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

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HORSE PLASMA VITAMIN D-BINDING ISOLATION AND STRUCTURAL  PROTEIN:  INVESTIGATION  By ROBERT CHARLES .Sc.  King's College,  A THESIS SUBMITTED OF THE  ROBINSON  London U n i v e r s i t y ,  IN PARTIAL  FULFILLMENT  REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  IN  THE  FACULTY  OF GRADUATE  (Department  We a c c e p t  this  of  STUDIES  Chemistry)  t h e s i s as c o n f o r m i n g  to the r e q u i r e d  standard  THE UNIVERSITY OF B R I T I S H  COLUMBIA  OCTOBER 1990  © Robert Charles  1987  Robinson  In  presenting this  degree at the  thesis in  University of  partial  fulfilment  of  of  department  this or  thesis for by  his  or  scholarly purposes may be her  representatives.  permission.  Department of The University of British Columbia Vancouver, Canada  for  an advanced  Library shall make it  agree that permission for extensive  It  publication of this thesis for financial gain shall not  DE-6 (2/88)  requirements  British Columbia, I agree that the  freely available for reference and study. I further copying  the  is  granted  by the  understood  that  head of copying  my or  be allowed without my written  Abstract Vitamin D-binding protein, sterols study,  secreted and i s  by t h e  part  of  (DBP)  liver,  four  chromatography,  and a n i o n e x c h a n g e 80 ml o f The circular  filtration, HPLC.  plasma w i t h secondary  an a b u n d a n t  an a c t i n s c a v e n g i n g  step procedure:  gel  is  which t r a n s p o r t s  DBP was i s o l a t e d f r o m h o r s e  reproducible,  system.  plasma i n a Affi-gel  Blue  DBP were  chromatography  obtained  of  21-25%.  structure  of  DBP was c a l c u l a t e d  A m o l e c u l a r mass o f  bonds  stabilization  to  of  studies  DBP c o n t r i b u t e  the m o l e c u l e w i t h  from  from 42% p - s h e e t  53,000 ± 3,000  was c a l c u l a t e d f r o m e l e c t r o p h o r e t i c  d i c h r o i s m and f l u o r e s c e n c e  this  affinity  d i c h r o i s m m e a s u r e m e n t s t o be 39% a - h e l i x ,  disulphide  In  highly  hydroxy1apatite  6-7 mg o f  serum  vitamin D  a yield  and 19% random c o i l . daltons  protein  gels.  revealed  that  substantial respect  to  Circular the  structural thermal  denaturation. Finally, fluorescence  acrylodan-labeled of  this  DBP was p r e p a r e d .  a d d u c t was s e n s i t i v e  a c t i n and t o t h e p r e s e n c e  of  The  to the b i n d i n g  dithiothreitol.  of  iii  Table of Contents Page Abstract  ii  List  of Tables  vi  List  of  Figures  vii  Acknowledgements  x  Abbreviations  U Part  Part  ix  Introduction 1:  Proteins  1.1.1  Functions  of A c t i n  1.1.2  Actin  1.1.3  A c t i n Scavenging  1.1.4  Review of  1.1.5  Physical Properties  1.1.6  Review of  1  Polymerization/Depolymerization  2: Spectroscopic  System i n Plasma  DBP  6 8 9  of  DBP  DBP P u r i f i c a t i o n Methods  11 13  Techniques  1.2.1  P r o t e i n Absorbance  Spectroscopy  1.2.3  Protein  1.2.3  P r o t e i n CD S p e c t r o s c o p y  Fluorescence  Spectroscopy  15 18 20  i v  2; M a t e r i a l s Part  1:  and M e t h o d s  Proteins  2.1.1  Preparation  of G - a c t i n  2.1.2  P r e p a r a t i o n of  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  Acrylodan-actin  25  2.1.5  Collection  2.1.6  P u r i f i c a t i o n of  2.1.7  C a l c u l a t i o n of  of  Coefficient  Part  23  Pyrene-actin  Assay  Horse  Plasma  DBP an  for  Extinction  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  Gel E l e c t r o p h o r e s i s  30  2: Spectroscopic  Methods  2.2.1  Absorbance  Spectra  2.2.2  Fluorescence  R e s u l t s and  Part  26 26  31  Spectra  31  2 . 2 . 3 CD S p e c t r a  3:  24  31  Discussion  1 : Y i e l d and P u r i t y  o f DBP  3.1.1  Yield  3.1.2  Optical  3.1.3  A c t i n Assays  37  3.1.4  C a l c u l a t i o n o f M o l e c u l a r Mass  40  3.1  C a l c u l a t i o n of  42  5  and P u r i t y  32  Spectra  34  Secondary  Structure  V  Part  Part  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  47  3.2.3  CD G u a n a d i n e . H C 1 I n d u c e d D e n a t u r a t i o n  3:  Denaturation  47  Acrylodan-DBP  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  Acrylodan-DBP/Actin Interaction  3.3.3  E f f e c t o f DTT on F l u o r e s c e n c e C o v a l e n t l y Bound t o DBP  of  53 Acrylodan  54  Part 4! Conclusions  55  Bibliography  57  vi  List  of  Tables  Table I  Page Physical  Properties  of  DBP  11  vii  List  of Figures Page  1  The  thin  f i l a m e n t of  2  The m a i n c o m p o n e n t s o f  3  The m e c h a n i s m o f  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 s t a i n e d w i t h a f l u o r e s c e n t antibody to a c t i n  4  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 s t a i n e d w i t h a f l u o r e s c e n t antibody to a c t i n  4  6  S c h e m a t i c d i a g r a m of  6  7  Schematic diagram showing the v a r i o u s a c t i v i t i e s 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  7  8  S c h e m a t i c d i a g r a m o f t h e c o m p l e x e s f o r m e d on t h e a d d i t i o n o f G - a c t i n o r F - a c t i n t o g e l s o l i n and DBP  8  9  Homology b e t w e e n t h e 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 i n r a t a l b u m i n and t h e p r e d i c t e d amino a c i d s e q u e n c e 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 in comparison to albumin  12  10  Absorbance  1  16  11  S e c o n d d e r i v a t i v e s p e c t r a of t h e a r o m a t i c amino a c i d s i n 6 M GuHCL, s o d i u m p h o s p h a t e , pH 6 . 8  16  12  Second d e r i v a t i v e s p e c t r u m of a l b u m i n i n GuHCl a t a r b i t r a r y  17  13  F l u o r e s c e n c e s p e c t r a of e x c i t a t i o n 278 nm  14  6-acryloyl-2-dimethylaminonaphthalene  19  15  N-(1-Pyrene)iodoacetamide  19  16  CD s p e c t r a o f p o l y - L - l y s i n e i n t h e 1, c r - h e l i c a l ; 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  21  17  Elution  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 t h e p u r i f i c a t i o n o f DBP  s k e l e t a l muscle  2  muscle c o n t r a c t i o n  s p e c t r a of  profile  s k e l e t a l muscle  3  F-actin  t h e a r o m a t i c amino a c i d s  human serum concentration  the a r o m a t i c a c i d s  f r o m HPLC c o l u m n gel  showing  19  33  vi i i 19a  Absorbance  s p e c t r u m of  DBP  34  19b  Absorbance  s p e c t r u m of  albumin  34  20  Second d e r i v a t i v e  21  Fluorescence emission spectrum, 278 nm, o f DBP  22  The  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 , 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  38  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 c a l c u l a t i o n o f m o l e c u l a r mass  40  25  G r a p h s h o w i n g t h e l o g of m o l e c u l a r mass a g a i n s t 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  41  26  CD s p e c t r u m o f  42  27  M e l t i n g c u r v e of by CD a t 220 nm  DBP, i n h i g h  28  M e l t i n g c u r v e of by CD a t 220 nm  DBP, i n  29  M e l t i n g c u r v e o f DBP f o l l o w e d by f l u o r e s c e n c e 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  48  30  Guanidine.HC1  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 ) . 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 a t 365 nm  51  32  The e f f e c t o f acrylodan-DBP  52  33  The e f f e c t o f a c t i n b i n d i n g on t h e e m i s s i o n 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  53  34  The e f f e c t o f acrylodan-DBP e x c i t a t i o n at  54  absorbance  acrylodan-actin  s p e c t r u m of  DBP  excitation  35 36  assay  37  gel  for  the  DBP salt,  low s a l t ,  denaturation  of  followed followed  DBP f o l l o w e d  44 46  by CD  DTT on t h e e m i s s i o n s p e c t r u m o f ( 5 : 1 l a b e l e d ) , e x c i t a t i o n a t 365 nm  DTT on t h e e m i s s i o n s p e c t r u m o f (0.35:1 labeled), 365 nm  ix  The  force  Drives  that  drives  my r e d b l o o d ;  T u r n s mine t o  the water that  dries  through  the  rocks  the mouthing streams  wax.  And I am dumb t o mouth u n t o my v e i n s How a t  t h e m o u n t a i n s p r i n g t h e same mouth s u c k s .  The f o r c e  that  through the green fuse d r i v e s  the  flower.  D y l a n Thomas  X  Acknowledgement  I  would  like  to  thank  P h a s t S y s t e m and D r .  Dr.  Leslie  Dana D e v i n e D.  Burtnick  for for  t h e use of everything  her else.  xi  Abbreviations Acrylodan  6-acryloyl-2-(dimethylamino)naphthalene  ATP  Adenosine  CD  Circular  CM-Sephadex  Carboxymethyl-Sephadex  DBP  Vitamin D-Binding  DEAE-Sephadex  Diethylaminoethyl-Sephadex  DMF  N,N-Dimethylformamide  DMSO  Dimethylsulphoxide  DTT  Dithiothreitol  EGTA  Ethylenebis(oxyethylenenitrilo)tetraacetic Acid  Gc  G r o u p S p e c i f i c Component o f  HPLC  High Pressure L i q u i d  MOPS  3-(N-morpho1ino)propanesulphonic  PMSF  Phenylmethy1 s u l p h o n y l  PIA  N-(1-pyrene)iodoacetamide  SDS  Sodium D o d e c y l  TEMED  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  Tris  T r i s ( h y d r o x y m e t h y l ) m e t h y l amine  Buffer A  2 mM T r i s - H C l , 0 . 2 mM C a C l , 1 . 0 mM DTT, pH 7 . 6  Buffer B  30 mM Na P h o s p h a t e B u f f e r , 0 . 1 % ( w / v ) S o d i u m A z i d e , 50 uM PMSF, pH 7 . 0  Buffer  25 mM T r i s - H C l , A T P , pH 8 . 4  E  Buffer H  5'-Triphosphate Dichroism  Protein  Plasma  Chromatography Acid  Fluoride  Sulphate  2  0 . 2 mM A T P ,  194 mM g l y c i n e ,  0 . 2 mM  150 mM K C l , 20 mM MOPS, 1 mM EGTA, pH 7 . 3  1  JLl  Introduction  Part  1.1.1  Functions Actin  cells,  of  Actin  i s one o f t h e most a b u n d a n t p r o t e i n s  often  c o m p r i s i n g up t o 20% o f t o t a l  a multiple functioned, major  role  motility  the  thin  interact  highly  eukaryotic  cell protein.  conserved p r o t e i n  with  (Stryer,  1981).  It  t r o p o m y o s i n and t h e t r o p o n i n  with  t h e m y o s i n heads o f t h e t h i c k  in  complex,  forms  filaments  filaments  causing  of the muscle ( f i g 2 ) .  Actin  ( I  Troponin complex  is  playing a  Actin,  f i l a m e n t of muscle ( f i g 1 ) . These t h i n  contraction  in  i n m u s c l e c o n t r a c t i o n and i n t h e s t r u c t u r e and  of non-muscle c e l l s  conjunction  1: Proteins  Tropomyosin  Fig 1: The thin filament of skeletal muscle. Stryer (1981).  2  H Z s' band line  A band  I band Myofibril  Z sarcomere Z  \ \ \  /  I  Z  IIIIII  IIIIII  I I I  \ \ .O-!.  A band  *  nCn IIIIII i IIIIII  IIIIII  IIIIII  nun nun  IIIIII i IIIIII  IIIIII  IIIIII  IIIIII  IIIIII  IIIIII i IIIIII  IIIIII  IIIIII  IIIIII  JIIIII  IIIIII i IIIIII  IIIIII  IIIIII  /  i  I  i  T h i c k filamenls (myosin)  7  Thin lilamcnls (actin)  if Z line  ^- Thin filament  L  ^ Thick filament  -a a a  Fig 2: The main components of s k e l e t a l muscle. Lenhinger (1975).  Z line  - Ji  ^ 1  3  The p r o c e s s  is driven  ATP t o ADP. M y o s i n f i r s t  energetically  binds  myosin-ATP b i n d i n g to a c t i n  t o ATP ( f i g  (fig  and t h i n  consequently  3A)  followed  (fig  f i l a m e n t s s l i d i n g over  i n the muscle c o n t r a c t i n g  3C)  resulting  each o t h e r (fig  Fig 3: The mechanism of muscle contraction. Stryer (1981).  3D).  and  of  by  3 B ) . ATP t h e n h y d r o l y s e s  ADP c a u s i n g t h e m y o s i n heads t o t i l t thick  by t h e h y d r o l y s i s  in  to the  4  The  control  ion r e g u l a t i o n . sarcoplasmic  of  contraction  Nerve  pulses  reticulum. This  which causes c o n f o r m a t i o n a l tropomyosin. expose  These  to  provided  release  changes  that  changes  myosin b i n d i n g  through  c a l c i u m from  calcium binds  conformational  the n e c e s s a r y  contraction  is  sites  calcium  the  to t r o p o n i n  C,  are  transmitted  in  tropomyosin  on a c t i n ,  to  allowing  proceed.  Fig 4: Immunofluorescence micrograph of a resting c e l l stained with a fluorescent antibody to a c t i n . Stryer (1981).  F l g  5 :  s c e n c e micrograph of a moving c e l l stained with a fluorescent antibody to a c t i n . The ruffled border is f a d i n g edge. Stryer (1981).  I m m u n o f l u o r e  t h e  5  Actin Some o f thin  i s a l s o a major c o n s t i t u e n t  this  of  the  microfilaments opposite  (fig  4).  i n one d i r e c t i o n the  functions  of  By a c t i v e l y  cells.  t o move ( f i g  actin outlined  rely  affair  and these  them i n  1.1.2  5). on i t s  ability  just  once.  in non-muscle c e l l s  However  (G-actin)  and t h e p o l y m e r  6)t  is  (F-actin)  as a d o u b l e - s t r a n d e d ,  a c t i n -units  can be v i e w e d  right-handed  i n each s t r a n d w i t h i n  and 2 . 1 6 s u b u n i t s  turn  polymerized  per  spontaneously  ( 5 0 - 1 5 0 mM) o r M g C l  directional,  favoured  (Pollard  but  occurring at  2  (Korn,  to F - a c t i n  helix  G-actin  two q u i t e d i f f e r e n t  ways  w i t h 13 pitch,  or  can be  i n the p r e s e n c e  ( 0 . 5 - 2 mM). T h i s  salt  i n two  w i t h a 5 . 9 nm p i t c h  1982).  a t b o t h ends o f  i n the absence of  globular  e a c h 72 nm h e l i x  left-handed helix  filament,  it  where  a 42,000 d a l t o n ,  as a s i n g l e - s t r a n d e d ,  NaCl  and  Polvmerization/DepolTmerization  Monomeric a c t i n  (fig  to  1982) .  Actin  protein  the  through  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 and C r a i g ,  the  the case of muscle c e l l s  the p o l y m e r i z a t i o n occurs  a more t r a n s i e n t  shape  shortening  and e l o n g a t i n g  and f o r m f i l a m e n t s . In  contraction,  to the  c e l l possesses the mechanism,  c o n f o r m a t i o n a l changes,  polymerize  is  cells  direction  The  non-muscle  a c t i n forms m i c r o f i l a m e n t s which resemble  f i l a m e n t s o f m u s c l e and c o n t r i b u t e  structure  actin  of  KCl  polymerization  a growing  rates.  of  is  actin  G-actin  is  and i n t h e p r e s e n c e  of  ATP.  or  Fig 6: Schematic diagram of F - a c t i n . Korn (1982).  7nm  The  control  concentration variety  of  proteins actin  is  of  a c t i n filament length via  not  specific  actin functions.  is  found  be c l a s s i f i e d by t h e i r  Hence a s e r i e s  1990).  for  of a c t i n  the binding  to G - a c t i n  provide  a buffering  capacity  G-actin  available for form F - a c t i n  of  These a c t i n b i n d i n g p r o t e i n s  function  bind only  proteins  enough t o a c c o u n t  i n c e l l s w h i c h can m o d i f y t h e a c t i v i t y  (Way and Weeds,  proteins  salt  (fig  and, for  7).  polymerization. aggregates,  actin structures.  Monomer b i n d i n g  i n the case of  actin,  can  ensuring  profilin, that  there  is  N e t w o r k and b u n d l i n g  i n c r e a s i n g the s t r e n g t h  diversity  of  Finally  the  filaments  c a n be r e g u l a t e d by s e v e r i n g  and  l e n g t h of  actin  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).  capping proteins polymerization, provide  w h i c h b r e a k up and s l o w down respectively,  a foundation  On t h e d e a t h o f  for  the  and by n u c l e a t i o n p r o t e i n s  rapid  polymerization.  c e l l s , whether  the r e s u l t  ageing,  G-actin  into  extracellular  fluids,  i n c l u d i n g blood plasma.  salt  conditions  favour  cause h i g h plasma v i s c o s i t y , (Haddad e t a l . , 1 9 9 0 ) . possesses  two F - a c t i n  and F - a c t i n  of  d i s e a s e or n a t u r a l  there  which  F-actin  released The  and c o n s e q u e n t l y  b l o c k i n g the  To a v o i d t h i s  are  injury,  high could  microcirculation  fate,  blood plasma  depolymerizing proteins,  DBP, w h i c h s c a v e n g e any a c t i n r e l e a s e d i n t o  the  gelsolin blood.  and  8  1.1.3  Actin  Scavenging  Gelsolin  i s an F - a c t i n  to b i n d to e i t h e r sites,  a  F-actin  one o f w h i c h i s  binding protein, K  System i n Plasma  = 1-2 x 1 0  depolymerize monomers,  8  Janmey and L i n d into  blood,  into  short  protein with  or G - a c t i n .  It  has two  calcium dependent.  M  _ 1  (Goldschmidt-Clermont indirectly  shifting  DBP b i n d s  a monomer  et a l . , 1 9 8 7 ) .  the G - a c t i n  f i l a m e n t s . Further  on t h e r e l e a s e o f  and g e l s o l i n s e v e r s  depolymerization  of  8). G-ACTIN  F-ACTIN  • •• • •• • •• • •• +  ODD D D D ODD D  G G G  D D D D D D D D D D  G G G  •  D* D* D* D» D* D» D» D* D» D»  D D D D D 0 D D D D  D» D» D» D* D* D* D» D» D» D  can  F-actin.  G G  •G •G •G  Fig 8: Schematic diagram of the complexes formed on the addition of G-actin or F-actin to g e l s o l i n (G) and DBP (D). Janmey and Lind (1987).  actin F-actin  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 to the m a j o r i t y b e i n g bound t o DBP ( f i g  It  actin  t h e e q u i l i b r i u m away f r o m that  ability  actin,  by s e q u e s t e r i n g  (1987) suggested  the  actin-binding  DBP i s  f o r m i n g a 1 : 1 complex w i t h  F-actin  thus  severing  of  actin  9  This that,  theory  is  supported  on a d d i t i o n o f  actin,  only  using  DBP and g e l s o l i n  a c t i n complexes  f r o m t h e p l a s m a by t h e alone,  and t h a t  gelsolin-actin showed t h a t  1.1.4  quicker  vitro  ( L i n d et  DBP and g e l s o l i n  are  cleared  studies  a l . , 1986)  are  t h a n DBP o r  Furthermore  sites, for  rendering  cleared  gelsolin faster  Janmey e t  al.  than  (1986)  it  inferring  that  the d i s p o s a l of inactive  to  DBP i s  the  the  main  extracellular  actin  as  polymerization.  R e v i e w o f PBP  which migrated  (1959) d i s c o v e r e d i n the  electrophoresis. System.  In  Daiger  et  fraction  He c a l l e d t h i s the  vitamin D-binding  same y e a r ,  a-globulin,  a p o s t a l b u m i n i n human serum on s t a r c h  t h e Group Thomas  but  it  et  gel  Specific  Component  a l . reported  was not  until  a  1975 t h a t  a l . showed Gc t o be DBP. DBP s i n c e has been f o u n d  other  body f l u i d s  fluid  (Hirschfeld,  al.,  In  found labeled  DBP can remove an a c t i n monomer f r o m one of  Hirschfeld  (Gc)  of  liver  complexes.  responsible  as f o r  to f l u o r e s c e n t l y  proteins  D B P - a c t i n complexes  two g e l s o l i n b i n d i n g protein  ( 1 9 8 6 ) who  t h e D B P - a c t i n c o m p l e x was f o r m e d .  radiolabeled actin-binding  showed t h a t  well  by Coue e t a l .  s u c h as u r i n e ,  ascitic  1962; N i e l s e n et  fluid  and  in  spinal  a l . , 1963; Berggard  et  1964). DBP b i n d s  1:1 to v i t a m i n D w i t h  a t 4 °C ( B o u i l l o n  et  transportation  vitamin D sterols  circulating  of  reservoir  al.  of  an a f f i n i t y  1986) c a r r y i n g  the  sterols  out  as w e l l (Cooke  K =10 a  1 0  M  two f u n c t i o n s ; as p r o v i d i n g and H a d d a d ,  _ 1  the  a 1989).  10  However,  the  concentration  of  DBP (~5  x 10  M) i s  in  vast  — ft  excess  of  v i t a m i n D (~5 x 10  alternate tested  function.  for  A l t h o u g h many t h o u s a n d s  DBP, no d e l e t i o n  gene h a s b e e n f o u n d , plasma.  competitive DBP i s  complex of  ^ and i s  of  s e r a have  alteration  to  DBP h a s a v i t a l  discovered  the adduct  DBP a r e  et  i n the  liver  t h e DBP role  in  1  DBP a d d u c t ,  had a p l a s m a t . i o f  1990).  Other p r o p e r t i e s  the  fatty  acids  (Ena  ions,  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  vitamin D (Cooke  of  DBP i n c l u d e  binding  a l . , 1989) and t h e  1:1 binding  of  (Goldschmidt-Clermont  et  al.,  1987).  the  of  same r a t e  1989). et  whilst  B o t h t h e apo and h o l o  c l e a r e d from plasma at  the  after  1-f h o u r s  and a c t i n had a p l a s m a t i  al.,  of  c i r c u l a t i o n by  and H a d d a d ,  binding  been  DBP s a c t i n -  at a rate  c l e a r e d from the  A radio-labeled  injection,  30 m i n ( D u e l a n d forms  (1980)  of  an  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 -  synthesized  and k i d n e y s .  intravenous  al.  suggesting  manner.  10 mg k g ^ day liver  or gross  implying that  Van B a e l e n e t  binding properties.  M) i n p l a s m a ,  with  calcium actin  11  1.1.5  Physical  Table  I:  P r o p e r t i e s o f DBP  Physical Properties  o f DBP  Gone . i n plasma  Man  59,000  Mouse  49,000  Rat  52,000  Some o f species  a r e shown i n t a b l e  (1986)  I.  has  9).  family thought  proteins  variance  species,  and t r a n s p o r t  amino a c i d s e q u e n c e f o r  i n the t r a n s p o r t DBP i s and c y s t e i n e  fatty of  of 3 0 0 -  f a m i l y of  1985) and protein  t h e same  proteins with  with  steroid  compounds  (Stryer,  in aspartic acid,  and l o w i n g l y c i n e a r e 28 c y s t e i n e  ago.  DBP, s u c h as t h e  a c i d s , and a l b u m i n s e r v e s  is  gene  between 3 0 0 - 5 0 0 m i l l i o n y e a r s  f o u n d t o be r i c h  DBP t h e r e  the  s e q u e n c e shows DBP t o be o f  some p r o p e r t i e s of  in  Bouillon  blood concentration  t o come f r o m a common a n c e s t r a l g e n e ,  Albumin shares  rat  DBP f r o m s e v e r a l  as a l b u m i n . T h i s  duplication occurring  In  a large  different  DBP cDNA c l o n e has been s e q u e n c e d ( C o o k e ,  The p r e d i c t e d  of  DBP f r o m  wide.  l e d to a p r e d i c t e d  (fig  of  There i s  s u g g e s t e d an a v e r a g e  400 mg/L s p e c i e s A rat  9 - 1 3 uM  the p h y s i c a l p r o p e r t i e s  m o l e c u l a r m a s s . On s t u d y i n g et a l .  10 uM  binding  as a b a c k up  1981). glutamic  w i t h no t r y p t o p h a n residues which,  acid  residues.  due t o  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  similarity 9),  of  t h e amino a c i d s e q u e n c e t o t h a t  have been p r o p o s e d t o a l l  (Cooke,  1985).  This  contrasts  DBP, where one d e e p l y  in forensic  relatively  1988).  The  stable  t h a n DBP a l o n e  1 . 1 . 6 Review of  evidence  cloth  1981).  DBP P u r i f i c a t i o n M e t h o d s  separating  A typical  DBP f r o m a l b u m i n .  by B o u i l l o n e t a l . 7 steps:  early  ( 1 9 7 8 ) u s e d 600 ml of dialysis,  method  rat  DEAE-cellulose  filtration,  1982, C h a p u i s - C e l l i e r  Blue  (a t r a d e  name f o r  Blue chromatography,  80 ml o f  97% p u r i t y  human p l a s m a .  from  of 0 . 1 % .  the h i g h a f f i n i t y  procedure:  73% y i e l d w i t h  DEAE-Sephadex  with a f i n a l y i e l d  l i n k e d to agarose b e a d s ) ,  S e p h a d e x G - 1 0 0 and D E A E - A f f i - g e l  proposed  chromatography,  b l u e dye c o v a l e n t l y Affi-gel  in  hydroxylapatite  et a l . u t i l i z e d  serum a l b u m i n f o r A f f i - g e l  for  serum and  C M - c e l l u l o s e chromatography, and g e l  schemes  due t o t h e d i f f i c u l t 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 ,  In  and  f o u n d t o be more  (Kawakami and Goodmann,  yields  chromatography  has many  (PStsch-Schneider  D B P - v i t a m i n D complex i s  DBP, most o f w h i c h g i v e p o o r  chromatography,  found  survive  T h e r e have been many p u b l i s h e d p u r i f i c a t i o n  involved  bonds  f r o m human  s c i e n c e as i t  s t a b l e , able to  2 weeks a t room t e m p e r a t u r e on d r y Klein,  earlier  (fig  1981).  DBP has p r o v e d u s e f u l and i s  with  albumin  in disulphide  b u r i e d m o n o t h i o l g r o u p was  (Kawakami and Goodmann,  phenotypes  be i n v o l v e d  of  of  cibacron  in a three  gel f i l t r a t i o n  Blue chromatography  step using  to give a  14  More r e c e n t l y , matrices et a l .  chromatographic  have been f a v o u r e d  despite  methods u s i n g their  small  (1986) designed a v i t a m i n D-Sepharose  affinity  capacity.  matrix,  from  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  followed  the o v e r a l l  by h y d r o x y l a p a t i t e  was 5 5 % . F i n a l l y ,  chromatography  B a e l e n and B o u i l l o n , purification  1986).  Gel  s t e p and p r o v i d e d  filtration a yield  DBP. T h i s method has been e m p l o y e d f o r s p e c i e s but including  has p r o d u c e d  horse.  s t e p was yield  a DNAase I - S e p h a r o s e m a t r i x was u s e d t o  a c t i n and t h e n DBP. DBP was t h e n e l u t e d w i t h  of  4 M MgCl2  served 70% f o r  several  Link  as a  bind  (van final  human p l a s m a  different  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 ,  15  Part 2: Spectroscopic 1.2.1  P r o t e i n Absorbance Absorbance  a s s e s s m e n t of clues  to  Spectroscopy  spectroscopy  protein  Techniques  provides  concentration  a convenient  as w e l l  t h e amino a c i d c o m p o s i t i o n o f  P r o t e i n absorbance 320 nm a r e  characteristic  contributions phenylalanine. electronic  spectra  of  three  These  025  spectra  result  0.80  040  0.20  250  X{nm)  300  250  A(nm)  300  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).  region 250-  tyrosine  from k to k  systems.  0.60 •  250 300 Mnml  some  the  tryptophan,  i n t.hese a r o m a t i c  0.18  of  a protein.  and a r e d o m i n a t e d by  absorbance  transitions  as g i v i n g  i n the wavelength  chroraophores;  method  and  16  Figure  10 shows t h e a b s o r b a n c e s p e c t r a o f  chromophores. all  the  Real protein  individual  the chromophores  (fig  11). Clearly  identify  the c o n t r i b u t i o n s  second d e r i v a t i v e  the c o n t r i b u t i o n  of  tryptophan  to  1 tryptophan  and 18  tyrosines.  Wavelength (nm)  Wavelength  (nm) 0.3  c  WW  A  0.0 —t>  > r>  V  /  >o o>  1 c  Oo M  N  260 1  Wavelength  j  270  i  280  (nm)  Fig 11: Second d e r i v a t i v e spectra in 6 M GuHCL, sodium phosphate, pH 6.8. Nozaki (1990). (a) AcTr NH , 2.51 x 10" M, (b) AcTyrNH , 7.6 x 1 0 M, (c) AcPheOEt, 1.78 x 10" M. 4  P  2  -4  2  3  each to  of  look  spectra  12) s p e c t r u m c a n be i d e n t i f i e d . A l b u m i n  250 1  of  absorbance spectrum i s  albumin ( f i g  240  sum o f  absorbances.  to the p r o t e i n  a t t h e more d i s t i n c t i v e  isolated  absorbance s p e c t r a are the  chromophore  A c l e a r e r method t o  the  the contains  17 0.25  -0.25 280  290  300  Wavelength (nm) Fig 12: Second d e r i v a t i v e spectrum of human serum albumin in GuHCl at a r b i t r a r y concentration. Nozoki (1990).  Absorbance measurements a l l o w the c a l c u l a t i o n of concentration  in solution  =  Where:  — 1 oS  A = I I = e = d = Q  via  1  o  the  C I  Beer-Lambert  calculation requires  extinction proteins .  c  absorbance 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 n t e n s i t y of i n c i d e n t l i g h t _^ _^ 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 ) c e l l p a t h l e n g t h (cm)  c = molar c o n c e n t r a t i o n The  law:  e. d  / I  prote  coefficient,  (M)  t h e k n o w l e d g e of which i s  usually  the  appropriate  quoted at  280 nm f  18  1.2.2 Fluorescence Fluorescence returns (S ).  from the  The  Q  (Stokes  shift)  Q  excitation  state w i l l  due t o  concerning  is  studies,  vibrational  solvents,  levels  of  emission.  On  dipole  the  polar  solvents.  This  a c c o m p a n i e d by a d r o p  quenching. Thus, environment  which i n t u r n ,  intrinsically  fluorescent;  tryptophan,  phenylalanine.  shift  in  can be g a i n e d  to  emission  from  protein  These f l u o r o p h o r e s  amino a c i d s w h i c h tyrosine  these  fluorophores  to energy t r a n s f e r  are  and  can be e x c i t e d  separately  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  t ryptophan.  in  information. three  of  the  information  provides  possess only  several  of  and hence a r e d s h i f t  Proteins  (fig  Additionally  l e a d i n g to a l o w e r i n g  and S^ l e v e l s  solvent  wavelength  lowest  on f l u o r e s c e n c e  In p o l a r  usually  state  energy.  be s t a b i l i z e d , Q  to a lower  becomes an i n s t a n t a n e o u s  a fluorophore's  fluorescence structural  in  comparison with non-polar  wavelength  intensity  loss  electron  to the ground  to e m i s s i o n .  decay to e x c i t e d  principle).  e n e r g y gap b e t w e e n S  longer  S^ p r i o r  a fluorophore  emission in  shifted  c a n have an e f f e c t  (Franck-Condon  (S^)  the r a p i d decay to the  a further  Solvents  Proteins  state  invariably  due t o of  excited  generally  producing  excited  first  level  fluorophores  of  e m i s s i o n may be o b s e r v e d when an  emission is  vibrational  S ,  Spectroscopy  containing  a r e d o m i n a t e d by t r y p t o p h a n  f r o m p h e n y l a l a n i n e and t y r o s i n e  to  due  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 e x c i t a t i o n 257 nm; Tyr, 6 uM tyrosine e x c i t a t i o n 274; Trp, 1 uM tryptophan e x c i t a t i o n 278 nm. Creighton (1989).  A more s p e c i f i c method t o s t u d y fluorescence  i s to introduce  protein  extrinsic  fluorophores.  (6-acryloyl-2-dimethylaminonaphthalene, particularly reacts  only  environment-sensitive with cysteine  N-(1-pyrene)iodoacetamide specific the  residues  polymerization  Acrylodan  f i g 14) i s a probe  (Pendergast  e t a l . , 1983)  ( f i g 15) i s a n o t h e r  of p y r e n e - l a b e l e d  via  fluorescent  p r o b e w h i c h has p r o v e d u s e f u l  fluorescence  structure  cysteine  in studying  actin  which  actin,  is sensitive  to  (Cooper et a l . , 1 9 8 3 ) . 0  II  NHCCH I 2  0  M« N a  Fig 14: 6-acryloyl-2-dimethylaminonaphthalene  Fig 15: N-(1-Pyrene)iodoacetamide  as  20  1 . 2 . 3 P r o t e i n CD Optically circularly  Spectroscopy  active  polarized  different  extents.  terms of  extinction  light  This  difference  and  right  respectively)  is usually  to  expressed  in  -  L  e  =  R  Ae  = 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 t h e 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 c o m b i n a t i o n of  of  the  the d i f f e r e n t two waves w i l l  polarized  light.  expressed  i n terms of  characterizes  This  fact  produce  where  [ 6 ]  [8]  6 , of  tan 8 i s of  L and R,  dichroism being  the  ellipse  equal to the  the e l l i p s e ,  that  ratio  AE i s  of  related  by:  =  3 3 0 0  the dependence of  Ae  8 on w a v e l e n g t h  is  O  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 , c a l c u l a t e d from observed  the  elliptically  has l e d t o c i r c u l a r  to the major a x i s  the molar e l l i p t i c i t y  A curve showing  a b s o r b a n c e s of  the e l l i p t i c i t y ,  such l i g h t ,  the minor a x i s to  (L and R,  left  coefficients.  e Where  substances absorb  [8]  ellipticity  i n deg cm  called a -1  dmol  at a s p e c i f i c  , can be wavelength:  21  [e] Where;  If  8 M d c  Me/iodo  i s the observed e l l i p t i c i t y (deg) i s t h e m o l e c u l a r mass (g mol ) i s t h e c e l l p a t h l e n g t h (cm) i s t h e c o n c e n t r a t i o n ( g ml )  M expresses  called  =  t h e 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  t h e mean r e s i d u e  (or  residual)  [8]  is  ellipticity.  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 t h e s i g n a l f r o m t h e p e p t i d e bond b a c k b o n e , t h e v a r i o u s characteristic  spectra  c o n f o r m a t i o n s of which  ( f i g 16).  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. F r e i f e l d e r (1976).  have  22  Using  these  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  and random c o i l e q u a t i o n at f  H  + f  f  +  B  [9] f  can be c a l c u l a t e d by s o l v i n g  two d i f f e r e n t  =  f  The  on d e n a t u r a t i o n  the  and u s i n g t h e  restraint,  +  H  f B  ellipticity  +  B  X  (deg  cm  f  R  dmol  ^  L  R  )  Xg, a r e t h e 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  proteins.  into  x  H  = mean r e s i d u e  CD a l s o p r o v i d e s  protein  following  = f r a c t i o n o f amino a c i d r e s i d u e s i n e a c h of t h e 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 structure).  Xfj,  of  wavelengths  the  (3-sheet  = I-  R  t e ] Where:  a-helix,  loss  of  (thermal  structure.  By m o n i t o r i n g t h i s  or  chemical)  structure,  loss  studying  the  stability  CD s i g n a l b e t w e e n 210 nm and 230 nm  The a - h e l i x  random c o i l  can be a s s e s s e d .  a good method f o r  of  and  is  fl-sheet  due t o l o s s structures  w h i c h has r e l a t i v e l y  signal  the  stability  of  a  of unfold low CD. protein  23  2: M a t e r i a l s and M e t h o d s  Part  2.1.1  The  P r e p a r a t i o n of  1: P r o t e i n s  G-Actin  The p r e p a r a t i o n o f G - a c t i n b a s e d on t h e method o f (10 g)  was e x t r a c t e d  0 . 2 mM C a C l , 2  ice.  The  S p u d i c h and Watt  with  buffer  0 . 2 mM A T P ,  and t h e n t h r o u g h  the  s o l i d r e s i d u e was d i s c a r d e d . The  (to  supernatant. temperature  The for  the p e l l e t dialysed  2 h,  rotor,  3 h,  A)  filter  and  filtrates  stirring  (1.5 h).  were  1 h,  combined,  4 °C,)  The  resuspended  room  to  3 h,  4 °C)  buffer  1 L of  and  A and  buffer  A,  ( 4 0 , 0 0 0 r p m , Beckman 4 5 T i  4 °C) and t h e s u p e r n a t a n t ,  immediately or d i a l y s e d a g a i n s t  rotor,  each of  s o l u t i o n was c e n t r i f u g e d  at  s o l u t i o n was  i n 30 ml o f  3 changes,  and  the  s o l i d K C l was added  ( 4 0 , 0 0 0 r p m , Beckman 4 5 T i (F-actin)  paper.  refiltered,  2 mM) were added t o  at which p o i n t  (64 h a g a i n s t  4 ° C ) . The  (100 m l , b u f f e r  (to  powder  of  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  0.8 M with gentle centrifuged  2  was  SO^min on  layer  grade  ( 4 0 , 0 0 0 r p m , Beckman 4 5 T i r o t o r ,  50 mM) and M g C l  for  a double  preparative  r e s i d u e was r e w a s h e d  KCl  (1971). Acetone  pH 7 . 6 )  through  The  centrifuged  m u s c l e powder  A ( 2 0 0 m l , 2 mM T r i s - H C l ,  1 . 0 mM DTT,  s o l u t i o n was f i l t e r e d  cheesecloth,  from r a b b i t  buffer  G-actin, A.  was  used  24  2.1.2  P r e p a r a t i o n of Pyrene-actin  method p r o p o s e d  was p r e p a r e d  by C o o p e r  Probes  Inc.,  Eugene,  (2:1).  This  solution  3 ml o f  actin  Pvrene-actin  against  II.  supernatant,  for  0 . 1 mM C a C l F-actin  by t h e  temperature.  A (3 c h a n g e s  each of  a minimum o f F-actin.  dialysed  addition  The  against  G-actin  of M g C l of  3h,  in buffer  al.  2  reagent  A.  in buffer  A)  buffer  t o 2 mM f o r  centrifuged  rotor,  4 *C)  to produce i n the  dialysed  to  2 hours  at  room  (5 x 10 "* M f i n a l  Inc.,  Eugene,  acrylodan-labeled  A (1L,  was  0 . 2 mM A T P ,  was r o c k e d i n t h e d a r k  resulting  acrylodan  (1988).  OR)  0 . 2 m l ) was added d i r e c t l y  being  (acrylodan-actin)  then  12 h ,  rotor,  was p o l y m e r i z e d  acrylodan  dark before 3 h,  et  2 mM T r i s - H C l ,  M o l e c u l a r Probes  DMF (~  product  was s t o r e d  fluorescent  Marriott  against  The m i x t u r e  temperature.  the  a 1 mg/ml s o l u t i o n  An e x c e s s  concentration,  polymerized  buffer  pH 7 . 9 . The  2 >  with  Preparation  t h e method of  12 h a t 4 ' C  been The  A c t i n was l a b e l e d w i t h  (1 ml o f  DMF/acetone  room t e m p . ) .  pyrene-actin,  the  room t e m p . )  ( 4 0 , 0 0 0 r p m , Beckman 4 5 T i  2.1.3 Acrvlodan-actin  G-actin  1 ml o f  of  Molecular  2 h,  2  4 °C) and c e n t r i f u g e d  following  (6 mg,  (1 mg/ml) w h i c h had p r e v i o u s l y  was d i a l y s e d  The  in  t h e n was s h a k e n (20 h ,  50 mM C a C l ,  4 °C).  e t a l . , 1 9 8 3 . PIA  OR) was d i s s o l v e d  (2 mM M g C l , 2  by a m o d i f i e d v e r s i o n  3 changes (40,000  in  (6 h)  60 h ,  in  the at  F-actin  room was  4 °C) i n  r p m , Beckman Type 50  acrylodan-labeled  supernatant.  to  dissolved  G-actin  the  25  2 . 1 . 4 The A c r v l o d a n - a c t i n The that  following  first  utilizes that  acrylodan-actin  u s e d by S a f e r  actin-binding the  proteins fact  can b i n d  that  was c o m b i n e d w i t h  included  to  DBP i s  facilitate gel.  of  the  pH 8 . 4 ]  loading  grade  into  the  sucrose  loaded onto  (0.1 g/ml),  gel  in buffer  a UV l i g h t  t h e band c o r r e s p o n d i n g  The  assay  in  plasma,  in buffer  0 . 2 mM A T P ) , 70 p i 10 p i  E at  of  which  pi), was  of  the  a non-  freshly  200 v o l t s  prepared  1 0 0 , 10 p i system for  (Bio-  45 m i n .  samples r e v e a l e d  D B P - a c t i n complex  E  7.5% a c r y l a m i d e ,  Triton-X  the p o s i t i v e  to uncomplexed  (10  A)  [6 ml b u f f e r  w h i c h was r u n on a M i n i - P r o t e a n I I  under  of  other  sample w e l l s  electrophoretic  s l o w m o v i n g band due t o t h e  G-actin.  solutions.  two p r o t e i n s ,  These t h e n were  Rad L a b s , R i c h m o n d , CA) When v e i w e d  DBP and  s a m p l e s t o be t e s t e d  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 ,  TEMED,  for  a modification  a 1 mg/ml s o l u t i o n  194 mM g l y c i n e ,  ammonium p e r s u l p h a t e  is  actin.  analytical  polyacrylamide  (25 mM T r i s - H C l ,  to t e s t  one of  to  (10 u l  of  assay  i n mixed p r o t e i n  e a c h of  a crystal  electrophoretic denaturing  (1989)  specifically  Acrylodan-actin  along with  Assay  in addition  a c r y 1 o d a n - 1 a b e 1ed  a to  26  2.1.5  The  Collection  The method o f procedure  Cote  platelets. (Alsask  of  and S m i l l i e  Blood  into  Horse  ( 1 2 0 L)  (1981)  leupeptin  ( 1 2 . 5 mg o f  each per  of  b l o o d was a l l o w e d  had f o r m e d , w i t h  cell-rich  lower  50 L p a i l s ,  Protease  M0) and PMSF ( 0 . 2 mM), were  layer.  2 L portions  2 . 1 . 6 The  1400 g  at  acid,  dextrose  inhibitors,  50 L b u c k e t ,  9 L  pepstatin,  Sigma, S t .  Louis,  to  stand  (~1 h)  until  (Sorvall  layer. -20  It  two  layer  and a r e d  as t h e  supernatant  RC-3 r o t o r ,  was f r o z e n  distinct  15 min a t  i n 500 ml  to  'C,  P u r i f i c a t i o n o f DBP  Equine  DBP was p r e p a r e d  method p r o p o s e d  DMSO; S i g m a , S t . solution  thawed h o r s e  using a modified version  by C h a p u i s - C e 1 1 i e r  human DBP. P e p s t a t i n  stock  89 g c i t r i c  P l a s m a was c o l l e c t e d  and s t o r e d  frequent  each c o n t a i n i n g  a p i a t e 1 e t - r i c h upper  the p l a t e l e t - r i c h  horses  added.  a 5 , 0 0 0 rpm c e n t r i f u g a t i o n  4 °C) o f  prepare  was c o l l e c t e d , w i t h  orthophosphate,  t o 27 L i n w a t e r ) .  the  slaughtered  (720 g s o d i u m c i t r a t e ,  dissolved  layers  employed to  from f r e s h l y  plastic  61 g s o d i u m d i h y d r o g e n  The  p l a s m a was b a s e d on  Edmonton)  three  anticoagulant  Plasma  collecting  Processors,  stirring,  of  (100 u l  Louis,  al.  (1982)  Sigma, S t .  p i asma (80 m l ) .  The  buffer,  to  (100 u l  Louis,  of  MO) were  p l a s m a was d i a l y s e d  0.1%(w/v)  50 pM PMSF, pH 7 . 0 , 4 ° C ) and c e n t r i f u g e d  sodium at  the  purify  a 2 mg/ml s t o c k s o l u t i o n  MO) and L e u p e p t i n  in water;  30 mM s o d i u m p h o s p h a t e  of  et  of  in  a 2 mg/ml added (12  to h,  azide,  1 8 , 0 0 0 rpm  for  27  30 min ( S o r v a l l supernatant Affi-Gel  SS34 r o t o r ) .  was a p p l i e d  Blue a f f i n i t y  The  pellet  (1 m l / m i n ) ,  at  chromatography  was d i s c a r d e d  and  room t e m p e r a t u r e , column ( 2 . 5  the to  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)  in buffer  (30 mM s o d i u m p h o s p h a t e ,  sodium a z i d e ,  pH 7 . 0 ) .  elution  of  protein  and t h e  region  was f o l l o w e d  by UV a b s o r b a n c e  at  assay  (also  chromatographic  employed to f o l l o w columns).  removed t h e m a j o r p l a s m a p r o t e i n ,  a l b u m i n , as w e l l  some f r a c t i o n a t i o n  of  (Gianazza  et  c o l u m n was r e g e n e r a t e d  applying buffer,  2 L of  The  potassium thiocyanate  as recommended by t h e  The  active  concentrated  fractions  to about  room t e m p e r a t u r e ,  to a B i o - G e l  (^250 m l )  u s i n g a YM30  MA) and t h e n a p p l i e d P100 g e l  (1 m l / m i n ) ,  filtration  active  applied  ( 0 . 5 ml/min)  the e l u a t e  directly  t h e n were  to a h y d r o x y l a p a t i t e  ( 2 . 0 x 20 cm, B i o - R a d L a b s , R i c h m o n d , CA) room t e m p e r a t u r e .  A 30 t o  B was u s e d t o e l u t e collected.  running  and  in buffer  of  by  ( 1 . 5 M) i n t h e  (5 x 110 cm, B i o - R a d L a b s , R i c h m o n d , CA) fractions  as  proteins  20 ml by u l t r a f i l t r a t i o n  Danvers,  column  manufacturer.  were p o o l e d  membrane ( A m i c o n ,  the  Blue  providing  a l . , 1982).  The  DBP t h r o u g h  The A f f i - G e l  o t h e r plasma  B  280 nm,  where DBP e l u t e d was d e t e r m i n e d u s i n g  acrylodan-actin subsequent  0.1%(w/v)  an  100 mM p h o s p h a t e  t h e bound p r o t e i n .  at  column B.  The  column again in buffer gradient  in  5 ml f r a c t i o n s  B at  buffer were  28  A Flow Chart  Showing  t h e 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  GEL  PlOO FILTRATION CHROMATOGRAPHY  1 ) A BEayed  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 buffer  active  B,  volumes  (2 m l )  size,  a single  were  4 °C) and f i l t e r e d  a n i o n exchange pore  fractions  were  dialysed  (12 h ,  ( 0 . 2 2 uM M i l l i p o r e  applied,  at  10 L filter).  room t e m p e r a t u r e ,  t o a HPLC  c o l u m n ( 2 0 0 x 4 . 6 mm, A q u a p o r e A X - 3 0 0 ,  Brownlee  peak w i t h  Labs, a salt  Santa C l a r a , gradient  CA).  Small  7 micron  DBP was e l u t e d  ( 0 - 0 . 2 5 M NaCl i n  as  buffer  B).  2.1.7  C a l c u l a t i o n of Using  the  coefficient  of  protein  and t h e  are the  only  range,  the  an E x t i n c t i o n  approximation DBP w i l l fact  following  coefficient  be e q u a l  that  absorbing  e-pyj.,  coefficients t y p e of  the molar to that  tyrosine,  species  in  £jrp  a n <  *  6  Cys  + be  a  r  e  t  n  e  DBP  denatured and  cysteine  t h e 2 7 6 - 2 8 2 nm w a v e l e n g t h  X r p  m  °l  +  a  extinction and von H i p p e l ,  c 6  r  molecule.  C  y  1989):  s  extinction  a t 280 nm and a , b and c a r e  amino a c i d p e r p r o t e i n  the  tryptophan  a t 280 nm can be made ( G i l l  X y r  for  extinction  of  e s t i m a t i o n of m o l a r  € = ae  Where:  that  Coefficient  t h e number o f  each  30  2.1.8 P r e p a r a t i o n DBP pH 7 . 3 )  of Acrylodan-DBP  (2 ml o f  a 1 mg/ml s o l u t i o n  was s h a k e n f o r  12 h o u r s  at  i n 20 mM MOPS, 0 . 1 % N a N , 3  room t e m p e r a t u r e  acrylodan  (5 x 10 "* M, M o l e c u l a r P r o b e s  dissolved  i n a minimum o f  dialysed  extensively  (4 c h a n g e s  each of  centrifuged The  pellet  2.1.9  DMF ( ~ 0 . 2 m l ) .  against  1 L for  Inc., The  Eugene, product  OR) was  20 mM MOPS, 0 . 1 % N a N 3 , pH 7 . 3  12 h a t 4 ° C ) b e f o r e  ( 4 0 , 0 0 0 r p m , Beckraan Type 50 r o t o r , was  with  being 1 h,  4 °C).  discarded.  Gel Electrophoresis Gel e l e c t r o p h o r e s i s  following  electrophoretic  (Bio-Rad  Labs,  Uppsala,  Sweden).  The  by D r .  Dana  available  was c a r r i e d systems:  R i c h m o n d , CA)  or  out  u s i n g one o f  M i n i - P r o t e a n II  system  PhastSystem (Pharmacia,  P h a s t S y s t e m was g e n e r o u s l y Devine.  the  made  31  Part  2.2.1  Absorbance  2: Spectroscopic  Methods  Spectra  A b s o r b a n c e m e a s u r e m e n t s and s e c o n d d e r i v a t i v e s p e c t r a were (Perkin  p e r f o r m e d on a Lambda 4B UV/VIS  Elmer,  Norwalk,  2.2.2 F l u o r e s c e n c e Fluorescence  Spectrophotometer  CT).  Spectra s p e c t r a were  Luminescence Spectrometer  recorded  in conjunction  spectrometer  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  (Haake,  equilibration  Berlin,  period  of  Elmer,  with  Computer  bath  (Perkin  on a L S - 5 B  Professional  water  absorbance  Germany),  Norwalk,  and a  a 7500 CT).  The  circulating  temperature  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  generated  Recording Spectro-Polarimeter equipped with chamber.  All  chamber f o r  a temperature s a m p l e s were  15 min b e f o r e  was s t a n d a r d i z e d 100  ml o f  H 0) 2  2 deg  cm  —  dmol  on a r e b u i l t (Jasco,  T o k y o ) w h i c h was  controlled,  allowed  J-20 Automatic  nitrogen  to e q u i l i b r a t e  m e a s u r e m e n t s were  using D-Pantolactone  in  of  t a k e n . The  -17.3 x 10  1 al.,  1977).  sample  the  (12 mg i n  w h i c h has an e l l i p t i c i t y a t 220 nm ( T u z i m u r a e t  purged  3  output  32  3; R e s u l t s a n d D i s c u s s i o n  3.1.1 Y i e l d a n d P u r i t y of DBP DBP  was e l u t e d  column i n the f i n a l of  in a single stage  of the p u r i f i c a t i o n  6 - 7 mg o f DBP were r e t r i e v e d  affording  a yield  concentration purification runs  peak ( f i g 17) f r o m t h e HPLC  of 2 1 - 2 5 % ,  scheme. A t o t a l  p e r 80 ml o f s t a r t i n g  a s s u m i n g an i n i t i a l  o f 350 mg/L ( B o u i l l o n  plasma  et a l . , 1986).  scheme was r e p r o d u c i b l e ,  yielding  plasma,  The  DBP f r o m s i x  in succession.  s  c  Time Fig  17: Elution p r o f i l e from HPLC column. Shaded area corresponds to DBP.  \  The solitary  peak c o n t a i n e d a s i n g l e  protein  band on a S D S - p o l y a c r y l a m i d e  that  m i g r a t e d as a  electrophoretic  gel  33  under single  reducing  (fig  band when s u b j e c t e d  polyacry1amide reducing  gel  18).  electrophoresis.  the major  DBP a l s o m i g r a t e d as a  to n o n - d e n a t u r i n g ,  SDS-polyacrylamide  constituted faint  conditions  Furthermore,  electrophoretic  species present  band due t o DBP d i m e r s  non-reducing  (Bouillon  on  non-  g e l s monomeric DBP  (>95%), w i t h et a l . ,  only  1977).  Fig 18: SDS polyacrylamide electrophoretic g e l . Lanes from left to r i g h t : 1, Plasma; 2, A f f i - g e l blue eluate; 3, P 100 eluate; 4, Hydroxylapatite eluate; and 5, HPLC eluate.  a very  34  3.1.2 Optical  Spectra  The a b s o r b a n c e s p e c t r u m of DBP r e v e a l e d a t y p i c a l tryptophan-free  profile  (fig  1 9 a ) . The peak a t 277 nm and  s h o u l d e r a t 284 nm a r e f e a t u r e s undulations underlying present  of  tyrosine  between 255 and 275 nm a r e i n d i c a t i v e phenylalanine  a shoulder  spectrum. If  tryptophan  albumin ( f i g  of  had been  280 nm, as  19b).  266  276  266  296  388  318  328  Wavelength/mo  Fig 19a: Absorbance spectrum of DBP (0.7 mg/ml).  27e"8  the  a t 290 nm w o u l d have been e x p e c t e d and t h e  a b s o r b a n c e maximum w o u l d have been n e a r e r  256  a b s o r b a n c e and t h e  Ml  288'. 8  388' .8 Havelength/nm  Pig 19b: Absorbance spectrum of albumin (0.7 mg/ml).  for  35  An e x t i n c t i o n calculated for (Cooke, The clearly have  coefficient  DBP f r o m t h e  of  22,560 M  reported  cm  was  sequence of  rat  DBP  1986). second d e r i v a t i v e the  l a c k of  a negative  is  seen  (fig  of  tyrosine.  tryptophan.  20). This  peak i s  Furthermore of  tyrosine  spectra  16).  the  s p e c t r u m shows  Proteins  peak a t 290 nm, h e r e  superposition (page  absorbance  containing  a positive  more tryptophan  peak a t 290 nm  characteristic  spectrum resembles  and p h e n y l a l a n i n e  Fig 20: Second d e r i v a t i v e absorbance spectrum of DBP ( 0.7  the  second  mg/ml).  derivative  36  The  fluorescence  emission spectrum, 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 no t r y p t o p h a n characteristic  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  ( f i g 2 1 ) . The maximum a t 307 nm i s of t y r o s i n e ,  l o w a r o u n d 3 3 0 - 3 5 0 nm where  and t h e f l u o r e s c e n c e tryptophan  that intensity  fluorescence  is  peaks.  0)  o c o  HftX=58.8  0)  o  Z3  i—f  U_  f  > V -p CO  /  •—i  0)  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 protein  three  optical  s p e c t r a a l l show t h a t  does n o t c o n t a i n t r y p t o p h a n  phenylalanine.  This  is consistent  the i s o l a t e d  b u t does have  t y r o s i n e and  w i t h the r e p o r t e d  s e q u e n c e f o r r a t p l a s m a DBP ( C o o k e ,  1986).  amino a c i d  37  3.1.3  Actin  assays  Evidence  of  DBP's a c t i n - b i n d i n g  from the a s s a y used to  isolate  the p r o t e i n  Acrylodan-actin  moves as a s i n g l e  electrophoretic  polyacrylamide  DBP, of  a second,  slower  DBP-acry1odan-actin  band on  gels,  m o v i n g band i s complex  properties  (fig  but  (page  was  gained  25).  non-denaturing i n the presence  s e e n due t o t h e  of  formation  22).  F i g 22: The a c r y l o d a n - a c t i n assay o f A f f i - g e l Blue f r a c t i o n s . Lanes from l e f t t o r i g h t see band a p p e a r i n g i n c e n t r e o f g e l due t o D B P - a c r y l o d a n - a c t i n complex. Lower band = G - a c t i n , upper band = F - a c t i n t h a t d i d not e n t e r t h e g e l .  38  Further arises  from  evidence  pyrene-actin  fluorescence greatly  f o r theactin-binding a c t i v i t y  intensity  increases  fluorescence  s t u d i e s . The  a t 3 8 6 nm o n e x c i t a t i o n  on p o l y m e r i z a t i o n  ( f i g 23a). In the presence  does  ( f i g 2 3 b ) . DBP s e q u e s t e r s  preventing actin is  a  results  i n a slow  fall  o f DBP t h i s actin  depo1ymerization,  fluorescence  level  characteristic  i nfluorescence  up t h e f r e e  e q u i l i b r i u m away f r o m  slow  to increase  monomers  p o l y m e r i z a t i o n . T h e a d d i t i o n o f DBP t o p y r e n e - F -  d u e t o DBP t y i n g  the  a t 3 4 4 nm  of pyrene-G-actin  pyrene-F-actin not occur  o f DBP  after  pyrene-G-actin,  thepolymerized returning to a 10 h o u r s .  o f an a c t i n  ( f i g 23c).  This  thus  state,  This  shifting  resulting i n  G-pyrene-actin effect i s  monomer-binding  protein.  (1nX=15.e  /  MH=J.6 8  286  488~  688  886  1888  1286  1486  1688  1866  Time/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 (1 H) added after 80 s. 2  2886  39  ftAX=15.8  00  niN=8.e  288  688  488  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 MgCl (1 M) added after 340 s. 2  t1nX=15.8  oo n C&4  MIH=e.6 8 288  486  686  888  1888  1288  1486  1688  1888 Time/s  c) G-actin (0.15 mg), Pyrene-G-actin ( 0.015 mg) incubated , 0.5 h, with 2 mM MgCl . At t = 0 DBP (0.2 mg) added, final volume 1 ml. 2  2686  40  3.1.4  Calculation  of Molecular  Mass  DBP and m o l e c u l a r mass s t a n d a r d s were r u n 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 t h e r e l a t i v e d i s t a n c e  moved by DBP i n c o m p a r i s o n  t o the standards, a molecular  o f 53,000 ± 3,000 was c a l c u l a t e d  ( f i g 25).  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.  mass  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 of  Secondary  The CD s p e c t r u m o f protein  high  in a-helix  a-helix  (39%),  B-sheet  Structure  DBP ( f i g  26)  and B - s h e e t (42%)  is  structure.  and random c o i l  c a l c u l a t e d u s i n g X J J , Xg and X R f a c t o r s 210 nm (Chen e t  3.2.1  Thermal The  al.,  c h a r a c t e r i s t i c of a  DBP f o l l o w e d bv CD  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  2 7 , open s q u a r e s ) .  s t a b l e up t o  55 ' C ,  at a g r e a t e r  rate.  structure returned  after  its  even a t  m o l e c u l e was a b l e t o r e f o l d . retain  actin-binding activity  started  15 ' C t h e  value,  This  was f o l l o w e d by CD  70 ' C a g r e a t  On c o o l i n g t o original  against  buffer  5 mM DTT  thermal denaturation effect  (fig  less  stable,  but  suggesting  that  its  the to assay.  room  a l s o was s u b j e c t e d  27, closed t r i a n g l e s ) .  on h e a t i n g ,  d e a l of  and t h e n d i a l y s e d a t 4 ' C  of b r e a k i n g d i s u l p h i d e b o n d s .  DTT,  unfold  r e c o o l e d DBP was shown  (12 h ) ,  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 without  to  u s i n g the a c r y l o d a n - a c t i n  (2 h)  H + 2 mM DTT  very  signal  A s e c o n d s a m p l e , w h i c h had been i n c u b a t e d a t temperature with  buffer H  DBP was s e e n t o be  which the p r o t e i n  However,  was r e t a i n e d . t o 97% o f  were  1972).  D e n a t u r a t i o n of  220 nm ( f i g  (19%)  for  a t 225 and  ( 1 5 0 mM K C l , 20 mM MOPS, 1 mM EGTA, pH 7 . 3 ) at  Values  Here,  f rom t h a t  of  the  DTT  to  has  the  i n i t i a l CD  t h e DBP s a m p l e  t h e p r o t e i n was s e e n t o be much  l o o s i n g more CD s i g n a l by 40 °C t h a n had t h e 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 nm. Open boxes, DBP; closed t r i a n g l e s , DBP + DTT. Relative e l l i p t i c i t y = 6/(6 at 16 *C).  A5  The to  recooled  63% o f  refold  the  protein,  as w e l l  as p l a y i n g  unfolded,  (fig  to  bonds  complete i t s  buffer  2 8 ) . The  buffer  H,  s t a b l e up t o  The  rate.  On c o o l i n g  beyond  to that  the h i g h  of  it to  92% o f  ionic  shape  i n a low  similar  salt  strength  its  the  a major  does not  this of  DTT  was  DBP and  + DTT  those  in  the  role  at a  CD s i g n a l .  t o be much l e s s  CD s i g n a l of  its  experiments  falling  ~40% a t  s a m p l e . The  t o 65% o f  were  relatively  70 *C,  low  b i n d a c t i n on c o o l i n g  greatly  salt  ± DTT  to  original  case, with  the h i g h s a l t  play  of  actin-binding  DTT was f o u n d  220 nm r e t u r n e d  bonds  this  refolding  that  DTT was a g a i n  retained  DBP s a m p l e d i d n o t at  on  thermal denaturation  T h e s e two CD t h e r m a l d e n a t u r a t i o n disulphide  out  30 °C and r e a c h i n g a v a l u e of  not  temperature  possibility  CD s p e c t r a were  and r e t u r n e d  rapidly  ellipticity  a t body  was c a r r i e d  sample i n c u b a t e d w i t h  + DTT  correct  55 °C beyond w h i c h CD s i g n a l was l o s t  as i n  salt  did  functions.  DBP s a m p l e w i t h o u t  stable,  close  i n the  L (20 mM MOPS, 1 mM EGTA, pH 7 . 3 ) initial  The  properties  it  stability  to m a i n t a i n i t s  and s i m i l a r p a t t e r n s  observed.  greater  a role  that  i n DBP a p p e a r ,  the unusual  r a i s i n g the  A s i m i l a r experiment buffer,  bonds  DBP. F u r t h e r m o r e ,  the d i s u l p h i d e  possibly,  for  suggesting  returned  DBP s a m p l e w h i c h had been i n c u b a t e d w i t h  significantly requires  The d i s u l p h i d e  denatured  °C) t h e  b i n d a c t i n and o n l y  CD s i g n a l ,  t o be r e s p o n s i b l e  partially (37  original  correctly.  evidence,  sample d i d not  and  original suggest  its  value. that  i n s t a b i l i z i n g DBP and  affect  stability.  the that  46  A *A  • n u  • ••• D  n  °nn  A  •  A  •  • •  •  •  • •  A  A  •  • • •  *  A A  •  10  20  30  40  50  60  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).  70  47  Thermal  3.2.2  D e n a t u r a t i o n of  A further  experiment  DBP F o l l o w e d bv  revealing  the  Fluorescence  stabilizing effect  t h e 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  out  of  by m o n i t o r i n g t h e  fluorescence generally  intensity  (fig  t e m p e r a t u r e on  2 9 ) . As p r o t e i n s  become more e x p o s e d t o t h e  fluorescence inherent  effect  intensity.  This  effect  t e m p e r a t u r e dependence of  is  intensity  at  temperature d i d not  307 nm, e x c i t a t i o n than the non-reduced  have  the s t r u c t u r a l  d i s u l p h i d e bonds, recooling original  the  without  with  DTT  lost  DTT  The  the  by  the On  t o 95% of  its  sample w i t h  and i n d i c a t i n g t h a t  d i s u l p h i d e bonds was u n a b l e t o  with  r e d u c e d DBP  temperature.  returned  compared t o 62% f o r  t h e CD d a t a ,  fluorescence  278 nm, more r a p i d l y  DBP s a m p l e .  the  The DBP  s t a b i l i z a t i o n provided  DBP s a m p l e w i t h o u t  their  superimposed over  and u n f o l d e d a t a l o w e r  fluorescence  consistent  at  tyrosines  lowering  fluorescence.  s a m p l e w h i c h had been i n c u b a t e d w i t h  carried  tyrosine  unfold,  solvent,  of  DTT,  t h e DBP  refold.  3 . 2 . 3 G u a n i d i n e . H C 1 I n d u c e d 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 buffer  of  DBP by g u a n i d i n e  hydrochloride  H ± DTT was f o l l o w e d by CD a t 220 nm ( f i g  experiment  d i d not  unfolding  patterns  CD s i g n a l  fell  hydrochloride  reveal of  sharply  a significant  30).  difference  reaching a value  The  between  t h e DBP s a m p l e s w i t h o r w i t h o u t between  in  DTT.  1 and 2 . 5 M g u a n i d i n e of  about  10% a t 4 M G u H C l .  the The  48 1.2  1.0"  •  o  • • •rl  m o  0.8  O  a H  o a • o  « u o  0.6-  9  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  • A  0.8-  •  •  o  CM CM  «  0.6-  a H  0.4"  2  0.2-  • •m 0.0  .  2  T4  Cone. GuHCl/M  Pig 30: Guanidine Denaturation of DBP followed by CD. Open boxes, DBP; closed t r i a n g l e s , DBP + DTT. Relative e l l i p t i c i t y = 6/(6 at 14 *C).  50  In  comparison to  presence  of  DTT  ± salt  denaturation  proceeds  denaturation.  Indeed  samples w i t h of  original  relatively state)  3.3.1  DTT  Acrvlodan  to a f a r t h e r at  point  cm  - 1  than  thermal  denatured  - 1  of  T h i s degree  coefficient at  (molten  the  of  fluorescent per  used i n the b i n d i n g  acrylodan  globular  DBP. The  1981, found o n l y  t h e a c r y l o d a n was n o t  This  the be  e m i s s i o n and  surprising  one  as  relatively  per m o l e c u l e of  v i t a m i n D.  interactions.  1988),  31.  known t o have a h i g h l y of  CA)  of  fluorescence  l a b e l i n g was somewhat  group  Richmond,  l a b e l was shown t o  a r e shown i n f i g u r e  i n a c c e s s i b l e monothiol DBP i s  for  (Bio-Rad,  365 nm ( L e h r e r and I s h i i ,  Kawakami and Goodman,  by h y d r o p h o b i c  state  reaching a  L a b e l i n g o f DBP  spectra  some o f  DBP  t o be r e a c h i n g a p l a t e a u a t a r o u n d 38%  partially-unfolded  5.0 ± 0.2 acrylodans  Furthermore,  the  guanidine  thermally  the B i o - R a d P r o t e i n Assay  incorporation  that  70 'C t h e  the  DBP i n  1989).  and an e x t i n c t i o n  excitation  27 + 2 8 ) ,  of  s i g n a l . T h i s may be due t o t h e p r o t e i n  (Kuwajima,  129,000 M  (figs  appear  stable,  Using  the t h e r m a l m e l t i n g curves  human DBP.  hydrophobic  l e d to the  covalently  pocket,  suspicion  bound,  but  held  51 1 MAX=180.8  in  J-  A}  OH  tiiH=e.6  458  468  358  588  Wavelength/nm  HAX=125.8  o e  <U  o CO QJ  o 3  /  QJ  J1IH=8.8  428  488  448  468  488  588  528  548  568  588  "• 688  Wavelength/nm 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 red (fig  shift  o f DTT  ( 5 mM)  i n the emission  to the acrylodan-DBP  maximum  i s immediately  3 2 ) . The i n c r e a s e i n i n t e n s i t y  hydrophobically acrylodan 1983).  bound  acrylodan  i s most  reacting  has low f l u o r e s c e n c e i n t e n s i t y  The r e d s h i f t  could  seen  likely  with  a large  due t o t h e  DTT, a s  (Pendergast.  be d u e t o t h e r e l e a s e o f  eta l . , this  52  acrylodan-DTT  into  the s o l v e n t .  by t h e o b s e r v a t i o n 2 mM DTT,  that,  labeling  of  acrylodans  last  on d i a l y s i s  pH 7 . 3 (3 c h a n g e s  diminished acryodan  This  point  against  i n 36 h ) ,  this  mixture  per  DBP. F u r t h e r m o r e ,  when t h i s  DTT~treated  treated acrylodan-DBP  front.  that  buffer  This  bound.  It  that  was t h i s  DTT-  experiments,  a g a i n s t a non-DTT  (20 mM MOPS, pH 7 . 3 , 3 c h a n g e s  i n 36  t1ftX=8e.8  h*IN=8.8 468  438  588  528  548  with  suggests  was u s e d i n s u b s e q u e n t  had been d i a l y s e d e x t e n s i v e l y  containing  electrophoretic  f l u o r e s c e n c e was s e e n t o m i g r a t e  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  it  of  a c r y l o d a n - D B P was t h e c a l c u l a t e d t o be 0 . 3 5 ± 0 . 2 9  a l l the a c r y l o d a n  after  had a much  e m i s s i o n s p e c t r a , and t h e new e x t e n t  DBP and none was d e t e c t e d a t t h e dye the  supported  20 mM MOPS,  a c r y l o d a n - D B P was r u n on an SDS p o l y a c r y l a m i . d e gel,  is  568 Wave  588  688  length/nm  Fig 32". The effect of DTT on the emission spectrum of acrylodan-DBP (5:1 labeled), excitation at 365 nm at 20 °C. A) acrylodan-DBP alone. B) acrylodan-DBP + 5 mM DTT.  h;.  53  The new e m i s s i o n s p e c t r u m had t h e same shape as t h e DBP with hydrophobically  bound a c r y l o d a n  although  the  emission  maximum 488 nm, e x c i t a t i o n a t 365 nm, had moved f r o m 495nra. This  i s blue  adduct other  shifted relative  ( 5 4 0 n m ) , and i s proteins:  that  slightly  acrylodan-mercaptoethanol  blue-shifted  relative  to  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 anhydrase suggesting  t o an  501 nm ( P e n d e r g a s t  the a c r y l o d a n  is  et a l . ,  in a very  1983),  hydrophobic  env i r o n m e n t .  3.3.2  Interaction  of Acrvodan-DBP w i t h  On a d d i t i o n of there  is  a c t i n to a c r y l o d a n - D B P i n a 1:1  a red-shift  488 nm t o 494 nm ( f i g  446  4bS  Fig 33: The of 365 B)  Actin  i n the f l u o r e s c e n c e 33),  486  506  e m i s s i o n maximum f r o m  combined w i t h a r e d u c t i o n  520  546  566  586  in  .668  Havelength/nm  effect of actin binding on the emission spectrum aerylodan DBP (0.35:1 labeled), e x c i t a t i o n at nm at 20 ' C . A) acrylodar.-PBP alone acrylodan-DBP + actin (1:1) -  ratio,  54  intensity,  i n d i c a t i n g that  hydrophobic  environment.  t h e a c r y l o d a n h a s moved t o a l e s s  These e f f e c t s  a c t i n binding at a s i t e d i f f e r e n t  c a n be e x p l a i n e d by  than that  labeled  with  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 exposes  the a c r y l o d a n probe  to the s o l v e n t .  3.3.3 E f f e c t o f DTT on F l u o r e s c e n c e Bound t o DBP  of Acrylodan  covalentlv  Two a c r y l o d a n - D B P s a m p l e s e q u i l i b r a t e d a g a i n s t buff ers,  one i n 20 mM T r i s — H C 1 ,  pH 8 . 0 and t h e o t h e r  i n c u b a t e d f o r 2 h i n t h e same b u f f e r and s u b s e q u e n t l y 8.0  dialysed against  initially  b u t c o n t a i n i n g 5 mM DTT,  2 mM DTT, 20 mM T r i s - H C l ,  pH  ( 3 c h a n g e s o v e r 36 h ) . The a b s o r b a n c e s a t 280 nm o f t h e  two s a m p l e s were w i t h i n for  different  2% o f e a c h o t h e r .  t h e a c r y l o d a n - D B P sample t h a t  was s l i g h t l y  blue-shifted  The e m i s s i o n maximum  had been i n c u b a t e d w i t h DTT  ( f i g 3 4 ) t o 483 nm and i t s  intensity  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  implying  that  environment. disulphide amount of  t h e a c r y l o d a n had moved i n t o A possible  explanation  bonds p r e s e n t strain  into  in  the  structure,  is  change a f f e c t s  that  is  protein  yielded  only  exchange  partly  DBP. W i t h o u t  unfolded  bonds g u i d e  s t a t e on  fluorescent  a c t i n and t o t h e groups. to  This  identify  techniques  product  state  initial the  anion  have  revealed  thermal  the  stability  of  t h e p r o t e i n w o u l d be  refolding  of  Furthermore,  h e a t e d DBP t o a  cooling.  DBP can be c o v a l e n t l y highly  daltons  purity.  a t body t e m p e r a t u r e .  the  It  p l a s m a DBP s p e c i e s  to a c h i e v e  investigations  (30%)  ensuing  polymerization.  other  these d i s u l p h i d e bonds,  the d i s u l p h i d e functional  its  t h e d i s u l p h i d e bonds t o t h e  significantly  is  plasma D B P ' s .  p l a s m a DBP. A f i n a l  HPLC s t e p was r e q u i r e d  i m p o r t a n c e of  strain  of m o l e c u l a r mass 5 3 , 0 0 0  horse  CD and f l u o r e s c e n c e  certain  probe.  resembles other  schemes f o r  pure  and t h i s  a  the  Conclusions  a b l e t o b i n d a c t i n and b l o c k  Published p u r i f i c a t i o n  that  introduce  the a c r y l o d a n  p l a s m a DBP c l o s e l y  a tryptophan-free  is  hydrophobic  bonds a r e c l e a v e d . The  P a r t 4: Horse  this  the p r o t e i n  r e l e a s e d when t h e d i s u l p h i d e conformational  for  a more  site  of  study of  labeled with acrylodan that  is  oxidation of  sensitive of  to produce a  to the b i n d i n g  the p r o t e i n  sulfhydryl  a c r y l o d a n - D B P s h o u l d be  extended  l a b e l i n g u s i n g p e p t i d e mapping  and by a p p l i c a t i o n of  other  fluorescence  of  methods  56  (polarization, characterize the b i n d i n g The  (such  the of  fact  covalently  quenching, site  of  energy  transfer)  to  l a b e l i n g and how i t  better is  affected  actin. that  acrylodan  t o DBP s u g g e s t s  appears  a study  to b i n d ,  in part,  and B r a n d ,  bound p r o b e  would p r o v i d e  the hydrophobic sequence  1979).  analogy  associated with  pocket with  Fluorescence further  properties  structural  on DBP t h a t  is  vitamin  D.  probe  acid, of  the  information  expected  serum a l b u m i n and t h a t  binding  non-  using a hydrophobic  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  Seliskar  by  from  probably  its is  about  57  References 1  B e r g g a r d , I., Cleve, H., Chim. 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