e c t r o p h o r e t i c t e c h n i q u e s . P r i o r t o t h e s e a n a l y s e s , CF and c o n t r o l p l a s m a s a m p l e s w e r e a c t i v a t e d w i t h e l l a g i c a c i d a n d f r a c t i o n a t e d w i t h 50 p e r c e n t s a t u r a t e d ammonium s u l p h a t e , pH 7 . 0 . T h e r e a s o n f o r i n c l u s i o n o f t h e s e d e t a i l s w i l l b e c o m e e v i d e n t i n t h e p a g e s w h i c h f o l l o w . F o l l o w i n g e l e c t r o -p h o r e s i s , t h e g e l s w e r e s t a i n e d f o r a r g i n i n e e s t e r a s e a c t i v i t y . One p r e c i p i t i n a r c , s o s t a i n e d , was r o u t i n e l y f o u n d t o be a b s e n t i n CF s a m p l e s . By c o m p a r i s o n w i t h c o n t r o l s a m p l e s , t h e m i s s i n g a r c i n CF s a m p l e s was i d e n t i f i e d as t h e f r a c t i o n o f a r g i n i n e e s t e r a s e a c t i v i t y u s u a l l y a s s o c i a t e d i n c o m p l e x w i t h a g M a c r o -g l o b u l i n ( c ^ M ) , a p l a s m a p r o t e a s e i n h i b i t o r . I t a p p e a r e d t o S h a p i r a a n d h i s c o l l e a g u e s t h a t , u n l i k e c o n t r o l a^M, CF c^M may h a v e f a i l e d t o c o m p l e x a r g i n i n e e s t e r a s e s i n a c t i v a t e d p l a s m a . To t e s t t h i s p o s s i b i l i t y , ammonium s u 1 f a t e - p r e c i p i t a t e d p l a s m a f r a c t i o n s f r o m CF and c o n t r o l s u b j e c t s w e r e a s s a y e d f o r t h e i r a b i l i t y t o b i n d b o v i n e t r y p s i n . T h e a m o u n t o f t r y p s i n PAGE 16 b o u n d was t a k e n t o be p r o p o r t i o n a l t o t h e l e v e l o f BAPNA e s t e r a s e a c t i v i t y m e a s u r e d i n t h e p r e s e n c e o f S T I . T h e t r y p s i n - b i n d i n g c a p a c i t y o f CF a^M was f o u n d t o be s i g n i f i c a n t l y d e p r e s s e d [ 1 ] . T h e s e r e s u l t s s u g g e s t e d t h a t i f t h e r e i s a d e f i c i e n c y o f o ^ M - " a r g i n i n e e s t e r a s e " p r o t e a s e c o m p l e x e s i n CF p l a s m a , i t m i g h t r e f l e c t a m o l e c u l a r d e f e c t i n t h e o^M o f CF p l a s m a , r a t h e r t h a n an a b s o l u t e d e f i c i e n c y o f p r o t e a s e m o l e c u l e s . D. T h e R o l e o f a^M i n N o r m a l P h y s i o l o g y a n d E v i d e n c e f o r an ggM D e f e c t i n C y s t i c F i b r o s i s : A l p h a 2 ~ M a c r o g 1 o b u 1 i n ( c^M) i s a u n i q u e p r o t e a s e i n h i b i t o r c a p a b l e o f b i n d i n g e n d o p e p t i d a s e s f r o m v i r t u a l l y a l l a n i m a l , p l a n t a nd b a c t e r i a l s o u r c e s a n d o f i n h i b i t i n g t h e i r p r o t e o l y t i c a c t i v i t y t o w a r d s h i g h m o l e c u l a r w e i g h t s u b s t r a t e s , w h i l e p r e s e r v i n g much o f t h e i r a c t i v i t y t o w a r d s l ow m o l e c u l a r w e i g h t s u b s t r a t e s [ 4 8 , 4 9 ] . F o r t h i s r e a s o n , some i n v e s t i g a t i o n s s u s p e c t t h a t c^M may a c t j_n v i v o as a p r o t e a s e m o d u l a t o r . H o w e v e r , t h e p r e c i s e r o l e o f t h i s m a c r o m o l e c u l e i n t h e b o d y i s s t i l l c o n j e c t u r a l . F u n c t i o n s a s c r i b e d t o o^M i n c l u d e ( a ) i n h i b i t i o n o f t h e Hageman f a c t o r - d e p e n d e n t p r o t e o l y t i c a c t i v i t i e s o f p l a s m a [ 4 8 , 5 0 ] , ( b ) r e m o v a l o f p r o t e a s e s b o u n d by o t h e r p l a s m a p r o t e a s e i n h i b i t o r s f r o m t h e c i r c u l a t o r y c o m p a r t m e n t [ 5 1 ] , ( c ) a r o l e i n t h e e n d o g e n o u s m e t a b o l i s m o f p l a s m a PAGE 17 f i b r i n o g e n [ 5 2 ] , ( d ) m o d u l a t i o n o f c e r t a i n immune r e s p o n s e s [ 5 3 ] , ( e ) i n a c t i v a t i o n o f p r o t e a s e s r e l e a s e d by i n v a s i v e p a t h o g e n s and p a r a s i t e s [ 4 9 ] , and ( f ) t r a n s p o r t o f s e r u m z i n c [ 5 4 ] . A l p f i ^ M i s f o u n d i n h i g h c o n c e n t r a t i o n s i n human p l a s m a ( a p p r o x i m a t e l y 250 m g / d l ) [ 5 5 ] , i s p r o d u c e d by a v a r i e t y o f c e l l t y p e s i n c l u d i n g h e p a t o c y t e s [ 5 5 ] , f i b r o b l a s t s [ 5 7 ] a n d m o n o c y t e s [ 5 8 ] and h a s b e e n shown t o l i n e t h e l u m e n a l s u r f a c e s o f e n d o -t h e l i a l t i s s u e s [ 5 9 ] , T h e i n t e r a c t i o n o f p r o t e a s e s w i t h n o r m a l o^M h a s b e e n i n v e s t i g a t e d t h o r o u g h l y . G e n e r a l c h a r a c t e r i s t i c s o f t h i s i n t e r -a c t i o n i n c l u d e t h e f o l l o w i n g [ 4 9 ] : ( 1 ) o n l y a c t i v e e n d o p e p -t i d a s e s r e a c t w i t h o^M; ( 2 ) u p o n c o m p l e x i n g , t h e e n z y m e i s i r r e v e r s i b l y b o u n d t o t^M; ( 3 ) t h e b i n d i n g o f a m o l e c u l e o f o^M w i t h two m o l e c u l e s o f p r o t e a s e s a t u r a t e s t h e i n h i b i t o r a n d p r e v e n t s s u b s e q u e n t b i n d i n g o f o t h e r p r o t e a s e m o l e c u l e s ; ( 4 ) t h e b o u n d e n z y m e i s m o s t i n h i b i t e d w i t h r e s p e c t t o l a r g e s u b s t r a t e m o l e c u l e s ; and ( 5 ) t h e b o u n d e n z y m e i s p r o t e c t e d f r o m h i g h m o l e c u l a r w e i g h t i n h i b i t o r s . A d d i t i o n a l c h a r a c t e r i s t i c s o f o^M o r i t s i n t e r a c t i o n s w i t h p r o t e a s e w i l l be d e s c r i b e d i n t h e a p p r o -p r i a t e c o n t e x t s i n t h e p a g e s w h i c h f o l l o w . PAGE 18 In t h e l a s t s e c t i o n , we r e v i e w e d t h e e v i d e n c e f o r a d e f i -c i e n c y i n CF p l a s m a a n d s a l i v a o f p r o t e o l y t i c a c t i v i t y o f t h e " a r g i n i n e e s t e r a s e " - t y p e . S i n c e p r e l i m i n a r y s t u d i e s by S h a p i r a et. a J L h a d s u g g e s t e d t h a t t h i s d e f i c i e n c y o f p r o t e o l y s i s m i g h t r e f l e c t a p r i m a r y d e f e c t i n CF a 2M, t h e s e w o r k e r s u n d e r t o o k a s e r i e s o f i n v e s t i g a t i o n s o f CF a 2 M [ 1 - 4 ] . F i r s t , t h e y m e a s u r e d t h e d e g r e e o f c o m p l e x i n g o f r a d i o - l a b e l l e d p r o t e a s e s f r o m e x o -. 1 p C 1 p c g e n o u s s o u r c e s ( x c J I - p a p a i n , x J I - b o v i n e t r y p s i n a n d 1 p c I - b o v i n e t h r o m b i n ) w i t h c*2M i n CF a n d c o n t r o l p l a s m a a n d f o u n d a 2 M - b i n d i n g o f t h e s e 3 p r o t e a s e s t o be s i g n i f i c a n t l y l o w e r i n CF s a m p l e s [ 2 ] , S h a p i r a and h i s c o l l e a g u e s s u g g e s t e d t h a t t h e m o l e c u l a r d e f e c t i n CF c*2M c o u l d b e : ( 1 ) a l o w e r a f f i n i t y f o r p r o t e a s e s ; ( 2 ) r e l e a s e o f p r o t e a s e f r o m t h e a 2 M - p r o t e a s e c o m p l e x e s i n CF p l a s m a b u t n o t i n c o n t r o l p l a s m a ( e v i d e n c e r e v i e w e d b e l o w s u g g e s t s t h a t n a t i v e a 2 M - p r o t e a s e c o m p l e x e s , o n c e f o r m e d , a r e n o t n o r m a l l y d i s s o c i a b l e ) o r ; ( 3 ) b l o c k a d e o f a p o r t i o n o f t h e b i n d i n g s i t e s o f CF a 2 M. T h e r e l a t i o n s h i p o f a d e c r e a s e d a r g i n i n e e s t e r a s e a c t i v i t y t o a ' d e f e c t i v e ' a 2 M h a s n e v e r b e e n s a t i s f a c t o r i l y e x p l a i n e d by S h a p i r a , N a d l e r and t h e i r c o l l e a g u e s , and one m u s t s u p p o s e t h a t t h e t h r e e e x p l a n a t i o n s s u g g e s t e d a b o v e c a r r y t h e i m p l i c a t i o n t h a t e s t e r a s e a c t i v i t y , n o t c o m p l e x e d , i s somehow ' d e s t r o y e d ' ( v i a b i n d i n g t o o t h e r p r o t e a s e i n h i b i t o r s o f p l a s m a ) . T h e p o s s i -b i l i t y o f o 2 M b l o c k a d e may s u g g e s t t h e p r e s e n c e i n CF p l a s m a o f PAGE 19 e n d o g e n o u s c t 2 M - p r o t e a s e c o m p l e x e s ( e . g . c ^ M - c o l 1 a g e n a s e o r o ^ M - e l a s t a s e ) . R e g a r d l e s s o f t h e r e a s o n f o r i m p a i r e d p r o t e a s e b i n d i n g by CF o^M, S h a p i r a e_t a j _ . [ 2 ] h y p o t h e s i z e d t h a t t h e d e f i c i e n c y o f a r g i n i n e e s t e r a s e a c t i v i t y i n CF p l a s m a p r e v i o u s l y r e p o r t e d by Rao a n d N a d l e r [ 7 - 1 0 ] c o u l d r e f l e c t t h e a b s e n c e o f an a 2 M - " a r g i n i n e e s t e r a s e " p r o t e a s e c o m p l e x . T h e l a c k o f s u c h a c o m p l e x i n CF p l a s m a and e x o c r i n e s e c r e t i o n s m i g h t , i n t u r n , e x p l a i n t h e a c c u m u l a t i o n o f a r g i n i n e a n d l y s i n e - r i c h CF f a c t o r s i n t h e s e f l u i d s . To a s s e s s t h e n a t u r e o f t h e s u s p e c t e d d e f e c t i n CF c^M, S h a p i r a e_t a j _ . [ 3 ] s o u g h t t o d e t e r m i n e t h e r a t e o f b r e a k d o w n o f o ^ M - b o v i n e t r y p s i n c o m p l e x e s j_n v i t r o , u s i n g o^M p u r i f i e d f r o m CF and c o n t r o l p l a s m a . T h e r a t e o f " a u t o d i g e s t i o n " o f o ^ M - t r y p s i n c o m p l e x e s was a s s e s s e d b y two c r i t e r i a : ( 1 ) t h e t i m e a t w h i c h h y d r o l y t i c a c t i v i t y f o r a h i g h m o l e c u l a r w e i g h t s u b s t r a t e ( c a s e i n ) b e g a n t o be m e a s u r a b l e a n d ( 2 ) t h e t i m e a t w h i c h i n h i b i t i o n o f BAEE e s t e r a s e a c t i v i t y b y a h i g h m o l e c u l a r w e i g h t i n h i b i t o r ( S T I ) b e g a n t o be m e a s u r a b l e . S h a p i r a e_t a l . [ 3 ] f o u n d t h a t t h e r a t e o f b r e a k d o w n o f n o r m a l c ^ M - t r y p s i n c o m p l e x e s was s e v e r a l t i m e s t h a t o f CF o ^ M - t r y p s i n c o m p l e x e s . O t h e r a u t h o r s c l a i m t h a t c o m p l e x e s , o n c e f o r m e d , do n o t b r e a k down [ 4 9 , 6 0 ] , a n d t h i s p o i n t i s d i s c u s s e d m o r e f u l l y b e l o w . H o w e v e r , S h a p i r a a nd h i s c o l l e a g u e s i n t e r p r e t e d t h i s f i n d i n g as an i n d i c a t i o n t h a t CF a 9 M i s a b n o r m a l . T h e f a i l u r e o f CF PAGE 20 o ^ M - t r y p s i n c o m p l e x e s t o show ' n o r m a l ' r e t u r n o f t h e a b i l i t y t o d i g e s t c a s e i n and t o be i n h i b i t e d by S T I was i n t e r p r e t e d as i n d i c a t i n g an a b n o r m a l b e h a v i o u r o f CF c*2M f r a g m e n t - p r o t e a s e c o m p l e x e s . S h a p i r a h a s a r r i v e d a t t h e f o l l o w i n g s e q u e n c e o f r e a c t i o n s i n t h e n o r m a l i n t e r a c t i o n o f a 2 M a n d t r y p s i n [ 4 ] : f a s t i ) a 2 M + t r y p s i n > t ^ M - t r y p s i n i i ) a 2 M - t r y p s i n s l o w • • • > [ a 2 ( v | _ t r y p s i n ] * i i i ) [ a 2 M - t r y p s i n ] * v e r y s 1 o w > ct 2M f r a g m e n t + t r y p s i n . S h a p i r a f e e l s t h a t s t e p 1 o c c u r s i n l e s s t h a n 30 s e c o n d s , r e s u l t i n g i n a c o m p l e x w i t h no a c t i v i t y t o h i g h m o l e c u l a r w e i g h t s u b s t r a t e s s u c h as c a s e i n o r i n h i b i t o r s s u c h as S T I . S t e p 2 t a k e s 30 - 60 m i n u t e s d u r i n g w h i c h t h e c o m p l e x u n d e r g o e s a c o n f o r m a t i o n a l c h a n g e r e s u l t i n g i n some a c t i v i t y t o c a s e i n a n d S T I . I n s t e p 3, o v e r a t l e a s t 2 h o u r s , t h i s c h a n g e d c o m p l e x d e c o m p o s e s t o c * 2 M f r a g m e n t s a n d t r y p s i n , p r e s u m a b l y d u e t o p r o t e o l y s i s b y t r y p s i n . S h a p i r a p o s t u l a t e s t h a t s t e p 2 d o e s n o t o c c u r i n c y s t i c f i b r o s i s d ue t o a d e f e c t i n a^w, i n s u p p o r t o f t h i s c o n c e p t , t h e work o f W i l s o n and F u d e n b e r g c o u l d be c i t e d [ 5 ] . U s i n g i s o e l e c t r i c f o c u s i n g , t h e s e i n v e s t i g a t o r s o b s e r v e d t h a t a p r o t e i n b a n d i s r o u t i n e l y m i s s i n g f r o m m o s t CF p l a s m a s a m p l e s . T h i s b a n d , p r e s e n t i n n o r m a l p l a s m a , was r e p o r t e d t o be PAGE 2 1 an a 2 M f r a g m e n t . On t h e b a s i s o f t h e s e f i n d i n g s , W i l s o n a n d F u d e n b e r g [ 5 ] s u g g e s t e d t h a t a 2 M f r a g m e n t s w e r e d e f i c i e n t i n CF and t h a t t h i s d e f i c i e n c y m i g h t be c o n n e c t e d w i t h t h e a c c u m u l a t i o n o f CF f a c t o r s r e p u t e d t o o c c u r i n t h i s d i s e a s e . A t v a r i a n c e w i t h t h e i d e a s o f S h a p i r a and h i s c o l l e a g u e s , m o s t p u b l i s h e d d a t a s u p p o r t t h e c o n c e p t t h a t t h e b i n d i n g o f p r o t e a s e s t o a 2 M i s i r r e v e r s i b l e u n d e r p h y s i o l o g i c a l c o n d i t i o n s [ 4 9 , 6 0 ] . H o w e v e r , m o s t a u t h o r s a g r e e t h a t , d u r i n g t h e p r o c e s s o f en z y m e b i n d i n g t o « 2 M , t h e a 2 M m o l e c u l e i s p a r t l y d i g e s t e d by t h e e n z y m e , p r o d u c i n g an a 2 M f r a g m e n t w i t h m o l e c u l a r w e i g h t o f 8 5 , 0 0 0 [ 4 8 , 4 9 , 5 2 , 6 1 ] . T h i s f r a g m e n t i s b o u n d t o t h e r e s t o f t h e a 2 M m o l e c u l e by d i s u l f i d e b o n d s a n d may be d e m o n s t r a t e d o n l y f o l l o w i n g c h e m i c a l r e d u c t i o n o f t h e m o l e c u l e [ 6 1 ] . T h e r e f o r e , t h e f i n d i n g s o f c o m p l e x d i s s o c i a t i o n b y S h a p i r a et_ a_l_. [ 3 ] may r e f l e c t some a r t i f a c t u a l i n s t a b i l i t y o f t h e i r a 2 M - t r y p s i n comp1 e x e s . I t s e e m s a p p r o p r i a t e h e r e t o p o i n t o u t t h a t t h e « 2 M p u r i f i c a t i o n p r o c e d u r e e m p l o y e d by S h a p i r a e t a]_. [ 1 , 3 , 4 ] i n v o l v e d e x p o s u r e o f a 2 M t o 1 . 6 M ammonium s u l f a t e , pH 7 . 0 . I t ha s b e e n d o c u m e n t e d by o t h e r s t h a t a 2 M h a s a s p e c i a l s e n s i -t i v i t y t o t h e NH 4+ c a t i o n . M e h l e t a j _ . [ 6 2 ] f o u n d t h a t i t s p r o t e a s e b i n d i n g a b i l i t y was a f f e c t e d by as l i t t l e as 0 . 2 M PAGE 22 ammonium s u l f a t e . U s i n g a p r e c i p i t a t i o n s t e p i n v o l v i n g 1M ammonium s u l f a t e , O h l s s o n a n d O l s s o n [ 6 3 ] f o u n d o n l y 61 p e r c e n t o f t h e i r p u r i f i e d c^M p r o d u c t r e t a i n e d p r o t e a s e b i n d i n g a b i l i t y . H e i m b u r g e r [ 6 4 ] h a s shown t h a t p r e c i p i t a t i o n o f <*2M w i t h t h i s s a l t a t pH 7.0 o r h i g h e r p r o d u c e s d e r a n g e m e n t o f t h e n a t i v e s t r u c t u r e o f c* 2M, as e v i d e n c e d by i t s i n c r e a s e d m o b i l i t y on 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 . T h e r e f o r e , i t h a s b e e n s u g g e s t e d t h a t ammonium s u l f a t e i s u n s u i t a b l e as a r e a g e n t i n c* 2M p u r i f i c a t i o n p r o c e d u r e s , w h e r e r e t e n t i o n o f n a t i v e p r o -p e r t i e s i s e s s e n t i a l [ 4 8 , 4 9 ] . In a r e c e n t p a p e r by B a r r e t t ejt a l . [ 6 5 ] , t h e a u t h o r s s h o w e d t h a t t r e a t m e n t o f p u r i f i e d a c t i v e c* 2M ( i . e . t* 2M c a p a b l e o f b i n d i n g p r o t e a s e s ) w i t h ammonium s a l t s i n c o n c e n t r a t i o n s as low as 100 mM p r o d u c e s a f o r m o f t h e m o l e c u l e m o r e m o b i l e i n e l e c t r o p h o r e s i s a n d l a c k i n g p r o t e a s e -- b i n d i n g a c t i v i t y . I n a d d i t i o n t o t h e s t u d i e s d e s c r i b e d a b o v e , S h a p i r a e_t a l . a t t e m p t e d t o c h a r a c t e r i z e t h e n a t u r e o f t h e s u p p o s e d d e f e c t i n <*2M b y a v a r i e t y o f o t h e r a p p r o a c h e s . When M i c h a e l i s - M e n t e n k i n e t i c a n a l y s e s o f t h e e n z y m e - s u b s t r a t e - i n h i b i t o r i n t e r a c t i o n w e r e p e r f o r m e d f o r n o r m a l a n d CF c* 2M, S h a p i r a e_t a l . [ 4 ] f o u n d t h a t CF a 2 M - t r y p s i n c o m p l e x e s s h o w e d s u b s t r a t e a f f i n i t y r e s e m b l i n g more c l o s e l y t h a t o f f r e e e n z y m e t h a n t h e a f f i n i t y o f n o r m a l c » 2 M - t r y p s i n c o m p l e x e s . In a d d i t i o n , t h e a p p a r e n t PAGE 2 3 b i n d i n g a f f i n i t y o f p r o t e a s e f o r CF o ^ M was f o u n d t o be 1 / 3 o f t h a t f o r n o r m a l c ^ M . When ( * 2 M - I - t r y p s i n c o m p l e x e s w e r e s u b j e c t e d t o g e l c h r o m a t o g r a p h y , 3 m a j o r p r o t e i n p e a k s w e r e s e e n i n t h e e f f l u e n t f o r n o r m a l s a m p l e s , b u t o n l y 2 m a j o r p e a k s w e r e s e e n f o r CF s a m p l e s [ 4 ] : P e a k I c o n t a i n e d a r g i n i n e e s t e r a s e a c t i v i t y , r a d i o -a c t i v i t y a n d a g M a n t i g e n i c d e t e r m i n a n t s . P e a k I p r o b a b l y r e p r e s e n t e d f r e e o ^ M a n d o ^ M - t r y p s i n c o m p l e x e s ; P e a k I I was a b s e n t i n CF s a m p l e s . I n n o r m a l s , t h i s p e a k c o n t a i n e d o n l y o ^ M a n t i g e n i c d e t e r m i n a n t s ( t h i s p e a k was p r o b a b l y o ^ M m o n o m e r s a n d t h e f r a g m e n t s a r i s i n g f r o m p r o t e o l y t i c c l e a v a g e o f m o n o m e r s o f o ^ M b y p r o t e a s e ) [ 4 ] . P e a k I I I , p r e s e n t i n c o n t r o l s a n d C F , p r o b a b l y r e p r e s e n t e d t h e l a b e l e d e n z y m e n o t b o u n d t o o ^ M . T h e f i n d i n g s o f a p p a r e n t azn f r a g m e n t s i n P e a k I I f o r n o r m a l s a m p l e s and t h e a b s e n c e o f t h e s e f o r CF s a m p l e s l e n d s u p p o r t t o W i l s o n a n d F u d e n b e r g ' s i s o e l e c t r i c f o c u s i n g d a t a [ 5 ] p r e v i o u s l y d e s c r i b e d , i n w h i c h CF p l a s m a was f o u n d t o be d e f i c i e n t i n o ^ M f r a g m e n t s by d i f f e r e n t t e c h n i q u e s . When t h e r a t i o o f t r y p s i n b i n d i n g t o o ^ M i n p e a k I was c a l c u l a t e d (cpm/mg a 2 M ) , CF o ^ M b o u n d a p p r o x i m a t e l y h a l f as much e n z y m e p e r m i l l i g r a m a s n o r m a l o ^ M . F i n a l l y , i n t h e same p a p e r o f S h a p i r a e_t a K [ 4 ] , S D S - g e l c h r o m a t o g r a p h y o f a 2 M - t r y p s i n c o m p l e x e s w i t h a n d w i t h -o u t p r e - t r e a t m e n t w i t h 8 M u r e a a n d d i t h i o t h r e i t o l g a v e r e s u l t s PAGE 24 l e a d i n g t o s i m i l a r c o n c l u s i o n s : 3 m a j o r p r o t e i n p e a k s w e r e d e t e c t a b l e i n t h e e f f l u e n t f o r n o r m a l s a m p l e s , b u t o n l y 2 f o r CF s a m p l e s [ 4 ] . The m i s s i n g p e a k i n CF s a m p l e s was a g a i n i n t e r -p r e t e d by t h e a u t h o r s t o r e f l e c t t h e a b s e n c e o f a^M f r a g m e n t s . To s u m m a r i z e , when S h a p i r a and h i s c o - w o r k e r s e x a m i n e d t h e i n t e r a c t i o n o f e x o g e n o u s p r o t e a s e s w i t h c^M d e r i v e d f r o m CF a n d c o n t r o l p l a s m a , s t r i k i n g d i f f e r e n c e s w e r e d e t e c t e d ( 1 ) i n t h e m o l a r b i n d i n g r a t i o s o f p r o t e a s e s t o c» 2M i n p l a s m a [ 2 , 4 ] , ( 2 ) i n t h e k i n e t i c i n t e r a c t i o n o f p u r i f i e d c * 2 M - b o v i n e t r y p s i n c o m p l e x e s w i t h t h e low m o l e c u l a r w e i g h t s u b s t r a t e b e n z o y l a r g i n i n e e t h y l e s t e r [ 4 ] , ( 3 ) i n t h e s t a b i l i t y o f t h e s e c o m p l e x e s o n c e f o r m e d [ 3 ] , and ( 4 ) i n t h e g e l c h r o m a t o g r a p h i c b e h a v i o r o f c o m p l e x e s 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 s o d i u m d o d e c y l s u l f a t e [ 4 ] . T h a t S h a p i r a and h i s c o l l e a g u e s d e m o n s t r a t e d d i f f e r e n c e s b e t w e e n t h e i r n o r m a l a n d CF a 2 M p r e p a r a t i o n s w i t h r e s p e c t t o t h e b i n d i n g o f p r o t e a s e s , c a n n o t be d i s p u t e d ; t h a t t h e y h a v e s u c c e e d e d i n p r o v i n g t h a t t h e a b i l i t y o f CF o^M t o b i n d p r o -t e a s e s i s d e f e c t i v e , c a n be c o n t e s t e d . In l i g h t o f t h e d o c u -m e n t a t i o n o f c*2M s e n s i t i v i t y t o a m i n e s a n d ammonium i o n p r e -v i o u s l y d e s c r i b e d , o n e s u s p e c t s t h a t t h e p u r i f i e d a 2 M ( e s p e c i a l l y t h e c o n t r o l p r e p a r a t i o n s ) u s e d by S h a p i r a et^ a j _ . may n o t h a v e b e e n s t r u c t u r a l l y a n d f u n c t i o n a l l y n a t i v e . PAGE 25 C l e a r l y , t h e w h o l e i s s u e o f p r o t e a s e - b i n d i n g b y a 2 M i n CF r e q u i r e d r e - i n v e s t i g a t i o n w i t h p a r t i c u l a r a t t e n t i o n b e i n g p a i d t o t h e q u a l i t y o f t h e « 2 M u s e d : a 2 M h a d t o be i s o l a t e d f r o m CF and c o n t r o l p l a s m a t o a h i g h s t a t e o f p u r i t y , w i t h o u t l o s s o f n a t i v e p r o t e a s e - b i n d i n g a c t i v i t y . I t i s o n l y by u s e o f s u c h m a t e r i a l t h a t a c a s e may b e g i n t o be made e i t h e r f o r o r a g a i n s t a r o l e o f a 2 M i n t h e p a t h o g e n e s i s o f C F . E. I s o 1 a t i o n o f P u r e , A c t i v e a 2 M f r o m S m a l l P 1 a s m a S a m p l e s : B e c a u s e CF i s a d i s o r d e r a f f e c t i n g c h i l d r e n , i s o l a t i o n o f n a t i v e a 2 M f r o m s m a l l v o l u m e s o f p l a s m a was d e s i r a b l e . Un-f o r t u n a t e l y , many c l a s s i c a l p r o t e i n p u r i f i c a t i o n t e c h n i q u e s c o u l d n o t be e m p l o y e d . F o r e x a m p l e , as d i s c u s s e d p r e v i o u s l y , s u b -j e c t i n g a 2 M t o an ammonium s u l f a t e p r e c i p i t a t i o n s t e p i s known t o i m p a i r t h e m o l e c u l e ' s p r o t e a s e b i n d i n g a b i l i t y [ 4 8 , 4 9 , 6 5 ] . I m m u n o a d s o r p t i o n c h r o m a t o g r a p h y w i t h a n t i - a 2 M i m m u n o g l o b u l i n s [ 6 6 ] w o u l d h a v e b e e n c o u n t e r p r o d u c t i v e as w e l l b e c a u s e t h e m a n o e u v r e a v a i l a b l e t o d e s o r b « 2 M f r o m a n t i b o d y a r e known t o i n a c t i v a t e o r d e n a t u r e t h e m o l e c u l e [ 6 7 ] . V i r c a e_t a j _ . h a d d e m o n s t r a t e d t h a t a f f i n i t y ( d y e - 1 i'gand) g e l f i l t r a t i o n c h r o m a t o g r a p h y o f human p l a s m a on a C i b a c r o n B l S e p h a r o s e C L - 6 B ( P h a r m a c i a ) ( C B S ) c o l u m n i s c a p a b l e o f i s o l a t i PAGE 26 40 p e r c e n t o f p l a s m a o^M i n a h i g h s t a t e o f p u r i t y i n a s i n g l e s t e p [ 6 8 ] . T h e m e t h o d o f V i r c a e_t a_l_. was d e v e l o p e d f o r c*2M i s o l a t i o n f r o m l a r g e p l a s m a s a m p l e s ( 1 0 0 m l ) , and f u r t h e r t h e t e c h n i q u e was h a n d i c a p p e d b y a p p l i c a b i l i t y o n l y t o p l a s m a s a m p l e s o f h a p t o g l o b i n t y p e 1-1, w h i c h w o u l d be f o u n d i n r o u g h l y o n l y 1 4.5 p e r c e n t o f human p l a s m a s a m p l e s [ 6 9 ] . Use o f p l a s m a s o f h a p t o g l o b i n t y p e s 2-1 and 2-2 i n t h e i s o l a t i o n s c h e m e r e s u l t e d i n g r o s s h a p t o g l o b i n c o n t a m i n a t i o n o f t h e a ^ M - r i c h CBS e f f l u e n t f r a c t i o n s [ 6 8 ] . O t h e r c*2M p u r i f i c a t i o n m e t h o d s a r e a v a i l a b l e [ 6 5 , 6 7 , 7 0 ] , w h i c h h a v e b e e n shown t o p r o d u c e a c t i v e a 2M, b u t a l l s t a r t w i t h l a r g e v o l u m e s o f p l a s m a ( 2 0 0 - 5 0 0 m l ) a n d g i v e c*2M y i e l d s o f l e s s t h a n 40 p e r c e n t . M o r e o v e r , u s e o f p l a s m a o f h a p t o g l o b i n t y p e 1-1 i s g e n e r a l l y p r e f e r r e d i n t h e s e t e c h n i q u e s . To m e e t t h e n e e d f o r an a 2 M p u r i f i c a t i o n t e c h n i q u e f o r p e d i a t r i c b l o o d s a m p l e s , a t w o - s t e p p r o c e d u r e was d e v e l o p e d f o r i s o l a t i o n o f a t l e a s t 98 p e r c e n t p u r e , p h y s i o l o g i c a l l y a c t i v e <*2M f r o m 5 ml p l a s m a s a m p l e s . T h e p r o c e d u r e i n v o l v e s CBS c h r o m a t o g r a p h y , f o l l o w e d by i m m u n o a d s o r p t i o n o f t h e c o n t a m i n a n t s o f t h e c * 2 M - r i c h CBS e f f l u e n t p o o l . T h i s i s o l a t i o n s c h e m e i s -a p p l i c a b l e t o a l l p l a s m a s a m p l e s i r r e s p e c t i v e o f h a p t o g l o b i n t y p e . PAGE 27 CF a n d c o n t r o l o^M p r e p a r a t i o n s w e r e made by t h i s p u r i -f i c a t i o n s c h e m e and w e r e u s e d t o r e - i n v e s t i g a t e t h e c l a i m s o f S h a p i r a e_t a]_. r e g a r d i n g c ^ M - p r o t e a s e b i n d i n g i n C F . T h e f i n d i n g s o f t h e s e s t u d i e s a r e r e p o r t e d and d i s c u s s e d i n t h e p a g e s w h i c h f o l l o w . PAGE 28 I I . M A T E R I A L S AND METHODS: A. M a t e r i a l s : A l l c h e m i c a l s u s e d w e r e o f a n a l y t i c a l o r r e a g e n t g r a d e . S o u r c e s o f c h e m i c a l s and m a t e r i a l s w e r e as f o l l o w s : S o y b e a n t r y p s i n i n h i b i t o r ( N o . T - 9 0 0 3 ) ( S I G M A ) ; P o l y b r e n e ( S I G M A ) ; a q u a s i l ( P I E R C E ) ; B l u e S e p h a r o s e C L - 6 B ( P H A R M A C I A ) ; C N B r - a c t i v a t e d S e p h a r o s e - 4 B ( P H A R M A C I A ) ; r a b b i t a n t i s e r a a g a i n s t human h a p t o g l o b i n , I g A , I g G , IgM, t r a n s f e r r i n , a l b u m i n , o ^ - a n t i t r y p s i n a n d c e r u l o p l a s m i n ( B E H R I N G / HOECHST C A N A D A ) ; V e r s a t o l p r o t e i n s t a n d a r d s ( G E N E R A L D I A G N O S T I C S ) ; M - P a r t i g e n ( R ) r a d i a l i m m u n o d i f f u s i o n p l a t e s f o r o^M, h a p t o g l o b i n a n d I g A ( B E H R I N G / H O E C H S T C A N A D A ) ; a g a r n o b l e ( D I F C O ) ; s o d i u m d i e t h y l -b a r b i t u r a t e ( F I S H E R S C I E N T I F I C ) ; b a r b i t u r i c a c i d ( E A S T M A N ) ; a c r y l a m i d e ( e l e c t r o p h o r e s i s g r a d e ) (BIORAD L A B O R A T O R I E S ) ; N , N ' - m e t h y l e n e b i s a e r y 1 a m i d e ( E A S T M A N ) ; r i b o f l a v i n ( S I G M A ) ; TEMED ( E A S T M A N ) ; h i g h m o l e c u l a r w e i g h t e l e c t r o p h o r e s i s c a l i b r a t i o n k i t (PHARMACIA F I N E C H E M I C A L S ) ; 2 X c r y s t a l l i z e d b o v i n e t r y p s i n ( T 8 2 5 3 ) ( S I G M A ) ; b e n z a m i d i n e ( S I G M A ) ; n i t r o p h e n y 1 g u a n i d i n o b e n z o a t e (NPGB) ( S I G M A ) ; BAEE ( S I G M A ) ; human c o n t r o l s e r u m f o r i m m u n o e l e c t r o p h o r e s i s ( H E L E N A ) ; h i d e p o w d e r a z u r e ( R e m a z o l b r i l l i a n t b l u e h i d e ) ( S I G M A ) ; p r o t e i n a s s a y d y e r e a g e n t c o n c e n t r a t e ( B I O - R A D ) ; YM30 u l t r a f i l t r a t i o n m e m b r a n e s ( A M I C O N ) . PAGE 29 B. E x p e r i m e n t a l S u b j e c t s : F i v e CF p a t i e n t s (mean a g e 1 3 . 0 y e a r s ; r a n g e 8-16 y e a r s ) and s i x a g e / s e x - m a t c h e d n o r m a l c o n t r o l s (mean a g e 12.4 y e a r s ; r a n g e 7-16 y e a r s ) w e r e s e l e c t e d f o r t h e s t u d y . A l l CF p a t i e n t s s h o w e d a b n o r m a l s w e a t e l e c t r o l y t e s and p a n c r e a t i c i n s u f f i c i e n c y , a n d a l l w e r e r e c e i v i n g o r a l p a n c r e a t i c e n z y m e r e p l a c e m e n t t h e r a p y . Two o f t h e p a t i e n t s s e l e c t e d w e r e r e c e i v i n g t r e a t m e n t w i t h a n t i b i o t i c s . C. I s o l a t i o n o f g ? M F r o m CF a n d C o n t r o l B l o o d S a m p l e s : F o l l o w i n g i n f o r m e d c o n s e n t by p a r e n t o r g u a r d i a n , t e n ml b l o o d s a m p l e s w e r e o b t a i n e d f r o m CF a n d c o n t r o l s u b j e c t s . B l o o d s a m p l e s w e r e o n l y e x p o s e d t o p l a s t i c o r s i l i c o n i z e d g l a s s s u r f a c e s t o p r e v e n t Hageman f a c t o r a c t i v a t i o n . E a c h 10 ml b l o o d s a m p l e was d r a w n i n t o 1 ml o f s t e r i l e 0.13M s o d i u m c i t r a t e . B l o o d s a m p l e s w e r e t h e n i m m e d i a t e l y t r a n s f e r r e d t o a c e n t r i f u g e t u b e c o n t a i n i n g 1 mg s o y b e a n t r y p s i n i n h i b i t o r a n d 4 mg P o l y b r e n e a n d m i x e d b y i n v e r s i o n . P l a s m a was h a r v e s t e d b y c e n t r i f u g a t i o n a t 500 X g a t 4" f o r 20 m i n u t e s and t h e n s u b j e c t e d t o CBS c h r o -m a t o g r a p h y [ 6 8 ] . F i v e ml p l a s m a - s o d i u m c i t r a t e m i x t u r e PAGE 30 ( w i t h o u t p r i o r d i a l y s i s a g a i n s t c o l u m n b u f f e r ) was a p p l i e d d i r e c t l y t o a s i l i c o n i z e d c o l u m n ( 1 . 5 X 90 cm) c o n t a i n i n g C i b a c r o n B l u e S e p h a r o s e C L - 6 B T h e c ^ M - r i c h f r a c t i o n o f p l a s m a was e l u t e d w i t h 50 mM T r i s HC1, pH 8.0 c o n t a i n i n g 3 mM s o d i u m a z i d e a t a r a t e o f 1.5 t o 2.0 m l / h o u r . Two ml f r a c t i o n s w e r e c o l l e c t e d and m o n i t o r e d f o r a b s o r b a n c e a t 280 nm. T h e f i r s t e f f l u e n t p e a k ( p o o l 1) c o n t a i n e d 90 p e r c e n t o f t h e p l a s m a o^M as v e r i f i e d b y r a d i a l i m m u n o d i f f u s i o n ( R I D ) a s s a y f o r a^M ( s e e F i g u r e 1) [ 7 1 ] . T h i s p o o l was t h e n s u b j e c t e d t o i m m u n o a d s o r p t i o n c h r o m a t o g r a p h y d i r e c t e d a g a i n s t c o n t a m i n a t i n g p r o t e i n s p e c i e s . P r e p a r a t i o n o f t h e i m m u n o a d s o r p t i o n c o l u m n i n v o l v e d t h e f o l l o w i n g s t e p s : C o m m e r c i a l l y - a v a i l a b l e a n t i s e r a r a i s e d i n r a b b i t s a g a i n s t human p l a s m a p r o t e i n s w e r e p o o l e d as f o l l o w s : 1 8.8 ml a n t i - h a p t o g 1 o b i n ( m a n u f a c t u r e r ' s q u o t e d t i t r e 1.52 m g / m l ) , 11.3 ml a n t i - I g A ( t i t r e 1.56 m g / m l ) , 2.5 ml a n t i - I g G ( t i t r e 1.87 m g / m l ) , 2.5 ml a n t i - I g M ( t i t r e 4.34 m g / m l ) , 2.5 ml a n t i - t r a n s f e r r i n ( t i t r e 1.3 m g / m l ) , 2.5 ml a n t i - a l b u m i n ( t i t r e 1.06 m g / m l ) , 2.0 ml a n t i - o ^ - a n t i t r y p s i n ( t i t r e 0.56 mg/ml) a n d 2.0 ml a n t i - c e r u l o p l a s m i n ( t i t r e 0.50 m g / m l ) . T h i s m a t e r i a l was t h e n t r e a t e d w i t h a f i n a l c o n c e n t r a t i o n o f 2.5 M^  ammonium s u l f a t e t o p r e c i p i t a t e t h e gamma g l o b u l i n f r a c t i o n o f t h e s e r u m p o o l . T h i s p r e c i p i t a t e was w a s h e d w i t h 3.0 M ammonium s u l f a t e a n d r e s u s p e n d e d i n p h o s p h a t e b u f f e r e d s a l i n e . T h e ammonium s u l f a t e PAGE 31 p r e c i p i t a t i o n was r e p e a t e d and t h e w a s h e d p e l l e t was r e s u s p e n d e d i n 100 mM s o d i u m b i c a r b o n a t e b u f f e r pH 8.0, c o n t a i n i n g 500 mM s o d i u m c h l o r i d e , and c o v a l e n t l y b o n d e d t o 18 g r a m s C N B r - a c t i v a t e d S e p h a r o s e 4B a c c o r d i n g t o t h e m a n u f a c t u r e r ' s i n s t r u c t i o n s . T h e gamma g l o b u l i n s w e r e a l l o w e d t o b i n d t o t h e a c t i v a t e d g e l f o r 2 h o u r s . T h e n t h e u n r e a c t e d C N B r m o i e t i e s i n t h e g e l w e r e b l o c k e d by t r e a t m e n t w i t h 200 mM g l y c i n e f o r 2 h o u r s a t r o o m t e m p e r -a t u r e . A f t e r e x h a u s t i v e w a s h i n g , t h e g e l was p a c k e d i n t o a 1.6 X 28 cm c o l u m n and e q u i l i b r a t e d w i t h 50 mM T r i s HC1, pH 7.5 c o n -t a i n i n g 250 mM s o d i u m c h l o r i d e a n d 3 mM s o d i u m a z i d e . A l i q u o t s o f CBS p o o l I, c o n t a i n i n g 6 mg a 2M b y r a d i a l i m m u n o d i f f u s i o n a s s a y , w e r e a p p l i e d t o t h e i m m u n o a d s o r p t i o n c o l u m n , and p u r i f i e d c*2M was e l u t e d w i t h t h e same b u f f e r a t a r a t e o f 15 m l / h o u r . A p p r o x i m a t e l y 70 p e r c e n t o f s t a r t i n g p l a s m a c*2M c o u l d be r e c o v e r e d i n t h i s e f f l u e n t . T h i s m a t e r i a l was c o n c e n t r a t e d t o 1 mg/ml by u l t r a f i l t r a t i o n i n s t i r r e d c e l l s u s i n g YM30 m e m b r a n e s and s t o r e d a t 4" u n t i l u s e (< 2 w e e k s ) . C o n t a m i n a n t s w e r e d e s o r b e d f r o m t h e i m m u n o a d s o r p t i o n g e l b y e l u t i o n w i t h s t a r t i n g b u f f e r a l t e r e d t o pH 2.5 u s i n g HC1. PAGE 32 D. P u r i f i c a t i o n o f B o v i n e C a t i o n i c T r y p s i n : B o v i n e c a t i o n i c t r y p s i n ( B C T ) f o r u s e i n o ^ M - p r o t e a s e b i n d i n g s t u d i e s was p u r i f i e d by t h e SE S e p h a d e x c h r o m a t o g r a p h i c m e t h o d o f S c h r o e d e r e_t a_l_. [ 7 2 ] . C o m m e r c i a l t w i c e c r y s t a l l i z e d b o v i n e t r y p s i n , a m i x t u r e o f a and & c a t i o n i c s p e c i e s a n d i n a c t i v e f r a g m e n t s , was u s e d as s t a r t i n g m a t e r i a l . On t h e 1 0 t h d a y o f c h r o m a t o g r a p h y , t h e p u r i f i e d e c a t i o n i c e n z y m e was e l u t e d f r o m t h e c o l u m n ( 2 . 6 X 70 cm) u s i n g s t a r t i n g b u f f e r ( e l u t i o n r a t e , 7 m l / h o u r ) . BCT so p u r i f i e d was d i a l y s e d a g a i n s t 1 mM h y d r o c h l o r i c a c i d w i t h 20 m_M c a l c i u m c h l o r i d e t o r e m o v e t h e c o l u m n b u f f e r and c o n c e n t r a t e d by u l t r a f i l t r a t i o n ( A m i c o n MMC w i t h YM10 m e m b r a n e s ) t o 5 mg/ml. T h i s m a t e r i a l was t h e n q u i c k f r o z e n and s t o r e d a t - 7 0 " u n t i l u s e . H o m o g e n e i t y o f p u r i f i e d BCT was d e m o n s t r a t e d by a g a r e l e c t r o p h o r e s i s ( a s d e s c r i b e d f o r I E P ) . BCT c o n t e n t o f s t o c k and w o r k i n g s o l u t i o n s was d e t e r m i n e d by e x t i n c t i o n a t 280 nm (1 g / d l e x t i n c t i o n c o e f f i c i e n t = 13.9 [ 7 3 ] ) . A c t i v i t y o f BCT s t o c k s o l u t i o n s was a s s e s s e d by a c t i v e s i t e t i t r a t i o n w i t h NPGB a c c o r d i n g t o t h e m e t h o d o f C h a s e a n d Shaw [ 7 4 ] . C o m m e r c i a l b o v i n e t r y p s i n was f o u n d t o be 5 0 - 6 0 p e r c e n t a c t i v e . P u r i f i e d & BCT was 7 5 - 8 0 p e r c e n t a c t i v e . BCT a c t i v i t y was n o t a f f e c t e d by f r e e z i n g and t h a w i n g n o r by s t o r a g e o f t h a w e d e n z y m e a t 4° f o r 1 w e e k . PAGE 33 E . E s t i m a t i o n o f P r o t e i n C o n t e n t o f C h r o m a t o g r a p h y F r a c t i o n s an_d_j* 2M I s o l a t e s : T h e p r o t e i n c o n t e n t o f c h r o m a t o g r a p h y f r a c t i o n s a n d a 2M i s o l a t e s was m e a s u r e d by e x t i n c t i o n a t 280 nm, u s i n g l g / d l e x t i n c t i o n c o e f f i c i e n t s o f 1 0 . 0 f o r CBS e f f l u e n t f r a c t i o n s a n d 9.07 f o r <x2M i s o l a t e s [ 7 5 ] . A c c u r a c y o f t h e r e s u l t s was c h e c k e d by a B r a d f o r d d y e b i n d i n g m i c r o a s s a y [ 7 6 ] u s i n g p r o t e i n s t a n d a r d s p r e p a r e d f r o m V e r s a t o l c o n t r o l s e r u m . W e i g h t s o f <*2M and BCT s t a t e d h e r e a f t e r a r e t h o s e d e t e r m i n e d by e x t i n c t i o n . T h e L o w r y p r o t e i n a s s a y [ 7 7 ] was a v o i d e d b e c a u s e o f i n t e r f e r e n c e s i n c o l o u r d e v e l o p m e n t p r o d u c e d by ' t r i s ' b u f f e r [ 7 8 ] . F. A s s e s s m e n t o f P u r i t y o f C h r o m a t o g r a p h y F r a c t i o n s a j i d _ o ^ 2 M I s o l a t e s : T h r e e t e s t s o f p u r i t y w e r e a p p l i e d t o a 2M i s o l a t e s : ( 1 ) i m m u n o e l e c t r o p h o r e s i s ( I E P ) w i t h p o l y s p e c i f i c and m o n o s p e c i f i c a n t i s e r a , ( 2 ) g r a d i e n t 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 ( g r a d i e n t - P A G E ) , and ( 3 ) s o d i u m d o d e c y l s u l f a t e 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 ( S D S - P A G E ) w i t h and w i t h o u t t h i o l r e d u c t i o n . PAGE 34 I m m u n o e l e c t r o p h o r e s i s ( I E P ) : I E P was p e r f o r m e d i n 1 g / d l a g a r g e l s u s i n g c o m m e r c i a l a p p a r a t u s (GELMAN INSTRUMENT CO.) [ 7 9 ] . A g a r s l i d e s w e r e made by d i s s o l v i n g 1 g r a m a g a r n o b l e a t 9 0 ° i n 100 ml o f 50 mM s o d i u m d i e t h y 1 b a r b i t u r a t e b u f f e r , pH 8.6 w i t h 14 mM b a r b i t u r i c a c i d , 50 mM s o d i u m a c e t a t e a n d 2 mM d i s o d i u m EDTA. Two p i s a m p l e s ( c o n t a i n i n g 12 pg p r o t e i n ) w e r e a p p l i e d t o a g a r w e l l s . A g a r g e l s w e r e e l e c t r o p h o r e s e d f o r 90 m i n u t e s a t 250 v o l t s ( a p p r o x i m a t e l y 40 ma p e r a g a r f r a m e ) . F o l l o w i n g e l e c t r o p h o r e s i s , l o n g i t u d i n a l t r o u g h s w e r e made i n e a c h g e l , and i m m u n o d i f f u s i o n o f 50 p i o f a p p r o p r i a t e p o l y o r m o n o - s p e c i f i c a n t i s e r u m was c a r r i e d o u t i n a m o i s t c h a m b e r a t a m b i e n t t e m p e r a t u r e f o r 16 h o u r s . A g a r g e l s w e r e w a s h e d w i t h n o r m a l s a l i n e , d e h y d r a t e d , s t a i n e d w i t h 2 mM C o o m a s s i e b r i l l i a n t b l u e G and d e s t a i n e d i n 30 E t 0 H : 6 5 d i s t i l l e d H 2 0 : 5 HOAc. A l p h a 2 M i s o l a t e s w e r e t e s t e d w i t h r a b b i t a n t i s e r a a g a i n s t w h o l e human s e r u m , a n d w i t h r a b b i t m o n o s p e c i f i c a n t i s e r u m a g a i n s t human a 2M, I g G , I g A , IgM, f i b r i n o g e n , B - 1 i p o p r o t e i n , o ^ - a n t i t r y p s i n , c e r u l o p l a s m i n , t r a n s f e r r i n , a l b u m i n , a n d h a p t o g l o b i n . PAGE 35 G r a d i e n t PAGE : G r a d i e n t PAGE o f o^M i s o l a t e s was p e r f o r m e d u s i n g 4 t o 26 g / d l p h o t o p o l y m e r i z e d a c r y l a m i d e g e l s a c c o r d i n g t o a m o d i f i c a t i o n o f t h e m e t h o d o f M a r g o l i s a n d K e n r i c k [ 8 0 ] . S l a b g e l s w e r e c a s t i n a c a s s e t t e , f o r m e d f r o m two t h i n g l a s s p l a t e s ( 1 2 5 x 2 6 0 x 1 mm) ( L K B ) s e p a r a t e d b y a 2 mm r u b b e r g a s k e t ( L K B ) . S a m p l e w e l l s w e r e made w i t h s t r i p s o f d y m o t a p e ( 0 . 5 X 5 X 6 mm) a f i x e d t o t h e t o p o f o n e o f t h e p l a t e s . G e l s w e r e p o u r e d i n a d a r k e n e d c o m p a r t m e n t w i t h t h e c a s s e t t e i n a v e r t i c a l p o s i t i o n . T h e a c r y l a m i d e s o l u t i o n was i n t r o d u c e d i n t o t h e b o t t o m o f t h e c a s s e t t e b y way o f two 21 g a u g e h y p o d e r m i c n e e d l e s i n s e r t e d t h r o u g h o p p o s i t e s i d e s o f t h e r u b b e r g a s k e t . T h e g r a d i e n t o f 4 t o 26 g / d l a c r y l a m i d e ( c o n t a i n i n g 5 p e r c e n t c r o s s - 1 i n k e r ) i n 90 mM T r i s HC1, pH 9.2 w i t h 9 mM b o r i c a c i d , 2.5 mM d i -s o d i u m e t h y l e n e d i ami ne t e t r a - a c e t i c a c i d ( E D T A ) a n d 13 phi r i b o f l a v i n was f o r m e d u s i n g a g r a d i e n t m a k e r ( L K B ) and p o u r e d u s i n g a p e r i s t a l t i c pump (BU C H L E R P O L Y S T A L T I C ) a t a r a t e o f 25 m l / h o u r . T h e a c r y l a m i d e g r a d i e n t > w a s s t a b i l i z e d w i t h a p a r a l l e l 30 t o 350 mM s u c r o s e d e n s i t y g r a d i e n t . To e n s u r e t h a t p o l y m e r i z a t i o n b e g a n a t t h e 4 g / d l e n d o f t h e g e l , an a n t i p a r a l l e l N , N , N 1 , N 1 - t e t r a m e t h y l e t h y 1 e n e d i a m i n e ( T E M E D ) c o n c e n t r a t i o n g r a d i e n t f r o m 0 t o 0.08 m l / d l was u s e d . G e l s w e r e o v e r l a i d w i t h b u f f e r c o n t a i n i n g 13 _u_M PAGE 36 r i b o f l a v i n and 0.1 m l / d l TEMED. P o l y m e r i z a t i o n was i n i t i a t e d by e x p o s u r e t o o v e r h e a d f l u o r e s c e n t l i g h t i n g . A f t e r 4 h o u r s , p h o t o p o l y m e r i z a t i o n was c o m p l e t e and g e l s w e r e r e m o v e d f r o m t h e c a s s e t t e a n d d i a l y s e d o v e r n i g h t t w i c e a g a i n s t 2 l i t e r c h a n g e s o f e l e c t r o p h o r e s i s b u f f e r ( 9 0 mM T r i s HC1, pH 9.2 w i t h 9 mM b o r i c a c i d a n d 2.5 mM d i s o d i u m EDTA) t o r e m o v e s u c r o s e . G r a d i e n t g e l s w e r e r u n i n a h o r i z o n t a l e l e c t r o p h o r e s i s c h a m b e r ( B R I N K M A N N / D E S A G A ) , e q u i p p e d w i t h a t h e r m o s t a t i c c o o l e r ( L A U D A ) . G e l s w e r e p r e - e 1 e c t r o p h o r e s e d f o r 30 m i n u t e s a t 70 v o l t s ( a p p r o x i -m a t e l y 2.5 ma) (BRINKMANN v o l t a g e and c u r r e n t r e g u l a t e d p o w e r s u p p l y ) . T w e n t y pg o 2 M s a m p l e s a n d s t a n d a r d p r o t e i n s w e r e a p p l i e d a t t h e c a t h o d e e n d o f t h e g e l i n d o u b l e s t r e n g t h e l e c t r o p h o r e s i s b u f f e r c o n t a i n i n g 0.75 mM b r o m o p h e n o l b l u e t r a c k i n g d y e . S a m p l e s w e r e e l e c t r o -p h o r e s e d a t 70 v o l t s f o r 30 m i n u t e s t o e n s u r e p r o t e i n e n t r y i n t o t h e 4 g / d l p o r t i o n o f t h e g e l . V o l t a g e was t h e n i n c r e a s e d t o 400 v o l t s ( a p p r o x i m a t e l y 15 m a ) , a n d g e l s w e r e r u n f o r 72 h o u r s w i t h b u f f e r c h a n g e s e v e r y 8 - 1 0 h o u r s . G e l s w e r e s t a i n e d f o r 2 h o u r s w i t h 3 mM C o o m a s s i e b r i l l i a n t b l u e G i n 5 m e t h a n o l : 1 a c e t i c a c i d : 5 d i s t i l l e d w a t e r , a n d t h e n d e s t a i n e d f o r 2 d a y s i n 2 m e t h a n o l : 1 a c e t i c a c i d : 5 d i s t i l l e d w a t e r p r i o r t o p h o t o g r a p h y . PAGE 37 S D S - P A G E : SDS-PAGE was p e r f o r m e d w i t h 5 g / d l p o 1 y a c r y 1 a m i d e t u b e g e l s , a c c o r d i n g t o a m o d i f i c a t i o n o f t h e m e t h o d o f W e b e r ejt a l . [ 8 1 ] . R e s e r v o i r and t u b e g e l b u f f e r w e r e t h e same as u s e d by t h e s e a u t h o r s , e x c e p t p o t a s s i u m p e r s u l f a t e was u s e d i n p l a c e o f ammonium p e r s u l f a t e f o r c h e m i c a l p o l y m e r i z a t i o n o f g e l s . T h e f i n a l c o n c e n t r a t i o n o f p o t a s s i u m p e r s u l f a t e i n 5 g / d l a c r y l a m i d e / 2 . 6 g / d l b i s a c r y l a m i d e s o l u t i o n was 3.3 mM. S a m p l e b u f f e r c o n t a i n e d t h e f o l l o w i n g : 35 mM SDS, 0.01 M N a P 0 4 pH 7.2, 0.14 M e - m e r c a p t o e t h a n o 1 , 10 m l / d l g l y c e r o l , a nd 30 _yM b r o m o p h e n o l b l u e . T w e n t y o r 100 yg s a m p l e s o f c^M w e r e m i x e d w i t h 100 y l o f s a m p l e b u f f e r , and i n c u b a t e d a t 37" f o r o n e h o u r , t h e n s a m p l e s w e r e a p p l i e d t o g e l s . T u b e g e l s w e r e r u n on a v e r t i c a l a p p a r a t u s ( B I O - R A D ) a t r o o m t e m p e r a t u r e . a^M s a m p l e s w e r e e 1 e c t r o p h o r e s e d a t 3 m a / t u b e f o r 16 h o u r s . G e l s w e r e t h e n r e m o v e d f r o m t u b e s and s t a i n e d and d e s t a i n e d by t h e m e t h o d d e s c r i b e d f o r g r a d i e n t s l a b g e l s . P u r i t y o f a^M i s o l a t e s was t h e n a s s e s s e d u s i n g a d e n s i t o m e t e r a t 595 nm. M o n o m e r i c c^M was d e m o n s t r a t e d by t h i s p r o c e d u r e . S a m p l e s u s e d t o d e m o n s t r a t e o^M d i m e r i c s t r u c t u r e w e r e h a n d l e d s i m i l a r l y , e x c e p t t h a t B - m e r c a p t o e t h a n o l was o m i t t e d f r o m t h e s a m p l e b u f f e r . S a m p l e s u s e d t o d e m o n s t r a t e p r o t e a s e - c o n v e r s i o n o f « , M t o the 85,000 PAGE 38 d a l t o n f r a g m e n t r e q u i r e d p r i o r i n c u b a t i o n o f 20 ug a 2 M w i t h t h e e q u i v a l e n t o f 1.31 y g 100 p e r c e n t a c t i v e b o v i n e c a t i o n i c t r y p s i n .in 50 mM T r i s HC1 pH 7.5, c o n t a i n i n g 5 mM s o d i u m c i t r a t e a n d 145 mM s o d i u m c h l o r i d e a t 37" C f o r 30 m i n u t e s , f o l l o w e d by p r o t e a s e i n a c t i v a t i o n by t r e a t m e n t w i t h 100 y l o f s a m p l e b u f f e r c o n t a i n i n g 10M u r e a a nd 140 mM e - m e r c a p t o e t h a n o l [ 6 1 ] . V G. A s s e s s m e n t o f P r o t e a s e B i n d i n g A b i l i t y o f g ^ M I s o l a t e s : T h e a b i l i t y o f a 2 M i s o l a t e s t o b i n d e n d o p e p t i d a s e s h a s b e e n t a k e n as t h e c r i t e r i o n o f n a t i v e a c t i v i t y . T h r e e i n d e p e n -d e n t t e s t s o f t h i s a c t i v i t y w e r e e m p l o y e d : ( 1 ) m o l a r p r o t e a s e b i n d i n g by a 2 M ; ( 2 ) g r a d i e n t PAGE c a l i b r a t e d w i t h a c t i v e a n d ammonium s u 1 f a t e - i n a c t i v a t e d a 2 M e 1 e c t r o p h o r e t i c s t a n d a r d s ; a nd ( 3 ) d e m o n s t r a t i o n by SDS-PAGE o f q u a n t i t a t i v e c o n v e r s i o n o f a 2 M t o t h e 8 5 , 0 0 0 d a l t o n f r a g m e n t by t r e a t m e n t w i t h b o v i n e c a t i o n i c t r y p s i n . ( 1 ) M o l a r P r o t e a s e B i n d i n g • S t u d i e s T h e m e t h o d o l o g y e m p l o y e d i n v o l v e d t i t r a t i o n o f « 2 M w i t h BCT and i s d e s c r i b e d i n s e c t i o n "H". PAGE 39 ( 2 ) G r a d i e n t PAGE: G r a d i e n t PAGE was p e r f o r m e d as d e s c r i b e d a b o v e on a^M i s o l a t e s a n d on a c t i v e ( " s l o w " ) a n d ammonium i o n -i n a c t i v a t e d ( " f a s t " ) a 2 M e l e c t r o p h o r e t i c s t a n d a r d s [ 6 5 , 8 2 ] . T h e p r o t e a s e b i n d i n g a c t i v i t y o f o^M e l e c t r o -p h o r e t i c s t a n d a r d s h a d b e e n p r e v i o u s l y e s t a b l i s h e d by t h e R B B - h i d e t i t r a t i o n a s s a y d e s c r i b e d i n s e c t i o n "H". Ammonium i o n i n a c t i v a t i o n o f o^M was a c c o m p l i s h e d by i n c u b a t i o n o f a 2 M w i t h 200 mM ammonium s u l f a t e f o r 30 m i n u t e s a t 3 7 " , f o l l o w e d by d i a l y s i s a g a i n s t CBS b u f f e r a n d c o n c e n t r a t i o n by u l t r a f i l t r a t i o n . ( 3 ) S D S - P A G E : S D S - P A G E w i t h t h i o l r e d u c t i o n was p e r f o r m e d a s d e s c r i b e d a b o v e on a 2 M r e a c t e d i n 1:1, 1:1.5 a n d 1:2 m o l a r r a t i o s w i t h BCT. H. E x p e r i m e n t s P e r f o r m e d t o C o m p a r e CF a n d C o n t r o l o t p M : ( 1 ) M o l a r P r o t e a s e B i n d i n g by CF a n d C o n t r o l a^M:. T h e t i t r a t i o n o f u n t r e a t e d « 2 M a n d a 2 M p r e - t r e a t e d w i t h 200 mM ammonium s u l f a t e f o r 30 m i n u t e s a t 37 " was PAGE 40 p e r f o r m e d u s i n g BCT and t h e i n s o l u b l e s u b s t r a t e R e m a z o l b r i l l i a n t b l u e - h i d e ( R B B - h i d e ) [ 8 3 ] as f o l l o w s : To a s e r i e s o f t u b e s c o n t a i n i n g 8 - 12 pg a 2M, t h e e q u i v a l e n t o f 0.1 t o 1.0 pg 100 p e r c e n t a c t i v e BCT was a d d e d i n a f i n a l i n c u b a t i o n v o l u m e o f 3.00 ml o f 50 mM T r i s - H C l , pH 8 . 0 . T u b e s w e r e m i x e d and p r e - i n c u b a t e d a t 3 7 ° f o r 30 m i n u t e s t o e n s u r e c o m p l e x i n g . T w e n t y m i l l i g r a m s R B B - h i d e was t h e n a d d e d t o e a c h t u b e , and t h e m i x t u r e was i n c u b a t e d a t 3 7 ° f o r 30 m i n u t e s w i t h f r e q u e n t v o r t e x i n g ( o n c e / t u b e / m i n u t e ) . T h e r e a c t i o n was t e r m i n a t e d by f i l t e r i n g o u t t h e r e m a i n i n g R B B - h i d e . T h e a m o u n t o f d y e p r o t e o l y t i c a l l y r e l e a s e d i n t o t h e m edium f r o m t h e h i d e was a s s e s s e d b y s p e c t r o p h o t o m e t r i c a s s a y o f t h e f i l t r a t e a t 595 nm. S u b s t r a t e K i n e t i c s o f CF and C o n t r o l £ ? M - B C T C o m p l e x e s : M i c h a e l i s - M e n t e n s u b s t r a t e k i n e t i c s [ 8 4 ] w e r e a s s e s s e d f o r CF a n d c o n t r o l <x2M-BCT c o m p l e x e s u s i n g t h e l o w m o l e -c u l a r w e i g h t s u b s t r a t e b e n z o y l a r g i n i n e e t h y l e s t e r ( B A E E ) as f o i l o w s : I n UV c u v e t t e s c o n t a i n i n g a f i n a l v o l u m e o f 2.00 ml 50 mM T r i s - H C l , pH 8.0 w i t h 20 mM c a l c i u m c h l o r i d e , 18 pg CF o r c o n t r o l a ?M a n d t h e e q u i v a l e n t o f 0.42 pg 100 p e r c e n t \ PAGE 41 a c t i v e BCT w e r e m i x e d and a l l o w e d t o c o m p l e x a t 37" f o r 2 m i n u t e s ; n e x t , BAEE was a d d e d t o t h e r e a c t i o n m i x t u r e t o g i v e a f i n a l c o n c e n t r a t i o n o f 0.05 t o 0.45 mM; t h e . c u v e t t e was m i x e d by i n v e r s i o n a n d p l a c e d i n t h e 3 7 ° h e a t e d a u t o m a t i c s a m p l e c h a n g i n g c o m p a r t m e n t o f a B e c k m a n m o d e l 25 s p e c t r o p h o t o m e t e r . T h e r a t e o f h y d r o l y s i s o f BAEE was f o l l o w e d f o r 3 m i n u t e s and a b s o r b a n c e c h a n g e a t 253 nm, a n d t h i s r a t e was p l o t t e d a g a i n s t s u b s t r a t e c o n c e n t r a t i o n . L i n e w e a v e r - B u r k d o u b l e r e c i p r o c a l p l o t s [ 8 5 ] o f t h e s e d a t a w e r e u s e d t o d e t e r m i n e t h e a p p a r e n t K a n d V f o r ^ r m max BAEE o f CF a n d c o n t r o l o ^ M - B C T c o m p l e x e s . S t a b i 1 i t y • o f C F a n d C o n t r o l a g M - B C T C o m p l e x e s : T h e s t a b i l i t y o f CF a n d c o n t r o l c ^ M - B C T c o m p l e x e s was t e s t e d a f t e r an 18 hour, i n c u b a t i o n a t 3 7 ° T h e c r i -t e r i o n c h o s e n t o i n d i c a t e c o m p l e x s t a b i l i t y was t h e a v a i l -a b i l i t y o f t h e a c t i v e s i t e o f o ^ M - b o u n d BCT t o h i g h m o l e c u l a r w e i g h t s u b s t r a t e ( R B B - h i d e ) and t o h i g h m o l e c u l a r w e i g h t i n h i b i t o r ( s o y b e a n t r y p s i n i n h i b i t o r [ S T I ] ) . P r o t e a s e s b o u n d i n s t a b l e c o m p l e x w i t h o ^ M w i l l show no a c t i v i t y t o w a r d s t h e s e l a r g e l i g a n d s [ 4 9 ] . D e t a i l s o f t h e s e a s s a y s w e r e as f o l l o w s : PAGE 42 N i n e t y m i c r o g r a m s o f c*2M i s o l a t e w e r e c o m p l e x e d w i t h t h e e q u i v a l e n t o f 4.4 y g 100 p e r c e n t a c t i v e BCT i n a f i n a l v o l u m e o f 1.0 ml 50 mM T r i s - H C l , pH 8 . 0 . I f t h e t o t a l BCT m o l a r b i n d i n g r a t i o o f t h e a 2 M i s o l a t e ( d e t e r m i n e d by t h e R B B - h i d e a s s a y d e s c r i b e d a b o v e ) d e v i a t e d f r o m t h e t h e o r -e t i c a l v a l u e o f 2 m o l e s B C T / m o l e c*2M ( n t h e o r e t ^ ' t n e n t h e w e i g h t o f a 2 M c o m p l e x e d w i t h 4.4 y g 100 p e r c e n t a c t i v e BCT was a d j u s t e d by ( 9 0 y g ) X ( n t n e o r e t / n e x p t ) . T h i s s o l u t i o n was i n c u b a t e d f o r 18 h o u r s a t 3 7 ° T h e s e w e i g h t s o f a 2 M a n d BCT e n s u r e d t h a t 75 p e r c e n t o f a 2 M was i n c o m p l e x w i t h a c t i v e BCT. To t e s t t h e s t a b i l i t y o f t h e o 2 M - B C T c o m p l e x i n -c u b a t e d f o r 18 h o u r s a t 3 7 ° t o w a r d s t h e h i g h m o l e c u l a r w e i g h t i n h i b i t o r S T I , t h e f o l l o w i n g p r o c e d u r e was f o l -l o w e d : I n t o a UV c u v e t t e c o n t a i n i n g 1.6 ml 50 m_M T r i s HC1, pH 8 . 0 / 2 0 m_M c a l c i u m c h l o r i d e , 200 y l o f 18 h o u r i n c u b a t e d c o m p l e x s o l u t i o n was m i x e d . One h u n d r e d m i c r o g r a m s S T I (SIGMA T - 9 0 0 3 ) was a d d e d , and t h e r e a c t i o n m i x t u r e was p r e - i n c u b a t e d a t 3 7 ° f o r 2 m i n u t e s . S u b s t r a t e B AEE was a d d e d t o g i v e a c o n c e n t r a t i o n o f 0.45 rnM i n a f i n a l r e a c t i o n v o l u m e o f 2.00 m l . T h e c u v e t t e was m i x e d by i n v e r s i o n a n d i n c u b a t e d a t 3 7 ° f o r 3 m i n u t e s . D u r i n g t h i s i n c u b a t i o n p e r i o d , t h e r a t e o f BAEE h y d r o l y s i s was f o l l o w e d PAGE 43 s p e c t r o p h o t o m e t r i c a l l y a t 253 nm. T h e s p e c i f i c a c t i v i t y ( y m o l e s BAEE h y d r o l y s e d / h o u r / m g a c t i v e BCT) o f 18 h o u r o^M-BCT c o m p l e x e s was c o m p a r e d w i t h t h e s p e c i f i c a c t i v i t y o f f r e s h l y p r e p a r e d c o m p l e x e s i n c u b a t e d f o r o n l y 2 m i n u t e s a t 3 7 ° a n d a s s a y e d i n t h e p r e s e n c e o f 100 pg S T I . To show t h a t a l l e s t e r o l y t i c a c t i v i t y e x p r e s s e d i n t h e p r e s e n c e o f S T I was a s s o c i a t e d w i t h t h e c^M-BCT c o m p l e x , an e q u i -v a l e n t w e i g h t o f BCT a l o n e ( 0 . 8 8 p g ) o r o^M a l o n e ( 1 8 p g ) was t r e a t e d w i t h S T I and a s s a y e d as b e f o r e . To t e s t t h e s t a b i l i t y o f 18 h o u r o^M-BCT c o m p l e x e s t o w a r d t h e h i g h m o l e c u l a r w e i g h t s u b s t r a t e R B B - h i d e , t h e f o l l o w i n g p r o c e d u r e was u t i l i z e d : T o e a c h t u b e , 133 p i o f 18 h o u r - c o m p l e x s o l u t i o n was a d d e d t o 50 mj^ T r i s - H C l , pH 8.0 t o g i v e a f i n a l i n c u b a t i o n v o l u m e o f 3.0 m l . To t h i s s o l u t i o n , 20 mg i n s o l u b l e R B B - h i d e s u b s t r a t e was a d d e d a n d t h e m i x t u r e was i n c u b a t e d a t 3 7 ° f o r 15 m i n u t e s w i t h f r e -q u e n t m i x i n g ( o n c e / m i n / t u b e ) . T h e r e a c t i o n was t e r m i n a t e d by f i l t e r i n g o u t t h e r e m a i n i n g R B B - h i d e . T h e a m o u n t o f p r o t e o l y t i c a l l y r e l e a s e d d y e was m e a s u r e d b y s p e c t r o -p h o t o m e t r y a s s a y o f t h e f i l t r a t e a t 595 nm. T h e a m o u n t o f d y e r e l e a s e d by t h e c o m p l e x was e x p r e s s e d as a p e r c e n t a g e o f d y e r e l e a s e d b y an e q u i v a l e n t a m o u n t o f f r e e 100 p e r c e n t a c t i v e BCT .(0.59 p g ) i n c u b a t e d w i t h s u b s t r a t e i n t h e a b s e n c e o f a 9 M . PAGE 44 ( 4 ) S u b u n i t S t r u c t u r e o f CF a n d C o n t r o l a , , M : T h e n a t i v e s u b - u n i t s t r u c t u r e o f a g M was demon-s t r a t e d b y t h e e l e c t r o p h o r e t i c t e c h n i q u e s d e s c r i b e d e a r l i e r ( s e c t i o n " F " ) c a l i b r a t e d w i t h a p p r o p r i a t e h i g h m o l e c u l a r w e i g h t e l e c t r o p h o r e s i s m a r k e r p r o t e i n s ( P H A R M A C I A ) . G r a d i e n t PAGE was u s e d t o show t h a t o u r <* 2M i s o l a t e s p o s s e s s e d n a t i v e t e t r a m e r i c s t r u c t u r e . S D S -PAGE was u s e d t o show t h e s t r u c t u r e o f o ^ M i s o l a t e s as f o l l o w s : SDS t r e a t m e n t p e r m i t t e d d e m o n s t r a t i o n o f d i m e r s ; e - m e r c a p t o -e t h a n o l + SDS t r e a t m e n t p e r m i t t e d d e m o n s t r a t i o n o f m o n o m e r s and 8 5 , 0 0 0 d a l t o n p r o t e o l y t i c f r a g m e n t s p r o d u c e d by t r y p s i n . F o r e x p l a n a t i o n s o f t h e t h e o r y b e h i n d t h e s e t r e a t m e n t s , s e e t h e D i s c u s s i o n s e c t i o n . I . S t a t i s t i c a l T r e a t m e n t o f D a t a : C o m p a r a t i v e d a t a g e n e r a t e d on CF a n d c o n t r o l o ^ M b y t h e f o r e g o i n g e x p e r i m e n t a l m e t h o d s was a n a l y s e d by t h e W i l c o x o n r a n k sum t e s t f o r u n p a i r e d s a m p l e s [ 8 6 ] . T h i s n o n - p a r a m e t r i c s t a t -i s t i c a l m e t h o d i s i d e a l f o r d a t a a n a l y s i s o f s m a l l s a m p l e s i z e s w h e r e p o p u l a t i o n c h a r a c t e r i s t i c s ( e . g . t y p e o f d i s t r i b u t i o n a n d h o m o g e n e i t y o f v a r i a n c e s ) a r e unknown [ 8 7 ] . PAGE 45 T h e S p e a r m a n r a n k c o r r e l a t i o n t e s t [ 8 6 ] was u s e d t o show t h a t t h e b i o c h e m i c a l p a r a m e t e r s m e a s u r e d i n t h e s e s t u d i e s d i d n o t c o r r e l a t e w i t h a g e o r s e x f o r c o n t r o l s u b j e c t s , n o r d i d t h e y c o r r e l a t e w i t h a g e , s e x , a n t i b i o t i c u s e o r s e v e r i t y o f d i s e a s e ( a s a s s e s s e d by Shwachman s c o r e s [ 9 7 ] ) f o r a f f e c t e d s u b j e c t s . PAGE 46 I I I . R E S U L T S : A. I s o l a t i o n o f a,M f r o m CF a n d C o n t r o l P l a s m a F i g u r e 1 s hows a t y p i c a l 2 80 nm a b s o r b a n c e p r o f i l e f o r e f f l u e n t f r a c t i o n s o b t a i n e d f r o m c h r o m a t o g r a p h i n g human p l a s m a on a c i b a c r o n b l u e s e p h a r o s e ( C B S ) c o l u m n . T h e f i r s t e f f l u e n t p e a k ( F i g u r e 1, p o o l 1) c o n t a i n e d 90 p e r c e n t o f t h e s t a r t i n g a 2 M o f p l a s m a as v e r i f i e d b y r a d i a l i m m u n o d i f f u s i o n ( R I D ) a s s a y f o r o^M. T h e a 2 M - r i c h CBS p o o l 1 was t h e n s u b j e c t e d t o a s e c o n d c h r o m a t o g r a p h i c s t e p : i m m u n o a d s o r p t i o n t o r e m o v e c o n t a m i n a t i n g p r o t e i n s p e c i e s . F i g u r e 2 s h o w s t y p i c a l 280 nm a b s o r b a n c e p r o f i l e s f o r (a) p u r i f i e d a 2 M e l u t e d u n d e r s t a r t i n g c o n d i t i o n s f r o m t h e i m m u n o a d s o r b a n c e c o l u m n and f o r ( b ) p r o t e i n c o n t a m i n a n t s d e s o r b e d f r o m t h e c o l u m n b y c h a n g i n g t o pH 2.5 c h r o m a t o g r a p h y b u f f e r . B. D e t e r m i n a t i o n o f P r o t e i n C o n t e n t s o f a,M P r e p a r a t i o n s : T h e p r o t e i n c o n t e n t s o f a 2 M 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 b y e x t i n c t i o n o f 280 nm, u s i n g t h e 1 g / d l e x t i n c t i o n c o e f f i c i e n t o f 9.07 [ 7 5 ] . T h e a c c u r a c y o f t h e s e r e s u l t s was c h e c k e d by a PAGE 47 F i g . 1 : CIBACRON BLUE SEP HARPSE ( C B S )  CHROMATOGRAPHY OF HUMAN PLATMA: E f f l u e n t P o o l 1 c o n t a i n s 90 p e r c e n t o f s t a r t i n g p l a s m a a 2 M as d e t e r -m i n e d b y r a d i a l i m m u n o d i f f u s i o n . PAGE 49 F i g . 2: IMMUNOADSORPTION CHROMATOGRAPHY OF CBS POOL 1: ( a ) e l u t i o n o f p u r e a 2 M u n d e r s t a r t i n g c o n d i t i o n s , a n d ( b ) e l u t i o n o f c o n t a m i n a t i n g p r o t e i n s p e c i e s w i t h pH 2.5 b u f f e r . ( a r r o w i n d i c a t e s c h a n g e t o a c i d i c c o n d i t i o n s ) (a) J l (b) 0.2 o oo C M < 0.1 1 40 5 0 6 0 0 " 4 0 F R A C T I O N N U M B E R 5 0 6 0 PAGE 52 F i g . 3: ASSAY OF P R O T E I N CONTENTS  OF g?M P R E P A R A T I O N S :  C o r r e l a t i o n o f D e t e r m i n a t i o n s  made by_28Q^nm E x t i n c t i o n a n d  b y t h e B r a d f o r d D y e - B i n d i n g - A s s a y : C o r r e l a t i o n c o e f f i c i e n t f o r b e s t l i n e , R= 0 . 9 0 0 (n = l l ) . ( p g 2 8 0 nm e x t i n c t i o n p r o t e i n ) = ( 1 . 4 5 ) X ( y g B r a d f o r d p r o t e i n ) PAGE 51 s e c o n d i n d i r e c t p r o t e i n e s t i m a t i o n m e t h o d , t h e B r a d f o r d d y e b i n d i n g a s s a y [ 7 6 ] . T h e m e t h o d s c o r r e l a t e d w e l l ( R = 0 . 9 0 , n = l l , [ s e e F i g u r e 3 ] ) and c o u l d be r e l a t e d as f o l l o w s : ( u g B r a d f o r d p r o t e i n ) X ( 1 . 4 5 ) = ( y g e x t i n c t i o n 2 8 0 nm p r o t e i n ) . C. P r o o f o f P u r i t y o f g,M I s o l a t e s : P u r i t y o f o^M i s o l a t e s was d e m o n s t r a t e d by t h r e e i n d e -p e n d e n t t e s t s : ( 1 ) IEP o f o^M i s o l a t e s r e v e a l e d a s i n g l e p r e c i p i t i n a r c when d e v e l o p e d by p o l y s p e c i f i c a n t i - s e r a ( F i g u r e 4 A ) . P o s i t i v e i d e n t i f i c a t i o n o f t h e a r c as o^M was a c c o m -p l i s h e d u s i n g m o n o s p e c i f i c r a b b i t a n t i h u m a n o^M s e r u m ( F i g u r e 4 B ) . T h e p r o d u c t was f r e e o f t e n o f t h e p o t e n t i a l c o n t a m i n a n t s t h e o r e t i c a l l y e x p e c t e d ( s e e M e t h o d s s e c t i o n ) a t l e v e l s w h i c h w o u l d h a v e d e t e c t e d a p p r o x i m a t e l y 5 p e r c e n t w/w c o n t a m i n a t i o n . ( 2 ) A s i n g l e h i g h m o l e c u l a r w e i g h t b a n d i s o b s e r v e d when 20 y g c^M i s a p p l i e d t o g r a d i e n t PAGE ( F i g u r e 5, t r a c k s 1, 2, 4, 5 ) . 0.6 1.2 1.8 2 4 C o n c D by B r a d f o r d a s s a y ( n W m l ) PAGE 54 F i g . 4: IMMUNOELECTROPHORESIS OF a 2Mj_ T h e t o p w e l l o f e a c h s l i d e c o n t a i n s human c o n t r o l s e r u m . T h e b o t t o m . w e l l c o n t a i n s a 2 M . S l i d e A was d e v e l o p e d a g a i n s t h o r s e a n t i - h u m a n s e r u m p r o t e i n s e r u m . S l i d e B a g a i n s t r a b b i t a n t i - h u m a n <* 2 M s e r u m . PAGE 55 PAGE 56 F i g 5. GRADIENT PAGE OF a ? M IN 4 TO  26 g / d l - G E L S : T r a c k s 1 , 4 , 5 - a c t i v e a 2 M i s o l a t e d f r o m c o n t r o l s u b j e c t s . T r a c k 2 - a c t i v e a 2 M i s o l a t e d f r o m a c y s t i c f i b r o s i s p a t i e n t . T r a c k 3 - i n a c t i v e (ammonium s u l f a t e - t r e a t e d ) a 2 M c o - e l e c -t r o p h o r e s e d w i t h P h a r m a c i a h i g h m o l e c u l a r w e i g h t c a l i b r a t i o n s t a n d a r d s ( t h y r o g 1 o b u 1 i n , f e r r i t i n , c a t a l a s e , l a c t a t e d e h y d r o g e n a s e , and b o v i n e s e r u m a l b u m i n ) . T h e b a n d n e a r e s t t h e o r i g i n ( s e e s o l i d l i n e ) i s t h e t r e a t e d a 2 M . PAGE 57 1 2 3 4 5 PAGE 58 S DS-PAGE w i t h t h i o l r e d u c t i o n o f 20 y g a 2 M i s o l a t e s r e v e a l e d l e s s t h a n 1 p e r c e n t c o n t a m i n a t i o n by w e i g h t when s t a i n e d g e l s w e r e a n a l y s e d by d e n s i t o m e t r y ( F i g u r e 6 A ) . A p p l i c a t i o n o f 5 t i m e s t h e u s u a l a m o u n t o f o^M t o t h e g e l a l l o w e d v i s u a l a s s e s s m e n t o f c o n t a m i n a t i o n ( F i g u r e 6 B ) . a 2 M i s o l a t e s w e r e f o u n d u n i f o r m l y t o be o f h i g h p u r i t y ( a t l e a s t 98 p e r c e n t ) r e g a r d l e s s o f h a p t o g l o b i n t y p e o f s t a r t i n g p l a s m a . P r o o f o f P r o t e a s e - B i n d i n g A b i l i t y o f c t g M l s o l a t e s : M o l a r P r o t e a s e B i n d i n g by a 2 M j ^ a 2 M - p r o t e a s e c o m p l e x e s p o s s e s s no r e s i d u a l p r o t e o -l y t i c a c t i v i t y t o w a r d s h i g h m o l e c u l a r w e i g h t s u b s t r a t e s [ 4 9 ] . P r o t e a s e a d d e d t o a 2 M i n e x c e s s o f t h e b i n d i n g c a p a c i t y w i l l h y d r o l y s e t h e h i g h m o l e c u l a r w e i g h t s u b s t r a t e R B B - h i d e . F i g u r e 7 s h o w s t y p i c a l t i t r a t i o n c u r v e s o f p r o -t e a s e a d d e d t o u n t r e a t e d a n d ammonium i o n - t r e a t e d t h e n r e a c t e d w i t h R B B - h i d e . T h e w e i g h t o f 100 p e r c e n t a c t i v e BCT r e q u i r e d t o s a t u r a t e t h e b i n d i n g s i t e s o f 8 - 12 y g <*2M c a n be f o u n d b y e x t r a p o l a t i o n . A s s u m i n g t h e s t o i c h i o m e t r y o f b i n d i n g t o be 2 m o l e s t r y p s i n p e r m o l e SDS-PAGE WITH T H I O L REDUCTION  OF gpM I S O L A T E D FROM HAPTO-GLOBIN T Y P E 2 - 2 PLASMA: T u b e g e l A c o n t a i n s 2 0 y g a g M . T u b e g e l B c o n t a i n s 1 0 0 yg o f t h e same c ^ M . PAGE 60 PAGE 61 F i g . 7: MOLAR P R O T E A S E B I N D I N G BY UNTREATED g ? M AND ct?M P R E - T R E A T E D WITH AMMONIUM ION: ( a ) T i t r a t i o n c u r v e f o r a2M p r e - t r e a t e d w i t h 200 mM ammonium s u l f a t e f o r 30 m i n u t e s a t 3 7 ° . W e i g h t o f c^M t i t r a t e d = 1 0 . 3 y g . W e i g h t o f BCT b o u n d b y i n a c t i v a t e d a 2 M=0.19 y g . M o l a r b i n d i n g r a t i o = 0 . 5 7 m o l e s B C T / m o l e i n a c t i v a t e d 0 2 M . ( b ) T i t r a t i o n c u r v e f o r u n t r e a t e d (*2M p r e p a r a t i o n . W e i g h t o f 02M t i t r a t e d = 1 0.3 y g . W e i g h t o f BCT b o u n d by a 2 M=0.61 y g . M o l a r b i n d i n g r a t i o = 1.80 m o l e s B C T / m o l e a2M. PAGE 63 oigM [ 6 0 , 6 5 , 7 0 ] , a n d a s s u m i n g m o l e c u l a r w e i g h t s o f 2 3 , 8 0 0 an d 7 2 5 , 0 0 0 r e s p e c t i v e l y [ 8 8 , 8 9 ] , one c a n c a l c u l a t e t h a t 1 vg BCT s h o u l d be b o u n d by 15.2 v q a 2 M . T h i s i n f o r m a t i o n and t h e t i t r a t i o n d a t a a l l o w s one t o c o m p u t e t h e a c t i v i t y o f t h e u n t r e a t e d a n d ammonium i o n - t r e a t e d o^M i s o l a t e s . C o m p a r a t i v e m o l a r p r o t e a s e b i n d i n g d a t a f o r CF and c o n t r o l <*2M p r e p a r a t i o n s a r e p r e s e n t e d i n t a b u l a r f o r m i n s e c t i o n " E " . G r a d i e n t P A G E o f a 2 M I s o 1 a t e s : When a 2 M b i n d s p r o t e a s e s t h e r e i s a c h a n g e i n i t s a b i l i t y t o p e n e t r a t e g r a d i e n t g e l s w h i c h h a v e i n c r e a s i n g p o l y a c r y l ami de c o n c e n t r a t i o n [ 8 2 , 6 5 ] . B a r r e t t e t a]_. [ 6 5 ] h a v e s u g g e s t e d t h a t t h e s u b u n i t s o f t h e a c t i v e t e t r a m e r a r e a r r a n g e d i n a c o p l a n a r c o n f i g u r a t i o n , w h i l e t h o s e o f t h e i n a c t i v a t e d m a c r o m o l e c u l e may f o r m t h e more c o m p a c t p s e u d o -t e t r a h e d r o n . T h i s w o u l d a l l o w t h e i n a c t i v e m a c r o m o l e c u l e t o p e n e t r a t e f u r t h e r i n t o t h e p o r e s o f a g r a d i e n t a c r y l -a m i d e g e l m a t r i x , a n d c o u l d a c c o u n t f o r i t s " f a s t " p o s i t i o n on t h e s e g e l s r e l a t i v e t o a c t i v e ( " s l o w " ) a 2 M . W h a t e v e r t h e r e l a t i v e c o n f i g u r a t i o n s o f a c t i v e a n d i n a c t i v e a 2 M, g r a d i e n t PAGE h a s b e c o m e an e s t a b l i s h e d t e c h n i q u e t o d e t e r -m i n e t h e p r o t e a s e b i n d i n g a c t i v i t y o f o 2 M i s o l a t e s [ 6 5 , 8 2 ] . I n t h e p r e s e n t s t u d i e s , ammonium i o n - i n a c t i v a t e d PAGE 64 ( " f a s t " ) was u s e d as an e l e c t r o p h o r e t i c m a r k e r p r o t e i n t o d e m o n s t r a t e t h a t o u r c t 2 M i s o l a t e s c o n t a i n o n l y t h e a c t i v e , e l e c t r o p h o r e t i c a l l y " s l o w " m o l e c u l a r s p e c i e s ( F i g u r e 5 ) . T r a c k s 1, 2, 4 a n d 5 show e l e c t r o p h o r e s i s o f t y p i c a l c tgM i s o l a t e s a l o n e . T r a c k 3 sh o w s c o - e l e c -t r o p h o r e s i s o f t h e f a s t a 2 M s t a n d a r d w i t h c o m m e r c i a l m o l e c u l a r w e i g h t m a r k e r p r o t e i n s ( P h a r m a c i a ) . S D S - P A G E W i t h T h i o l R e d u c t i o n o f a 2 M I s o l a t e s a n d a 0 M - B C T C o m p l e x e s : T r e a t m e n t o f p u r i f i e d c ^ M w i t h SDS a n d 8 - m e r c a p t o -e t h a n o l r e s u l t e d i n p r o d u c t i o n o f a s i n g l e b a n d , i . e . mono-m e r i c a 2 M ( F i g u r e 8 a ) a s s e s s e d by PA G E . P r e - t r e a t m e n t o f <* 2M w i t h i n c r e a s i n g a m o u n t s o f BCT up t o a 1:2 m o l a r r a t i o c a u s e s s t o i c h i o m e t r i c c o n v e r s i o n o f t h i s monomer t o t h e 8 5 , 0 0 0 d a l t o n f r a g m e n t ( F i g u r e 8 b - d ) . Had t h e a 2 M i s o l a t e s b e e n p a r t i a l l y i n a c t i v a t e d b y p r o t e a s e s d u r i n g t h e p u r i f i c a t i o n p r o c e s s , F i g u r e 8 a w o u l d h a v e s hown c o n t a m -i n a t i o n by t h e 8 5 , 0 0 0 d a l t o n f r a g m e n t . Had t h e a 2 M i s o -l a t e s n o t b e e n a c t i v e , BCT t r e a t m e n t w o u l d h a v e p r o d u c e d p r o t e o l y t i c f r a g m e n t s o f a 2 M , a n d t h e s e w o u l d h a v e a p p e a r e d as a s m e a r , r a t h e r t h a n as a s i n g l e d i s c r e t e b a n d o f 8 5 , 0 0 0 d a l t o n s ( F i g u r e 8 d ) . PAGE 65 F i g . 8: SDS-PAGE o f a ? M SHOWING THE S T O I C H I O M E T R I C  CONVERSION BY BCT OF THE g?M MONOMER TO THE a?M HALF-MONOMER (85K F R A G M E N T ) : Ja) u n t r e a t e d a 2 M when e x p o s e d t o SDS a n d t h i o l r e d u c t i o n shows one m o n o m e r i c b a n d ; ( b - d ) C12M p r e - t r e a t e d w i t h BCT ( 1 . 0 , 1.5 a n d 2.0 m o l e s p r o t e a s e / m o l e a 2 M , r e s p e c -t i v e l y ) shows i n c r e a s i n g p r o t e o l y t i c c o n v e r s i o n o f m o n o m e r s t o h a l f m o n o m e r s . • • m b d K A G E 67 E x p e r i m e n t s P e r f o r m e d t o C o m p a r e C F a n d C o n t r o l a ^ H : M o l a r P r o t e a s e B i n d i n g by U n t r e a t e d g , M a n d by  Ammonium I o n - t r e a t e d g^M: T a b l e s I a n d I I show t h e r e s u l t s o f t h e s e s t u d i e s . U n t r e a t e d CF g 2 M c o u l d b i n d 1.97 ± 0.18 m o l e s B C T / m o l e g 2 M c o m p a r e d t o 1.72 ± 0.29 f o r c o n t r o l s . Ammonium i o n - i n a c t i v a t e d CF g 2 M c o u l d b i n d 0.69 ± 0.05 m o l e s B C T / m o l e a 2 M c o m p a r e d t o 0.74 ± 0.15 f o r c o n t r o l s . None o f t h e s e d i f f e r e n c e s w e r e f o u n d t o be s t a t i s t i c a l l y s i g n i -f i c a n t b y t h e W i l c o x o n Rank Sum t e s t f o r u n p a i r e d s a m p l e s [ 8 6 ] . I n t e r a c t i o n o f CF a n d C o n t r o l g 2 M - B o v i n e C a t i o n i c  T r y p s i n ( B C T ) C o m p l e x e s and N o n - C o m p 1 e x e d BCT w i t h B e n z o y l A r g i n i n e E t h y l E s t e r ( B A E E ) : F i g u r e 9 shows t h e M i c h a e l i s - M e n t e n v e l o c i t y y_s s u b s t r a t e c o n c e n t r a t i o n p l o t o f t h e i n t e r a c t i o n o f t y p i c a l a 2 M - B C T c o m p l e x e s w i t h t h e low m o l e c u l a r w e i g h t s u b s t r a t e B A E E . F i g u r e 10 shows t h e L i n e w e a v e r - B u r k d o u b l e r e c i p r o c a l r e p r e s e n t a t i o n [ 8 5 ] o f t h i s d a t a f r o m w h i c h a p p a r e n t K a n d V p a r a m e t e r s w e r e d e r i v e d . PAGE 68 T A B L E I . MOLAR P R O T E A S E B I N D I N G R A T I O S FOR CF AND CONTROL « 2 M NOT EXPOSED TO AMMONIUM S U L F A T E TREATMENT PAGE 69 S U B J E C T S : MOLES BCT/MOLE a 2 M CF ( n = 5 ) 1.97 ± 0 . 1 8 * CONTROL (n = 6) 1 .72 ± 0 . 2 9 * N.S. * mean ± S.D. v a l u e s b a s e d u p o n q u a n t i t a t i o n o f o^M b y e x t i n c t i o n a t 2 8 0 nm (1 g / d l e x t i n c t i o n c o e f f i c i e n t = 9.07 [ 7 5 ] ) ** s t a t i s t i c a l s i g n i f i c a n c e t e s t e d a t t h e p < 0.05 l e v e l . PAGE 70 T A B L E I I . MOLAR P R O T E A S E B I N D I N G R A T I O S FOR CF AND CONTROL a 2 M T R E A T E D WITH 200 mM AMMONIUM S U L F A T E FOR 30 MINUTES AT 37" P A G E 71 S U B J E C T S : M O L E S B C T / M O L E I N A C T I V A T E D a 2 M C F ( n = 5 ) 0.69 ± 0 . 0 5 * ( N . S . * * ( C O N T R O L ( n = 6 ) 0.74 ± 0 . 1 5 * * mean ± S . D . ** s t a t i s t i c a l s i g n i f i c a n c e t e s t e d a t t h e p < 0.05 l e v e l . PAGE 72 F i g . 9: S U B S T R A T E K I N E T I C S OF g?M-BCT C O M P L E X E S :  V e l o c i t y v s S u b s t r a t e Con c e n t r a t i o n • P 1 o t eZ 39Vd PAGE 74 F i g . 10: S U B S T R A T E K I N E T I C S OF g?M-BCT COMPLEXES: L i n e w e a v e r - ^ - B u r k P l o t : 1 / V E L O C I T Y (JJ moles B A E E hydrol/h.^ig active B C T ) o O - L -1 CD > m m 9Z 39Vd PAGE 76 T a b l e I I I shows t h e a p p a r e n t a f f i n i t y a n d m a x i m a l v e l o c i t y o f a c t i v a t i o n p a r a m e t e r s f o r CF a n d c o n t r o l agM-BCT c o m p l e x e s as w e l l as K a n d V m o „ d a t a f o r n o n - c o m p l e x e d m ifia x B C T . I n a d d i t i o n , t h e r e s i d u a l e s t e r o l y t i c a c t i v i t y o f o^M-BCT c o m p l e x e s t o BAEE ( e x p r e s s e d as a p e r c e n t o f f r e e BCT a c t i v i t y ) i s l i s t e d i n T a b l e I I I . T h e r e s i d u a l e s t e r o l y t i c a c t i v i t y o f c o m p l e x e s i s a m e a s u r e o f t h e d e g r e e o f i n h i b i t i o n o f BCT a c t i v i t y t o BAEE u p o n b i n d i n g o f p r o t e a s e t o o^M. None o f t h e d i f f e r e n c e s f o r CF a n d c o n t r o l o^M-BCT c o m p l e x e s t a b u l a t e d w e r e f o u n d t o be s t a t i s t i c a l l y s i g n i f i c a n t . S t a b i l i t y o f CF a n d C o n t r o l c t g M - B C T C o m p l e x e s t o H i g h M o l e c u l a r W e i g h t S u b s t r a t e s and I n h i b i t o r s : T h e i n t e r a c t i o n o f t h e a c t i v e s i t e o f t h e a^M-BCT c o m p l e x w i t h t h e h i g h m o l e c u l a r w e i g h t i n h i b i t o r s o y b e a n t r y p s i n i n h i b i t o r ( S T I ) was e x a m i n e d . agM-BCT c o m p l e x e s l e f t a t 37" f o r 18 h o u r s w e r e c o m p a r e d w i t h f r e s h l y p r e -p a r e d (2 m i n u t e ) c o m p l e x e s i n t e r m s o f a b i l i t y t o h y d r o l y s e BAEE i n t h e p r e s e n c e o f S T I . I t was f o u n d t h a t t h e d e g r e e o f i n t e r a c t i o n o f CF o r c o n t r o l c o m p l e x e s w i t h S T I d o e s n o t c h a n g e e v e n a f t e r 18 h o u r s i n c u b a t i o n ( s e e T a b l e I V ) . PAGE 77 T A B L E I I I . I N T E R A C T I O N OF CF AND CONTROL a 2 M - B C T COMPLEXES AND NON-COMPLEXED BCT WITH BAEE APPARENT Km SUBJECTS (UM) Vmax (yMOLES BAEE HYDROLYSED/ HOUR/yg ACTIVE BCT) RESIDUAL ESTEROLYTIC ACTIVITY OF COMPLEXES ( p e r c e n t f r e e BCT a c t i v i t y ) CF (n=5) 43 ± 6* 1.54 ± 0.03* 67.2 p e r c e n t ± 2.9* CONTROL (n=6) 52 ± 10* ,S.** N.S.** 1.51 ± 0.08* 66.1 p e r c e n t ± 3.6* N.S. ** NON-COM PLEXED 20 ± 4* BCT (n=4) 2.53 ± 0.20* mean ± S.D. s t a t i s t i c a l s i g n i f i c a n c e t e s t e d a t the p < 0.05 l e v e l . T A B L E I V . S T A B I L I T Y O F C F A N D C O N T R O L a 2 M - B C T C O M P L E X E S TO A H I G H M O L E C U L A R W E I G H T I N H I B I T O R ( S O Y B E A N T R Y P S I N I N H I B I T O R ) S P E C I F I C A C T I V I T Y (uMOLES BAEE HYDROLYSED/HOUR/u g A C T I V E B C T ) S U B J E C T S 18 HOUR COMPLEX + S T I 2 MINUTE COMPLEX + S T I CF ( n = 4 ) 1.09 ± 0 . 0 3 * 1.04 ± 0 . 0 1 * N.S. ** N.S.** CONTROL ( n - 6 ) 1.08 ± 0 . 1 0 * 1.06 ± 0 . 0 5 * mean ± S.D. s t a t i s t i c a l s i g n i f i c a n c e t e s t e d a t t h e p < 0.05 l e v e l PAGE 81 No s t a t i s t i c a l l y 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 e s t e r o l y t i c a c t i v i t i e s o f CF a n d c o n t r o l a g M - B C T c o m p l e x e s w e r e o b s e r v e d a t e i t h e r 2 m i n u t e s o r 18 h o u r s i n c u b a t i o n . T h e s e r e s u l t s i n d i c a t e t h a t b o t h CF a n d c o n t r o l a 2 M - B C T c o m p l e x e s show a h i g h d e g r e e o f s t a b i l i t y . As a n o t h e r t e s t o f t h e i r s t a b i l i t y , CF a n d c o n t r o l a g M - B C T c o m p l e x e s i n c u b a t e d f o r 18 h o u r s a t 3 7 " w e r e a s s a y e d f o r t h e i r a b i l i t y t o h y d r o l y s e t h e h i g h m o l e c u l a r w e i g h t s u b -s t r a t e R B B - h i d e . P r o t e o l y t i c a c t i v i t y o f t h e s e c o m p l e x e s was c o m p a r e d t o R B B - h i d e p r o t e o l y s i s p r o d u c e d b y an e q u i v a l e n t w e i g h t o f f r e e BCT ( s e e T a b l e V ) . CF a n d c o n t r o l 18 h o u r a 2 M - B C T c o m p l e x e s w e r e o b s e r v e d t o p r o d u c e n e g l i g i b l e l e v e l s o f R B B - h i d e p r o t e o l y s i s ( 2 . 4 p e r c e n t a n d 0 . 3 p e r c e n t o f f r e e BCT p r o t e o l y t i c a c t i v i t y , r e s p e c t i v e l y ) . CF and c o n t r o l v a l u e s o v e r l a p p e d s u b -s t a n t i a l l y , s o t h i s d i f f e r e n c e was n o t f o u n d t o be s t a t i s t i c a l l y s i g n i f i c a n t . ( 4 ) S u b u n i t S t r u c t u r e o f CF a n d C o n t r o l a ^ M : S u b u n i t s t r u c t u r e c a n be d e m o n s t r a t e d by S D S -PAGE as f o l l o w s : t r e a t m e n t o f a^M w i t h SDS a l o n e p e r m i t s d e m o n s t r a t i o n o f d i m e r i c a ? M ; t r e a t m e n t o f a ? M w i t h SDS a n d t h i o l r e a g e n t s P A G E 8 2 T A B L E V . S T A B I L I T Y O F C F A N D C O N T R O L c t 2 M - B C T C O M P L E X E S TO A H I G H M O L E C U L A R W E I G H T S U B S T R A T E ( R E M A Z O L B R I L L I A N T B L U E - H I D E ) PAGE 83 R E S I D U A L H Y D R O L Y T I C A C T I V I T Y OF COMPLEXES FOR RBB-HIDE S U B J E C T S ( p e r c e n t f r e e BCT a c t i v i t y ) CF (n=4) 2.4 p e r c e n t ± 2 . 4 * N.S.** CONTROL (n-6) 0.3 p e r c e n t ± 0.2* mean ± S.D. s t a t i s t i c a l s i g n i f i c a n c e t e s t e d . a t t h e p < 0.05 l e v e l . PAGE 84 ( e . g . , d i t h i o t h r e i t o l o r e - m e r c a p t o e t h a n o l ) p e r m i t s d e m o n s t r a t i o n o f m o n o m e r i c o ^ M ; a n d SDS a n d t h i o l r e a g e n t t r e a t m e n t o f c o m p l e x e s f o r m e d by r e a c t i o n o f 2 m o l e s t r y p s i n w i t h one m o l e c^M p e r m i t s d e m o n s t r a t i o n o f 85 ,000 d a l t o n f r a g m e n t s ( h a l f -m o n o m e r i c o ^ M ) • U s i n g t h i s t e c h n i q u e , t h e s u b u n i t s t r u c t u r e o f CF c ^ M a n d CF o ^ M - p r o t e a s e c o m p l e x e s was f o u n d t o be l i k e t h a t o f c o n t r o l m a t e r i a l . F i g u r e 11 s hows 3 s e t s o f S D S -PAGE t u b e g e l s : g e l s e t s 1 a n d 2 show c o n t r o l c^M; g e l s e t 3 s h o w s CF o^M. T r e a t m e n t o f CF a n d c o n t r o l o^M w i t h SDS a l o n e p r o d u c e d d i m e r i c o^M ( F i g u r e 1 1 : l a , 2a a n d 3 a ) . T r e a t m e n t o f CF a n d c o n t r o l o^M w i t h e - m e r c a p t o e t h a n o l and SDS p r o d u c e d m o n o m e r i c a 2 M ( F i g u r e 1 1 : l b , 2b a n d 3 b ) . F i n a l l y , r e a c t i o n o f CF a n d c o n t r o l c ^ M w i t h BCT a t 2 m o l e s t r y p s i n / m o l e a 2 M a n d t h e n t r e a t m e n t w i t h B - m e r c a p t o e t h a n o l and SDS p r o d u c e d h a l f - m o n o -m e r i c a ? M ( F i g u r e 1 1 : l c , 2c a n d 3 c ) . [^AGE 85 F i g . 1 1 : S U B U N I T STRUCTURE OF CF AND  CONTROL g?M: G e l s e t s 1 and 2 show s u b u n i t s t r u c t u r e o f c o n t r o l c^M: ( a ) d i m e r , ( b ) monomer, and ( c ) h a l f - m o n o m e r i c f r a g m e n t f r o m t r y p s i n t r e a t m e n t . G e l s e t 3 shows s u b u n i t s t r u c t u r e o f CF ogM: d i t t o . PAGE 86 b c 2 a b c 3 PAGE 87 I V . D I S C U S S I O N : A. I s o l a t i o n o f P u r e , A c t i v e a p M : T h e a ^ M i s o l a t i o n m e t h o d o f V i r c a e t a j _ . [ 6 8 ] was m o d i f i e d as f o l l o w s : w h o l e p l a s m a , t a k e n i n t o a m i x t u r e o f a n t i -p r o t e a s e r e a g e n t s , was a p p l i e d d i r e c t l y t o t h e C i b a c r o n B l u e S e p h a r o s e ( C B S ) c o l u m n w i t h o u t p r i o r d i a l y s i s a g a i n s t c o l u m n b u f f e r . By d e l e t i n g t h e d i a l y s i s s t e p , we s o u g h t t o d e c r e a s e t h e i n v i t r o e x p o s u r e t i m e o f « 2 M t o t h e p r o t e o l y t i c f a c t o r s o f p l a s m a and t o a v o i d p o s s i b l e d e l e t e r i o u s e f f e c t s o f d i a l y s i s . J u d g i n g f r o m t h e p h o t o g r a p h o f t h e g r a d i e n t PAGE g e l (4 t o 8 g / d l a c r y l a m i d e ) w h i c h a p p e a r s i n t h e i r p a p e r [ 6 8 ] , t h e a 2 M p r o d u c e d b y V i r c a e_t a_K a p p e a r s t o be a m i x t u r e o f t h e " f a s t " ( i n a c t i v e ) a n d " s l o w " ( a c t i v e ) e l e c t r o p h o r e t i c f o r m s o f t h e m o l e c u l e . I f s o , t h e n some i n a c t i v a t i o n o f o ^ M may h a v e o c c u r r e d d u r i n g i s o l a t i o n . T h e a p p l i c a t i o n o f w h o l e p l a s m a t o t h e CBS c o l u m n d e s c r i b e d h e r e a v o i d s t h i s a n d d o e s n o t a f f e c t t h e p r o t e i n r e s o l u t i o n ( F i g u r e l ) . A n o t h e r m o d i f i c a t i o n o f t h e CBS m e t h o d o f V i r c a e_t a j _ . [ 6 8 ] was a 50 p e r c e n t r e d u c t i o n i n t h e v o l u m e o f p l a s m a a p p l i e d t o t h e c o l u m n . By t h e s e m a n e u v e r s a n d e m p l o y i n g a v e r y s l o w e l u t i o n r a t e ( 1 . 5 m l / h r ) i t was p o s s i b l e t o b r i n g 90 p e r c e n t o f s t a r t i n g PAGE 88 p l a s m a c^M t o an a v e r a g e s t a t e o f 60 p e r c e n t p u r i t y ( r a n g e , 4 0 - 9 0 p e r c e n t d e p e n d i n g u p o n p l a s m a h a p t o g l o b i n t y p e ) i n a s i n g l e s t e p . V i r c a e t a_l_. a c h i e v e d a p p a r e n t p u r i t y ( a s a s s e s s e d by G r a d i e n t P A G E ) o f 40 p e r c e n t s t a r t i n g p l a s m a a^W i n a s i n g l e CBS s t e p ; h o w e v e r , o n l y p l a s m a h a p t o g l o b i n t y p e 1-1 c o u l d be u s e d , s i n c e u s e o f t y p e 2-1 a n d 2-2 p l a s m a s r e s u l t e d i n g r o s s h a p t o g l o b i n c o n t a m i n a t i o n o f t h e i r c^M i s o l a t e s [ 6 8 ] . By s u b j e c t i n g t h e c ^ M - r i c h CBS p o o l I ( f r a c t i o n s No. 2 4 - 3 4 , F i g u r e 1) t o a s e c o n d p u r i f i c a t i o n s t e p , t h e a p p l i c a b i l i t y o f t h e m e t h o d h a s b e e n b r o a d e n e d t o i n c l u d e a l l p l a s m a s a m p l e s , r e g a r d l e s s o f h a p t o g l o b i n t y p e . CBS p o o l I i s a p p l i e d , w i t h o u t p r i o r c o n c e n t r a t i o n , t o an i m m u n o a d s o r p t i o n c o l u m n d i r e c t e d a g a i n s t e i g h t r e c o g n i z e d c o n t a m i n a n t s o f t h e p o o l ( F i g u r e 2 ) . o^M o f a t l e a s t 98 p e r c e n t p u r i t y i s e l u t e d f r o m t h e c o l u m n ( F i g u r e 6 ) . T h e a v e r a g e a 2M y i e l d i s 71 p e r c e n t ( r a n g e , 4 8 - 9 9 p e r c e n t , n=9) o f s t a r t i n g p l a s m a a 2M. T h i s i s c o m p a r a b l e w i t h t h e 75' p e r c e n t y i e l d f r o m t h e two s t e p m e t h o d o f V i r c a e_t aj_ [ 6 8 ] . H o w e v e r , l o s s e s o f up t o 50 p e r c e n t o f s t a r t i n g p l a s m a a2 M w e r e e n c o u n t e r e d by t h i s a u t h o r u p o n u l t r a f i l t r a t i o n c o n c e n t r a t i o n o f i s o l a t e s t o 1 mg/ml f o r s u b s e q u e n t c h a r a c t e r i z a t i o n . T h e s e l o s s e s w e r e d u e t o a d s o r p t i o n t o u l t r a f i l t r a t i o n m embrane s u r f a c e s . U l t r a f i l t r a t i o n i n s t i r r e d c e l l s (AMICON MMC) u s i n g a v a r i e t y o f d i f f e r e n t m e m b r a n e t y p e s (AMICON XM, PM, UM and YM m e m b r a n e s and M I L L I P O R E PT and PS PAGE 89 m e m b r a n e s ) o f a p p r o p r i a t e l y s m a l l m o l e c u l a r w e i g h t c u t - o f f l i m i t s g a v e v a r i a b l e l o s s e s o f p u r i f i e d a^M, r a n g i n g f r o m 20 - 50 p e r c e n t . C o n c e n t r a t i o n d i a l y s i s a g a i n s t p o l y e t h y l e n e g l y c o l o r p o l y a c r y l a m i d e a l s o g a v e s i m i l a r p o o r r e c o v e r i e s . A t p r e s e n t , t h e r e a p p e a r s t o be no e a s y way t o c o n c e n t r a t e s m a l l q u a n t i t i e s o f t h i s p r o t e i n . L y o p h i l i z a t i o n was n o t a t t e m p t e d f o r f e a r o f i n a c t i v a t i n g o^M. CF a n d c o n t r o l a 2 M s o i s o l a t e d c o u l d be shown t o be p h y s i o l o g i c a l l y " a c t i v e " ( i . e . c a p a b l e o f b i n d i n g e n d o p e p t i d a s e s ) b y 3 d i f f e r e n t t e c h n i q u e s : ( 1 ) On g r a d i e n t P AGE, t h e s e o^M p r e p a r a t i o n s w e r e f o u n d t o be e l e c t r o p h o r e t i c a l l y " s l o w " r e l a t i v e t o ammonium i o n - o r p r o t e a s e - i n a c t i v a t e d a 2 M ( s e e F i g u r e 5) [ 6 5 ] ; ( 2 ) T h e t i t r a t i o n o f CF a n d c o n t r o l a 2 M p r e p a r a t i o n s w i t h BCT c o u l d be f o l l o w e d by S D S - P A G E , w i t h c o m p l e t e c o n v e r s i o n o f <*2M m o n o m e r i c s u b u n i t s t o p r o t e o 1 y t i c a l 1 y - d e r i v e d h a l f - m o n o m e r s o c c u r r i n g a t 2 m o l e s B C T / m o l e <*2M ( s e e F i g u r e 8 ) ; a n d ( 3 ) M o l a r p r o t e a s e b i n d i n g r a t i o s f o r t h e b i n d i n g o f t r y p s i n w i t h CF and c o n t r o l « 2 M p r e p a r a t i o n s c o u l d be d e t e r m i n e d b y u s e o f t h e R B B - h i d e t i t r a t i o n a s s a y d e s c r i b e d e a r l i e r ( s e e F i g u r e 7 and T a b l e I ) . By t h e s e s e m i - q u a n t i t a t i v e a n d q u a n t i t a t i v e c r i t e r i a , t h e CF a n d c o n t r o l c*2M p r e p a r a t i o n s u s e d f o r t h e f o l l o w i n g c o m p a r a t i v e s t u d i e s w e r e f o u n d t o be " a c t i v e " . PAGE 90 B. T h e R o l e o f g , H i n t h e P a t h o g e n e s i s o f C F : As d i s c u s s e d i n t h e i n t r o d u c t i o n , t h e p a t h o g e n e s i s o f c y s t i c f i b r o s i s may i n v o l v e a d e f i c i e n c y o f p r o t e o l y t i c e n z y m e a c t i v i t y w i t h a r g i n i n e e s t e r a s e s p e c i f i c i t y i n human e x o c r i n e g l a n d s a n d p l a s m a , l e a d i n g t o t h e a c c u m u l a t i o n o f h i g h l y m e m b r a n e a c t i v e , c a t i o n i c p o l y p e p t i d e s , c a l l e d "CF f a c t o r s " . T h e p r o t e a s e i n h i b i t o r o ^ M a c r o g 1 o b u 1 i n ( a 2 M ) may be d e f e c t i v e i n CF a n d may u n d e r l i e t h i s d e f i c i e n c y o f p r o t e o l y s i s . When S h a p i r a a n d h i s c o - w o r k e r s e x a m i n e d t h e i n t e r a c t i o n o f e x o g e n o u s p r o t e a s e s w i t h c^M d e r i v e d f r o m CF a n d c o n t r o l p l a s m a , s t r i k i n g d i f f e r e n c e s w e r e d e t e c t e d ; ( 1 ) i n t h e m o l a r b i n d i n g r a t i o s o f p r o t e a s e s t o a 2 M i n p l a s m a [ 2 , 4 ] ; ( 2 ) i n t h e s u b s t r a t e k i n e t i c s o f p u r i f i e d a 2 M - b o v i n e t r y p s i n c o m p l e x e s f o r t h e low m o l e c u l a r w e i g h t s u b s t r a t e b e n z o y l a r g i n i n e e t h y l e s t e r [ 4 ] ; ( 3 ) i n t h e s t a b i l i t y o f t h e s e c o m p l e x e s o n c e f o r m e d [ 3 ] ; a n d ( 4 ) i n t h e g e l c h r o m a t o g r a p h i c b e h a v i o r o f c o m p l e x e s 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 s o d i u m d o d e c y l s u l f a t e [ 4 ] . H o w e v e r , some o f t h e p u r i f i e d a 2 M p r e p a r a t i o n s ( p a r t i c u l a r l y t h e c o n t r o l m a t e r i a l ) u s e d by S h a p i r a et_ a j _ . a p p e a r t o be s t r u c t u r a l l y a n d f u n c t i o n a l l y n o n - n a t i v e [ 3 , 4 ] . T h e s t u d i e s o f S h a p i r a e_t a j _ . on CF a 2 M w e r e o f s u f f i c i e n t i m p o r t a n c e t o r e q u i r e v e r i f i c a t i o n , PAGE 91 and s e v e r a l l a b o r a t o r i e s , i n c l u d i n g t h e a u t h o r ' s own, h a v e u n d e r t a k e n t h i s w o r k : T h e d i s c u s s i o n w h i c h f o l l o w s r e p o r t s on t h e f i n d i n g s o f t h e p r e s e n t i n v e s t i g a t i o n , as w e l l as t h e f i n d i n g s o f o t h e r r e s e a r c h g r o u p s : ( 1 ) M o l a r P r o t e a s e B i n d i n g by CF a n d C o n t r o l « 2 M : S h a p i r a e_t a_l_. r e p o r t e d t h a t t h e o^M o f CF p l a s m a s h o w e d a r e d u c e d a b i l i t y t o b i n d e x o g e n o u s p r o t e a s e s ( p a p a i n , b o v i n e t r y p s i n a n d b o v i n e t h r o m b i n ) [ 2 ] . In t h e i r r e p o r t s , t h e b i n d i n g o f L C D 1 - 1 a b e l 1 ed b o v i n e t r y p s i n b y <x2M i n CF p l a s m a s a m p l e s c o r r e s p o n d e d t o a m o l a r p r o t e a s e b i n d i n g r a t i o o f a p p r o x i m a t e l y 0.4 - 0.5 m o l e s t r y p s i n / m o l e a 2M, w h i l e b i n d i n g by a 2 M i n c o n t r o l p l a s m a s a m p l e s was 0.8 - 0.9 m o l e s t r y p s i n / m o l e a 2M. T h e s e d a t a w e r e i n t e r p r e t e d as i n d i c a t i n g d e f e c t i v e p r o t e a s e b i n d i n g by CF a 2M [ 2 ] . H o w e v e r , human p l a s m a i s a v e r y c o m p l e x f l u i d c o n t a i n i n g a number o f p r o t e a s e i n h i b i t o r y s y s t e m s - , i n -c l u d i n g a t l e a s t f i v e i n h i b i t o r s o f t r y p s i n . a ^ _ a n t i t r y p s i n , i n t e r - a - t r y p s i n i n h i b i t o r , a n t i t h r o m b i n I I I , CI i n a c t i v a t o r a n d a 2M a l l c o m p e t e t o b i n d t r y p s i n [ 6 4 ] . T h u s , t h e f i n d i n g s o f 12 5 S h a p i r a e t a j _ . o f l o w e r I - r a d i o a c t i v i t y i n t h e a n t i - a 2 M i m m u n o p r e c i p i t a t e s o f CF p l a s m a do n o t n e c e s s a r i l y i n d i c a t e a d e f i c i e n t a b i l i t y o f CF a 2M t o b i n d t r y p s i n . A l t e r n a t i v e l y , t h e s e d a t a c o u l d r e f l e c t i n c r e a s e d t r y p s i n b i n d i n g by o n e o r m o r e PAGE 92 o f t h e o t h e r p r o t e a s e i n h i b i t o r s o f CF p l a s m a . M o l a r p r o t e a s e b i n d i n g c a p a b i l i t i e s o f CF a n d c o n t r o l c^M c a n o n l y be a s s e s s e d w i t h c e r t a i n t y by u s e o f p u r i f i e d n a t i v e a 2 M , i s o l a t e d f r o m o t h e r c o m p e t i n g p r o t e a s e i n h i b i t o r y s y s t e m s . S c h i d l o w a n d K u e p p e r s a t t e m p t e d t o a s s e s s m o l a r t r y p s i n b i n d i n g b y a 2 M i n s e r u m [ 9 0 ] . A f t e r e s t a b l i s h i n g t h a t t h e c o n c e n t r a t i o n s o f a 2 M i n CF a n d c o n t r o l s e r a w e r e t h e s ame, t h e s e w o r k e r s q u a n t i f i e d t h e a m o u n t o f t r y p s i n b o u n d t o a 2 M b y m e a s u r i n g t h e a m o u n t o f BAPNA a m i d a s e a c t i v i t y o f s e r u m e x p r e s s e d i n t h e p r e s e n c e o f s o y b e a n t r y p s i n i n h i b i t o r . T h e y f o u n d t h a t <*2M o f t h e s e r a o f CF a n d r e s p i r a t o r y d i s e a s e c o n t r o l p a t i e n t s b o u n d m o d e r a t e l y g r e a t e r a m o u n t s o f b o v i n e t r y p s i n t h a n s e r u m <*2M o f n o r m a l c o n t r o l s . H o w e v e r , 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 s e f i n d i n g s and t h e a c t u a l i n v i v o p r o t e a s e b i n d i n g c a p a b i l i t i e s o f a 2 M i s u n c e r t a i n , s i n c e a 2 M i n s e r u m i s p a r t i a l l y " i n a c t i v a t e d " b y e n d o g e n o u s p r o t e a s e b i n d i n g o c c u r r i n g d u r i n g t h e p r o c e s s o f b l o o d c l o t f o r m a t i o n [ 4 9 ] . T h i s c o u l d r e s u l t i n a s u b s t a n t i a l l y r e d u c e d c a p a c i t y t o b i n d e x o g e n o u s p r o t e a s e s j_n v i t r o . P a r s o n s a n d Romeo r e p o r t e d a c o m p a r a t i v e s t u d y o f p r o t e a s e b i n d i n g by CF a n d c o n t r o l a,>M [ 9 1 ] . As i n t h e p r e s e n t i n v e s -t i g a t i o n , t h e s e w o r k e r s u s e d p u r i f i e d c*2M i s o l a t e d f r o m CF a n d c o n t r o l p l a s m a . T h e y e m p l o y e d a r a d i o - i m m u n o p r e c i p i t a t i o n a s s a y s i m i l a r t o t h a t u s e d by S h a p i r a e_t a_l_. a n d f o u n d t h a t CF a n d c o n t r o l a 2 M a p p e a r e d t o b i n d s i m i l a r a m o u n t s o f I - t r y p s i n . PAGE 93 H o w e v e r , "a p o r t i o n " o f t h e - ^ I - t r y p s i n f o r m e d " a g g r e g a t e s " u n d e r c o n d i t i o n s u s e d i n t h e i r i m m u n o p r e c i p i t a t i o n a s s a y [ 9 1 ] . I n t h e a u t h o r ' s e x p e r i e n c e , t r y p s i n a g g r e g a t i o n o c c u r s as a r e s u l t o f d e n a t u r a t i o n o f t h e m o l e c u l e . I t i s n o t c l e a r i f t h i s a f f e c t e d t h e r e s u l t s o f P a r s o n s a n d Romeo by m a k i n g i t d i f f i c u l t t o d i s c r i m i n a t e t r u e L C 0 1 - t r y p s i n b i n d i n g by <* 2M f r o m p o t e n -t i a l c o n t a m i n a t i o n o f i m m u n o p r e c i p i t a t e s w i t h I - t r y p s i n a g g r e g a t e s . I n a d d i t i o n , h i g h c o n c e n t r a t i o n s o f 1 2 5 I - t r y p s i n ( t h r e e - f o l d i n e x c e s s o f t h e p r o t e a s e b i n d i n g c a p a b i l i t i e s o f a 2 M ) w e r e u s e d t o m a x i m i z e t h e a m o u n t o f r a d i o - l a b e l i n t h e i m m u n o p r e c i p i t a t e s and c o u l d h a v e c a u s e d n o n - s p e c i f i c h y d r o l y s i s o f a 2 M i t s e l f . An i n v e s t i g a t i o n o f p r o t e a s e b i n d i n g b y o g M was u n d e r -t a k e n u s i n g a d i f f e r e n t a p p r o a c h f r o m t h o s e a l r e a d y m e n t i o n e d . B o v i n e c a t i o n i c t r y p s i n ( B C T ) o f e s t a b l i s h e d p u r i t y a n d a c t i v i t y was u s e d t o t i t r a t e CF a n d c o n t r o l a 2 M s a m p l e s o f e s t a b l i s h e d p u r i t y and a n t i p r o t e a s e a c t i v i t y . I n s o l u b l e , h i g h m o l e c u l a r w e i g h t R e m a z o l b r i l l i a n t b l u e h i d e ( R B B - h i d e ) [ 8 3 ] was c h o s e n as t h e i n d i c a t o r o f t h e end p o i n t o f t i t r a t i o n . L i k e o t h e r h i g h m o l e c u l a r w e i g h t s u b s t r a t e s , R B B - h i d e i s a c c e s s i b l e o n l y t o f r e e BCT a n d n o t t o BCT b o u n d i n c o m p l e x t o a^. T h e r e f o r e , t h e p o i n t a t w h i c h h y d r o l y s i s o f R B B - h i d e b e c o m e s m e a s u r a b l e i s an i n d i c a t i o n o f t h e p o i n t a t w h i c h a 2 M " b i n d i n g s i t e s " h a v e be'come s a t u r a t e d w i t h B C T . PAGE 94 T a b l e I shows t h e r e s u l t s o f t h e s e s t u d i e s . CF a 2 M c o u l d b i n d 1.97 ± 0.18 m o l e s B C T / m o l e a 2M c o m p a r e d t o 1.72 ± 0.29 f o r c o n t r o l s . T h i s d i f f e r e n c e was n o t s t a t i s t i c a l l y s i g n i f i c a n t . T h e s e m o l a r b i n d i n g r a t i o s a r e i n a g r e e m e n t w i t h l i t e r a t u r e v a l u e s o f a p p r o x i m a t e l y 2 m o l e s t r y p s i n / m o l e <*2M [ 6 0 , 6 5 , 7 0 ] . T h e s e d a t a s u g g e s t t h a t t h e m o l a r p r o t e a s e b i n d i n g r a t i o s o f CF an d c o n t r o l <x 2M a r e t h e s a m e . I n many o f t h e i r c o m p a r a t i v e s t u d i e s o f p r o t e a s e b i n d i n g by CF a n d c o n t r o l a 2M, S h a p i r a e_t a_l_. h a d e x p o s e d t h e i r a 2M t o a f r a c t i o n a t i o n s t e p i n v o l v i n g ammonium s u l f a t e a t pH 7.0 [ 1 , 3 , 4 ] . H o w e v e r , t r e a t m e n t o f a 2 M w i t h ammonium s a l t s a n d c e r t a i n p r i -m a r y a m i n e s ( e . g . m e t h y l a m i n e , e t h y l a m i n e , h y d r a z i n e , a - m e t h y l -h y d r o x y 1 a m i n e , e t c . ) i s known t o p r o d u c e f r o m p a r t i a l t o c o m p l e t e i n a c t i v a t i o n o f t h e m o l e c u l e s ' p r o t e a s e b i n d i n g a b i l i t y [ 4 8 , 4 9 , 6 2 , 6 5 ] . T h e e x t e n t d e p e n d s u p o n c o n d i t i o n s o f t r e a t m e n t . B e c a u s e o f t h e i n s t a b i l i t y o f a 2 M t o ammonium i o n , i t h a s b e e n r e c o m m e n d e d t h a t ammonium s u l f a t e s h o u l d n o t be u s e d a t n e u t r a l o r a l k a l i n e pH's t o f r a c t i o n a t e p l a s m a d u r i n g a 2 M i s o l a t i o n p r o c e d u r e s [ 6 4 ] . I t h a s b e e n s u g g e s t e d t h a t e x p o s u r e o f p h y s i o -l o g i c a l l y " a c t i v e " <* 2M t o ammonium i o n u n d e r t h e s e c o n d i t i o n s may r e s u l t i n c l e a v a g e o f one o r more o f t h e f o u r a l k y l -a m i n e - r e a c t i v e g l u t a m y l r e s i d u e s r e p u t e d t o be p r e s e n t i n t h e m o l e c u l e [ 9 2 ] . T h i s i n t u r n may p r o d u c e a c o n f o r m a t i o n a l PAGE 95 r e a r r a n g e m e n t s i m i l a r t o t h a t s e e n when a 2 M c o m p l e x e s e n d o -p e p t i d a s e s [ 6 5 ] . A t t e n d a n t w i t h t h e s e c h a n g e s i s l o s s o f some o r a l l o f t h e p r o t e a s e b i n d i n g a b i l i t y o f t h e a 2 M [ 6 5 ] . U s i n g g r a d i e n t PAGE o n e c a n f o l l o w t h i s ammonium i o n - m e d i a t e d i n a c t i -v a t i o n o f a 2 M : b y t h i s t r e a t m e n t , e l e c t r o p h o r e t i c a l l y " s l o w " ( a c t i v e ) a2M i s c o n v e r t e d t o e l e c t r o p h o r e t i c a l l y " f a s t " ( i n a c t i v e ) a 2 M [ 8 2 , 6 5 ] ( s e e F i g u r e 5 ) . S i n c e S h a p i r a e_t a j _ . h a d u s e d ammonium s a l t p r e c i p i t a t i o n t o p u r i f y a2M, o n e m i g h t e x p e c t t h e i r CF a n d c o n t r o l a2M t o show a r e d u c e d p r o t e a s e b i n d i n g c a p a c i t y r e l a t i v e t o n a t i v e a2M. I n t h e p a p e r i n w h i c h S h a p i r a e_t a j _ . u s e d p u r i f i e d a2M t o i n v e s t i g a t e m o l a r p r o t e a s e b i n d i n g [ 4 ] , CF a 2 M b o u n d 12 pg t r y p s i n / m g a2M w h i l e c o n t r o l a 2 M b o u n d 25 pg t r y p s i n / m g a 2 M ; t h i s c o r r e s p o n d s t o 0.37 m o l e s B C T / m o l e a 2 M f o r CF a n d 0.76 f o r c o n t r o l . P r e s u m a b l y , t h e « 2 M i s o l a t e d b y S h a p i r a e t a l . was n o t f u l l y a c t i v e . To e x p l o r e t h e p o s s i b i l i t y t h a t t h e d i f f e r e n c e s o b s e r v e d by S h a p i r a a n d h i s c o l l e a g u e s i n m o l a r p r o t e a s e b i n d i n g b y CF a n d c o n t r o l a2M [ 2 , 4 ] r e f l e c t e d a g r e a t e r ammonium i o n - s e n s i t i v i t y o f CF a2M, t h e m o l a r p r o t e a s e b i n d i n g o f t h e a u t h o r ' s own p u r i f i e d CF a n d c o n t r o l « 2 M p r e p a r a t i o n s was s t u d i e d f o l l o w i n g i n a c t i v a t i o n w i t h 2 0 0 rnJ4 ammonium s u l f a t e , pH 8.0 f o r 30 m i n u t e s a t 3 7 " . T a b l e I I shows t h e r e s u l t s : i n a c t i v a t e d CF a ^ M c o u l d PAGE 96 b i n d 0.69 ± 0.05 m o l e s B C T / m o l e a 2 M c o m p a r e d t o 0.74 ± 0.15 f o r c o n t r o l s . T h i s d i f f e r e n c e was n o t s t a t i s t i c a l l y s i g n i f i c a n t T h e s e d a t a p r e s u m a b l y r e f l e c t s p e c i f i c b i n d i n g t o a 2 M s i t e s r e l a t i v e l y r e s i s t a n t t o t h e d e l e t e r i o u s e f f e c t s o f ammonium i o n s . T h i s a u t h o r ' s i n a c t i v a t e d CF a n d c o n t r o l c^M b o u n d t h e same a m o u n t o f p r o t e a s e as t h e c o n t r o l c*2M i s o l a t e d by S h a p i r a e_t a K u s i n g ammonium s u l f a t e f r a c t i o n a t i o n . T h i s s t u d y s u g g e s t s ( b u t d o e s n o t p r o v e ) t h a t p u r i f i e d CF a n d c o n t r o l a 2 M p r e p -a r a t i o n s a r e e q u a l l y s e n s i t i v e t o ammonium i o n . I n l i g h t o f t h e s e f i n d i n g s , t h e m o l a r p r o t e a s e b i n d i n g d i f f e r e n c e s b e t w e e n CF an d c o n t r o l c*2M r e p o r t e d by S h a p i r a ' s g r o u p r e q u i r e e x p l a n -a t i o n . T h e y may r e f l e c t : ( a ) d i f f e r e n c e s i n t h e l e n g t h o f t i m e CF a n d c o n t r o l p l a s m a s a m p l e s o r p u r i f i e d c^M p r e p a r a t i o n s w e r e s t o r e d ; ( b ) d i f f e r e n c e s i n t h e ammonium i o n s e n s i t i v i t i e s o f CF an d c o n t r o l o^M p r e p a r a t i o n s a p p a r e n t a f t e r t r e a t m e n t w i t h 1.6 M s a l t b u t n o t a f t e r t r e a t m e n t w i t h 2 0 0 mM s a l t ; o r ( c ) d i f f e r -e n c e s i n t h e ammonium i o n s e n s i t i v i t i e s o f CF a n d c o n t r o l a 2 M p r e p a r a t i o n s o b s e r v a b l e when ammonium s a l t t r e a t m e n t i s c a r r i e d o u t on w h o l e p l a s m a , b u t n o t o b s e r v a b l e when t r e a t m e n t i s c a r r i e d o u t on p u r i f i e d a 2 M . B a s e d u p o n c o n v e r s a t i o n s w i t h D r . S h a p i r a c o n c e r n i n g t h e d a t e s o f a c q u i s i t i o n o f CF and c o n t r o l p l a s m a s a m p l e s u s e d i n h i s s t u d i e s , a n d b a s e d u p o n t h e f a c t t h a t a^w t e n d s t o g r a d u a l l y l o s e a n t i p r o t e a s e a c t i v i t y d u r i n g s t o r a g e [ 6 5 ] , t h i s a u t h o r b e l i e v e s t h a t s y s t e m a t i c d i f f e r e n c e s i n PAGE 97 t h e d u r a t i o n o f s t o r a g e o f CF and c o n t r o l p l a s m a s and p u r i f i e d <*2M p r e p a r a t i o n s may b e s t e x p l a i n t h e a 2 M - p r o t e a s e b i n d i n g d i f f e r e n c e s b e t w e e n t h e s e two g r o u p s o f s u b j e c t s o b s e r v e d b y S h a p i r a e_t a j _ . [ 1 - 4 ] . ( 2 ) I n t e r a c t i o n o f C F a n d C o n t r o l a ^ M - T r y p s i n C o m p l e x e s w i t h B A E E : S h a p i r a e_t a j _ . s t u d i e d t h e s u b s t r a t e k i n e t i c s when CF a n d c o n t r o l a g M - t r y p s i n c o m p l e x e s a n d n o n - c o m p 1 e x e d t r y p s i n i n t e r -a c t e d w i t h t h e low m o l e c u l a r w e i g h t s u b s t r a t e b e n z o y l a r g i n i n e e t h y l e s t e r ( B A E E ) [ 4 ] . T h e y r e p o r t e d s t r i k i n g d i f f e r e n c e s i n t h e a f f i n i t y o f CF a n d c o n t r o l a 2 M - t r y p s i n c o m p l e x e s f o r B A E E . T h e a p p a r e n t K m o f CF a g M - t r y p s i n c o m p l e x e s was s i m i l a r t o t h a t o f f r e e t r y p s i n ( 1 5 0 and 1 1 0 _p_M, r e s p e c t i v e l y ) b u t 4 - f o l d l o w e r t h a n t h a t o f c o n t r o l c o m p l e x e s ( 6 3 0 J J M ) . 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 t o mean t h a t t h e modes o f b i n d i n g o f t r y p s i n t o CF a n d c o n t r o l a 2 M w e r e d i f f e r e n t [ 4 ] . T h e s e r e s u l t s p r o m p t e d t h i s a u t h o r t o c o m p a r e t h e a c t i v i t i e s o f CF a n d c o n t r o l a 2 M - b o v i n e c a t i o n i c t r y p s i n ( B C T ) c o m p l e x e s t o t h e a c t i v i t y o f n o n - c o m p l e x e d BCT u s i n g B A E E . T h e a u t h o r ' s f i n d i n g s a r e s u m m a r i z e d i n T a b l e I I I . T h e a p p a r e n t K PAGE 98 o f CF c o m p l e x e s f o r BAEE ( 4 3 ± 6 yM) 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 t h a t o f c o n t r o l c o m p l e x e s (52 ± 10 _yMj. T h e a p p a r e n t K m o f f r e e BCT ( 2 0 ± 4 yM) was f o u n d t o be l o w e r t h a n t h a t f o r c o m p l e x e d BCT as e x p e c t e d . T h i s v a l u e i s i n l i n e w i t h a p p a r e n t K m d a t a o f o t h e r s f o r f r e e e n z y m e ( r a n g e : 4 t o 50 yM) [ 8 8 , 9 3 ] . T h e low a f f i n i t i e s o f CF a n d c o n t r o l o ^ M - t r y p s i n c o m p l e x e s f o r BAEE o b s e r v e d b y S h a p i r a and h i s c o l l e a g u e s may r e f l e c t p a r t i a l i n a c t i v a t i o n o f o 2 M b y e x p o s u r e t o ammonium i o n . T h e a c t u a l K m d i f f e r e n c e s b e t w e e n CF a n d c o n t r o l o i g M - t r y p s i n c o m p l e x e s r e p o r t e d by S h a p i r a e t a]_. may r e f l e c t d i f f e r e n c e s i n t h e a g e a n d / o r mode o f s t o r a g e o f c o n t r o l a n d CF a 2 M p r e p a r a t i o n s u s e d b y t h e s e w o r k e r s . I f t h e i r d a t a do r e f l e c t a c t u a l d i f f e r e n c e s i n t h e k i n e t i c s o f CF and c o n t r o l a 2 M - t r y p s i n c o m p l e x e s o n l y a p p a r e n t a f t e r e x p o s u r e t o h i g h l e v e l s o f ammonium s a l t s , i t i s n o t c l e a r w h a t r e l e v a n c e t h i s p h e n o m e n o n h a s t o t h e J_n v i v o p e r f o r m a n c e o f <X 2M. T a b l e I I I a l s o s u m m a r i z e s t h e a u t h o r ' s f i n d i n g s on t h e m a x i m a l v e l o c i t y ( V m a x ) o f BCT b o t h i n t h e f r e e s t a t e a n d i n c o m p l e x w i t h CF o r c o n t r o l a 2 M . T h e V m a x f o r BAEE o f CF c o m p l e x e s was v i r t u a l l y i d e n t i c a l t o t h a t o f c o n t r o l c o m p l e x e s ( 1 . 5 4 y m o l e s BAEE h y d r o 1 y s e d / h o u r / y g a c t i v e BCT a n d 1 . 5 1 , r e s p e c t i v e l y ) . A t a BAEE c o n c e n t r a t i o n o f 0.45 mM, CF a 2 M was f o u n d t o d e p r e s s t h e r a t e o f BAEE h y d r o l y s i s b y BCT t o PAGE 99 67.2 p e r c e n t o f t h e f r e e BCT h y d r o l y s i s r a t e ( r e s i d u a l e s t e r o l y t i c a c t i v i t y ) . T h i s c o m p a r e d w i t h a v a l u e o f 66.1 p e r c e n t f o r c o n t r o l a 2 M . None o f t h e s e d i f f e r e n c e s a r e s t a t i s t i c a l l y s i g n i f i c a n t . S h a p i r a e_t a j _ . d i d n o t r e p o r t V o r r e s i d u a l a c t i v i t y v a l u e s f o r CF an d c o n t r o l a 2 M - B C T comp1 e x e s [ 4 ] . ( 3 ) S t a b i l i t y o f CF a n d C o n t r o l o t g M ^ T r y p s i n Comp1 e x e s : S h a p i r a e t a l _ . r e p o r t e d a b n o r m a l b r e a k d o w n o f o ^ M - t r y p s i n c o m p l e x e s i n CF [ 3 ] . T h e y f o u n d t h a t t h e a b i l i t y o f anti-t r y p s i n c o m p l e x e s t o d i s c r i m i n a t e s t e r i c a l l y b e t w e e n h i g h a nd l o w m o l e c u l a r w e i g h t s u b s t r a t e s a n d i n h i b i t o r s b e g a n t o be l o s t w i t h i n 10 m i n u t e s when c o n t r o l a 2 M was c o m p l e x e d w i t h t r y p s i n . T h i s o c c u r r e d much more s l o w l y ( w i t h i n 6 0 - 9 0 m i n u t e s ) when CF <* 2M was u s e d . T h e s e r e s u l t s s u g g e s t e d t o S h a p i r a et_ a_l_. t h a t c o n t r o l a 2 M - t r y p s i n c o m p l e x e s w e r e g r a d u a l l y c o n v e r t e d i n t o a 2 M f r a g m e n t - t r y p s i n c o m p l e x e s , b u t t h i s d i d n o t o c c u r as r e a d i l y t o CF a 2 M - t r y p s i n c o m p l e x e s . H o w e v e r , o t h e r p u b l i s h e d s t u d i e s on t h e s t a b i l i t y o f n o r m a l o t 2 M - p r o t e a s e c o m p l e x e s i n -d i c a t e t h a t o n c e t h e c o m p l e x i s f o r m e d t h e e n z y m e i s i r r e v e r s i b l y b o u n d t o a 2 M [ 4 8 , 4 9 , 6 0 ] , a n d t h a t a 2 M f r a g m e n t - p r o t e a s e com-p l e x e s a r e o n l y d e t e c t a b l e when a 2 M - p r o t e a s e c o m p l e x e s a r e d e n a t u r e d [ 4 8 , 9 4 ] . PAGE 1 0 0 T h i s was r e - i n v e s t i g a t e d b y P a r s o n s a nd Romeo, who e x a m i n e d t h e s t a b i l i t y o f CF a n d c o n t r o l a 2 M - p o r c i n e t r y p s i n c o m p l e x e s a g a i n s t i n h i b i t i o n by S T I [ 9 1 ] . To do t h i s t h e y s t u d i e d t h e r e s i d u a l a m i d o l y t i c a c t i v i t y t o w a r d BAPNA o f < * 2 M - t r y p s i n com-p l e x e s f o r m e d a t n o n - s a t u r a t i n g a n d s a t u r a t i n g t r y p s i n c o n c e n -t r a t i o n s a n d a s s a y e d i n t h e p r e s e n c e o f S T I . T h e y r e p o r t e d 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 r e s i d u a l a m i d o l y t i c a c t i v i t y o f CF and c o n t r o l o t 2 M - t r y p s i n c o m p l e x e s a f t e r i n c u b a t i o n o f a 2 M a n d p o r c i n e t r y p s i n f o r p e r i o d s o f 5 and 6 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 . H o w e v e r , t h e a s s a y c o n d i t i o n s e m p l o y e d by P a r s o n s a n d Romeo f o r n o n - s a t u r a t i n g t r y p s i n c o n c e n t r a t i o n s w e r e n o t o p t i -m i z e d t o a l l o w d e t e c t i o n o f d i f f e r e n c e s i n t h e r e s i d u a l a m i d o -l y t i c a c t i v i t i e s b e t w e e n t h e s e i n c u b a t i o n t i m e s [ 9 1 ] : ( a ) c o m p l e x e s w e r e i n c u b a t e d a t r o o m t e m p e r a t u r e i n s t e a d o f 3 8 ° ( t h e t e m p e r a t u r e e m p l o y e d by S h a p i r a ejt a j _ . ) , a n d ( b ) BAPNA h y d r o l y s i s b y c o m p l e x e s as m e a s u r e d b y a b s o r b a n c e a t 4 1 0 nm b a r e l y r o s e a b o v e b a c k g r o u n d a m i d o l y t i c l e v e l s . T h u s , h a d t h e < * 2 M - t r y p s i n c o m p l e x e s b e c o m e l e a k y t o S T I a f t e r 1 h o u r ' s i n c u b a t i o n , t h e r e s u l t i n g i n h i b i t i o n o f r e s i d u a l e s t e r o l y t i c a c t i v i t y w o u l d h a v e g o n e u n d e t e c t e d . T h e l e v e l s o f a m i d o l y t i c a c t i v i t y m e a s u r e d a t s a t u r a t i n g t r y p s i n c o n c e n t r a t i o n s w e r e an o r d e r o f m a g n i t u d e h i g h e r t h a n e x p e c t e d , and t h i s may r e f l e c t i n c o m p l e t e i n h i b i t i o n o f f r e e t r y p s i n b y S T I . PAGE 101 T h i s a u t h o r u n d e r t o o k a r e - i n v e s t i g a t i o n o f t h e s t a b i l i t y o f CF a n d c o n t r o l a 2 M - b o v i n e c a t i o n i c t r y p s i n ( B C T ) c o m p l e x e s . S h a p i r a e_t a j _ . , h a d f o r m e d o ^ M - t r y p s i n c o m p l e x e s w i t h o n l y e n o u g h b o v i n e t r y p s i n t o e n s u r e 8 p e r c e n t o c c u p a n c y o f t h e t h e o r -e t i c a l l y a v a i l a b l e p r o t e a s e b i n d i n g s i t e s o f t h e [ 3 ] . H o w e v e r , i n t h e p r e s e n t i n v e s t i g a t i o n a q u a n t i t y o f BCT s u f f i -c i e n t t o e n s u r e 75 p e r c e n t o c c u p a n c y o f p r o t e a s e b i n d i n g s i t e s o f was s e l e c t e d ( s e e ' M a t e r i a l s a n d M e t h o d s ' ) . T h e h i g h m o l e c u l a r w e i g h t i n h i b i t o r S T I and t h e h i g h m o l e c u l a r w e i g h t s u b s t r a t e R B B - h i d e w e r e c h o s e n as p r o b e s o f t h e s t a b i l i t y o f t h e s e c*2M-BCT c o m p l e x e s . I t s h o u l d be a p p r e c i a t e d t h a t t h e r e i s a d i f f e r e n c e b e t w e e n ( a ) t h e b r e a k d o w n o f 0 2 M - p r o t e a s e c o m p l e x e s w i t h t h e r e l e a s e o f p r o t e a s e s o r p r o t e a s e - o ^ M f r a g m e n t s a n d ( b ) a c h a n g e i n t h e c o n f o r m a t i o n o f t h e c o m p l e x w i t h t h e l o s s o f s t e r i c s e l e c t i v i t y t o w a r d s s u b s t r a t e s a n d i n h i b i t o r s o f d i f f e r i n g m o l e c u l a r s i z e s . T h e m e t h o d s c h o s e n t e s t b o t h p o s s i b i l i t i e s a n d do n o t d i s t i n g u i s h b e t w e e n t h e m . ( a ) S t a b i l i t y o f C o m p l e x e s t o S T I : CF a n d c o n t r o l o ^ M - B C T c o m p l e x e s i n c u b a t e d a t 3 7 " f o r 18 h o u r s w e r e c o m p a r e d w i t h f r e s h l y p r e p a r e d c o m p l e x e s ( i n c u b a t i o n o f o^M a n d BCT f o r 2 m i n u t e s ) i n t e r m s o f t h e i r a b i l i t y t o h y d r o l y s e BAEE i n t h e p r e s e n c e o f a 1 0 0 - f o l d m o l a r e x c e s s o f PAGE 102 S T I . S p e c i f i c a c t i v i t y v a l u e s f o r 2 m i n u t e as w e l l as 18 h o u r CF an d c o n t r o l c o m p l e x e s a r e p r e s e n t e d i n T a b l e I V . We f o u n d no s t a t i s t i c a l l y 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 e s t e r o l y t i c a c t i v i t i e s o f CF a n d c o n t r o l « 2 M - B C T c o m p l e x e s a t e i t h e r t i m e p e r i o d . F u r t h e r m o r e , t h e d e g r e e o f i n t e r a c t i o n o f S T I w i t h CF o r c o n t r o l c o m p l e x e s d o e s n o t c h a n g e e v e n a f t e r 18 h o u r s i n c u b a t i o n a t 3 7 " , i n d i c a t i n g a h i g h d e g r e e o f c o m p l e x s t a b i l i t y f o r b o t h CF an d c o n t r o l a 2 M p r e p a r a t i o n s . ( b ) S t a b i l i t y o f C o m p l e x e s t o R B B - h i d e : CF a n d c o n t r o l a 2 M - B C T c o m p l e x e s i n c u b a t e d f o r 18 h o u r s a t 37" w e r e a s s a y e d f o r t h e i r a b i l i t y t o h y d r o l y s e t h e h i g h m o l e c u l a r w e i g h t s u b s t r a t e R B B - h i d e . P r o t e o l y t i c a c t i v i t y o f t h e s e c o m p l e x e s was e x p r e s s e d as a p e r c e n t a g e o f p r o t e o l y s i s p r o d u c e d b y an e q u i v a l e n t a m o u n t o f f r e e BCT ( s e e T a b l e V ) . CF a n d c o n t r o l 18 h o u r a^M-BCT c o m p l e x e s w e r e o b s e r v e d t o p r o d u c e n e g l i g i b l e l e v e l s o f R B B - h i d e p r o t e o l y s i s ( 2 . 4 p e r c e n t a n d 0.3 p e r c e n t o f f r e e BCT p r o t e o l y t i c a c t i v i t y , r e s p e c t i v e l y ) . CF a n d c o n t r o l v a l u e s o v e r l a p p e d s u b s t a n t i a l l y . T h i s d i f f e r e n c e was n o t s t a t i s t i c a l l y s i g n i f i c a n t . In o t h e r w o r d s , t h e a u t h o r o b s e r v e d t h a t CF a n d c o n t r o l a^M-BCT c o m p l e x e s r e m a i n s t a b l e f o r a t l e a s t 18 h o u r s a t 3 7 " . T h e s e d a t a s u p p o r t t h e c o n t e n t i o n t h a t p r o t e a s e s a r e b o u n d i r r e v e r s i b l y b y n o r m a l a n d CF a 9 M . T h e PAGE 103 i n s t a b i l i t y o f b o t h CF a n d c o n t r o l a 2 M - t r y p s i n c o m p l e x e s o b s e r v e d by S h a p i r a and h i s c o - w o r k e r s [ 3 ] may r e f l e c t t h e i r u s e o f 1.6 M ammonium s u l f a t e i n t h e p u r i f i c a t i o n o f a^ M. T h e i r CF a 2 M p r e p a r a t i o n s s h o w e d a m o r e n o r m a l p a t t e r n o f s t a b i l i t y t h a n t h e i r c o n t r o l s . T h e r e a s o n f o r t h i s i s u n c l e a r b u t may r e f l e c t l a c k o f p a i r i n g o f c l i n i c a l a n d c o n t r o l s a m p l e s d u r i n g i s o l a t i o n a n d / o r a s s a y o f a 2 M p r e p a r a t i o n s . S i n c e a 2 M - B C T c o m p l e x e s do n o t a p p e a r t o d i s s o c i a t e , t h i s a u t h o r d i d n o t a t t e m p t ( a ) t h e d e t e r m i n a t i o n o f i n h i b i t i o n c o n s t a n t s ( ) f o r t h e i n t e r a c t i o n o f BCT w i t h CF a n d c o n t r o l a 2 M , n o r ( b ) t h e c h a r a c t e r i z a t i o n o f t h i s i n h i b i t i o n as com-p e t i t i v e o r n o n - c o m p e t i t i v e . S u c h k i n e t i c c h a r a c t e r i z a t i o n s o f t h e i n t e r a c t i o n o f i n h i b i t o r , e n z y m e a n d s u b s t r a t e r e q u i r e t h a t t h e s e r e a c t i o n c o m p o n e n t s be i n r e v e r s i b l e e q u i l i b r i u m w i t h t h e i r c o m p l e x e s [ 4 9 ] , a c o n d i t i o n n o t s a t i s f i e d by a 2 M - p r o t e a s e c o m p l e x e s i n t h i s a u t h o r ' s h a n d s . ( 4 ) S u b u n i t - S t r u c t u r e - o f C F a n d C o n t r o l a 2 M : S h a p i r a and h i s c o - w o r k e r s u s e d g e l c h r o m a t o g r a p h y c a l i -b r a t e d w i t h m o l e c u l a r w e i g h t m a r k e r p r o t e i n s t o s t u d y t h e s u b u n i t s t r u c t u r e o f CF a n d c o n t r o l a 0 M b e f o r e a n d a f t e r t r e a t m e n t w i t h PAGE 104 b o v i n e t r y p s i n [ 4 ] . T h e y r e p o r t e d t h a t p u r i f i e d CF and c o n t r o l a 2 M p r e p a r a t i o n s c h r o m a t o g r a p h e d on B i o - G e l A-1.5m g a v e an e l u t i o n p r o f i l e c o n s i s t e n t w i t h t e t r a m e r i c a 2 M (= 7 2 5 , 0 0 0 d a l t o n s [ 8 9 ] ) , b u t t h e s e same p r e p a r a t i o n s , when c h r o m a t o g r a p h e d i n t h e p r e s e n c e o f s o d i u m d o d e c y l s u l f a t e ( S D S ) on B i o - G e l A-0.5m, g a v e m o n o m e r i c a 2 M . T h i s e l u t i o n p a t t e r n was o b s e r v e d r e g a r d l e s s o f w h e t h e r o r n o t CF a n d c o n t r o l a 2 M h a d b e e n r e d u c e d and a l k y l a t e d p r i o r t o l o a d i n g . T h e s e f i n d i n g s w e r e i n t e r p r e t e d as i n d i c a t i n g t h a t d i s u l f i d e b o n d s do n o t p a r t i c i p a t e i n t e t r a m e r f o r m a t i o n . H o w e v e r , p r e v i o u s i n v e s t i g a t i o n s o f t h e s u b u n i t s t r u c t u r e o f a 2 M h a d f o u n d t h a t a 2 M t e t r a m e r s a r e c o m p o s e d o f d i m e r s h e l d t o g e t h e r b y h y d r o p h o b i c i n t e r a c t i o n s , a n d d i m e r s a r e f o r m e d f r o m a 2 M m o n o m e r s l i n k e d by d i s u l f i d e b o n d s [ 4 8 ] . A c c o r d i n g l y , t r e a t m e n t o f t e t r a m e r i c a 2 M w i t h d e t e r g e n t r e l e a s e s a 2 M d i m e r s a n d n o t m o n o m e r s . When S h a p i r a e_t [ 4 ] i n c u b a t e d CF a n d c o n t r o l a 2 M w i t h e q u i m o l a r a m o u n t s o f b o v i n e t r y p s i n and t h e n c h r o m a t o g r a p h e d t h e r e s u l t i n g a 2 M - t r y p s i n c o m p l e x e s on B i o - G e l A-1.5m, a l l o f t h e CF a 2 M b u t o n l y a f r a c t i o n o f t h e c o n t r o l agM e l u t e d i n t h e t e t r a m e r i c p o s i t i o n . M o s t o f t h e c o n t r o l a 2 M was c o n v e r t e d i n t o l o w e r m o l e c u l a r w e i g h t s p e c i e s e l u t i n g as a b r o a d p e a k j u s t b e f o r e , a n d t r a i l i n g j u s t b e h i n d , t h e i r I g G c a l i b r a t i o n m a r k e r ( 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 ) . T h i s p e a k was a b s e n t f r o m 7 PAGE 105 c h r o m a t o g r a p h i c c o n d i t i o n s e m p l o y e d by S h a p i r a and h i s c o l -l e a g u e s ; d i s s o c i a t i o n o n l y o c c u r s u n d e r d e n a t u r i n g c o n d i t i o n s ( e . g . t r e a t m e n t w i t h d e t e r g e n t s and r e d u c i n g a g e n t s ) [ 4 8 ] . T h i s s u g g e s t s t h a t i t i s t h e c o n t r o l ( ^ M - t r y p s i n c o m p l e x e s p r o d u c e d by S h a p i r a e_t a j _ . ( b u t n o t t h e CF c o m p l e x e s ) t h a t s h o w e d a n o -m a l o u s b e h a v i o r . A s i m i l a r p a t t e r n was s e e n when CF a n d c o n t r o l c ^ M - t r y p s i n c o m p l e x e s w e r e c h r o m a t o g r a p h e d on B i o - G e l A-0.5m i n t h e p r e s e n c e o f SDS : c o n t r o l c o m p l e x e s s h o w e d an e x t r a p e a k n o t s e e n i n t h e e l u t i o n p r o f i l e o f CF c o m p l e x e s [ 4 ] . T h i s p e a k e l u t e d b e t w e e n t h e IgG ( 1 5 0 , 0 0 0 d a l t o n s ) and t h e BSA ( 6 8 , 0 0 0 d a l t o n s ) c a l i - b r a t i o n m a r k e r s and may r e p r e s e n t t h e p r o t e o l y t i c a l l y - d e r i v e d h a l f - m o n o m e r s ( 8 5 , 0 0 0 d a l t o n f r a g m e n t s ) o b s e r v e d by o t h e r s o n l y a f t e r d i s u l f i d e b o n d r e d u c t i o n o f d e t e r g e n t - t r e a t e d c o m p l e x e s [ 4 8 ] . A g a i n , CF o ^ M i n t h i s s t u d y a p p e a r s m o r e n o r m a l t h a n c o n t r o l o ^ M . H o w e v e r , t h e s e d a t a w e r e i n t e r p r e t e d by S h a p i r a and h i s c o - w o r k e r s as e v i d e n c e t h a t i n d i v i d u a l m o n o m e r i c u n i t s ( 1 8 5 , 0 0 0 d a l t o n s ) o f CF o ^ M c a n n o t be c l e a v e d i n h a l f by t r y p s i n . A n u m b e r o f r e s e a r c h g r o u p s i n c l u d i n g t h e a u t h o r ' s h a v e u n d e r t a k e n a r e - e x a m i n a t i o n o f t h e s u b u n i t s t r u c t u r e o f CF o ^ M as a c o n s e q u e n c e o f t h e s e r e p o r t s [ 9 1 , 9 4 , 9 5 ] . I n s t e a d o f u s i n g PAGE 106 a n d h i s c o - w o r k e r s as e v i d e n c e t h a t i n d i v i d u a l m o n o m e r i c u n i t s ( 1 8 5 , 0 0 0 d a l t o n s ) o f CF a 2 M c a n n o t be c l e a v e d i n h a l f b y t r y p s i n . A n u m b e r o f r e s e a r c h g r o u p s i n c l u d i n g t h e a u t h o r ' s h a v e u n d e r t a k e n a r e - e x a m i n a t i o n o f t h e s u b u n i t s t r u c t u r e o f CF a^M as a c o n s e q u e n c e o f t h e s e r e p o r t s [ 9 1 , 9 5 , 9 6 ] . I n s t e a d o f u s i n g t h e g e l c h r o m a t o g r a p h i c t e c h n i q u e s e m p l o y e d by S h a p i r a e t a l . , o t h e r i n v e s t i g a t o r s h a v e c h o s e n t o a n a l y s e o^M s u b u n i t s t r u c -t u r e by S D S - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s ( P A G E ) . T h i s t e c h n i q u e i s p o p u l a r b e c a u s e o f i t s s p e e d , s e n s i t i v i t y a n d 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 d e t e r m i n a t i o n . F u r t h e r m o r e , SDS-PAGE i s f a r l e s s w a s t e f u l o f o^M t h a n i s g e l c h r o m a t o g r a p h y . T h e s u b u n i t s t r u c t u r e o f a 2 M c a n be d e m o n s t r a t e d b y S DS-PAGE as f o l l o w s [ 6 1 ] : t r e a t m e n t o f a 2 M w i t h SDS a l o n e p e r m i t s d e mon-s t r a t i o n o f d i m e r i c a 2 M ; t r e a t m e n t o f a 2 M w i t h SDS a n d t h i o l r e a g e n t s ( e . g . , d i t h i o t h r e i t o l o r e - m e r c a p t o e t h a n o l ) p e r m i t s d e m o n s t r a t i o n o f m o n o m e r i c <x2M; a n d SDS a n d t h i o l r e a g e n t t r e a t m e n t o f c o m p l e x e s f o r m e d b y r e a c t i o n o f 2 m o l e s t r y p s i n w i t h o n e m o l e « 2 M p e r m i t s d e m o n s t r a t i o n o f 8 5 , 0 0 0 d a l t o n f r a g m e n t s ( h a l f m o n o m e r i c a 2 M ) . U s i n g t h i s t e c h n i q u e , o t h e r i n v e s t i g a t o r s h a v e f o u n d t h a t t h e s u b u n i t s t r u c t u r e o f CF « 2 M a n d CF <x 2M-p r o t e a s e c o m p l e x e s i s l i k e t h a t o f c o n t r o l p r e p a r a t i o n s [ 9 1 ,95 , 9 6 ] . PAGE 1 0 7 T h e a u t h o r h a s r e - e x a m i n e d t h e s u b u n i t s t r u c t u r e o f CF a 2 M u s i n g S D S - P A G E . F i g u r e 1 1 s h o w s 3 s e t s o f S DS-PAGE t u b e g e l s : g e l s e t s 1 a n d 2 show c o n t r o l e t 2 M ; g e l s e t 3 s h o w s CF a 2 M . T r e a t m e n t o f CF a n d c o n t r o l a 2 M w i t h SDS a l o n e p r o d u c e d d i m e r i c a 2 M ( F i g u r e 1 1 : l a , 2 a a n d 3 a ) . T r e a t m e n t o f CF a n d c o n t r o l a 2 M w i t h e - m e r c a p t o e t h a n o l a n d SDS p r o d u c e d m o n o m e r i c a 2 M ( F i g u r e 1 1 : l b , 2b a n d 3 b ) . F i n a l l y , r e a c t i o n o f CF a n d c o n t r o l a 2 M w i t h BCT a t 2 m o l e s t r y p s i n / m o l e a 2 M a n d t h e n t r e a t m e n t w i t h e - m e r c a p t o e t h a n o l and SDS p r o d u c e d h a l f - m o n o m e r i c a 2 M ( F i g u r e 1 1 : l c , 2 c a n d 3 c ) . S l o w l y m i g r a t i n g m i n o r b a n d s w e r e l o c a t e d n e a r t o t h e t o p o f t h e g e l s ( F i g u r e 1 1 ) and r e p r e s e n t e d n o n - d i s s o c i a t e d a 2 M , n o t h i g h m o l e c u l a r w e i g h t c o n t a m i n a n t s . S i m i l a r b a n d s w e r e o b s e r v e d b y H a r p e l [ 6 1 ] . T h e s e d a t a s u g g e s t no d i f f e r e n c e s b e t w e e n t h e s u b - u n i t s t r u c t u r e s o f CF a n d c o n t r o l a 2 M , c o n f i r m i n g t h e w o r k o f o t h e r i n v e s t i g a t o r s [ 9 1 , 9 5 , 9 6 ] . £ . C o n c l u s i o n : C o n t r a r y t o t h e r e p o r t s o f S h a p i r a a n d h i s c o l l e a g u e s [ 1 - 4 ] , t h i s a u t h o r f o u n d no d i f f e r e n c e s b e t w e e n t h e s u b u n i t s t r u c t u r e o f CF a n d c o n t r o l a 0 M n o r b e t w e e n t h e a b i l i t i e s o f CF PAGE 108 and c o n t r o l a 2 M t o i n t e r a c t w i t h b o v i n e c a t i o n i c t r y p s i n . B a s e d u p o n t h e s e f i n d i n g s , t h e a u t h o r b e l i e v e s t h a t no f i r m e v i d e n c e e x i s t s t o i m p l i c a t e an o^M d e f e c t i n t h e p a t h o g e n e s i s o f C y s t i c F i b r o s i s . PAGE 109 V. B I B L I O G R A P H Y S H A P I R A , E . , RAO, G . J . S . , WESSEL, H.V. a n d NADLER, H.L. [ 1 9 7 6 ] " A b s e n c e o f an c ^ m a c r o g 1 o b u 1 i n - p r o t e a s e c o m p l e x i n c y s t i c f i b r o s i s " . P e d i a t Res 1 0 : 8 1 2 - 8 1 7 S H A P I R A , E . , B E N - Y O S E P H , Y., a n d NADLER, H.L. [ 1 9 7 6 ] " D e c r e a s e d f o r m a t i o n o f a g m a c r o g l o b u l i n - p r o t e a s e c o m p l e x e s i n p l a s m a o f p a t i e n t s w i t h c y s t i c f i b r o s i s " . B i o c h e m B i o p h y s Res Commun 7 1 : 8 6 4 - 8 7 0 S H A P I R A , E . , B E N - Y O S E P H , Y., and NADLER, H.L. [ 1 9 7 7 ] " A b n o r m a l b r e a k d o w n o f a g m a c r o g 1 o b u 1 i n - t r y p s i n c o m p l e x i n c y s t i c f i b r o s i s " . C l i n C h i m A c t a 7 8 : 3 5 9 - 3 6 3 S H A P I R A , E . , MARTIN, C . L . a n d NADLER, H.L. [ 1 9 7 7 ] " C o m p a r i s o n b e t w e e n p u r i f i e d o ^ m a c r o g 1 o b u 1 i n p r e p a r a t i o n s f r o m n o r m a l c o n t r o l s a nd p a t i e n t s w i t h c y s t i c f i b r o s i s - k i n e t i c a n d s t r u c t u r a l p r o p e r t i e s o f c o m p l e x w i t h b o v i n e t r y p s i n " . J B i o l Chem 2 5 2 : 7 9 2 3 - 7 9 2 9 WILSON, G.B. a n d FUDENBERG, H.H. [ 1 9 7 6 ] " S t u d i e s on c y s t i c f i b r o s i s u s i n g i s o e l e c t r i c f o c u s i n g : I I D e m o n s t r a t i o n o f d e f i c i e n t p r o t e o l y t i c c l e a v a g e o f a 2 m a c r o g l o b u l i n i n c y s t i c f i b r o s i s p l a s m a " . P e d i a t R e s 1 0 : 8 7 - 9 6 WILSON, G. 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[ 1 9 7 3 ] " C h a r a c t e r i z a t i o n o f c y s t i c f i b r o s i s f a c t o r a n d i t s i n t e r a c t i o n w i t h human i m m u n o g l o b u l i n " . J Exp Med 1 3 7 : 1 5 3 8 - 1 5 4 3 PAGE 112 BARNETT, D.R., SCHANFI EL D, M.S., McCOMBS, M.L. e t a j _ . [ 1 9 7 3 ] " I s o e l e c t r i c f o c u s i n g a n d IgG a l l o t y p i n g o f t h e s e r u m f r a c t i o n c o n t a i n i n g t h e c y s t i c f i b r o s i s c i l i a r y i n h i b i t o r " . T e x Rep B i o l Med 31^:709-714 H E R Z B E R G , V., C A L V E R T , L. and BOWMAN, B.H. [ 1 9 7 3 ] " E v i d e n c e t h a t t h e c i l i a r y i n h i b i t o r o f c y s t i c f i b r o s i s i s n o t an a n t i b o d y " . J C l i n I n v e s t 5 2 : 2 7 3 2 - 2 7 3 6 B E R A T I S , N.G., CONOVER, J . H . , CONOD, E . J . e t a j _ . [ 1 9 7 4 ] " C y s t i c f i b r o s i s c i l i a r y i n h i b i t o r " . P e d i a t r Res 8 : 6 8 7 ( o n l y ) BOWMAN, B.H., LANKFORD, B . J . , F U L L E R , G.M., CARSON, S.D., KUROSKY, A. and BARNETT, D.R. [ 1 9 7 5 ] " C y s t i c f i b r o s i s : T h e c i l i a r y i n h i b i t o r i s a s m a l l p o l y p e p t i d e a s s o c i a t e d w i t h i m m u n o g l o b u l i n G". B i o c h e m B i o p h y s Res Comm 6 4 : 1 3 1 0 - 1 3 1 5 DANES, B.S. and BEARN, A.G. [ 1 9 7 2 ] " O y s t e r c i l i a r y i n h i b i t i o n by c y s t i c f i b r o s i s c u l t u r e m e d i u m " . J E x p Med 1 3 6 : 1 3 1 3 - 1 3 1 7 CONOVER, J . H . , B E R A T I S , N.G., CONOD, E . J . , A I N B E N D E R , E. and HIRSCHHORN, K. [ 1 9 7 3 ] " S t u d i e s on c i l i a r y d y s k i n e s i a f a c t o r i n c y s t i c f i b r o s i s : I I . S h o r t t e r m l e u k o c y t e c u l t u r e s a n d l o n g t e r m l y m p h o i d l i n e s " . P e d i a t Res 7 : 2 2 4 - 2 2 8 BOWMAN, B.H., BARNETT, D.R., MATALON, R., DANES, B.S. and BEARN, A.G. 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P o o l e , N o r t h H o l l a n d P u b l i s h i n g C o . , A m s t e r d a m a n d L o n d o n ( 5 2 ) H A R P E L , P . C , M 0 S E S S 0 N , M.W. a n d COOPER, N.R. [ 1 9 7 5 ] " S t u d i e s on t h e s t r u c t u r e and f u n c t i o n o f o ^ M a c r o g l o b u l i n a n d CT i n a c t i v a t o r " . i n : P r o t e a s e s and B i o l o g i c a l C o n t r o l ( e d s . , E. R e i c h , D. R i f k i n a nd E . Shaw) C o l d S p r i n g H a r b o r L a b o r a t o r y , C o l d S p r i n g H a r b o r , N.Y. pp 3 8 7 - 4 0 4 ( 5 3 ) JAMES, K. [ 1 9 8 0 ] " A l p h a 2 - m a c r o g 1 o b u 1 i n a n d i t s p o s s i b l e i m p o r t a n c e i n immune s y s t e m s " . T I B S F e b r u a r y : 4 3 - 4 7 , E l s e v i e r / N o r t h H o l l a n d B i o m e d i c a l P r e s s ( 5 4 ) P A R I S I , A . F . and V A L L E E , B . L . [ 1 9 7 0 ] " I s o l a t i o n o f a z i n c - o ^ m a c r o g 1 o b u 1 i n f r o m human s e r u m " . B i o c h e m i s t r y 9 : 2 4 2 1 - 2 4 2 6 PAGE 116 ( 5 5 ) JAMES, K., JOHNSON, G. a n d FUDENBERG, H.H. [ 1 9 6 6 ] "Th e q u a n t i t a t i v e e s t i m a t i o n o f a 2 m a c r o g l o b u l i n i n n o r m a l , p a t h o l o g i c a l and c o r d s e r a " . C l i n C h i m A c t a 1 4 : 2 0 7 - 2 1 4 ( 5 6 ) G I T L I N , D. a n d B I A S U C C I , A. [ 1 9 6 9 ] " D e v e l o p m e n t o f a G, aA, aM, e, C p , A, C, e s t e r a s e i n h i b i t o r , c e r u 1 o p l a s m i n , t r a n s f e r i n , h e m o p e x i n , h a p t o g l o b i n , f i b r i n o g e n , p l a s m i n o g e n , c q - a n t i t r y p s i n o r o s o m u c o i d , 3 - 1 i p o p r o t e i n , c t 2 m a c r o g l o b u 1 i n , a n d p r e a l b u m i n i n t h e human c o n c e p t u s " . J C l i n I n v e s t 4 8 : 1 4 3 3 - 1 4 4 6 ( 5 7 ) MOSHER, D.F. a n d WING, D.A. [ 1 9 7 6 ] " S y n t h e s i s a n d s e c r e t i o n o f a 2 m a c r o g l o b u l i n by c u l t u r e d human f i b r o b l a s t s " . J Exp Med 1 4 3 : 4 6 2 - 4 6 7 ( 5 8 ) HOVI, T., MOSHER, D. a n d V A H E R I , A. [ 1 9 7 7 ] " C u l t u r e d human m o n o c y t e s s y n t h e s i z e and s e c r e t e a 2 - m a c r o g l o b u l i n " . J . E x p . M e d . 1 4 5 : 1 5 8 0 - 1 5 8 9 ( 5 9 ) H A R P E L , P.C. [ 1 9 7 9 ] p a p e r p r e s e n t e d a t t h e 3 r d C a n a d i a n C y s t i c F i b r o s i s P r o f e s s i o n a l C o n f e r e n c e , H a r r i s o n , B.C. O c t . 1 7 - 2 0 t h ( 6 0 ) SAUNDERS, R., DYCE, B . J . , VANNIER, W.E. a n d HAVERBACH, B . J . [ 1 9 7 1 ] "T h e s e p a r a t i o n o f a l p h a - 2 m a c r o g 1 o b u 1 i n i n t o f i v e c o m p o n e n t s w i t h d i f f e r i n g e l e c t r o p h o r e t i c and e n z y m e - b i n d i n g p r o p e r t i e s " . J C l i n I n v e s t 5 0 : 2 3 7 6 - 2 3 8 3 ( 6 1 ) H A R P E L , P.C. [ 1 9 7 3 ] " S t u d i e s on human p l a s m a a 2 m a c r o g 1 o b u 1 i n e n z y m e . i n t e r a c t i o n s ; E v i d e n c e f o r p r o t e o l y t i c m o d i f i c a t i o n o f t h e s u b u n i t c h a i n s t r u c t u r e " . J E x p t l Med 1 3 8 : 5 0 8 - 5 2 1 ( 6 2 ) MEHL, J.W., O'CONNELL, W. and DeGROOT, J . [ 1 9 6 4 ] " M a c r o g 1 o b u 1 i n f r o m human p l a s m a w h i c h f o r m s an e n z y m a t i c a 1 1 y a c t i v e c o m p o u n d w i t h t r y p s i n " . S c i e n c e 1 4 5 : 8 2 1 - 8 2 2 PAGE 117 OHLSSON, K. and OLSSON, I . [ 1 9 7 4 ] " N e u t r a l p r o t e a s e s o f human g r a n u l o c y t e s : I I I I n t e r a c t i o n b e t w e e n human g r a n u l o c y t e e l a s t a s e and p l a s m a p r o t e a s e i n h i b i t o r s " . S c a n d J C l i n L a b I n v e s t 3 4 : 3 4 9 - 3 5 5 HE IMBURGER, N. P r o c e e d i n g s o f t h e I n t e r n a t i o n a l R e s e a r c h C o n f e r e n c e on P r o t e i n a s e - I n h i b i t o r s : 1 - 2 2 ( M u n i c h , 1 9 7 0 ) ( e d s . H. F r i t z a n d H. T s c h e s c h e ) , W a l t e r d e G r u y t e r , B e r l i n a n d New Y o r k [ 1 9 7 1 ] B A R R E T T , A . J . , BROWN, M.A. and S A Y E R S , C.A. [ 1 9 7 9 ] " T h e e l e c t r o p h o r e t i c a l 1 y ' s l o w ' a n d ' f a s t ' f o r m s o f t h e c ^ m a c r o g l o b u l i n m o l e c u l e " . B i o c h e m J 1 8 1 : 4 0 1 - 4 1 8 M c E N T I R E , J . E . [ 1 9 7 8 ] " P u r i f i c a t i o n o f a 2 m a c r o g 1 o b u 1 i n b y i m m u n o a d s o r b e n t c h r o m a t o g r a p h y " . J Immunol M e t h 2 4 : 3 9 - 4 5 H A R P E L , P.C. [ 1 9 7 6 ] "Human o 2 m a c r o g l o b u l i n " . M e t h E n z y m o l 4 5 : 6 3 9 - 6 5 2 V I R C A , G.D., T R A V I S , J . , H A L L , P.K. a n d ROBE R T S , R.C. [ 1 9 7 8 ] " P u r i f i c a t i o n o f human c ^ m a c r o g l o b u l i n by c h r o m a t o g r a p h y on c i b a c r o n b l u e s e p h a r o s e " . A n a l B i o c h e m 8 9 : 2 7 4 - 2 7 8 QUEEN, K.G. a n d PEACOCK, A . C . [ 1 9 6 6 ] " H a p t o g l o b i n t y p e d e t e r m i n a t i o n u s i n g n o n - c a r c i n o g e n i c r e a g e n t s " . C l i n C h i m A c t a 1^3 :47-51 SWENSON, R.P. and HOWARD, J . B . [ 1 9 7 9 ] " S t r u c t u r a l c h a r a c t e r i s t i c s o f human c ^ m a c r o g l o b u 1 i n s u b u n i t s " . J B i o l Chem 2 5 4 : 4 4 5 2 - 4 4 5 6 M ANCINI, G., CARBONARA, A.O. a n d HEREMANS, J . F . [ 1 9 6 5 ] " I m m u n o c h e m i c a l q u a n t i t a t i o n o f a n t i g e n s b y s i n g l e r a d i a l i m m u n o d i f f u s i o n " . I m m u n o c h e m i s t r y 2 : 2 3 5 - 2 5 4 PAGE 118 ( 7 2 ) SCHROEDER, D.D. and SHAW, E . [ 1 9 6 8 ] " C h r o m a t o g r a p h y o f t r y p s i n a n d i t s d e r i v a t i v e s " . J B i o l Chem 2 4 3 : 2 9 4 3 - 2 9 4 9 ( 7 3 ) LOUVARD, M.N. a n d P U I G S E R V E R , A. [ 1 9 7 4 ] "On b o v i n e and p o r c i n e a n i o n i c t r y p s i n o g e n s ". B i o c h i m B i o p h y s A c t a 3 7 1 : 1 7 7 - 1 8 5 ( 7 4 ) C H A S E , T. J r . a n d SHAW, E . [ 1 9 7 0 ] " T i t r a t i o n o f t r y p s i n , p l a s m i n a n d t h r o m b i n w i t h p - n i t r o p h e n y 1 p - g u a n i d i n o b e n z o a t e H C 1 " . M e t h E n z y m o l 1 9 : 2 0 - 2 7 ( 7 5 ) DUNN, J . T . and S P I R O , R.G. [ 1 9 6 7 ] " T h e c ^ m a c r og 1 o b u 1 i n o f human p l a s m a : I I s o l a t i o n a n d c o m p o s i t i o n " . J B i o l Chem 2 4 2 : 5 5 4 9 - 5 5 5 5 ( 7 6 ) BRADFORD, M.M. [ 1 9 7 6 ] "A r a p i d a n d s e n s i t i v e m e t h o d f o r t h e q u a n t i t a t i o n o f m i c r o g r a m q u a n t i t i e s o f p r o t e i n u t i l i z i n g t h e p r i n c i p l e o f p r o t e i n - d y e b i n d i n g " . A n a l B i o c h e m 72_:248-254 ( 7 7 ) LOWRY, O.H., ROSEBROUGH, N . J . , FARR, A . L . a n d RANDALL, R . J . [ 1 9 5 1 ] " P r o t e i n m e a s u r e m e n t w i t h f o l i n p h e n o l r e a g e n t " . J B i o l Chem 1 9 3 : 2 6 5 - 2 7 5 ( 7 8 ) P E T E R S O N , G.L. [ 1 9 7 9 ] " R e v i e w o f t h e f o l i n p h e n o l p r o t e i n q u a n t i t a t i o n m e t h o d o f L o w r y , R o s e b r o u g h , F a r r and R a n d a l l " . A n a l B i o c h e m 1 0 0 : 2 0 1 - 2 2 0 ( 7 9 ) GELMAN E l e c t r o p h o r e s i s P r o d u c t M a n u a l , T e c h n i c a l B u l l e t i n No. 18, G e l m a n I n s t r u m e n t s C o m p a n y , L a b o r a t o r y P r o d u c t s D i v i s i o n ( 8 0 ) MARGOLIS, J . a n d K E N R I C K , K.G. [ 1 9 6 8 ] " 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 a c o n t i n u o u s m o l e c u l a r s i e v e g r a d i e n t " . A n a l B i o c h e m 25_:347-362 PAGE 119 ( 8 1 ) WEBER, K., P R I N G L E , J.R. and OSBORN, M. [ 1 9 7 2 ] " M e a s u r e m e n t o f m o l e c u l a r w e i g h t s b y e l e c t r o p h o r e s i s on S D S - a c r y l a m i d e g e l " . M e t h E n z y m o l 2 6 : 3 - 2 7 ( 8 2 ) Z A I S , D.P. a n d ROBERTS, R.C. [ 1 9 7 7 ] " S y s t e m f o r s i m p l i f i e d d i s c o n t i n u o u s - g r a d i e n t p o l y a c r y 1 a m i d e g e l e l e c t r o p h o r e s i s " . C l i n Chem 2 3 : 5 9 0 - 5 9 2 ( 8 3 ) R I N D E R K N E C H T , H. , GEOKAS, M.C., S I L V E R M A N , P. a n d HAVERBACH, B . J . [ 1 9 6 8 ] "A new u l t r a s e n s i t i v e m e t h o d f o r t h e d e t e r m i n a t i o n o f p r o t e o l y t i c a c t i v i t y " . C l i n i c a C h i m i c a A c t a 2 1 : 1 9 7 - 2 0 3 ( 8 4 ) M I C H A L , G. [ 1 9 7 4 ] " D e t e r m i n a t i o n o f M i c h a e l i s C o n s t a n t s a n d I n h i b i t o r C o n s t a n t s " . i n : M e t h o d s o f  E n z y m a t i c A n a l y s i s ( e d . H.V. B e r g m e y e r ) V o l . 1, 2 n d E d i t i o n , A c a d e m i c P r e s s , New Y o r k , p p . 1 4 4 - 1 5 6 ( 8 5 ) L INEWEAVER, H. and BURK, J . [ 1 9 3 4 ] " T h e d e t e r m i n a t i o n o f e n z y m e d i s s o c i a t i o n c o n s t a n t s " . J Am Chem S o c 5 6 : 6 5 8 - 6 6 6 ( 8 6 ) C 0 L T 0 N , T. [ 1 9 7 4 ] " N o n p a r a m e t r i c M e t h o d s " i n : S t a t i s t i c s i n M e d i c i n e , L i t t l e , Brown a nd C o . , B o s t o n , p p . 2 1 9 - 2 2 7 ( 8 7 ) A R M I T A G E , P. [ 1 9 7 1 ] " S t a t i s t i c a l m e t h o d s i n m e d i c a l r e s e a r c h " . B l a c k w e l l S c i e n t i f i c P u b l i c a t i o n s , O x f o r d , pp 3 9 4 - 4 0 7 . ( 8 8 ) WALSH, K.A. [ 1 9 7 0 ] " T r y p s i n o g e n s a n d t r y p s i n s o f v a r i o u s s p e c i e s " . M e t h E n z y m o l 1 9 : 4 1 - 6 3 ( 8 9 ) J O N E S , J.M., C R E E T H , J.M. a n d KEKWICK, R.A. [ 1 9 7 2 ] " T h i o l r e d u c t i o n o f human o ^ m a c r o g l o b u 1 i n : T h e s u b u n i t s t r u c t u r e " . B i o c h e m J 1 2 7 : 1 8 7 - 1 9 7 PAGE 120 ( 9 0 ) SHIDLOW, D.V. and K E U P P E R S , F. [ 1 9 8 0 ] " T r y p s i n b i n d i n g a c t i v i t y o f a l p h a 2 - m a c r o g l o b u l i n i n c y s t i c f i b r o s i s and o t h e r l u n g d i s e a s e s " . A m e r i c a n R e v i e w o f R e s p i r a t o r y D i s e a s e 1 2 1 : 3 1 - 3 7 ( 9 1 ) PARSONS, M. and ROMEO, G. [ 1 9 8 0 ] " C y s t i c f i b r o s i s o ^ m a c r o g 1 o b u 1 i n - p r o t e a s e i n t e r a c t i o n i n v i t r o " . C l i n C h i m A c t a 1 0 0 : 2 1 5 - 2 2 4 ( 9 2 ) SWENSON, R.P. a n d HOWARD, J . B . [ 1 9 8 0 ] " A m i n o a c i d s e q u e n c e o f t h e t r y p t i c p e p t i d e c o n t a i n i n g t h e a l k y 1 a m i n e - r e a c t i v e s i t e f r o m human c ^ m a c r o g l o b u l i n " . J B i o l Chem 2 5 5 : 8 0 8 7 - 8 0 9 1 ( 9 3 ) BERGMEYER, H.V. [ 1 9 7 4 ] " M e t h o d s o f e n z y m a t i c a n a l y s i s " . V o l . I , 2 n d e d i t i o n A c a d e m i c P r e s s , New Y o r k , S a n F r a n c i s c o , L o n d o n , pp 5 1 5 - 5 1 6 ( 9 4 ) A B E , S. a n d NAGAI, Y. [ 1 9 7 3 ] " E v i d e n c e f o r t h e p r e s e n c e o f a c o m p l e x o f c o l l a g e n a s e w i t h o ^ m a c r o g 1 o b u 1 i n i n human r h e u m a t o i d s y n o v i a l f l u i d " . J B i o c h e m ( T o k y o ) 7 3 : 8 9 7 - 9 0 0 ( 9 5 ) ROBERTS, R.C., N E L L E S , L . P . , H A L L , P., a n d M I S C H L E R , E. [ 1 9 7 9 ] " C o m p a r i s o n o f c y s t i c f i b r o t i c and n o r m a l o ^ M a c r o g 1 o b u 1 i n : P h y s i c o c h e m i c a l a n d p r i m a r y s t r u c t u r e s t u d i e s " . CF C l u b A b s t r a c t s , May 1, 1 9 7 9 , A t l a n t a , pp 8 ( 9 6 ) BURDON, M.G. [ 1 9 8 0 ] " N o r m a l s u b u n i t c l e a v a g e o f O 2 m a c r o g 1 o b u 1 i n i n c y s t i c f i b r o s i s " . C l i n C h i m A c t a 1 0 0 : 2 2 5 - 2 2 9 ( 9 7 ) G u i d e t o D i a g n o s i s a n d M a n a g e m e n t o f C y s t i c F i b r o s i s : A S y l l a b u s f o r P h y s i c i a n s , p r e p a r e d by t h e P r o f e s s i o n a l E d u c a t i o n C o m m i t t e e , C y s t i c F i b r o s i s F o u n d a t i o n , A t l a n t a , G e o r g i a , 1977 pp 9 3 - 5 APPENDIX: PUBLICATIONS RESULTING FROM THESE STUDIES ISOLATION OF PURE, ACTIVE c t g M A C R O G I O B U L I N FROM SMALL-SCALE PLASMA SAMPLES M i c h a e l A. B r i d g e s Derek A. A p p l e g a r t h * Joanne Johannson A. George F. D a v i d s o n Lawrence T.K. Wong B i o c h e m i c a l D i s e a s e s L a b o r a t o r y C h i I d r e n ' s H o s p i t a l 250 West 59th Avenue Vancouver, B.C. Canada V5X 1X2 whom c o r r e s p o n d e n c e s h o u l d be a d d r e s s e d . KEYWORDS a^Macroglobul plasma i s o l a t i o n a c t i ve B r i d g e s , A p p l e g a r t h , Johannson, D a v i d s o n , and Wong Page 1 124 SUMMARY P h y s i o l o g i c a l l y " a c t i v e " a p M a c r o g l o b u l i n (apM) o f a t l e a s t 98 p e r c e n t p u r i t y c an be i s o l a t e d i n h i g h y i e l d f r o m s m a l l s c a l e plasma samples r e g a r d -l e s s o f plasma h a p t o g l o b i n t y p e . The t e c h n i q u e d e s c r i b e d h e r e i n , i n v o l v i n g c i b a c r o n b l u e s e p h a r o s e chromatography and immunoadsorption i s s u i t a b l e f o r p u r i f i c a t i o n o f agM from plasma samples o f 5 ml. INTRODUCTION a ^ M a c r o g l o b u l i n { a ^ ) i s a unique p r o t e a s e i n h i b i t o r f o u n d i n p l a s m a : I t i s c a p a b l e o f b i n d i n g e n d o p e p t i d a s e s from a n i m a l , p l a n t and b a c t e r i a l s o u r c e s and o f i n h i b i t i n g t h e i r p r o t e o l y t i c a c t i v i t y towards h i g h m o l e c u l a r weight s u b s t r a t e s , w h i l e p r e s e r v i n g much o f t h e i r a c t i v i t y towards low mole-c u l a r weight s u b s t r a t e s [ 1 ] . B r i d g e s , A p p l e g a r t h , Johannson, D a v i d s o n , and Wong Page 2 125 A p r i m a r y r o l e f o r c^M i n t h e p a t h o g e n e s i s o f c y s t i c f i b r o s i s was p o s t u l a t e d by S h a p i r a e t aj_. [ 2 ] . These a u t h o r s p u b l i s h e d d a t a which seemed t o show t h a t t h e k i n e t i c s o f t h e c^M-protease i n t e r -a c t i o n were abnormal i n c y s t i c f i b r o s i s ( C F ) . T h i s may have r e f l e c t e d a g e n e t i c d e f e c t o f t h e a^M m o l e c u l e . W i l s o n and Fudenberg s u g g e s t e d t h a t such an a b n o r m a l i t y i n t h e b i n d i n g a f f i n i t y o f c^M f o r plasma p r o t e a s e s might a c c o u n t f o r t h e p r e s e n c e o f "CF f a c t o r s " , i m p l i c a t e d i n t h e p a t h o g e n e s i s o f CF [ 3 ] . c^M a b n o r m a l i t i e s have a l s o been r e p o r t e d i n m u l t i p l e s c l e r o s i s [ 4 ] , E h l e r s - D a n T o s Syndrome [ 5 ] and D y g g v e - M e l c h i o r - C l a u s e n Syndrome [ 6 ] . Because o f t h e p o s s i b l e i n v o l v e m e n t o f a^M i n p a t h o l o g i c a l s t a t e s , e s p e c i a l l y t h o s e i n v o l v i n g c h i l d r e n , i s o l a t i o n o f n a t i v e c^M from s m a l l volumes o f plasma has become' d e s i r a b l e . U n f o r t u n -a t e l y , many c l a s s i c a l p r o t e i n p u r i f i c a t i o n t e c h n i q u e s may not be employed. F o r example, s u b j e c t i o n of a ^ l t o an ammonium s u l f a t e p r e c i p i t a t i o n s t e p i s known t o i m p a i r t h e m o l e c u l e ' s p r o t e a s e b i n d i n g a b i l i t y [ 7 ] . Immunoadsorption chromatography w i t h B r i d g e s , A p p l e g a r t h , Johannson, D a v i d s o n , and Wong Page 3 126 anti-a^M immunoglobulins [ 8 ] i s a l s o i n e f f e c t u a l b e cause the maneuvers a v a i l a b l e t o desorb o^M from a n t i b o d y a r e known t o i n a c t i v a t e o r d e n a t u r e t h e m o l e c u l e [ 1 , 7 ] . V i r c a e t aj_. have demon s t r a t e d t h a t a f f i n i t y (dye l i g a n d ) g e l f i l t r a t i o n c hromatography o f human plasma on a c i b a c r o n b l u e s e p h a r o s e CL-6B (Pharmacia) (CBS) column i s c a p a b l e o f i s o l a t i n g 40 p e r c e n t o f plasma a^M i n a h i g h s t a t e o f p u r i t y i n a s i n g l e s t e p [9]. The method o f V i r c a e t aj_. as p u b l i s h e d was d e v e l o p e d f o r a^ M i s o l a t i o n f r o m l a r g e - s c a l e " p l a s m a samples (100 m l ) , and t h e t e c h n i q u e i s h a n d i -capped by a p p l i c a b i l i t y o n l y t o p l a s m a samples o f h a p t o g l o b i n t y p e 1-1, which would be f o u n d i n r o u g h l y o n l y 14.5 p e r c e n t o f human plasma samples [ 1 0 ] . Use o f plasmas o f h a p t o g l o b i n t y p e s 2-1 and 2-2 i n t h e i s o l a t i o n scheme r e s u l t s i n g r o s s h a p t o g l o b i n contam-i n a t i o n o f t h e a2M-rich CBS e f f l u e n t f r a c t i o n s [9]. O t h e r p u r i f i c a t i o n methods a r e a v a i l a b l e [ 7 , 1 1 , 1 2 ] , which have been shown t o p r o d u c e a c t i v e a^, b u t a l l s t a r t w i t h l a r g e volumes o f plasma B r i d g e s , A p p l e g a r t h , Johannson, D a v i d s o n , and Wong Page 4 (200-500 ml) and g i v e y i e l d s o f l e s s t h a n 40 p e r c e n t . M o r e o v e r , use o f plasma o f h a p t o g l o b i n t y p e 1-1 i s g e n e r a l l y p r e f e r r e d i n t h e s e t e c h n i q u e s . In t h i s r e p o r t we d e s c r i b e a two-step p r o c e d u r e f o r i s o l a t i o n i n h i g h y i e l d of a t l e a s t 98 p e r c e n t p u r e , p h y s i o l o g i c a l l y a c t i v e a^M from 5 ml plasma sam p l e s . The p r o c e d u r e i n v o l v e s CBS chromatography, f o l l o w e d by immunoadsorption o f t h e c o n t a m i n a n t s o f t h e c ^ M - r i c h CBS e f f l u e n t p o o l . T h i s i s o l a t i o n scheme i s a p p l i c a b l e t o a l l plasma samples r e g a r d -l e s s o f h a p t o g l o b i n t y p e . MATERIALS AND METHODS I s o l a t i o n of g^M from Human Plasma: F o l l o w i n g i n f o r m e d c o n s e n t by p a r e n t or g u a r d -i a n , b l o o d samples were o b t a i n e d from 12 c h i l d r e n (mean age 12.6 y e a r s ; range 7-16 y e a r s ) . B l o o d samples were o n l y e x p o s e d t o p l a s t i c o r s i l i c o n i z e d g l a s s s u r f a c e s t o p r e v e n t Hageman f a c t o r a c t i v a t i o n . B r i d g e s , A p p l e g a r t h , Jo.hannson, D a v i d s o n , and Wong Page 5 128 Each 10 ml b l o o d sample was drawn i n t o 1 ml o f s t e r i l e 0.13M sodium c i t r a t e . B l o o d samples were t h e n i m m e d i a t e l y t r a n s f e r r e d t o a c e n t r i f u g e tube c o n t a i n i n g 1 mg soybean t r y p s i n i n h i b i t o r (SIGMA) and 4 mg p o l y b r e n e (SIGMA) and mixed by i n v e r s i o n . Plasma was h a r v e s t e d by c e n t r i f u g a t i o n a t 500 X g a t 4° f o r 20 minutes and t h e n s u b j e c t e d t o CBS chromato-graphy. F i v e ml p l a s m a - sodium c i t r a t e m i x t u r e ( w i t h o u t p r i o r d i a l y s i s a g a i n s t column b u f f e r ) was a p p l i e d d i r e c t l y t o a s i l i c o n i z e d column (1.5 X 90 cm) c o n t a i n i n g c i b a c r o n b l u e s e p h a r o s e CL-6B (PHARMACIA). The a 2 M - r i c h f r a c t i o n o f plasma was e l u t e d w i t h 50 nM T r i s HC1, pH 8.0 c o n t a i n i n g 3 nM sodium a z i d e a t a r a t e o f 1.5 t o 2.0 ml/hour. Two ml f r a c t i o n s were c o l l e c t e d and m o n i t o r e d f o r absorbance a t 280 nm. The f i r s t e f f l u e n t peak ( p o o l 1) c o n -t a i n e d 90 p e r c e n t o f the plasma a^A as v e r i f i e d by r a d i a l i m m u n o d i f f u s i o n (RID) a s s a y f o r a^M (BEHRING/HOECHST) (see F i g . 1 ) . T h i s p o o l was t h e n s u b j e c t e d t o immunoadsorption chromatography d i r e c t e d a g a i n s t c o n t a m i n a t i n g p r o t e i n s p e c i e s . B r i d g e s , A p p l e g a r t h , Johannson, D a v i d s o n , and Wong Page 6 129 P r e p a r a t i o n o f t h e immunoadsorption column i n v o l v e d t h e f o l l o w i n g s t e p s : C o m m e r c i a l l y - a v a i l a b l e a n t i s e r a r a i s e d i n r a b b i t s a g a i n s t human p l a s m a p r o -t e i n s (BEHRING/HOECHST) were p o o l e d as f o l l o w s : 18.8 ml a n t i - h a p t o g l o b i n ( m a n u f a c t u r e r ' s q u o t e d t i t r e 1.52 mg/ml), 11.3 ml a n t i - I g A ( t i t r e 1.56 mg/ml), 2.5 ml a n t i - I g G ( t i t r e 1.87 mg/ml), 2.5 ml a n t i - I g M ( t i t r e 4.34 mg/ml), 2.5 ml a n t i - t r a n s f e r r i n ( t i t r e 1.3 mg/ml), 2.5 ml a n t i - a l b u m i n ( t i t r e 1.06 mg/ml), 2.0 ml a n t i - a j - a n t i t r y p s i n ( t i t r e 0.56 mg/ml) and 2.0 ml a n t i - c e r u l o p l a s m i n ( t i t r e 0.50 mg/ml). T h i s m a t e r i a l was t h e n t r e a t e d w i t h a f i n a l c o n c e n t r a t i o n o f 2.5 M ammonium s u l f a t e t o p r e c i p i t a t e t h e gamma g l o b u l i n f r a c t i o n o f t h e serum p o o l . T h i s p r e c i -p i t a t e was washed w i t h 3.0 M ammonium s u l f a t e and r e s u s p e n d e d i n p h o s p h a t e b u f f e r e d s a l i n e . The ammonium s u l f a t e p r e c i p i t a t i o n was r e p e a t e d and t h e washed p e l l e t was r e s u s p e n d e d i n 100 mM sodium b i c a r b o n a t e b u f f e r pH 8.0, c o n t a i n i n g 500 mM sodium c h l o r i d e , and c o v a l e n t l y bonded t o 18 grams C N B r - a c t i v a t e d s e p h a r o s e 4B (PHARMACIA) a c c o r d i n g t o t h e m a n u f a c t u r e r ' s i n s t r u c t i o n s . The gamma g l o b u l i n s were a l l o w e d t o b i n d t o t h e a c t i v a t e d g e l f o r 2 B r i d g e s , A p p l e g a r t h , Johannson, D a v i d s o n , and Wong Page 7 h o u r s . The g e l was t h e n p a c k e d i n t o a 1.6 X 28 cm column and e q u i l i b r a t e d w i t h 50 mM T r i s HC1, pH 7.5 c o n t a i n i n g 250 mM sodium c h l o r i d e and 3 nM sodium a z i d e . A l i q u o t s o f CBS p o o l I , c o n t a i n i n g 6 mg c^M by r a d i a l i m m u n o d i f f u s i o n a s s a y (BEHRING/HOECHST), were a p p l i e d t o t h e immunoadsorption column, and p u r i f i e d c^M was e l u t e d w i t h t h e same b u f f e r a t a r a t e o f 15 ml/hour. A p p r o x i m a t e l y 70 p e r c e n t of s t a r t i n g plasma a^M c o u l d be r e c o v e r e d i n t h i s e f f l u e n t . T h i s m a t e r i a l was c o n c e n t r a t e d t o 1 mg/ml by u l t r a f i l t r a t i o n i n s t i r r e d c e l l s u s i n g -YM30-membranes (AMICON) and s t o r e d a t 4° u n t i l use (< 2 weeks). Contaminants were d e s o r b e d f r o m t h e immunoadsorption g e l by e l u t i o n w i t h s t a r t i n g buffer a l t e r e d t o pH 2.5 u s i n g HC1. E s t i m a t i o n o f P r o t e i n C o n t e n t o f Chromatography F r a c t i o n s and g^M I s o l a t e s : The p r o t e i n c o n t e n t o f chromatography f r a c t i o n s and g^M i s o l a t e s was measured by e x t i n c t i o n a t 280 nm, u s i n g 1 g / d l e x t i n c t i o n c o e f f i c i e n t s o f 10.0 f o r CBS e f f l u e n t f r a c t i o n s and 9.07 f o r g^M i s o l a t e s [ 1 3 ] , A c c u r a c y o f t h e r e s u l t s was c h e c k e d by a B r i d g e s , A p p l e g a r t h , Johannson, D a v i d s o n , and Wong Page 8 131 B r a d f o r d dye b i n d i n g m i c r o a s s a y (BIORAD) u s i n g p r o t e i n s t a n d a r d s p r e p a r e d f r o m V e r s a t o l c o n t r o l serum (GENERAL DIAGNOSTICS). The methods c o r r e l a t e d w e l l (R = 0.90) and c o u l d be r e l a t e d as f o l l o w s : (yg B r a d f o r d p r o t e i n ) X (1.45) = ( y g e x t i n c t i o n 280 nm p r o t e i n ) . Weights o f c^M and BCT s t a t e d h e r e a f t e r a r e t h o s e d e t e r m i n e d by e x t i n c t i o n . The Lowry p r o t e i n a s s a y was a v o i d e d because o f i n t e r f e r e n c e s i n c o l o u r development p r o d u c e d by ' t r i s ' b u f f e r [ 1 4 ] . Assessment o f P u r i t y o f Chromatography F r a c t i o n s and qoM I s o l a t e s : T h r e e t e s t s o f p u r i t y were a p p l i e d t o c^M i s o l a t e s : ( a ) i m m u n o e l e c t r o p h o r e s i s ( I E P ) w i t h p o l y -s p e c i f i c and m o n o s p e c i f i c a n t i s e r a , (b) g r a d i e n t 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 (PAGE), and ( c ) sodium d o d e c y l s u l f a t e - P A G E (SDS-PAGE) w i t h and w i t h o u t t h i o l r e d u c t i o n . ( a ) I m m u n o e l e c t r o p h o r e s i s ( I E P ) : IEP o f 12 yg a l i q u o t s o f c^M was p e r f o r m e d i n 1 g / d l agar g e l s u s i n g commercial a p p a r a t u s (GELMAN INSTRUMENT CO.). i s o l a t e s were t e s t e d w i t h r a b b i t a n t i s e r a a g a i n s t whole human serum, and w i t h B r i d g e s , A p p l e g a r t h , Johannson, D a v i d s o n , and Wong Page 9 132 r a b b i t m o n o s p e c i f i c a n t i s e r u m a g a i n s t human a^M, IgG, IgA, IgM, f i b r i n o g e n , e - l i p o p r o t e i n , ^ - a n t i -t r y p s i n , c e r u l o p l a s m i n , t r a n s f e r r i n , a l b u m i n , and h a p t o g l o b i n (BEHRING/HOECHST). (b) G r a d i e n t PAGE: G r a d i e n t PAGE o f a^M i s o l a t e s was p e r f o r m e d u s i n g 4 t o 26 g / d l p h o t o p o l y m e r i z e d a c r y l a m i d e g e l s a c c o r d i n g t o a m o d i f i c a t i o n o f t h e method o f M a r g o l i s and K e n r i c k [ 1 5 ] . The g r a d i e n t o f 4 t o 26 g / d l a c r y l a m i d e ( c o n t a i n i n g 5 p e r c e n t c r o s s - l i n k e r ) i n 90 mM T r i s HC1, pH 9.2 w i t h 9 mM b o r i c a c i d , 2.'5'rtM d i s o d i u m 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 (EDTA) and 13 yM^  r i b o f l a v i n was formed u s i n g a g r a d i e n t maker (LKB). G e l s were p o u r e d i n a darkened room u s i n g a p e r i s t a l t i c pump (BUCHLER POLYSTALTIC) a t a r a t e o f 25 ml/hour. The a c r y l a m i d e g r a d i e n t was s t a b i l i z e d w i t h a p a r a l l e l 30 t o 350 rnM s u c r o s e d e n s i t y g r a d -i e n t . To e n s u r e t h a t p o l y m e r i z a t i o n began a t the 4 g / d l end o f the g e l , an a n t i p a r a l l e l N,N,N',N'-tetra-m e t h y l e t h y l e n e d i a m i n e (TEMED) c o n c e n t r a t i o n g r a d i e n t f r o m 0 t o 0.08 m l / d l was used. P o l y m e r i z a t i o n was B r i d g e s , A p p l e g a r t h , Johannson, D a v i d s o n , and Wong Page 10 i n i t i a t e d by e x p o s u r e t o overhead f l u o r e s c e n t l i g h t -i n g . A f t e r p h o t o p o l y m e r i z a t i o n , g e l s were d i a l y s e d o v e r n i g h t t w i c e a g a i n s t 2 l i t e r changes o f e l e c t r o -p h o r e s i s b u f f e r (90 mM T r i s HC1, pH 9.2 w i t h 9 mM b o r i c a c i d and 2.5 mM d i s o d i u m EDTA) t o remove s u c -r o s e . G r a d i e n t g e l s were r un i n a h o r i z o n t a l e l e c t r o p h o r e s i s chamber (BRINKMANN/DESAGA), e q u i p p e d w i t h a t h e r m o s t a t i c c o o l e r (LAUDA). G e l s were p r e - e l e c t r o p h o r e s e d f o r 30 minutes a t 70 v o l t s ( a p p r o x i m a t e l y 2.5 ma) (BRINKMANN v o l t a g e and c u r r e n t r e g u l a t e d power s u p p l y ) . 20 yg o^M samples and s t a n d a r d p r o t e i n s were a p p l i e d at t h e c a t h o d a l end o f t h e g e l i n double s t r e n g t h e l e c t r o p h o r e s i s b u f f e r c o n t a i n i n g 0.75 mM bromophenol b l u e t r a c k i n g dye. Samples were e l e c t r o p h o r e s e d a t 70 v o l t s f o r 30 minutes t o e n s u r e p r o t e i n e n t r y i n t o t h e 4 g / d l p o r t i o n o f t h e g e l . V o l t a g e was t h e n i n c r e a s e d t o 400 v o l t s ( a p p r o x i m a t e l y 15 ma), and g e l s were r u n f o r 72 hours w i t h b u f f e r changes e v e r y 8 - 1 0 h o u r s . G e l s were s t a i n e d f o r 2 hours with 3 mM Coomassie b r i l l i a n t b l u e G i n 5 methanol : 1 a c e t i c a c i d : 5 d i s t i l l e d water, and t h e n d e s t a i n e d f o r 2 days i n 2 methanol : 1 a c e t i c a c i d : 5 d i s t i l l e d water p r i o r t o p h o t o g r a p h y . B r i d g e s , A p p l e g a r t h , Johannson, D a v i d s o n , and Wong Page 11 134 ( c ) SDS-PAGE: SDS-PAGE was p e r f o r m e d w i t h 5 g / d l p o l y a c r y l -amide t u b e g e l s , a c c o r d i n g t o a m o d i f i c a t i o n o f t h e method o f Weber e t al _ . [ 1 6 ] . Sample, r e s e r v o i r and t u b e g e l b u f f e r were t h e same as used by t h e s e a u t h o r s , e x c e p t t h a t p o t a s s i u m p e r s u l f a t e (3.3 mM) was used i n p l a c e o f ammonium p e r s u l f a t e f o r c h e m i c a l p o l y m e r i z a t i o n o f g e l s . Twenty or 100 yg samples o f a^M were mixed w i t h 100 y l o f sample b u f f e r , and h e a t e d t o 37° f o r one hour, t h e n samples were a p p l i e d t o g e l s . Tube g e l s were r u n on a v e r t i c a l a p p a r a t u s (BIO-RAD) a t room t e m p e r a t u r e . - a^M samples were e l e c t r o p h o r e s e d a t 3 ma/tube f o r 16 h o u r s . G e l s were t h e n removed f r o m tubes and s t a i n e d and d e s t a i n e d by t h e method d e s c r i b e d f o r g r a d i e n t s l a b g e l s . P u r i t y o f a.£\ i s o l a t e s was t h e n a s s e s s e d u s i n g a d e n s i t o -meter a t 595 nm. Monomeric a^M was d e m o n s t r a t e d by t h i s p r o c e d u r e . Samples used t o d e m o n s t r a t e c^M d i m e r i c s t r u c t u r e were h a n d l e d s i m i l a r l y , e x c e p t t h a t B - m e r c a p t o e t h a n o l was o m i t t e d f r o m t h e sample b u f f e r . Samples used t o demonstrate p r o t e a s e - c o n -v e r s i o n o f a^M t o t h e 85,000 d a l t o n f r a g m e n t r e q u i r e d p r i o r i n c u b a t i o n o f 20 yg c^M w i t h t h e B r i d g e s , A p p l e g a r t h , Johannson, D a v i d s o n , and Wong Page 12 135 e q u i v a l e n t o f 1.31 pg 100 p e r c e n t a c t i v e b o v i n e c a t i o n i c t r y p s i n i n 50 mM T r i s HC1 pH 7.5, c o n t a i n i n g 5 mM sodium c i t r a t e and 145 mM sodium c h l o r i d e a t 37° C f o r 30 m i n u t e s , f o l l o w e d by p r o t e a s e i n a c t i v a t i o n by t r e a t m e n t w i t h 100 p i o f sample b u f f e r c o n t a i n i n g 10M ure a and 140 mM e - m e r c a p t o e t h a n o l [ 1 7 ] . Assessment o f S t r u c t u r e o f P u r i f i e d g^M: N a t i v e s t r u c t u r e o f c^M was d e m o n s t r a t e d by t h e e l e c t r o p h o r e t i c t e c h n i q u e s j u s t d e s c r i b e d , c a l i -b r a t e d w i t h a p p r o p r i a t e h i g h m o l e c u l a r weight e l e c -t r o p h o r e s i s marker p r o t e i n s (PHARMACIA). G r a d i e n t PAGE was used t o show t h a t our i s o l a t e s p o s s e s s e d n a t i v e t e t r a m e r i c s t r u c t u r e . SDS-PAGE was used t o show t h e s t r u c t u r e o f o^M i s o l a t e s as f o l l o w s : SDS t r e a t m e n t p e r m i t t e d d e m o n s t r a t i o n o f dim e r s ; e - m e r c a p t o e t h a n o l + SDS t r e a t m e n t p e r m i t t e d d e m o n s t r a t i o n o f monomers and 85,000 d a l t o n p r o t e o -l y t i c f r a g m ents p r o d u c e d by t r y p s i n . F o r e x p l a n a -t i o n s o f t h e t h e o r y b e h i n d t h e s e t r e a t m e n t s see t h e D i s c u s s i o n s e c t i o n . B r i d g e s , A p p l e g a r t h , Johannson, D a v i d s o n , and Wong Page 13 Assessment o f P r o t e a s e B i n d i n g A b i l i t y o f g^M I s o l a t e s : The a b i l i t y o f g^M i s o l a t e s t o b i n d endo-p e p t i d a s e s has been t a k e n as the c r i t e r i o n o f n a t i v e a c t i v i t y . T h r e e i n d e p e n d e n t t e s t s o f t h i s a c t i v i t y were employed: ( a ) t r e a t m e n t o f a^M i s o l a t e s w i t h p u r i f i e d b o v i n e c a t i o n i c t r y p s i n f o l l o w e d by e x p o s u r e o f t h e complex so formed t o a h i g h m o l e c u l a r weight s u b s t r a t e , Remazol b r i l l i a n t b l u e h i d e (RBBhide) (SIGMA No. H4631); (b) g r a d i e n t PAGE c a l i b r a t e d w i t h a c t i v e and ammonium s u l f a t e - i n a c t i v a t e d o^M e l e c -t r o p h o r e t i c s t a n d a r d s ; and ( c ) D e m o n s t r a t i o n by SDS-PAGE o f q u a n t i t a t i v e c o n v e r s i o n o f a^^ t o t h e 85,000 d a l t o n fragment by t r e a t m e n t w i t h b o v i n e c a t i o n i c t r y p s i n . ( a ) ggM T i t r a t i o n With B o v i n e C a t i o n i c T r y p s i n : P u r i f i e d e b o v i n e c a t i o n i c t r y p s i n (BCT) was p r e p a r e d by t h e SE Sephadex c h r o m a t o g r a p h i c method o f S c h r o e d e r et_ a_[. [ 1 8 ] , u s i n g commercial t w i c e c r y s t -a l l i z e d b o v i n e t r y p s i n (SIGMA No. T8253) as s t a r t i n g m a t e r i a l . BCT so p u r i f i e d was d i a l y s e d a g a i n s t 1 nM h y d r o c h l o r i c a c i d w i t h 20 mM c a l c i u m c h l o r i d e t o B r i d g e s , A p p l e g a r t h , Johannson, D a v i d s o n , and Wong Page 14 remove t h e column b u f f e r and c o n c e n t r a t e d b y u l t r a -f i l t r a t i o n (Amicon MMC w i t h YM10 membranes) t o 5 mg/ml. T h i s m a t e r i a l was t h e n q u i c k f r o z e n and s t o r e d a t -70° u n t i l use. Homogeneity o f p u r i f i e d BCT was d e m o n s t r a t e d by a g a r e l e c t r o p h o r e s i s (as d e s c r i b e d f o r I E P ) . BCT c o n t e n t o f s t o c k and w o r k i n g s o l u t i o n s was d e t e r m i n e d by e x t i n c t i o n a t 280 nm (1 g / d l e x t i n c t i o n c o e f f i c i e n t = 13.9 [ 1 9 ] ) . A c t i v i t y o f BCT s t o c k s o l u t i o n s was a s s e s s e d by a c t i v e s i t e t i t r a t i o n w i t h NP6B a c c o r d i n g t o t h e method o f Chase and Shaw [ 2 0 ] . Commercial b o v i n e t r y p s i n was f o u n d t o be 50-60 p e r c e n t a c t i v e . P u r i f i e d B BCT was 75-80 p e r c e n t a c t i v e . The t r e a t m e n t o f c^M w i t h BCT was p e r f o r m e d u s i n g t h e i n s o l u b l e s u b s t r a t e RBB-hide as f o l l o w s : T o a s e r i e s o f t u b e s c o n t a i n i n g 8 - 12 yg a^M, t h e e q u i v a l e n t o f 0.1 t o 1.0 yg 100 p e r c e n t a c t i v e BCT was added i n a f i n a l i n c u b a t i o n volume o f 3.00 ml o f 50 nM T r i s - H C l , pH 8.0. Tubes were mixed and p r e -i n c u b a t e d a t 37° f o r 30 minutes t o e n s u r e complex-i n g . Twenty m i l l i g r a m s RBB-hide was t h e n added t o each t u b e , and t h e m i x t u r e was i n c u b a t e d a t 37° f o r 30 minutes w i t h f r e q u e n t v o r t e x i n g ( o n c e / t u b e / m i n . ) . B r i d g e s , A p p l e g a r t h , Johannson, D a v i d s o n , and Wong Page 15 The r e a c t i o n was t e r m i n a t e d by f i l t e r i n g out t h e r e m a i n i n g RBB-hide. The amount o f dye p r o t e o l y t i c a l l y r e l e a s e d i n t o t h e medium f r o m t h e h i d e was a s s e s s e d by s p e c t r o p h o t o m e t r y a s s a y o f the f i U r a t e a t 595 nm. (b) G r a d i e n t PAGE: G r a d i e n t PAGE was p e r f o r m e d as d e s c r i b e d above on a^M i s o l a t e s and on a c t i v e ("slow") and ammonium i o n - i n a c t i v a t e d ( " f a s t " ) a^M e l e c t r o p h o r e t i c s t a n -d a r d s . The p r o t e a s e b i n d i n g a c t i v i t y o f c^M e l e c -t r o p h o r e t i c s t a n d a r d s had been p r e v i o u s l y e s t a b l i s h e d by t h e t i t r a t i o n a s s a y . Ammonium i o n - i n a c t i v a t i o n of c^M was a c c o m p l i s h e d by i n c u b a t i o n o f c^M w i t h 200 mM ammonium s u l f a t e f o r 30 minutes a t 3 7 ° , f o l l o w e d by d i a l y s i s a g a i n s t CBS b u f f e r and c o n c e n -t r a t i o n by u l t r a f i l t r a t i o n . ( c ) SDS-PAGE: SDS-PAGE w i t h t h i o l r e d u c t i o n was p e r f o r m e d as d e s c r i b e d above on c^M r e a c t e d i n a 1:2 molar r a t i o w i t h BCT. B r i d g e s , A p p l e g a r t h , Johannson, D a v i d s o n , and Wong Page 16 I I I . RESULTS AND DISCUSSION: I s o l a t i o n o f q^M We have m o d i f i e d t h e method o f V i r c a e t aJL [ 9 ] by a p p l y i n g whole p l a s m a , t a k e n i n t o a m i x t u r e o f a n t i p r o t e a s e r e a g e n t s , d i r e c t l y t o a C i b a c r o n b l u e s e p h a r o s e (CBS) column. V i r c a e t a l . had d i a l y s e d ^ t h e i r plasma samples a g a i n s t column b u f f e r p r i o r t o chromatography. By a v o i d i n g d i a l y s i s , we sought t o d e c r e a s e t h e j_n v i t r o e x p o s u re t i m e o f a^M t o t h e p r o t e o l y t i c f a c t o r s o f plasma and t o a v o i d p o s s i b l e d e l e t e r i o u s e f f e c t s o f d i a l y s i s . J u d g i n g from t h e p h o t o g r a p h o f t h e g r a d i e n t PAGE g e l (4 t o 8 g / d l a c r y l a m i d e ) which appears i n t h e i r p a p e r [ 9 ] , t h e p r o d u c e d by V i r c a e_t a l _ . may be a m i x t u r e o f t h e " f a s t " and "slow" e l e c t r o p h o r e t i c forms o f t h e m o l e c u l e . I f s o , t h e n some i n a c t i v a t i o n o f a^M may have o c c u r r e d d u r i n g i s o l a t i o n . Our a p p l i c a t i o n of whole plasma t o t h e CBS column a v o i d s t h i s and does not a f f e c t t h e p r o t e i n r e s o l u t i o n . ( F i g . 1; compare t h i s w i t h CBS e l u t i o n p r o f i l e o f V i r c a e t a_l_. [ 9 ] ) . B r i d g e s , A p p l e g a r t h , Johannson, D a v i d s o n , and Wong Page 17 A n o t h e r m o d i f i c a t i o n o f the CBS method o f V i r c a e t a_h [ 9 ] was a 50 p e r c e n t r e d u c t i o n i n t h e volume o f plasma a p p l i e d t o t h e column. By t h e s e maneuvers and employing a v e r y s l o w e l u t i o n r a t e (1.5 m l / h r ) we were a b l e t o b r i n g 90 p e r c e n t o f s t a r t i n g plasma a^ M t o an a v e r a g e s t a t e o f 60 p e r c e n t p u r i t y ( r a n g e , 40-90 p e r c e n t depending upon plasma h a p t o -g l o b i n t y p e ) i n a s i n g l e s t e p . V i r c a e t al_. a c h i e v e d a p p a r e n t p u r i t y (as a s s e s s e d by G r a d i e n t PAGE) of 40 p e r c e n t s t a r t i n g plasma o^ M i n a s i n g l e CBS s t e p ; however, o n l y p l a s m a h a p t o g l o b i n t y p e 1-1 c o u l d be used, s i n c e use o f t y p e 2-1 and 2-2 plasmas r e s u l t e d i n g r o s s h a p t o g l o b i n c o n t a m i n a t i o n o f t h e i r c^M i s o l a t e s [ 9 ] . By s u b j e c t i n g our a^M-rich CBS p o o l I ( f r a c t i o n s No. 24-34, F i g . 1) t o a second p u r i f i -c a t i o n s t e p , we have broadened the a p p l i c a b i l i t y o f th e method so t h a t a l l plasma samples, r e g a r d l e s s of h a p t o g l o b i n t y p e , c an be used. CBS p o o l I i s a p p l i e d , w i t h o u t p r i o r c o n c e n t r a t i o n , t o an immuno-a d s o r p t i o n column d i r e c t e d a g a i n s t 8 r e c o g n i z e d c o n t a m i n a n t s o f t h e p o o l . a^ M o f a t l e a s t 98 p e r c e n t p u r i t y i s e l u t e d from t h e column. The B r i d g e s , A p p l e g a r t h , Johannson, D a v i d s o n , and xWong Page 18 a v e r a g e c^M y i e l d i s 71 p e r c e n t ( r a n g e , 48-99 p e r c e n t , n=9) o f s t a r t i n g plasma a^M. T h i s compares f a v o r a b l y w i t h t h e 75 p e r c e n t y i e l d from the two s t e p method o f V i r c a e t al_ [ 9 ] . However, l o s s e s of up t o 50 p e r c e n t o f our m a t e r i a l were e n c o u n t e r e d upon u l t r a f i l t r a t i o n c o n c e n t r a t i o n o f i s o l a t e s t o 1 mg/ml f o r s u b s e q u e n t c h a r a c t e r i z a t i o n . U l t r a f i l t r a t i o n i n s t i r r e d c e l l s (AMICON MMC) u s i n g a v a r i e t y o f d i f f e r e n t membrane t y p e s (AMICON XM, PM, UM and YM membranes and MILLI PORE PT and PS membranes) o f a p p r o p r i a t e l y s m a l l m o l e c u l a r w e i g h t c u t - o f f l i m i t s gave v a r i a b l e l o s s e s o f p u r i f i e d d^M, r a n g i n g from 20 - 50 p e r c e n t . C o n c e n t r a t i o n d i a l y s i s a g a i n s t p o l y e t h y l e n e g l y c o l or p o l y a c r y l a m i d e a l s o gave s i m i l a r p o o r r e c o v e r i e s . At p r e s e n t , t h e r e a p p e a r s t o be no e a s y way t o c o n c e n -t r a t e s m a l l q u a n t i t i e s o f t h i s p r o t e i n . L y o p h i l -i z a t i o n was not a t t e m p t e d f o r f e a r o f i n a c t i v a t i n g o^M. P r o o f o f P u r i t y o f « ? M I s o l a t e s : P u r i t y o f i s o l a t e s was d e m o n s t r a t e d by B r i d g e s , A p p l e g a r t h , Johannson, D a v i d s o n , and Wong Page 19 t h r e e i n d e p e n d e n t t e s t s : (a) IEP o f i s o l a t e s r e v e a l e d a s i n g l e p r e -c i p i t i n a r c when d e v e l o p e d by p o l y s p e c i f i c a n t i s e r a . P o s i t i v e i d e n t i f i c a t i o n o f t h e a r c as c^M was a c c o m p l i s h e d u s i n g m o n o s p e c i f i c r a b b i t antihuman a^M serum; t h e p r o d u c t was f r e e o f t e n o f t h e p o t e n t i a l c o n t a m i n a n t s t h e o r e t i c a l l y e x p e c t e d (see Methods s e c t i o n ) a t l e v e l s which would have d e t e c t e d a p p r o x i m a t e l y 5 p e r c e n t w/w c o n t a m i n a t i o n . (b) A s i n g l e h i g h m o l e c u l a r weight band i s o b s e r v e d when 20 yg a^M i s a p p l i e d t o g r a d i e n t PAGE ( F i g 2, t r a c k s 1, 2, 4, 5 ) . ( c ) SDS-PAGE w i t h t h i o l r e d u c t i o n o f 20 y g a 2M i s o l a t e s r e v e a l e d l e s s t h a n 1 p e r c e n t contam-i n a t i o n by weight when s t a i n e d g e l s were a n a l y s e d by d e n s i t o m e t r y ( F i g . 3 A ) . A p p l i -c a t i o n o f 5 t i m e s t h e u s u a l amount o f c^M t o t h e g e l a l l o w e d v i s u a l assessment o f contam-i n a t i o n ( F i g . 3B). a^M i s o l a t e s were f o u n d u n i f o r m l y t o be o f h i g h p u r i t y ( a t l e a s t 98 p e r c e n t ) r e g a r d l e s s o f h a p t o g l o b i n t y p e o f s t a r t i n g p l a s m a . B r i d g e s , A p p l e g a r t h , Johannson, D a v i d s o n , and Wong Page 2 0 143 P r o o f o f S u b u n i t S t r u c t u r e o f g ^M I s o l a t e s : In v i v o g,,M i s a t e t r a m e r o f about 7 2 5 , 0 0 0 d a l t o n s m o l e c u l a r weight [ 2 1 ] . Monomeric u n i t s ( 1 8 5 , 0 0 0 d a l t o n s ) a r e j o i n e d t o f o r m dimers by i n t e r -c h a i n d i s u l f i d e bonds, and each monomer i s l i n k e d i n t e r n a l l y by i n t r a c h a i n d i s u l f i d e bonds [ 1 7 ] . Dimers a r e l i n k e d t o g e t h e r by n o n - c o v a l e n t i n t e r -a c t i o n s [ 1 , 1 7 ] . One can , t h e r e f o r e , c h e m i c a l l y d i s s e c t t h e s u b u n i t s t r u c t u r e o f p u r i f i e d g^M. I f t h e m a c r o m o l e c u l e does not fragment i n t o dimers a f t e r t r e a t m e n t w i t h u r e a , d e t e r g e n t s o r c h a o t r o p i c agents and i f t h e dimers do not f o r m monomers a f t e r t h i o l r e d u c t i o n , t h e n t h e g^M i s o l a t e s do not c o r r e s p o n d t o c u r r e n t l y a c c e p t e d p r o o f s f o r n a t i v e i n t a c t g ^M s t r u c t u r e . F o r example, one group o f workers f o u n d t e t r a m e r i c g^M i s o l a t e d from normal plasma c o n -v e r t e d c o m p l e t e l y t o t h e monomeric f o r m upon exposure t o SDS [ 2 ] . However, t h i s g ^ M had been exposed t o a t l e a s t one r e c o g n i z e d d e n a t u r a n t d u r i n g t h e i s o l a t i o n p r o c e s s (ammonium s u l f a t e ) [ 7 ] . F i g u r e 2 shows t h a t g^M i s o l a t e d by our p r o c e d u r e r e t a i n s i t s t e t r a m e r i c s t r u c t u r e , m i g r a t i n g B r i d g e s , A p p l e g a r t h , Johannson, D a v i d s o n , and Wong 144 Page 21 j u s t b e h i n d p o r c i n e t h y r o g l o b u l i n (669,000 d a l t o n s m o l e c u l a r w e i g h t ) i n a g r a d i e n t PAGE g e l . F i g u r e 4 demonstrates t h a t our g ^ i s o l a t e s e x h i b i t normal s u b u n i t s t r u c t u r e s i n c e SDS t r e a t m e n t o f a^A p r o d u c e s t h e d i m e r i c f o r m o f t h e m o l e c u l e ( a p p r o x i -m a t e l y 360,000 d a l t o n s ) ( F i g 4 a ) , and t r e a t m e n t o f a^M w i t h SDS and B-mercaptoethanol p r o d u c e s t h e monomeric f o r m o f a 2M (185,000 d a l t o n s ) ( F i g 4 b ) . P r o o f o f P r o t e a s e - B i n d i n g A b i l i t y o f g^M I s o l a t e s : (a.) g 2M T i t r a t i o n With BCT: g 2 M - p r o t e a s e complexes p o s s e s s no r e s i d u a l p r o t e o l y t i c a c t i v i t y towards h i g h m o l e c u l a r weight s u b s t r a t e s . P r o t e a s e added t o g,,M i n e x c e s s o f t h e b i n d i n g c a p a c i t y w i l l h y d r o l y s e t h e h i g h m o l e c u l a r weight s u b s t r a t e RBB-hide. F i g . 5 shows a t y p i c a l g 2M t i t r a t i o n c u r v e o f p r o t e a s e added t o g,,M t h e n r e a c t e d w i t h RBB-hide. The weight o f 100 p e r c e n t a c t i v e BCT r e q u i r e d t o s a t u r a t e t h e b i n d i n g s i t e s o f 8 - 12 yg g ^ c a n be f o u n d by e x t r a p o l a t i o n . Assuming t h e s t o i c h i o m e t r y o f b i n d i n g t o be 2 moles t r y p s i n p e r mole a 2M [ 7 , 1 1 ] , and assuming m o l e c u l a r w e i g h t s o f 23,800 and 725,000 r e s p e c t i v e l y [22, 2 1 ] , B r i d g e s , A p p l e g a r t h , Johannson, D a v i d s o n , and Wong 145 Page 22 one c a n c a l c u l a t e t h a t 1 pg BCT s h o u l d be bound by 15.2 pg a ^ M . T h i s i n f o r m a t i o n and t h e t i t r a t i o n d a t a a l l o w s one t o compute t h e a c t i v i t y o f t h e o ^ M i s o l a t e . The a^M i s o l a t e s i n t h i s s t u d y showed a mean (± S.D.) b i n d i n g r a t i o o f 1.92 moles BCT/mole (± 0.21) (n = 1 2 ) . ( b j G r a d i e n t PAGE o f I s o l a t e s ; When a ^ M b i n d s p r o t e a s e s t h e r e i s a change i n i t s a b i l i t y t o p e n e t r a t e g r a d i e n t g e l s which have i n c r e a s i n g p o l y a c r y l a m i d e c o n c e n t r a t i o n [ 7 ] . B a r r e t t e t al_. [ 7 ] have s u g g e s t e d t h a t t h e s u b u n i t s o f t h e a c t i v e t e t r a m e r a r e a r r a n g e d i n a c o p l a n a r c o n f i g -u r a t i o n , w h i l e t h o s e o f t h e i n a c t i v a t e d macromolecule may form t h e more compact p s e u d o t e t r a h e d r o n . T h i s w o u ld a l l o w t h e i n a c t i v e macromolecule t o p e n e t r a t e f u r t h e r i n t o t h e p o r e s o f a g r a d i e n t a c r y l a m i d e g e l m a t r i x , and c o u l d a c c o u n t f o r i t s " f a s t " p o s i t i o n on t h e s e g e l s r e l a t i v e t o a c t i v e ("slow") c ^ M . What-e v e r t h e r e l a t i v e c o n f i g u r a t i o n s o f a c t i v e and i n -a c t i v e a,>M, g r a d i e n t PAGE has become an e s t a b l i s h e d t e c h n i q u e t o dete r m i n e the p r o t e a s e b i n d i n g a c t i v i t y o f a ^ M i s o l a t e s [ 7 , 9 ] . In t h e p r e s e n t s t u d i e s , B r i d g e s , A p p l e g a r t h , Johannson, D a v i d s o n , and Wong Page 23 146 ammonium i o n - i n a c t i v a t e d ( " f a s t " ) c ^ M was used as an e l e c t r o p h o r e t i c marker p r o t e i n t o demonstrate t h a t o u r a ^M i s o l a t e s c o n t a i n o n l y t h e a c t i v e , e l e c t r o -p h o r e t i c a l l y "slow" m o l e c u l a r s p e c i e s ( F i g . 2 ) . T r a c k s 1, 2, 4 and 5 show e l e c t r o p h o r e s i s o f t y p i c a l <*2M i s o l a t e s a l o n e . T r a c k 3 shows ^ - e l e c t r o -p h o r e s i s o f t h e f a s t o^M s t a n d a r d w i t h commercial m o l e c u l a r weight marker p r o t e i n s ( P h a r m a c i a ) . ( c ) SDS-PAGE With T h i o l R e d u c t i o n o f c ^ M I s o l a t e s  and gpM-BCT Complexes: T r e a t m e n t o f p u r i f i e d a w i t h SDS and B-m e r c a p t o e t h a n o l r e s u l t e d i n p r o d u c t i o n o f a s i n g l e band, i . e . monomeric o^M ( F i g . 4b) a s s e s s e d by PAGE. P r e - t r e a t m e n t o f a 2 M w i t h BCT i n a 1:2 molar r a t i o c auses s t o i c h i o m e t r i c c o n v e r s i o n o f t h i s mono-mer t o t h e 85,000 d a l t o n fragment ( F i g . 4 c ) . Had t h e i s o l a t e s been p a r t i a l l y i n a c t i v a t e d by p r o t e a s e s d u r i n g t h e p u r i f i c a t i o n p r o c e s s , F i g . 4b would have shown c o n t a m i n a t i o n by t h e 85,000 d a l t o n f r a g m e n t . Had t h e a 2 M i s o l a t e s not been f u l l y a c t i v e , BCT t r e a t m e n t would have p r o d u c e d p r o t e o l y t i c f r a g m e n t s o f a 2 M , and t h e s e would have appeared as B r i d g e s , A p p l e g a r t h , Johannson, D a v i d s o n , and Wong Page 24 a smear, r a t h e r t h a n as a s i n g l e d i s c r e t e band o f 85,000 d a l t o n s ( F i g . 4c).. In summary we have p r e s e n t e d methods which a l l o w i s o l a t i o n o f a^M o f a t l e a s t 98 p e r c e n t p u r i t y i n h i g h y i e l d from 5 ml plasma samples. By a l l o f t h e c r i t e r i a t e s t e d , t h e c^M i s o l a t e i s i n i t s p h y s i o l o g i c a l l y a c t i v e form. B r i d g e s , A p p l e g a r t h , Johannson, D a v i d s o n , and Wong Page 25 148 ACKNOWLEDGEMENTS: We wish t o thank Dr. P. C. H a r p e l f o r s h a r i n g h i s e x p e r i e n c e i n c i b a c r o n b l u e s e p h a r o s e chromato-gr a p h y w i t h us and f o r o t h e r h e l p f u l d i s c u s s i o n s . We wis h t o acknowledge t h e k i n d c o - o p e r a t i o n o f Dr. L.T. K i r b y and Mrs. M. F r o h l i c h and t h e f i n a n c i a l s u p p o r t o f The C a n a d i a n C y s t i c F i b r o s i s F o u n d a t i o n . B r i d g e s , A p p l e g a r t h , Johannson, D a v i d s o n , and Wong 149 Page 26 REFERENCES (1) STARKEY, P.M. and BARRETT, A . J . [1977] a 2 m a c r o g l o b u l i n , a p h y s i o l o g i c a l r e g u l a t o r o f p r o t e i n a s e a c t i v i t y , i n : R e s e a r c h Mono-graphs In C e l l And T i s s u e P h y s i o l o g y V o l . J_I_:663-696. ed. J.T. D i n g l e , N o r t h - H o l l a n d P u b l i s h i n g Co., Amsterdam (2) SHAPIRA, E., MARTIN, C.L. and NADLER, H.L. [1977] Comparison between p u r i f i e d a 2macro-g l o b u l i n p r e p a r a t i o n s f r o m normal c o n t r o l s and p a t i e n t s w i t h c y s t i c f i b r o s i s - k i n e t i c and s t r u c t u r a l p r o p e r t i e s o f complex w i t h b o v i n e t r y p s i n . J . B i o l . C h e m . 252:7923-7929 (3) WILSON, G.B. and FUDENBERG, H.H. [1976] S t u d i e s on c y s t i c f i b r o s i s u s i n g i s o e l e c t r i c f o c u s i n g : II D e m o n s t r a t i o n o f d e f i c i e n t p r o t e o l y t i c c l e a v a g e o f a 2 m a c r o g l o b u l i n i n c y s t i c f i b r o s i s p l a s m a . P e d i a t . R e s . 10:87-96 (4) RASTOGI, S.D. and CLAUSEN, J . [1980] Abnor-m a l i t i e s i n n a t i v e and d i s s o c i a t e d a 2macro-g l o b u l i n i s o l a t e d from m u l t i p l e s c l e r o s i s serum. C l i n Chim A c t a 107:141-144. (5) MAHOUR, G.H., SONG, M.K., ADHAM, N.F. and RINDERKNECHT, H. [1978] a 2 m a c r o g l o b u l i n d e f i c i e n c y i n a p a t i e n t w i t h E h l e r s - D a n l o s syndrome. P e d i a t r i c s 61:894-897 (6) RASTOGI, S.C. CLAUSEN, J . , MELCHIOR, J.C. and DYGGVE, H.V. [1980] Abnormal serum a 2macro-g l o b u l i n i n D y g g v e - M e l c h i o r - C l a u s e n syndrome. J . C l i n . C h e m . C l i n . B i o c h e m . 18:67-68 B r i d g e s , A p p l e g a r t h , Johannson, D a v i d s o n , and Wong Page 27 (7) BARRETT, A . J . , BROWN, M.A. and SAYERS, C.A. [1979] The e l e c t r o p h o r e t i c a l l y 'slow' and ' f a s t ' forms o f t h e c t 2 m a c r o g l o b u l i n mole-c u l e . Biochem.J. 181:401-418 (8) McENTIRE, J . E . [1978] P u r i f i c a t i o n o f c ^ m a c r o g l o b u l i n by immunoadsorbent chroma-t o g r a p h y . J.Immunol.Meth. 24:39-45 (9) VIRCA, G.D., TRAVIS, J . , HALL, P.K. and ROBERTS, R.C. [1978] P u r i f i c a t i o n o f human a 2 m a c r o g l o b u l i n by chromatography on c i b a -c r o n b l u e s e p h a r o s e . A n a l . B i o c h e m . 89:274-278 (10) QUEEN, K.G. and PEACOCK, A.C. [1966] Hapto-g l o b i n t y p e d e t e r m i n a t i o n u s i n g n o n - c a r c i n o -g e n i c r e a g e n t s . C l i n . C h i m . A c t a 13:47-51 (11) SWENSON, R.P. and HOWARD, J.B. [1979] S t r u c t u r a l c h a r a c t e r i s t i c s o f human c^macro-g l o b u l i n s u b u n i t s . J . B i o l . C h e m . 254:4452-4456 (12) HARPEL, P.C. [1976] Human a 2 m a c r o g l o b u l i n . Meth.Enzymol. 45:639-652 (13) DUNN, J.T. and SPIRO, R.G. [1967] The a 2 m a c r o g l o b u l i n o f human p l a s m a : I . I s o l a t i o n and c o m p o s i t i o n . J . B i o l . C h e m . 242:5549-5555 (14) PETERSON, G.L. [1979] Review o f t h e f o l i n p h e n o l p r o t e i n q u a n t i t a t i o n method o f Lowry, Rosebrough, F a r r and R a n d a l l . A n a l . B i o c h e m . 100:201-220 B r i d g e s , A p p l e g a r t h , Johannson, D a v i d s o n , and Wong Page 28 151 (15) MARGOLIS, J . and KENRICK, K.G. [1968] 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 a c o n t i n u o u s m o l e c u l a r s i e v e g r a d i e n t . A n a l . B i o c h e m . 25:347-362 (16) WEBER, K., PRINGLE, J.R. and OSBORN, M. [1972] Measurement o f m o l e c u l a r w e i g h t s b y e l e c t r o -p h o r e s i s on SD S - a c r y l a m i d e g e l . Meth.Enzymol. 26:3-27 (17) HARPEL, P.C. [1973] S t u d i e s on human plasma c^macrog1obu1i n-enzyme i n t e r a c t i o n s . J.Exp.Med. 138:508-521 (18) SCHROEDER, D.D. and SHAW, E. [1968] J . B i o l . C h e m . 243:2943 (19) LOUVARD, M.N. and PUIGSERVER, A. [1974] On b o v i n e and p o r c i n e a n i o n i c t r y p s i n o g e n s . B i o c h i m . B i o p h y s . A c t a 371:177-185 (20) CHASE, T. J r . and SHAW, E. [1970] T i t r a t i o n o f t r y p s i n , p l a s m i n and th r o m b i n w i t h p - n i t r o -p h e n y l p - g u a n i d i n o b e n z o a t e HC1. Meth. Enzymol. 19:20-27 (21) JONES, J.M., CREETH, J.M. and KEKWICK, R.A. [1972] T h i o l r e d u c t i o n o f human c^macro-g l o b u l i n : The s u b u n i t s t r u c t u r e . Biochem.J. 127:187-197 (22) WALSH, K.A. [1970] T r y p s inogens and t r y p s i n s o f v a r i o u s s p e c i e s . Meth.Enzymol. 19:41-63 MB/gm:2242A B r i d g e s , A p p l e g a r t h , Johannson, D a v i d s o n , and Wong Page 29 F i g . 1: CIBACRON BLUE SEPHAROSE (CBS) CHROMATOGRAPHY OF HUMAN PLASMA B r i d g e s , A p p l e g a r t h , Johannson, D a v i d s o n , and Wong Page 30 F i g 2. G r a d i e n t PAGE i n a 4 t o 26 g / d l g e l T r a c k s 1, 4, 5 - a c t i v e a2M i s o l a t e d from c o n t r o l s u b j e c t s . T r a c k 2 - a c t i v e a2M i s o l a t e d from a c y s t i c f i b r o s i s p a t i e n t . T r a c k 3 - i n a c t i v e (ammonium s u l f a t e -t r e a t e d ) a;?M c o - e l e c t r o p h o r e s e d w i t h Pharmacia h i g h m o l e c u l a r weight c a l i b r a t i o n s t a n d a r d s ( t h y r o g l o b u l i n , f e r r i t i n , c a t a -l a s e , l a c t a t e dehydrogenase, and b o v i n e serum a l b u m i n ) . The band n e a r e s t t he o r i g i n i s t h e t r e a t e d a2M. 155 1 2 3 4 5 B r i d g e s , A p p l e g a r t h , Johannson, D a v i d s o n , and Wong 1 5 6 Page 31 F i g . 3: SDS-PAGE WITH THIOL REDUCTION OF a ? M ISOLATED FROM HAPTOGLOBIN TYPE 2-2 "PLASMA: Tube g e l A c o n t a i n s 20 pg c^M. Tube g e l B c o n t a i n s 100 pg o f t h e same o^M. B r i d g e s , A p p l e g a r t h , Johannson, D a v i d s o n , and Wong 1 5 8 Page 32 F i g . 4: SDS-PAGE o f g?M I s o l a t e s on 5 g / d l G e l s : (a) a 2M w i t h no t h i o l r e d u c t i o n . (b) a 2M w i t h t h i o l r e d u c t i o n . ( c ) w i t h p r i o r t r e a t m e n t w i t h BCT (1:2 m o l a r r a t i o ) , f o l l o w e d by p r o t e a s e i n a c t i -v a t i o n w i t h 10 M u r e a and t h i o l r e d u c t i o n . 159 0 ' J, . o I O B r i d g e s , A p p l e g a r t h , Johannson, D a v i d s o n , and Wong Page 33 160 F i g . 5: TITRATION OF a ? M WITH BCT ( S u b s t r a t e : RBB-hide) Weight o f c^M t i t r a t e d = 10.3 v g E q u i v a l e n t weight o f BCT = 0.61 vg M o l a r b i n d i n g r a t i o : 1.80 moles BCT/mole ot;?M 0.4 0.6 pg active BCT PROTEASE BINDING BY a 2 M A C R 0 G L 0 B U L I N IN CYSTIC FIBROSIS M i c h a e l A. B r i d g e s Derek A. A p p l e g a r t h * Joanne Johannson Lawrence T.K. Wong and A. George F. Davidson B i o c h e m i c a l D i s e a s e s L a b o r a t o r y C h i l d r e n ' s H o s p i t a l 250 West 59th Avenue Vancouver, B.C. Canada V5X 1X2 *To whom co r r e s p o n d e n c e s h o u l d be a d d r e s s e d . KEYWORDS C y s t i c F i b r o s i s a ^ M a c r o g l o b u l i n a 2M p r o t e a s e B r i d g e s , A p p l e g a r t h , Johannson, Wong, and Davidson Page 1 SUMMARY The i n t e r a c t i o n of a 2 M a c r o g l o b u l i n (ct2M) with exogenous p r o t e a s e s has been r e p o r t e d by o t h e r s t o be abnormal i n c y s t i c f i b r o s i s ( C F ) . We have re-examined t h e s e c l a i m s . Four parameters were c o n s i d e r e d : (1) the molar p r o t e a s e b i n d i n g o f c*2M; (2) the i n t e r a c t i o n o f bovi n e c a t i o n i c t r y p s i n (BCT), complexed t o a 2M, with low m o l e c u l a r weight s u b s t r a t e , Benzoyl a r g i n i n e e t h y l e s t e r (BAEE); (3) the s t a b i l i t y o f formed a 2M-BCT complexes; and (4) th e s u b u n i t s t r u c t u r e o f a 2M. We have found CF a 2M t o be s i m i l a r t o c o n t r o l « 2 M i n e v e r y r e s p e c t s t u d i e d . INTRODUCTION C y s t i c f i b r o s i s (CF) i s a g e n e t i c a l l y t r a n s -m i t t e d e x o c r i n o p a t h y , c l i n i c a l l y c h a r a c t e r i z e d by pulmonary d i s e a s e , abnormal sweat e l e c t r o l y t e s and p a n c r e a t i c i n s u f f i c i e n c y l e a d i n g t o m a l a b s o r p t i o n [ 1 ] . The e t i o l o g y and p a t h o g e n e s i s o f t h i s syndrome i s u n c l e a r but i t has been su g g e s t e d t h a t i t may B r i d g e s , A p p l e g a r t h , Johannson, Wong, and Davidson Page 2 i n v o l v e a p r o t e o l y t i c enzyme d e f i c i e n c y i n human e x o c r i n e glands and plasma, l e a d i n g t o the accum-u l a t i o n o f h i g h l y membrane a c t i v e p o l y c a t i o n i c pep-t i d e s , c a l l e d "CF f a c t o r s " [ 2 ] . A d e f i c i e n c y of p r o t e a s e a c t i v i t y w i t h t r y p s i n - l i k e s u b s t r a t e s p e c i -f i c i t y i n CF s a l i v a and plasma i s w e l l documented [ 2 ] . The p r o t e a s e i n h i b i t o r c ^ M a c r o g l o b u l i n (o^M) i s an i m p o r t a n t r e g u l a t o r of plasma p r o t e a s e a c t i v i t y [ 3 ] , and i t was su g g e s t e d by W i l s o n and Fudenberg t h a t a d e f e c t i n might u n d e r l i e t h i s d e f i c i e n c y o f p r o t e o l y s i s i n CF [ 4 ] . S h a p i r a and h i s co-workers examined the i n t e r -a c t i o n o f exogenous p r o t e a s e s with c^M d e r i v e d from CF and c o n t r o l plasma [ 5 - 7 ] . S t r i k i n g d i f f e r e n c e s were r e p o r t e d (1) i n the molar b i n d i n g r a t i o s o f p r o t e a s e s t o c^M i n plasma [ 5 ] , (2) i n the sub-s t r a t e k i n e t i c s o f p u r i f i e d c^M-bovine t r y p s i n complexes f o r the low m o l e c u l a r weight s u b s t r a t e b e n z o y l a r g i n i n e e t h y l e s t e r [ 6 ] , (3) i n the s t a b -i l i t y o f t h e s e complexes once formed [ 7 ] , and (4) i n the s u b - u n i t s t r u c t u r e o f a^A when a s s e s s e d by g e l chromatography i n the p r e s e n c e and absence o f sodium dodec y l s u l f a t e [ 6 ] . 166 B r i d g e s , A p p l e g a r t h , Johannson, Wong, and Davidson Page 3 The p r e s e n t i n v e s t i g a t i o n s r e p r e s e n t a r e -e x a m i n a t i o n o f the i n t e r a c t i o n o f CF and c o n t r o l a^M with b o v i n e t r y p s i n u s i n g agM p u r i f i e d and c h a r a c t e r i z e d by methods d e s c r i b e d i n a companion paper [ 8 ] . MATERIALS AND METHODS A) E x p e r i m e n t a l S u b j e c t s : F i v e CF p a t i e n t s (mean age 13.0 y e a r s ; range 8-16 y e a r s ) and s i x age/sex-matched normal c o n t r o l s (mean age 12.4 y e a r s ; range 7-16 y e a r s ) were s e l e c t e d f o r the s t u d y . A l l CF p a t i e n t s showed abnormal sweat e l e c t r o l y t e s and p a n c r e a t i c i n s u f f i c i e n c y , and a l l were r e c e i v i n g o r a l p a n c r e a t i c enzyme replacement t h e r a p y . The c l i n i c a l s t a t u s o f the CF p a t i e n t s v a r i e d ; t h e i r Shwachman s c o r e s were as f o l l o w s : 45, 60, 75, 80 and 90. Two o f the p a t i e n t s s e l e c t e d were r e c e i v i n g t r e a t m e n t with a n t i b i o t i c s . B) I s o l a t i o n o f o^M from CF and C o n t r o l B l o o d  Samples: F o l l o w i n g i n f o r m e d consent by p a r e n t or B r i d g e s , A p p l e g a r t h , Johannson, Wong, and Davidson Page 4 g u a r d i a n , t e n ml b l o o d samples were o b t a i n e d from CF and c o n t r o l s u b j e c t s . The h a n d l i n g o f b l o o d s p e c i -mens and the method of i s o l a t i o n of n a t i v e o^M from 5 ml plasma samples has been d e s c r i b e d elsewhere i n t h i s j o u r n a l [ 8 ] . A l l CF and c o n t r o l o^M i s o l a t e s were at l e a s t 98 p e r c e n t p u r e as c o n f i r m e d by SDS-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 (SDS-PAGE). The a n t i p r o t e a s e a c t i v i t y and t e t r a m e r i c form of p u r i f i e d o^M specimens was v e r i f i e d by gradientPAGE [ 8 ] . C) C h a r a c t e r i z a t i o n o f CF and C o n t r o l a ^ M : 1) M o l a r P r o t e a s e B i n d i n g by CF and C o n t r o l a 2 M : The t e c h n i q u e s used t o p r e p a r e the b o v i n e c a t i o n i c t r y p s i n (BCT) employed i n t h e s e s t u d i e s and t o a s s e s s i t s p r o t e o l y t i c a c t i v i t y have been o u t l i n e d i n a companion paper [ 8 ] . The molar b i n d i n g r a t i o s o f a 2 M and BCT were determined by p r e - i n c u b a t i o n o f otgM with v a r i o u s amounts of BCT f o l l o w e d by i n c u b a t i o n with the i n s o l u b l e s u b s t r a t e Remazol b r i l l i a n t b l u e - h i d e (RBB-hide) as d e s c r i b e d b e f o r e [ 8 ] . B r i d g e s , A p p l e g a r t h , Johannson, Wong, and Davidson Page 5 Weights o f a 2M and BCT were based upon q u a n t i t a t i o n o f <*2M and BCT by e x t i n c t i o n a t 280 nm u s i n g 1 g / d l e x t i n c t i o n c o e f f i c i e n t s of 9.07 [9] and 13.9 [ 1 0 ] , r e s p e c t i v e l y . 2) S u b s t r a t e K i n e t i c s of CF and C o n t r o l a 2M-BCT Complexes: M i c h a e l i s - M e n t e n s u b s t r a t e k i n e t i c s were a s s e s s e d f o r CF and c o n t r o l a 2M-BCT complexes u s i n g the low m o l e c u l a r weight s u b s t r a t e benz-o y l a r g i n i n e e t h y l e s t e r (BAEE) as f o l l o w s : In s i l i c a c u v e t t e s c o n t a i n i n g a f i n a l volume o f 2.00 ml 50 m T r i s - H C l , pH 8.0 with 20 rrM c a l c i u m c h l o r i d e , 18 yg CF or c o n t r o l a 2M and the e q u i v a l e n t of 0.42 yg 100 p e r c e n t a c t i v e BCT were mixed and a l l o w e d t o complex a t 37° f o r 2 minutes; next, BAEE was added t o the r e a c t i o n m i x t u r e t o g i v e a f i n a l c o n c e n t r a t i o n of 0.05 t o 0.45 mM; the s o l u t i o n was mixed by i n v e r s i o n o f the c u v e t t e and p l a c e d i n the 37° hea t e d a u t o m a t i c sample changing compartment o f a Beckman model 25 s p e c t r o p h o t o m e t e r . The r a t e of h y d r o l y s i s of BAEE was f o l l o w e d f o r 3 minutes by an absorbance change at 253 nm. 169 B r i d g e s , A p p l e g a r t h , Johannson, Wong, and Davidson Page 6 T h i s r a t e was p l o t t e d a g a i n s t s u b s t r a t e c o n c e n t r a t i o n . Lineweaver-Burk double r e c i -p r o c a l p l o t s of t h e s e data were used t o determine t he apparent K and V f o r BAEE m max of CF and c o n t r o l o^M-BCT complexes. 3) S t a b i l i t y o f CF and C o n t r o l g^M-BCT  Complexes: The s t a b i l i t y of CF and c o n t r o l c^M-BCT complexes was t e s t e d a f t e r an 18 hour i n c u b a -t i o n a t 37° The c r i t e r i o n chosen t o i n d i c a t e complex s t a b i l i t y was the a v a i l a b i l i t y o f the a c t i v e s i t e o f g,>M-bound BCT t o h i g h mole-c u l a r weight s u b s t r a t e (RBB-hide) and t o high m o l e c u l a r weight i n h i b i t o r (soybean t r y p s i n i n h i b i t o r [ S T I ] ) . P r o t e a s e s bound i n s t a b l e complex with a^A w i l l show no a c t i v i t y towards t h e s e l a r g e l i g a n d s [ 3 ] . D e t a i l s o f th e s e a s s a y s were as f o l l o w s : N i n e t y yg of a^A i s o l a t e were complexed with t h e e q u i v a l e n t o f 4.4 yg 100 p e r c e n t a c t i v e BCT i n a f i n a l volume of 1.0 ml 50 mM T r i s - H C l , pH 8.0. I f the t o t a l BCT molar B r i d g e s , A p p l e g a r t h , Johannson, Wong, and Davidson Page 7 b i n d i n g r a t i o o f the « 2 M i s o l a t e ( d e t e r m i n e d by the RBB-hide a s s a y d e s c r i b e d above) d e v i a t e d from t he t h e o r e t i c a l v a l u e o f 2 moles BCT/mole a 2 M ( " t h e o r e t ) ' t n e n t h e w e i 9 h t o f o^M complexed w i t h 4.4 pg 100 p e r c e n t a c t i v e BCT was a d j u s t e d by (90 w g ) X ( n t h e o r e t / n e x p t ) . T h i s s o l u t i o n was i n c u b a t e d f o r 18 hours at 37° These weights o f o^M and BCT e n s u r e d t h a t 75 p e r c e n t o f a^M was i n complex with a c t i v e BCT. To t e s t t h e s t a b i l i t y o f t h e a ?M-BCT complex i n c u b a t e d f o r 18 hours a t 37° towards the h i g h m o l e c u l a r weight i n h i b i t o r soybean t r y p s i n i n h i b i t o r ( S T I ) , the f o l l o w i n g p r o c e -dure was f o l l o w e d : Two hundred p i o f 18 hour i n c u b a t e d complex s o l u t i o n was added t o a s i l i c a c u v e t t e c o n t a i n i n g 1.6 ml 50 mM T r i s HC1 (pH 8.0) w i t h 20 mM c a l c i u m c h l o r i d e . One hundred micrograms STI (SIGMA T-9003) was added, and the r e a c t i o n m i x t u r e was p r e - i n c u -b a t e d a t 37° f o r 2 minutes. S u b s t r a t e BAEE was added t o g i v e a c o n c e n t r a t i o n o f 0.45 B r i d g e s , A p p l e g a r t h , Johannson, Wong, and Davidson Page 8 mM i n a f i n a l r e a c t i o n volume o f 2.00 ml. The s o l u t i o n was mixed by i n v e r s i o n and i n c u b a t e d a t 37° f o r 3 minutes. D u r i n g t h i s i n c u b a t i o n p e r i o d , the r a t e of BAEE h y d r o l y s i s was f o l -lowed s p e c t r o p h o t o m e t r i c a l l y at 253 nm. The s p e c i f i c a c t i v i t y (pmoles BAEE h y d r o l y s e d / hour/pg a c t i v e BCT) o f 18 hour c^M-BCT com-p l e x e s was compared with t he s p e c i f i c a c t i v i t y of f r e s h l y p r e p a r e d complexes i n c u b a t e d f o r o n l y 2 minutes a t 37° and assay e d i n the p r e -sence o f 100 pg STI. To show t h a t a l l e s t e r o -l y t i c a c t i v i t y e x p r e s s e d i n the p r e s e n c e of STI was a s s o c i a t e d with t he c^M-BCT complex, an e q u i v a l e n t weight of BCT alo n e (0.88 pg) or o^M a l o n e (18 pg) was t r e a t e d with STI and assaye d as b e f o r e . To t e s t t h e s t a b i l i t y o f 18 hour a2M-BCT complexes toward the hi g h m o l e c u l a r weight s u b s t r a t e RBB-hide, the f o l l o w i n g p r o c e d u r e was u t i l i z e d : To each tube, 133 p i o f 18 hour-complex s o l u t i o n was added t o 50 mM T r i s - H C l , pH 8.0 t o g i v e a f i n a l i n c u b a t i o n B r i d g e s , A p p l e g a r t h , Johannson, Wong, and Davidson Page 9 volume of 3.0 ml. To t h i s s o l u t i o n , 20 mg i n s o l u b l e RBB-hide s u b s t r a t e was added and the m i x t u r e was i n c u b a t e d a t 37° f o r 15 minutes with f r e q u e n t m i x i n g ( o n c e / m i n / t u b e ) . The r e a c t i o n was t e r m i n a t e d by f i l t e r i n g out the r e m a i n i n g RBB-hide. The amount of p r o t e o -l y t i c a l l y r e l e a s e d dye was measured by s p e c t r o -p h o t o m e t r y a s s a y o f the f i l t r a t e a t 595 nm. The amount o f dye r e l e a s e d by the complex was e x p r e s s e d as a p e r c e n t a g e o f dye r e l e a s e d by an e q u i v a l e n t amount o f f r e e 100 p e r c e n t a c t i v e BCT (0.59 ug) i n c u b a t e d with s u b s t r a t e i n the absence o f a 0 M . 4) S u b u n i t S t r u c t u r e o f CF and C o n t r o l a^: SDS-PAGE can be used t o demonstrate a 2 M s u b u n i t s t r u c t u r e . Depending upon whether SDS a l o n e or SDS and e-mercaptoethanol i s used t o p r e - t r e a t a ^M samples, c ^ M dimers, monomers and p r o t e o l y t i c a l l y - d e r i v e d half-monomers can be demonstrated. D e t a i l s o f SDS-PAGE o f agM and agM-BCT complexes a r e d e s c r i b e d elsewhere [ 8 ] . B r i d g e s , A p p l e g a r t h , Johannson, Wong, and Davidson Page 1 0 5) S t a t i s t i c a l Treatment of Data: Comparative data g e n e r a t e d on CF and c o n t r o l o^M by the f o r e g o i n g e x p e r i m e n t a l methods was a n a l y s e d by t h e W i l c o x o n rank sum t e s t f o r un-p a i r e d samples [ 1 1 ] . T h i s n o n - p a r a m e t r i c s t a t i s t i c a l method i s i d e a l f o r data a n a l y s i s o f s m a l l sample s i z e s where p o p u l a t i o n c h a r a c t e r i s t i c s ( e . g . t y p e o f d i s t r i b u t i o n and homogeneity o f v a r i a n c e s ) a r e un-known. I I I . RESULTS AND DISCUSSION: Our a s s a y s were d e s i g n e d t o c l a r i f y d i f f e r e n c e s i n r e s u l t s r e p o r t e d by two groups of workers. The d e t a i l s a r e d i s c u s s e d under the s e p a r a t e headings below. The Spearman rank c o r r e l a t i o n t e s t [ 1 1 ] was used t o show t h a t the b i o c h e m i c a l parameters measured i n t h e s e s t u d i e s d i d not c o r r e l a t e with age or sex f o r c o n t r o l s u b j e c t s , nor d i d t h e y c o r r e l a t e with age, sex, a n t i b i o t i c use or s e v e r i t y o f d i s e a s e (as a s s e s s e d by Shwachman s c o r e s ) f o r a f f e c t e d s u b j e c t s . B r i d g e s , A p p l e g a r t h , Johannson, Wong, and Davidson Page 11 A) M o l a r P r o t e a s e B i n d i n g by CF and C o n t r o l g^M: S h a p i r a et a±. r e p o r t e d t h a t the g^ M o f CF plasma showed a r e d u c e d a b i l i t y t o b i n d exogenous p r o t e a s e s ( p a p a i n , b o v i n e t r y p s i n and b o v i n e thrombin) [ 5 ] . In t h e i r r e p o r t s , the b i n d i n g o f 125 I - l a b e l l e d b o v i n e t r y p s i n by g^ M i n CF plasma samples c o r r e s p o n d e d t o a molar p r o t e a s e b i n d i n g r a t i o of a p p r o x i m a t e l y 0.4 - 0.5 moles t r y p s i n / m o l e g^M, w h i l e b i n d i n g by i n c o n t r o l plasma samples was 0.8 - 0.9 moles t r y p s i n / m o l e ggM. These data were i n t e r p r e t e d as i n d i c a t i n g d e f e c t i v e p r o t e a s e b i n d i n g by CF g^ M [ 5 ] . However, s i n c e a t l e a s t f i v e i n h i b i t o r s of t r y p s i n are p r e s e n t i n human plasma [ 1 2 ] , the data c o u l d a l s o r e f l e c t i n c r e a s e d t r y p s i n b i n d i n g by one or more of the o t h e r p r o t e a s e i n h i b i t o r s o f CF plasma, and molar p r o t e a s e b i n d i n g c a p a b i l i t i e s o f CF and c o n t r o l a2M can o n l y be a s s e s s e d with c e r t a i n t y by use o f p u r i f i e d n a t i v e ggM, i s o l a t e d from o t h e r competing p r o t e a s e i n h i b -i t o r y systems. Bridges, Applegarth, Johannson, Wong, and Davidson Page 1 2 Parsons and Romeo reported a comparative study of protease binding by pu r i f i ed CF and control o^ M [ 1 3 ] . They used a radio-immunoprecipitation assay s im i la r to that employed by Shapira ejt a_K, and found that CF and control o^ M appeared to bind s im i la r 1 ?5 amounts of I - t ryps in . However, "a por t ion" of 1 pc the I - t rypsin formed aggregates under condit ions used in the i r immunoprecipitation assay [ 1 3 ] . I t was not c lear i f th is affected the i r resu l ts by making i t 125 d i f f i c u l t to discr iminate true I - t rypsin binding by o^ M from potent ia l contamination of immunopre-1 oc c ip i ta tes with I - t ryps in aggregates. In add i -1?5 t i o n , high concentrations of I - t rypsin ( three-fo ld in excess of the protease binding capab i l i t i es of a^) were used to maximize the amount of rad io -label in the immunoprecipitates and could have caused non-speci f ic hydrolysis of c^ M i t s e l f . We undertook to invest igate protease binding by a^ M using a d i f ferent approach from ei ther of the previous workers mentioned. Bovine cat ion ic t ryps in (BCT) of establ ished pur i t y and a c t i v i t y was used to t i t r a t e CF and control a?M samples of establ ished 176 B r i d g e s , A p p l e g a r t h , Johannson, Wong, and Davidson Page 13 p u r i t y and a n t i p r o t e a s e a c t i v i t y . I n s o l u b l e , h i g h m o l e c u l a r weight Remazol b r i l l i a n t b l u e h i d e (RBB-h i d e ) was chosen as the i n d i c a t o r o f the end p o i n t o f t i t r a t i o n . S i n c e t h i s s u b s t r a t e i s a c c e s s i b l e o n l y t o f r e e BCT and not t o BCT bound i n complex t o o ^ M , RBB-hide h y d r o l y s i s i n d i c a t e s t he p o i n t a t which a^M " b i n d i n g s i t e s " have been s a t u r a t e d with BCT. T a b l e I shows the r e s u l t s of t h e s e s t u d i e s . CF o 2 M c o u l d b i n d 1 . 9 7 ± 0 . 1 8 moles BCT/mole a 2 M compared t o 1 . 7 2 ± 0 . 2 9 f o r c o n t r o l s . T h i s d i f f e r -ence was not s t a t i s t i c a l l y s i g n i f i c a n t . These molar b i n d i n g r a t i o s a r e i n agreement with l i t e r a t u r e v a l u e s of a p p r o x i m a t e l y 2 moles t r y p s i n / m o l e a 2 M [ 1 4 , 1 5 ] . These data s u g g e s t t h a t t he molar p r o t e a s e b i n d i n g r a t i o s of CF and c o n t r o l a 2 M a r e the same. In many o f t h e i r comparative s t u d i e s o f p r o -t e a s e b i n d i n g by CF and c o n t r o l a 2 M , S h a p i r a e t a l . had exposed t h e i r a 2 M t o a f r a c t i o n a t i o n step i n v o l v i n g ammonium s u l f a t e a t pH 7 . 0 [ 6 , 7 , 1 6 ] . However, t r e a t m e n t o f with ammonium s a l t s and c e r t a i n p r i m a r y amines i s known t o produce from 177 B r i d g e s , A p p l e g a r t h , Johannson, Wong, and Davidson Page 14 p a r t i a l t o complete i n a c t i v a t i o n o f the p r o t e a s e b i n d i n g a b i l i t y [ 1 4 ] . Because o f the i n s t a b i l i t y o f c^M t o ammonium i o n , i t has been recommended t h a t ammonium s u l f a t e s h o u l d not be used a t n e u t r a l or a l k a l i n e pH's t o f r a c t i o n a t e plasma d u r i n g o^M i s o l a t i o n p r o c e d u r e s [ 3 ] . We reasoned t h a t s i n c e S h a p i r a e t al_. had used ammonium s a l t p r e c i p i t a t i o n t o p u r i f y one might expect t h e i r CF and c o n t r o l a^M t o show a r e d u c e d p r o t e a s e b i n d i n g c a p a c i t y r e l a t i v e t o n a t i v e a^M. In the paper i n which S h a p i r a e t a l . used p u r i f i e d c^M t o i n v e s t i g a t e molar p r o t e a s e b i n d i n g [ 6 ] , CF a^M bound 12 pg t r y p s i n / m g c^M w h i l e c o n t r o l bound 25 pg t r y p s i n / m g a^; t h i s c o r r e s p o n d s t o 0.37 moles BCT/mole f o r CF and 0.76 f o r c o n t r o l . P r e -sumably, the a^ M i s o l a t e d by S h a p i r a e t al_. was not f u l l y a c t i ve. To r u l e out the p o s s i b i l i t y t h a t the d i f f e r -ences o b s e r v e d by S h a p i r a e t al_. i n molar p r o t e a s e b i n d i n g by CF and c o n t r o l a^ M [ 6 ] r e f l e c t e d a g r e a t e r ammonium i o n - s e n s i t i v i t y o f CF a ?M, we B r i d g e s , A p p l e g a r t h , Johannson, Wong, and Davidson Page 15 s t u d i e d the molar p r o t e a s e b i n d i n g o f our own p u r i -f i e d CF and c o n t r o l c^M p r e p a r a t i o n s , a f t e r i n -a c t i v a t i o n by p r e - t r e a t m e n t with 200 mM ammonium s u l f a t e a t pH 8.0 f o r 30 minutes a t 3 7 ° . T a b l e I shows the r e s u l t s : i n a c t i v a t e d CF c^M c o u l d b i n d 0.69 ± 0.05 moles BCT/mole « 2 M compared t o 0.74 ± 0.15 f o r c o n t r o l s . T h i s d i f f e r e n c e was not s t a t i s -t i c a l l y s i g n i f i c a n t . These data presumably r e f l e c t b i n d i n g t o s i t e s which are r e l a t i v e l y r e s i s t a n t t o t he d e l e t e r i o u s e f f e c t s o f ammonium i o n s . T h i s s t u d y s u g g e s t s t h a t CF and c o n t r o l o^M a r e e q u a l l y s e n s i t i v e t o ammonium i o n . B) I n t e r a c t i o n o f CF and C o n t r o l a^M-Trypsin  Complexes with BAEE: S h a p i r a e t a l . s t u d i e d the s u b s t r a t e k i n e t i c s when CF and c o n t r o l ( ^ M - t r y p s i n complexes a r e i n t e r a c t e d with the low m o l e c u l a r weight s u b s t r a t e b e n z o y l a r g i n i n e e t h y l e s t e r (BAEE) [ 6 ] . They r e p o r t e d s t r i k i n g d i f f e r e n c e s i n the a f f i n i t y o f CF and c o n t r o l a ? M - t r y p s i n complexes f o r BAEE. The B r i d g e s , A p p l e g a r t h , Johannson, Wong, and Davidson Page 16 apparent K m o f CF o^M complexes was s i m i l a r t o t h a t of f r e e t r y p s i n (150 and 110 yM, r e s p e c t i v e l y ) but 4 - f o l d lower than t h a t o f c o n t r o l a^A complexes (630 yM). These r e s u l t s were i n t e r p r e t e d t o mean t h a t the modes o f b i n d i n g o f t r y p s i n t o CF and c o n -t r o l o^M were d i f f e r e n t [ 6 ] . These r e s u l t s prompted us t o compare the a c t i v i t y of CF and c o n t r o l c^M-bovine c a t i o n i c t r y p s i n (BCT) complexes t o the a c t i v i t y o f non-complexed BCT u s i n g BAEE. Our f i n d i n g s a r e summar-i z e d i n T a b l e I I . The apparent K m of CF complexes f o r BAEE (43 ± 6 yM) was not s i g n i f i c a n t l y d i f f e r e n t from t h a t o f c o n t r o l complexes (52 ± 10 yM). The apparent K m of f r e e BCT (20 ± 4 yM) was found t o be lower than t h a t f o r complexed BCT as e x p e c t e d . T h i s v a l u e i s i n l i n e w i t h apparent K m data o f o t h e r s f o r f r e e enzyme (4 t o 50 yM) [17, 18]. The low a f f i n i t i e s o f CF and c o n t r o l o ^ M - t r y p s i n complexes f o r BAEE o b s e r v e d by S h a p i r a e t a_]_. may r e f l e c t p a r t i a l i n a c t i v a t i o n o f o^M by exposure t o ammonium i o n . B r i d g e s , A p p l e g a r t h , Johannson, Wong, and Davidson Page 17 T a b l e II a l s o summarizes our f i n d i n g s on the maximal v e l o c i t y o f a c t i v a t i o n ( V r n a w ) o f BCT both i n the f r e e s t a t e and i n complex with CF or c o n t r o l agM. The V m a x f o r BAEE o f CF complexes was v i r t -u a l l y i d e n t i c a l t o t h a t o f c o n t r o l complexes (1.54 pmoles BAEE h y d r o l y s e d / h o u r / p g a c t i v e BCT and 1.51, r e s p e c t i v e l y ) . At a BAEE c o n c e n t r a t i o n o f 0.45 nM, CF was found t o depress the r a t e of BAEE h y d r o -l y s i s by BCT t o 67.2 p e r c e n t o f the f r e e BCT h y d r o -l y s i s r a t e ( r e s i d u a l e s t e r o l y t i c a c t i v i t y ) . T h i s compared with a v a l u e o f 66.1 p e r c e n t f o r c o n t r o l ogM. None o f t h e s e d i f f e r e n c e s a r e s t a t i s t i c a l l y s i g n i f i c a n t . C) S t a b i l i t y o f CF and C o n t r o l g ^ - T r y p s i n  Complexes: S h a p i r a e t aJL r e p o r t e d abnormal breakdown o f o^M-trypsin complexes i n CF [ 7 ] . They found t h a t t h e a b i l i t y o f o^M-trypsin complexes t o d i s c r i m -i n a t e s t e r i c a l l y between high and low m o l e c u l a r weight s u b s t r a t e s and i n h i b i t o r s was begun t o be l o s t B r i d g e s , A p p l e g a r t h , Johannson, Wong, and Davidson Page 18 w i t h i n 10 minutes when c o n t r o l c^M was complexed with t r y p s i n . T h i s o c c u r r e d much more s l o w l y ( w i t h i n 60-90 minutes) when CF a^l was used. These r e s u l t s s u g g e s t e d t o S h a p i r a e_t a_L t h a t c o n t r o l c ^ M - t r y p -s i n complexes were g r a d u a l l y c o n v e r t e d i n t o o^M fragm e n t s , but t h i s d i d not o c c u r as r e a d i l y t o CF a ^ - t r y p s i n complexes. However, o t h e r p u b l i s h e d s t u d i e s on the s t a b i l i t y of normal c^M-protease complexes i n d i c a t e t h a t once the complex i s formed the enzyme i s i r r e v e r s i b l y bound t o c^M [ 3 ] . T h i s was r e - i n v e s t i g a t e d by Parsons and Romeo, who examined the s t a b i l i t y o f CF and c o n t r o l o^M-porcine t r y p s i n complexes a g a i n s t i n h i b i t i o n by STI [ 1 3 ] . To do t h i s , t h e y s t u d i e d the r e s i d u a l e s t e r o l y t i c a c t i v i t y toward BAPNA o f c ^ M - t r y p s i n complexes formed at n o n - s a t u r a t i n g and s a t u r a t i n g t r y p s i n c o n c e n t r a t i o n s and a s s a y e d i n t h e p r e s e n c e o f STI. They r e p o r t e d 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 r e s i d u a l e s t e r o l y t i c a c t i v i t y of CF and c o n t r o l o^M-porcine t r y p s i n complexes a f t e r i n c u b a t i o n o f afl and p o r c i n e t r y p s i n f o r p e r i o d s o f 5 and 60 minutes at room temperature [ 1 3 ] . However, the a s s a y B r i d g e s , A p p l e g a r t h , Johannson, Wong, and Davidson Page 19 c o n d i t i o n s employed by Parsons and Romeo f o r non-s a t u r a t i n g t r y p s i n c o n c e n t r a t i o n s were not o p t i m i z e d t o a l l o w d e t e c t i o n of d i f f e r e n c e s i n r e s i d u a l e s t e r o -l y t i c a c t i v i t i e s between t h e s e i n c u b a t i o n t i m e s , and the l e v e l s of e s t e r o l y t i c a c t i v i t y measured a t s a t u r -a t i n g t r y p s i n c o n c e n t r a t i o n s were an o r d e r o f magni-tude h i g h e r than e x p e c t e d . T h i s may r e f l e c t incom-p l e t e i n h i b i t i o n o f f r e e t r y p s i n by STI. We undertook a r e - i n v e s t i g a t i o n o f the s t a b -i l i t y o f CF and c o n t r o l a^M-bovine c a t i o n i c t r y p s i n (BCT) complexes. The hi g h m o l e c u l a r weight i n h i b i t o r STI and the high m o l e c u l a r weight s u b s t r a t e RBB-hide were chosen as probes of the s t a b i l i t y of t h e s e o^M-BCT complexes. ( i ) S t a b i l i t y of Complexes t o STI: CF and c o n t r o l agM-BCT complexes i n c u b a t e d a t 37° f o r 18 hours were compared with f r e s h l y p r e p a r e d complexes ( i n c u b a t i o n o f ct^NI and BCT f o r 2 minutes) i n terms of t h e i r a b i l i t y t o h y d r o l y s e BAEE i n the p r e s e n c e of a 1 0 0 - f o l d molar excess o f ST I . S p e c i f i c B r i d g e s , A p p l e g a r t h , Johannson, Wong, and Davidson Page 20 a c t i v i t y v a l u e s f o r 2 minute and 18 hour CF and c o n t r o l complexes a re p r e s e n t e d i n T a b l e I I I . We found no s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s i n the e s t e r o l y t i c a c t i v i t i e s of CF and c o n t r o l o^M-BCT complexes a t e i t h e r time p e r i o d . F u r t h e r m o r e , t he degree o f i n t e r a c t i o n o f STI with CF or c o n t r o l complexes does not change even a f t e r 18 hours i n c u -b a t i o n a t 3 7 ° , i n d i c a t i n g a h i g h degree o f complex s t a b i l i t y f o r both CF and c o n t r o l c^M p r e p a r a t i o n s . ( i i ) S t a b i l i t y o f Complexes t o RBB-hide: CF and c o n t r o l a^M-BCT complexes i n c u b a t e d f o r 18 hours a t 37° were a s s a y e d f o r t h e i r a b i l i t y t o h y d r o l y s e t he h i g h m o l e c u l a r weight s u b s t r a t e RBB-hide. P r o t e o l y t i c a c t i v i t y o f t h e s e complexes was e x p r e s s e d as a p e r c e n t a g e of p r o t e o l y s i s p r o d u c e d by an e q u i v a l e n t amount o f f r e e BCT (see T a b l e I I I ) . CF and c o n t r o l 18 hour a-pM-BCT complexes were ob s e r v e d t o produce n e g l i g i b l e l e v e l s of RBB-hide p r o t e o l y s i s (2.4 p e r c e n t and 0.3 p e r c e n t o f f r e e BCT p r o t e o l y t i c a c t i v i t y , r e s p e c t i v e l y ) . CF and c o n t r o l v a l u e s o v e r l a p p e d s u b s t a n t i a l l y . T h i s d i f f e r e n c e was B r i d g e s , A p p l e g a r t h , Johannson, Wong, and Davidson Page 21 not s t a t i s t i c a l l y s i g n i f i c a n t . In o t h e r words, we obse r v e d t h a t CF and c o n t r o l ctgM-BCT complexes remain s t a b l e f o r a t l e a s t 18 hours a t 3 7 ° . These data s u p p o r t t h e c o n t e n t i o n t h a t p r o t e a s e s a re bound i r r e v e r s i b l y by normal and CF a^M. In the r e p o r t o f S h a p i r a e t a]_. CF o^M p r e p a r a t i o n s showed a more normal p a t t e r n o f s t a b i l i t y than c o n t r o l o ^ M . The re a s o n f o r t h i s i s u n c l e a r . S i n c e o^M-BCT complexes do not appear t o d i s s o c i a t e , we d i d not attempt t h e d e t e r m i n a t i o n of i n h i b i t i o n c o n s t a n t s (K.) f o r the i n t e r a c t i o n of BCT with CF and c o n t r o l a^, nor have we t r i e d t o c h a r a c t e r i z e t h i s i n h i b i t i o n as of t h e c o m p e t i t i v e or no n - c o m p e t i t i v e t y p e . Such k i n e t i c c h a r a c t e r i z a t i o n s of the i n t e r a c t i o n o f i n h i b i t o r , enzyme and s u b s t r a t e r e q u i r e t h a t t h e s e r e a c t i o n components be i n r e v e r -s i b l e e q u i l i b r i u m with t h e i r complexes [ 3 ] , a con -d i t i o n not s a t i s f i e d by a ^ M-protease complexes i n our hands. B r i d g e s , A p p l e g a r t h , Johannson, Wong, and Davidson Page 22 D) S u b u n i t S t r u c t u r e of CF and C o n t r o l a^A: The s u b u n i t s t r u c t u r e o f c ^ M can be demon-s t r a t e d by SDS-PAGE as f o l l o w s : t r e a t m e n t o f c ^ M with SDS a l o n e p e r m i t s d e m o n s t r a t i o n o f d i m e r i c c ^ M ; t r e a t m e n t o f c ^ M with SDS and t h i o l r e a g e n t s ( e . g . , d i t h i o t h r e i t o l or 8-mercaptoethanol) p e r m i t s d e m o n s t r a t i o n o f monomeric a^A; and SDS and t h i o l r e a g e n t t r e a t m e n t o f complexes formed by r e a c t i o n of 2 moles t r y p s i n w i t h one mole c ^ M p e r m i t s demon-s t r a t i o n of 85,000 d a l t o n fragments (half-monomeric ogM) [19]. F i g u r e 1 shows 3 s e t s o f SDS-PAGE tube g e l s : g e l s e t s 1 and 2 show c o n t r o l c ^ M ; g e l s e t 3 shows CF a^A. A l l s t a r t i n g m a t e r i a l f o r t h e s e e x p e r i -ments was v e r i f i e d t o be p u r e , a c t i v e c ^ M i n the t e t r a m e r i c form [ 8 ] . We found, as have o t h e r s [13, 20, 2 1 ] , t h a t t r e a t m e n t o f CF and c o n t r o l c ^ M with SDS a l o n e p r o d u c e d d i m e r i c a^A ( F i g u r e 1: l a , 2a and 3 a ) . Treatment o f CF and c o n t r o l a^A with B-mercaptoethanol and SDS p r o d u c e d monomeric a^A ( F i g u r e 1: l b , 2b and 3 b ) . F i n a l l y , r e a c t i o n o f CF B r i d g e s , A p p l e g a r t h , Johannson, Wong, and Davidson Page 23 and c o n t r o l o^M with BCT at 2 moles t r y p s i n / m o l e ct^M and then t r e a t m e n t with e-mercaptoethanol and SDS produced half-monomeric c ^ M ( F i g u r e 1: l c , 2c and 3 c ) . S l o w l y m i g r a t i n g minor bands were l o c a t e d near t o the top o f the g e l s and r e p r e s e n t e d n o n - d i s -s o c i a t e d a 2 M , not h i g h m o l e c u l a r weight c o n t a m i -n a n t s . S i m i l a r bands were observed by H a r p e l [ 1 9 ] . The data suggest no d i f f e r e n c e s i n s u b - u n i t s t r u c t u r e between CF and c o n t r o l a g M . In 1976 W i l s o n and Fudenberg [ 4 ] r e p o r t e d a s t u d y of plasma from c y s t i c f i b r o s i s p a t i e n t s and c o n t r o l s u s i n g i s o - e l e c t r i c f o c u s i n g . T h e i r s t u d y r e p o r t e d e v i d e n c e f o r a d e f i c i e n t p r o t e o l y t i c c l e a -vage of cigM i n c y s t i c f i b r o s i s plasmas. These i n v e s t i g a t o r s s u g g e s t e d s e v e r a l p o s s i b l e mechanisms by which t h i s d i f f e r e n t b e h a v i o u r of c y s t i c f i b r o s i s o^M might o c c u r . Our data o n l y touch on one o f t h e i r s u g g e s t i o n s which was t h a t t h e r e might be a g e n e t i c a l l y - d e t e r m i n e d a b n o r m a l i t y i n the s t r u c t u r e o f o ^ M , p r i m a r i l y at the a c t i v e s i t e , d e c r e a s i n g i t s b i n d i n g a f f i n i t y f o r plasma p r o t e a s e s . Our f i n d i n g s do not bear t h i s o u t . Other work on has s u g g e s t e d p o s s i b l e a b n o r m a l i t i e s i n the c a r b o -B r i d g e s , A p p l e g a r t h , Johannson, Wong, and Davidson Page 24 h y d r a t e c o m p o s i t i o n [ 2 2 , 2 3 ] . T h i s would not be l i k e l y t o a f f e c t the i n t e r a c t i o n o f the a c t i v e s i t e with b o v i n e c a t i o n i c t r y p s i n , so our data might be expe c t e d t o have l i t t l e b e a r i n g on s p e c u l a t i o n s c o n c e r n i n g t h i s t y p e o f s t r u c t u r a l a b n o r m a l i t y i n In c o n c l u s i o n , we looked a t f o u r parameters o f o^M f u n c t i o n or s t r u c t u r e : ( 1 ) the molar p r o t e a s e b i n d i n g of c ^ M ; ( 2 ) the i n t e r a c t i o n of c ^ M - B C T complexes with B A E E ; ( 3 ) the s t a b i l i t y o f formed o ^ M - B C T complexes; and, ( 4 ) the s u b u n i t s t r u c t u r e of o ^ M . We found no d i f f e r e n c e s between CF and c o n t r o l ctpM p r e p a r a t i o n s . B r i d g e s , A p p l e g a r t h , Johannson, Wong, and Davidson Page 25 ACKNOWLEDGEMENTS: We wish t o acknowledge the k i n d c o - o p e r a t i o n o f Dr. L.T. K i r b y and Mrs. M. 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[1973] " S t u d i e s on human plasma a 2 M a c r o g l o b u l i n - enzyme i n t e r a c t i o n s ; E v i d e n c e f o r p r o t e o l y t i c m o d i f i c a t i o n of the s u b u n i t c h a i n s t r u c t u r e . J . E x p t l . M e d . 138:508-521. (20) ROBERTS, R.C., NELLES, L.P., HALL, P., and MISCHLER, E. [1979] Comparison o f c y s t i c f i b r o t i c and normal c ^ M a c r o g l o b u l i n : P h y s i c o c h e m i c a l and p r i m a r y s t r u c t u r e s t u d i e s . CF Club A b s t r a c t s , May 1, 1979, A t l a n t a , p.8. (21) BURDON, M.G. [1980] Normal s u b u n i t c l e a v a g e of <x2Macroglobulin i n c y s t i c f i b r o s i s . C l i n . C h i m . A c t a 100:225-229. (22) BEN-YOSEPH, Y., DEFRANCO, C.L. and NADLER, H.L. [1979] Decreased s i a l i c a c i d and a l t e r e d b i n d i n g t o l e c t i n s o f p u r i f i e d o ^ M a c r o g l o b u l i n from p a t i e n t s with c y s t i c f i b r o s i s . C l i n . C h i m . A c t a 99:31-35. B r i d g e s , A p p l e g a r t h , Johannson, Wong, and Davidson Page 29 (23) SHAPIRA, E. and MENENDEZ, R. [1980] I n c r e a s e d b i n d i n g o f c o n c a n a v a l i n A t o c ^ M a c r o g l o b u l i n , IgM and IgG from c y s t i c f i b r o s i s plasma. Bi ochem.Biophys.Res.Commun. 93:50-56. MB/gm2386A J u l y , 1981 B r i d g e s , A p p l e g a r t h , J o h a n n s o n , Wong a n d D a v i d s o n P a g e 30 T A B L E I MOLAR PROTEASE BINDING R A T I O S FOR UNTREATED CF AND CONTROL <*2M AND FOR CF AND CONTROL a 2 M I N A C T I V A T E D BY TREATMENT WITH 200 mM AMMONIUM S U L F A T E FOR 30 M I N U T E S AT 3 7 ° S U B J E C T S : M O L E S B C T / M O L E a 2 M M O L E S B C T / M O L E I N A C T I V A T E D a 2 M CF ( n = 5 ) CONTROL ( n = 6 ) 1.97 ± 0 . 1 8 * N.S.** 1.72 ± 0 . 2 9 1 0.69 ± 0 . 0 5 * 0.74 ± 0 . 1 5 * N.S ** mean ± S.D. v a l u e s b a s e d u p o n q u a n t i t a t i o n o f a 2 M b y e x t i n c t i o n a t 280 nm (1 g / d l e x t i n c t i o n c o e f f i c i e n t = 9.07 [ 9 ] ) s t a t i s t i c a l s i g n i f i c a n c e t e s t e d a t t h e a t t h e p < 0.05 l e v e l . gm0569B:1 J u l y / 8 1 B r i d g e s , A p p l e g a r t h , Johannson, Wong and Davidson Page 31 TABLE I I . INTERACTION OF CF AND CONTROL c^M-BCT COMPLEXES AND NON-COMPLEXED BCT WITH BAEE SUBJECTS APPARENT ,Km (uM) Vmax (uMOLES BAEE HYDROLYSED/ HOUR/yg ACTIVE BCT) RESIDUAL ESTEROLYTIC ACTIVITY OF COMPLEXES ( p e r c e n t f r e e BCT a c t i v i t y ) CF (n=5) 43 ± 6* ) V N.S.** 1.54 ± 0.03* ) \ N.S.** 67.2 p e r c e n t ± 2.9* ) V N.S.** CONTROL (n=6) 52 ± 10* ) 1.51 ± 0.08* ) 66.1 p e r c e n t ± 3.6* ) NON-COMPLEXED BCT (n=4) 20 ± 4* 2.53 ± 0.20* mean ± S.D. s t a t i s t i c a l s i g n i f i c a n c e t e s t e d a t the p < 0.05 l e v e l . gm2394A J u l y / 8 1 B r i d g e s , A p p l e g a r t h , J o h a n n s o n , Wong a n d D a v i d s o n P a g e 32 T A B L E I I I . S T A B I L I T Y OF CF AND CONTROL o^M-BCT COMPLEXES TO THE HIGH MOLECULAR WEIGHT I N H I B I T O R SOYBEAN T R Y P S I N I N H I B I T O R ( S T I ) AND TO THE HIGH MOLECULAR WEIGHT S U B S T R A T E REMAZOL B R I L L I A N T B L U E - H I D E ( R B B - H I D E ) S P E C I F I C A C T I V I T Y IN THE P R E S E N C E OF S T I (y M O L E S BAEE HYDROLYSED/ HOUR/yg A C T I V E B C T ) R E S I D U A L H Y D R O L Y T I C A C T I V I T Y OF 18 HOUR CO M P L E X E S FOR RBB-HIDE ( p e r c e n t f r e e BCT a c t i v i t y ) 18 HOUR 2 MINUTE COMPLEX COMPLEX S U B J E C T S + S T I + S T I CF ( n = 4 ) 1.09 ± 0 . 0 3 * ) V N . S . * * 1.04 ± 0 . 0 1 * ) S N . S . * * 2.4 p e r c e n t ± 2 . 4 * ) >N.S.** CONTROL ( n = 6 ) 1.08 ± 0 . 1 0 * ) 1.06 ± 0 . 0 5 * ) 0.3 p e r c e n t ± 0.2*) mean ± S.D. s t a t i s t i c a l s i g n i f i c a n c e t e s t e d a t t h e a t t h e p < 0.05 l e v e l . gm0569B : 2 J u l y / 8 1 196 B r i d g e s , A p p l e g a r t h , Johannson, Wong, and Davidson Page 3 3 F i g . 1: SUBUNIT STRUCTURE OF CF AND  CONTROL g?M: G e l s e t s 1 and 2 show s u b u n i t s t r u c t u r e o f c o n t r o l a2M: (a) dimer, (b) monomer, and ( c ) half-monomeric fragment from t r y p s i n t r e a t m e n t . G e l s e t 3 shows s u b u n i t s t r u c t u r e o f CF « 2 M : d i t t o . 1 c a b c 

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