UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Horse plasma vitamin D-binding protein : isolation and structural investigation Robinson, Robert Charles 1990

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1991_A6_7 R62.pdf [ 3.34MB ]
Metadata
JSON: 831-1.0059797.json
JSON-LD: 831-1.0059797-ld.json
RDF/XML (Pretty): 831-1.0059797-rdf.xml
RDF/JSON: 831-1.0059797-rdf.json
Turtle: 831-1.0059797-turtle.txt
N-Triples: 831-1.0059797-rdf-ntriples.txt
Original Record: 831-1.0059797-source.json
Full Text
831-1.0059797-fulltext.txt
Citation
831-1.0059797.ris

Full Text

HORSE PLASMA VITAMIN D-BINDING PROTEIN: ISOLATION AND STRUCTURAL INVESTIGATION By ROBERT CHARLES ROBINSON . S c . K i n g ' s C o l l e g e , London U n i v e r s i t y , 1987 A THESIS SUBMITTED IN OF THE REQUIREMENTS MASTER OF PARTIAL FULFILLMENT FOR THE DEGREE OF SCIENCE IN THE FACULTY OF GRADUATE STUDIES (Depar tment of C h e m i s t r y ) We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA OCTOBER 1990 © R o b e r t C h a r l e s R o b i n s o n In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada DE-6 (2/88) A b s t r a c t V i t a m i n D - b i n d i n g p r o t e i n (DBP) i s an abundant serum p r o t e i n , s e c r e t e d by the l i v e r , w h i c h t r a n s p o r t s v i t a m i n D s t e r o l s and i s p a r t of an a c t i n s c a v e n g i n g s y s t e m . In t h i s s t u d y , DBP was i s o l a t e d f rom h o r s e p lasma i n a h i g h l y r e p r o d u c i b l e , f o u r s t e p p r o c e d u r e : A f f i - g e l B l u e a f f i n i t y c h r o m a t o g r a p h y , g e l f i l t r a t i o n , h y d r o x y 1 a p a t i t e c h r o m a t o g r a p h y and a n i o n exchange HPLC. 6-7 mg of DBP were o b t a i n e d f rom 80 ml of p l a s m a w i t h a y i e l d of 2 1 - 2 5 % . The s e c o n d a r y s t r u c t u r e of DBP was c a l c u l a t e d f rom c i r c u l a r d i c h r o i s m measurements t o be 39% a - h e l i x , 42% p - s h e e t and 19% random c o i l . A m o l e c u l a r mass of 5 3 , 0 0 0 ± 3 , 0 0 0 d a l t o n s was c a l c u l a t e d f rom e l e c t r o p h o r e t i c g e l s . C i r c u l a r d i c h r o i s m and f l u o r e s c e n c e s t u d i e s r e v e a l e d t h a t the d i s u l p h i d e bonds of DBP c o n t r i b u t e s u b s t a n t i a l s t r u c t u r a l s t a b i l i z a t i o n to the m o l e c u l e w i t h r e s p e c t t o t h e r m a l d e n a t u r a t i o n . F i n a l l y , a c r y l o d a n - l a b e l e d DBP was p r e p a r e d . The f l u o r e s c e n c e of t h i s adduct was s e n s i t i v e to the b i n d i n g of a c t i n and t o t h e p r e s e n c e of d i t h i o t h r e i t o l . i i i T a b l e of C o n t e n t s Page A b s t r a c t i i L i s t o f T a b l e s v i L i s t o f F i g u r e s v i i Acknowledgements x A b b r e v i a t i o n s i x U I n t r o d u c t i o n P a r t 1 : P r o t e i n s 1 . 1 . 1 F u n c t i o n s of A c t i n 1 1 . 1 . 2 A c t i n P o l y m e r i z a t i o n / D e p o l y m e r i z a t i o n 6 1 . 1 . 3 A c t i n S c a v e n g i n g System i n P lasma 8 1 . 1 . 4 Rev iew of DBP 9 1 . 1 . 5 P h y s i c a l P r o p e r t i e s of DBP 11 1 . 1 . 6 Rev iew of DBP P u r i f i c a t i o n Methods 13 P a r t 2 : S p e c t r o s c o p i c T e c h n i q u e s 1 . 2 . 1 P r o t e i n A b s o r b a n c e S p e c t r o s c o p y 15 1 . 2 . 3 P r o t e i n F l u o r e s c e n c e S p e c t r o s c o p y 18 1 . 2 . 3 P r o t e i n CD S p e c t r o s c o p y 20 i v 2; M a t e r i a l s and Methods P a r t 1 : P r o t e i n s 2 . 1 . 1 P r e p a r a t i o n of G - a c t i n 23 2 . 1 . 2 P r e p a r a t i o n of P y r e n e - a c t i n 24 2 . 1 . 3 P r e p a r a t i o n of A c r y l o d a n - a c t i n 24 2 . 1 . 4 A c r y l o d a n - a c t i n A s s a y 25 2 . 1 . 5 C o l l e c t i o n of Horse P l a s m a 26 2 . 1 . 6 P u r i f i c a t i o n of DBP 26 2 . 1 . 7 C a l c u l a t i o n of an E x t i n c t i o n C o e f f i c i e n t f o r DBP 29 2 . 1 . 8 P r e p a r a t i o n of A e r y l o d a n - D B P 30 2 . 1 . 9 G e l E l e c t r o p h o r e s i s 30 P a r t 2 : S p e c t r o s c o p i c Methods 2 . 2 . 1 A b s o r b a n c e S p e c t r a 31 2 . 2 . 2 F l u o r e s c e n c e S p e c t r a 31 2 . 2 . 3 CD S p e c t r a 31 3 : R e s u l t s and D i s c u s s i o n P a r t 1 : Y i e l d and P u r i t y of DBP 3 . 1 . 1 Y i e l d and P u r i t y 32 3 . 1 . 2 O p t i c a l S p e c t r a 34 3 . 1 . 3 A c t i n A s s a y s 37 3 . 1 . 4 C a l c u l a t i o n of M o l e c u l a r Mass 40 3 . 1 5 C a l c u l a t i o n of S e c o n d a r y S t r u c t u r e 42 V P a r t 2 : S t a b i l i t y o f DBP 3 . 2 . 1 CD T h e r m a l D e n a t u r a t i o n 43 3 . 2 . 2 F l u o r e s c e n c e Thermal D e n a t u r a t i o n 47 3 . 2 . 3 CD G u a n a d i n e . H C 1 Induced D e n a t u r a t i o n 47 P a r t 3 : A c r y l o d a n - D B P 3 . 3 . 1 A c r y l o d a n L a b e l i n g of DBP 50 3 . 3 . 2 A c r y l o d a n - D B P / A c t i n I n t e r a c t i o n 53 3 . 3 . 3 E f f e c t of DTT on F l u o r e s c e n c e of A c r y l o d a n C o v a l e n t l y Bound to DBP 54 P a r t 4 ! C o n c l u s i o n s 55 B i b l i o g r a p h y 5 7 v i L i s t o f T a b l e s T a b l e Page I P h y s i c a l P r o p e r t i e s of DBP 11 v i i L i s t of F i g u r e s Page 1 The t h i n f i l a m e n t of s k e l e t a l m u s c l e 1 2 The main components of s k e l e t a l m u s c l e 2 3 The mechanism of musc le c o n t r a c t i o n 3 4 I m m u n o f l u o r e s c e n c e m i c r o g r a p h of a r e s t i n g c e l l 4 s t a i n e d w i t h a f l u o r e s c e n t a n t i b o d y t o a c t i n 5 I m m u n o f l u o r e s c e n c e m i c r o g r a p h of a moving c e l l 4 s t a i n e d w i t h a f l u o r e s c e n t a n t i b o d y t o a c t i n 6 S c h e m a t i c d i a g r a m of F - a c t i n 6 7 S c h e m a t i c d i a g r a m showing the v a r i o u s a c t i v i t i e s 7 of a c t i n b i n d i n g p r o t e i n s a l o n g w i t h examples 8 S c h e m a t i c d i a g r a m of the complexes formed on the 8 a d d i t i o n of G - a c t i n or F - a c t i n t o g e l s o l i n and DBP 9 Homology between the d i s u l p h i d e - b o n d i n g p a t t e r n s 12 i n r a t a l b u m i n and the p r e d i c t e d amino a c i d sequence i n r a t DBP. S m a l l c i r c l e s show amino a c i d s d e l e t e d i n c o m p a r i s o n t o a l b u m i n 10 A b s o r b a n c e s p e c t r a of the a r o m a t i c amino a c i d s 16 11 Second d e r i v a t i v e s p e c t r a of the a r o m a t i c amino 16 a c i d s i n 6 M GuHCL, sod ium p h o s p h a t e , pH 6 . 8 12 Second d e r i v a t i v e s p e c t r u m of human serum 17 a l b u m i n i n GuHCl a t a r b i t r a r y c o n c e n t r a t i o n 13 F l u o r e s c e n c e s p e c t r a of the a r o m a t i c a c i d s 19 e x c i t a t i o n 278 nm 14 6 - a c r y l o y l - 2 - d i m e t h y l a m i n o n a p h t h a l e n e 19 15 N - ( 1 - P y r e n e ) i o d o a c e t a m i d e 19 16 CD s p e c t r a of p o l y - L - l y s i n e i n the 1, c r - h e l i c a l ; 21 2, B - s h e e t ; 3 , random c o i l c o n f o r m a t i o n s 17 E l u t i o n p r o f i l e f rom HPLC column 32 18 SDS p o l y a c r y l a m i d e e 1 e c t r o p h o r e t i c g e l showing 33 the p u r i f i c a t i o n of DBP v i i i 19a A b s o r b a n c e s p e c t r u m of DBP 34 19b A b s o r b a n c e s p e c t r u m of a l b u m i n 34 20 Second d e r i v a t i v e a b s o r b a n c e s p e c t r u m of DBP 35 21 F l u o r e s c e n c e e m i s s i o n s p e c t r u m , e x c i t a t i o n 36 278 nm, of DBP 22 The a c r y l o d a n - a c t i n a s s a y 37 23 Time r e l a t e d p y r e n e - a c t i n f l u o r e s c e n c e s t u d i e s , 38 e x c i t a t i o n a t 344 nm and e m i s s i o n a t 386 nm 24 SDS p o l y a c r y l a m i d e e l e c t r o p h o r e t i c g e l f o r the 40 c a l c u l a t i o n of m o l e c u l a r mass 25 Graph showing the l o g of m o l e c u l a r mass a g a i n s t 41 d i s t a n c e moved on a e l e c t r o p h o r e t i c g e l 26 CD s p e c t r u m of DBP 42 27 M e l t i n g c u r v e of DBP, i n h i g h s a l t , f o l l o w e d 44 by CD a t 220 nm 28 M e l t i n g c u r v e of DBP, i n low s a l t , f o l l o w e d 46 by CD a t 220 nm 29 M e l t i n g c u r v e of DBP f o l l o w e d by f l u o r e s c e n c e 48 e m i s s i o n a t 307 nm e x c i t a t i o n o n a t 278 nm 30 G u a n i d i n e . H C 1 d e n a t u r a t i o n of DBP f o l l o w e d by CD 49 31 F l u o r e s c e n c e s p e c t r a of a e r y l o d a n - D B P ( 5 : 1 l a b e l e d ) . 51 A) E x c i t a t i o n s p e c t r u m , e m i s s i o n a t 510 nm B) E m i s s i o n s p e c t r u m , e x c i t a t i o n at 365 nm 32 The e f f e c t of DTT on the e m i s s i o n s p e c t r u m of 52 a c r y l o d a n - D B P ( 5 : 1 l a b e l e d ) , e x c i t a t i o n a t 365 nm 33 The e f f e c t of a c t i n b i n d i n g on the e m i s s i o n 53 s p e c t r u m of a c r y l o d a n - D B P ( 0 . 3 5 : 1 l a b e l e d ) , e x c i t a t i o n a t 365 nm 34 The e f f e c t of DTT on the e m i s s i o n s p e c t r u m of 54 a c r y l o d a n - D B P ( 0 . 3 5 : 1 l a b e l e d ) , e x c i t a t i o n a t 365 nm i x The f o r c e t h a t d r i v e s the w a t e r t h r o u g h the r o c k s D r i v e s my r e d b l o o d ; t h a t d r i e s the m o u t h i n g s t r e a m s Turns mine t o wax. And I am dumb t o mouth unto my v e i n s How a t the m o u n t a i n s p r i n g the same mouth s u c k s . The f o r c e t h a t t h r o u g h the g r e e n f u s e d r i v e s the f l o w e r . D y l a n Thomas X Acknowledgement I wou ld l i k e t o thank D r . Dana Dev ine f o r the use of her P h a s t S y s t e m and D r . L e s l i e D. B u r t n i c k f o r e v e r y t h i n g e l s e . xi A b b r e v i a t i o n s A c r y l o d a n ATP CD CM-Sephadex DBP DEAE-Sephadex DMF DMSO DTT EGTA Gc HPLC MOPS PMSF PIA SDS TEMED T r i s 6 - a c r y l o y l - 2 - ( d i m e t h y l a m i n o ) n a p h t h a l e n e A d e n o s i n e 5 ' - T r i p h o s p h a t e C i r c u l a r D i c h r o i s m C a r b o x y m e t h y l - S e p h a d e x V i t a m i n D - B i n d i n g P r o t e 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 N , N - D i m e t h y l f o r m a m i d e D i m e t h y l s u l p h o x i d e D i t h i o t h r e i t o l E t h y l e n e b i s ( o x y e t h y l e n e n i t r i l o ) -t e t r a a c e t i c A c i d Group S p e c i f i c Component of P lasma H i g h P r e s s u r e L i q u i d Chromatography 3 - ( N - m o r p h o 1 i n o ) p r o p a n e s u l p h o n i c A c i d P h e n y l m e t h y 1 s u l p h o n y l F l u o r i d e N - ( 1 - p y r e n e ) i o d o a c e t a m i d e Sodium D o d e c y l S u l p h a t e N , N , N ' , N* - T e t r a n t e t h y l e t h y l e n e d i a m i n e T r i s ( h y d r o x y m e t h y l ) m e t h y l amine B u f f e r A B u f f e r B B u f f e r E B u f f e r H 2 mM T r i s - H C l , 0 . 2 mM C a C l 2 , 0 . 2 mM ATP, 1 . 0 mM DTT, pH 7 . 6 30 mM Na P h o s p h a t e B u f f e r , 0.1%(w/v) Sodium A z i d e , 50 uM PMSF, pH 7 . 0 25 mM T r i s - H C l , 194 mM g l y c i n e , 0 . 2 mM ATP, pH 8 . 4 150 mM K C l , 20 mM MOPS, 1 mM EGTA, pH 7 . 3 1 JLl I n t r o d u c t i o n P a r t 1 : P r o t e i n s 1 . 1 . 1 F u n c t i o n s of A c t i n A c t i n i s one of the most abundant p r o t e i n s i n e u k a r y o t i c c e l l s , o f t e n c o m p r i s i n g up to 20% of t o t a l c e l l p r o t e i n . I t i s a m u l t i p l e f u n c t i o n e d , h i g h l y c o n s e r v e d p r o t e i n p l a y i n g a majo r r o l e i n musc le c o n t r a c t i o n and i n the s t r u c t u r e and m o t i l i t y of n o n - m u s c l e c e l l s ( S t r y e r , 1 9 8 1 ) . A c t i n , i n c o n j u n c t i o n w i t h t r o p o m y o s i n and the t r o p o n i n c o m p l e x , fo rms the t h i n f i l a m e n t of musc le ( f i g 1 ) . These t h i n f i l a m e n t s i n t e r a c t w i t h the myos in heads of the t h i c k f i l a m e n t s c a u s i n g c o n t r a c t i o n of the musc le ( f i g 2 ) . Troponin I complex Tropomyosin Actin ( Fig 1: The thin filament of skeletal muscle. Stryer (1981). 2 H Z A I s' band l ine band band Myo f ib r i l Z sarcomere Z / \ I \ I \ I \ I A band \ * .O-!. Z I I I I I I I I I I I I nun nun I I I I I I I I I I I I I I I I I I J I I I I I / nCn I I I I I I i I I I I I I I I I I I I i I I I I I I i I I I I I I i I I I I I I I I I I I I i I I I I I I i IIIIII I I I I I I I IIIII I I I I I I I IIIII I I I I I I IIIIII I I I I I I I Th ick f i lamenls (myosin) 7 T h i n l i lamcn ls (actin) Z line ^- Thin filament ^ Thick filament if Z line - - - Ji L a a a ^ 1 Fig 2: The main components of skele ta l muscle. Lenhinger (1975). 3 The p r o c e s s i s d r i v e n e n e r g e t i c a l l y by the h y d r o l y s i s of ATP t o ADP. M y o s i n f i r s t b i n d s t o ATP ( f i g 3A) f o l l o w e d by m y o s i n - A T P b i n d i n g t o a c t i n ( f i g 3 B ) . ATP t h e n h y d r o l y s e s to ADP c a u s i n g the myos in heads to t i l t ( f i g 3C) r e s u l t i n g i n the t h i c k and t h i n f i l a m e n t s s l i d i n g o v e r each o t h e r and c o n s e q u e n t l y i n the musc le c o n t r a c t i n g ( f i g 3 D ) . Fig 3: The mechanism of muscle contraction. Stryer (1981). 4 The c o n t r o l of c o n t r a c t i o n i s p r o v i d e d t h r o u g h c a l c i u m i o n r e g u l a t i o n . Nerve p u l s e s r e l e a s e c a l c i u m f rom the s a r c o p l a s m i c r e t i c u l u m . T h i s c a l c i u m b i n d s to t r o p o n i n C, w h i c h causes c o n f o r m a t i o n a l changes t h a t a re t r a n s m i t t e d to t r o p o m y o s i n . These c o n f o r m a t i o n a l changes i n t r o p o m y o s i n expose the n e c e s s a r y m y o s i n b i n d i n g s i t e s on a c t i n , a l l o w i n g c o n t r a c t i o n to p r o c e e d . Fig 4: Immunofluorescence micrograph of a F l g 5 : I m m u n o f l u o r e s c e n c e micrograph of a resting ce l l stained with a moving c e l l stained with a fluorescent fluorescent antibody to ac t in . antibody to ac t in . The ruffled border is Stryer (1981). t h e f ad ing edge. Stryer (1981). 5 A c t i n i s a l s o a major c o n s t i t u e n t of n o n - m u s c l e c e l l s . Some of t h i s a c t i n forms m i c r o f i l a m e n t s w h i c h r e s e m b l e the t h i n f i l a m e n t s of m u s c l e and c o n t r i b u t e to the shape and s t r u c t u r e of the c e l l s ( f i g 4 ) . By a c t i v e l y s h o r t e n i n g t h e s e m i c r o f i l a m e n t s i n one d i r e c t i o n and e l o n g a t i n g them i n the o p p o s i t e d i r e c t i o n the c e l l p o s s e s s e s the m e c h a n i s m , t h r o u g h a c t i n c o n f o r m a t i o n a l c h a n g e s , to move ( f i g 5 ) . The f u n c t i o n s of a c t i n o u t l i n e d r e l y on i t s a b i l i t y t o p o l y m e r i z e and fo rm f i l a m e n t s . In the case of m u s c l e c e l l s and c o n t r a c t i o n , the p o l y m e r i z a t i o n o c c u r s j u s t o n c e . However i t i s a more t r a n s i e n t a f f a i r i n n o n - m u s c l e c e l l s where p o l y m e r i z a t i o n and d e p o l y m e r i z a t i o n o c c u r many t i m e s ( P o l l a r d and C r a i g , 1982) . 1 . 1 . 2 A c t i n P o l v m e r i z a t i o n / D e p o l T m e r i z a t i o n Monomeric a c t i n ( G - a c t i n ) i s a 4 2 , 0 0 0 d a l t o n , g l o b u l a r p r o t e i n and the p o l y m e r ( F - a c t i n ) can be v iewed i n two ways ( f i g 6 ) t as a d o u b l e - s t r a n d e d , r i g h t - h a n d e d h e l i x w i t h 13 a c t i n - u n i t s i n each s t r a n d w i t h i n each 72 nm h e l i x p i t c h , or as a s i n g l e - s t r a n d e d , l e f t - h a n d e d h e l i x w i t h a 5 . 9 nm p i t c h and 2 . 1 6 s u b u n i t s per t u r n ( K o r n , 1 9 8 2 ) . G - a c t i n can be p o l y m e r i z e d s p o n t a n e o u s l y to F - a c t i n i n the p r e s e n c e of K C l or N a C l ( 5 0 - 1 5 0 mM) or M g C l 2 ( 0 . 5 - 2 mM). T h i s p o l y m e r i z a t i o n i s d i r e c t i o n a l , o c c u r r i n g at b o t h ends of a g r o w i n g a c t i n f i l a m e n t , but a t two q u i t e d i f f e r e n t r a t e s . G - a c t i n i s f a v o u r e d i n the absence of s a l t and i n the p r e s e n c e of ATP. Fig 6: Schematic diagram of F-act in . Korn (1982). 7 n m The c o n t r o l o f a c t i n f i l a m e n t l e n g t h v i a s a l t c o n c e n t r a t i o n i s not s p e c i f i c enough to a c c o u n t f o r the v a r i e t y of a c t i n f u n c t i o n s . Hence a s e r i e s of a c t i n b i n d i n g p r o t e i n s i s found i n c e l l s w h i c h can m o d i f y the a c t i v i t y of a c t i n (Way and Weeds, 1 9 9 0 ) . These a c t i n b i n d i n g p r o t e i n s can be c l a s s i f i e d by t h e i r f u n c t i o n ( f i g 7 ) . Monomer b i n d i n g p r o t e i n s b i n d o n l y t o G - a c t i n a n d , i n the case of p r o f i l i n , p r o v i d e a b u f f e r i n g c a p a c i t y f o r a c t i n , e n s u r i n g t h a t t h e r e i s G - a c t i n a v a i l a b l e f o r p o l y m e r i z a t i o n . Network and b u n d l i n g p r o t e i n s fo rm F - a c t i n a g g r e g a t e s , i n c r e a s i n g the s t r e n g t h and d i v e r s i t y of a c t i n s t r u c t u r e s . F i n a l l y the l e n g t h of a c t i n f i l a m e n t s can be r e g u l a t e d by s e v e r i n g and 7 Fig 7: Schematic diagram showing the various a c t i v i t i e s of actin binding proteins along with examples. Way and Heeds (1990). c a p p i n g p r o t e i n s w h i c h b r e a k up and s l o w down the p o l y m e r i z a t i o n , r e s p e c t i v e l y , and by n u c l e a t i o n p r o t e i n s w h i c h p r o v i d e a f o u n d a t i o n f o r r a p i d p o l y m e r i z a t i o n . On the d e a t h of c e l l s , whether the r e s u l t of i n j u r y , d i s e a s e or n a t u r a l a g e i n g , G - a c t i n and F - a c t i n a r e r e l e a s e d i n t o e x t r a c e l l u l a r f l u i d s , i n c l u d i n g b l o o d p l a s m a . The h i g h s a l t c o n d i t i o n s t h e r e f a v o u r F - a c t i n and c o n s e q u e n t l y c o u l d cause h i g h p lasma v i s c o s i t y , b l o c k i n g the m i c r o c i r c u l a t i o n (Haddad et a l . , 1 9 9 0 ) . To a v o i d t h i s f a t e , b l o o d p lasma p o s s e s s e s two F - a c t i n d e p o l y m e r i z i n g p r o t e i n s , g e l s o l i n and DBP, w h i c h scavenge any a c t i n r e l e a s e d i n t o the b l o o d . 8 1.1.3 A c t i n Scavenging System i n Plasma G e l s o l i n i s an F - a c t i n s e v e r i n g p r o t e i n w i t h the a b i l i t y to b i n d t o e i t h e r F - a c t i n or G - a c t i n . I t has two a c t i n - b i n d i n g s i t e s , one of w h i c h i s c a l c i u m d e p e n d e n t . DBP i s a monomer b i n d i n g p r o t e i n , f o r m i n g a 1 : 1 complex w i t h a c t i n , K a = 1 -2 x 1 0 8 M _ 1 ( G o l d s c h m i d t - C l e r m o n t et a l . , 1 9 8 7 ) . I t can d e p o l y m e r i z e F - a c t i n i n d i r e c t l y by s e q u e s t e r i n g a c t i n monomers, thus s h i f t i n g the e q u i l i b r i u m away f rom F - a c t i n . Janmey and L i n d (1987) s u g g e s t e d t h a t on the r e l e a s e of a c t i n i n t o b l o o d , DBP b i n d s the G - a c t i n and g e l s o l i n s e v e r s F - a c t i n i n t o s h o r t f i l a m e n t s . F u r t h e r d e p o l y m e r i z a t i o n of the ge1so1 i n - c a p p e d a c t i n f i l a m e n t s l e a d s t o the m a j o r i t y of a c t i n b e i n g bound t o DBP ( f i g 8 ) . F-ACTIN O D D D D D O D D D • D D D D D 0 D D D D D» D» D» D* D* D* D» D» D» D G G G •G •G •G G-ACTIN • • • • • • • • • • • • + D D D D D D D D D D G G G D* D* D* D» D* D» D» D* D» D» G G Fig 8: Schematic diagram of the complexes formed on the addition of G-actin or F-act in to ge lso l in (G) and DBP (D). Janmey and Lind (1987). 9 T h i s t h e o r y i s s u p p o r t e d by Coue et a l . (1986) who found t h a t , on a d d i t i o n of DBP and g e l s o l i n t o f l u o r e s c e n t l y l a b e l e d a c t i n , o n l y the D B P - a c t i n complex was f o r m e d . In v i t r o s t u d i e s u s i n g r a d i o l a b e l e d a c t i n - b i n d i n g p r o t e i n s ( L i n d e t a l . , 1986) showed t h a t a c t i n complexes of DBP and g e l s o l i n a r e c l e a r e d f r o m the p lasma by the l i v e r q u i c k e r t h a n DBP or g e l s o l i n a l o n e , and t h a t D B P - a c t i n complexes a r e c l e a r e d f a s t e r t h a n g e l s o l i n - a c t i n c o m p l e x e s . F u r t h e r m o r e Janmey et a l . (1986) showed t h a t DBP can remove an a c t i n monomer f rom one of the two g e l s o l i n b i n d i n g s i t e s , i n f e r r i n g t h a t DBP i s the main p r o t e i n r e s p o n s i b l e f o r the d i s p o s a l of e x t r a c e l l u l a r a c t i n as w e l l as f o r r e n d e r i n g i t i n a c t i v e t o p o l y m e r i z a t i o n . 1.1.4 Review of PBP H i r s c h f e l d (1959) d i s c o v e r e d a p o s t a l b u m i n i n human serum w h i c h m i g r a t e d i n the f r a c t i o n on s t a r c h g e l e l e c t r o p h o r e s i s . He c a l l e d t h i s the Group S p e c i f i c Component (Gc) S y s t e m . In the same y e a r , Thomas et a l . r e p o r t e d a v i t a m i n D - b i n d i n g a - g l o b u l i n , but i t was not u n t i l 1975 t h a t D a i g e r et a l . showed Gc t o be DBP. DBP s i n c e has been found i n o t h e r body f l u i d s such as u r i n e , a s c i t i c f l u i d and s p i n a l f l u i d ( H i r s c h f e l d , 1 9 6 2 ; N i e l s e n et a l . , 1 9 6 3 ; B e r g g a r d et a l . , 1 9 6 4 ) . DBP b i n d s 1 : 1 t o v i t a m i n D w i t h an a f f i n i t y K a = 1 0 1 0 M _ 1 at 4 °C ( B o u i l l o n et a l . 1986) c a r r y i n g out two f u n c t i o n s ; the t r a n s p o r t a t i o n of v i t a m i n D s t e r o l s as w e l l as p r o v i d i n g a c i r c u l a t i n g r e s e r v o i r o f the s t e r o l s (Cooke and Haddad, 1 9 8 9 ) . 10 However , the c o n c e n t r a t i o n of DBP (~5 x 10 M) i s i n v a s t — ft e x c e s s of v i t a m i n D (~5 x 10 M) i n p l a s m a , s u g g e s t i n g an a l t e r n a t e f u n c t i o n . A l t h o u g h many t h o u s a n d s of s e r a have been t e s t e d f o r DBP, no d e l e t i o n o r g r o s s a l t e r a t i o n t o the DBP gene has been f o u n d , i m p l y i n g t h a t DBP has a v i t a l r o l e i n p l a s m a . Van B a e l e n et a l . (1980) d i s c o v e r e d DBP 1 s a c t i n -b i n d i n g p r o p e r t i e s . V i t a m i n D and a c t i n b i n d t o DBP i n a n o n -c o m p e t i t i v e manner . DBP i s s y n t h e s i z e d i n the l i v e r a t a r a t e of 10 mg kg ^ day ^ and i s c l e a r e d f r o m t h e c i r c u l a t i o n by the l i v e r and k i d n e y s . A r a d i o - l a b e l e d DBP a d d u c t , a f t e r i n t r a v e n o u s i n j e c t i o n , had a p lasma t . i of 1-f hours w h i l s t the complex of the a d d u c t and a c t i n had a p lasma t i of 30 min ( D u e l a n d et a l . , 1 9 9 0 ) . B o t h the apo and h o l o v i t a m i n D fo rms of DBP a r e c l e a r e d f rom p lasma at the same r a t e (Cooke and Haddad , 1 9 8 9 ) . O ther p r o p e r t i e s of DBP i n c l u d e b i n d i n g f a t t y a c i d s (Ena et a l . , 1989) and the 1 : 1 b i n d i n g of c a l c i u m i o n s , w h i c h has been shown t o be n o n - c o o p e r a t i v e w i t h a c t i n b i n d i n g ( G o l d s c h m i d t - C l e r m o n t et a l . , 1 9 8 7 ) . 11 1.1.5 P h y s i c a l P r o p e r t i e s of DBP T a b l e I: P h y s i c a l P r o p e r t i e s of DBP Gone . i n plasma Man 5 9 , 0 0 0 10 uM Mouse 4 9 , 0 0 0 Rat 5 2 , 0 0 0 9 - 1 3 uM Some of the p h y s i c a l p r o p e r t i e s of DBP f r o m d i f f e r e n t s p e c i e s a r e shown i n t a b l e I. There i s a l a r g e v a r i a n c e i n m o l e c u l a r m a s s . On s t u d y i n g DBP f rom s e v e r a l s p e c i e s , B o u i l l o n et a l . (1986) s u g g e s t e d an a v e r a g e b l o o d c o n c e n t r a t i o n of 3 0 0 -400 mg/L s p e c i e s w i d e . A r a t DBP cDNA c l o n e has been sequenced ( C o o k e , 1985) and has l e d to a p r e d i c t e d amino a c i d sequence f o r the p r o t e i n ( f i g 9 ) . The p r e d i c t e d sequence shows DBP t o be of the same f a m i l y of p r o t e i n s as a l b u m i n . T h i s f a m i l y of p r o t e i n s i s t h o u g h t t o come f r o m a common a n c e s t r a l g e n e , w i t h gene d u p l i c a t i o n o c c u r r i n g between 3 0 0 - 5 0 0 m i l l i o n y e a r s a g o . A l b u m i n s h a r e s some p r o p e r t i e s w i t h DBP, such as the b i n d i n g and t r a n s p o r t of f a t t y a c i d s , and a l b u m i n s e r v e s as a back up i n t h e t r a n s p o r t o f s t e r o i d compounds ( S t r y e r , 1 9 8 1 ) . DBP i s found t o be r i c h i n a s p a r t i c a c i d , g l u t a m i c a c i d and c y s t e i n e and low i n g l y c i n e w i t h no t r y p t o p h a n r e s i d u e s . In r a t DBP t h e r e a r e 28 c y s t e i n e r e s i d u e s w h i c h , due to the 12 Fig 9: Homology between the disulphide-bonding patterns in rat albumin and the predicted amino acid sequence in rat DBP. Small c i r c l e s show amino acids deleted in comparison to albumin. Cooke (1986). 13 s i m i l a r i t y of the amino a c i d sequence t o t h a t of a l b u m i n ( f i g 9 ) , have been p r o p o s e d t o a l l be i n v o l v e d i n d i s u l p h i d e bonds ( C o o k e , 1 9 8 5 ) . T h i s c o n t r a s t s w i t h e a r l i e r e v i d e n c e f rom human DBP, where one d e e p l y b u r i e d m o n o t h i o l g roup was f o u n d (Kawakami and Goodmann, 1 9 8 1 ) . DBP has p r o v e d u s e f u l i n f o r e n s i c s c i e n c e as i t has many p h e n o t y p e s and i s r e l a t i v e l y s t a b l e , a b l e t o s u r v i v e 2 weeks at room t e m p e r a t u r e on d r y c l o t h ( P S t s c h - S c h n e i d e r and K l e i n , 1 9 8 8 ) . The D B P - v i t a m i n D complex i s found t o be more s t a b l e t h a n DBP a l o n e (Kawakami and Goodmann, 1 9 8 1 ) . 1 . 1 . 6 Rev iew of DBP P u r i f i c a t i o n Methods There have been many p u b l i s h e d p u r i f i c a t i o n schemes f o r DBP, most of w h i c h g i v e poor y i e l d s due t o the d i f f i c u l t y i n s e p a r a t i n g DBP f rom a l b u m i n . A t y p i c a l e a r l y method p r o p o s e d by B o u i l l o n et a l . (1978) used 600 ml of r a t serum and i n v o l v e d 7 s t e p s : d i a l y s i s , D E A E - c e l l u l o s e c h r o m a t o g r a p h y , p r e c i p i t a t i o n w i t h ammonium s u l p h a t e , h y d r o x y l a p a t i t e c h r o m a t o g r a p h y , C M - c e l l u l o s e c h r o m a t o g r a p h y , DEAE-Sephadex c h r o m a t o g r a p h y and g e l f i l t r a t i o n , w i t h a f i n a l y i e l d of 0 . 1 % . In 1 9 8 2 , C h a p u i s - C e l l i e r et a l . u t i l i z e d the h i g h a f f i n i t y of serum a l b u m i n f o r A f f i - g e l B l u e (a t r a d e name f o r c i b a c r o n b l u e dye c o v a l e n t l y l i n k e d t o a g a r o s e b e a d s ) , i n a t h r e e s t e p p r o c e d u r e : A f f i - g e l B l u e c h r o m a t o g r a p h y , g e l f i l t r a t i o n u s i n g Sephadex G -100 and D E A E - A f f i - g e l B l u e c h r o m a t o g r a p h y t o g i v e a 73% y i e l d w i t h 97% p u r i t y f r o m 80 ml of human p l a s m a . 14 More r e c e n t l y , c h r o m a t o g r a p h i c methods u s i n g a f f i n i t y m a t r i c e s have been f a v o u r e d d e s p i t e t h e i r s m a l l c a p a c i t y . L i n k et a l . (1986) d e s i g n e d a v i t a m i n D - S e p h a r o s e m a t r i x , f rom w h i c h DBP c o u l d be removed by 60% a c e t o n e . When t h i s s t e p was f o l l o w e d by h y d r o x y l a p a t i t e c h r o m a t o g r a p h y the o v e r a l l y i e l d was 55%. F i n a l l y , a DNAase I - S e p h a r o s e m a t r i x was used t o b i n d a c t i n and then DBP. DBP was t h e n e l u t e d w i t h 4 M MgCl2 (van B a e l e n and B o u i l l o n , 1 9 8 6 ) . G e l f i l t r a t i o n s e r v e d as a f i n a l p u r i f i c a t i o n s t e p and p r o v i d e d a y i e l d of 70% f o r human p lasma DBP. T h i s method has been employed f o r s e v e r a l d i f f e r e n t s p e c i e s but has p r o d u c e d c o n t a m i n a t e d m a t e r i a l i n some c a s e s , i n c l u d i n g h o r s e . 15 P a r t 2 : S p e c t r o s c o p i c T e c h n i q u e s 1 . 2 . 1 P r o t e i n A b s o r b a n c e S p e c t r o s c o p y A b s o r b a n c e s p e c t r o s c o p y p r o v i d e s a c o n v e n i e n t method of a s s e s s m e n t of p r o t e i n c o n c e n t r a t i o n as w e l l as g i v i n g some c l u e s t o the amino a c i d c o m p o s i t i o n of a p r o t e i n . P r o t e i n a b s o r b a n c e s p e c t r a i n the w a v e l e n g t h r e g i o n 2 5 0 -320 nm are c h a r a c t e r i s t i c and are d o m i n a t e d by the c o n t r i b u t i o n s of t h r e e ch ro raophores ; t r y p t o p h a n , t y r o s i n e and p h e n y l a l a n i n e . These a b s o r b a n c e s p e c t r a r e s u l t f r o m k t o k e l e c t r o n i c t r a n s i t i o n s i n t.hese a r o m a t i c s y s t e m s . 025 0.18 0.80 0.60 • 040 0.20 250 300 Mnml 250 300 X{nm) 250 300 A(nm) Fig 10: Absorbance spectra of the aromatic amino acids in a 1 cm c e l l in 0.01 H potassium phosphate buffer, pH 7.0 at 25' C. a, 1 mM phenylalanine; b, 0.1 mM tyrosine; c, 0.1 mM tryptophan. Creighton (1989). 16 F i g u r e 10 shows the a b s o r b a n c e s p e c t r a of the i s o l a t e d c h r o m o p h o r e s . R e a l p r o t e i n a b s o r b a n c e s p e c t r a a r e t h e sum of a l l the i n d i v i d u a l chromophore a b s o r b a n c e s . A c l e a r e r method t o i d e n t i f y the c o n t r i b u t i o n s o f each of the chromophores t o the p r o t e i n a b s o r b a n c e s p e c t r u m i s t o l o o k a t the more d i s t i n c t i v e second d e r i v a t i v e s p e c t r a ( f i g 1 1 ) . C l e a r l y the c o n t r i b u t i o n of t r y p t o p h a n to the a l b u m i n ( f i g 12) s p e c t r u m can be i d e n t i f i e d . A l b u m i n c o n t a i n s 1 t r y p t o p h a n and 18 t y r o s i n e s . Wavelength (nm) Wavelength (nm) 0.3 0.0 —-t> > r> 240 250 260 270 280 Wavelength (nm) Fig 11: Second derivat ive spectra in 6 M GuHCL, sodium phosphate, pH 6.8. Nozaki (1990). (a) AcTr P NH 2 , 2.51 x 10" 4 M, (b) AcTyrNH 2, 7.6 x 1 0 - 4 M, (c) AcPheOEt, 1.78 x 10" 3 M. c A N WW >o 1 o> c Oo j 1 M 1 i V / 17 0.25 - 0 . 2 5 280 290 300 Wavelength (nm) Fig 12: Second derivative spectrum of human serum albumin in GuHCl at arbi t rary concentration. Nozoki (1990). A b s o r b a n c e measurements a l l o w the c a l c u l a t i o n of p r o t e c o n c e n t r a t i o n i n s o l u t i o n v i a the B e e r - L a m b e r t l a w : = — 1 o S 1 o C I / I e. d c Where: A = a b s o r b a n c e I - i n t e n s i t y of t r a n s m i t t e d l i g h t I Q = i n t e n s i t y of i n c i d e n t l i g h t _^ _^ e = m o l a r e x t i n c t i o n c o e f f i c i e n t (M cm ) d = c e l l p a t h l e n g t h (cm) c = m o l a r c o n c e n t r a t i o n (M) The c a l c u l a t i o n r e q u i r e s the knowledge of the a p p r o p r i a t e e x t i n c t i o n c o e f f i c i e n t , w h i c h i s u s u a l l y q u o t e d a t 280 nm f p r o t e i n s . 18 1 . 2 . 2 F l u o r e s c e n c e S p e c t r o s c o p y o f P r o t e i n s F l u o r e s c e n c e e m i s s i o n may be o b s e r v e d when an e l e c t r o n r e t u r n s f r o m the f i r s t e x c i t e d s t a t e (S^) to the g round s t a t e ( S Q ) . The e m i s s i o n i s i n v a r i a b l y s h i f t e d to a l o w e r w a v e l e n g t h ( S t o k e s s h i f t ) due t o the r a p i d decay to the l o w e s t v i b r a t i o n a l l e v e l of S^ p r i o r to e m i s s i o n . A d d i t i o n a l l y f l u o r o p h o r e s g e n e r a l l y decay to e x c i t e d v i b r a t i o n a l l e v e l s of S Q , p r o d u c i n g a f u r t h e r l o s s i n e n e r g y . S o l v e n t s can have an e f f e c t on f l u o r e s c e n c e e m i s s i o n . On e x c i t a t i o n a f l u o r o p h o r e becomes an i n s t a n t a n e o u s d i p o l e ( F r a n c k - C o n d o n p r i n c i p l e ) . In p o l a r s o l v e n t s , the p o l a r e x c i t e d s t a t e w i l l be s t a b i l i z e d , l e a d i n g to a l o w e r i n g of the e n e r g y gap between S Q and S^ l e v e l s and hence a r e d s h i f t i n e m i s s i o n i n c o m p a r i s o n w i t h n o n - p o l a r s o l v e n t s . T h i s s h i f t to l o n g e r w a v e l e n g t h i s u s u a l l y accompanied by a d rop i n e m i s s i o n i n t e n s i t y due t o s o l v e n t q u e n c h i n g . Thus , i n f o r m a t i o n c o n c e r n i n g a f l u o r o p h o r e ' s e n v i r o n m e n t can be g a i n e d f rom f l u o r e s c e n c e s t u d i e s , w h i c h i n t u r n , p r o v i d e s p r o t e i n s t r u c t u r a l i n f o r m a t i o n . P r o t e i n s p o s s e s s o n l y t h r e e amino a c i d s w h i c h a r e i n t r i n s i c a l l y f l u o r e s c e n t ; t r y p t o p h a n , t y r o s i n e and p h e n y l a l a n i n e . These f l u o r o p h o r e s can be e x c i t e d s e p a r a t e l y ( f i g 1 3 ) , however the e m i s s i o n s p e c t r a of p r o t e i n s c o n t a i n i n g s e v e r a l o f t h e s e f l u o r o p h o r e s a r e dominated by t r y p t o p h a n due t o energy t r a n s f e r f rom p h e n y l a l a n i n e and t y r o s i n e t o t r y p t o p h a n . 19 Fig 13: Fluorescence spectra of the aromatic acids in 0.01 M potassium phosphate buffer, pH 7.0, at 25° C. Phe, 100 uM phenylalanine exci tat ion 257 nm; Tyr, 6 uM tyrosine exci ta t ion 274; Trp, 1 uM tryptophan exci tat ion 278 nm. Creighton (1989). A more s p e c i f i c method to s t u d y p r o t e i n s t r u c t u r e v i a f l u o r e s c e n c e i s to i n t r o d u c e e x t r i n s i c f l u o r o p h o r e s . A c r y l o d a n ( 6 - a c r y l o y l - 2 - d i m e t h y l a m i n o n a p h t h a l e n e , f i g 14) i s a p a r t i c u l a r l y e n v i r o n m e n t - s e n s i t i v e f l u o r e s c e n t p robe w h i c h r e a c t s o n l y w i t h c y s t e i n e r e s i d u e s ( P e n d e r g a s t et a l . , 1983) N - ( 1 - p y r e n e ) i o d o a c e t a m i d e ( f i g 15) i s a n o t h e r c y s t e i n e s p e c i f i c p robe w h i c h has p r o v e d u s e f u l i n s t u d y i n g a c t i n , as the f l u o r e s c e n c e of p y r e n e - l a b e l e d a c t i n i s s e n s i t i v e t o p o l y m e r i z a t i o n (Cooper et a l . , 1 9 8 3 ) . 0 II NHCCH2I M « a N 0 Fig 14: 6-acryloyl-2-dimethylaminonaphthalene Fig 15: N-(1-Pyrene)iodoacetamide 20 1 . 2 . 3 P r o t e i n CD S p e c t r o s c o p y O p t i c a l l y a c t i v e s u b s t a n c e s a b s o r b l e f t and r i g h t c i r c u l a r l y p o l a r i z e d l i g h t (L and R, r e s p e c t i v e l y ) t o d i f f e r e n t e x t e n t s . T h i s d i f f e r e n c e i s u s u a l l y e x p r e s s e d i n te rms of e x t i n c t i o n c o e f f i c i e n t s . e L - e R = A e Where = e x t i n c t i o n c o e f f i c i e n t f o r L (M_|cm J ) = e x t i n c t i o n c o e f f i c i e n t f o r R (M cm ) AE i s c a l l e d the c i r c u l a r d i c h r o i s m . As a r e s u l t of the d i f f e r e n t a b s o r b a n c e s of L and R, the c o m b i n a t i o n of the two waves w i l l p r o d u c e e l l i p t i c a l l y p o l a r i z e d l i g h t . T h i s f a c t has l e d t o c i r c u l a r d i c h r o i s m b e i n g e x p r e s s e d i n te rms of the e l l i p t i c i t y , 6 , of the e l l i p s e t h a t c h a r a c t e r i z e s such l i g h t , where t a n 8 i s e q u a l t o the r a t i o of the m i n o r a x i s t o the majo r a x i s of the e l l i p s e , AE i s r e l a t e d to the m o l a r e l l i p t i c i t y [8] b y : [ 6 ] = 3 3 0 0 A e A c u r v e showing the dependence of 8 on w a v e l e n g t h i s c a l l e d a O -1 CD s p e c t r u m . M o l a r e l l i p t i c i t y , [8 ] i n deg cm dmol , can be c a l c u l a t e d f r o m o b s e r v e d e l l i p t i c i t y a t a s p e c i f i c w a v e l e n g t h : 21 [ e ] = M e / i o d o Where; 8 i s the o b s e r v e d e l l i p t i c i t y (deg) M i s the m o l e c u l a r mass (g mol ) d i s the c e l l p a t h l e n g t h (cm) c i s the c o n c e n t r a t i o n (g ml ) I f M e x p r e s s e s the mean r e s i d u e m o l e c u l a r m a s s , t h e n [8] i s c a l l e d the mean r e s i d u e (o r r e s i d u a l ) e l l i p t i c i t y . P r o t e i n CD s p e c t r a a r e d o m i n a t e d by the s i g n a l f r o m the p e p t i d e bond b a c k b o n e , the v a r i o u s c o n f o r m a t i o n s of w h i c h have c h a r a c t e r i s t i c s p e c t r a ( f i g 1 6 ) . Fig 16: CD spectra of p o l y - W y s i n e in the 1, o r h e l i c a l ; 2, B-sheet; 3, random c o i l conformations. Freifelder (1976). 22 U s i n g t h e s e s t a n d a r d s p e c t r a the p e r c e n t a g e a - h e l i x , (3-sheet and random c o i l can be c a l c u l a t e d by s o l v i n g the f o l l o w i n g e q u a t i o n at two d i f f e r e n t w a v e l e n g t h s and u s i n g the r e s t r a i n t , f H + f B + f R = I -t e ] = f H x H + f B X B + f R ^ R Where: [9] = mean r e s i d u e e l l i p t i c i t y (deg cm dmol L ) f = f r a c t i o n of amino a c i d r e s i d u e s i n each of the f o l l o w i n g c o n f o r m a t i o n s : H, a - h e l i x ; B, (3-sheet and R, random c o i l (o r more p r o p e r l y u n d e f i n e d s t r u c t u r e ) . Xf j , X g , a re the mean r e s i d u e e l l i p t i c i t i e s of 100% a - h e l i c a l p r o t e i n s , 100% p - s h e e t p r o t e i n s and 100% random c o i l p r o t e i n s r e s p e c t i v e l y CD a l s o p r o v i d e s a good method f o r s t u d y i n g the s t a b i l i t y of p r o t e i n s . The l o s s of CD s i g n a l between 210 nm and 230 nm on d e n a t u r a t i o n ( t h e r m a l or c h e m i c a l ) i s due to l o s s of p r o t e i n s t r u c t u r e . The a - h e l i x and fl-sheet s t r u c t u r e s u n f o l d i n t o the random c o i l s t r u c t u r e , w h i c h has r e l a t i v e l y low CD. By m o n i t o r i n g t h i s l o s s of s i g n a l the s t a b i l i t y of a p r o t e i n can be a s s e s s e d . 23 2: M a t e r i a l s and Methods P a r t 1: P r o t e i n s 2 . 1 . 1 The P r e p a r a t i o n o f G - A c t i n The p r e p a r a t i o n of G - a c t i n f rom r a b b i t m u s c l e powder was based on t h e method of S p u d i c h and Watt ( 1 9 7 1 ) . A c e t o n e powder (10 g) was e x t r a c t e d w i t h b u f f e r A (200 m l , 2 mM T r i s - H C l , 0 . 2 mM C a C l 2 , 0 . 2 mM ATP, 1 . 0 mM DTT, pH 7 . 6 ) f o r SO^min on i c e . The s o l u t i o n was f i l t e r e d t h r o u g h a d o u b l e l a y e r of c h e e s e c l o t h , and then t h r o u g h p r e p a r a t i v e grade f i l t e r p a p e r . The r e s i d u e was rewashed (100 m l , b u f f e r A) and r e f i l t e r e d , the s o l i d r e s i d u e was d i s c a r d e d . The f i l t r a t e s were c o m b i n e d , c e n t r i f u g e d ( 4 0 , 0 0 0 rpm, Beckman 4 5 T i r o t o r , 1 h , 4 ° C , ) and K C l ( t o 50 mM) and M g C l 2 ( t o 2 mM) were added t o the s u p e r n a t a n t . The c r u d e a c t i n was a l l o w e d t o p o l y m e r i z e at room t e m p e r a t u r e f o r 2 h , a t w h i c h p o i n t s o l i d K C l was added t o 0 . 8 M w i t h g e n t l e s t i r r i n g ( 1 . 5 h ) . The s o l u t i o n was c e n t r i f u g e d ( 4 0 , 0 0 0 rpm, Beckman 4 5 T i r o t o r , 3 h , 4 °C) and the p e l l e t ( F - a c t i n ) r e s u s p e n d e d i n 30 ml of b u f f e r A and d i a l y s e d (64 h a g a i n s t 3 c h a n g e s , each of 1 L of b u f f e r A , 4 ° C ) . The s o l u t i o n was c e n t r i f u g e d ( 4 0 , 0 0 0 rpm, Beckman 4 5 T i r o t o r , 3 h , 4 °C) and the s u p e r n a t a n t , G - a c t i n , was used i m m e d i a t e l y o r d i a l y s e d a g a i n s t b u f f e r A . 24 2 . 1 . 2 P r e p a r a t i o n o f P v r e n e - a c t i n P y r e n e - a c t i n was p r e p a r e d by a m o d i f i e d v e r s i o n of the method p r o p o s e d by Cooper et a l . , 1 9 8 3 . PIA (6 mg, M o l e c u l a r P r o b e s I n c . , Eugene , OR) was d i s s o l v e d i n 1 ml of DMF/acetone ( 2 : 1 ) . T h i s s o l u t i o n then was shaken (20 h , room t e m p . ) w i t h 3 ml of a c t i n (1 mg/ml) w h i c h had p r e v i o u s l y been p o l y m e r i z e d (2 mM M g C l 2 , 50 mM C a C l 2 , 2 h , room t e m p . ) . The p r o d u c t then was d i a l y s e d a g a i n s t II. b u f f e r A (3 changes each of 12 h , 4 °C) and c e n t r i f u g e d ( 4 0 , 0 0 0 rpm, Beckman 4 5 T i r o t o r , 3 h , 4 ° C ) . The s u p e r n a t a n t , p y r e n e - a c t i n , was s t o r e d i n b u f f e r A . 2 . 1 . 3 A c r v l o d a n - a c t i n P r e p a r a t i o n A c t i n was l a b e l e d w i t h the f l u o r e s c e n t r e a g e n t a c r y l o d a n f o l l o w i n g the method of M a r r i o t t e t a l . ( 1 9 8 8 ) . G - a c t i n (1 ml of a 1 mg/ml s o l u t i o n i n b u f f e r A) was d i a l y s e d f o r 12 h at 4 'C a g a i n s t 2 mM T r i s - H C l , 0 . 2 mM ATP, 0 . 1 mM C a C l 2 > pH 7 . 9 . The G - a c t i n was p o l y m e r i z e d t o F - a c t i n by the a d d i t i o n of M g C l 2 t o 2 mM f o r 2 hours at room t e m p e r a t u r e . An e x c e s s of a c r y l o d a n (5 x 10 "* M f i n a l c o n c e n t r a t i o n , M o l e c u l a r P robes I n c . , Eugene , OR) d i s s o l v e d i n a minimum of DMF (~ 0 . 2 ml ) was added d i r e c t l y t o the F - a c t i n . The m i x t u r e was r o c k e d i n the d a r k (6 h) a t room t e m p e r a t u r e . The r e s u l t i n g a c r y l o d a n - l a b e l e d F - a c t i n was d i a l y s e d a g a i n s t b u f f e r A ( 1 L , 3 changes i n 60 h , 4 °C) i n the d a r k b e f o r e b e i n g c e n t r i f u g e d ( 4 0 , 0 0 0 rpm, Beckman Type 50 r o t o r , 3 h , 4 *C) t o p roduce a c r y l o d a n - l a b e l e d G - a c t i n ( a c r y l o d a n - a c t i n ) i n the s u p e r n a t a n t . 25 2 . 1 . 4 The A c r v l o d a n - a c t i n A s s a y The f o l l o w i n g a c r y l o d a n - a c t i n a s s a y i s a m o d i f i c a t i o n of t h a t f i r s t used by S a f e r (1989) to t e s t f o r DBP and o t h e r a c t i n - b i n d i n g p r o t e i n s i n mixed p r o t e i n s o l u t i o n s . The a s s a y u t i l i z e s the f a c t t h a t DBP i s one of two p r o t e i n s , i n p l a s m a , t h a t can b i n d s p e c i f i c a l l y to a c t i n . A c r y l o d a n - a c t i n (10 u l of a 1 mg/ml s o l u t i o n i n b u f f e r A) was combined w i t h each of the samples to be t e s t e d (10 p i ) , a l o n g w i t h a c r y s t a l of a n a l y t i c a l grade s u c r o s e w h i c h was i n c l u d e d t o f a c i l i t a t e l o a d i n g i n t o the sample w e l l s of the e l e c t r o p h o r e t i c g e l . These then were l o a d e d onto a n o n -d e n a t u r i n g p o l y a c r y l a m i d e e l e c t r o p h o r e t i c g e l [6 ml b u f f e r E (25 mM T r i s - H C l , 194 mM g l y c i n e , 0 . 2 mM A T P ) , 7 .5% a c r y l a m i d e , 0 . 2 % N , N ' - m e t h y l e n e b i s a c r y l a m i d e , 70 p i of f r e s h l y p r e p a r e d ammonium p e r s u l p h a t e ( 0 . 1 g / m l ) , 10 p i T r i t o n - X 1 0 0 , 10 p i TEMED, pH 8 . 4 ] w h i c h was run on a M i n i - P r o t e a n I I s y s t e m ( B i o -Rad L a b s , R i c h m o n d , CA) i n b u f f e r E at 200 v o l t s f o r 45 m i n . When ve iwed under a UV l i g h t the p o s i t i v e samples r e v e a l e d a s l o w moving band due to the D B P - a c t i n complex i n a d d i t i o n t o the band c o r r e s p o n d i n g to uncomplexed a c r y 1 o d a n - 1 a b e 1ed G - a c t i n . 26 2 . 1 . 5 The C o l l e c t i o n of Horse P lasma The method of c o l l e c t i n g p lasma was based on the p r o c e d u r e Cote and S m i l l i e (1981) employed t o p r e p a r e p l a t e l e t s . B l o o d (120 L) f r o m f r e s h l y s l a u g h t e r e d h o r s e s ( A l s a s k P r o c e s s o r s , Edmonton) was c o l l e c t e d , w i t h f r e q u e n t s t i r r i n g , i n t o t h r e e p l a s t i c 50 L p a i l s , each c o n t a i n i n g 9 L of a n t i c o a g u l a n t (720 g sod ium c i t r a t e , 89 g c i t r i c a c i d , 61 g sod ium d i h y d r o g e n o r t h o p h o s p h a t e , 1400 g d e x t r o s e d i s s o l v e d t o 27 L i n w a t e r ) . P r o t e a s e i n h i b i t o r s , p e p s t a t i n , l e u p e p t i n ( 1 2 . 5 mg of each per 50 L b u c k e t , S i g m a , S t . L o u i s , M0) and PMSF ( 0 . 2 mM), were a d d e d . The b l o o d was a l l o w e d to s t a n d (~1 h) u n t i l two d i s t i n c t l a y e r s had f o r m e d , w i t h a p i a t e 1 e t - r i c h upper l a y e r and a r e d c e l l - r i c h l o w e r l a y e r . P lasma was c o l l e c t e d as the s u p e r n a t a n t of a 5 , 0 0 0 rpm c e n t r i f u g a t i o n ( S o r v a l l R C - 3 r o t o r , 15 min at 4 °C) of the p l a t e l e t - r i c h l a y e r . I t was f r o z e n i n 500 ml t o 2 L p o r t i o n s and s t o r e d at - 2 0 ' C , 2 . 1 . 6 The P u r i f i c a t i o n of DBP E q u i n e DBP was p r e p a r e d u s i n g a m o d i f i e d v e r s i o n of the method p r o p o s e d by C h a p u i s - C e 1 1 i e r et a l . (1982) t o p u r i f y human DBP. P e p s t a t i n (100 u l of a 2 mg/ml s t o c k s o l u t i o n i n DMSO; S i g m a , S t . L o u i s , MO) and L e u p e p t i n (100 u l of a 2 mg/ml s t o c k s o l u t i o n i n w a t e r ; S i g m a , S t . L o u i s , MO) were added t o thawed h o r s e p i asma (80 m l ) . The p lasma was d i a l y s e d (12 h , 30 mM sod ium p h o s p h a t e b u f f e r , 0.1%(w/v) sodium a z i d e , 50 pM PMSF, pH 7 . 0 , 4 ° C ) and c e n t r i f u g e d at 1 8 , 0 0 0 rpm f o r 27 30 min ( S o r v a l l SS34 r o t o r ) . The p e l l e t was d i s c a r d e d and the s u p e r n a t a n t was a p p l i e d (1 m l / m i n ) , a t room t e m p e r a t u r e , t o an A f f i - G e l B l u e a f f i n i t y c h r o m a t o g r a p h y column ( 2 . 5 x 100 cm, 5 0 - 1 0 0 mesh, B i o - R a d L a b o r a t o r i e s , R i c h m o n d , CA) i n b u f f e r B (30 mM sod ium p h o s p h a t e , 0.1%(w/v) sod ium a z i d e , pH 7 . 0 ) . The e l u t i o n of p r o t e i n was f o l l o w e d by UV a b s o r b a n c e a t 280 nm, and the r e g i o n where DBP e l u t e d was d e t e r m i n e d u s i n g the a c r y l o d a n - a c t i n a s s a y ( a l s o employed t o f o l l o w DBP t h r o u g h s u b s e q u e n t c h r o m a t o g r a p h i c c o l u m n s ) . The A f f i - G e l B l u e column removed the majo r p lasma p r o t e i n , a l b u m i n , as w e l l as p r o v i d i n g some f r a c t i o n a t i o n of o t h e r p lasma p r o t e i n s ( G i a n a z z a et a l . , 1 9 8 2 ) . The column was r e g e n e r a t e d by a p p l y i n g 2 L of p o t a s s i u m t h i o c y a n a t e ( 1 . 5 M) i n the r u n n i n g b u f f e r , as recommended by the m a n u f a c t u r e r . The a c t i v e f r a c t i o n s were p o o l e d (^250 ml ) and c o n c e n t r a t e d to about 20 ml by u l t r a f i l t r a t i o n u s i n g a YM30 membrane ( A m i c o n , D a n v e r s , MA) and t h e n a p p l i e d (1 m l / m i n ) , a t room t e m p e r a t u r e , t o a B i o - G e l P100 g e l f i l t r a t i o n column (5 x 110 cm, B i o - R a d L a b s , R i c h m o n d , CA) i n b u f f e r B. The a c t i v e f r a c t i o n s of the e l u a t e then were a p p l i e d ( 0 . 5 m l / m i n ) d i r e c t l y t o a h y d r o x y l a p a t i t e column ( 2 . 0 x 20 cm, B i o - R a d L a b s , R i c h m o n d , CA) a g a i n i n b u f f e r B a t room t e m p e r a t u r e . A 30 t o 100 mM p h o s p h a t e g r a d i e n t i n b u f f e r B was used t o e l u t e the bound p r o t e i n . 5 ml f r a c t i o n s were c o l l e c t e d . 28 A F low C h a r t Showing the P u r i f i c a t i o n o f DBP FROZEN PLASMA 1) Thawed 2) D i a l y s e d 3) C e n t r i f u g e d AF F I - G E L BLUE A F F I N I T Y CHROMATOGRAPHY 1) A s s a y e d 2) C o n c e n t r a t e d PlOO GEL FILTRATION CHROMATOGRAPHY 1 ) A B E a y e d HYDROXYLAPATITE CHROMATOGRAPHY 1) A s s a y e d 2) D i a l y s e d ANION EXCHANGE H.P.L.C. 1) D i a l y s e d 2) A s s a y e d DBF 29 The a c t i v e f r a c t i o n s were d i a l y s e d (12 h , 10 L b u f f e r B, 4 °C) and f i l t e r e d ( 0 . 2 2 uM M i l l i p o r e f i l t e r ) . S m a l l vo lumes (2 ml ) were a p p l i e d , a t room t e m p e r a t u r e , t o a HPLC a n i o n exchange column (200 x 4 . 6 mm, Aquapore A X - 3 0 0 , 7 m i c r o n pore s i z e , B rown lee L a b s , S a n t a C l a r a , C A ) . DBP was e l u t e d as a s i n g l e peak w i t h a s a l t g r a d i e n t ( 0 - 0 . 2 5 M N a C l i n b u f f e r B ) . 2 . 1 . 7 C a l c u l a t i o n o f an E x t i n c t i o n C o e f f i c i e n t f o r DBP U s i n g the a p p r o x i m a t i o n t h a t the m o l a r e x t i n c t i o n c o e f f i c i e n t of DBP w i l l be e q u a l t o t h a t of the d e n a t u r e d p r o t e i n and the f a c t t h a t t y r o s i n e , t r y p t o p h a n and c y s t e i n e a r e the o n l y a b s o r b i n g s p e c i e s i n the 2 7 6 - 2 8 2 nm w a v e l e n g t h r a n g e , the f o l l o w i n g e s t i m a t i o n of m o l a r e x t i n c t i o n c o e f f i c i e n t a t 280 nm can be made ( G i l l and von H i p p e l , 1 9 8 9 ) : € = a e X y r + b e X r p + c 6 C y s Where: e-pyj., £ j r p a n < * 6 C y s a r e t n e m ° l a r e x t i n c t i o n c o e f f i c i e n t s a t 280 nm and a , b and c a r e the number of each t y p e of amino a c i d per p r o t e i n m o l e c u l e . 30 2.1.8 P r e p a r a t i o n of Acrylodan-DBP DBP (2 ml of a 1 mg/ml s o l u t i o n i n 20 mM MOPS, 0 . 1 % N a N 3 , pH 7 . 3 ) was shaken f o r 12 hours a t room t e m p e r a t u r e w i t h a c r y l o d a n (5 x 10 "* M, M o l e c u l a r P r o b e s I n c . , E u g e n e , OR) d i s s o l v e d i n a minimum of DMF ( ~ 0 . 2 m l ) . The p r o d u c t was d i a l y s e d e x t e n s i v e l y a g a i n s t 20 mM MOPS, 0 . 1 % NaN3, pH 7 . 3 (4 changes each of 1 L f o r 12 h a t 4 ° C ) b e f o r e b e i n g c e n t r i f u g e d ( 4 0 , 0 0 0 rpm, Beckraan Type 50 r o t o r , 1 h , 4 ° C ) . The p e l l e t was d i s c a r d e d . 2.1.9 G e l E l e c t r o p h o r e s i s G e l e l e c t r o p h o r e s i s was c a r r i e d out u s i n g one of the f o l l o w i n g e l e c t r o p h o r e t i c s y s t e m s : M i n i - P r o t e a n I I s y s t e m ( B i o - R a d L a b s , R i c h m o n d , CA) or P h a s t S y s t e m ( P h a r m a c i a , U p p s a l a , Sweden) . The P h a s t S y s t e m was g e n e r o u s l y made a v a i l a b l e by D r . Dana D e v i n e . 31 P a r t 2 : S p e c t r o s c o p i c Methods 2 . 2 . 1 A b s o r b a n c e S p e c t r a A b s o r b a n c e measurements and second d e r i v a t i v e a b s o r b a n c e s p e c t r a were p e r f o r m e d on a Lambda 4B UV/VIS S p e c t r o p h o t o m e t e r ( P e r k i n E l m e r , N o r w a l k , C T ) . 2.2.2 F l u o r e s c e n c e S p e c t r a F l u o r e s c e n c e s p e c t r a were r e c o r d e d on a L S - 5 B L u m i n e s c e n c e S p e c t r o m e t e r i n c o n j u n c t i o n w i t h a 7500 P r o f e s s i o n a l Computer ( P e r k i n E l m e r , N o r w a l k , C T ) . The s p e c t r o m e t e r was e q u i p p e d w i t h a t h e r m o s t a t e d c i r c u l a t i n g w a t e r b a t h ( H a a k e , B e r l i n , G e r m a n y ) , and a t e m p e r a t u r e e q u i l i b r a t i o n p e r i o d of 15 min was a l l o w e d b e f o r e each measurement . 2.2 .3 CP S p e c t r a CD s p e c t r a were g e n e r a t e d on a r e b u i l t J - 2 0 A u t o m a t i c R e c o r d i n g S p e c t r o - P o l a r i m e t e r ( J a s c o , Tokyo) w h i c h was e q u i p p e d w i t h a t e m p e r a t u r e c o n t r o l l e d , n i t r o g e n purged sample chamber . A l l samples were a l l o w e d to e q u i l i b r a t e i n the chamber f o r 15 min b e f o r e measurements were t a k e n . The o u t p u t was s t a n d a r d i z e d u s i n g D - P a n t o l a c t o n e (12 mg i n 100 ml of H 2 0 ) w h i c h has an e l l i p t i c i t y of - 1 7 . 3 x 1 0 3 2 — 1 deg cm dmol a t 220 nm ( T u z i m u r a et a l . , 1 9 7 7 ) . 32 3; R e s u l t s and D i s c u s s i o n 3.1.1 Y i e l d and P u r i t y of DBP DBP was e l u t e d i n a s i n g l e peak ( f i g 17) f r o m the HPLC column i n the f i n a l s t a g e of the p u r i f i c a t i o n scheme. A t o t a l of 6 - 7 mg of DBP were r e t r i e v e d per 80 ml of s t a r t i n g p l a s m a , a f f o r d i n g a y i e l d of 2 1 - 2 5 % , a s s u m i n g an i n i t i a l p l a s m a c o n c e n t r a t i o n of 350 mg/L ( B o u i l l o n et a l . , 1 9 8 6 ) . The p u r i f i c a t i o n scheme was r e p r o d u c i b l e , y i e l d i n g DBP f r o m s i x runs i n s u c c e s s i o n . s c Time Fig 17: Elut ion prof i l e from HPLC column. Shaded area corresponds to DBP. \ The peak c o n t a i n e d a s i n g l e p r o t e i n t h a t m i g r a t e d as a s o l i t a r y band on a S D S - p o l y a c r y l a m i d e e l e c t r o p h o r e t i c g e l 33 under r e d u c i n g c o n d i t i o n s ( f i g 1 8 ) . DBP a l s o m i g r a t e d as a s i n g l e band when s u b j e c t e d t o n o n - d e n a t u r i n g , n o n - r e d u c i n g 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 . F u r t h e r m o r e , on n o n -r e d u c i n g S D S - p o l y a c r y l a m i d e e l e c t r o p h o r e t i c g e l s monomeric DBP c o n s t i t u t e d the major s p e c i e s p r e s e n t (>95%), w i t h o n l y a v e r y f a i n t band due t o DBP d i m e r s ( B o u i l l o n et a l . , 1 9 7 7 ) . Fig 18: SDS polyacrylamide electrophoretic ge l . Lanes from left to r igh t : 1, Plasma; 2, A f f i - g e l blue eluate; 3, P 100 eluate; 4, Hydroxylapatite eluate; and 5, HPLC eluate. 34 3 . 1 . 2 O p t i c a l S p e c t r a The absorbance spec t rum of DBP r e v e a l e d a t y p i c a l t r y p t o p h a n - f r e e p r o f i l e ( f i g 1 9 a ) . The peak at 277 nm and s h o u l d e r at 284 nm are f e a t u r e s of t y r o s i n e absorbance and the u n d u l a t i o n s between 255 and 275 nm are i n d i c a t i v e of the u n d e r l y i n g p h e n y l a l a n i n e s p e c t r u m . I f t r y p t o p h a n had been p r e s e n t a s h o u l d e r at 290 nm would have been e x p e c t e d and the a b s o r b a n c e maximum would have been n e a r e r 280 nm, as f o r a l b u m i n ( f i g 1 9 b ) . 256 266 276 266 296 388 318 328 Wavelength/mo Fig 19a: Absorbance spectrum of DBP (0.7 mg/ml). 27e"8 Ml 288'. 8 388' .8 H a v e l e n g t h / n m Pig 19b: Absorbance spectrum of albumin (0.7 mg/ml). 35 An e x t i n c t i o n c o e f f i c i e n t of 2 2 , 5 6 0 M cm was c a l c u l a t e d f o r DBP f r o m the r e p o r t e d sequence of r a t DBP ( C o o k e , 1 9 8 6 ) . The second d e r i v a t i v e a b s o r b a n c e s p e c t r u m shows more c l e a r l y the l a c k of t r y p t o p h a n . P r o t e i n s c o n t a i n i n g t r y p t o p h a n have a n e g a t i v e peak a t 290 nm, h e r e a p o s i t i v e peak a t 290 nm i s seen ( f i g 2 0 ) . T h i s peak i s c h a r a c t e r i s t i c of t y r o s i n e . F u r t h e r m o r e the s p e c t r u m r e s e m b l e s the s u p e r p o s i t i o n of t y r o s i n e and p h e n y l a l a n i n e second d e r i v a t i v e s p e c t r a (page 1 6 ) . Fig 20: Second derivative absorbance spectrum of DBP ( 0.7 mg/ml). 36 The f l u o r e s c e n c e e m i s s i o n s p e c t r u m , e x c i t a t i o n at 278 nm, i s a g a i n t y p i c a l of a p r o t e i n c o n t a i n i n g t y r o s i n e and no t r y p t o p h a n ( f i g 2 1 ) . The maximum at 307 nm i s t h a t c h a r a c t e r i s t i c of t y r o s i n e , and the f l u o r e s c e n c e i n t e n s i t y i s low a round 3 3 0 - 3 5 0 nm where t r y p t o p h a n f l u o r e s c e n c e p e a k s . 0) o c o 0) o Z3 i—f U_ > -p CO •—i 0) HftX=58.8 f / V HIM=8.8 388 318 328 338 348 358 Wavelength/nm Fig 21: Fluorescence emission spectrum, excitation 278 nm, of DBP (0.7 mg/ml) at 20 *C. These t h r e e o p t i c a l s p e c t r a a l l show t h a t the i s o l a t e d p r o t e i n does not c o n t a i n t r y p t o p h a n but does have t y r o s i n e and p h e n y l a l a n i n e . T h i s i s c o n s i s t e n t w i t h the r e p o r t e d amino a c i d sequence f o r r a t p lasma DBP ( C o o k e , 1 9 8 6 ) . 37 3 . 1 . 3 A c t i n a s s a y s E v i d e n c e of D B P ' s a c t i n - b i n d i n g p r o p e r t i e s was g a i n e d f rom the a s s a y used to i s o l a t e the p r o t e i n (page 2 5 ) . A c r y l o d a n - a c t i n moves as a s i n g l e band on n o n - d e n a t u r i n g e l e c t r o p h o r e t i c p o l y a c r y l a m i d e g e l s , but i n the p r e s e n c e of DBP, a s e c o n d , s l o w e r mov ing band i s seen due t o the f o r m a t i o n of D B P - a c r y1 o d a n - a c t i n complex ( f i g 2 2 ) . F i g 22: The a c r y l o d a n - a c t i n assay of A f f i - g e l Blue f r a c t i o n s . Lanes from l e f t to r i g h t see band appearing i n centre of g e l due to DBP-acrylodan-actin complex. Lower band = G - a c t i n , upper band = F - a c t i n that d i d not enter the g e l . 38 F u r t h e r e v i d e n c e f o r t h e a c t i n - b i n d i n g a c t i v i t y o f DBP a r i s e s f r o m p y r e n e - a c t i n f l u o r e s c e n c e s t u d i e s . The f l u o r e s c e n c e i n t e n s i t y a t 386 nm on e x c i t a t i o n a t 344 nm g r e a t l y i n c r e a s e s on p o l y m e r i z a t i o n o f p y r e n e - G - a c t i n t o p y r e n e - F - a c t i n ( f i g 2 3 a ) . I n t h e p r e s e n c e o f DBP t h i s i n c r e a s e does n o t o c c u r ( f i g 2 3 b ) . DBP s e q u e s t e r s a c t i n monomers p r e v e n t i n g p o l y m e r i z a t i o n . The a d d i t i o n o f DBP t o p y r e n e - F -a c t i n r e s u l t s i n a s l o w f a l l i n f l u o r e s c e n c e ( f i g 2 3 c ) . T h i s i s due t o DBP t y i n g up t h e f r e e p y r e n e - G - a c t i n , t h u s s h i f t i n g t h e e q u i l i b r i u m away f r o m t h e p o l y m e r i z e d s t a t e , r e s u l t i n g i n a s l o w d e p o 1 y m e r i z a t i o n , r e t u r n i n g t o a G - p y r e n e - a c t i n f l u o r e s c e n c e l e v e l a f t e r 10 h o u r s . T h i s e f f e c t i s c h a r a c t e r i s t i c o f an a c t i n m o n o m e r - b i n d i n g p r o t e i n . (1nX=15.e / MH=J.6 8 286 488~ 688 886 1888 1286 1486 1688 1866 2886 T ime/s Figure 23: Time related pyrene-actin fluorescence studies, excitation at 344 nm and emission at 386 nm at 20 °C. a) G-actin (0.2 mg), pyrene-G-actin (0.02 mg) in buffer A (1 ml total). 2 pi of «gCl 2 (1 H) added after 80 s. 39 00 ftAX=15.8 niN=8.e 288 488 688 688 1868 1288 I486 it.ee 1888 2868 Time/s b) G-actin (0.2 mg), pyrene-G-actin (0.02 mg), DBP (0.27 mg) in buffer A (1 ml total). (1:1 ratio of actin to DBP). 2 pi of MgCl2 (1 M) added after 340 s. oo n C&4 t1nX=15.8 MIH=e.6 8 288 486 686 888 1888 1288 1486 1688 1888 2686 Time/s c) G-actin (0.15 mg), Pyrene-G-actin ( 0.015 mg) incubated , 0.5 h, with 2 mM MgCl2. At t = 0 DBP (0.2 mg) added, final volume 1 ml. 40 3.1.4 Calculation of M o l e c u l a r Mass DBP and mo l e c u l a r mass s t a n d a r d s were run on a d e n a t u r i n g SDS e l e c t r o p h o r e t i c g e l ( f i g 2 4 ) . From the r e l a t i v e d i s t a n c e moved by DBP i n comparison t o the s t a n d a r d s , a mol e c u l a r mass of 53,000 ± 3,000 was c a l c u l a t e d ( f i g 2 5 ) . Fig 24: SDS polyacrylamide electrophoretic gel. Lanes 1 and 3 top to bottom molecular mass standards: 95,000; 55,000; 43,000; 36,000; 29,000; 18,400; and 12,400. Lane 2, DBP. 41 Fig 25: Graph showing the log of molecular mass against distance moved on electrophoretic gel, from the results of 2 runs. 1 = 95,000, 2 = 55,000, 3 = 43,000, 4 = 36,000, 5 = 29,000, 6 = 18,400, and 7 = 12,400 (Pharmacia). 42 Fig 26,: CD spectrum of DBP in 20 m« MOPS, 1 mM EGTA, pH 7.3 at 25 *C. A) DBP, B) buffer. 43 3 . 1 . 5 C a l c u l a t i o n o f S e c o n d a r y S t r u c t u r e The CD s p e c t r u m of DBP ( f i g 26) i s c h a r a c t e r i s t i c of a p r o t e i n h i g h i n a - h e l i x and B - s h e e t s t r u c t u r e . V a l u e s f o r a - h e l i x ( 3 9 % ) , B - s h e e t (42%) and random c o i l (19%) were c a l c u l a t e d u s i n g XJJ , Xg and XR f a c t o r s at 225 and 210 nm (Chen et a l . , 1 9 7 2 ) . 3 . 2 . 1 Thermal D e n a t u r a t i o n of DBP f o l l o w e d bv CD The t h e r m a l d e n a t u r a t i o n of DBP i n h i g h s a l t b u f f e r H (150 mM K C l , 20 mM MOPS, 1 mM EGTA, pH 7 . 3 ) was f o l l o w e d by CD a t 220 nm ( f i g 2 7 , open s q u a r e s ) . DBP was seen to be v e r y s t a b l e up t o 55 ' C , a f t e r w h i c h the p r o t e i n s t a r t e d t o u n f o l d at a g r e a t e r r a t e . However , even at 70 'C a g r e a t d e a l of i t s s t r u c t u r e was r e t a i n e d . On c o o l i n g t o 15 'C the s i g n a l r e t u r n e d t o 97% of i t s o r i g i n a l v a l u e , s u g g e s t i n g t h a t the m o l e c u l e was a b l e t o r e f o l d . T h i s r e c o o l e d DBP was shown to r e t a i n a c t i n - b i n d i n g a c t i v i t y u s i n g the a c r y l o d a n - a c t i n a s s a y . A second s a m p l e , w h i c h had been i n c u b a t e d at room t e m p e r a t u r e w i t h 5 mM DTT (2 h) and then d i a l y s e d at 4 'C a g a i n s t b u f f e r H + 2 mM DTT (12 h ) , a l s o was s u b j e c t e d t o t h e r m a l d e n a t u r a t i o n ( f i g 2 7 , c l o s e d t r i a n g l e s ) . DTT has the e f f e c t of b r e a k i n g d i s u l p h i d e b o n d s . H e r e , the i n i t i a l CD s p e c t r u m was i n d i s t i n g u i s h a b l e f rom t h a t of the DBP sample w i t h o u t DTT, but on h e a t i n g , the p r o t e i n was seen t o be much l e s s s t a b l e , l o o s i n g more CD s i g n a l by 40 °C t h a n had the DBP sample w i t h o u t DTT by 70 °C. 44 Temp/°C Pig 27: Melting curve of DBP, at high ionic strength, followed by CD at 220 Open boxes, DBP; closed tr iangles, DBP + DTT. Relative e l l i p t i c i t y = 6/(6 at 16 *C). nm. A5 The r e c o o l e d sample d i d not b i n d a c t i n and o n l y r e t u r n e d t o 63% of the o r i g i n a l CD s i g n a l , s u g g e s t i n g t h a t i t d i d not r e f o l d c o r r e c t l y . The d i s u l p h i d e bonds i n DBP a p p e a r , on t h i s e v i d e n c e , t o be r e s p o n s i b l e f o r the u n u s u a l s t a b i l i t y of t h i s p r o t e i n , as w e l l as p l a y i n g a r o l e i n the c o r r e c t r e f o l d i n g of p a r t i a l l y d e n a t u r e d DBP. F u r t h e r m o r e , a t body t e m p e r a t u r e (37 °C) the DBP sample w h i c h had been i n c u b a t e d w i t h DTT was s i g n i f i c a n t l y u n f o l d e d , r a i s i n g the p o s s i b i l i t y t h a t DBP r e q u i r e s the d i s u l p h i d e bonds t o m a i n t a i n i t s shape and p o s s i b l y , t o c o m p l e t e i t s f u n c t i o n s . A s i m i l a r e x p e r i m e n t was c a r r i e d out i n a low s a l t b u f f e r , b u f f e r L (20 mM MOPS, 1 mM EGTA, pH 7 . 3 ) ± DTT ( f i g 2 8 ) . The i n i t i a l CD s p e c t r a were s i m i l a r to t h o s e i n b u f f e r H, and s i m i l a r p a t t e r n s of t h e r m a l d e n a t u r a t i o n were o b s e r v e d . The DBP sample w i t h o u t DTT was a g a i n r e l a t i v e l y s t a b l e up t o 55 °C beyond w h i c h CD s i g n a l was l o s t at a g r e a t e r r a t e . On c o o l i n g i t r e t a i n e d i t s a c t i n - b i n d i n g p r o p e r t i e s and r e t u r n e d to 92% of the o r i g i n a l CD s i g n a l . The sample i n c u b a t e d w i t h DTT was found to be much l e s s s t a b l e , as i n the h i g h s a l t c a s e , w i t h the CD s i g n a l f a l l i n g r a p i d l y beyond 30 °C and r e a c h i n g a v a l u e of ~40% a t 70 *C, c l o s e t o t h a t of the h i g h s a l t + DTT s a m p l e . The low s a l t + DTT DBP sample d i d not b i n d a c t i n on c o o l i n g and i t s e l l i p t i c i t y a t 220 nm r e t u r n e d to 65% of i t s o r i g i n a l v a l u e . These two CD t h e r m a l d e n a t u r a t i o n e x p e r i m e n t s s u g g e s t t h a t the d i s u l p h i d e bonds p l a y a major r o l e i n s t a b i l i z i n g DBP and t h a t i o n i c s t r e n g t h does not g r e a t l y a f f e c t s t a b i l i t y . 46 A • n *A u • • • • n D°nn • A • • A • • • • • AA • • • • * A • A 10 20 30 40 50 60 70 Temp/°C Fig 28: Melting curve of DBP, at low ionic strength, followed by CD at 220 Open boxes, DBP; closed triangles, DBP + DTT. Relative e l l i p t i c i t y = 8/(6 at 16 °C). 47 3.2.2 Thermal D e n a t u r a t i o n of DBP F o l l o w e d bv F l u o r e s c e n c e A f u r t h e r e x p e r i m e n t r e v e a l i n g the s t a b i l i z i n g e f f e c t of the d i s u l p h i d e bonds a g a i n s t t h e r m a l d e n a t u r a t i o n was c a r r i e d out by m o n i t o r i n g the e f f e c t of t e m p e r a t u r e on t y r o s i n e f l u o r e s c e n c e i n t e n s i t y ( f i g 2 9 ) . As p r o t e i n s u n f o l d , t y r o s i n e s g e n e r a l l y become more exposed t o the s o l v e n t , l o w e r i n g t h e i r f l u o r e s c e n c e i n t e n s i t y . T h i s e f f e c t i s s u p e r i m p o s e d o v e r the i n h e r e n t t e m p e r a t u r e dependence of f l u o r e s c e n c e . The DBP sample w h i c h had been i n c u b a t e d w i t h DTT l o s t f l u o r e s c e n c e i n t e n s i t y a t 307 nm, e x c i t a t i o n at 278 nm, more r a p i d l y w i t h t e m p e r a t u r e t h a n the n o n - r e d u c e d DBP s a m p l e . The r e d u c e d DBP d i d not have the s t r u c t u r a l s t a b i l i z a t i o n p r o v i d e d by the d i s u l p h i d e b o n d s , and u n f o l d e d at a l o w e r t e m p e r a t u r e . On r e c o o l i n g the DBP sample w i t h o u t DTT r e t u r n e d t o 95% of i t s o r i g i n a l f l u o r e s c e n c e compared t o 62% f o r the sample w i t h DTT, c o n s i s t e n t w i t h the CD d a t a , and i n d i c a t i n g t h a t the DBP w i t h o u t d i s u l p h i d e bonds was u n a b l e t o r e f o l d . 3 . 2 . 3 G u a n i d i n e . H C 1 Induced D e n a t u r a t i o n F o l l o w e d bv CD The d e n a t u r a t i o n of DBP by g u a n i d i n e h y d r o c h l o r i d e i n b u f f e r H ± DTT was f o l l o w e d by CD a t 220 nm ( f i g 3 0 ) . The e x p e r i m e n t d i d not r e v e a l a s i g n i f i c a n t d i f f e r e n c e between the u n f o l d i n g p a t t e r n s of t h e DBP samples w i t h o r w i t h o u t DTT. The CD s i g n a l f e l l s h a r p l y between 1 and 2 . 5 M g u a n i d i n e h y d r o c h l o r i d e r e a c h i n g a v a l u e of about 10% a t 4 M G u H C l . 48 1.2 1.0" o •rl m o a H • • • 0.8 O o a • o « u o 9 0.6-0.4-0.2 10 —i— 20 30 40 —i— 50 60 70 Temp/°C Fig 29: Melting curve of DBP followed by fluorescence emission at 307 nm, excitation at 278 nm. Open boxes, DBP; closed triangles, DBP + DTT Relative fluorescence = P/( Y at 14 *C). 49 1.2 1.0 i I 0.8-o CM CM « a H 2 0.6-0.4" 0.2-0.0 • A • • • • m . 2 T-4 Cone. GuHCl/M Pig 30: Guanidine Denaturation of DBP followed by CD. Open boxes, DBP; closed tr iangles, DBP + DTT. Relative e l l i p t i c i t y = 6/(6 at 14 *C). 50 In c o m p a r i s o n t o the t h e r m a l m e l t i n g c u r v e s of DBP i n the p r e s e n c e of DTT ± s a l t ( f i g s 27 + 2 8 ) , the g u a n i d i n e d e n a t u r a t i o n p r o c e e d s t o a f a r t h e r p o i n t t h a n t h e r m a l d e n a t u r a t i o n . Indeed a t 70 'C the t h e r m a l l y d e n a t u r e d DBP s a m p l e s w i t h DTT appear t o be r e a c h i n g a p l a t e a u a t a round 38% of o r i g i n a l s i g n a l . T h i s may be due t o the p r o t e i n r e a c h i n g a r e l a t i v e l y s t a b l e , p a r t i a l l y - u n f o l d e d s t a t e ( m o l t e n g l o b u l a r s t a t e ) ( K u w a j i m a , 1 9 8 9 ) . 3 . 3 . 1 A c r v l o d a n L a b e l i n g of DBP U s i n g the B i o - R a d P r o t e i n A s s a y ( B i o - R a d , R i c h m o n d , CA) and an e x t i n c t i o n c o e f f i c i e n t f o r a c r y l o d a n of 1 2 9 , 0 0 0 M - 1 c m - 1 a t 365 nm ( L e h r e r and I s h i i , 1 9 8 8 ) , the i n c o r p o r a t i o n of the f l u o r e s c e n t l a b e l was shown to be 5 . 0 ± 0 . 2 a c r y l o d a n s per DBP. The f l u o r e s c e n c e e m i s s i o n and e x c i t a t i o n s p e c t r a a r e shown i n f i g u r e 3 1 . T h i s degree of l a b e l i n g was somewhat s u r p r i s i n g as Kawakami and Goodman, 1 9 8 1 , found o n l y one r e l a t i v e l y i n a c c e s s i b l e m o n o t h i o l group per m o l e c u l e of human DBP. F u r t h e r m o r e , DBP i s known to have a h i g h l y h y d r o p h o b i c p o c k e t , used i n the b i n d i n g of v i t a m i n D. T h i s l e d t o the s u s p i c i o n t h a t some of the a c r y l o d a n was not c o v a l e n t l y b o u n d , but h e l d by h y d r o p h o b i c i n t e r a c t i o n s . 51 1 MAX=180.8 J- A} in OH tiiH=e.6 358 468 458 588 W a v e l e n g t h / n m o e <U o CO QJ o 3 QJ HAX=125.8 / J1IH=8.8 488 428 448 468 488 588 528 548 568 588 "• 688 W a v e l e n g t h / n m Fig 31: Fluorescence spectra of acrylodan-DBP (5:1 labeled) at 20 "C. A) Excitation spectrum, emission at 510 nm. B) Emission spectrum, excitation at 365 nm. On a d d i t i o n o f DTT (5 mM) t o t h e a c r y l o d a n - D B P a l a r g e r e d s h i f t i n t h e e m i s s i o n maximum i s i m m e d i a t e l y s e e n ( f i g 3 2 ) . The i n c r e a s e i n i n t e n s i t y i s most l i k e l y due t o t h e h y d r o p h o b i c a l l y bound a c r y l o d a n r e a c t i n g w i t h DTT, as a c r y l o d a n has low f l u o r e s c e n c e i n t e n s i t y ( P e n d e r g a s t . e t a l . , 1 9 8 3 ) . The r e d s h i f t c o u l d be due t o t h e r e l e a s e o f t h i s 52 a c r y l o d a n - D T T i n t o the s o l v e n t . T h i s l a s t p o i n t i s s u p p o r t e d by the o b s e r v a t i o n t h a t , on d i a l y s i s a g a i n s t 20 mM MOPS, 2 mM DTT, pH 7 . 3 (3 changes i n 36 h ) , t h i s m i x t u r e had a much d i m i n i s h e d a c r y o d a n e m i s s i o n s p e c t r a , and the new e x t e n t of l a b e l i n g of a c r y l o d a n - D B P was the c a l c u l a t e d to be 0 . 3 5 ± 0 . 2 9 a c r y l o d a n s per DBP. F u r t h e r m o r e , when t h i s DTT~t rea ted a c r y l o d a n - D B P was run on an SDS p o l y a c r y l a m i . d e e l e c t r o p h o r e t i c g e l , a l l the a c r y l o d a n f l u o r e s c e n c e was seen t o m i g r a t e w i t h DBP and none was d e t e c t e d at the dye f r o n t . T h i s s u g g e s t s t h a t the r e m a i n i n g a c r y l o d a n was c o v a l e n t l y bound . I t was t h i s DTT-t r e a t e d a c r y l o d a n - D B P t h a t was used i n subsequent e x p e r i m e n t s , a f t e r i t had been d i a l y s e d e x t e n s i v e l y a g a i n s t a non-DTT c o n t a i n i n g b u f f e r (20 mM MOPS, pH 7 . 3 , 3 changes i n 36 h ; . t1ftX=8e.8 h*IN=8.8 468 438 588 528 548 568 588 688 W a v e l e n g t h / n m Fig 32". The effect of DTT on the emission spectrum of acrylodan-DBP (5:1 labeled), excitat ion at 365 nm at 20 °C. A) acrylodan-DBP alone. B) acrylodan-DBP + 5 mM DTT. 5 3 The new e m i s s i o n s p e c t r u m had the same shape as the DBP w i t h h y d r o p h o b i c a l l y bound a c r y l o d a n a l t h o u g h the e m i s s i o n maximum 488 nm, e x c i t a t i o n a t 365 nm, had moved f rom 495nra. T h i s i s b l u e s h i f t e d r e l a t i v e t o an a c r y l o d a n - m e r c a p t o e t h a n o l a d d u c t (540 nm), and i s s l i g h t l y b l u e - s h i f t e d r e l a t i v e t o o t h e r p r o t e i n s : p a p a i n , 491 nm; p a r v a l b u m i n , 498 nm; and c a r b o n i c a n h y d r a s e 501 nm ( P e n d e r g a s t et a l . , 1 9 8 3 ) , s u g g e s t i n g t h a t the a c r y l o d a n i s i n a v e r y h y d r o p h o b i c env i r o n m e n t . 3 . 3 . 2 I n t e r a c t i o n o f A c r v o d a n - D B P w i t h A c t i n On a d d i t i o n of a c t i n to a c r y l o d a n - D B P i n a 1 : 1 r a t i o , t h e r e i s a r e d - s h i f t i n the f l u o r e s c e n c e e m i s s i o n maximum f rom 488 nm to 494 nm ( f i g 3 3 ) , combined w i t h a r e d u c t i o n i n 446 4bS 486 506 520 546 566 586 .668 Have length/nm Fig 33: The effect of actin binding on the emission spectrum of aerylodan -DBP (0.35:1 labeled), exci tat ion at 365 nm at 20 ' C . A) acrylodar.-PBP alone B) acrylodan-DBP + actin (1:1) 54 i n t e n s i t y , i n d i c a t i n g t h a t the a c r y l o d a n has moved t o a l e s s h y d r o p h o b i c e n v i r o n m e n t . These e f f e c t s can be e x p l a i n e d by a c t i n b i n d i n g a t a s i t e d i f f e r e n t than t h a t l a b e l e d w i t h a c r y l o d a n and p r o d u c i n g a c o n f o r m a t i o n a l change i n DBP w h i c h e x p o s e s the a c r y l o d a n probe t o the s o l v e n t . 3.3.3 E f f e c t of DTT on Fluorescence of Acrylodan c o v a l e n t l v Bound to DBP Two a c r y l o d a n - D B P samples e q u i l i b r a t e d a g a i n s t d i f f e r e n t b u f f e r s , one i n 20 mM T r i s — H C 1 , pH 8 . 0 and the o t h e r i n i t i a l l y i n c u b a t e d f o r 2 h i n the same b u f f e r but c o n t a i n i n g 5 mM DTT, and s u b s e q u e n t l y d i a l y s e d a g a i n s t 2 mM DTT, 20 mM T r i s - H C l , pH 8 . 0 (3 changes o v e r 36 h ) . The a b s o r b a n c e s a t 280 nm of the two s a m p l e s were w i t h i n 2% of each o t h e r . The e m i s s i o n maximum f o r t h e a c r y l o d a n - D B P sample t h a t had been i n c u b a t e d w i t h DTT was s l i g h t l y b l u e - s h i f t e d ( f i g 34) to 483 nm and i t s i n t e n s i t y enhanced 30%, 588 Ntl 688 Fig 34: The effect of DTT on the emission spectrum of c acrylodan-DBP (0.35:1 labeled), excitation at 365 nm, 20 'C. A) acrylodan-DBP alone B) acrylodan-DBP + DTT 55 i m p l y i n g t h a t the a c r y l o d a n had moved i n t o a more h y d r o p h o b i c e n v i r o n m e n t . A p o s s i b l e e x p l a n a t i o n f o r t h i s i s t h a t the d i s u l p h i d e bonds p r e s e n t i n the p r o t e i n i n t r o d u c e a c e r t a i n amount of s t r a i n i n t o the s t r u c t u r e , and t h i s s t r a i n i s r e l e a s e d when the d i s u l p h i d e bonds a r e c l e a v e d . The e n s u i n g c o n f o r m a t i o n a l change a f f e c t s the a c r y l o d a n p r o b e . P a r t 4 : C o n c l u s i o n s Horse p lasma DBP c l o s e l y r e s e m b l e s o t h e r p lasma D B P ' s . I t i s a t r y p t o p h a n - f r e e p r o t e i n of m o l e c u l a r mass 5 3 , 0 0 0 d a l t o n s t h a t i s a b l e to b i n d a c t i n and b l o c k i t s p o l y m e r i z a t i o n . P u b l i s h e d p u r i f i c a t i o n schemes f o r o t h e r p lasma DBP s p e c i e s y i e l d e d o n l y p a r t l y pure h o r s e p lasma DBP. A f i n a l a n i o n exchange HPLC s t e p was r e q u i r e d to a c h i e v e p u r i t y . CD and f l u o r e s c e n c e i n v e s t i g a t i o n s have r e v e a l e d the i m p o r t a n c e of the d i s u l p h i d e bonds t o the t h e r m a l s t a b i l i t y of DBP. W i t h o u t t h e s e d i s u l p h i d e b o n d s , the p r o t e i n would be s i g n i f i c a n t l y u n f o l d e d (30%) at body t e m p e r a t u r e . F u r t h e r m o r e , the d i s u l p h i d e bonds g u i d e the r e f o l d i n g of h e a t e d DBP to a f u n c t i o n a l s t a t e on c o o l i n g . DBP can be c o v a l e n t l y l a b e l e d w i t h a c r y l o d a n to p roduce a h i g h l y f l u o r e s c e n t p r o d u c t t h a t i s s e n s i t i v e to the b i n d i n g of a c t i n and to the s t a t e of o x i d a t i o n of the p r o t e i n s u l f h y d r y l g r o u p s . T h i s i n i t i a l s t u d y of a c r y l o d a n - D B P s h o u l d be e x t e n d e d to i d e n t i f y the s i t e of l a b e l i n g u s i n g p e p t i d e mapping t e c h n i q u e s and by a p p l i c a t i o n of o t h e r f l u o r e s c e n c e methods 56 ( p o l a r i z a t i o n , q u e n c h i n g , energy t r a n s f e r ) to b e t t e r c h a r a c t e r i z e the s i t e of l a b e l i n g and how i t i s a f f e c t e d by the b i n d i n g of a c t i n . The f a c t t h a t a c r y l o d a n a p p e a r s t o b i n d , i n p a r t , n o n -c o v a l e n t l y t o DBP s u g g e s t s a s t u d y u s i n g a h y d r o p h o b i c probe ( s u c h as 2 - ( N ~ m e t h y 1 a n i 1 i n o ) n a p h t h a 1 e n e - 6 - s u l p h o n i c a c i d , S e l i s k a r and B r a n d , 1 9 7 9 ) . F l u o r e s c e n c e p r o p e r t i e s of the bound probe wou ld p r o v i d e f u r t h e r s t r u c t u r a l i n f o r m a t i o n about the h y d r o p h o b i c p o c k e t on DBP t h a t i s e x p e c t e d f rom i t s sequence a n a l o g y w i t h serum a l b u m i n and t h a t p r o b a b l y i s a s s o c i a t e d w i t h b i n d i n g v i t a m i n D. 57 R e f e r e n c e s 1 B e r g g a r d , I . , C l e v e , H . , and B e a m , A . G . ( 1964) C l i n . C h i m . A c t a 1 0 , 1 - 1 1 . 2 B o r k e , J . L . , L i t w i l l e r , R . D . , B e l l , M . P . , P a s s , D . N . , McKean, D . J . , and Kumar, R. (1989) I n t . J . B i o c h e m . 2 0 , 1 3 4 3 - 1 3 4 9 . 3 B o u i l l o n , R . , van B a e l e n , H . , Rombauts , W . , and de Moor , P . (1977) J . B i o l Chem. 2 5 3 , 4 4 2 6 - 4 4 3 1 . 4 B o u i l l o n , R . , van B a e l e n , H . , and de Moor , P . (1986) B i n d i n g P r o t e i n s of S t e r i o d Hormones. C o l l o q u e NSERM/John L i b b e y E u r o t e x t L t d . 1 4 9 , 3 3 3 - 3 5 6 . 5 C h a p u i s - C e 1 1 i e r , C . , G i a n a z z a , E . , and A r n a u d , P . (1982) B i o c h i m . B i o p h y s . A c t a 7 0 9 , 3 5 3 - 3 5 7 . 6 Chen , Y . H . , Y a n g , J . T . , and M a r t i n e z , H .M. (1972) B i o c h e m . 1 1 , 4 1 2 0 - 4 1 3 1 . 7 C o o k e , N . E . (1986) J . B i o l . Chem. 2 6 1 , 3 4 4 1 - 3 4 5 0 . 8 C o o k e , N . E . , and Haddad, J . G . (1989) E n d o c r i n e R e v . 1 0 , 2 9 4 - 3 0 7 . 9 C o o p e r , J . A . , W a l k e r , S . B . , and P o l l a r d , T . D . (1983) J . M u s c l e R e s . C e l l M o t i l i t y 4 , 2 5 3 - 2 6 2 . 10 C o t e , G . P . , and S m i l l i e , L . B . (1981) J . B i o l . Chem. 2 5 6 , 7 2 5 7 - 7 2 6 1 . 11 Coue , M . , C o n s t a n s , J . , and O l m u c k i , A . (1986) E u r . J . B i o c h e m . 1 6 0 , 2 7 3 - 2 7 7 . 12 C r e i g h t o n , T . E . (1989) P r o t e i n S t r u c t u r e a P r a c t i c a l A p p r o a c h . O x f o r d U n i . P r e s s 2 5 1 - 2 8 4 . 13 D a i g e r , S . P . , S c h a n f i e l d , M . S . , and C a v a l l i - S f o r z a , L . L . (1975) P r o c . N a t l . A c a d . S c i . USA. 7 2 , 2 0 7 6 - 2 0 8 0 . 14 D u e l a n d , S . , B l o m h o f f , R . , and P e n d e r s e n , J . I . 1990) B i o c h e m . J . 7 2 1 - 7 2 5 . 15 E n a , J . M . , E s t e r b a n , C , P e r e z , M . D . , U r i e l , J . , and C a l v o , M. (1989) B i o c h e m . I n t . 1 9 , 1 - 7 . 16 F r e i f e l d e r , D. (1976) P h y s i c a l B i o c h e m i s t r y . W.H. Freeman and C o . 4 4 4 - 4 7 4 . 58 17 G i a n a z z a , E . , and A r n a u d , P. (1982) B i o c h e m . J . 2 0 3 , 6 3 7 - 6 4 1 . 18 G i l l , S . C . , and von H i p p e l , P . H . (1989) A n a l . B i o c h e m . 1 8 2 , 3 1 9 - 3 2 6 . 19 G o l d s c h m i d t - C l e r m o n t , P . J . , W i l l i a m s , M . H . , and G a l b r a i t h , R . M . (1987) B i o c h e m . B i o p h y s . R e s . Com. 146 , 6 1 1 - 6 1 7 . 20 Haddad , J . G . , H a r p e r , K . D . , G u o t h , M . , P i e t r a , G . G . , and S a n g e r , S .W. (1990) P r o c N a t l . A c a d . S c i . USA. 8 7 , 1 3 8 1 - 1 3 8 5 . 21 H i r s c h f e l d , J . (1959) A c t a . P a t h o l . M i c r o b i o l . S c a n d . 4 7 , 1 6 0 - 1 6 8 . 22 H i r s c h f e l d , J . (1962) P r o g . A l l e r g y . 6 , 1 5 5 - 1 8 6 . 23 Janmey, P . A . , and L i n d , S . E . (1987) B l o o d 7 0 , 5 2 4 - 5 3 0 . 24 Janmey, P . A . , S t o s s e l , T . P . , and L i n d , S . E . (1986) B i o c h e m . B i o p h y s . R e s . Com. 1 3 6 , 7 2 - 7 9 . 25 K u w a j i m a , K. (1989) P r o t i e n s I S t r u c t u r e , F u n c t i o n and G e n e t i c s 6 , 8 7 - 1 0 3 . 26 K a w a k a m i , M . , and Goodmann, D . S . (1981) B i o c h e m . 2 0 , 5 8 8 1 - 5 8 8 7 . 27 K o r n , E . D . (1982) P h y s i o l . R e v . 6 2 , 6 7 2 - 7 3 7 . 28 L a e m m l i , U . K . (1970) N a t u r e . 2 2 7 , 6 8 0 - 6 8 5 . 29 L e h r e r , S . S . , and I s h i i , Y . (1988) B i o c h e m . 2 7 , 5 8 9 9 - 5 9 0 6 . 30 L i n d , S . E . , S m i t h , B . B . , Janmey, P . A . , and S t o s s e l , T . P . (1986) J . C l i n . I n v e s t . 7 8 , 7 3 6 - 7 4 2 . 31 L i n k , R . P . , P e r l m a n , K . L . , P i e r c e , E . A . , S c h n o e s , H . K . , and D e L u c a , H . F . (1986) A n a l . B i o c h e m . 1 5 7 , 2 6 2 - 2 6 9 . 32 M a r r i o t t , G . , Z e c h e l , K . , and J o v i n , T . M . (1988) B i o c h e m . 2 7 , 6 2 1 4 - 6 2 2 0 . 33 N i e l s e n , J . C . , N e r s t r o m , B . , and F e l d b o , M. (1963) A c t a P a t h o l . M i c r o b i o l . S c a n d . 5 8 , 2 6 4 - 2 7 1 . 34 N o z a k i , Y . (1990) A r c h . B i o c h e m . B i o p h y s . 2 7 7 , 3 2 4 - 3 3 . 35 P e n d e r g a s t , F . G . , M e y e r , M . , C a r l s o n , G . L . , I i d a , S . , and P o t t e r , J . D . (1983) J . B i o l . Chem. 2 5 8 , 7 5 4 1 - 7 5 4 4 . 59 36 P o l l a r d , T . D . , and C r a i g , S .W. (1982) TIBS 7 , 5 5 - 5 8 . 37 P o t s c h - S c h n e i d e r , L . , and K l e i n , H. (1988) E l e c t r o p h o r e s i s 9 , 6 0 2 - 6 0 5 . 38 S a f e r , D. (1989) A n a l . B i o c h e m . 1 7 8 , 3 2 - 3 7 . 39 S e l i s k a n , C . J . , and B r a n d , L . (1971) J . Amer. Chem. S o c . 9 3 , 5 4 0 5 - 5 4 2 0 . 40 S p u d i c h , J . A . , and W a t t , S . (1971) J . B i o l . Chem. 2 4 6 , 4 8 6 6 - 4 8 7 1 . 41 S t r y e r , L . (1981) B i o c h e m i s t r y . W.H. Freeman and C o . , 2nd e d . 8 1 5 - 8 3 7 . 42 Thomas, W . C . , M o r g a n , H . G . , C o n n e r s , T . B . , Haddock , L . , B i l l s , C . E . , and Howard, J . E . (1959) J . C l i n . I n v e s t . 3 8 , 1 0 7 8 - 1 0 8 5 . 43 T u z i m u r a , K . , Konno , T . , Meguro , H . , H a t a n o , M . , M a r a k a m i , T . , K a s h i w a b a r a , K . , S a i t o , K . , Kondo , Y . , and S u z u k i , T . M . (1977) A n a l . B i o c h e m . 8 1 , 1 6 7 - 1 7 4 . 44 Van B a e l e n , H . , B o u i l l o n , R . , and de Moor , P. (1980) J . B i o l . Chem. 2 5 5 , 2 2 7 0 - 2 2 7 2 . 45 Van B a e l e n , H . , and B o u i l l o n , R. (1986) B i n d i n g P r o t e i n s of S t e r o i d Hormones. C o l l o q u e NSERM/John L i b b e y E u r o t e x t L t d . 1 4 9 , 6 3 - 8 3 . 46 Way, M . , and Weeds, A . (1990) N a t u r e 3 4 4 , 2 9 2 - 2 9 4 . 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0059797/manifest

Comment

Related Items