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Isolation and amino acid sequence of neurohypophysial hormones or Pacific chinook salmon (Oncorhynchus… Wilson, Nadine 1968

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ISOLATION  AND AMINO A C I D  NEUROHYPOPHYSIAL PACIFIC  HORMONES OF  CHINOOK  (Oncorhynchus  S E Q U E N C E OF  SALMON  tschawytscha) by  Nadine B.A.,  University  A THESIS THE  of  SUBMITTED  British  in  Columbia,  1955  I N P A R T I A L F U L F I L M E N T OF  REQUIREMENTS Doctor  Wilson  F O R T H E D E G R E E OF  of  the  Philosophy  Department of Zoology  We  accept  required  this  thesis  as  conforming  standard  THE UNIVERSITY  OF B R I T I S H  August,  COLUMBIA  1968  Nadine Wilson  1969  to  the  In p r e s e n t i n g  for  that  this  thesis  an a d v a n c e d d e g r e e  the  Study.  thesis  Library shall  I further  for  agree  at  in p a r t i a l  the U n i v e r s i t y of  make i t  that  freely  for  the  requirements  Columbia,  I agree  reference  and  e x t e n s i v e copying of  this  s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e Head o f my  or p u b l i c a t i o n  of  my w r i t t e n  Department  for  of  British  available  permission  D e p a r t m e n t o r b y h.iis r e p r e s e n t a t i v e s .  without  fulfilment  this  thesis  for  permission.  of  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada  August 9,  1968  Columbia  It  is  financial  understood  gain  shall  that  not  be  copying  allowed  ABSTRACT The n e u r o h y p o p h y s i a l  hormones o f P a c i f i c  salmon (Oncorhynchus tschawytscha)  chinook  were p u r i f i e d and  amino a c i d sequence o f both hormones  the  determined.  The e x t r a c t i o n and p u r i f i c a t i o n procedure  was  de-  v e l o p e d i n an e f f o r t t o maximize the y i e l d s o f pure hormones.  P i t u i t a r y g l a n d s were e x t r a c t e d a t 4°C u s i n g 0.2  acetic acid.  P u r i f i c a t i o n c o n s i s t e d of g e l f i l t r a t i o n ,  u l t r a f i l t r a t i o n , and i o n exchange. Sephadex G-15  columns was  Gel f i l t r a t i o n  used t o s e p a r a t e  o f the two hormones was  accomplished  on  neurohypophysial  p e p t i d e s from h i g h m o l e c u l a r weight m a t e r i a l .  exchangers:  M  Separation  on one o f t h r e e c a t i o n  c a r b o x y m e t h y l c e l l u l o s e , phosphocellulose or  sulfoethyl-Sephadex.  The hormones were e l u t e d from c a t i o n  exchange columns u s i n g a sodium or ammonium i o n c o n c e n t r a t i o n g r a d i e n t a t c o n s t a n t pH; pH 5 was  used f o r chromato-  graphy on c a r b o x y m e t h y l c e l l u l o s e and on p h o s p h o c e l l u l o s e ; pH 2.45  was  used f o r chromatography on  sulfoethyl-Sephadex.  The two hormones were p u r i f i e d f u r t h e r by rechromatography of i n d i v i d u a l hormones on another c a t i o n exchange medium.  The m a t e r i a l o b t a i n e d by rechromatography  pure as determined  by amino a c i d a n a l y s e s .  was  The y i e l d s o f  pure hormones a t t h e end o f p u r i f i c a t i o n p r o c e d u r e was 48% of t h e s t a r t i n g m a t e r i a l .  The s p e c i f i c a c t i v i t i e s o f t h e  two p u r i f i e d hormones were 145 and 229 o x y t o c i c u n i t s p e r m i l l i g r a m f o r Hormone I and Hormone I I r e s p e c t i v e l y . The  amino a c i d sequence o f Hormone I was d e t e r m i n e d  by a c o m b i n a t i o n o f t h r e e methods:  s u b t r a c t i v e Edman  d e g r a d a t i o n , p a r t i a l a c i d h y d r o l y s i s , and Dansyl-Edman technique.  The amino a c i d sequence o f Hormone I was found  t o be t h a t o f 4 - s e r i n e , The  8-isoleucine  oxytocin.  amino a c i d sequence o f Hormone I I was d e t e r m i n e d  by t h e Edman s u b t r a c t i v e method, t h e Dansyl-Edman t e c h n i q u e , and t h e m o b i l i t y o f t h e C - t e r m i n a l r e s i d u e on h i g h voltage  electrophoresis.  The amino a c i d sequence o f  Hormone I I was found t o be t h a t o f 8 - a r g i n i n e The  oxytocin.  two n e u r o h y p o p h y s i a l hormones d e s c r i b e d  salmon have amino a c i d sequences i d e n t i c a l w i t h described  from  those  from f o u r s p e c i e s o f Gadiformes and one s p e c i e s  of C y p r i n i f o r m e s  by o t h e r w o r k e r s .  Sa'lmoniformes' on t h e e v o l u t i o n a r y  The p o s i t i o n o f tree of t e l e o s t fishes  suggests t h a t t h e s e s t r u c t u r e s a r e c h a r a c t e r i s t i c o f a wide range o f t e l e o s t s .  iv T A B L E  I.  OF  C O N T E N T S  INTRODUCTION 1.  Background  2.  Characterization  3.  N e u r o h y p o p h y s i a l hormones o f v e r t e b r a t e s other than f i s h e s  4.  1 o f n e u r o h y p o p h y s i a l hormones . .  4  N e u r o h y p o p h y s i a l hormones o f f i s h e s o t h e r than t e l e o s t s  II.  3  6  5.  N e u r o h y p o p h y s i a l hormones o f t e l e o s t f i s h e s . .  6.  P r e s e n t s t u d y : n e u r o h y p o p h y s i a l hormones i n Oncorhynchus t s c h a w y t s c h a  8 10  EXPERIMENTAL PROCEDURES 1.  C o l l e c t i o n of p i t u i t a r y glands  13  2. 3.  B i o l o g i c a l assays A n a l y t i c a l methods  14  i. : ii. :  4.  E x t r a c t i o n of oxytocic pituitaries i. ii. iii.  5. 6.  Measurement o f p r o t e i n c o n c e n t r a t i o n . . 17 S p e c i f i c c o n d u c t i v i t y measurements . . . 18 a c t i v i t y from salmon  E x t r a c t i o n w i t h 0.2 M a c e t i c a c i d a t 4°C E x t r a c t i o n a t 100°C i n 0.25% a c e t i c a c i d o f acetone powder p r e p a r e d from frozen p i t u i t a r i e s A d d i t i o n a l e x t r a c t i o n methods  20 21 22  C o n c e n t r a t i o n o f crude and p a r t i a l l y purified extracts  23  Ultrafiltration  24  V  7.  Gel f i l t r a t i o n i. ii.  8.  ii. iii. iv.  27  P r e p a r a t i o n o f exchangers and column packing . . . . . . . B u f f e r s used i n c a t i o n exchange. . . . L o a d i n g and e l u t i o n o f c a t i o n exchange columns Repeated use o f t h e same column. . . .  28 31 31 32  Performic acid oxidation  33  10.  A c i d h y d r o l y s i s of peptides  34  11.  Amino a c i d i. ii.  analyses  Procedure  .  Quantitation of r e s u l t s  36 37  12.  P a r t i a l acid hydrolysis  38  13.  High v o l t a g e e l e c t r o p h o r e s i s  38  14.  Edman d e g r a d a t i o n  40  15.  S u b t r a c t i v e Edman method i n amino a c i d sequence d e t e r m i n a t i o n D a n s y l a t i o n method i n amino a c i d sequence determination i. D a n s y l a t i o n r e a c t i o n and h y d r o l y s i s . . ii. I d e n t i f i c a t i o n o f d a n s y l amino a c i d s by t h i n l a y e r chromatography  16.  III.  26  I o n exchange chromatography i.  9.  P r e p a r a t i o n o f t h e g e l and column packing L o a d i n g and e l u t i o n o f g e l f i l t r a t i o n columns  42  43 44  RESULTS A.  S t a b i l i t y of oxytocic a c t i v i t y i n the p i t u i t a r y t i s s u e and t i s s u e e x t r a c t s o f Oncorhynchus t schawytscha 1.  S t a b i l i t y of oxytocic a c t i v i t y s t o r a g e o f g l a n d s a t -196°C  during 42  vi 2.  B. C.  S t a b i l i t y o f o x y t o c i c a c t i v i t y i i n crude and p a r t i a l l y p u r i f i e d p i t u i t a r y extracts  E x t r a c t i o n o f n e u r o h y p o p h y s i a l hormones from salmon p i t u i t a r i e s  51  P u r i f i c a t i o n o f n e u r o h y p o p h y s i a l hormones of salmon  51  1.  G e l f i l t r a t i o n o f crude salmon pituitary extracts  54  2.  D e - s a l t i n g o f n e u r o h y p o p h y s i a l hormones .  61  3.  i . D e - s a l t i n g on Sephadex G-10 i i . D e - s a l t i n g by u l t r a f i l t r a t i o n . . . . S e p a r a t i o n o f n e u r o h y p o p h y s i a l hormones of salmon by i o n exchange chromatography  61 62  4.  5.  D.  49  64  p u r i f i c a t i o n o f salmon n e u r o h y p o p h y s i a l Hormone I by i o n exchange chromatography  74  P u r i f i c a t i o n o f salmon n e u r o h y p o p h y s i a l Hormone I I by i o n exchange chromatography  77  Amino a c i d sequence o f n e u r o h y p o p h y s i a l hormones o f Oneorhynchus t s c h a w y t s c h a 1.  ...  84  Amino a c i d sequence o f Hormone I i.  2.  Determination of N-terminal pentapeptide ii. C h a r a c t e r i z a t i o n of C-terminal tetrapeptide Amino a c i d sequence o f Hormone I I i. ii.  3.  Determination of N-terminal pentapeptide Determination of C-terminal tetrapeptide  R e a c t i o n y i e l d s encountered i n t h e c o u r s e o f amino a c i d sequence work. . .  84 89  92 94 97  vii IV.  DISCUSSION 1.  N e u r o h y p o p h y s i a l hormone c o n t e n t o f salmon pituitaries  2.  B i o l o g i c a l assays used i n i s o l a t i o n o f  100  n e u r o h y p o p h y s i a l hormones o f salmon  103  3.  E x t r a c t i o n o f n e u r o h y p o p h y s i a l hormones. . . . 104  4.  P u r i f i c a t i o n o f n e u r o h y p o p h y s i a l hormones. . . 106  5.  S t r u c t u r a l a n a l y s i s of neurohypophysial hormones o f t e l e o s t s i. Q u a n t i t a t i v e amino a c i d a n a l y s i s . . . . ii. Amino a c i d sequence s t u d i e s o f t h e l e s s b a s i c t e l e o s t hormone iii. Amino a c i d sequence s t u d i e s o f t h e more b a s i c t e l e o s t hormone . . . . . . . iv. S t r u c t u r e o f t h e 0. t s c h a w y t s c h a n e u r o h y p o p h y s i a l hormones v. Comments on t h e a n a l y t i c a l methods . . .  6. 7. 8.  113 113 113 117 118 119  S p e c i f i c a c t i v i t i e s o f salmon neurohypop h y s i a l hormones  119  R e l a t i v e p r o p o r t i o n s of neurohypophysial hormones i n t e l e o s t s  120  Evolutionary  125  LITERATURE CITED  considerations  . 132  viii L I S T I. II.  III. IV.  V.  VI.  VII. VIII. IX. X. XI. XII. XIII.  OF  T A B L E S  Munsick's b u f f e r e d t i s s u e b a t h s o l u t i o n  16  Comparison of the s p e c i f i c a c t i v i t i e s of the p i t u i t a r y samples of Oncorhynchus t s c h a w y t s c h a c o l l e c t e d at three d i f f e r e n t locations  48  E x t r a c t i o n o f o x y t o c i c a c t i v i t y from the p i t u i t a r i e s o f 0. t s c h a w y t s c h a  52-53  U l t r a f i l t r a t i o n of p a r t i a l l y p u r i f i e d p i t u i t a r y e x t r a c t s of 0. t s c h a w y t s c h a u s i n g D i a f l o UM-2 membranes  65  U l t r a f i l t r a t i o n of p a r t i a l l y p u r i f i e d p i t u i t a r y e x t r a c t s of 0. t s c h a w y t s c h a u s i n g D i a f l o UM-3 membranes  66-67  Ion exchange chromatography of n e u r o h y p o p h y s i a l hormones o f 0. t s c h a w y t s c h a : s p e c i f i c conduct i v i t i e s of e l u t e d hormones  73  Amino a c i d c o m p o s i t i o n ( r e s i d u e s per mole) o f n e u r o h y p o p h y s i a l Hormone I of 0. t s c h a w y t s c h a . .  79  Amino a c i d c o m p o s i t i o n ( r e s i d u e s p e r mole) of n e u r o h y p o p h y s i a l Hormone I I o f 0.tschawytscha.  83  .  The amino a c i d sequence of N - t e r m i n a l t e t r a p e p t i d e o f Hormone I of 0. t s c h a w y t s c h a  85  P a r t i a l a c i d h y d r o l y s i s of Hormone I f o l l o w i n g f i v e Edman d e g r a d a t i o n c y c l e s  87  I d e n t i f i c a t i o n o f d a n s y l d e r i v a t i v e s of amino a c i d s 6 and 7 o f Hormone I  90  The amino a c i d sequence of N - t e r m i n a l p e n t a p e p t i d e o f Hormone I I o f 0. t s c h a w y t s c h a  93  i d e n t i f i c a t i o n of d a n s y l d e r i v a t i v e s of amino a c i d s 6, 7 and 8 of Hormone I I  95  ix XIV. XV.  XVI. XVII.  Comparison o f n e u r o h y p o p h y s i a l hormone c o n t e n t s of t e l e o s t p i t u i t a r i e s  102  Comparison o f y i e l d s o b t a i n e d a t d i f f e r e n t stages of p u r i f i c a t i o n of neurohypophysial hormones  112  Comparison o f q u a n t i t a t i v e amino a c i d a n a l y s e s of t e l e o s t n e u r o h y p o p h y s i a l hormones  114  The G e n e t i c Code  127  X  L I S T  OF  I L L U S T R A T I O N S  FIGURE 1.  N e u r o h y p o p h y s i a l hormones o f v e r t e b r a t e s  2.  Monitoring of the e f f l u e n t of a g e l f i l t r a t i o n column by t h r e e d i f f e r e n t methods  19  3.  Loss o f o x y t o c i c a c t i v i t y i n crude salmon p i t u i t a r y e x t r a c t a t n e u t r a l and s l i g h t l y b a s i c pH a t 25°C  50  4.  P u r i f i c a t i o n sequence o f l a r g e s c a l e salmon pituitary extracts  55  5.  G e l f i l t r a t i o n o f crude salmon p i t u i t a r y e x t r a c t on B i o g e l P-2  56  Comparison o f g e l f i l t r a t i o n on Sephadex G-25 and Sephadex G-15  57  6.  2  7.  G e l f i l t r a t i o n o f crude salmon p i t u i t a r y ext r a c t u s i n g a tandem s e r i e s o f Sephadex G-15. . . 59  8.  Rechromatography o f p a r t i a l l y p u r i f i e d salmon p i t u i t a r y e x t r a c t s on Sephadex G-15 colums o f e q u a l bed volumes and d i f f e r e n t d i m e n s i o n s . . . . 60  9.  D e - s a l t i n g o f o x y t o c i n on Sephadex G-10  63  S e p a r a t i o n o f salmon n e u r o h y p o p h y s i a l hormones on Whatman CM-32  69  S e p a r a t i o n o f salmon n e u r o h y p o p h y s i a l hormones on S e l e c t a c e l p h o s p h o c e l l u l o s e  70  S e p a r a t i o n o f salmon n e u r o h y p o p h y s i a l hormones on SE-Sephadex.  72  10. 11. 12. 13.  Rechromatography o f Hormone I on SE-Sephadex.  . . 76  14.  Rechromatography o f Hormone I on Whatman CM-22. . 78  15.  Rechromatography o f Hormone I I on Whatman CM-22 . 82  xi 16. 17. 18.  I d e n t i f i c a t i o n o f amino a c i d 7 o f Hormone I as D N S - p r o l i n e  91  I d e n t i f i c a t i o n o f amino a c i d 7 o f Hormone I I as D N S - p r o l i n e  96  Family tree of t e l e o s t orders  131  xii A B B R E V I A T I O N S U S E D A.a. ala arg asn asp cys-cys  amino a c i d alanine arginine asparagine aspartic acid - cystine  A  CyS03H gin glu gly gly-NR^ his ileu leu lys met phe pro ser trp tyr thr val -  - cysteic acid glutamine glutamic acid glycine - glycinamide histidine isoleucine leucine lysine methionine phenylalanine proline serine tryptophan tyrosine threonine valine  DEAE  U C A G  uridine cytosine adenosine guanosine  - absorbance a t 280 nm*  o o n  2 80 nm CM - c a r b o x y m e t h y l CDNS-dansyl - t e r m i n a l - c a1-dimethylaminorboxyterminal naphthalene-5sulfonyl-  diethylaminoethyl  mho - 1/ohm ymole, ym - micromole nm  -  nanometer  N-terminal  -  amino-terminal  p.s.i. - pounds p e r square i n c h PTC-peptide - p h e n y l t h i o c a r b a m y l peptide SE - s u l f o e t h y l TCA  -  trichloroacetic acid  one absorbance u n i t i s t h a t amount o f substance which when d i s s o l v e d i n 1 ml o f s o l v e n t has an absorbance o f 1.0 i n a c e l l w i t h a 10 mm l i g h t path..(A;U.)'."  ACKNOWLEDGEMENTS  Foremost, I w i s h t o e x p r e s s my thanks t o Dr. M i c h a e l Smith f o r h i s e n t h u s i a s m , i n t e r e s t and f r i e n d s h i p as w e l l as f o r t h e s h a r i n g of h i s knowledge which I have enjoyed t h r o u g h o u t the p a s t t h r e e y e a r s .  P a r t i c u l a r thanks a r e due  t o M i s s Dorothy Kauffman, p r e s e n t l y of the U n i v e r s i t y of Washington, f o r her guidance i n the amino a c i d t e c h n i q u e s , and t o Dr. G.H.  D i x o n of the Department  of Biochemistry,  U n i v e r s i t y o f B r i t i s h Columbia, and Dr. John B l a c k ,  now  w i t h the U n i v e r s i t y of Oregon M e d i c a l S c h o o l f o r much h e l p and a d v i c e .  I am i n d e b t e d t o Dr. C. Finnegan of the D e p a r t -  ment o f Zoology f o r h i s c o n t i n u i n g i n t e r e s t and encouragement t h r o u g h o u t my g r a d u a t e work.  A word of s p e c i a l a p p r e c i a t i o n  i s extended t o Mr. S. F a l l e r t and the s t a f f o f Green R i v e r H a t c h e r y , Washington, f o r t h e i r c o - o p e r a t i o n d u r i n g the c o l l e c t i o n of p i t u i t a r y glands. K. Henze of the Department  I would a l s o l i k e t o thank Mr.  of Physiology, U n i v e r s i t y of  B r i t i s h Columbia, f o r drawing the i l l u s t r a t i o n s , and Mr.  V.  W y l i e and my o t h e r c o l l e a g u e s f o r a s s i s t a n c e i n c o l l e c t i n g o f the g l a n d s and f o r many o t h e r i n s t a n c e s of h e l p and encouragement. The r e s e a r c h work was done i n i t i a l l y i n the Vancouver L a b o r a t o r y o f the F i s h e r i e s Research Board o f Canada, and  xiv  l a t t e r l y i n t h e Department o f B i o c h e m i s t r y a t t h i s u n i v e r sity.  F i n a n c i a l s u p p o r t was p r o v i d e d by t h e M e d i c a l  Research  C o u n c i l o f Canada through a g r a n t t o Dr. M i c h a e l  Smith. L a s t , I would l i k e t o acknowledge t h e encouragement, h e l p and p a t i e n c e o f my husband, S t u a r t , throughout p e r i o d o f my graduate  work.  the  To my c h i l d r e n G r e g o r y , K a t y a , and Duncan  I.  (1)  INTRODUCTION  Background Hormones h a v i n g o x y t o c i c , p r e s s o r and a n t i d i u r e t i c  a c t i v i t y have been found i n t h e neurohypophyses o f r e p r e sentatives of a l l classes of vertebrates.  The neurohypo-  p h y s i s i s d e f i n e d as t h e a n a t o m i c a l s t r u c t u r e c o n s i s t i n g o f hypothalamic  n u c l e i o f neurons, t h e i r axons p a s s i n g down  the i n f u n d i b u l a r s t a l k , and t h e i n f u n d i b u l a r p r o c e s s o r p a r s nervosa o f t h e p i t u i t a r y g l a n d the n e u r o h y p o p h y s i a l  (1).  hormones i s thought t o occur i n t h e  c e l l b o d i e s o f neurons which comprise nuclei  (2).  The b i o s y n t h e s i s o f  the hypothalamic  I n mammals n u c l e i s u p r a o p t i c u s and paraven-  t r i c u l a r i s have been d e s c r i b e d and, i n f i s h e s , praeopticus (3).  nucleus  The hormones a r e t r a n s p o r t e d i n a s s o c i a -  t i o n w i t h a p r o t e i n o r p r o t e i n s by a x o p l a s m i c  flow along  the i n f u n d i b u l u m f o r s t o r a g e i n , and l a t e r r e l e a s e from, the p a r s n e r v o s a ( 4 ) . These p e p t i d e hormones c o n t a i n a c h a i n o f n i n e amino a c i d s , which i n g e n e r a l s t r u c t u r e f o l l o w a u n i f o r m p a t t e r n (Figure 1).  They have a d i s u l f i d e b r i d g e between c y s t e i n e  r e s i d u e s a t p o s i t i o n 1 (the amino end o f t h e p e p t i d e  chain)  2  1 2  (b)  2  T • 1  (e)  2  T— 1  (d)  4  5  2  T—  6  O-O—  1 2 3 4 5 6 7 8 9 •-•-ILEU-GLN-#-*-#-LEU-#  1  (c)  3  t  (a)  •-  3 4 5 6 PHE -- G L N - * -  7  •  8 9 ARG-  -•- -•  GLN-#-Y~  3 ILEU -  4 5 6 7 GLN-#-^-  4  5  6  7  9 8 LYS-  -•-  8 •ARG  8  9  O-  •  1 2 3 4 5 6 7 8 9 (f) y-0-lLEU-GLN—#— ^ - » - I L E U - +  (0) 3 PHE- -  7  1 2 3 4 5 6 7 8 9 • • I L E U S E R # « # I L E U - # ^-•-ILEU-SER-#-J-  1 2 3 4 5 6 7 8 9 (h) y• - I L E U - S E R - » - ^ - » - G L N - »  9  F i g u r e 1: N e u r o h y p o p h y s i a l hormones o f v e r t e b r a t e s Filled circles: p o s i t i o n s o c c u p i e d by i d e n t i c a l amino a c i d s t h r o u g h o u t v e r t e b r a t e s (1 - 1/2 Cys; 2 - T y r ; 5 - Asn; 6 1/2 Cys; 7 - P r o ; 9 - G l y - N H ) • Empty c i r c l e s : positions of amino a c i d s u b s t i t u t i o n s . 2  (a) g e n e r a l i z e d s t r u c t u r e ; (b) o x y t o c i n ; (c) 3-phe, 8-arg o x y t o c i n ( a r g i n i n e v a s o p r e s s i n ) ; (d) 3-phe, 8 - l y s o x y t o c i n ( l y s i n e v a s o p r e s s i n ) ; (e) 8-arg o x y t o c i n ( a r g i n i n e v a s o t o c i n ) ; (f) 8 - i l e u o x y t o c i n ( m e s o t o c i n ) ; (g) 4-ser, 8 - i l e u o x y t o c i n ; ( i s o t o c i n ) ; (h) 4 - s e r , 8-gln o x y t o c i n ( g l u m i t o c i n ) .  3 and p o s i t i o n 6.  In a l l n a t u r a l l y o c c u r r i n g neurohypophysial  hormones f o r which amino a c i d sequence has been  determined,  t y r o s i n e , a s p a r a g i n e , p r o l i n e , and g l y c i n a m i d e have been found i n p o s i t i o n s 2, 5, 7, and 9, r e s p e c t i v e l y .  Variations  i n s t r u c t u r e i n v o l v e amino a c i d s u b s t i t u t i o n s i n p o s i t i o n s 3, 4, and 8. (2)  C h a r a c t e r i z a t i o n of Neurohypophysial Neurohypophysial  Hormones  hormones a r e o f t e n c h a r a c t e r i z e d  on t h e b a s i s o f t h e i r p h a r m a c o l o g i c a l a c t i v i t i e s .  Compari-  son o f t h e a c t i v i t y r a t i o s i n a number o f b i o l o g i c a l  assays  ( p h a r m a c o l o g i c a l p r o f i l e ) o f an unknown hormone w i t h t h e a c t i v i t i e s o f known p e p t i d e s has been used t o p r e d i c t t h e c h e m i c a l i d e n t i t y o f t h e unknown w i t h c o n s i d e r a b l e p r e cision  ( 5 ) . T h i s procedure  i s o f t e n used because t h e  s e n s i t i v i t y o f t h e b i o l o g i c a l assays and t h e i r  specificity  a l l o w s t h e e x a m i n a t i o n o f v e r y s m a l l amounts of impure hormones.  However, p h a r m a c o l o g i c a l s t u d i e s can g i v e e r r o n e -  ous o r ambiguous r e s u l t s  ( 5 ) . The d e t e r m i n a t i o n o f t h e  amino a c i d c o n t e n t o f a hormone and t h e d e f i n i t i o n o f the sequence i n which t h e amino a c i d s a r e arranged i s conseq u e n t l y t h e most fundamental T h i s has been attempted  approach  to characterization.  l e s s o f t e n than p h a r m a c o l o g i c a l  c h a r a c t e r i z a t i o n because i t r e q u i r e s more hormone m a t e r i a l and t h i s must be i n a c o m p l e t e l y pure form.  4 Studies  by e i t h e r  pharmacological  or  chemical  methods have l e d t o t h e c o n c l u s i o n t h a t i n each species  there  exceptions  a r e two n e u r o h y p o p h y s i a l  to this  hormones.  has been d e t e c t e d  i n which the presence of three  i n  (6) a n d  neurohypophy-  sial  hormones c a n n o t be r u l e d o u t a t t h e p r e s e n t  (3)  N eu r o h y p o p h y s i a 1 than  Possible  g e n e r a l i z a t i o n are the cyclostomes  which o n l y one a c t i v e p r i n c i p l e elasmobranchs  vertebrate  Hormones i n V e r t e b r a t e s  time ( 7 ) .  Other  Fishes  • The f i r s t  structural  analysis of  neurohypophysial  hormones was done b y d u V i g n e a u d ' s g r o u p o n 3-phe,  8-arg  oxytocin  (9) o f  ox  (arginine vasopressin)(8)  pituitaries.  closely  (sheep,  substances  Studies horse,  and on o x y t o c i n  o n f o u r o t h e r mammals  man, a n d w h a l e )  i n each species w i t h  logical by  survey  hormones  (11).  confirmed  (lysine vasopressin)  replaces  3-phe, 8-arg o x y t o c i n  i n one amino a c i d  Ferguson and H e l l e r oxytocin  A  pharmaco-  i n order  o f mammals  t h e wide d i s t r i b u t i o n o f  One e x c e p t i o n  oxytocin  differ  (10).  composition  o f r e p r e s e n t a t i v e s o f most f a m i l i e s  Ferguson and H e l l e r  these  and r e v e a l e d two  t h e amino a c i d  o f o x y t o c i n and 3-phe, 8-arg o x y t o c i n  followed  found  i s 3-phe,  from hog p i t u i t a r i e s (12).  i n position  8-lys where i t  The two p e p t i d e s 8.  The s u r v e y  i n d i c a t e d t h e p r e s e n c e o f 3-phe,  Suiformes (11).  of 8-lys  5 O x y t o c i n has been found i n b i r d s ( 1 3 ) , ( 1 4 ) , b u t 3-phe, 8-arg o x y t o c i n i s r e p l a c e d by 8-arg o x y t o c i n (arginine vasotocin)  (10,  (15).  A s y n t h e t i c p r e p a r a t i o n o f 8 - a r g i n i n e o x y t o c i n was r e p o r t e d b e f o r e t h e hormone was found i n n a t u r e ( 1 6 ) . I t subsequently  has been found i n p i t u i t a r i e s o f r e p r e s e n -  t a t i v e s of a l l vertebrate c l a s s e s with the exception of mammals ( 1 7 ) . I n mammals, however, 8 - a r g i n i n e o x y t o c i n has been r e p o r t e d from t h e p i n e a l g l a n d o f beef on t h e b a s i s of c h r o m a t o g r a p h i c and p h a r m a c o l o g i c a l sequently of p i g s  evidence (18).  Sub-  8 - l y s o x y t o c i n was found i n t h e p i n e a l b o d i e s  (19). I t i s d i f f i c u l t t o assess the s i g n i f i c a n c e  of these f i n d i n g s , because a subsequent attempt t o i s o l a t e 8-arg o x y t o c i n from beef p i n e a l was n o t s u c c e s s f u l ( 2 0 ) . According  t o the data a v a i l a b l e a t present,  pitui-  t a r y 8 - a r g i n i n e o x y t o c i n appears t o have remained unchanged t h r o u g h o u t v e r t e b r a t e e v o l u t i o n u n t i l Mammalia.  The o t h e r  hormone, on t h e c o n t r a r y , has been s u b j e c t t o e v o l u t i o n a r y change. chicken  O x y t o c i n has been c h e m i c a l l y i d e n t i f i e d (13) and p h a r m a c o l o g i c a l l y from t u r k e y  two o t h e r b i r d s (14) . i n lower v e r t e b r a t e s disputed subject.  from  (21) and  The p o s s i b l e e x i s t e n c e o f o x y t o c i n ( r e p t i l e s , amphibians, f i s h e s ) i s a  Another neurohypophysial  homologue,  8 - i l e u o x y t o c i n (mesotocin) has been c h e m i c a l l y c h a r a c t e r i z e d i n s e v e r a l amphibian s p e c i e s  (22), (23), (24),  (25).  However, w h i l e Acher d e s c r i b e d 8 - i l e u o x y t o c i n from Rana e s c u l e n t a i n c h e m i c a l terms  ( 2 5 ) , Munsick  identified  o x y t o c i n p h a r m a c o l o g i c a l l y from Rana p i p i e n s (26) .  Acher  has i d e n t i f i e d 8 - i l e u o x y t o c i n on t h e b a s i s o f amino a c i d a n a l y s i s from some r e p t i l i a n s p e c i e s  (27), (28), y e t a  r e c e n t r e p o r t on c o b r a , N a j a n a j a , suggests t h e presence of b o t h o x y t o c i n and 8 - i l e u o x y t o c i n ( 2 9 ) . (4)  N e u r o h y p o p h y s i a l Hormones o f F i s h e s Other than T e l e o s t s I n c y c l o s t o m e s o n l y one n e u r o h y p o p h y s i a l hormone,  8-arg o x y t o c i n , has been i n d i c a t e d p h a r m a c o l o g i c a l l y ( 6 ) , (17) . The hormone, 4-ser, 8-gln o x y t o c i n ( g l u m i t o c i n ) was c h a r a c t e r i z e d c h e m i c a l l y by A c h e r s group i n 1965 from 1  elasmobranch  f i s h e s ( 3 0 ) , and s u b s e q u e n t l y found by t h e  same group i n a d d i t i o n a l elasmobranch  species  (31),  (32).  The s t r u c t u r e o f 4-ser, 8-gln o x y t o c i n was c o n f i r m e d by s n y t h e s i s by K l i e g e r ( 3 3 ) . A c h e r s work i n d i c a t e s t h e 1  presence o f s m a l l amounts o f t h e second hormone, 8-arg o x y t o c i n , i n elasmobranchs.  The presence o f o x y t o c i c  p r i n c i p l e s o t h e r than 4-ser, 8-gln o x y t o c i n has n o t y e t been e x c l u d e d f o r some elasmobranchs  ( 7 ) , ( 3 4 ) . The study  of Sawyer on Hydrolagus c o l l i e i f a i l e d t o f i n d  4-ser,  8-gln o x y t o c i n i n t h i s h o l o c e p h a l i a n r a t f i s h , and i n d i c a t e d t h a t o x y t o c i n may be p r e s e n t t o g e t h e r w i t h 8-arg o x y t o c i n (34) .  7 In D i p n o i , i n a d d i t i o n t o 8-arg o x y t o c i n , b o t h o x y t o c i n and 8 - i l e u o x y t o c i n have been c h a r a c t e r i z e d pharmacologically.  F o l l e t and H e l l e r have t e n t a t i v e l y i d e n t i -  f i e d 8 - i l e u o x y t o c i n from a s p e c i e s o f A f r i c a n l u n g f i s h ( P r o t o p t e r u s a e t h i o p i c u s ) and from an A u s t r a l i a n s p e c i e s , Neoceratodus f o r s t e r i  ( 2 3 ) . P i c k e r i n g and McWatters,  however, r e p o r t e d o x y t o c i n from a South American s p e c i e s , L e p i d o s i r e n paradoxa ( 3 5 ) . Sawyer and van Dyke a l s o found o x y t o c i n i n t h e A f r i c a n P. a e t h i o p i c u s  ( 3 6 ) . Sawyer's  specimens were c o l l e c t e d from a d i f f e r e n t l a k e than were those o f H e l l e r .  Sawyer i s a l s o r e p o r t e d t o have found 8-  i l e u o x y t o c i n i n another b a t c h o f g l a n d s from t h e same A f r i c a n species  (35). A l l of the i d e n t i f i c a t i o n s of the  n e u r o h y p o p h y s i a l hormones o f D i p n o i were on a  pharmacological  basis. Sawyer and van Dyke i n d i c a t e t h e presence o f 8-arg o x y t o c i n as w e l l as 8 - i l e u o x y t o c i n i n t h e p r i m i t i v e b i c h i r , Polypterus  ( 3 6 ) . The c l a s s i f i c a t i o n o f P o l y p t e r u s  by d i f f e r e n t a u t h o r s v a r i e s . with the Chondrostei w i t h i n the Chondrostei  Polypterus  (37); C o l b e r t a l s o placed P o l y p t e r i n i (38) ; N i k o l s k i i i n c l u d e d them i n t h e  A c t i n o p t e r y g i i , but separated F o l l e t and H e l l e r s e p a r a t e B r a c h i o p t e r y g i i (40).  Romer c l a s s i f i e d  adopted  them from C h o n d r o s t e i ( 3 9 ) ;  them from t h e A c t i n o p t e r y g i i as  8 P r e s e n t knowledge of the n e u r o h y p o p h y s i a l hormones of the o t h e r C h o n d r o s t e i r e l i e s e x c l u s i v e l y on the pharmacol o g i c a l d a t a of F o l l e t and detected  o n l y one  H e l l e r (40).  studies  n e u r o h y p o p h y s i a l hormone i n the  Polyodon s p a t h u l a .  paddlefish,  On the o t h e r hand, they found two  mones i n t h r e e A c i p e n s e r s p e c i e s t i v e l y i d e n t i f i e d as 8 - a r g i n i n e the p a d d l e f i s h .  Their  The  examined.  One  was  hortenta-  oxytocin, also present i n  second hormone remained u n i d e n t i f i e d  s i n c e i t r e p r e s e n t e d o n l y a s m a l l f r a c t i o n (under 4%)  of  the  t o t a l a c t i v i t y found. The  members of H o l o s t e i i n c l u d e d i n the survey of  F o l l e t and H e l l e r i n d i c a t e d the p r e s e n c e , i n a d d i t i o n t o 8-arg o x y t o c i n , of a p r i n c i p l e i n d i s t i n g u i s h a b l e chromatog r a p h i c a l l y or p h a r m a c o l o g i c a l l y 4-ser, 8 - i l e u o x y t o c i n (5)  from the t e l e o s t hormone,  (isotocin, ichthyotocin)(40).  Neurohypophys1a1 Hormones o f T e l e o s t  Fishes  I n t e l e o s t f i s h e s examined up t o p r e s e n t two have been found: tocin.  The  4-ser, 8 - i l e u o x y t o c i n , and  hormones  8-arg oxy-  presence of the then s t i l l u n i d e n t i f i e d 4-ser,  8 - i l e u o x y t o c i n was  suggested by H e l l e r e t a l . (41) on  b a s i s of b i o l o g i c a l assays of p o l l a c k p i t u i t a r i e s ,  the  Pollachius  virehs. The  f i r s t c h e m i c a l d a t a on the s t r u c t u r e of t h i s  hormone were p u b l i s h e d  by Acher e t 'al. i n 1962  group s t u d i e d t h r e e marine s p e c i e s  (42) .  new  This  of the f a m i l y Gadidae:  p o l l a c k P o l l a c h i u s v i r e n s , hake M e r l u c c i u s m e r l u c c i u s  and  b i b cod Gadus l u s c u s .  The  o x y t o c i n was  s h o r t l y a f t e r Acher's r e p o r t by  confirmed  s t r u c t u r e of 4-ser, 8 - i l e u  independent s y n t h e s e s of Guttman (4) and J o h l (44).  the  Sawyer's  study of t h i s hormone from P o l l a c h i u s v i r e n s demonstrated pharmacological  i d e n t i t y of the p o l l a c k hormone and  s y n t h e t i c standard  (45).  the  8-Arg o x y t o c i n has a l s o been i n -  d i c a t e d , but not i d e n t i f i e d c h e m i c a l l y i n the above s t u d i e s of n e u r o h y p o p h y s i a l hormones of marine Gadidae. I n 1965,  4-ser, 8 - i l e u o x y t o c i n and  8-arg o x y t o c i n  were r e p o r t e d from a f r e s h water t e l e o s t , C y p r i n u s c a r p i o (Family C y p r i n i d a e )  by Acher's group.  of both hormones was  The  identification  based on q u a n t i t a t i v e amino a c i d  a n a l y s i s and on b i o l o g i c a l assays  (46).  8-Arg o x y t o c i n appears t o be as u b i q u i t o u s  in  t e l e o s t f i s h e s as i t i s i n a l l v e r t e b r a t e c l a s s e s w i t h  the  e x c e p t i o n of mammals (17) .  was  Shortly a f t e r t h i s peptide  f i r s t s y n t h e s i z e d i n the l a b o r a t o r y  (16), i t was  i n p o l l a c k by H e l l e r and P i c k e r i n g (41) , (47) . amino a c i d a n a l y s e s  identified The  of 8-arg o x y t o c i n from t e l e o s t f i s h  were r e p o r t e d i n 1961  from Gadus l u s c u s by Acher (48)  by Rasmussen from U r o p h y c i s t e n u i s  (49) , (50).  P o l l a c h i u s v i r e n s and M e r l u c c i u s m e r l u c c i u s  Two  (51).  and  species,  have been used  as s o u r c e s f o r amino a c i d sequence s t u d i e s r e p o r t e d Acher  first  by  10 Thus i t can be s a i d t h a t f i v e s p e c i e s of t e l e o s t f i s h have so f a r been examined on a c h e m i c a l b a s i s w i t h r e s p e c t t o n e u r o h y p o p h y s i a l hormones.  These s p e c i e s a r e :  P o l l a c h l u s v i r e n s , M e r l u c c i u s m e r l u c c i u s , Gadus l u s c u s , C y p r i n u s c a r p i o and U r o p h y c i s t e n u i s (one hormone o n l y i d e n t i f i e d from the l a t t e r s p e c i e s ) as w e l l as Scombridae s p e c i e s r e f e r r e d t o by Acher i n 1967  (32) .  The pharmaco-  l o g i c a l s u r v e y of F o l l e t and H e l l e r d e a l t w i t h the f o l l o w i n g s p e c i e s of t e l e o s t f i s h :  Gadus c a l l a r i a s , Esox l u s c i u s ,  A n g u i l l a a n g u i l l a , C y p r i n u s sp. and Salmo i r r i d e u s .  The  t e n t a t i v e i d e n t i f i c a t i o n s o f 4-ser, 8 - i l e u o x y t o c i n and 8-arg o x y t o c i n i n t h i s s u r v e y are based on b i o a s s a y s of f r a c t i o n s o b t a i n e d by paper chromatography of p i t u i t a r y e x t r a c t s (40). I n t h e i r survey of n e u r o h y p o p h y s i a l hormones of  non-  mammalian v e r t e b r a t e s , H e l l e r and P i c k e r i n g have i d e n t i f i e d , p h a r m a c o l o g i c a l l y , the presence of 8-arg o x y t o c i n i n the rainbow t r o u t , Salmo i r r i d e u s  (52).  I n another s u r v e y , on  bony f i s h e s and c y c l o s t o m e s , the same group of workers p h a r m a c o l o g i c a l l y i d e n t i f i e d 4-ser, 8 - i l e u o x y t o c i n from S. i r r i d e u s (40). (6)  P r e s e n t Study:  Neurohypophys1a1 Hormones i n  'Oneorhync'h'us t s c h a w y t s c h a The p o s i t i o n of s a l m o n i d f i s h e s on the e v o l u t i o n a r y t r e e of the t e l e o s t s makes the s a l m o n i f o r m o r d e r p a r t i c u l a r l y  11 s i g n i f i c a n t f o r study.  Romer c o n s i d e r s Salmoniformes  p r i m i t i v e s t o c k a t the base of the e v o l u t i o n a r y  as a  line  l e a d i n g toward more advanced forms of t e l e o s t s (37)  .  The  f a m i l i e s of t e l e o s t s whose n e u r o h y p o p h y s i a l hormones have been s t u d i e d p r e v i o u s l y  (Gadidae, C y p r i n i d a e and Scombridae)  a r e p o s i t i o n e d by Romer m o d e r a t e l y w e l l a l o n g the of  the t e l e o s t e v o l u t i o n a r y t r e e  (37).  branches  Because of t h i s  the c h a r a c t e r i z a t i o n of n e u r o h y p o p h y s i a l hormones i n salmonids may  be c o n s i d e r e d an i m p o r t a n t s t e p i n the study of e v o l u t i o n  of t e l e o s t hormones. The o b j e c t i v e of the p r e s e n t work was  the i s o l a t i o n  and c h a r a c t e r i z a t i o n of the n e u r o h y p o p h y s i a l hormones of the P a c i f i c chinook salmon, Oncorhynchus t s c h a w y t s c h a .  Deter-  m i n a t i o n of the s t r u c t u r e by c h e m i c a l r a t h e r than pharmacol o g i c a l a n a l y s i s was  chosen, because,  as i n d i c a t e d e a r l i e r ,  t h e method i s p o t e n t i a l l y l e s s ambiguous.  Exhaustive  c h e m i c a l s t u d i e s of t e l e o s t n e u r o h y p o p h y s i a l hormones have n o t been found i n the l i t e r a t u r e .  T h i s a l s o made i t d e s i -  r a b l e t o do a c h e m i c a l study i n the p r e s e n t i n v e s t i g a t i o n . The study of amino a c i d c o m p o s i t i o n and sequence of the two n e u r o h y p o p h y s i a l hormones which have been i s o l a t e d i n the c o u r s e o f t h i s work from salmon was p o s s i b l e f o r two major r e a s o n s . was  F i r s t , a l a r g e number of p i t u i t a r y g l a n d s  a v a i l a b l e because o f the s l a u g h t e r i n g o f s e x u a l l y mature  f i s h which accompanies h a t c h e r y c u l t u r e of P a c i f i c salmon  12 (53).  Secondly, the s t r u c t u r a l work was made p o s s i b l e by  such r e c e n t advances i n p e p t i d e sequencing methods as the Dansyl method of Gray and H a r t l e y (54) and by the i n c r e a s e d s e n s i t i v i t y of automatic amino a c i d a n a l y z e r s which became a v a i l a b l e i n the course of the l a s t decade.  I t was  thus  p o s s i b l e to determine the sequence of amino a c i d s i n one to two micromoles  of neurohypophysial hormones, which i n  terms of weight r e p r e s e n t s one t o two m i l l i g r a m s of p u r i f i e d material. The d e t e r m i n a t i o n of amino a c i d sequence r e q u i r e s m a t e r i a l of a t l e a s t 90% p u r i t y .  In the p r e s e n t study an  e x t r a c t i o n and p u r i f i c a t i o n procedure on the p r e p a r a t i v e s c a l e was  developed w i t h the aim of m i n i m i z i n g l o s s e s of  m a t e r i a l a t each s u c c e s s i v e step of p r o c e s s i n g . methods, m o l e c u l a r e x c l u s i o n , u l t r a f i l t r a t i o n and  Extraction cation  exchange techniques were e x t e n s i v e l y examined l e a d i n g t o a r e l a t i v e l y simple procedure f o r i s o l a t i o n of pure hormones i n good y i e l d s .  13  II.  (1)  Collection  EXPERIMENTAL PROCEDURES  of P i t u i t a r y  Glands  P i t u i t a r y g l a n d s o f spawning Oncorhynchus t s c h a w y t s c h a were c o l l e c t e d  a t h a t c h e r i e s i n Washington S t a t e .  The  used i n the f i r s t p a r t of t h i s work were c o l l e c t e d 1963,  1964,  Hatchery  and 1965 a t t h r e e l o c a t i o n s :  glands  during  Green R i v e r S t a t e  (near Auburn) and S p r i n g Creek and L i t t l e White  F e d e r a l H a t c h e r i e s (near B i n g e n ) . were c o l l e c t e d  o n l y a t Green R i v e r .  I n 1966  and 1967  glands  The c o l l e c t i o n took  p l a c e i n September and October, the spawning season i n those a r e a s . The f i s h were k i l l e d by h a t c h e r y workers and p r o d u c t s removed.  gonadal  The s l a u g h t e r i n g t e c h n i q u e s differed'.:  from one h a t c h e r y t o a n o t h e r .  A t Green R i v e r f i s h were  k i l l e d by severance o f the s p i n a l chord a t the l e v e l of the operculum.  A t S p r i n g Creek and L i t t l e White h a t c h e r i e s ,  the f i s h were k i l l e d by a blow on the head, which o f t e n r e s u l t e d i n s k u l l f r a c t u r e and c e r e b r a l haemmorhage. U s i n g an e l e c t r i c b o r e r , a c y l i n d r i c a l c o r e was r e moved from the head of the f i s h i n such a manner as t o i n c l u d e the p i t u i t a r y g l a n d s .  The d e t a i l s of the  procedure  have been d e s c r i b e d by T s u y u k i , Schmidt and Smith Male and female c o r e s were k e p t s e p a r a t e l y .  The  (53). intact  14 g l a n d s were removed from i s o l a t e d c o r e s u s i n g a p o i n t e d laboratory spatula.  The g l a n d s were p l a c e d i n t o screw cap  p l a s t i c j a r s , l a b e l l e d and k e p t i n s o l i d carbon d i o x i d e u n t i l a r r i v a l a t Vancouver, B.C.  I n t h i s manner t h e g l a n d s  were f r o z e n a t most w i t h i n two hours a f t e r t h e death of t h e fish. The l e n g t h o f s t o r a g e time i n s o l i d carbon d i o x i d e v a r i e d w i t h t h e s i t e and date of c o l l e c t i o n . years  (1963 t o 1965) t h e g l a n d s remained  In the e a r l i e r  i n solid  carbon  d i o x i d e up t o f i v e days, and upon a r r i v a l a t t h e l a b o r a t o r y were s t o r e d i n l i q u i d n i t r o g e n (-196°C). years  (1966 and 1967), t h e g l a n d s remained  In  subsequent  i n solid  d i o x i d e f o r a maximum p e r i o d o f t w e l v e hours.  carbon  Upon a r r i v a l  i n Vancouver t h e s e g l a n d s were s t o r e d i n a -80°C f r e e z e r (Revco, U l t r a Low  Temperature).  The number o f g l a n d s c o l l e c t e d v a r i e d from y e a r t o year.  About 7,800 g l a n d s were c o l l e c t e d i n 1966 and 3,900  g l a n d s i n 1967.  T h i s r e p r e s e n t e d a p p r o x i m a t e l y 780 and 390  grams o f t i s s u e ,  respectively.  (2)  Biological  Assays  The a s s a y s , based on c o n t r a c t i o n o f r a t u t e r u s i n presence of o x y t o c i c s u b s t a n c e s , were performed  according  t o H o l t o n (55) on v i r g i n W i s t a r l a b o r a t o r y r a t s , w e i g h i n g from 180 t o 230 gm.  The stage o f t h e e s t r o u s c y c l e was de-  t e r m i n e d by m i c r o s c o p i c e x a m i n a t i o n of an u n s t a i n e d v a g i n a l  smear, and o n l y a n i m a l s i n p r o - e s t r u s o r e s t r u s were used f o r t h e experiments  (5).  Rats were k i l l e d w i t h an overdose  of e t h e r , whole u t e r i e x c i s e d and one horn used f o r t h e assay.  The t i s s u e was suspended  i n the b u f f e r e d p h y s i o l o -  g i c a l s a l i n e recommended by Munsick  (Table 1 ) . The c e r v i c a l  end o f t h e u t e r i n e horn was anchored t o a g l a s s a i r l e a d a t the bottom o f t h e t i s s u e b a t h , w h i l e t h e ovary was a t t a c h e d by means o f a t h r e a d t o t h e arm o f a kymograph (C.F. Palmer Ltd.).  The l e v e r arm o f t h e kymograph was e i t h e r n o t  w e i g h t e d , o r was weighted w i t h a p i e c e o f p l a s t i c i n e w e i g h i n g up t o 0.5 gm.  The temperature o f t h e j a c k e t e d g l a s s  t i s s u e b a t h (volume 10 ml) was m a i n t a i n e d c o n s t a n t a t 35°C by a c i r c u l a t i n g water pump (Haake, Model F ) . A t t h e end o f each c o n t r a c t i o n t h e e n t i r e  solution  was removed through an o u t l e t a t t h e bottom o f t h e t i s s u e b a t h u s i n g t h e s u c t i o n o f a water a s p i r a t o r .  The u t e r u s was  r i n s e d i m m e d i a t e l y w i t h Munsick's s o l u t i o n and then r e suspended  i n t h e same s o l u t i o n i n p r e p a r a t i o n f o r t h e n e x t  bioassay.  Munsick's s o l u t i o n was d i s p e n s e d from a p l a s t i c  squeeze b o t t l e k e p t i n a water b a t h a t 35°C.  The pH o f  b o t h t h e t i s s u e b a t h and t h e s t o c k Munsick's s o l u t i o n was m a i n t a i n e d a t 7.4 - 7.5 by c o n s t a n t b u b b l i n g o f 5% CO2 i n a i r mixture.  T h i s a l s o s e r v e d t o mix t h e s o l u t i o n s i n t h e  t i s s u e bath. Syntocinon-10 a standard.  (Sandoz P h a r m a c e u t i c a l s ) was used as  A one ml ampule c o n t a i n i n g 10 I n t e r n a t i o n a l  16 T A B L E Munsick's  A.  Stock  I  B u f f e r e d T i s s u e Bath S o l u t i o n (56)  solution:  NaCl 120.67 gm  (18 L)  NaHCO-j 46.62 gm  K C l 8.275 gm 0.02% B.  phenol r e d 270 ml  Phosphate b u f f e r , s t o c k : (a) 22.714 gm N a H P 0 to 1,000 ml 2  4  d i l u t e d i n hot d i s t i l l e d  water  (b) 5.520 gm Nal^PO^ d i l u t e d as above (a) and (b) were mixed i n a p p r o x i m a t e l y e q u a l q u a n t i t i e s u n t i l pH o f 7.4 was o b t a i n e d and t h e r e s u l t i n g s o l u t i o n was r e f r i g e r a t e d .  C.  Working s o l u t i o n :  988 m l o f A 10 m l o f B 1 ml o f 0.5 M C a C l 500 mg g l u c o s e  F o r t h e sake o f convenience made more c o n c e n t r a t e d  t h e s t o c k s o l u t i o n A was o f t e n  ( i n three instead of eighteen l i t e r s )  and a 165 ml q u a n t i t y d i l u t e d t o 988 ml as r e q u i r e d .  17 U n i t s of o x y t o c i n per ml was  d i l u t e d t o 100 ml w i t h the  same b u f f e r as f o r the unknown. Q u a l i t a t i v e assays:  one p o i n t assays were used t o  m o n i t o r f r a c t i o n a t i o n of m o l e c u l a r e x c l u s i o n and i o n exchange columns, and u l t r a f i l t r a t i o n f i l t r a t e s . a s s a y s , however, were performed active  on a l l p o o l e d  Quantitative biologically  fractions. Q u a n t i t a t i v e assays:  f o u r p o i n t assays were p e r -  formed a c c o r d i n g t o the method o f H o l t o n l i m i t of the s t a n d a r d e r r o r was  (55).  When the  t o be c a l c u l a t e d , f i v e t o  t e n groups of f o u r doses were used f o r each unknown.  For  a q u i c k check of t h e a c t i v i t y of an unknown sample (e.g. when the e f f l u e n t peaks of chromatographic  s e p a r a t i o n s were  t o be d e f i n e d ) o n l y one t o t h r e e groups of f o u r assays were performed. F o r assays of t h e o x y t o c i c e f f e c t of hormone p r e p a r a t i o n s i n the presence o f magnesium i o n (57), magnesium c h l o r i d e was  added t o Munsick's  s o l u t i o n i n o r d e r t o make  i t 0.5 mM w i t h r e s p e c t t o magnesium i o n . p e c t s , the assay was  performed  In a l l other res-  i n the i d e n t i c a l manner as  the assay w i t h o u t magnesium. (3)  A n a l y t i c a l Methods  (i)  Measurement of p r o t e i n c o n c e n t r a t i o n P r o t e i n c o n c e n t r a t i o n was measured by the F o l i n Lowry  method (58) and by absorbance a t 280 nm.  Absorbance a t  18 260 nm was a l s o measured r o u t i n e l y .  Figure 2 i l l u s t r a t e s  e l u t i o n p r o f i l e s o f t h e same column measured by t h e s e t h r e e methods.  I t can be seen t h a t t h e e l u t i o n p r o f i l e based on  260 nm r e a d i n g s i s s h a r p e r , b u t o t h e r w i s e p a r a l l e l s the 280 nm p r o f i l e .  The p r o f i l e o b t a i n e d by F o l i n Lowry measure-  ments i s e n t i r e l y d i f f e r e n t :  most of the F o l i n Lowry p o s i t i v e  m a t e r i a l i s eluted i n the l a r g e r molecular weight f r a c t i o n . The F o l i n Lowry method was n o t used r o u t i n e l y ,  since  i t i r r e v o c a b l y uses an a l i q u o t o f t h e sample f o r measurements. The amount o f hormone m a t e r i a l l o s t by t h i s method i s i n s i g n i f i c a n t a t t h e s t a r t o f the i s o l a t i o n p r o c e d u r e , but i n c r e a s e s r a p i d l y as the p u r i f i c a t i o n p r o g r e s s e s . The s p e c t r o p h o t o m e t e r s used f o r measurements o f absorbance i n t h e c o u r s e of t h i s i n v e s t i g a t i o n were: models DU, DK and DB, and Unicam SP 820, s e r i e s 2.  Beckman The l a t t e r  i n s t r u m e n t was used w i t h and w i t h o u t an e x p a n s i o n s c a l e . (ii)  Specific conductivity  measurements  S a l t c o n c e n t r a t i o n was m o n i t o r e d by c o n d u c t i v i t y measurements.  A Radiometer Type CDM 2d c o n d u c t i v i t y meter  was used f o r t h i s p u r p o s e . t r o d e was 0.5586.  The c e l l c o n s t a n t of t h e e l e c -  Thus the conductance measurements ob-  t a i n e d by d i r e c t r e a d i n g were d i v i d e d by t h i s number t o obtain s p e c i f i c conductivity  values.  19  A.  B.  C.  14. On  Fraction No.  F i g u r e 2:  Monitoring of the e f f l u e n t of a g e l f i 1 t r a t i o n column by t h r e e d i f f e r e n t methods"!  L o a d i n g sample: A  ' - - 2 8 0 nm U  ;  8 3 0  10 m l , 600 mg F o l i n - L o w r y p e p t i d e ; 6 80 A  " - 2 6 0 nm* U  B  i  o  9  e  l  p  " ' 2  A.  F o l i n - L o w r y p e p t i d e , absorbance a t 660  B.  Absorbance a t 280  nm  C.  Absorbance a t 260  nm  nm  20 (4)  E x t r a c t i o n o f O x y t o c i c A c t i v i t y from Salmon P i t u i t a r i e s  (i)  E x t r a c t i o n w i t h 0.2 M a c e t i c a c i d a t 4°C The 0.2 M a c e t i c a c i d and t h e g l a s s w a r e t o be used  f o r t h e e x t r a c t i o n were p r e c o o l e d .  The e x t r a c t i o n  was  c a r r i e d o u t i n a c o l d room (temperature a p p r o x i m a t e l y 4°C). Frozen glands  (100 gm) were weighed q u i c k l y and then p l a c e d  i n t o 0.2 M a c e t i c a c i d disrupted  (1,000 m l , pH 2.6).  The t i s s u e was  i m m e d i a t e l y i n a Waring b l e n d e r a t maximum speed  u n t i l t h e s u s p e n s i o n was o f even c o n s i s t e n c y . 0.5 t o 1.0 m i n u t e s .  This  required  F u r t h e r d i s r u p t i o n o f t h e t i s s u e was  c a r r i e d o u t u s i n g an e l e c t r i c homogenizer o f P o t t e r - E l v e h j e m type (TRI-R STIR-R, Model S-63, s e t t i n g 5) f o r a p p r o x i m a t e l y one minute. The t i s s u e homogenate was then c e n t r i f u g e d  a t 48,200  g f o r 30 min a t 0°C, u s i n g a S e r v a l l RC 2B c e n t r i f u g e SS-34 head. ice.  and  The s u p e r n a t a n t l i q u i d was decanted and k e p t on  The sediment was resuspended  i n 100 - 200 ml o f c o l d  0.2 M a c e t i c a c i d and t h e s u s p e n s i o n c e n t r i f u g e d  a t 48,000 g  f o r 45 t o 60 min a t 0°C, u s i n g t h e S e r v a l l RC 2B  centrifuge  and SS-34 head.  The two s u p e r n a t a n t s o l u t i o n s were p o o l e d  t o y i e l d t h e crude e x t r a c t .  An a l i q u o t was saved f o r u l t r a -  v i o l e t absorbance measurements and f o r b i o a s s a y . An attempt t o p r e p a r e t h e crude e x t r a c t by c e n t r i fugation  a t 27,300 g f o r 45 minutes a t 0°C, u s i n g t h e  21 S e r v a l l RC 2B c e n t r i f u g e  and GSA head was n o t s u c c e s s f u l  because t h e r e s u l t i n g crude e x t r a c t was opaque and r e q u i r e d re-centrifuging. The s u p e r n a t a n t s o l u t i o n o b t a i n e d by r e - e x t r a c t i o n o f the sediment was b i o a s s a y e d on s e v e r a l o c c a s i o n s , and found to contain extract.  from 5.9 t o 17.2% o f t h e t o t a l a c t i v i t y o f t h e A t h i r d e x t r a c t i o n o f sediment d i d n o t y i e l d  detectable oxytocic (ii)  activity.  E x t r a c t i o n a t 100°C i n 0.25% a c e t i c a c i d o f acetone powder p r e p a r e d from f r o z e n  pituitaries  The acetone was d r i e d over c a l c i u m c h l o r i d e f o r f i v e days w i t h s e v e r a l changes o f d e s s i c a n t .  The acetone was  then c o o l e d i n an i c e b a t h and t h e f r o z e n p i t u i t a r i e s p l a c e d i n t h e acetone.  Following  t h i s , t h e acetone was r e p l a c e d  t h r e e t i m e s a t h a l f hour i n t e r v a l s , a f t e r t h e t h i r d change.  and once a g a i n s i x hours  The g l a n d s were then l e f t i n  acetone a t 4°C f o r an a d d i t i o n a l 18 h o u r s . this period  t h e acetone was decanted and t h e g l a n d s d r i e d on  a s l i g h t l y warm P e t r i d i s h . 0.25%  A t t h e end o f  Dry g l a n d s were homogenized i n  a c e t i c a c i d a t 25°C u s i n g 10 ml o f e x t r a c t a n t  of wet t i s s u e .  An e l e c t r i c homogenizer,  p e r gm  TRI-R STIR-R  (Model S-63, s e t t i n g 5) was used f o r d i s r u p t i o n o f t h e t i s sue.  The t i s s u e homogenate was then p l a c e d i n a b o i l i n g  water b a t h f o r 3 m i n u t e s , c o o l e d on i c e and c e n t r i f u g e d a t 48,200 g f o r 30 min a t 0°C, u s i n g t h e S e r v a l l RC 2B c e n t r i -  fuge and an SS-34 head.  The s u p e r n a t a n t l i q u i d was decanted  and k e p t on i c e . The sediment was resuspended i n 20% o f t h e o r i g i n a l extractant  volume, and t h e e x t r a c t i o n , c o o l i n g , and  c e n t r i f u g a t i o n repeated.  The two s u p e r n a t a n t s o l u t i o n s  were p o o l e d t o y i e l d t h e crude e x t r a c t .  An a l i q u o t was  saved f o r u l t r a v i o l e t absorbance measurements and f o r b i o assay. T h i s e x t r a c t i o n procedure was i n t e n d e d t o approximate the method r e p o r t e d by Kamm (59) and t o compare t h e y i e l d o b t a i n e d by Kamm's procedure w i t h t h e e x t r a c t i o n  procedure  d e v e l o p e d f o r salmon p i t u i t a r i e s ( i ) . (iii)  A d d i t i o n a l e x t r a c t i o n methods Other e x t r a c t i o n methods c o n s i s t e d  the two p r o c e d u r e s d e s c r i b e d above.  o f v a r i a t i o n s on  V a r i a t i o n s on t h e f i r s t  e x t r a c t i o n p r o c e d u r e were as f o l l o w s : (a)  L e n g t h e n i n g t h e time o f e x t r a c t i o n .  I n these  e x p e r i m e n t s t h e t i s s u e homogenate was l e f t on a magnetic s t i r r e r f o r the e n t i r e period  o f e x t r a c t i o n a t 4°C o r a t  25°C. (b)  Three v a r i a t i o n s i n e x t r a c t a n t  were done.  G l a c i a l a c e t i c a c i d , 2 M a c e t i c a c i d , and 1 M sodium  acetate  (pH 5) were each.used f o r t h e e x t r a c t i o n o f t h e t i s s u e . G l a c i a l a c e t i c a c i d e x t r a c t s were i n c o n v e n i e n t t o handle because t h e excess a c i d had t o be removed by l y o p h y l i z a t i o n . This could  o n l y be done a f t e r d i l u t i o n w i t h water t o a p p r o x i -  mately 1 M a c e t i c a c i d .  Such d i l u t i o n produced l a r g e volumes  of e x t r a c t f o r l y o p h y l i z i n g and c o n s e q u e n t l y consumed a g r e a t deal of time.  2 M a c e t i c a c i d e x t r a c t s a l s o required the  removal o f excess a c i d by l y o p h y l i z a t i o n , b u t t h e d i l u t i o n r e q u i r e d f o r t h i s p r o c e s s was t w o - f o l d  r a t h e r than  approxi-  m a t e l y s i x t e e n - f o l d as i n t h e case o f g l a c i a l a c e t i c a c i d . (c)  Omission o f t h e P o t t e r - E l v e h j e m homogenized  step  from t h e e x t r a c t i o n p r o c e d u r e , t h e d i s r u p t i o n b e i n g c a r r i e d out u s i n g Waring b l e n d e r (d)  only.  Use o f l y o p h y l i z e d t i s s u e f o r e x t r a c t i o n  freeze-drying  o f whole f r o z e n g l a n d s .  tated the t i s s u e d i s r u p t i o n The  involved  Lyophylization  facili-  step.  o n l y v a r i a n t on e x t r a c t i o n p r o c e d u r e ( i i ) con-  s i s t e d i n o m i s s i o n o f t h e acetone powder s t e p and subsequent e x t r a c t i o n i n b o i l i n g water b a t h f o r 5 minutes r a t h e r 3 minutes.  than  The t i m i n g began when t h e temperature o f t h e  homogenate reached a p l a t e a u a t a p p r o x i m a t e l y 92°C. The  e x t r a c t i o n p r o c e d u r e s used i n t h e c o u r s e o f t h i s  study and t h e y i e l d s o f o x y t o c i c a c t i v i t y o b t a i n e d a r e summarized i n T a b l e I I I , p. 52-53. (5)  Concentration  o f t h e Crude and P a r t i a l l y P u r i f i e d  Extracts Crude e x t r a c t s d i d n o t have t o be c o n c e n t r a t e d i n large scale Figure  (100 gm) e x t r a c t s i n 0.2 M a c e t i c a c i d ( c f .  7 ) . P r i o r t o t h i s , crude e x t r a c t was c o n c e n t r a t e d t o  remove for  e x c e s s a c i d and t o  reduce  the  further p u r i f i c a t i o n steps.  volume o f  the  Concentration of  sample crude  e x t r a c t s by u l t r a f i l t r a t i o n was  found u n s a t i s f a c t o r y ,  cause p r e c i p i t a t i o n  took p l a c e  and s u b s t a n t i a l l y partially de-salt  of m a t e r i a l  reduced the  purified extracts  them was  discussed  6 of  rate.  i n order to  c a r r i e d out  in section  flow  cedure.  repeatedly  bottom  lyophylization  Losses  of  of  this  excess  Corp.  of  isolation  freeze  hormonal a c t i v i t y  were n o t  observed  material  i n the  hormones, flask  the  however,  It  that  be b r o u g h t i n t o  this  was  purpose. i n the  essential  all  pro-  of  the  solution  by  course to  the  dry repeated  flask.  Ultrafiltration  (6)  The D i a - f l o u l t r a f i l t r a t i o n a p p a r a t u s carry  out  fast  The h i g h r a t e the  is  d r y e r and r o u n d -  for  the  to  a c i d by l y o p h y l i -  was u s e d  of  and  Ultrafiltration  course  lyophylization procedure.  of  concentrate  flask  recovery  rinsing  i n the  A Thermovac I n d u s t r i e s  membrane  chapter.  C o n c e n t r a t i o n and r e m o v a l o f z a t i o n was done  the  U l t r a f i l t r a t i o n of  routinely.  this  above  be-  selective is  filtration  achieved  chamber c o n t a i n i n g  i n t r o d u c e d by membranes  the of  of  is  aqueous  designed solutions.  by a p p l y i n g n i t r o g e n p r e s s u r e sample,  and t h e  v a r y i n g pore  selectivity  sizes  to  to is  through which  the s o l u t i o n i s f i l t e r e d . available:  UM-1,  UM-2,  Three types o f membranes were  and UM-3,  w i t h molecular weight  c u t - o f f ranges o f 10,000, 1,000, and 350, r e s p e c t i v e l y . The m o l e c u l a r w e i g h t s of n e u r o h y p o p h y s i a l hormones a r e i n the 1,000 range, w h i l e the m o l e c u l a r weight o f s a l t s i n the e x t r a c t i s below 350.  Thus a UM-3 membrane was  expected  to d e - s a l t and t o c o n c e n t r a t e a s o l u t i o n o f n e u r o h y p o p h y s i a l hormones. The u l t r a f i l t r a t i o n apparatus was used a c c o r d i n g t o the d i r e c t i o n s of t h e m a n u f a c t u r e r  (60). U l t r a f i l t r a t i o n  chambers 450 and 600 ml i n volume and 7.5 cm D i a f l o u l t r a f i l t r a t i o n membranes were used.  P r e s s u r e a p p l i e d was 80  p . s . i . and f l o w r a t e s r a n g i n g from 1.2 t o 2.0 ml/min were obtained. tration  By v a r y i n g t h e p r e s s u r e a p p l i e d and t h e concen-  o f t h e s o l u t i o n i n t h e chamber, i t was observed t h a t  the f l o w r a t e appeared  t o depend on t h e c o n c e n t r a t i o n o f t h e  s o l u t e i n t h e chamber r a t h e r than on the p r e s s u r e a p p l i e d t o t h a t chamber. In o r d e r t o m i n i m i z e l o s s e s of hormone d u r i n g u l t r a f i l t r a t i o n , a compromise was reached between l o w e r i n g o f the i o n i c  s t r e n g t h by u l t r a f i l t r a t i o n and by d i l u t i o n .  This  c o n s i s t e d i n c o n c e n t r a t i n g t h e sample above t h e u l t r a f i l t r a t i o n membrane and t h e r e b y r e d u c i n g t h e s a l t c o n t e n t o f the sample by a f a c t o r dependent on t h e r a t i o between the s t a r t i n g volume and t h a t o f t h e f i n a l c o n c e n t r a t e (10 t o 15 ml).  The t o t a l s a l t c o n t e n t was t h e r e b y reduced, b u t u s u a l l y  not s u f f i c i e n t l y t o p e r m i t c a t i o n exchange chromatography. The  s o l u t i o n was a d j u s t e d l a t e r t o t h e d e s i r e d conductance  f o r l o a d i n g t h e c a t i o n exchange column.by d i l u t i o n . (7)  Gel F i l t r a t i o n  (i)  P r e p a r a t i o n o f t h e g e l and column p a c k i n g Two t y p e s o f g e l f i l t r a t i o n media were used i n t h e  course o f t h e s e e x p e r i m e n t s :  crosslinked dextran  (Sephadex,  Pharmacia) and c r o s s l i n k e d p o l y a c r y l a m i d e ( B i o g e l , B i o r a d ) . Both g e l s were t r e a t e d i d e n t i c a l l y i n r e s p e c t t o s w e l l i n g and i n p a c k i n g o f t h e column. o n l y a t one p o i n t .  The t r e a t m e n t d i f f e r e d  slightly  More c a r e had t o be e x e r c i s e d i n sus-  pending t h e d r y B i o g e l because i t tended t o form l a r g e aggregates.  Consequently  t h e s u s p e n s i o n had t o be s t i r r e d  v i g o r o u s l y and t h e d r y g e l added s l o w l y . The s w e l l i n g u s u a l l y was a l l o w e d t o t a k e p l a c e o v e r n i g h t o r f o r t w e n t y - f o u r h o u r s , and f i n i n g o f t h e g e l was accomplished during t h a t time.  The s w e l l i n g and p a c k i n g  was done i n t h e same m o l a r i t y o f a c e t i c a c i d i n which t h e column was t o be r u n .  O c c a s i o n a l l y t h e Sephadex g e l was  s w o l l e n by t h e f a s t method recommended by t h e m a n u f a c t u r e r ; f o r one hour i n a 60°C b a t h ( 6 1 ) . S m a l l columns ( i . e . 200 mm x 12 mm diameter) were packed by p o u r i n g t h e t h i n g e l s l u r r y by hand.  The column  was h a l f f i l l e d w i t h b u f f e r when t h e p o u r i n g began and t h e  o u t l e t was  opened s l i g h t l y when the bed was  depth of 2 0 t o 30 mm.  packed t o a  L a r g e r columns (25 t o 50 mm  in  d i a m e t e r ) were packed u s i n g a f u n n e l i n which the g e l susp e n s i o n was  continuously s t i r r e d .  f u n n e l w i t h a s h o r t wide stem was  A l a r g e (500 ml)  i n s e r t e d i n t o a rubber  s t o p p e r f i t t e d t o the top o f the column. blade s t i r r e r  An e l e c t r i c  ( G e r a l d T. H e l l e r Company, GT  s t i r the s u s p e n s i o n  i n the f u n n e l .  f i l l e d with gel slurry.  The  21) was  f u n n e l was  rotary  used t o kept  When the column bed reached the  d e s i r e d h e i g h t the f u n n e l was p l a c e d on top of the g e l bed.  removed and a p e r l o n d i s c The  two bed volumes of e l u a n t and was of a l l columns was  glass  column was  washed w i t h  ready t o use.  The  packing  c a r r i e d out a t 25°C.  B l u e D e x t r a n (Pharmacia) s o l u t i o n was  run through  newly packed Sephadex columns t o m o n i t o r the q u a l i t y of packing.  Blue D e x t r a n c o u l d not be used w i t h B i o g e l , s i n c e  i t was  r e t a i n e d by the g e l .  spread  the B l u e D e x t r a n band.  Sephadex G - 1 0 a l s o tended t o P  On G-15,  however, B l u e  D e x t r a n behaved as d e s c r i b e d by the m a n u f a c t u r e r (ii)  (62).  L o a d i n g and e l u t i o n of g e l f i l t r a t i o n columns E x p e r i m e n t s were g e n e r a l l y c a r r i e d out a t 25°C.  A f t e r removal of the l i q u i d above the column bed, s o l u t i o n s of hormone p r e p a r a t i o n s were loaded i n the  appropriate  volume and then washed i n t o ti^gelswifn3 0..5 to,* li0j 0.i/ ml of b u f f e r . v  28 (10 m l was used f o r 450 mm x 50 mm d i a m e t e r columns).  The  column was then e l u t e d w i t h t h e b u f f e r and f r a c t i o n s c o l l e c t e d using  a m e c h a n i c a l f r a c t i o n c o l l e c t o r ( G i l s o n , model V-10,  equipped w i t h  timer).  I n o r d e r t o p r o c e s s a l a r g e volume o f e x t r a c t ( a p p r o x i m a t e l y 1 l i t e r ) , a Sephadex G-15 column (450 mm x 50 mm diameter) was used r e p e a t e d l y batches of the e x t r a c t  t o f r a c t i o n a t e 225 ml  (tandem g e l f i l t r a t i o n ) .  When t h i s  t e c h n i q u e was employed a f r e s h sample o f e x t r a c t was a p p l i e d t o t h e column as soon as t h e b i o l o g i c a l l y a c t i v e  material  was e l u t e d and f r a c t i o n s c o l l e c t e d c o n t i n u a l l y .  The t o t a l  time r e q u i r e d  f o r a tandem 5-column r u n was between 20 and  25 h o u r s . The absorbance  o f f r a c t i o n s a t 260 and 280 nm, t h e i r  c o n d u c t i v i t y , and o x y t o c i c a c t i v i t y , were determined.  The  b i o l o g i c a l l y a c t i v e f r a c t i o n s were then p o o l e d and e i t h e r p r o c e s s e d f u r t h e r i m m e d i a t e l y o r s t o r e d a t 5°C. Packed Sephadex columns were s t o r e d a t 25°C w i t h 0.02% sodium a z i d e a f t e r washing w i t h s e v e r a l bed volumes o f buffer. (8)  I o n Exchange Chromatography  (i)  Preparation  o f t h e exchangers  and column p a c k i n g  P r e c y c l i n g o f Whatman CM-32 and o f Whatman CM-22 was c a r r i e d out according t o the d i r e c t i o n s of the manufacturers (63).  U s u a l l y t h e amount p r e c y c l e d  was about 30% more than  r e q u i r e d by the column.  The d r y exchanger was s t i r r e d  into  0.5 N sodium h y d r o x i d e (15 ml per gm of exchanger) and  left  f o r 30 m i n u t e s .  A t the end o f t h i s p e r i o d t h e l i q u i d  was  decanted and the exchanger washed w i t h d i s t i l l e d water i n a 500 ml Buchner f u n n e l under s u c t i o n u n t i l the washings were a t pH 8.  The exchanger was then s t i r r e d i n t o 0.5 N hydro-  chloric acid  (15 ml per gm o f exchanger) and a g a i n l e f t f o r  30 m i n u t e s .  The l i q u i d was decanted and the exchanger  washed on a Buchner f u n n e l u n t i l t h e washings were n e u t r a l . The h y d r o c h l o r i c a c i d t r e a t m e n t was r e p e a t e d once.  Equili-  b r a t i o n w i t h the a p p r o p r i a t e b u f f e r was c a r r i e d out a t 2M i o n i c s t r e n g t h and the c o r r e c t pH v a l u e (pH 5 ) .  I t was  found c o n v e n i e n t t o c a r r y out t h i s s t e p i n a 500 ml g r a d u a t e d cylinder.  The exchanger was s t i r r e d w i t h the b u f f e r and  a l l o w e d t o s e t t l e f o r 10 m i n u t e s .  A t the end of t h i s p e r i o d  the l i q u i d was removed by s u c t i o n , the c y l i n d e r  refilled  w i t h b u f f e r , i n v e r t e d s e v e r a l t i m e s and a g a i n a l l o w e d t o settle.  T h i s s t e p was r e p e a t e d 7 t o 8 t i m e s .  The  conduc-  t a n c e and pH o f t h e l i q u i d were checked f o r e q u i l i b r a t i o n . The e q u i l i b r a t e d exchanger was then s t i r r e d i n t o 20  volumes  of t h e s t a r t i n g b u f f e r and a l l o w e d t o s e t t l e f o r 30 m i n u t e s . The l i q u i d was removed by s u c t i o n and the s t e p r e p e a t e d once. Most of the removal o f f i n e p a r t i c l e s was a c c o m p l i s h e d d u r i n g the e q u i l i b r a t i o n s t e p . changer  A t h i c k s l u r r y of the p r e p a r e d ex-  ( a p p r o x i m a t e l y 20% w/v)  was degassed u s i n g a water  30 pump.  I t was found t o be more c o n v e n i e n t t o p r e p a r e a t h i c k  s l u r r y and pour i t i n t o t h e column i n one p a s s , because t h e f l o w r a t e o f t h i s exchanger i s low and p a c k i n g o f t h e column would t a k e t o o l o n g when a t h i n s l u r r y was used.  For the  same r e a s o n i t was found d e s i r a b l e t o have t h e s t a r t i n g b u f f e r t o a depth o f o n l y 20 t o 30 mm a t t h e bottom o f t h e column p r i o r t o p o u r i n g .  The exchanger was a l l o w e d t o  s e t t l e t o a h e i g h t o f about 20 mm b e f o r e t h e o u t l e t was opened.  The column was u s u a l l y packed i n t h e l a t e  after-  noon and l e f t t o wash w i t h s t a r t i n g b u f f e r o v e r n i g h t .  By  morning complete e q u i l i b r a t i o n was a c h i e v e d w i t h r e g a r d t o b o t h c o n d u c t i v i t y and pH. Phosphocellulose (Selectacel) d i d not require s p e c i a l precycling.  I t was e q u i l i b r a t e d i n 0.2 M b u f f e r and packed  i n t o t h e column i n t h e s t a r t i n g  0.002 M) b u f f e r .  Because  of t h e f i b r o u s n a t u r e o f t h e exchanger i t was packed i n a thin slurry. starting  The column was e q u i l i b r a t e d o v e r n i g h t w i t h t h e  buffer.  SE-Sephadex C-25 (Pharmacia) was p r e c y c l e d a c c o r d i n g to the d i r e c t i o n s of the manufacturer (64). changer  The d r y ex-  (30 gm) s w e l l e d t o a volume o f 300 ml w h i l e s o a k i n g  i n water.  No a p p r e c i a b l e s h r i n k a g e was n o t i c e d as t h e  column was packed. The exchanger was a l l o w e d t o s w e l l i n water f o r a t l e a s t one hour and then f i n e p a r t i c l e s were removed by  31 decantation.  The exchanger was washed on a Buchner f u n n e l  w i t h 500 ml o f 0.5 N sodium h y d r o x i d e  followed with d i s -  t i l l e d water u n t i l n e u t r a l i t y was reached. was c a r r i e d o u t i n 2 M b u f f e r o f d e s i r e d pH.  Equilibration S e v e r a l changes  of b u f f e r were r e q u i r e d u n t i l complete e q u i l i b r a t i o n was achieved.  The column was packed and washed o v e r n i g h t as i n  the p r e p a r a t i o n o f p h o s p h o c e l l u l o s e  columns.  A l l cation  exchange columns were p r e c y c l e d , e q u i l i b r a t e d and packed a t 25°C. (ii)  B u f f e r s used i n c a t i o n exchange Sodium a c e t a t e , ammonium a c e t a t e , and ammonium formate  b u f f e r s were made by t i t r a t i o n o f t h e a c i d a t d e s i r e d molari t y w i t h t h e a p p r o p r i a t e base t o t h e r e q u i r e d pH.  A  Radiometer 26 pH meter was used f o r t h e t i t r a t i o n .  A 2 M  s t o c k s o l u t i o n was f r e s h l y made b e f o r e use and lower s t r e n g t h b u f f e r s o b t a i n e d by d i l u t i o n .  ionic  I n ammonium formate  b u f f e r s , t h e pH had t o be a d j u s t e d w i t h f o r m i c a c i d f o l l o w i n g dilution. (iii)  L o a d i n g and e l u t i o n o f c a t i o n exchange columns L o a d i n g and e l u t i o n were conducted a t 25°C.  f r a c t i o n s were c o l l e c t e d u s i n g t h e G i l s o n f r a c t i o n  The collector.  U s u a l l y t h e sample was loaded onto t h e column from a r e s e r v o i r and t h e c o l l e c t i o n o f f r a c t i o n s s t a r t e d  immediately.  32 The  c o n d u c t i v i t y o f t h e l o a d i n g sample was a d j u s t e d by  d i l u t i o n w i t h d i s t i l l e d water t o make i t i l o w e r than t h e c o n d u c t i v i t y needed t o e l u t e Hormone I . A f t e r a l l o f t h e sample p e r c o l a t e d i n t o t h e column bed,  the w a l l s of the  column above t h e exchanger bed were r i n s e d w i t h s e v e r a l m i l l i l i t e r s o f s t a r t i n g b u f f e r and t h e r i n s i n g s a l l o w e d t o pass i n t o t h e bed. The column was washed w i t h  starting  b u f f e r u n t i l t h e u l t r a v i o l e t absorbance o f t h e e l u a t e decreased.  The e l u t i o n w i t h l i n e a r l y i n c r e a s i n g s a l t concen-  t r a t i o n s was then c a r r i e d o u t u s i n g a g r a d i e n t  forming  system w i t h e q u a l volumes o f a p p r o p r i a t e b u f f e r s i n a m i x i n g chamber and i n a r e s e r v o i r o f e q u a l dimensions ( 6 5 ) . I n most c a s e s , b i o a s s a y and t h e u l t r a v i o l e t  absor-  bance measurements were made as f r a c t i o n s emerged from t h e column.  The g r a d i e n t was checked by measuring t h e conduc-  tance o f t h e f r a c t i o n s . (iv)  Repeated use o f t h e same c a t i o n column Unless  was  i t c o u l d be re-used  d i s c a r d e d a f t e r use.  immediately,  On a few o c c a s i o n s  t h e exchanger  a l l t h r e e ex-  changers were re-used w i t h i n a day o r two o f t h e f i r s t r u n . A l l r e q u i r e d complete r e - e q u i l i b r a t i o n , which c o n s i s t e d i n washing t h e column w i t h h i g h i o n i c s t r e n g t h (1 - 2 M) b u f f e r f o l l o w e d by a two t o t h r e e l i t e r wash w i t h t h e s t a r t i n g buffer.  SE-Sephadex, p h o s p h o c e l l u l o s e ,  c o u l d be re-used w i t h o u t r e - p a c k i n g .  and Whatman CM-22  Whatman CM-32 r e q u i r e d  r e - p a c k i n g because of d e c r e a s i n g f l o w r a t e . i n wet form was  Phosphocellulose  never k e p t l o n g e r than a b s o l u t e l y e s s e n t i a l  because of p o s s i b l e phosphatase a c t i v i t y  i n m i c r o b i a l con-  taminants . (9)  Performic Acid Oxidation C y s t i n e and c y s t e i n e r e s i d u e s were c o n v e r t e d  c y s t e i c a c i d r e s i d u e s by p e r f o r m i c o x i d a t i o n (66).  to Cysteic  a c i d g i v e s b e t t e r q u a n t i t a t i v e d a t a than c y s t e i n e on the a u t o m a t i c amino a c i d a n a l y s e r .  A d d i t i o n a l l y , conversion to  c y s t e i c a c i d removes the i n t r a m o l e c u l a r d i s u l f i d e b r i d g e i n neurohypophysial  hormones thus f a c i l i t a t i n g sequence d e t e r -  mination . The  sample of p e p t i d e i n t e n d e d f o r o x i d a t i o n , the  f r e s h l y p r e p a r e d p e r f o r m i c a c i d s o l u t i o n , and the p i p e t t e f o r measuring the l a t t e r were c o p i e d i n i c e t o 0°C. a c i d was  added t o the p e p t i d e sample, the t e s t tube  Performic stoppered  w i t h a ground g l a s s s t o p p e r and the r e a c t i o n a l l o w e d t o proceed  a t 0°C  f o r two t o t h r e e hours.  removed by l y o p h y l i z a t i o n .  The  Excess r e a g e n t s were  f i r s t lyophylization  was  c a r r i e d out u n t i l no p e r o x i d e c o u l d be d e t e c t e d by s m e l l . The  sample was  then r e d i s s o l v e d i n water and r e l y o p h y l i z e d .  A l a r g e excess of o x i d a n t was a c i d t o 0.05  used, i . e . 0.2 ml of p e r f o r m i c  ymoles o f p e p t i d e .  P e r f o r m i c a c i d was p r e p a r e d by m i x i n g 88% f o r m i c a c i d and 30% hydrogen p e r o x i d e i n 9 : 1 p r o p o r t i o n s and a l l o w i n g t h e m i x t u r e t o s t a n d a t room temperature hour.  A mixture of formic acid t o peroxide  f o r one  (9.5 : 0.5)  warmed f o r f i v e minutes i n a 50°C b a t h has a l s o been used and found (10)  satisfactory.  Acid Hydrolysis of Peptides T o t a l h y d r o l y s i s o f t h e sample was r e q u i r e d i n t h r e e  types o f e x p e r i m e n t s : molecule;  (1) amino a c i d a n a l y s i s o f t h e whole  (2) amino a c i d a n a l y s i s f o l l o w i n g Edman  degrada-  t i o n c y c l e ( s ) f o r s u b t r a c t i v e Edman method o f amino a c i d sequence d e t e r m i n a t i o n ; and (3) i d e n t i f i c a t i o n o f d a n s y l d e r i v a t i v e s o f N - t e r m i n a l r e s i d u e s o f whole o r p a r t i a l l y degraded hormones. The sample t o be a n a l y z e d was p l a c e d i n a t e s t  tube  and d r i e d i n a vacuum d e s s i c a t o r over phosphorus p e n t o x i d e and sodium h y d r o x i d e .  Kimax c u l t u r e t u b e s , 10 x 75 mm,  were used f o r t o t a l h y d r o l y s e s and f o r s u b t r a c t i v e Edmans. H y d r o l y s i s o f d a n s y l a t e d p e p t i d e s was c a r r i e d o u t i n t h e same t e s t tube, 6 x 30 mm, used f o r d a n s y l a t i o n r e a c t i o n . Twice d i s t i l l e d 50% h y d r o c h l o r i c a c i d 0.01  (20 u l f o r  ymole o f p e p t i d e ) was added t o t h e d r i e d sample and  the t e s t tube p u l l e d o u t i n a h o t flame t o form a 1 - 2 mm d i a m e t e r neck i n t h e m i d d l e .  The h o t g l a s s was c o o l e d i n  a stream o f a i r o r a t room t e m p e r a t u r e , and t h e c o n t e n t s f r o z e n i n an acetone and s o l i d carbon d i o x i d e b a t h .  The  mouth o f t h e tube was then a t t a c h e d t o an o i l vacuum pump and t h e tube evacuated.  The s o l u t i o n was a l l o w e d t o m e l t  and e v a c u a t i o n c o n t i n u e d u n t i l b u b b l i n g ceased.  When  b u b b l i n g was t o o i n t e n s e , as was t h e case w i t h t h e hydrol y z a t e s o f d a n s y l a t e d p e p t i d e s , i t was c o n t r o l l e d by r e p e a t e d f r e e z i n g and thawing o f t h e sample.  The tube was  s e a l e d under vacuum u s i n g a s m a l l f l a m e . The h y d r o l y s i s was conducted i n a c o n t r o l l e d tempera t u r e h e a t i n g b l o c k (Temp-Blok Module H e a t e r , L a b - L i n e I n s t r u m e n t s ) which was covered w i t h s e v e r a l t h i c k n e s s e s o f aluminium f o i l w i t h a h o l e f o r a thermometer stem.  The  h y d r o l y s i s was a l l o w e d t o proceed f o r 16 hours a t 105°C, unless otherwise indicated i n the text. F o l l o w i n g h y d r o l y s i s , t h e s e a l e d tube was opened and the h y d r o c h l o r i c a c i d e v a p o r a t e d under vacuum over hydroxide p e l l e t s .  sodium  D e s s i c a t i o n was a l l o w e d t o proceed  l o n g e r than would be w a r r a n t e d f o r t h e appearance  of the  sample i n o r d e r t o remove a l l t r a c e s o f h y d r o c h l o r i c a c i d . When 0.1 ml h y d r o c h l o r i c a c i d had been added f o r h y d r o l y s i s , t h e sample was l e f t under vacuum f o r 1.5 h o u r s .  The d r y  sample was then ready f o r t h e n e x t s t e p ; e i t h e r amino a c i d a n a l y s i s o r t h i n l a y e r chromatography.  I f t h e n e x t s t e p was  not done i m m e d i a t e l y t h e sample was s t o r e d a t -20°C.  (11)  Amino A c i d A n a l y s e s On o c c a s i o n  during t h i s i n v e s t i g a t i o n , three  of amino a c i d a n a l y z e r were used. Model 120, a T e c h n i c o n a n a l y z e r , equipped w i t h an i n t e g r a t o r .  models  They were a Beckman and a Beckman Model 120 C,  The b u l k o f t o t a l  hydrblyzates  and a l l o f t h e s u b t r a c t i v e Edman r e s u l t s were a n a l y z e d on the Beckman Model 120 C, and t h e e x p e r i m e n t a l p r o c e d u r e described (i)  i s f o r t h i s model.  Procedure The sample c o n t a i n i n g  0.01 t o 0.03 umoles o f p e p t i d e  was d i s s o l v e d i n 100 o r 200 y l o f pH 2.2 sodium c i t r a t e buffer.  When o n l y t h e l o n g column was t o be used, as i n  sequencing o f Hormone I , t h e sample was d i s s o l v e d i n 100 y l and t h e t o t a l amount a p p l i e d t o t h e column.  When t h e s h o r t  column was t o be r u n i n a d d i t i o n t o t h e l o n g column, t h e sample was d i s s o l v e d i n 200 y l o f b u f f e r and 100 y l o f s o l u t i o n was a p p l i e d t o each column.  The l o n g column on  Beckman 120 C i s used f o r r e s o l u t i o n o f a c i d i c and n e u t r a l amino a c i d s ; t h e s h o r t column i s used f o r r e s o l u t i o n o f basic  residues. The r u n was performed a c c o r d i n g  the m a n u f a c t u r e r (67) , (68) .  t o the d i r e c t i o n s of  The sample was a p p l i e d  using  n i t r o g e n p r e s s u r e and t h e w a l l s o f t h e column r i n s e d  twice  with the s t a r t i n g buffer  (pH 3.52 o r pH 5.18).  The space  above t h e r e s i n was f i l l e d t o t h e t o p o f t h e column w i t h t h e  s t a r t i n g b u f f e r , the l e a d from the a p p r o p r i a t e pump a t t a c h e d and the run s t a r t e d .  The e l u t i o n of b a s i c amino a c i d s  c a r r i e d out w i t h a pH 5.18 time r e q u i r e d was  sodium c i t r a t e b u f f e r and  50 m i n u t e s .  The e l u t i o n of a c i d i c  was  the and  n e u t r a l amino a c i d s r e q u i r e d a b u f f e r change f o r the e l u t i o n o f the l a t t e r group.  The b u f f e r change was  s t a r t i n g b u f f e r t o pH 4.25  b u f f e r and was  from the  set to begin  a u t o m a t i c a l l y 25 t o 30 minutes a f t e r the run began.  This  d i f f e r e n c e i n time depended on the l e n g t h o f the column used. was  S i m i l a r l y , the d u r a t i o n of the t o t a l l o n g column run  125 t o 130  (ii)  minutes.  Q u a n t i t a t i o n of r e s u l t s The i n t e g r a t e d v a l u e s f o r each amino a c i d were e i t h e r  r e a d o f f the i n t e g r a t o r t a p e , o r c a l c u l a t e d by m u l t i p l y i n g the n e t h e i g h t of each peak by i t s w i d t h . p r o l i n e was the 570 nm  Quantitation for  o b t a i n e d from the 440 nm r e c o r d e r t r a c i n g , w h i l e l i n e was  used f o r i n t e g r a t i o n o f a l l o t h e r amino  acids. The n u m e r i c a l v a l u e o b t a i n e d above was  d i v i d e d by  the  c o n s t a n t s o b t a i n e d from a s t a n d a r d amino a c i d run t o g i v e v a l u e s f o r the amounts o f amino a c i d s i n micromoles.  In  o r d e r t o t r a n s p o s e the number of micromoles i n t o r e s i d u e s per mole, a l l v a l u e s f o r amino a c i d s found i n a p p r o x i m a t e l y e q u a l amounts i n the m o l e c u l e were averaged and the average was  equated w i t h one r e s i d u e per mole.  38 To e x p r e s s t h e s p e c i f i c a c t i v i t y o f p u r i f i e d h o r mones i n terms o f o x y t o c i c u n i t s p e r m i l l i g r a m hormone, t h e t o t a l number o f o x y t o c i c u n i t s c o n t a i n e d  i n the a l i q u o t  used f o r amino a c i d a n a l y s i s was d i v i d e d by t h e average v a l u e o f micromoles o b t a i n e d  from t h i s a n a l y s i s and then  d i v i d e d by t h e number o f m i l l i g r a m s  e q u a l t o one micromole  of t h e hormone (Tables V I I and V I I I ) . (12)  P a r t i a l Acid  Hydrolysis  The method o f p a r t i a l a c i d h y d r o l y s i s d e s c r i b e d CM.  Tsung and F r a e n k e l - C o n r a t (69) was f o l l o w e d .  by  This  method i s i n t e n d e d t o e f f e c t p r e f e r e n t i a l r e l e a s e o f a s p a r t i c acid  (and a s p a r a g i n y l )  residues.  The p e p t i d e was  d i s s o l v e d i n 0.03 N h y d r o c h l o r i c a c i d t o a f i n a l of 1 mg/ml and h y d r o l y z e d tube.  a t 105°C i n a s e a l e d  concentration  evacuated  The h a l f time f o r h y d r o l y s i s o f a s p a r t i c a c i d r e s i -  dues i s r e p o r t e d 11 h o u r s .  t o be 5.5 hours and o f a s p a r a g i n e  residues  I n c o n t r o l e x p e r i m e n t s i t was found t h a t 22 hour  h y d r o l y s i s of 3-phe, 8 - l y s o x y t o c i n r e s u l t e d i n s i x fragments in addition to aspartic acid.  F o r Hormone I , an 18 hour  h y d r o l y s i s was used. (13)  High V o l t a g e High v o l t a g e  Electrophoresis e l e c t r o p h o r e s i s was used t o m o n i t o r t h e  p u r i t y o f hormone p r e p a r a t i o n s ments o b t a i n e d  and t o i d e n t i f y p e p t i d e f r a g -  e i t h e r by p a r t i a l a c i d h y d r o l y s i s o r as a  r e s u l t o f Edman d e g r a d a t i o n  cycles.  The e x p e r i m e n t s were  c a r r i e d o u t on Whatman 3 MM paper a t e i t h e r pH 6.5 dine  (pyri-  : a c e t i c a c i d : water; 25 : 1 : 225) o r pH 3.6  ( p y r i d i n e : a c e t i c a c i d : w a t e r ; 1 : 10 : 289) .  Either a  f l a t - p l a t e apparatus (Savant Instruments) o r a v e r t i c a l l i q u i d c o o l e d apparatus ( M i c h l - R y l e type) equipped w i t h a h i g h v o l t a g e D.C. power s u p p l y  (Canadian Research I n s t i t u t e ,  Model EP5K-200) was used f o r e l e c t r o p h o r e t i c s e p a r a t i o n s . R e f e r e n c e amino a c i d s (6.5 umoles/ml i n 0.1 M sodium bicarbonate) 5 mm l o n g .  were each a p p l i e d a t t h e o r i g i n i n a s t r e a k P e p t i d e was d i s s o l v e d i n b u f f e r and a p p l i e d as  a s t r e a k 20 - 25 mm l o n g a t t h e o r i g i n i n such a manner t h a t about one f i f t h o f t h e p e p t i d e band c o u l d be c u t o f f and s t a i n e d a l o n g w i t h amino a c i d m a r k e r s , w h i l e t h e r e m a i n i n g p e p t i d e a r e a ( s ) c o u l d be used f o r e l u t i o n . A f t e r s p o t t i n g , t h e o r i g i n was d r i e d i n a stream o f warm a i r , and t h e paper w e t t e d w i t h b u f f e r u s i n g  appropriate  p r e c a u t i o n s t o p r e v e n t movement o r d i f f u s i o n o f s u b s t a n c e s at the o r i g i n .  Following the e l e c t r o p h o r e t i c separation,  the paper was d r i e d and t h e edge b e a r i n g t h e amino a c i d markers and some o f t h e unknown was c u t o f f f o r s t a i n i n g . The cadmium a c e t a t e / n i n h y d r i n s t a i n was made up  immediately  p r i o r t o use from 1% n i n h y d r i n i n a c e t o n e , 15 m i s , and 85 mis o f a s o l u t i o n o f cadmium a c e t a t e  (5 gm) and g l a c i a l  a c e t i c a c i d (250 ml) i n d i s t i l l e d water (500 m l ) .  40 Areas marked f o r e l u t i o n on t h e u n s t a i n e d  s t r i p were  c u t o u t and t h e e l u t i o n c a r r i e d o u t u s i n g t h e method o f Heppel (70) . The v e r t i c a l M i c h l - R y l e type h i g h v o l t a g e apparatus was found t o be p r e f e r a b l e t o t h e f l a t p l a t e type i n p e p t i d e work because c o n s i d e r a b l e s p r e a d i n g was observed w i t h t h e latter  (14)  apparatus.  Edman  Degradation  The Edman r e a c t i o n e s s e n t i a l l y c o n s i s t s o f two s t e p s : formation of a phenylthiocarbamyl s p l i t t i n g o f f of the N-terminal  peptide  (PTC-peptide) and  amino a c i d as an a n i l i n o -  t h i a z o l i n o n e , thus r e s u l t i n g i n a s h o r t e n i n g o f t h e p e p t i d e c h a i n by one amino a c i d r e s i d u e .  Edman i n 1950 d e s c r i b e d  the a p p l i c a t i o n o f t h i s r e a c t i o n t o t h e d e t e r m i n a t i o n o f amino a c i d sequence i n p r o t e i n s ( 7 1 ) . The method used i n the p r e s e n t work was t h a t o f B l a c k , Kauffman and Dixon (72) w i t h some m o d i f i c a t i o n s . The p e p t i d e  (0.1 ymole) was d i s s o l v e d i n 200 y l o f  50% p y r i d i n e ( r e d i s t i l l e d )  i n water i n a c o n i c a l g r a d u a t e d  15 ml c e n t r i f u g e tube equipped w i t h a g l a s s s t o p p e r .  25 y l  of N - e t h y l m o r p h o l i n e ( d i s t i l l e d ) and 10 y l o f p h e n y l i s o - . thiocyanate  ( d i s t i l l e d ) were added, t h e tube f l u s h e d w i t h  n i t r o g e n , stoppered,  and t h e c o u p l i n g r e a c t i o n a l l o w e d t o  p r o c e e d f o r 3 hours a t 37°C.  41 A t t h e end o f t h i s t i m e , 2 drops o f d e - i o n i z e d water were added and t h e m i x t u r e e x t r a c t e d w i t h an e q u a l volume o f benzene t o remove excess r e a g e n t s . e x t r a c t i o n s t e p was r e p e a t e d  I n i t i a l l y , t h e benzene  three times, but losses of  PTC-peptide were o b s e r v e d , and t h e benzene e x t r a c t i o n s t e p was t h e r e a f t e r o n l y done once on each sample.  Benzene ex-  t r a c t i o n c o u l d be o m i t t e d a l t o g e t h e r when o n l y t h e s u b t r a c t i v e Edman method was used i n amino a c i d sequence d e t e r m i n a t i o n . The overnight.  aqueous phase was d r i e d i n a vacuum d e s s i c a t o r T r i f l u o r o a c e t i c a c i d (0.1 ml) was added t o t h e  d r y r e s i d u e and t h e c l e a v a g e  r e a c t i o n a l l o w e d t o proceed a t  room temperature f o r one hour.  T r i f l u o r o a c e t i c a c i d was  removed under vacuum, e i t h e r a t 50°C f o r 30 minutes o r a t 25°C f o r 90 m i n u t e s . The d r y r e s i d u e was i n i t i a l l y d i s s o l v e d i n water and e x t r a c t e d t h r e e times w i t h 1 ml o f b u t y l a c e t a t e .  Because  l o s s e s o f p e p t i d e m a t e r i a l were observed t o o c c u r , t h e ext r a c t i o n w i t h b u t y l a c e t a t e was performed once on t h e d r y r e s i d u e i n subsequent e x p e r i m e n t s u s i n g 0.5 ml o f b u t y l acetate. Excess b u t y l a c e t a t e was removed i n a vacuum d e s i c c a t o r and t h e r e s i d u e r e d i s s o l v e d i n 5'0%=&pyridine i n water.  (distilled)  (15)  S u b t r a c t i v e Edman Method i n Amino A c i d Sequence Determination The s u b t r a c t i v e Edman sequence method i n v o l v e d amino  a c i d a n a l y s i s o f t h e r e s i d u a l p e p t i d e a t t h e end o f each deg r a d a t i o n s t e p d e s c r i b e d i n s e c t i o n 14 o f t h i s c h a p t e r .  A  s e r i e s o f amino a c i d a n a l y s e s was thus o b t a i n e d showing a g r a d u a l l y d e c r e a s i n g p e p t i d e c h a i n as t h e s u c c e s s i v e amino a c i d s were removed from t h e N-terminus by t h e Edman degradation reaction.  The n e x t Edman d e g r a d a t i o n c y c l e was n o t  begun u n t i l t h e r e s u l t s o f t h e p r e v i o u s c y c l e were o b t a i n e d from amino a c i d a n a l y s i s .  I t was found t h a t t h e sequence o f  f o u r t o f i v e N - t e r m i n a l r e s i d u e s c o u l d be d e f i n e d by t h e s u b t r a c t i v e Edman method.  (16)  D a n s y l a t i o n Method i n Amino A c i d Sequence D e t e r m i n a t i o n W h i l e t h e s u b t r a c t i v e Edman method used  comparative  amino a c i d a n a l y s i s t o e s t a b l i s h which amino a c i d was removed, and hence which was t h e t e r m i n a l , d a n s y l a t i o n i n v o l v e s p o s i t i v e i d e n t i f i c a t i o n o f each s u c c e s s i v e N-terminus as i t becomes exposed f o l l o w i n g an Edman d e g r a d a t i o n c y c l e ( 5 4 ) . Dansyl c h l o r i d e (l-dimethylaminonaphthalene-5-sulfonyl c h l o r i d e ) i s r e a c t e d w i t h an a l i q u o t o f t h e p e p t i d e f o l l o w i n g each Edman d e g r a d a t i o n c y c l e t o form a d a n s y l a t e d p e p t i d e ( d a n s y l c h l o r i d e r e a c t s n o t o n l y w i t h a l p h a amino groups, b u t  a l s o w i t h t h e e p s i l o n amino group o f l y s i n e and w i t h t h e p h e n o l i c hydroxy1 g r o u p s ) .  D a n s y l a t e d p e p t i d e i s then  h y d r o l y z e d and d a n s y l a t e d amino a c i d s i d e n t i f i e d .  The method  f o l l o w e d i n t h e p r e s e n t work i s a p p r o x i m a t e l y t h a t d e s c r i b e d by B l a c k , Kauffman and Dixon ( 7 2 ) . (i)  D a n s y l a t i o n r e a c t i o n and h y d r o l y s i s An a l i q u o t c o n t a i n i n g 0.01 ymole o f p e p t i d e was r e -  moved from t h e p y r i d i n e - w a t e r s o l u t i o n o f t h e p e p t i d e a t the end o f each Edman d e g r a d a t i o n c y c l e ( s e c t i o n 1'A o f t h i s chapter).  The a l i q u o t was p l a c e d i n a 6 x 30 mm t e s t  tube  and d r i e d i n a vacuum d e s s i c a t o r over phosphorus p e n t o x i d e and sodium h y d r o x i d e .  20 y l o f sodium b i c a r b o n a t e was added  and t h e p e p t i d e d r i e d a g a i n .  10 y l o f d e - i o n i z e d water was  added t o t h e d r y p e p t i d e and t h e pH o f t h e r e s u l t i n g t i o n checked w i t h :pir:; ': paper. t h e s t e p was r e p e a t e d .  solu-  I f t h e pH was l e s s than 8,  10 y l o f d a n s y l c h l o r i d e  (3 mg/ml i n  acetone) was added t o t h e aqueous s o l u t i o n o f p e p t i d e , t h e tube s e a l e d w i t h P a r a f i l m , and t h e r e a c t i o n a l l o w e d t o p r o ceed a t 37°C f o r . 1 . 5 t o 3 h o u r s , o r u n t i l t h e y e l l o w c o l o u r of the reagent disappeared.  The sample was then d r i e d i n a  vacuum d e s s i c a t o r and h y d r o l y z e d w i t h 50% h y d r o c h l o r i c a c i d ( r e - d i s t i l l e d t w i c e ) f o r 16 hours a t 105°C a f t e r s e a l i n g t h e tube under vacuum, as d e s c r i b e d i n s e c t i o n 9 o f t h i s c h a p t e r . A s h o r t e r h y d r o l y s i s time  (6 hours) was used where a d a n s y l -  p r o l i n e r e s i d u e was s u s p e c t e d , d e s t r o y e d by p r o l o n g e d (ii)  since dansyl-proline i s  acid hydrolysis.  I d e n t i f i c a t i o n o f d a n s y l amino a c i d s by t h i n l a y e r chromatography D a n s y l amino a c i d standards were p r e p a r e d  by t h e  method o f B o u l t o n and Bush (73) and checked a g a i n s t s t a n dards purchased from Calbiochem. were used.  The c o m m e r c i a l l y  from d a n s y l s u l f o n i c a c i d .  Both s e t s o f s t a n d a r d s  o b t a i n e d standards were f r e e The l a t t e r p r o v i d e d a u s e f u l  r e f e r e n c e compound i n some t h i n l a y e r systems, w h i l e i n o t h e r s i t was found t o obscure t h e amino a c i d d e r i v a t i v e s w i t h s i m i l a r Rf v a l u e s . Three s o l v e n t systems were used r o u t i n e l y i n i d e n t i f i c a t i o n o f d a n s y l amino a c i d s ( 7 2 ) : system A  (chloroform  : methanol : g l a c i a l a c e t i c a c i d ; 95 : 10 : 1 ) , system B (n-propanol  : ammonia; 80 : 20) and system C (n-propanol :  w a t e r ; 80 : 2 0 ) .  Systems A and B s e p a r a t e d most o f t h e  d a n s y l d e r i v a t i v e s o f amino a c i d s p r e s e n t i n neurohypophysi hormones, w h i l e system C was used t o improve t h e s e p a r a t i o n of DNS-aspartic  and D N S - c y s t e i c  acids.  Because i t i s b a s i c  System B enhanced f l u o r e s c e n c e and was an e x c e l l e n t medium t o b r i n g o u t weak f l u o r e s c e n c e o f some p e p t i d e  derivatives.  Kontes t h i n l a y e r p l a t e s , 200 x 200 mm, were  spread  w i t h S i l i c a g e l (5 gm suspended i n 9.5 ml o f water) u s i n g  the g l a s s a p p l i c a t o r r o d a c c o r d i n g t o t h e d i r e c t i o n s o f t h e m a n u f a c t u r e r (74).  A f t e r d r y i n g s u f f i c i e n t l y t o t u r n opaque,  the g l a s s p l a t e was t r a n s f e r r e d t o an 85°C oven f o r one hour and t h e n s t o r e d i n a d e s s i c a t o r over c a l c i u m c h l o r i d e . to  Prior  s p o t t i n g , t h e p l a t e was d i v i d e d i n t o 7 - 1 0 mm channels  u s i n g a sharp o b j e c t t o mark t h e c o a t i n g on t h e p l a t e .  The  d r y sample o f d a n s y l a t e d p e p t i d e h y d r o l y z a t e was d i s s o l v e d i n 1 H ammonium h y d r o x i d e  (5 y l ) and s p o t t e d r e p e a t e d l y  until  a l l f l u o r e s c e n c e was t r a n s f e r r e d from t h e t e s t tube as observed i n t h e U.V. v i e w e r Products  (Chromato-Vue, U l t r a v i o l e t  L t d . , San G a b r i e l , C a l i f . ) .  The s t a n d a r d s o l u t i o n s  were such t h a t one t o two a p p l i c a t i o n s y i e l d e d a spot o f suitable intensity.  S m a l l compact s p o t s improved s e p a r a t i o n .  For t h i s r e a s o n , a t h i n c a p i l l a r y tube (1 mm i n o u t s i d e d i a m e t e r ) was used f o r a p p l i c a t i o n , and t h e spot d r i e d immediately  i n a c u r r e n t o f warm a i r .  Kontes t h i n l a y e r chromatography tanks  (250 x 250 mm)  were l i n e d w i t h Whatman No. 1 f i l t e r paper and e q u i l i b r a t e d w i t h t h e s o l v e n t system. for  each r u n .  A f r e s h s o l v e n t m i x t u r e was used  When s u f f i c i e n t m a t e r i a l was a v a i l a b l e two  p l a t e s were s p o t t e d and chromatographed s i m u l t a n e o u s l y i n two s o l v e n t systems.  When t h e amount o f m a t e r i a l was l i m i t e d ,  the same p l a t e was r e - r u n i n a d i f f e r e n t system a f t e r a short r e a c t i v a t i o n period.  When t h e s p o t s t o be i d e n t i f i e d  were slow r u n n i n g i n t h e f i r s t s o l v e n t system, t h e second  chromatography was done i n the same d i r e c t i o n as the f i r s t . When the s p o t s were f a s t r u n n i n g i n the f i r s t system, i t was found c o n v e n i e n t t o r e v e r s e the p l a t e and t o do the second chromatography i n the o p p o s i t e d i r e c t i o n .  Chromato-  graphy i n system A r e q u i r e d a p p r o x i m a t e l y 1 hour; i n B, 2-1/2 the  h o u r s ; and i n C, 3-1/2  hours.  Following  chromatography,  p l a t e s were d r i e d and photographed. The photography was done u s i n g a P o l a r o i d Model  camera equipped w i t h a copy l e n s and U.V. P h o t a r , U.V.,  filter  160  (Tiffen  1-b s e r i e s 7) and P o l a r o i d Land P i c t u r e R o l l  Type 47, 3,000 speed f i l m and No. 10 a p e r t u r e on the camera. The p l a t e was i l l u m i n a t e d w i t h a M i n e r a l i g h t UVS  11 u l t r a -  v i o l e t l i g h t s o u r c e ( U l t r a v i o l e t P r o d u c t s , San G a b r i e l , Calif.).  The exposure time v a r i e d from 18 seconds f o r the  p l a t e s chromatographed i n p r o p a n o l : ammonia t o 45 seconds for  t h e p l a t e s r u n i n c h l o r o f o r m : methanol : a c e t i c .  The  time of exposure was determined i n each case by the i n t e n s i t y of  fluorescence.  The development of the photograph was  a l l o w e d t o proceed f o r 10 seconds.  47  III. A.  (1)  RESULTS  S t a b i l i t y of Oxytocic A c t i v i t y i nthe P i t u i t a r y T i s s u e and T i s s u e E x t r a c t s of Oncorhynchus tschawytscha  S t a b i l i t y of Oxytocic A c t i v i t y  During  Storage of  G l a n d s a t -196°C The o x y t o c i c a c t i v i t y o f p i t u i t a r y  glands  stored i n  l i q u i d n i t r o g e n (-196°C) f o r p e r i o d s r a n g i n g f r o m two t o t w e n t y - s i x m o n t h s was s u r v e y e d  i n order t o determine  group o f glands would g i v e t h e b e s t y i e l d hormones.  I n t h e same e x p e r i m e n t  of  neurohypophysial  a comparison of glands  c o l l e c t e d a t t h r e e d i f f e r e n t l o c a t i o n s w e r e made. mately  which  Approxi-  one gram o f e a c h t i s s u e was e x t r a c t e d i n i d e n t i c a l  m a n n e r , u s i n g 2.5 m l o f 0.25% a c e t i c a c i d p e r gram a n d f o l lowing the procedure  p r e s c r i b e d by t h e B r i t i s h Pharmacopeia  (1958)  ( 5 9 ) . The b i o a s s a y was done u s i n g t h e m e t h o d o f  Holton  ( 5 5 ) . The t i m e  the glands  interval  between t h e e x t r a c t i o n o f  a n d t h e p e r f o r m a n c e o f t h e b i o a s s a y was  identical.  The r e s u l t s a r e shown i n T a b l e I I . The d i f f e r e n c e s i n s p e c i f i c  a c t i v i t i e s observed  were  minor, c o n s i d e r i n g t h a t the average e r r o r o f the bioassay was 1 0 % .  A l l p r e p a r a t i v e e x t r a c t s were from  c o l l e c t e d a t Green R i v e r Hatchery.  pituitaries  Oxytocic a c t i v i t y sur-  48 T A B L E  II  Comparison o f t h e S p e c i f i c A c t i v i t i e s o f t h e P i t u i t a r y Samples o f Oncorhynchus  tschawytscha c o l l e c t e d a t  Three D i f f e r e n t L o c a t i o n s  S i t e of Collection  Months Stored at -196°C  S e x  Specific Activity Oxytocic units per A . U . ^ 2 8 0  L i m i t of E r r o r of Bioassay  L i t t l e White  26  female  0.0437  6.0  L i t t l e White  14  female  0.0471  10.9  L i t t l e White  2  female  0.0463  7.7  S p r i n g Creek  2  female  0.0582  12.3  Green R i v e r  2  female  0.0593  14.3  Green R i v e r  2  male  0.0512  8.1  Green R i v e r H a t c h e r y ( S t a t e ) , near Auburn, Wash. S p r i n g Creek and L i t t l e White H a t c h e r i e s near B i n g e n , Wash.  (Federal),  49 v i v e d extended c o l d s t o r a g e o f t h e i n t a c t g l a n d s .  This  method o f p r e s e r v a t i o n a t -196°C, o r a l t e r n a t i v e l y , a t -80°C, was adopted as t h e s t a n d a r d (2)  procedure.  S t a b i l i t y o f O x y t o c i c A c t i v i t y i n Crude and P a r t i a l l y Purified Pituitary Extracts F i g u r e 3 demonstrates t h e l o s s o f o x y t o c i c  activity  i n impure e x t r a c t s under n e u t r a l and s l i g h t l y b a s i c c o n d i tions.  I t can be seen t h a t a c t i v i t y i s l o s t g r a d u a l l y a t  pH 7.6 (50% o f i n i t i a l v a l u e found a f t e r 80 m i n u t e s ) ,  while  a t pH 8.1 o x y t o c i c a c t i v i t y o f a crude e x t r a c t i s l o s t a t a higher r a t e . I n t h e c o u r s e o f l o n g term s t o r a g e  (21 days) o f crude  p i t u i t a r y e x t r a c t s i n 0.002 M a c e t i c a c i d a t 4°C l o s s e s up t o 68% o f t h e i n i t i a l o x y t o c i c a c t i v i t y were observed. S t o r a g e i n 0.05 M a c e t i c a c i d under t h e same c o n d i t i o n s r e s u l t e d i n l o s s e s o f o x y t o c i c a c t i v i t y up t o 42%.  Losses o f  a c t i v i t y were n o t observed when t h e crude p i t u i t a r y e x t r a c t s were s t o r e d i n 0.2 M a c e t i c a c i d a t 4°C f o r 21 days.  Similar  l o s s e s were n o t observed i n c o n t r o l experiments u s i n g Parke-Davis oxytocin. A partially purified extract (biologically active eff l u e n t o f g e l f i l t r a t i o n ) c o n t a i n i n g 557* 79 u n i t s o f oxyt o c i c a c t i v i t y was s t o r e d f o r two months a t -20°C as a l y o p h y l i z e d powder.  The b i o a s s a y  following t h i s period of  50  Each p o i n t on the graph r e p r e s e n t s one r a t u t e r u s w i t h o u t magnesium (55).  f o u r p o i n t assay on  s t o r a g e showed t h a t 530-57 u n i t s o f o x y t o c i c a c t i v i t y  sur-  vived the storage period.  B.  E x t r a c t i o n o f N e u r o h y p o p h y s i a l Hormones from Salmon P i t u i t a r i e s  The e f f e c t on t h e y i e l d o f o x y t o c i c a c t i v i t y o f varying  s e v e r a l o f t h e parameters i n t h e e x t r a c t i o n p r o -  cedure a r e r e c o r d e d i n T a b l e I I I . vestigated  included  concentration  Factors  which were i n -  t h e temperature o f e x t r a c t i o n , pH,  of e x t r a c t a n t ,  t h e r e l a t i v e amounts o f s o l v e n t  and t i s s u e , t h e s t a t e o f t h e t i s s u e ( f r o z e n , l y o p h y l i z e d , o r acetone d r i e d g l a n d s ) and t h e method o f d i s r u p t i n g t h e tissue.  Factors  a c t i v i t y included  which l e d t o i n c r e a s e d  y i e l d of oxytocic  adequate d i s i n t e g r a t i o n o f t i s s u e , low  t e m p e r a t u r e , low pH and adequate amounts o f e x t r a c t i n g vent.  The c o n c e n t r a t i o n  sol-  o f a c e t i c a c i d above 0.2 M, t h e  s t a t e of t h e t i s s u e ( f r o z e n , l y o p h y l i z e d o r acetone were r e l a t i v e l y u n i m p o r t a n t i n o b t a i n i n g  dried)  optimum y i e l d s .  The p r o c e d u r e s u b s e q u e n t l y adopted f o r e x t r a c t i o n o f hormones i n v o l v e d d i s r u p t i o n o f t i s s u e i n 0.2 M a c e t i c a c i d a t 4°C.  C.  P u r i f i c a t i o n of Neurohypophysial Hormones o f Salmon  Twenty f o u r p i t u i t a r y e x t r a c t s were made i n t h e c o u r s e o f t h i s work.  The f i r s t twenty e x t r a c t s s e r v e d t o  T A B L E Extraction of Oxytocic A c t i v i t y State of tissue  Method o f disruption*  Frozen  P o t t e r - -Elvehjem  0.25% HOAc  2 .5  100  5 min.  Acetone powder  P o t t e r - -Elvehjem  0.25% HOAc  .- 5 .0  100  5 min.  Acetone powder  TRI-R  0.25% HOAc  . 10.0  100  3 min.  Frozen  TRI-R  g l a c i a l HOAc  10 .0  25  24 h r .  Lyophylized Lyophylized  P o t t e r - -Elvehjem  2M HOAc  . 10.0  25  24 h r .  P o t t e r - -Elvehjem  2 M HOAc  - 10 .0  4  24 h r .  Frozen  TRI-R  2 M HOAc  .. 10 .0  4  5 min.  Frozen  TRI-R  1 M NaOAc  r  10 .0  4  5 min.  Frozen  Waring + TRI-R  0.2 M HOAc  - 10 .0  4  5 min.  Frozen  Waring  0.2 M HOAc  10 .0  4  5 min.  Volume o f extractant Extraction (ml) p e r gm Temp. D u r a t i o n wet t i s s u e wt.(°C)  Extractant  r  * P o t t e r - E l v e h j e m g l a s s t i s s u e homogenizer. S-63) e l e c t r i c t i s s u e homogenizer, full  speed.  TRI-R STIR-R  s e t t i n g 5.  (model  Waring b l e n d e r ,  from t h e _ P i t u i t a r i e s o f Oncorhynchus Crude e x t r a c t . ^ o b t a i n e d Absorbance Oxytocic u n i t s per r a t i o nm - '280nm ' 280/260 A  U  G  m  w  e  t  w t  tschawytscha L i m i t of error of bioassay  0.67  0.0593  1.8  6.0  0.81  0.0765  2.5  13.0  0.1040  4.5  10.2  0.0360  6.3  8.6  0.83  0.144  9.5  7.3  0.81  0.148  8.5  11.0  0.93  0.076  8.2  6.2  0.88  0.010  1.4  12.9  0.87  0.079  10.0  10.6  0.90  0.098  7.3  9.3  0.84  _  d e v e l o p o p t i m a l c o n d i t i o n s f o r p u r i f i c a t i o n s t e p s , and u t i l i z e d from one t o t e n grams o f t i s s u e each.  The l a s t  f o u r e x t r a c t s were done on p r e p a r a t i v e s c a l e each u t i l i z i n g 100 gm o f t i s s u e , and p r o v i d e d pure hormones f o r t h e analyses  o f amino a c i d c o m p o s i t i o n  and sequence.  The f l o w  c h a r t i l l u s t r a t e d i n F i g u r e 4 summarizes t h e p u r i f i c a t i o n s c h e d u l e s developed f o r l a r g e s c a l e e x t r a c t s .  (1)  G e l F i l t r a t i o n o f Crude Salmon P i t u i t a r y E x t r a c t s Two t y p e s o f g e l f i l t r a t i o n media were used i n p u r i -  f y i n g t h e n e u r o h y p o p h y s i a l hormones o f salmon: Sephadex.  B i o g e l and  Only Sephadex G-15 was used i n p r e p a r a t i v e work.  F i g u r e 5 i l l u s t r a t e s a t y p i c a l e l u t i o n p r o f i l e obt a i n e d u s i n g B i o g e l P-2 and 0.2 M a c e t i c a c i d as t h e e l u t i n g buffer.  S p r e a d i n g o f hormonal a c t i v i t y was observed when  0.002 M a c e t i c a c i d was used f o r e l u t i o n .  Similar  spreading  of s y n t h e t i c o x y t o c i n took p l a c e under i d e n t i c a l c o n d i t i o n s . A comparison o f Sephadex G-25 and G-15 i s shown i n F i g u r e 6.  A spreading  of hormonal a c t i v i t y , s i m i l a r t o t h a t  observed on B i o g e l P-2, d i d n o t take p l a c e a l t h o u g h i o n i c s t r e n g t h b u f f e r was used.  a low  The p u r i f i c a t i o n on  Sephadex G-15 i s almost 100% b e t t e r t h a n t h a t o b t a i n e d on G-25 columns.  T h i s o b s e r v a t i o n - w a s made c o n s i s t e n t l y , s i n c e  Sephadex G-25 columns were used i n p u r i f y i n g t h e f i r s t t e n salmon p i t u i t a r y e x t r a c t s .  Sephadex G-15 was used i n a l l  55 Oncorhynchus t s c h a w y t s c h a , f r o z e n p i t u i t a r i e s , 100 g 0.2 M HOAc, c o l d , 10:1 v/w TISSUE HOMOGENATE 48,200 g, 30 min. I  CRUDE EXTRACT  PRECIPITATE re-extracted  (1 L) Sephadex G-15 5 tandem runs ACTIVE EFFLUENT (1 L) UM-2 o r UM-3 ultrafiltrations (lyophylization) CONCENTRATE one o f : Whatman CM-32 SE-Sephadex G-25 Phosphocellulose  FILTRATE  i HORMONE I  :  HORMONE I I  I  (ultrafiltration; lyophylization) SE-Sephadex C-25 o r Whatman CM-32 HORMONE I Spec.activity:* F i g u r e 4:  *  1.25-1.45 x 10'  Ultrafiltration; lyophylization  HORMONE I I 2.09-2.29 x 10'  P u r i f i c a t i o n Sequence o f Large S c a l e Salmon Pituitary Extract  O x y t o c i c u n i t s p e r mg  P r o c e d u r e shown i n b r a c k e t s was n o t always employed.  56  3.00-1  Salt Hormone  2.50-  2.00-  E c O CO OJ  1.50-  <  1.00-  1 0.50  -  10  —i— 20  I 30  I— 40  —  i  50  Fraction No.  F i g u r e 5:  G e l f i l t r a t i o n o f crude salmon p i t u i t a r y on B i o g e l P-2.  extract  Coliamn 320 mm x 30 mm d i a m e t e r ; e l u e n t 0.2 M a c e t i c a c i d ; f l o w r a t e 1.0 ml/min; f r a c t i o n volume 5.0 ml. L o a d i n g sample: units.  5.0 m l ; 94.5 A . U .  2 g Q  ; 15.9JI1.2 o x y t o c i c  P u r i f i c a t i o n 3.4 f o l d ; r e c o v e r y o f o x y t o c i c a c t i v i t y  89.5%.  57  to  4.03  3.87  3.50-  3.00-  A_  B.  G-25  G-15  2.50 -  £  2.00-  0  10  20  30  40  Fraction No.  F i g u r e 6:  50  0  10  20  30  40  50  F r a c t i o n No.  Comparison o f g e l f i l t r a t i o n on Sephadex and Sephadex G-15.  G-25  Columns 550 mm x 10 mm d i a m e t e r ; e l u e n t 0.002 M a c e t i c a c i d ; f l o w r a t e 0.5 ml/min (A) and 0.3 ml/min ( B ) ; f r a c t i o n volume 1.5 ml (A) and 1.0 ml ( B ) . L o a d i n g sample (A and B ) : 0.8 m l ; 51.0 A . U . o 0.7610.051 o x y t o c x c u n i t s .  ; Dn  P u r i f i c a t i o n 2.6 f o l d ( A ) , and 4.7 f o l d ( B ) ; r e c o v e r y o f b i o l o g i c a l a c t i v i t y 53% (A) and 48% ( B ) . S o l i d l i n e : absorbance a t 280 nm; c r o s s - h a t c h e d : containing effluent fractions.  hormone  58 l a t e r e x p e r i m e n t s , b u t t h e m o l a r i t y o f e l u t i n g b u f f e r was i n c r e a s e d t o 0.2 M.  T h i s was done i n o r d e r t o p r e s e r v e  maximum o x y t o c i c a c t i v i t y o f t h e e x t r a c t , as d i s c u s s e d i n section 2 of t h i s  chapter.  F u r t h e r a d a p t a t i o n o f t h e Sephadex G-15 g e l f i l t r a t i o n i n v o l v e d u s i n g t h e same column r e p e a t e d l y separations) extract  (tandem  t o a l l o w r a p i d p r o c e s s i n g o f l a r g e volumes o f  (Figure 7 ) . This permitted  gelfiltration  f o l l o w i n g the e x t r a c t i o n of the t i s s u e without concentration.  immediately  t h e need f o r  The e l u t i o n o f hormones c o i n c i d e d w i t h t h e  e l u t i o n of s a l t i n a l l g e l f i l t r a t i o n experiments.  The  usual y i e l d of oxytocic a c t i v i t y f o l l o w i n g g e l f i l t r a t i o n on Sephadex G-15 w i t h 0.2 M a c e t i c a c i d as e l u e n t ranged from 90 t o 100%. However, i n one l a r g e s c a l e e x p e r i m e n t , the s a l t peak was used i n s t e a d o f t h e b i o a s s a y the hormones, and t h e r e c o v e r y  of the i n i t i a l  to locate oxytocic  acti-  v i t y was 78%. Rechromatography o f t h e b i o l o g i c a l l y a c t i v e f r a c t i o n from Sephadex G-15 on Sephadex G-10 o r G-15 r e s u l t e d i n two-fold p u r i f i c a t i o n .  P u r i f i c a t i o n a f f o r d e d a t t h i s stage  by G-10 columns was comparable w i t h t h a t o f G-15 columns. Ah i n c r e a s e i n l e n g t h o f t h e column had no e f f e c t on p u r i f i c a t i o n o b t a i n e d by t h i s rechromatography ( F i g u r e 8 ) . A t w o ^ f o l d p u r i f i c a t i o n a t t h i s e a r l y stage o f t h e i s o l a t i o n sequence was r e g a r d e d t o be o f l i t t l e v a l u e t o t h e o v e r a l l  I  • .  .  _  _  F i g u r e 7:  . .  _  Fraction No.  _  '  G e l f i l t r a t i o n o f crude salmon p i t u i t a r y e x t r a c t u s i n g a tandem s e r i e s o f Sephadex G-15.  Column 450 mm x 50 mm d i a m e t e r ; e l u e n t 0.2 M a c e t i c a c i d ; f l o w r a t e 4 ml/min; f r a c t i o n volume 20 m l . T o t a l l o a d i n g sample:  1050 m l ; 12060  P u r i f i c a t i o n 4.4 f o l d ; r e c o v e r y  A . U .  2  8  q  954-103 o x y t o c i c u n i t s .  o f o x y t o c i c a c t i v i t y 96%.  S o l i d l i n e : absorbance a t 280 nm; d o t t e d l i n e : b a r s : hormone c o n t a i n i n g e f f l u e n t f r a c t i o n s .  specific conductivity;  Ul  60  0-15 . 2 . 5 x 9 2  Bl  6-15.  5x44 salt  Salt I  8.00-1  1  Hormone  Hormone  I  I  6.00-  E c O CO CM  4.00 -  1 ft  2.00-  -T— 20  T" 30  T  T  —i  40  90  10  A.  20  Fraction  Fraction No.  F i g u r e 8:  1—  T"  30  40  -1 90  No.  Rechroma t o g raphy o f p a r t i a l l y p u r i f i e d salmon p i t u i t a r y e x t r a c t on Sephadex G-15 columns o f e q u a l g e l bed volumes and d i f f e r e n t d i m e n s i o n s .  Column 920 mm x 25 mm d i a m e t e r ; e l u e n t 0.2 M a c e t i c a c i d ; f l o w r a t e 0.6 ml/min; f r a c t i o n volume 10.0 m l . L o a d i n g solution: 12.0 m l ; 723 A.U.280 ' 435144 o x y t o c i c u n i t s . P u r i f i c a t i o n 1.9 f o l d ; r e c o v e r y o f o x y t o c i c a c t i v i t y 93%. 11111  B.  Column 440 mm x 50 mm d i a m e t e r ; e l u e n t 0.2 M a c e t i c a c i d ; f l o w r a t e 4.0 ml/min; f r a c t i o n volume 20.0 m l . L o a d i n g s o l u t i o n : 41 m l ; 800 A.U. go > 480+49 o x y t o c i c u n i t s . P u r i f i c a t i o n 2.1 f o l d ; r e c o v e r y o f o x y t o c i c a c t i v i t y 94%. 1)1X1  2  61 p u r i f i c a t i o n o f the e x t r a c t .  On t h e o t h e r hand, e v e r y  a d d i t i o n a l s t e p i n c l u d e d i n t h e i s o l a t i o n sequence was a p o t e n t i a l source f o r l o s s o f v a l u a b l e m a t e r i a l . r e a s o n rechromatography  For t h i s  o f t h e a c t i v e e l u e n t from t h e f i r s t  g e l f i l t r a t i o n s t e p on another g e l f i l t r a t i o n column was not adopted f o r r o u t i n e use.  (2)  D e - s a l t i n g o f N e u r o h y p o p h y s i a l Hormones D e - s a l t i n g of p a r t i a l l y p u r i f i e d neurohypophysial  hormones i s a n e c e s s a r y p r e l i m i n a r y t o i o n exchange chromatography.  I t was observed d u r i n g g e l f i l t r a t i o n  on salmon p i t u i t a r y e x t r a c t s  experiments  ( c f . s e c t i o n 4(a) o f t h i s  c h a p t e r ) t h a t the s a l t peak c o i n c i d e d w i t h t h e peak o f oxyt o c i c a c t i v i t y on B i o g e l P-2, Sephadex G-25 and Sephadex G-15.  Two methods o f d e - s a l t i n g were examined i n t h e c o u r s e  of t h i s work:  g e l f i l t r a t i o n on Sephadex G-10 and u l t r a -  filtration. (i)  D e - s a l t i n g on Sephadex  G-10  Commercial p r e p a r a t i o n s o f o x y t o c i n (Sigma, s y n t h e t i c powder, Grade IV) and b o v i n e albumen (Armour, c r y s t a l i n e ) were used f o r t h e s e e x p e r i m e n t s .  Bovine albumen was used as  the i n d i c a t o r of the e l u t i o n f r o n t i n p l a c e of Blue Dextran (Pharmacia), because the l a t t e r e x h i b i t s a h i g h degree o f s p r e a d i n g on Sephadex G-10 g e l .  A Sephadex G-10 ( l * l m x 10 mm d i a m e t e r ) column  pro-  v i d e s adequate s e p a r a t i o n o f o x y t o c i n and sodium c h l o r i d e ( F i g u r e 9,A). However, sodium a c e t a t e and ammonium a c e t a t e c o u l d n o t be s e p a r a t e d  from o x y t o c i n i n t h e same system  ( F i g u r e 9,B). Attempts were made t o o b t a i n s e p a r a t i o n by v a r y i n g t e m p e r a t u r e , f l o w r a t e , and t h e c o m p o s i t i o n eluting buffer.  of the  The f l o w r a t e was v a r i e d from 0.5 t o 13.7  ml/min, t h e temperature from 9.5°C t o 73.0°C, and t h e e l u t i n g b u f f e r was v a r i e d by i n c r e a s i n g t h e c o n c e n t r a t i o n of a c e t i c a c i d from 0.002 M t o 0.200 M and by a d d i t i o n o f 20% m e t h y l a l c o h o l . or i n c o m b i n a t i o n , (ii)  None o f t h e c o n d i t i o n s t r i e d , achieved  the desired  singly  separation.  D e - s a l t i n g by u l t r a f i l t r a t i o n D i a f l o UM-2 and UM-3 membranes were used f o r de-  s a l t i n g o f p i t u i t a r y e x t r a c t s o f salmon.  The b i o l o g i c a l l y  a c t i v e e l u e n t from Sephadex G-15 was s u b j e c t e d t o u l t r a f i l t r a t i o n p r i o r t o t h e s e p a r a t i o n o f hormones on a c a t i o n exchanger.  F o l l o w i n g t h i s s e p a r a t i o n and b e f o r e  rechroma-  tography o f i n d i v i d u a l hormones on another c a t i o n exchanger, d e - s a l t i n g was a l s o r e q u i r e d .  The degree t o which t h e sample  was d e - s a l t e d by u l t r a f i l t r a t i o n a t each s t e p was d e t e r mined by t h e i o n i c s t r e n g t h a t which t h e d e s i r e d substance was e x p e c t e d t o be e l u t e d from t h e i o n exchange  column.  -|2.00  2°  30  40  90  60  70  20  Fraction No.  30  40  SO  60  70  Fraction No.  F i g u r e 9: D e - s a l t i n g o f o x y t o c i n on Sephadex G-10. Column 1.1 m x 10 mm d i a m e t e r ; e l u e n t 0.002 M a c e t i c f l o w r a t e 0.3 ml/min; f r a c t i o n volume 1.0 m l .  acid;  Peal; 1 ( s o l i d l i n e ) b o v i n e albumen, Armour c r y s t a l i n e ; peak 2 ( s o l i d l i n e ) o x y t o c i n , Sigma s y n t h e t i c powder, Grade I V ; peak 3 ( d o t t e d l i n e ) - s a l t L e f t - h a n d s c a l e : A 280 nm. ?  A:  peak 3 i s sodium c h l o r i d e .  B:  peak 3 i s sodium a c e t a t e .  64 T a b l e s I V and V i l l u s t r a t e t h e r e c o v e r i e s o f m a t e r i a l following u l t r a f i l t r a t i o n .  The r e s u l t s o b t a i n e d appeared  t o be independent o f t h e c o n t e n t s o r o f t h e volume o f t h e sample, and s u b j e c t t o i n d i v i d u a l p e c u l i a r i t i e s o f t h e membranes used. the v e r y poor.  The r e c o v e r i e s ranged from t h e v e r y good t o I n former c a s e s , a c o n s i d e r a b l e c o n c o m i t a n t  p u r i f i c a t i o n o f t h e sample was observed as can be seen from the absorbance measurements.  I t can be seen from t h e s e  r e s u l t s t h a t two t y p e s o f l o s s e s o f m a t e r i a l were e x p e r ienced:  p a s s i n g o f t h e hormones i n t o t h e f i l t r a t e and  complete l o s s o f t h e hormones.  The second t y p e o f l o s s  c o u l d n o t be e x p l a i n e d s i n c e t h e d e t a i l s o f membrane s t r u c t u r e a r e n o t r e p o r t e d by t h e m a n u f a c t u r e r s ( 6 0 ) . Some e x p e r i m e n t s w i t h UM-1 membranes, d i r e c t e d a t removing h i g h m o l e c u l a r w e i g h t contaminants were l a r g e l y negative.  Sandoz " S y n t o c i n o n " was passed q u a n t i t a t i v e l y  i n t o t h e u l t r a f i l t r a t e , as was e x p e c t e d from t h e m o l e c u l a r w e i g h t s c u t - o f f range o f t h i s t y p e o f membrane (10,000) and the m o l e c u l a r w e i g h t o f o x y t o c i n (1,000).  Oxytocic a c t i v i t y  of crude e x t r a c t s o f salmon p i t u i t a r i e s , on t h e o t h e r hand, was n o t passed by t h e UM-1 membranes. (3)  S e p a r a t i o n o f Neur ohypophy s i a1 Hormones o f Salmon by I o n Exchange  Chromatpgraphy  F o l l o w i n g g e l f i l t r a t i o n and d e - s a l t i n g , t h e two n e u r o h y p o p h y s i a l hormones o f salmon were s e p a r a t e d by i o n  T A B L E U l t r a f i l t r a t i o n of P a r t i a l l y P u r i f i e d  IV Pituitary  t s c h a w y t s c h a U s i n g D i a f l o UM-2 Volume of sample (ml) 100  (Syntocinon)  % of i n i t i a l o x y t o c i c a c t i v i t y i n concentrate in filtrate  E x t r a c t s of Oncorhynchus  Membranes % o f i n i t i a l absorbance @ 280 nm i n concentrate in filtrate (a)  97.5  nil  70  92.0  7.2  105  107.8  nil  225  41.2  13.7  44.6  50.4  300  80.4  15.7  49.4  54.4  100  83.8  12.5  72.8  30.4  (a)  absorbance too low f o r measurement  (b)  not t e s t e d :  70.5  26.8 (b)  10.5  p r e c i p i t a t i o n of m a t e r i a l i n c o n c e n t r a t e  A l l samples were f i l t e r e d u n t i l 10 - 15 ml remained above the membrane. A l l samples, w i t h the e x c e p t i o n o f S y n t o c i n o n , were p u r i f i e d by passage through a Sephadex G-15 column. P r e s s u r e of 80 p . s . i . was used i n a l l e x p e r i m e n t s .  T A B L E U l t r a f i l t r a t i o n of P a r t i a l l y  V  P u r i f i e d P i t u i t a r y E x t r a c t s of  Oncorhynchus t s c h a w y t s c h a U s i n g D i a f l o UM-3 Membranes Loading S o l u t i o n Contents  Volume, mli  % i n i t i a l oxytocic a c t i v i t y concentrate filtrate  % i n i t i a l absorbance @ 280 nm concentrate filtrate (a)  (a)  (a)  (a)  Sandoz 'Syntocinon' pH 4.8  160  22.8  23.0  Sandoz ' S y n t o c i n o n pH 2.1  160  25.6  23.2  600  77.8  20.6  92.0  10.6  250  89.1  12.6  80.3  18.8  Sephadex G-15 effluent  1335  83.0  7.9  Sephadex G-15 effluent  1050  103.0  nil  66.8  45.5  70  90.5  5.8  65.2  28.0  Sephadex G-15 effluent Ultrafiltration concentrate of Sephadex G-15 effluent  Hormone I a f t e r i o n exchange separation  1  (continued)  55.0  T A B L E  V  (continued)  % i n i t i a l oxytocic a c t i v i t y concentrate filtrate  % i n i t i a l absorbance § 280 nm concentrate filtrate  Loading S o l u t i o n Contents  Volume, mfc  Hormone I a f t e r i o n exchange separation  57  56.4  5.4  50.0  Hormone I I a f t e r i o n exchange separation  600  103.0  nil  31.0  Hormone I I a f t e r i o n exchange separation  250  32.4  68.4  Hormone I I a f t e r i o n exchange rechromatography  350  96.0  15.8  (a)  absorbance t o o low f o r measurement.  (b)  not tested:  All  55.0 (a)  (a)  (a)  (a)  (a)  p r e c i p i t a t i o n of m a t e r i a l i n concentrate.  samples were f i l t e r e d u n t i l 10 - 15 ml remained above t h e membrane.  68 exchange chromatography ( c f . F i g u r e 4 ) .  Three types o f  c a t i o n exchangers were used f o r t h i s purpose: carboxymethylcellulose phosphocelluose CM-25).  Whatman  (CM-32, m i c r o g r a n u l a r ) , S e l e c t a c e l  and S u l f o e t h y l Sephadex (SE-Sephadex,  The i o n exchange media were f i r s t  w i t h known s y n t h e t i c n e u r o h y p o p h y s i a l  hormones (Sigma  o x y t o c i n , Grade I V o r Sandox S y n t o c i n o n , 8-arg o x y t o c i n ) .  standardized  The n e u r o h y p o p h y s i a l  and N.B.C. 3-phe,  hormones o f salmon  were e l u t e d from these c a t i o n exchangers a t t h e i o n i c strengths corresponding  t o those a t which o x y t o c i n and  3-phe, 8-arg o x y t o c i n were e l u t e d , r e s p e c t i v e l y .  This  i n d i c a t e d t h a t a t a g i v e n pH t h e charge borne by t h e salmon neurohypophysial  hormones were i d e n t i c a l w i t h t h e charges  of o x y t o c i n and 3-phe, 8-arg o x y t o c i n , r e s p e c t i v e l y . I n i t i a l l y t h e s e p a r a t i o n o f hormones on Whatman CM-32 and on p h o s p h o c e l l u l o s e r e p o r t e d by Sawyer (45).  was a c h i e v e d u s i n g an e l u t i o n method The method i n v o l v e s t h e e l u t i o n  of t h e l e s s b a s i c hormone u s i n g 0.02 M ammonium a c e t a t e b u f f e r , pH 5, f o l l o w e d by a g r a d i e n t t o 0.2 M b u f f e r o f pH 7.5 t o e l u t e t h e more b a s i c hormone. method was s u b s t i t u t e d by a c o n t i n u o u s  Subsequently t h i s s a l t g r a d i e n t from  0.002 M t o 0.200 M sodium o r ammonium a c e t a t e a t c o n s t a n t pH  (pH 5) f o r e l u t i o n o f both hormones.  Representative  e l u t i o n p r o f i l e s f o r Whatman CM-32 and p h o s p h o c e l l u l o s e , using a continuous  s a l t g r a d i e n t a t pH o f 5, a r e i l l u s t r a -  t e d i n F i g u r e s 10 and 11 r e s p e c t i v e l y .  69  Loading and A I  A  • B  H  IM NaOA H  c  H  Fraction No.  F i g u r e 10: S e p a r a t i o n o f salmon n e u r o h y p o p h y s i a l hormones on Whatman CM-3 2. Column 390 mm x 12 mm d i a m e t e r ; f l o w r a t e 0.6 ml/min; volume 5.0 m l ; sodium a c e t a t e , pH 5.  fraction  B u f f e r A ( t o f r a c t i o n 30) 0.196 mmho, 0.002 M. B u f f e r B (A — B g r a d i e n t , f r a c t i o n s 31 — 200) 14.8 mmho, 0.200 M. Loading s o l u t i o n : per A.U^so  11111  42 m l ; s p e c i f i c  activity  (oxytocic units  ) 1*4.  Hormone I , s p e c i f i c a c t i v i t y 65.0.  a c t i v i t y 25.8; Hormone I I , s p e c i f i c  Recovery o f o x y t o c i c a c t i v i t y : 98%. L i g h t b l a c k l i n e : absorbance a t 280 nm; heavy b l a c k l i n e : o x y t o c i c a c t i v i t y (each p o i n t = one f o u r - p o i n t a s s a y ) ; dashed l i n e : s p e c i f i c c o n d u c t i v i t y .  70  Load A  A  |  GO  '  100  B  ISO  ^ I M NH.OA^  200  Fraction No.  F i g u r e 11:  S e p a r a t i o n o f salmon n e u r o h y p o p h y s i a l hormones on S e l e c t a c e l p h o s p h o c e 1 l u l o s e .  Column 420 mm x 12 mm d i a m e t e r ; f l o w r a t e 1.7 ml/min; f r a c t i o n volume 5.0 m l ; ammonium a c e t a t e , pH 5. B u f f e r A ( t o f r a c t i o n 30) 0.59 mmho, approx. 0.004 M; B u f f e r B (A — B g r a d i e n t , f r a c t i o n s 31 — 200) 9.3 mmho, 0.200 M. Loading s o l u t i o n : u n i t s p e r A.U.280 Hormone I , s p e c i f i c a c t i v i t y 93.0.  97 m l ; s p e c i f i c 1*6. activity  (oxytocic  8.9; Hormone I I , s p e c i f i c  Recovery o f o x y t o c i c a c t i v i t y : Light black l i n e : oxytocic a c t i v i t y  activity  88%.  absorbance a t 280 nm; heavy b l a c k l i n e : (each p o i n t = one f o u r - p o i n t a s s a y ) .  71 Chromatography developed  in  Hormone  I.  applied  to  file  a  in  of  a  broad  course  SE-Sephadex at of  Subsequently the  12.  scale While  In  a  Hormone  II  was  done  acetate  at  pH 5,  work  system  of  the  separation the  narrow peak, band.  this  the  separation  large  Figure  metrical  the  on  elution  the  in  a  was  (2.45)  an e f f o r t  to  scaled  and  up  SE-Sephadex i s  of  Hormone I  of  the  was  pro-  a  sym-  results  elution  manner w i t h  activity  purify  shown  gives  Hormone I I  experiment  was  An e l u e n t  on  stepwise  and o x y t o c i c  in  pH  hormones.  elution  subsequent  low  in  of  2 M ammonium  eluted  in  three  fractions. The initial  separation  for  which  end  of  were  choice  the  this  hormone. the  used. by  well  The r e s u l t s  columns  and a  did  appear  not  to  from  affect  three that  pH 5, in  for  the  purpose  Toward Hormone on  obtaining  strengths  of  I the this  which  was  best  successively  Whatman  at  and  CM-32  ammonium a c e t a t e strength  the  exchange  Hormone I and  at  by p h o s p h o c e l l u l o s e ,  length  ionic  the  of  cation  2.45,  sodium to the  by  for  and s e p a r a t i o n  ionic  a pH o f  Increase  change  the  used  intended.  method  the  indicate  SE-Sephadex at  b y Whatman C M - 3 2 .  were  fast  from  SE-Sephadex at  by  be  governed  structural studies,  eluted  to  a d d i t i o n a l amounts  VI i l l u s t r a t e s  were  was  hormones  column p r o v i d e d a Table  retained less  for  exchanger  hormones  investigation  hormones  media  of  cation  separated  required  sulfoethyl  of  buffer  w h i c h Hormone  i Loading i  O  A  H  B  A H  10  SO  100  150  H  B  i  200  250  Fraction No.  F i g u r e 12:  S e p a r a t i o n o f salmon n e u r o h y p o p h y s i a l hormones on SE-Sephadex.  Column 430 mm x 25 mm d i a m e t e r ; f l o w r a t e 0.7 ml/min; f r a c t i o n ammonium formate pH 2.45.  volume 10 m l ;  B u f f e r A ( t o f r a c t i o n 40) 1.75 mmho, 0.085 M. B u f f e r B (A — B g r a d i e n t , f r a c t i o n s 41 — 220; B, f r a c t i o n s 221 — 250) 22.0 mmho,0.2 M. Loading s o l u t i o n : Hormone I , s p e c i f i c  130 m l ; s p e c i f i c  activity  (oxytocic u n i t s per A.U^SO ™)  a c t i v i t y 2.7; Hormone I I , s p e c i f i c  1  0.42.  a c t i v i t y 3.3.  Recovery o f o x y t o c i c a c t i v i t y : 92%. L i g h t b l a c k l i n e : absorbance a t 280 nm; heavy b l a c k l i n e : a c t i v i t y (each p o i n t = one f o u r - p o i n t assay) .  oxytocic  ^, nj  T A B L E  VI  Ion Exchange Chromatography o f N e u r o h y p o p h y s i a l Hormones o f Oncorhynchus tschawytscha: C a t i o n Exchanger  S p e c i f i c C o n d u c t i v i t i e s f o r E l u t e d Hormones E l u t i n g b u f f e r used  Specific conductivity  Salt  pH  Hormone I  % initial oxytocic Hormone I I a c t i v i t y recovered  12  sodium a c e t a t e  5.0-  0.8 - 1.7  8.2 - 9. 2  87  2 7.1 - 1 1 .  70  Column length x diameter mm  Whatman CM-32  90  X  Whatman CM-32  90  X  12  sodium a c e t a t e  5.0  0.8 - 1.0  Whatman CM-32  190  X  12  ammon. a c e t a t e  5.0  0.8 - 1.9  Whatman CM-32  390  X  12  ammon. a c e t a t e  5.0  0.9 - 1.0  5.7 - 6. 7  98  Phosphocellulose  420  X  12  ammon. a c e t a t e  5.0  1.3 - 2.0  8.0 - 8. 8  88  SE-Sephadex  120  X  12  ammon. a c e t a t e  5.0  2.2 - 2.5  10.7 -11. 2  77  SE-Sephadex  120  X  12  ammon. formate  2.45  3.7 = 4.0  SE-Sephadex  430  X  25  ammon. formate  2.45  9.6 -11. 1  7/ . R->  89  68 20.0  92  M i x i n g c h a m b e r / r e s e r v o i r volumes used were as f o l l o w s : 250 : 250 c c f o r 90 x 12 columns; 450 : 450 c c f o r 420 x 12 columns; 950 : 950 c c f o r 420 x 25 columns. Ammonium and sodium a c e t a t e g r a d i e n t s were from 0.002 M t o 0.2 M; ammonium formate from 0.085 M t o 2.0 M. A pH g r a d i e n t was a p p l i e d o n l y i n t h e f i r s t e x p e r i m e n t i n the T a b l e .  74 I was  eluted.  The  recoveries  of i n i t i a l o x y t o c i c  activity  did  not appear t o v a r y w i t h the c a t i o n exchange media used,  and  f l u c t u a t e d from 68 t o 98 p e r c e n t . A t t h i s s t a g e of the p u r i f i c a t i o n , the e x t e n t of  p o t e n t i a t i o n w i t h magnesium i o n  (57) was  measured i n an  e f f o r t t o p r e d i c t the hormones' c h e m i c a l i d e n t i t y . p o t e n t i a t i o n v a l u e s ranged from 2.1 and  from 1.5  t o 2.5  f o r Hormone I I .  t o 3.8 I t was  The  f o r Hormone I not  found  p o s s i b l e t o p r e d i c t the i d e n t i t y of the hormones on b a s i s of t h e s e r e s u l t s and  the  the p o t e n t i a t i o n measurements  were not c a r r i e d on r o u t i n e l y .  On the o t h e r hand, the  r a t i o of t o t a l o x y t o c i c a c t i v i t y of Hormone I I compared t o t h a t of Hormone I was 2.0  and  the two  (4)  3.0  c o n s i s t e n t l y observed t o l i e between  i n each of the c a t i o n exchange s e p a r a t i o n s  of  hormones.  P u r i f i c a t i o n of Salmon N e u r o h y p o p h y s i a l Hormone I_ by Ion Exchange Chromatography I n o r d e r t o determine the sequence of amino a c i d s  w i t h i n the m o l e c u l e i t was  necessary to prepare a s u f f i c i e n t  amount of the salmon hormone of 90%, Quantitative was  or g r e a t e r ,  amino a c i d a n a l y s i s of the hormone  used as a c r i t e r i o n of p u r i t y .  of the Hormone I was  An amino a c i d  purity. preparation analysis  not attempted from the i n i t i a l  separa-  75  t i o n of the two hormones by c a t i o n exchange chromatography ( c f . p r e c e e d i n g s e c t i o n ) , because n e i t h e r the  specific  a c t i v i t y of the hormone a t t h i s stage of p u r i f i c a t i o n ,  nor  the appearance of the e l u t i o n p r o f i l e w a r r a n t e d an a n a l y s i s . Rechromatography of Hormone I on Whatman CM-32 o r on p h o s p h o c e l l u l o s e d i d not y i e l d a pure p r e p a r a t i o n as judged by amino a c i d a n a l y s e s .  A better purification  was  o b t a i n e d by rechromatography on SE-Sephadex a t pH 5, y e t the l e v e l of c o n t a m i n a t i n g amino a c i d s was t o attempt sequence s t u d i e s :  s t i l l too high  g l u , l y s , a l a , v a l , l e u , phe,  h i s , and a r g were p r e s e n t i n amounts r a n g i n g from 0.1 0.4  to  r e s i d u e s per m o l e c u l e . A r e d u c t i o n i n pH o f the b u f f e r used f o r rechroma-  tography  (pH 2.45  ammonium formate) y i e l d e d a pure p r e p a r -  a t i o n of Hormone I . tography s t e p .  F i g u r e 13 i l l u s t r a t e s t h i s rechroma-  The absorbance r e a d i n g s c o r r e s p o n d i n g t o  t h e peak of b i o l o g i c a l a c t i v i t y approached the l i m i t of e r r o r of the spectrophotometer  and, as such, c o u l d not be  used f o r e s t i m a t i o n of s p e c i f i c a c t i v i t y o f the Hormone I .  The s p e c i f i c a c t i v i t y was  purified  c a l c u l a t e d u s i n g the  d a t a o b t a i n e d from amino a c i d a n a l y s i s of the p r e p a r a t i o n (cf. Experimental Procedures). F u r t h e r s t u d i e s on the i s o l a t i o n of Hormone I showed t h a t the sequence i n which the chromatographies ducted a t pH 5 and pH 2.45  were con-  r e s p e c t i v e l y was n o t r e l e v a n t t o  76  B  t o tube 185  8.0-i  6.0-  Hormone J_ x  2  <u  E E 4.0 - -0.2 E c  O O CO 3 "D CM C o 2.0 -4-0.1 < o  1  x— x—  F i g u r e 13:  $  x•x - i — 60 90 t X—X—X—X— F r a c t i o n No.  30  L  L  -i 120  J  6  0  Rechromatography o f Hormone I_ on SE-Sephadex,  Column 120 mm x 12 mm d i a m e t e r ; f l o w r a t e 2.5 ml/min; f r a c t i o n volume 5.0 m l ; ammonium formate pH 2.45. B u f f e r A ( t o f r a c t i o n 10) 1.72 mmho, 0.085 M. B u f f e r B (A — B g r a d i e n t , f r a c t i o n s 1 1 — 185) 11.4 mmho, 1 M. Loading s o l u t i o n : per  A.U. g nni) 2  0  4 ml; s p e c i f i c a c t i v i t y  (oxytocic units  20.0.  Hormone I ( p u r i f i e d ) , s p e c i f i c a c t i v i t y 145 o x y t o c i c u n i t s per m i l l i g r a m . Recovery o f o x y t o c i c a c t i v i t y : 68%. Light black l i n e : absorbance a t 280 nm; heavy b l a c k l i n e : o x y t o c i c a c t i v i t y (each p o i n t = one f o u r - p o i n t a s s a y ) ; dashed l i n e : specific conductivity.  the p u r i f i c a t i o n .  Thus t h e i n i t i a l s e p a r a t i o n o f Hormone I  and Hormone I I c o u l d be done on SE-Sephadex a t pH 2.45, and the subsequent rechromatography o f Hormone I on Whatman CM-32 a t pH 5.  F i g u r e 14 i l l u s t r a t e s a rechromatography on  Whatman CM-32 and which r e s u l t e d i n p r e p a r a t i o n o f pure hormone, f o l l o w i n g t h e i n i t i a l s e p a r a t i o n o f hormones on an SE-Sephadex column. Amino a c i d a n a l y s e s o f p u r i f i e d Hormone I from t h r e e separate  i s o l a t i o n p r o c e d u r e s a r e g i v e n i n T a b l e V I I and  are compared w i t h t h e t h e o r e t i c a l v a l u e s f o r 4-ser, 8 - i l e u o x y t o c i n which have been r e p o r t e d from o t h e r t e l e o s t s .  On  the b a s i s o f q u a n t i t a t i v e amino a c i d a n a l y s i s d a t a i n t h i s t a b l e , Hormone I o f 0. t s c h a w y t s c h a and 4-ser, 8 - i l e u oxyt o c i n appear t o have i d e n t i c a l amino a c i d The  compositions.  s p e c i f i c a c t i v i t y c a l c u l a t e d f o r Hormone I o f salmon  was 125, 138 and 145 o x y t o c i c u n i t s p e r mg, and t h e s p e c i f i c a c t i v i t y v a l u e r e p o r t e d i n t h e l i t e r a t u r e u s i n g t h e same assay method on t h e s y n t h e t i c p r e p a r a t i o n o f t h e hormone i s 150 o x y t o c i c u n i t s p e r m i l l i g r a m (Table V I I ) .  (5)  P u r i f i c a t i o n o f Salmon Neurohypophysia1 Hormone I I by I o n Exchange Chromatography Amino a c i d a n a l y s e s o f Hormone I I f o l l o w i n g o n l y one  c a t i o n exchange chromatography (the i n i t i a l s e p a r a t i o n o f Hormone I and Hormone I I on Whatman CM-32) appeared t o be  78  Loading  *•  A  A  B  —H  to tube 214  0.80  Hormone T 0.60  0)  E c O CO CM  a. 0.40  0.20  3 X  J  O  30  - -X->*9< —r- $<XX 60  -1 o 120 J  90  F r a c t i o n No.  F i g u r e 14: Rechromatography o f Hormone X on. Whatman CM-32. Column 190 mm x 12 mm d i a m e t e r ; f l o w r a t e 0.8 ml/min; f r a c t i o n volume 5.0 m l ; ammonium a c e t a t e pH 5. B u f f e r A ( t o f r a c t i o n 20) 0.215 mmho, 0.002 M; B u f f e r B (A — B g r a d i e n t , f r a c t i o n s 21 — 214) 6.9 mmho, 0.1 M. L o a d i n g s o l u t i o n : 53 m l ; s p e c i f i c a c t i v i t y u n i t s p e r A.U. Qnm) 3.0.  (oxytocic  28  Hormone I ( p u r i f i e d ) , s p e c i f i c a c t i v i t y 125 o x y t o c i c u n i t s per m i l l i g r a m . Recovery o f o x y t o c i c a c t i v i t y : 89%. Light black l i n e : oxytocic a c t i v i t y  absorbance a t 280 nm; heavy b l a c k l i n e : (each p o i n t = one f o u r - p o i n t a s s a y ) .  79 T A B L E  VII  Amino A c i d C o m p o s i t i o n (Residues per Mole) of N e u r o h y p o p h y s i a l Hormone I of Oncorhynchus t s c h a w y t s c h a Amino A c i d  4- s e r , 8 - i l e u oxytocin theoretical  Ammonia Cysteic acid Aspartic acid Serine Proline 1/2 Cystine Glycine Isoleucine T y r o s i n e (c) Other amino a c i d s : Lysine Valine Glutamic a c i d Leucine Oxytocic u n i t s per  ym  Oxytocic u n i t s per mg L i m i t of b i o a s s a y error, %  2.00 2.00 1.00 1.00 1.00 2.00 1.00 2.00 1.00  P r e p No. 24 (a)  a r a t i o n No.23 No. 22 (b) (b)  2.19  3.38 1.59 0.96 1.01 1.03  2.34 1.64 1.03 1.00 0.90  1.22 1.71 0.21  1.34 1.80 0.42  —  (b)  1.04 1.02 0.90 1.69 1.21 1.87 0.83  (a)  —  —  nil nil nil nil  0.06 0.04 0.16 trace  0.14 0.04 trace trace  nil trace trace trace  ——  133  121  140  150  (75)  138  125  145  8.0  (75)  10.2  14.0  8.0  (a)  H y d r o l y z a t e of u n o x i d i z e d  hormone  (b)  H y d r o l y z a t e of the hormone f o l l o w i n g p e r f o r m i c a c i d oxidation.  (c)  L o s s of t y r o s i n e was observed f r e q u e n t l y , i n b o t h the o x i d i z e d and the u n - o x i d i z e d h y d r o l y s a t e s , of b o t h the salmon hormones and the s t a n d a r d s .  O x y t o c i c a s s a y s a c c o r d i n g t o H o l t o n . M o l e c u l a r w e i g h t of 4-ser, 8 - i l e u o x y t o c i n was c a l c u l a t e d as 965. Hormone I from salmon e x t r a c t s 23 and 24 was p u r i f i e d on Whatman c a r b o x y m e t h y l c e l l u l o s e (CM-32, m i c r o g r a n u l a r ) a t pH 5 f o l l o w i n g the i n i t i a l s e p a r a t i o n of hormones on s u l f o e t h y l Sephadex (SE-Sephadex C-25), a t pH 2.45. The r e v e r s e of t h i s p r o c e d u r e was used i n p u r i f i c a t i o n of Hormone I from e x t r a c t 22.  80 warranted  on t h e b a s i s o f t h r e e c r i t e r i a :  specific  activity  o f t h e p o o l e d hormone peak, appearance o f t h e e l u t i o n p r o f i l e , and b e h a v i o u r o f t h e m o l e c u l e phoresis.  on h i g h v o l t a g e e l e c t r o -  The s p e c i f i c a c t i v i t y o f Hormone I I o b t a i n e d by  separation of neurohypophysial  hormones o f salmon on  Whatman CM-32 was i n t h e range o f 100 o x y t o c i c u n i t s p e r m i l l i g r a m , while the highest values reported i n the l i t e r a t u r e f o r 8-arg o x y t o c i n were 115 and 125 o x y t o c i c u n i t s per m i l l i g r a m ( 7 5 ) , ( 6 ) .  The 280 nm absorbance peak c o r -  r e s p o n d i n g t o t h e o x y t o c i c a c t i v i t y peak o f Hormone I I appeared s y m m e t r i c a l . preparation presence  High v o l t a g e e l e c t r o p h o r e s i s o f t h e  (pH 3.6, 3,000 v o l t s , 35 min) r e v e a l e d t h e  o f o n l y one n i n h y d r i n p o s i t i v e substance  and t h i s ,  upon e l u t i o n from t h e paper, was found t o have o x y t o c i c activity.  Amino a c i d a n a l y s i s , however, r e v e a l e d an impure  preparation:  l y s (0.43); h i s (0.52); ammonia (3.53);  arg  (1.36); asp (0.95); t h r (0.21); s e r (0.53); g l u (1.52);  pro  (1.30) ; 1/2 c y s (1.7=4); g l y (1.55); a l a (0.26); v a l  (0.31); met (0.13); i l e u phe  (0.09).  (1.07); l e u (0.20); t y r (0.93);  The u n d e r l i n e d amino a c i d r e s i d u e s r e p r e s e n t  those r e s i d u e s l a t e r found t o c o n s t i t u t e t h e m o l e c u l e o f Hormone I I . F u r t h e r p u r i f i c a t i o n o f Hormone I I by rechromatography was n e x t i n v e s t i g a t e d . F o l l o w i n g an i n i t i a l s e p a r a t i on SE-Sephadex a t pH 2.45 an attempt was made t o p u r i f y  81 Hormone I I u s i n g t h e same system as f o r Hormone I , t h a t i s , by rechromatography on Whatman CM-32.  However, a pure  p r e p a r a t i o n o f Hormone I I was n o t a c h i e v e d by t h i s method: ammonia  (6.12); a r g (0.80); Cy_SO_ (1.55); asp (0.94); g l u 3  (1.10); p r o (2.44); g_ly (1.11); i l e u lys  (1.43); v a l (1.69).  (0.86); t y r ; (0.40);  The r e m a i n i n g m a t e r i a l was sub-  j e c t e d t o rechromatography on p h o s p h o c e l l u l o s e and a pure p r e p a r a t i o n o f hormone was o b t a i n e d . F u r t h e r s t u d i e s on t h e rechromatography o f Hormone I I showed t h a t p h o s p h o c e l l u l o s e a t pH 5.0 p r o v i d e d t h e b e s t p u r i f i c a t i o n , i r r e s p e c t i v e o f whether t h e i n i t i a l a t i o n was done a t pH 5.0 o r a t pH 2.45.  fraction-  Satisfactory  p u r i f i c a t i o n was a l s o o b t a i n e d on Whatman CM-22 ( f i b r o u s ) at  pH 5.0 a f t e r i n i t i a l f r a c t i o n a t i o n under s i m i l a r con-  ditions  (Figure 15). The amino a c i d a n a l y s e s o b t a i n e d f o r t h r e e  separate  p r e p a r a t i o n s o f Hormone I I a r e comparable t o t h e t h e o r e t i c a l v a l u e s f o r 8-arg o x y t o c i n (Table V I I I ) .  However, t h e  s p e c i f i c a c t i v i t i e s o f t h e p r e p a r a t i o n s o f Hormone I I were a l m o s t t w i c e those r e p o r t e d f o r 8-arg o x y t o c i n (Table V I I I ) .  82  Hormone II  Fraction No.  Figure  15:  Rechromatography o f Hormone I I on Whatman CM-22.  Column 440 mm x 12 mm d i a m e t e r ; f l o w r a t e 2.8 ml/min; f r a c t i o n volume 5.0 m l , ammonium a c e t a t e pH 5. B u f f e r A ( t o f r a c t i o n 10) 1.65 mmho, 0.03 M; B u f f e r B (A — B g r a d i e n t , f r a c t i o n s 11 — 200) 8.5 mmho, 0.2 M. Loading s o l u t i o n : u n i t s p e r A.U^gg  11111  16.5 m l ; s p e c i f i c a c t i v i t y ) 12.3.  (oxytocic  Hormone I I ( p u r i f i e d ) , s p e c i f i c a c t i v i t y 201 o x y t o c i c per m i l l i g r a m .  units  Recovery o f o x y t o c i c a c t i v i t y : 63%. Light black l i n e : absorbance a t 2 80 nm; heavy b l a c k l i n e : o x y t o c i c a c t i v i t y ; dashed l i n e : specific conductivity.  83 T A B L E  VIII  Amino A c i d C o m p o s i t i o n (Residues p e r Mole) o f N e u r o h y p o p h y s i a l Hormone I I o f Oncorhynchus t s c h a w y t s c h a Amino A c i d  8-arginineoxytocin theoretical  Ammonia 3.00 Arginine 1.00 Cysteic acid 2.00 Aspartic acid 1.00 Glutamic a c i d 1.00 Proline 1.00 1/2 C y s t i n e 2.00 Glycine 1.00 Isoleucine 1.00 T y r o s i n e (C) 1.00 Other Amino A c i d s : Lysine nil Histidine nil Serine nil Alanine nil Valine nil Leucine nil Oxytocic u n i t s — per ym Oxytocic u n i t s per mg 125 115 L i m i t or bioassay error, % 13.0 (a) (b) (C)  (b)  (a)  (6) (75} (75)  P r e p a r a t i o n No. 23 No. 22 No. 22 No. 21 (b) (b) (a) (a) 5.20 0.79 1.71 1.02 1.04 1.05  3.98 0.83 1.96 1.09 1.00 0.80  0.94 0.90 0.25  1.14 0.87 0.43  3.68 0.82  2.98 0.68  1.06 1.08 0.84 1.54 1.27 0.92 0.55  0.96 1.02 0.92 1.40 1.01 0.65 0.40  nil nil trace nil nil trace  nil nil nil nil nil nil  nil nil nil nil nil nil  0.25 0.18 0.20 0.16 0.14 nil  220  241  238  192  209  229  227  201  6.0  6.0  6.5  13.0  H y d r o l y z a t e o f u n o x i d i z e d hormone. H y d r o l y z a t e s o f o x i d i z e d hormone ( p e r f o r m i c L o s s o f t y r o s i n e observed f r e q u e n t l y .  acid)  O x y t o c i c assays a c c o r d i n g t o H o l t o n (55). M o l e c u l a r w e i g h t o f 8-arg o x y t o c i n was c a l c u l a t e d as 1050. Hormone I I from salmon e x t r a c t 23 was p u r i f i e d by rechromatography on p h o s p h o c e l l u l o s e (Selectacel) following the i n i t i a l s e p a r a t i o n o f hormones on s u l f o e t h y l Sephadex (SE-Sephadex C-25). Hormone I I from salmon e x t r a c t 22 was p u r i f i e d by rechromatography on p h o s p h o c e l l u l o s e (Selectacel) following the s e p a r a t i o n o f hormones on Whatman CM-32. Hormone I I from salmon e x t r a c t 21 was p u r i f i e d by rechromatography on Whatman CM-22 ( c a r b o x y m e t h y l c e l l u l o s e , f i b r o u s ) f o l l o w i n g s e p a r a t i o n o f hormones on Whatman CM-32 ( c a r b o x y m e t h y l c e l l u l o s e , microgranular).  84 D.  Amino A c i d Sequence o f N e u r o h y p o p h y s i a l Hormones o f O n c o r h y n c h u s t s c h a w y t s c h a  The s e q u e n c e o f physial  amino a c i d s  of  hormones was d e t e r m i n e d f o l l o w i n g p e r f o r m i c a c i d  oxidation cysteic  to  convert cystine  and c y s t e i n e  Amino A c i d  (i)  Determination of N - t e r m i n a l  Sequence  of  a dansyl derivative  of  t o g r a p h y i n two s o l v e n t chloroform this  acids  pentapeptide  systems  : methyl a l c o h o l  result,  was  cysteic  and t h e  (propanol  : acetic  sequence of  o b t a i n e d by t h e  Edman m e t h o d .  c a n be s e e n t h a t  cycle  one o f  the after  tyrosine  is  lost  and h a l f  of  the  cycle. four  Using this  times  N-terminal S e r was  serine  results  acid). the  second,  residue  method,  the  thus  assigned  to  the  residues  three  amino  Edman.  subtractive first  degrada-  is  lost,  isoleucine  after  after  the  has been  and t w i c e  The s e q u e n c e o f the N - t e r m i n u s .  the  third,  fourth degradation  sequencing  on t h e N - t e r m i n a l t r i p e p t i d e tetrapeptide.  next  after  and  Confirmation  o b t a i n e d by t h e  two c y s t e i c the  identified  : water,  s u b t r a c t i v e method o f  the  It  Hormone I was  a c i d by t h i n l a y e r c h r o m a -  T a b l e IX p r e s e n t s  tion  into  Hormone I_ of_ Salmon  The N - t e r m i n a l amino a c i d o f  of  residues  a c i d (66).  (1)  as  b o t h salmon n e u r o h y p o -  repeated on  the  CySC^H-Tyr-Ileu-  T A B L E  IX  The Amino A c i d Sequence o f N - t e r m i n a l  Tetrapeptide  of Hormone I o f Oncorhynchus t s c h a w y t s c h a R e s u l t s o f s u b t r a c t i v e Edman d e g r a d a t i o n method:  Amino A c i d  Amino a c i d a n a l y s i s ( r e s i d u e s p e r mole) a f t e r s t e p no. 0  1  2  3  4  Ammonia  2. 19  Cysteic acid  1. 69  1..00  0 .97  0. 91  0. 91  Aspartic acid  1. 04  1..00  1 . 00  0. 90  0. 94  Serine  1. 02  0..91  0 .85  0. 84  0. 49  Proline  0. 90  0..89  0 .90  0. 95  1. 00  Glycine  1. 21  1..20  1 .22  1. 18  1. 22  Isoleucine  1. 87  1,.69  1 .63  1. 14  1. 10  Tyrosine  0. 83  0,.78  0  0  0  These a r e r e s u l t s o f d u p l i c a t e  analyses.  86 F o l l o w i n g t h e f i f t h Edman d e g r a d a t i o n c y c l e , 22% o f the a s p a r t i c r e s i d u e was l o s t from t h e p e p t i d e , i n d i c a t i n g that a s p a r t i c residue probably occupied p o s i t i o n 5 i n the molecule.  However, due t o t h e r i s i n g b a s e l i n e o f incom-  p l e t e l y r e a c t e d N - t e r m i n i i t was n o t p o s s i b l e t o c o n t i n u e w i t h t h e Edman d e g r a d a t i o n c y c l e s w i t h o u t p u r i f i c a t i o n o f the m a t e r i a l .  An attempt t o s e p a r a t e t h e c o m p l e t e l y r e -  a c t e d m a t e r i a l , r e p r e s e n t e d a t t h i s n s t a g e by t h e C - t e r m i n a l t e t r a p e p t i d e , from t h e u n r e a c t e d p e p t i d e s p e c i e s , on a DEAE-cellulose  column (18 mm x 12 mm d i a m e t e r  i n pyridinium-  a c e t a t e , pH 5) f a i l e d t o r e c o v e r t h e d e s i r e d m a t e r i a l . I n a subsequent experiment use was made o f p a r t i a l a c i d h y d r o l y s i s i n o r d e r t o o b t a i n a fragment f o r f u r t h e r sequence d e t e r m i n a t i o n .  The p e p t i d e f o l l o w i n g f i v e Edman  d e g r a d a t i o n c y c l e s a g a i n r e v e a l e d o n l y a 20% l o s s i n a s p a r t i c residues.  P a r t i a l acid h y d r o l y s i s of t h i s m a t e r i a l  u s i n g 0.01 N h y d r o c h l o r i c a c i d a t 105°C, f o r 18 h o u r s , was c a r r i e d o u t t o c l e a v e t h e a s p a r t y l (or a s p a r a g i n y l ) r e s i d u e a t t h e a d j o i n i n g N- and C - t e r m i n a l p e p t i d e bonds ( 6 9 ) . Table X i l l u s t r a t e s t h e p e p t i d e s e r i e s which c o u l d be env i s a g e d t o be p r e s e n t i n t h e p a r t i a l h y d r o l y z a t e on t h e b a s i s of t h e r e s u l t s o b t a i n e d f o r t h e N - t e r m i n a l sequence o f t h e hormone.  Cleavage was expected  t o r e l e a s e t h e r e m a i n i n g 80%  of t h e i n a c c e s s i b l e C - t e r m i n u s , as w e l l as t o c o n f i r m t h e presence o f a s p a r t i c a c i d i n p o s i t i o n f i v e .  Table X (2)  i l l u s t r a t e s t h e r e s u l t s o b t a i n e d by amino a c i d a n a l y s i s o f  87 T A B L E P a r t i a l Acid Hydrolysis  X  o f Hormone I F o l l o w i n g  F i v e Edman D e g r a d a t i o n  (1)  Peptide species  Cycles  and f r e e amino a c i d s i n p a r t i a l  hydrolyzate  As  Ser Ileu-Ser  aspartic acid  Tyr-Ileu-Ser  (CyS0 H, 3  Pro,lieu,Gly)  CyS0 -Tyr-lleu-Ser 3  P e p t i d e s and f r e e amino a c i d s r e p r e s e n t e d above a r e t h o s e which are expected t o be produced by p a r t i a l a c i d h y d r o l y s i s o f p a r t l y degraded m o l e c u l e o f Hormone I . V e r t i c a l arrows r e p r e s e n t h y d r o l y t i c c l e a v a g e s i t e s e x p e c t e d . U n d e r l i n e d amino a c i d s and p e p t i d e s a r e those which have been i d e n t i f i e d by e l e c t r o p h o r e t i c s e p a r a t i o n and amino a c i d a n a l y s e s f o l l o w i n g p a r t i a l h y d r o l y s i s . (2)  F r e e amino a c i d s  ( r e s i d u e s p e r mole o f hormone) found on  amino a c i d a n a l y s i s o f p a r t i a l Cysteic acid Aspartic acid Serine Glycine Isoleucine  hydrolyzate  0.19 1.00 0.20 0.39 0.17  L i b e r a t i o n o f a s p a r t i c a c i d was 85% o f t h e t h e o r e t i c a l v a l u e .  88 the p a r t i a l h y d r o l y z a t e . a c i d was r e l e a s e d smaller  I t can be seen t h a t f r e e  aspartic  by t h e r e a c t i o n as e x p e c t e d , t o g e t h e r w i t h  amounts o f f r e e c y s t e i c a c i d , s e r i n e , g l y c i n e , and  isoleucine.  The r e l e a s e  o f f r e e s e r i n e was a l s o expected  from t h e p a r t i a l l y r e a c t e d h e x a p e p t i d e s e r i e s but t h e r e l e a s e  (Table X ( 1 ) ) ,  o f o t h e r amino a c i d s i n d i c a t e d t h a t t h e  h y d r o l y s i s was more e x t e n s i v e than a n t i c i p a t e d s t u d i e s on s y n t h e t i c  3-phe, 8 - l y s o x y t o c i n .  from p i l o t  The r e l e a s e o f  a s p a r t i c a c i d was c a l c u l a t e d t o be 85% o f t h a t  remaining  a f t e r f i v e Edman d e g r a d a t i o n c y c l e s . Electrophoretic  separation  of p a r t i a l hydrolyzate  (pH 6.5,4,000 v o l t s , 35 min) c o n f i r m e d t h e presence o f aspartame i n p o s i t i o n 5.  Aspartic  a c i d , c y s t e i c a c i d , and  one u n i d e n t i f i e d n e u t r a l amino a c i d were r e v e a l e d by ninhydrin, peptide.  i n a d d i t i o n t o two minor p e p t i d e s and one major The p e p t i d e s were e l u t e d from t h e paper and sub-  j e c t e d t o amino a c i d a n a l y s e s .  The f i r s t minor p e p t i d e ,  i d e n t i f i e d as '0' (from t h e n e u t r a l amino a c i d r e g i o n ) d i d not p r o v i d e d e t e c t a b l e amounts o f amino a c i d s .  The second  minor p e p t i d e , i d e n t i f i e d as '1' ( m o b i l i t y 100 mm toward the anode) c o n t a i n e d e q u i m o l a r amounts o f c y s t e i c a c i d , i s o l e u c i n e , t y r o s i n e and s e r i n e , which suggested t h a t i t was d e r i v e d  from t h e N-terminus o f t h e hormone.  m a i n i n g 5.0% o f t h e e l u t e d m a t e r i a l  The r e -  from '1' was s u b j e c t e d  t o t h r e e c o n s e c u t i v e Edman d e g r a d a t i o n c y c l e s and p l a c e d  89 on the amino a c i d a n a l y z e r t o r e v e a l the presence of one  f r e e amino a c i d , s e r i n e , thus r e - c o n f i r m i n g  ment of s e r i n e i n t o p o s i t i o n 4. ( m o b i l i t y 116 mm  the  The major p e p t i d e  toward anode) was  only place-  '2'  found on amino a c i d  a n a l y s i s t o c o n t a i n e q u i m o l a r amounts of c y s t e i c a c i d , p r o l i n e , g l y c i n e and Thus '2' was (ii)  i s o l e u c i n e , and a t r a c e of s e r i n e .  i d e n t i f i e d as the C - t e r m i n a l  C h a r a c t e r i z a t i o n of C - t e r m i n a l  tetrapeptide.  tetrapeptide  The m a t e r i a l r e m a i n i n g a f t e r the amino a c i d a n a l y s i s of p e p t i d e 0.015  1  2' contained  enough m a t e r i a l  (approximately  umoles) f o r two dansyl-Edman c y c l e s .  of t h i s t e t r a p e p t i d e m o l e c u l e ) was  N-terminus  ( p o s i t i o n 6 i n o x i d i z e d hormone  i d e n t i f i e d as d a n s y l - c y s t e i c a c i d i n two  l a y e r chromatography systems. m o b i l i t i e s obtained  T a b l e XI  (1) l i s t s  the  was  identified  i n t e r v e n i n g Edman c y c l e as d a n s y l - p r o l i n e i n  t h i n l a y e r chromatography systems used. shown i n F i g u r e 16.  thin  in thissseparation.  P o s i t i o n 7 (second amino a c i d i n '2') a f t e r one  The  T a b l e XI  The  two  separation i s  (2) l i s t s the m o b i l i t i e s of  the f i r s t chromatography system; the same t h i n - l a y e r p l a t e was  r e - r u n i n the second system i n the o p p o s i t e d i r e c t i o n ,  t h e r e f o r e the m o b i l i t i e s of the second s e p a r a t i o n are  not  given. Thus p o s i t i v e i d e n t i f i c a t i o n was  obtained  f o r seven  out of n i n e amino a c i d s i n the sequence of Hormone I .  The  90 T A B L E  XI  I d e n t i f i c a t i o n o f D a n s y l D e r i v a t i v e s o f Amino A c i d s 6 and 7 o f Hormone I  D e t e r m i n a t i o n o f t h e N-terminus o f t h e C - t e r m i n a l t e t r a p e p t i d e : amino a c i d 6 o f Hormone I . M o b i l i t y o f d a n s y l amino a c i d s i n s o l v e n t systems A DNS-NH DNS-pro DNS-CyS0 H Unknown DNS-OH DNS-ser DNS-gly DNS-ileu 2  3  B 1.00 0.99 0 0 0.03 (blue) 0.05 0.42 1.00  1.00 1.00 0.13 0.12 0.56 (blue) 0.40 0.71 1.00  D e t e r m i n a t i o n o f t h e second amino a c i d ficom t h e C - t e r m i n a l t e t r a p e p t i d e : amino a c i d 7 o f Hormone I . M o b i l i t y o f d a n s y l amino a c i d s i n s o l v e n t system DNS-NH2 DNS-ser DNS-pro Unknown DNS-ileu DNS-gly  1.00 0.05 1.00 1.00 1.00 0.42  M o b i l i t i e s a r e expressed r e l a t i v e t o dansyl-ammonia. S o l v e n t systems:  A  -  acid  c h l o r o f o r m : methanol : a c e t i c (95 : 10 : 1 ) ; B  ammonia (80 : 20) .  - n-propanol  A F i g u r e 16: I d e n t i f i c a t i o n o f amino a c i d 1_ o f Hormone Jas D N S - p r o l i n e . A:  c h l o r o f o r m : methanol : a c e t i c a c i d (95 : 10 : 1) l e f t t o r i g h t : DNS-ser; DNS-NH ; DNS-OH and DNS-a.a. ; DNS-pro; DNS-OH; D N S - i l e u ; DNS-gly. 2  B:  n-propanol : ammonia (80 : 2 0 ) , P l a t e r e v e r s e d , l e f t t o r i g h t : DNS-gly; D N S - i l e u ; n i l ; DNS-pro; DNS-a.a. ; DNS-NH ; n i l . 7  0  7  assignment o f t h e l a s t two amino a c i d s glycine)  ( i s o l e u c i n e and  was by homology w i t h t h e o t h e r n e u r o h y p o p h y s i a l  hormones which u n i v e r s a l l y c o n t a i n  g l y c i n e as r e s i d u e 9.  The complete sequence o f Hormone I f o l l o w i n g t h e o x i d a t i o n w i t h p e r f o r m i c a c i d was thus determined a s : CyS0 H-Tyr-Ileu-Ser-Asp-CyS0 H-Pro-Ileu-Gly 3  3  (2)  Amino A c i d Sequence o f Hormone I I o f Salmon  (i)  Determination of N-terminal pentapeptide The N - t e r m i n a l t r i p e p t i d e was i d e n t i f i e d as C y S 0 3  T y r - I l e u u s i n g t h e dansyl-Edman t e c h n i q u e .  The i d e n t i f i c a -  t i o n o f d a n s y l d e r i v a t i v e s o f t y r o s i n e and i s o l e u c i n e was found t o be d i f f i c u l t  as t h e s p o t s o b t a i n e d were o f f a i n t  f l u o r e s c e n c e and t h e experiments had t o be r e p e a t e d  twice,  thus l o s i n g t h e advantage o f t h e s e n s i t i v i t y o f t h e method. I d e n t i f i c a t i o n of the N-terminal pentapeptide using the Edman s u b t r a c t i v e method, on t h e o t h e r hand, p r e s e n t e d no difficulties.  The r e s u l t s o f t h e Edman s u b t r a c t i v e  are summarized i n T a b l e X I I .  analysis  I t can be seen from t h i s  t a b l e t h a t one c y s t e i c a c i d r e s i d u e was l o s t a f t e r t h e f i r s t Edman c y c l e , t y r o s i n e a f t e r t h e second, i s o l e u c i n e a f t e r t h e t h i r d , g l u t a m i c a c i d a f t e r t h e f o u r t h , and a s p a r t i c a f t e r the f i f t h c y c l e .  acid  The background o f u n r e a c t e d N-  t e r m i n i , s i m i l a r l y t o t h e d e g r a d a t i o n o f Hormone I , began  93 T A B L E  XII  The Amino A c i d Sequence o f N - t e r m i n a l P e n t a p e p t i d e o f Hormone I I o f Oncorhynchus t s c h a w y t s c h a  R e s u l t s o f s u b t r a c t i v e Edman d e g r a d a t i o n method:  Amino A c i d  Amino a c i d a n a l y s i s ( r e s i d u e s p e r mole) a f t e r s t e p no.: 0  1  Ammonia  3.98  3.20  Arginine  0.83  0.79  4  5  3.19  2. 27  2. 04  0.85  0.75  0. 69  0. 68  1.96  1.18  0.99  0.93  1. 02  0. 94  Aspartic acid  1.09  1.06  1.03  1.02  1. 00  0. 59  Glutamic a c i d  1.00  1.03  1.03  1.00  0. 45  0. 47  Proline  0.80  1.00  0.99  1.01  0. 89  0. 97  Glycine  1.14  1.04  0.99  1.05  0. 91  1. 03  Isoleucine  0.87  0.86  0.92  0.41  0. 20  0. 26  Tyrosine  0.43  0.44  0.13  0. 05  0  Cysteic  acid  2 —  3  —  These a r e t h e r e s u l t s o f d u p l i c a t e a n a l y s e s e x c e p t for residue  5 which was a s i n g l e a n a l y s i s .  t o r i s e a f t e r the t h i r d d e g r a d a t i o n c y c l e . p e n t a p e p t i d e was  The  N-terminal  i d e n t i f i e d from t h e s e experiments as  CyS0 H-Tyr-Ileu-Glu-Asp. 3  (ii)  D e t e r m i n a t i o n of C - t e r m i n a l  tetrapeptide  Amino a c i d a n a l y s i s of the C - t e r m i n a l sequence of Hormone I I was  c a r r i e d out on the m a t e r i a l which remained  a f t e r f i v e Edman d e g r a d a t i o n c y c l e s used f o r  subtractive  method a n a l y s i s .  (positions  6, 7, and nique.  The  n e x t t h r e e amino a c i d s  8) were i d e n t i f i e d u s i n g the dansyl-Edman t e c h -  The  m o b i l i t i e s of d a n s y l d e r i v a t i v e s i n t h i n l a y e r  chromatography systems are l i s t e d i n T a b l e X I I I and  the  i d e n t i f i c a t i o n of amino a c i d 7 i l l u s t r a t e d i n F i g u r e On and  The was  the b a s i s of the e x p e r i m e n t s , amino a c i d s 6,  8, were i d e n t i f i e d as c y s t e i c a c i d , p r o l i n e ,  arginine,  17. 7,  and  respectively. i d e n t i f i c a t i o n o f the amino a c i d i n p o s i t i o n 8  not e n t i r e l y s a t i s f a c t o r y .  a n a l y s i s o n l y two a r g i n i n e and  At t h i s p o i n t of sequence  amino a c i d s remained t o be  glycine.  W h i l e i n the chromatographic system  A the unknown behaved l i k e the d a n s y l a r g i n i n e a separation  of the unknown i n t o two  standard,  fluorescent  one o f which corresponded t o d a n s y l - a r g i n i n e , i n system B, and  placed:  even more pronounced i n C  bands,  became e v i d e n t  (Table X I I I  (3).  95 T A B L E  XIII  I d e n t i f i c a t i o n o f D a n s y l D e r i v a t i v e s o f Amino A c i d s 6, 7, and 8 o f Hormone I I (1)  D e t e r m i n a t i o n o f amino a c i d 6 o f Hormone I I : M o b i l i t i e s o f d a n s y l amino a c i d s i n s o l v e n t system. A DNS-NH 1.00 DNS-pro 0.95 DNS-gly 0.46 DNS-arg 0 DNS-CyS0 H 0 Unknown ,, 0 No. 6 o f LVP ' 0  C 1.00 0.64 0.74 0.52 0.60 0.59 0.59  2  3  (a  (2)  D e t e r m i n a t i o n o f amino a c i d 7 o f Hormone I I : M o b i l i t i e s o f d a n s y l amino a c i d s i n s o l v e n t systems B DNS-NH DNS-pro Unknown DNS-gly DNS-arg 0  (3)  1.00 1.00 1.00 0.41 0  1.00 0.64 0.6 4  D e t e r m i n a t i o n o f amino a c i d 8 o f Hormone I I : M o b i l i t i e s o f d a n s y l amino a c i d s i n s o l v e n t systems A DNS-NHDNS-arg Unknown DNS-gly  B  1.00 0 0 0.50  1.00 0.37 0.39+0.41 0.67  C: same p l a t e r e - r u n through  1.00 0.74 0.76+0.79 0.89  t h r e e systems i n (3)  (a) S y n t h e t i c 3-phe, 8 - l y s o x y t o c i n ( l y s i n e M o b i l i t i e s a r e expressed  C  vasopressin)  r e l a t i v e t o dansyl-ammonia.  S o l v e n t systems: A - c h l o r o f o r m : methanol : a c e t i c a c i d (95: 10 : 1 ) ; B - n - p r o p a n o l : ammonia (80 : 2 0 ) ; C n - p r o p a n o l : water (80 : 2 0 ) .  F i g u r e 17:  I den t i f i ca t1 on o f amino a c i d 1_ o f Hormone as D N S - p r o l i n e .  L e f t t o r i g h t ( s t a r t i n g i n channel 4 ) : DNS-a.a. ; n i l ; DNS-pro; DNS-NH^ • 7  DNS-gly; n i l ;  However, t h e second band d i d n o t have the m o b i l i t y o f d a n s y l - g l y c i n e o r o f a d a n s y l - d e r i v a t i v e o f any amino a c i d i n the hormone. The placement o f a r g i n i n e i n t o p o s i t i o n 8 was subs t a n t i a t e d by p o s i t i v e i d e n t i f i c a t i o n o f t h e C - t e r m i n a l amino a c i d .  The r e s i d u e o f 8 Edman d e g r a d a t i o n  c y c l e s was  s u b j e c t e d t o e l e c t r o p h o r e s i s (pH 6.5, 15 m i n u t e s , 4,000 v o l t s ) with glycinamide  and a r g i n i n e s t a n d a r d s .  No f r e e  a r g i n i n e was r e v e a l e d i n t h e r e s i d u e o f Hormone I I , w h i l e a strong glycinamide  band was observed (212 mm  toward  cathode). P o s i t i v e i d e n t i f i c a t i o n was thus o b t a i n e d f o r t h e complete amino a c i d sequence o f Hormone I I .  Following oxi-  d a t i o n w i t h p e r f o r m i c a c i d the hormone m o l e c u l e was found t o have t h e f o l l o w i n g amino a c i d sequence: CyS0 H-Tyr-Ileu-Glu-Asp-CyS0 H-Pro-Arg-GlyNH 3  (3)  3  2  .  R e a c t i o n Y i e l d s Encountered i n t h e Course o f Amino A c i d Sequence Work I n t h e course o f s u c c e s s i v e Edman r e a c t i o n s the t o t a l  amount o f m a t e r i a l r e m a i n i n g  f o r the next degradation  cycle  was found t o d i m i n i s h i n excess o f t h e amounts t h a t c o u l d be accounted f o r , through t h e removal o f a l i q u o t s f o r amino a c i d analyses  i n t h e s u b t r a c t i v e Edman d e g r a d a t i o n ,  and f o r  98 c h r o m a t o g r a p h i c i d e n t i f i c a t i o n of d a n s y l d e r i v a t i v e s i n the dansyl-Edman method. the two  The  l o s s e s may  be a s c r i b e d  to  e x t r a c t i o n s t e p s t h a t the m i x t u r e undergoes w i t h  each s u c c e s s i v e  Edman d e g r a d a t i o n c y c l e :  e x t r a c t i o n f o l l o w i n g the c o u p l i n g  the benzene  s t e p and  the  butyl  a c e t a t e e x t r a c t i o n f o l l o w i n g the c l e a v a g e s t e p of  the  Edman r e a c t i o n . D u r i n g dansyl-Edman sequence a n a l y s i s of Hormone I I and  of s y n t h e t i c 3-phe, 8 - l y s o x y t o c i n , benzene e x t r a c t i o n  and  b u t y l a c e t a t e e x t r a c t i o n were r e p e a t e d t h r e e t i m e s .  Hormone I I (0.2 urn)  became exhausted a f t e r t h r e e , and  8-arg o x y t o c i n  urn) a f t e r s i x , d e g r a d a t i o n c y c l e s .  (1.0  3-phe,  Attempts t o omit the benzene e x t r a c t i o n e n t i r e l y r e s u l t e d i n l a r g e amounts of e x t r a n e o u s v i o l e t - b l u e f l u o r e s c e n c e chromatography of d a n s y l d e r i v a t i v e s which obscured fluorescence The ponsible  on  the  of the d a n s y l amino a c i d . b u t y l a c e t a t e e x t r a c t i o n was  for a considerable  Edman method was  loss.  found t o be  When the  res-  subtractive  a p p l i e d t o sequence d e t e r m i n a t i o n of  Hormone I I , the benzene e x t r a c t i o n was  omitted e n t i r e l y ,  w h i l e b u t y l a c e t a t e e x t r a c t i o n of aqueous p e p t i d e s o l u t i o n was  performed t h r e e times a f t e r each c y c l i z a t i o n s t e p .  In  t h i s e x p e r i m e n t , the amount of m a t e r i a l l e f t a f t e r second, t h i r d , and  f o u r t h Edman d e g r a d a t i o n c y c l e was  63.5,  and  20.3  15.6%  found t o  of t h e o r e t i c a l v a l u e r e s p e c t i v e l y .  be In  subsequent experiments on Hormone I I one benzene e x t r a c t i o n was a g a i n i n t r o d u c e d a f t e r t h e c o u p l i n g s t e p  (to allow  i d e n t i f i c a t i o n of dansyl d e r i v a t i v e s ) , but the b u t y l acetate e x t r a c t i o n s t e p was performed o n l y once. y i e l d of remaining peptide:  T h i s improved t h e  62% o f p e p t i d e remained a f t e r  f o u r Edman d e g r a d a t i o n c y c l e s o f Hormone I I .  When t h e  b u t y l a c e t a t e e x t r a c t i o n s t e p was m o d i f i e d so as t o e x t r a c t the c y c l i z a t i o n r e s i d u e i n d r y r a t h e r than i n aqueous form, the y i e l d o f b a s i c p e p t i d e a f t e r f o u r Edman c y c l e s was found t o be 65%. D e g r a d a t i o n o f Hormone I r e s u l t e d i n h i g h e r l o s s e s than t h a t o f Hormone I I , p o s s i b l y because t h e p o l a r s i d e c h a i n o f t h e a r g i n i n e r e s i d u e found i n Hormone I I makes i t less susceptible to e x t r a c t i o n with organic solvents. highest y i e l d obtained a f t e r performing  four  The  degradation  c y c l e s on Hormone I was 50% o f t h e t h e o r e t i c a l v a l u e , even though benzene e x t r a c t i o n was o m i t t e d a l t o g e t h e r and b u t y l a c e t a t e e x t r a c t i o n c a r r i e d o u t o n l y once on d r y r e s i d u e s .  100  IV.  (1)  DISCUSSION  Neu'rohypophys1a 1 Hormone Content o f Salmon P i t u i t a r i e s A major d i f f i c u l t y i n comparing t h e amounts o f hor-  mones i s o l a t e d from 0. t s c h a w y t s c h a  w i t h those  isolated  from o t h e r t e l e o s t s by o t h e r workers l i e s i n t h e v a r i a t i o n i n experimental conditions.  The c o n t e n t o f n e u r o h y p o p h y s i a l  hormones o f t e l e o s t p i t u i t a r i e s may be e x p r e s s e d of b i o l o g i c a l a c t i v i t y p e r u n i t body weight (40), o r p e r u n i t o f g l a n d weight a c t i v i t y i s expressed  i n units  (76), p e r g l a n d  ( 1 0 ) . The b i o l o g i c a l  i n terms o f u n i t s o f o x y t o c i c , p r e s s o r ,  or a n t i d i u r e t i c a c t i v i t y  ( 5 ) . A l s o a v a r i e t y o f s p e c i e s of  a n i m a l s can be used f o r a g i v e n type o f b i o a s s a y .  Thus oxy-  t o c i c a c t i v i t y i s estimated using the uterus of guinea p i g , r a t , or cat i n v i v o or i n v i t r o  ( 5 ) . The b i o a s s a y on r a t  u t e r u s in. v i t r o can be done w i t h o r w i t h o u t magnesium i o n (57).  Thus, t h e v a l u e s o b t a i n e d by L e d e r i s i n h i s work  w i t h Salmo i r i d e u s were g i v e n i n m i l l i u n i t s of o x y t o c i c a c t i v i t y p e r 100 gm o f body weight o f t h e t r o u t , and t h e o x y t o c i c a c t i v i t y was measured on r a t u t e r u s i n v i t r o i n the presence  o f magnesium i o n (76) .  For t h i s reason, h i s  d a t a were n o t i n c l u d e d i n t h e p r e s e n t comparison.  Addition-  a l l y , a d i s t i n c t i o n must be made between t h e b i o l o g i c a l  101 a c t i v i t i e s r e p o r t e d f o r whole t i s s u e powders, as i n the work of Acher and o f Chauvet ( 4 6 ) , (48), ( 7 7 ) , and  the  b i o l o g i c a l a c t i v i t i e s of the e x t r a c t s of those powders or of f r e s h t i s s u e s , as i n the work of F o l l e t and H e l l e r (40). T a b l e XIV attempts t o summarize o x y t o c i c a c t i v i t i e s of t e l e o s t p i t u i t a r i e s r e p o r t e d i n the l i t e r a t u r e and t o compare them w i t h those of the p r e s e n t s t u d y . The hormone c o n t e n t per g l a n d of Oncorhynchus tschawytscha i s r e l a t i v e l y high. t h i s was  A p r a c t i c a l advantage of  t h a t a p p r o x i m a t e l y 4,000 f i s h p r o v i d e d s u f f i c i e n t  pure hormone f o r c h e m i c a l s t u d i e s .  Ten thousand  fish  g l a n d s were used by Acher e t a l . f o r d e t e r m i n a t i o n o f amino a c i d c o m p o s i t i o n o f 8-arg o x y t o c i n of Gadus l u s c u s (4 8 ) . On t h e o t h e r hand, the a c t i v i t y per u n i t w e i g h t o f salmon g l a n d was  low and c o n s e q u e n t l y r e l a t i v e l y l a r g e amounts of  t i s s u e had t o be p r o c e s s e d t o o b t a i n m i l l i g r a m q u a n t i t i e s of pure hormones. P e r k s mentions  t h a t i n the elasmobranch  fishes  n e u r o h y p o p h y s i a l hormone per p i t u i t a r y does not appear t o v a r y w i t h the s i z e of the f i s h , and t h a t c a r e must be t a k e n not t o o v e r e s t i m a t e d i f f e r e n c e s observed as unknown f a c t o r s , such as s e a s o n a l v a r i a t i o n , may  be i n v o l v e d ( 7 ) .  The apparent v a r i a t i o n i n the amounts of hormone per p i t u i t a r y i n d i f f e r e n t s p e c i e s of f i s h  (Table XIV) may  d i f f e r e n c e s due t o v a r i a t i o n s i n the e x t r a c t i o n  reflect  procedure,  T A B L E  XIV  Comparison o f N e u r o h y p o p h y s i a l  Hormone Content o f  Teleost P i t u i t a r i e s Oxytocic a c t i v i t y ^ m y / f i s h my/mg d r y wt.  Species  Extraction  Author  81  hot 0.25% HOAc o f acetone powder  F o l l e t and H e l l e r (40)  81  hot 0.25% HOAc o f acetone powder  F o l l e t and H e l l e r (40)  114  hot 0.25% HOAc o f acetone powder  F o l l e t and H e l l e r (40)  hot 0.25% HOAc o f frozen glands  F o l l e t and H e l l e r (40)  500  whole acetone powder  Acher (48)  200  whole acetone powder  Chauvet (77)  70  whole acetone powder  Acher (46)  50  c o l d 0.2 M HOAc o f frozen glands  Salmo i r i d e u s (trout) Esox l u c i u s (pike) Anquilla (eel)  anguilla  Gadus c a l l a r i a s (cod) Gadus l u s c u s (bid cod) Merluccius merluccius (hake)  185 97  (b) (b)  Cyprinus c a r p i o (carp) Oncorhynchus tschawytscha ( P a c i f i c chinook salmon) (a) (b)  750 - 1,000  p r e s e n t study  O x y t o c i c a c t i v i t y measured u s i n g r a t u t e r u s w i t h o u t magnesium method o f H o l t o n ( 5 5 ) . A c t i v i t y p e r f i s h c a l c u l a t e d from t h e d a t a o f t h e a u t h o r s on t h e number o f f i s h g l a n d s used t o o b t a i n a g i v e n weight o f acetone powder. Such v a l u e was n o t g i v e n f o r C. c a r p i o .  103 or may  be b i o l o g i c a l l y s i g n i f i c a n t .  F u r t h e r study of  neurohypophysial hormones i n elasmobranchs i s r e q u i r e d to c l a r i f y this point.  (2)  B i o l o g i c a l Assays Used i n I s o l a t i o n of  Neurohypophysial  Hormones of Salmon S e v e r a l c r i t e r i a were used i n choosing the assay f o r i s o l a t i o n of salmon hormones.  An o x y t o c i c assay was  desir-  able s i n c e a l l neurohypophysial hormones of animals have some o x y t o c i c p r o p e r t i e s , w h i l e not a l l of them have p r e s s o r or antidiuretic activity itself  ( 7 5 ) . The assay method had to lend  to a c c u r a t e q u a n t i t a t i o n i n order t h a t  isolation  methods c o u l d be compared and examined i n terms of y i e l d s of  hormones.  A r a p i d method was  r e q u i r e d so t h a t the  e f f l u e n t f r a c t i o n s of chromatographic monitored without d e l a y . u t e r i n e horn i n  vitro  columns c o u l d be  The method of Holton u s i n g a r a t  (55) f u l f i l l e d  these  requirements.  A v a r i a n t of the Holton method, r a t uterus c o n t r a c t i l i t y i n presence of magnesium i o n , was  used i n s e v e r a l  experiments  f o l l o w i n g the stage of s e p a r a t i o n of the two hormones.  The  assay i n presence of magnesium has been d e s c r i b e d by Munsick as an a d d i t i o n a l t o o l f o r p h a r m a c o l o g i c a l c h a r a c t e r i z a t i o n of  neurohypophysial hormones, because the magnesium i o n  appears  to p o t e n t i a t e the a c t i o n of a l l neurohypophysial  hormones, w i t h the e x c e p t i o n of o x y t o c i n , on the u t e r i n e  104 tissue.  The p o t e n t i a t i o n r a t i o i s d i f f e r e n t f o r d i f f e r e n t  n e u r o h y p o p h y s i a l hormones (57).  The p o t e n t i a t i o n s ob-  t a i n e d f o r t h e two salmon hormones were w i t h i n t h e range of those r e p o r t e d  f o r 4-ser, 8 - i l e u o x y t o c i n  and 8-arg  oxytocin.  (3)  E x t r a c t i o n o f Neurohypophy s i a1 Hormones F r e s h f r o z e n g l a n d s o f f i s h e s were e x t r a c t e d by  F o l l e t and H e l l e r  (40), l y o p h y l i z e d g l a n d s were  by Sawyer (45), b u t t h e m a j o r i t y  extracted  o f workers appear t o use  acetone powders o f p i t u i t a r y g l a n d s (10),  ( 4 0 ) . The w i d e l y  used e x t r a c t i o n method o f Kamm recommended by t h e B r i t i s h Pharmacopeia (1958) (59) i n v o l v e s h e a t i n g acetone powder i n 0.25% a c e t i c a c i d .  of p i t u i t a r y  I n a d d i t i o n t o Kamm's  method, o t h e r e x t r a c t i o n p r o c e d u r e s have been s u c c e s s f u l l y used by some i n v e s t i g a t o r s .  Among them a r e t h e d i l u t e  sul-  f u r i c a c i d e x t r a c t i o n o f Acher e t a_l. (78) and t h e p y r i d i n i u m acetate e x t r a c t i o n of Lindner e t a l . The  (79).  e x t r a c t i o n method o f Acher was f i r s t d e s i g n e d f o r  e x t r a c t i o n o f mammalian neurohypophyses.  I t i n v o l v e s ex-  t r a c t i o n w i t h c o l d 0.01 N s u l f u r i c a c i d i n o r d e r t o p r e s e r v e t h e neurophysin-hormone complex found i n t h e e x t r a c t s of mammalian neurohypophyses (10),  (78). Following the  e x t r a c t i o n , t h e n e u r o p h y s i n complex i s s e p a r a t e d from t h e s m a l l m o l e c u l e s i n t h e e x t r a c t by s a l t p r e c i p i t a t i o n and  105 dialysis.  The n e x t s t e p i n v o l v e s d i s r u p t i o n of the complex  and s e p a r a t i o n of the s m a l l m o l e c u l a r w e i g h t neurohypophys i a l hormones from the h o r m o n a l l y i n e r t neurophysine  (78).  Acher was unable t o d e t e c t a n e u r o p h y s i n complex i n the p i t u i t a r y e x t r a c t s of lower v e r t e b r a t e s , and adapted h i s method t o t h i s group o f a n i m a l s by i n t r o d u c i n g mammalian n e u r o p h y s i n i n t o the e x t r a c t  (15).  I t has been p r e v i o u s l y  shown by him t h a t i n t e r - s p e c i f i c neurophysin-hormone comp l e x e s can i n d e e d be formed  (80).  The a d d i t i o n o f  'foreign'  n e u r o p h y s i n can produce an erroneous r e s u l t i f the p u r i f i c a t i o n o f n e u r o p h y s i n i s i n c o m p l e t e (26).  For t h i s  reason Acher's e x t r a c t i o n method was not attempted i n the c o u r s e o f t h i s work. The method o f L i n d n e r a l s o i n v o l v e s the n e u r o p h y s i n hormone complex.  The e x t r a c t i o n o f acetone d r i e d  neurohy-  pophyses i s c a r r i e d out a t 4°C u s i n g p y r i d i n i u m a c e t a t e buffer.  Under t h e s e c o n d i t i o n s the mammalian n e u r o p h y s i n -  hormone complex i s p r e s e r v e d .  The h i g h m o l e c u l a r w e i g h t  complex i s s e p a r a t e d by g e l f i l t r a t i o n and then d i s r u p t e d using 1 N formic acid  (79) .  T h i s method was  two o c c a s i o n s i n the e a r l y p a r t of t h i s  attempted  on  investigation  u s i n g f r o z e n salmon g l a n d s , but a h i g h m o l e c u l a r w e i g h t hormone complex was not d e t e c t e d . I t has been shown by du Vigneaud's forms a r e v e r s i b l e adduct  group t h a t acetone  w i t h n e u r o h y p o p h y s i a l hormones  106 which r e s u l t s i n d e - a c t i v a t i o n o f t h e hormones ( 8 1 ) , The adduct, b e l i e v e d t o be a m o n o i s o p r o p y l i d e n e forms a t room temperature i n 48 h o u r s . l o g i c a l a c t i v i t y i s almost t o t a l  (82).  derivative,  The l o s s o f b i o -  (3% o f l y s i n e v a s o p r e s s i n  a c t i v i t y was d e t e c t e d a f t e r t h e acetone t r e a t m e n t ) , b u t can be r e s t o r e d by h e a t i n g t h e i s o p r o p y l i d e n e d e r i v a t i v e t o 90°C f o r 30 minutes i n 0.25% a c e t i c a c i d . I n v i e w o f these • / ebrie.d , , r e s u l t s , acetone p i t u i t a r y powders were n o t used r o u t i n e l y i n t h e c o u r s e o f t h e p r e s e n t work. The e x t r a c t i o n method developed f o r salmon p i t u i t a r i e s i n v o l v e d d i s r u p t i o n o f t h e t i s s u e i m m e d i a t e l y upon removal from t h e f r e e z e r 4°C.  (-80°C) i n 0.2 M a c e t i c a c i d a t  I t has been shown i n t h e c o u r s e o f t h i s work t h a t  o x y t o c i c a c t i v i t y o f salmon p i t u i t a r i e s s u r v i v e d l o n g p e r iods of cold storage.  The y i e l d o f o x y t o c i c  activity  o b t a i n e d by t h i s e x t r a c t i o n method was somewhat b e t t e r than t h a t o b t a i n e d by t h e method o f Kamm.  The m o l a r i t y o f t h e  r e s u l t i n g crude e x t r a c t p e r m i t t e d t h e n e x t s t e p o f i s o l a t i o n procedure t o f o l l o w i m m e d i a t e l y a f t e r  centrifugation  o f t i s s u e homogenate.  (4)  P u r i f i c a t i o n of Neurohypophysial  Hormones  A number o f p u r i f i c a t i o n methods have been a p p l i e d , s i n g l y o r i n c o m b i n a t i o n w i t h each o t h e r , i n neurohypophys i a l hormone work.  These i n c l u d e d s a l t p r e c i p i t a t i o n ,  107 c o u n t e r - c u r r e n t d i s t r i b u t i o n , a d s o r p t i o n on s i l i c a , p h o r e s i s on a c e l l u l o s e  column, p a r t i t i o n  electro-  chromatography,  g e l f i l t r a t i o n , paper chromatography, and i o n exchange chromatography ( 1 0 ) . C h e m i c a l l y pure n e u r o h y p o p h y s i a l hormones have been o b t a i n e d from p i t u i t a r i e s by du Vigneaud u s i n g c o u n t e r current d i s t r i b u t i o n  ( 8 3 ) , by Acher u s i n g s a l t  f o l l o w e d by i o n exchange chromatography  precipitation  ( 1 0 ) , by Rasmussen  u s i n g c o u n t e r - c u r r e n t d i s t r i b u t i o n and g e l f i l t r a t i o n ( 4 9 ) , and by V l i e g e n t h a r t u s i n g g e l f i l t r a t i o n , i o n exchange, and paper chromatography  (84).  C r i t e r i a applied i n develop-  i n g t h e p u r i f i c a t i o n s c h e d u l e used f o r t h e p r e s e n t work were a d a p t a b i l i t y o f t h e p u r i f i c a t i o n sequence t o work on a p r e p a r a t i v e s c a l e , and p o t e n t i a l f o r maximum o v e r a l l yields  c o u p l e d w i t h maximum p u r i f i c a t i o n o f b o t h hormones. The p u r i f i c a t i o n p r o c e d u r e w h i c h was adopted f o r  r o u t i n e use o r i g i n a t e d from t h e work o f Sawyer and van Dyke on p o l l a c k p i t u i t a r y  ( 4 5 ) , which was m o d i f i e d and ex-  tended i n t h e c o u r s e o f p r e s e n t s t u d i e s .  Sawyer's  iso-  l a t i o n p r o c e d u r e c o n s i s t e d o f g e l f i l t r a t i o n on Sephadex G-25 and subsequent s e p a r a t i o n o f two p o l l a c k hormones on a c a r b o x y m e t h y l c e l l u l o s e column  (CMC).  I n a subsequent  r e p o r t on H y d r o l a g u s c o l l i e i Sawyer s u b s t i t u t e d  partition  chromatography on Sephadex f o r g e l f i l t r a t i o n , and i o n exchange on CM-Sephadex f o r i o n exchange on CMC ( 3 4 ) .  108 The p u r i f i c a t i o n p r o c e d u r e used r e p e a t e d l y by Acher's group t o i s o l a t e n e u r o h y p o p h y s i a l hormones of t e l e o s t s , has been adapted from the o r i g i n a l p r o c e d u r e f o r beef g l a n d s by A c h e r , L i g h t and du Vigneaud  (7 8) by a d d i -  t i o n of f o r e i g n n e u r o p h y s i n , as d i s c u s s e d i n t h e p r e c e e d i n g section.  The d e t a i l s of the p r o c e d u r e e v o l v e d by A c h e r ,  L i g h t and du Vigneaud c o n s i s t e d of the f o l l o w i n g s t e p s : p r e c i p i t a t i o n o f n e u r o p h y s i n complex w i t h sodium  a  chloride,  r e p e a t e d d i a l y s i s a g a i n s t w a t e r , d i s s o c i a t i o n of the comp l e x w i t h TCA, TCA,  chromatography  on A m b e r l i t e IR-45 t o remove  and s e p a r a t i o n o f the two hormones on A m b e r l i t e  IRC-50 (78). The p r o c e d u r e d e v e l o p e d f o r t h e p u r i f i c a t i o n of salmon n e u r o h y p o p h y s i a l hormones i n the p r e s e n t s t u d y cons i s t e d of g e l f i l t r a t i o n , u l t r a f i l t r a t i o n , the  separation of  two hormones by i o n exchange, u l t r a f i l t r a t i o n ,  rechromatography of the i n d i v i d u a l hormones.  and  Gel f i l t r a t i o n  was used t o s e p a r a t e the h o r m o n e - c o n t a i n i n g low m o l e c u l a r w e i g h t f r a c t i o n from the h i g h m o l e c u l a r w e i g h t s u b s t a n c e s i n the crude e x t r a c t .  Sephadex G-25  has been used by  o t h e r workers s u c c e s s f u l l y f o r t h i s purpose In t h e p r e s e n t i n v e s t i g a t i o n Sephadex G-15  (45) , (85). and B i o g e l  P-2  were examined, because t h e i r m o l e c u l a r w e i g h t e x c l u s i o n l i m i t i s lower than t h a t o f Sephadex G-25. of  On the b a s i s  t h e s e p r e l i m i n a r y experiments Sephadex G-15  l a r g e s c a l e i s o l a t i o n s of salmon hormones.  was used i n  The use o f a  tandem s e r i e s of s e p a r a t i o n s on a Sephadex G-15  column  109 was found t o be a h i g h l y e f f i c i e n t p u r i f i c a t i o n s t e p f o r p r o c e s s i n g l a r g e volumes o f e x t r a c t .  The y i e l d o f hormones  was good, t h e e x p e r i m e n t a l time s h o r t , and d i r e c t a p p l i c a t i o n of crude e x t r a c t was p o s s i b l e . C a t i o n exchange has been proved an e f f e c t i v e t e c h n i q u e f o r s e p a r a t i n g two n e u r o h y p o p h y s i a l hormones on t h e b a s i s o f t h e i r d i f f e r e n t i o n i c charges by Acher's using a c r y l i c resins methylcellulose  group  (10) and by Sawyer u s i n g carboxy-  (45) o r carbomethyl-Sephadex  columns ( 3 4 ) .  I n t h e p r e s e n t s t u d y c e l l u l o s e - b a s e d and d e x t r a n - b a s e d c a t i o n exchange media such as p h o s p h o c e l l u l o s e , SE-Sephadex and Whatman CM-32 were used i n p r e f e r e n c e t o c a r b o x y m e t h y l cellulose.  P r i o r i o n exchange s e p a r a t i o n s o f neurohypo-  p h y s i a l hormones on c a t i o n exchangers by Acher's and Sawyer's group u t i l i z e d b o t h changes i n s a l t c o n c e n t r a t i o n and i n pH (from pH 5.0 t o pH 7.5 o r 7.7) f o r e l u t i n g t h e more b a s i c o f t h e two hormones (10) , (34) .  P u b l i s h e d work  on d i s u l f i d e i n t e r c h a n g e (86) suggests t h a t n e u t r a l pH i s n o t a p p r o p r i a t e f o r p r e s e r v a t i o n o f i n t e g r i t y o f -S-Sc o n t a i n i n g p e p t i d e s such as n e u r o h y p o p h y s i a l hormones.  In  the p r e s e n t work i t was found p o s s i b l e t o omit a change t o n e u t r a l pH and t o e l u t e t h e more b a s i c o f t h e two hormones (Hormone I I ) a t pH o f 5, u s i n g o n l y a s a l t g r a d i e n t . Salmon hormones o b t a i n e d by s e p a r a t i o n on a c a t i o n exchange column were n o t p u r e , as judged by t h e amino a c i d a n a l y s e s , s p e c i f i c a c t i v i t i e s , and e l u t i o n p r o f i l e s o f t h e  110 hormones.  F o r t h i s r e a s o n , rechromatography o f i n d i v i d u a l  hormones was c a r r i e d o u t .  F o r t h i s purpose i t was found  advantageous t o use a d i f f e r e n t c a t i o n exchanger from t h a t used f o r t h e s e p a r a t i o n o f hormones.  Thus, i f t h e hormones  were f i r s t s e p a r a t e d on a c a r b o x y m e t h y l i o n exchanger, p u r i f i c a t i o n o f t h e more b a s i c salmon hormone was a c h i e v e d b e s t by rechromatography on P u r i f i c a t i o n o f t h e l e s s b a s i c hormone  (Hormone I I )  phosphocellulose.  (Hormone I) r e q u i r e d  rechromatography a t a d i f f e r e n t pH from t h a t used i n t h e s e p a r a t i o n o f hormones.  The sequence i n which t h e two  c h r o m a t o g r a p h i e s were done appeared n o t t o m a t t e r :  i f the  i n i t i a l s e p a r a t i o n was done a t pH 2.45 on SE-Sephadex, t h e n t h e subsequent rechromatography o f t h e l e s s b a s i c hormone was on Whatman CM-32 a t pH 5 o r v i c e v e r s a . I n summary, rechromatography o f t h e two i n d i v i d u a l hormones o n l y had t o be performed once t o o b t a i n pure preparations  p r o v i d i n g t h a t t h e c o n d i t i o n s o u t l i n e d above  were o b s e r v e d : for  a pH change from 5 t o 2.45 o r v i c e  t h e l e s s b a s i c hormone  on p h o s p h o c e l l u l o s e  versa  (Hormone I ) and a rechromatography  o f t h e more b a s i c o f t h e two hormones  (Hormone I I ) i r r e s p e c t i v e o f whether t h e s e p a r a t i o n of t h e two hormones was done on SE-Sephadex o r on Whatman CM-32. A complete l i s t o f y i e l d s p e r s t e p o f p u r i f i c a t i o n and o f o v e r a l l y i e l d s has been r e p o r t e d by A c h e r , L i g h t and du Vigneaud on t h e i r work w i t h beef p i t u i t a r i e s  (78). Table  I l l  XV p r e s e n t s a comparison between the y i e l d s they r e p o r t e d and the y i e l d s o b t a i n e d i n the course of p u r i f y i n g i t a r y e x t r a c t s of 0. t s c h a w y t s c h a •  The procedure  pituemployed  i n the p r e s e n t i n v e s t i g a t i o n r e s u l t e d i n p r e p a r a t i o n of two pure hormones a t an o v e r a l l y i e l d l e v e l comparable t o t h a t o f Acher, L i g h t and du Vigneaud. The  l o s s e s e x p e r i e n c e d i n p u r i f y i n g salmon hormones  can be l a r g e l y a t t r i b u t e d t o the u l t r a f i l t r a t i o n  steps.  I t s h o u l d be p o s s i b l e t o improve y i e l d s by e i t h e r r e d u c i n g the e x t e n t o f u l t r a f i l t r a t i o n o r by u s i n g a l t e r n a t e p r o cedures  t o d e - s a l t and t o c o n c e n t r a t e the m a t e r i a l .  requirement  f o r u l t r a f i l t r a t i o n may  The  be p o s s i b l y reduced  by  s e p a r a t i o n o f the hormones on SE-Sephadex a t pH 5, and r e chromatography o f the l e s s b a s i c hormone on the same column a t pH 2.45.  The i o n i c s t r e n g t h s a t which t h i s hormone i s  e l u t e d from v a r i o u s c a t i o n exchange columns are g i v e n i n Table VI.  C o n c e n t r a t i o n of m a t e r i a l c o u l d be a c h i e v e d  lyophylization. observed  Losses due t o l y o p h y l i z a t i o n were not  d u r i n g the p r e s e n t work.  d e - s a l t i n g procedure  On the o t h e r hand, a  such as has been r e p o r t e d by  and L i n d n e r on Sephadex G-25 ultrafiltration.  by  Porath  (87) c o u l d be s u b s t i t u t e d f o r  The d i s a d v a n t a g e  o f the method used by  P o r a t h and L i n d n e r i s t h a t the hormones are e l u t e d from the column u s i n g a m i x t u r e of a c e t i c a c i d , p y r i d i n e and water (15 : 60 : 25) which i s t o x i c t o t i s s u e s and would have t o be removed p r i o r t o c o n d u c t i n g the b i o a s s a y .  T A B L E  XV  Comparison o f Y i e l d s O b t a i n e d a t D i f f e r e n t Stages o f P u r i f i c a t i o n o f N e u r o h y p o p h y s i a l Hormones A c h e r , L i g h t and du Vigneaud Purification step  Specific activity per •A'-'U'' Folin(a)  Extraction  ( 7 8 ) : beef Hormone y i e l d %  Purification step  Specific activity per A 280nm  Hormone y i e l d % per s t e p o v e r a l l 100 (b)  per step  overall  85-90  85-90  Extraction  0.08 - 0.10  Gel f i l t r a t i o n tandem series  0.4  Precipitation of complex and d i a l y s i s  2.5  70-80  60-70  A f t e r removal of p r o t e i n  10.0  75-90  45-63  oxytocin: 50 70-90 vasopressin: 100  30-55  S e p a r a t i o n by chromatography  P r e s e n t s t u d y : Oncorhynchus t s c h a w y t s c h a  - 1.4  Ultrafiltrat i o n and 10 - 100 s e p a r a t i o n by chromatography Ultrafiltrap e r mg: t i o n Re-chro- Hormone I matography 125-145 Hormone I I 209-229  78-97  98-97  69-91  54-88  63-74  34-65  (a) c a l c u l a t e d from d a t a of Acher, L i g h t and du Vigneaud ( 7 8 ) . (b) o x y t o c i c a c t i v i t y measured by method o f H o l t o n ( 5 5 ) . (c) no o x y t o c i c a c t i v i t y r e m a i n i n g i n t h e a c i d i n s o l u b l e f r a c t i o n o f t i s s u e homogenate,  113 (5)  S t r u c t u r a l A n a l y s e s o f N e u r o h y p o p h y s i a l Hormones of T e l e o s t s Chemical a n a l y s e s of n e u r o h y p o p h y s i a l hormones of  lower v e r t e b r a t e s , w i t h one e x c e p t i o n , have been done by a s i n g l e group o f w o r k e r s , Acher, e t a l . from L a b o r a t o i r e de Chimie B i o l o g i q u e , F a c u l t e des S c i e n c e s , P a r i s .  The  excep-  t i o n i s the q u a n t i t a t i v e amino a c i d a n a l y s i s of 8-arg o x y t o c i n from U r o p h y c i s t e n u i s  (50) p u b l i s h e d by Rasmussen  (49) . (i)  Q u a n t i t a t i v e amino a c i d a n a l y s e s To d a t e n e u r o h y p o p h y s i a l hormones of f i v e  teleost  f i s h e s have been i d e n t i f i e d on a c h e m i c a l b a s i s ; however, a q u a n t i t a t i v e amino a c i d a n a l y s i s has been p u b l i s h e d f o r o n l y two o f the f i v e s p e c i e s : Urophycis tenuis  (49).  Cyprinus carpio  (46) and  These d a t a are compared w i t h those  o b t a i n e d i n the p r e s e n t study o f Oncorhynchus t s c h a w y t s c h a i n T a b l e XVI.  I t can be seen from t h i s T a b l e t h a t q u a n t i -  t a t i v e amino a c i d a n a l y s e s of the salmon n e u r o h y p o p h y s i a l hormones are comparable (ii)  t o those o b t a i n e d by o t h e r w o r k e r s .  Amino a c i d sequence s t u d i e s of the l e s s b a s i c t e l e o s t hormone The amino a c i d sequence of 4-ser, 8 - i l e u o x y t o c i n  i s o l a t e d from the p i t u i t a r y g l a n d s of t h r e e marine  teleosts  T A B L E  XVI  Comparison o f Q u a n t i t a t i v e Amino A c i d A n a l y s e s o f T e l e o s t N e u r o h y p o p h y s i a l Hormones Amino A c i d  C. c a r p i o 0.tschawytscha 4-ser,8-ileu o x y t o c i n ( 4 6 ) Hormone I  Ammonia  Amino A c i d  2.19  Ammonia  C. c a r p i o 8-arg oxytocin(46)  U.tenuis O.tschawytscha 8-arg Hormone I I oxytocin(49) 3.80  3.96  Cys  1.46*  1.69  Arg  1.06  0.82  0.83  Asp  1.00  1.04  Cys  2.04*  1.65  1.96*  Ser  0.90  1.02  Asp  1.00  1.00  1.09  Pro  1.06  0.90  Glu  0.96  0.89  1.00  Gly  1.14  1.21  Pro  0.89  1.15  0.80  lieu  1.73  1.87  Gly  1.37  Tyr  0.61  0.83  lieu  0.92  1.12  0.87  Tyr  0.85  0.77  0.43  Lys  0.06  Val  0.04  Leu  0.15  0.16  Ser  0.19  Glu  0.20  Leu  0.13  Arg  0.08  *  v a l u e f o r Cys o b t a i n e d f o l l o w i n g p e r f o r m i c o x i d a t i o n .  1.14  0.19  115 was  f i r s t p o s t u l a t e d by Acher i n 1962, a l t h o u g h q u a n t i t a t i v e  amino a c i d a n a l y s e s of the new  hormone were n o t g i v e n .  The  amino a c i d sequence of the f i r s t f o u r N - t e r m i n a l amino a c i d r e s i d u e s was  a s s i g n e d on the b a s i s of d e g r a d a t i o n w i t h the  enzyme l e u c i n e - a m i n o p e p t i d a s e . m a i n i n g f i v e amino a c i d s was hormones.  a s s i g n e d by analogy w i t h known  The new hormone was  the replacement  The sequence of the r e -  named ' i s o t o c i n ' t o i n d i c a t e  of 8 - l e u c i n e w i t h i s o l e u c i n e  (42).  A d d i t i o n a l s t r u c t u r a l work on 4-ser, 8 - i l e u o x y t o c i n was  r e p o r t e d by Acher.  The experiment  c o n s i s t e d i n sub-  j e c t i n g the hormone, p r e v i o u s l y o x i d i z e d w i t h p e r f o r m i c a c i d , t o the a c t i o n of s u b t i l i s i n .  The enzyme r e l e a s e d two  fragments whose amino a c i d c o m p o s i t i o n p r o v i d e d c o n f i r mation f o r the a s s i g n e d s t r u c t u r e : (Tyr,  one fragment  was  CySO^H., l i e u , Ser) , and the o t h e r (Asp, P r o ,  lieu,  CyS0 H, G l y ) ( 1 0 ) . 3  The p r e s e n t work on Hormone I o f salmon employed c h e m i c a l r a t h e r than enzymic d e g r a d a t i o n methods.  The  Edman d e g r a d a t i o n method was  degra-  employed f o r s t e p w i s e  d a t i o n o f the m o l e c u l e , and d i l u t e h y d r o c h l o r i c a c i d used f o r p a r t i a l h y d r o l y s i s .  was  The sequence o f the N - t e r m i n a l  t e t r a p e p t i d e CySO^H-Tyr-Ileu-Ser, was  e s t a b l i s h e d by the  s u b t r a c t i v e method of Edman (72); p o s i t i o n 5, a s p a r t i c , by p a r t i a l a c i d h y d r o l y s i s and a d d i t i o n a l d a t a from a f i f t h Edman d e g r a d a t i o n c y c l e ; and p o s i t i o n s 6 and 7, CySC^-pro by dansyl-Edman t e c h n i q u e  (72).  Thus p o s i t i v e  identifi-  116 c a t i o n was o b t a i n e d f o r seven o u t o f n i n e amino a c i d r e s i dues.  The placement o f amino a c i d s 8 and 9, i l e u and g l y  can be done on t h e b a s i s o f a n a l o g y . Hormone I o f salmon  The sequence o f  (following oxidation with performic  a c i d ) was thus e s t a b l i s h e d t o be as f o l l o w s : CyS0 H-Tyr-Ileu-Ser-Asp-CyS0 H-Pro-Ileu-Gly 3  3  R e p o r t s on c h e m i c a l d e g r a d a t i o n o f n e u r o h y p o p h y s i a l hormones i n lower v e r t e b r a t e s have n o t been found i n t h e literature.  The s u b t r a c t i v e method o f Edman has been used  by du Vigneaud i n c o n f i r m i n g t h e sequence o f t h e f o u r N - t e r m i n a l amino a c i d r e s i d u e s o f beef o x y t o c i n ( 9 ) .  Du  Vigneaud's d a t a on s t e p w i s e Edman d e g r a d a t i o n o f o x y t o c i n show t h e i n c r e a s i n g l y i n c o m p l e t e removal o f subsequent amino a c i d r e s i d u e s observed i n t h e c o u r s e o f t h e p r e s e n t s t u d y o f salmon hormones. the  In the degradation of oxytocin,  removal o f N - t e r m i n a l c y s t e i c r e s u l t e d i n a d e c r e a s e  from 1.99 t o 1.12 r e s i d u e s p e r mole; t h e removal o f t y r o s i n e produced a d e c r e a s e from 0.69 t o 0.17 r e s i d u e s p e r mole; the  removal o f i s o l e u c i n e a f t e r t h e t h i r d Edman degrada-  t i o n c y c l e r e s u l t e d i n r e d u c t i o n from 0.74 t o 0.34 r e s i d u e s per  mole, and t h e removal o f g l u t a m i c a f t e r t h e f o u r t h  d e g r a d a t i o n s t e p produced a drop from 0.90 t o 0.55 r e s i d u e s per  mole.  Edman r e p o r t s almost 100% c o m p l e t i o n o f de-  g r a d a t i o n c y c l e s i n h i s e x p e r i m e n t s ( 8 8 ) , p r o b a b l y because  1*1=7 of t h e p u r i t y o f t h e r e a g e n t s used and because d e g r a d a t i o n s were done under n i t r o g e n atmosphere. (iii)  Amino a c i d sequence s t u d i e s o f t h e more b a s i c t e l e o s t hormone The s t r u c t u r e o f 8 - a r g i n i n e o x y t o c i n from  teleost  f i s h was s t u d i e d by Acher ( 5 1 ) . Amino a c i d a n a l y s i s r e v e a l e d t h e presence o f s t o i c h i o m e t r i c q u a n t i t i e s o f t h e e i g h t c o n s t i t u e n t amino a c i d s :  cystine, tyrosine,  isoleucine,  a s p a r t i c a c i d , g l u t a m i c a c i d , p r o l i n e , a r g i n i n e , and g l y c i n e . L e u c i n e aminopeptidase  r e l e a s e d t y r o s i n e and i s o l e u c i n e w h i l e  the N - t e r m i n a l h a l f - c y s t i n e remained a t t a c h e d t o t h e m o l e c u l e by t h e d i s u l f i d e b r i d g e .  T t was concluded from t h i s e x p e r i -  ment t h a t t h e N - t e r m i n a l t r i p e p t i d e o f 8 - a r g i n i n e o x y t o c i n was C y s - T y r - I l e u .  I n o r d e r t o study t h e C-terminus o f t h e  m o l e c u l e , t r y p s i n was a l l o w e d t o a c t on t h e hormone.  The  r e l e a s e o f g l y c i n a m i d e suggested t h a t t h e C - t e r m i n a l d i p e p t i d e was a r g i n y l - g l y c i n a m i d e ( 5 1 ) . The p r e s e n t study o f t h e more b a s i c hormone  (Hormone  I I ) o f salmon u t i l i z e d c h e m i c a l d e g r a d a t i o n o f t h e m o l e c u l e only.  The s u b t r a c t i v e Edman method was a p p l i e d t o t h e N-  t e r m i n a l p e n t a p e p t i d e , CySO^H-Tyr-Ileu-Glu-Asp.  Positions  6, 7, and 8 CySO^H-Pro-Arg, were i d e n t i f i e d u s i n g t h e dansyl-Edman method, and t h e C - t e r m i n a l g l y c i n a m i d e was i d e n t i f i e d by e l e c t r o p h o r e t i c s e p a r a t i o n o f t h e r e s i d u e r e -  118 m a i n i n g a f t e r e i g h t Edman c y c l e s .  Thus p o s i t i v e  identifi-  c a t i o n of a l l n i n e amino a c i d r e s i d u e s c o n t a i n e d i n the m o l e c u l e of Hormone I I was (iv)  obtained.  S t r u c t u r e of the 0. t s c h a w y t s c h a  neurohypophysial  hormones The  f o r e g o i n g s t r u c t u r a l d e t e r m i n a t i o n has l e d t o  complete amino a c i d sequences f o r the p e r f o r m i c o x i d i z e d salmon hormones.  The o x i d i z e d Hormone I has the sequence  H NCyS0 H-Tyr-Ileu-Ser-Asp-CyS0 H-Pro-Ileu-Gly 2  3  3  where the sequence of amino a c i d s 8 and 9 ( l i e u and  Gly)  i s a s s i g n e d by analogy w i t h o t h e r n e u r o h y p o p h y s i a l  hormones.  O x i d i z e d Hormone I I has the sequence H NCyS0 H-Tyr-Ileu-Glu-Asp-CyS0 H-Pro-Arg-GlyNH 2  3  3  By analogy w i t h o t h e r n e u r o h y p o p h y s i a l  2  hormones, the  u n o x i d i z e d hormones can be c o n s i d e r e d t o have i n t r a m o l e c u l a r d i s u l f i d e bonds.  From the ammonia a n a l y s e s , and more p a r -  t i c u l a r l y from the b e h a v i o u r on c a t i o n exchangers,  i t is  r e a s o n a b l e t o assume t h a t the a s p a r t i c a c i d and g l y c i n e r e s i d u e s i n Hormone I are i n the form of amides.  Hence the  s t r u c t u r e 4-ser, 8 - i l e u o x y t o c i n can be a s s i g n e d t o t h i s hormone.  By analogous r e a s o n i n g the s t r u c t u r e 8-arg oxy-  t o c i n can be a s s i g n e d t o Hormone I I .  119 (v)  Comments on Until  method had It  not  fourth  tractive cause  or  been a p p l i e d the  fifth  study  on  of  partial  4-ser,  p r e v i o u s l y on  from  the p a r t i a l  position  hydrolysis  used  released the  by  electrophoresis, for  analysis.  (6)  Specific The  fied  from  from  125  by  residues or  these  and  du  of  activity  the p i t u i t a r i e s t o 145  because  N-  acid  The  4-ser,  (89)  acid  i n that  hormones), tool  (69).  occurs  for  at  this complete  two t e t r a p e p t i d e s high  C-termini  voltage  available  Hormones  8-ileu oxytocin  of Oncorhynchus  oxytocic units  been  differed  Salmon N e u r o h y p o p h y s i a l of  this  residue  s e p a r a b l e on and  in  It  Vigneaud  powerful  hormones.  have both  Activities  specific  be-  seem t o h a v e  (aspartic  s h o u l d be  used  hormones.  aspartic  position  t h i s method  sub-  continued either  5 i n a l l known n e u r o h y p o p h y s i a l  sequence s t u d i e s of  when t h e  procedure  neurohypophysial  central  past  material.  method r e p r e s e n t s a p o t e n t i a l l y  obtained  be  of unreacted  hydrolysis  preferentially the  acid  hormones.  salmon hormones  8 - i l e u o x y t o c i n does not  used  Because of  of  could not  remaining  sequence  to neurohypophysial  N - t e r m i n a l amino a c i d  of high background  scarcity  methods  sequencing  Edman m e t h o d  The  it  analytical  the present work, the dansyl-Edman  made p o s s i b l e  the  of  the  tschawytscha  (assay of Holton)  per  puriranged  milli-  120 gram (Table V I I ) .  The h i g h e s t v a l u e o b t a i n e d was  from  a p r e p a r a t i o n which c o n t a i n e d p r a c t i c a l l y no contaminant amino a c i d s .  The r e s u l t agrees w i t h the r e p o r t e d v a l u e of  150 o x y t o c i c u n i t s per m i l l i g r a m (assay of H o l t o n ) ( 7 5 ) . The s p e c i f i c a c t i v i t y of 8-arg o x y t o c i n  isolated  from salmon glands was almost t w i c e as h i g h as the v a l u e s p r e v i o u s l y r e p o r t e d f o r t h i s hormone o b t a i n e d by s y n t h e s i s . A s p e c i f i c a c t i v i t y v a l u e of 115 u n i t s per m i l l i g r a m (method o f H o l t o n ) has been quoted r e p e a t e d l y  (5), (75).  A s l i g h t l y h i g h e r v a l u e , 125 o x y t o c i c u n i t s p e r m i l l i g r a m (assay o f H o l t o n ) , has been r e p o r t e d by Sawyer ( 6 ) .  The  v a l u e s o b t a i n e d i n the p r e s e n t work range up t o 229 oxyt o c i c u n i t s per m i l l i g r a m on t h e b a s i s of t h e same assay. The h i g h e s t v a l u e s were o b t a i n e d from the p r e p a r a t i o n of hormone which d i d n o t c o n t a i n any contaminant amino a c i d s (Table V I I I ) .  The d i f f e r e n c e i n s p e c i f i c a c t i v i t i e s of the  8-arg o x y t o c i n from P a c i f i c salmon and those o f s y n t h e t i c p r e p a r a t i o n s of t h i s p e p t i d e c o u l d be a s c r i b e d t o the p r e sence o f b i o l o g i c a l l y i n a c t i v e dimers (90) i n the l a t t e r .  (7)  R e l a t i v e P r o p o r t i o n s of Neurohypophysia1 Hormones in  Teleosts S e p a r a t i o n o f the two n e u r o h y p o p h y s i a l  hormones of  salmon by i o n exchange chromatography c o n s i s t e n t l y showed t h a t the o x y t o c i c a c t i v i t y was h i g h e r i n the 8-arg o x y t o c i n  121 peak than i n the 4-ser, 8 - i l e u o x y t o c i n peak. r a t i o v a r i e d between 2.0  and  3.0.  The  actual  I n s e p a r a t i o n s of  the  two t e l e o s t hormones d e s c r i b e d i n the l i t e r a t u r e , the  rela-  t i v e amount of the f i r s t hormone i s e i t h e r g r e a t e r than or e q u a l t o t h a t of the second hormone.  S i n c e the neurohypo-  p h y s i a l hormones of salmon do not appear t o d i f f e r i n s t r u c t u r e from those of o t h e r t e l e o s t s , the v a r i a t i o n s i n a c t i v i t y r e f l e c t d i f f e r e n c e s i n the amounts of hormones. For example, i n s e p a r a t i o n of n e u r o h y p o p h y s i a l mones of C y p r i n u s 'Carpio, a f r e s h w a t e r  t e l e o s t , the o x y t o c i c  a c t i v i t y c o n t a i n e d i n the 4-ser, 8 - i l e u o x y t o c i n peak approximately  The p r o f i l e of o x y t o c i c a c t i v i t y  t a i n e d i n the s e p a r a t i o n of n e u r o h y p o p h y s i a l  ob-  hormones of  a marine t e l e o s t , shows t h a t the amounts of  the two hormone a c t i v i t i e s are a p p r o x i m a t e l y Two  was  two times h i g h e r than t h a t of the 8-arg  o x y t o c i n peak (46).  Gadus l u s c u s ,  hor-  equal  independent s e p a r a t i o n s o f n e u r o h y p o p h y s i a l  (48).  hormones  of M e r l u c c i u s m e r l u c c i u s , a n o t h e r marine t e l e o s t , show the o x y t o c i c a c t i v i t y of the 4-ser, 8 - i l e u o x y t o c i n peak as s e v e r a l times g r e a t e r t h a n the a c t i v i t y a s s o c i a t e d w i t h 8-arg o x y t o c i n  (77), (91).  A s i m i l a r separation for a  t h i r d marine t e l e o s t , P o l l a c h i u s v i r e n s , shows the 8 - i l e u o x y t o c i n a c t i v i t y t o be a p p r o x i m a t e l y 8-arg o x y t o c i n  (91).  I n the f i v e s e p a r a t i o n s  4-ser,  t w i c e t h a t of described  above p o l y a c r y l i c a c i d c a t i o n exchangers were used and  the  122 e l u t i o n o f b o t h hormones was  c a r r i e d out a t pH 7.7.  Sawyer  and van Dyke a l s o have r e p o r t e d the s e p a r a t i o n of neurohypop h y s i a l hormones of P o l l a c h i u s v i r e n s . o x y t o c i n peak was peak a t pH 7.5,  The  4-ser,  8-ileu  e l u t e d a t pH 5, and the 8-arg o x y t o c i n  from a c a r b o x y m e t h y l c e l l u l o s e column.  Approxi-  m a t e l y e q u a l amounts of o x y t o c i c a c t i v i t y were p r e s e n t i n t h e s e hormone peaks (45). I n view of the p r e s e n t f i n d i n g s of 2 : 1 t o 3 : 1 r a t i o of o x y t o c i c a c t i v i t y of 8-arg o x y t o c i n t o 4-ser, 8 - i l e u oxyt o c i n i n the spawning P a c i f i c salmon, i t i s of i n t e r e s t t o note the work o f L e d e r i s on the exposure water  (76) .  o f Salmo i r i d e u s t o sea  The measurements o f L e d e r i s o f  neurohypophysial  hormones i n the p i t u i t a r i e s o f e x p e r i m e n t a l t r o u t were based on o x y t o c i c and p r e s s o r assays o f the g l a n d e x t r a c t s .  The  r a t i o s o f p i t u i t a r y 8-arg o x y t o c i n t o 4-ser, 8 - i l e u o x y t o c i n o b t a i n e d by t h i s method (3.0 f o r c o n t r o l f i s h , d e c r e a s i n g t o 1.5  f o r t h e f i s h exposed t o sea water) were s i m i l a r t o  the r a t i o s observed i n the p r e s e n t study f o l l o w i n g s e p a r a t i o n of the two salmon hormones on c a t i o n  exchangers.  O x y t o c i c assays c a r r i e d out by L e d e r i s , however, were done u s i n g r a t u t e r u s i n the presence of magnesium i o n , w h i l e those of the p r e s e n t i n v e s t i g a t i o n were performed  by the  s t a n d a r d method o f H o l t o n (55) w i t h o u t the a d d i t i o n of magnesium.  For t h i s r e a s o n , the v a l u e of the above com-  parison i s limited. There are t h r e e p o s s i b l e e x p l a n a t i o n s f o r the c o m p a r a t i v e l y l a r g e amount o f 8-arg o x y t o c i n o b t a i n e d from  123 salmon p i t u i t a r i e s i n t h e p r e s e n t work.  The f i r s t , and  the s i m p l e s t , e x p l a n a t i o n i s t h a t i s o l a t i o n o f t h i s hormone a t an a c i d i c pH p r e s e r v e s i t s f u l l b i o l o g i c a l a c t i v i t y . Loss o f b i o l o g i c a l a c t i v i t y o f a n e u r o h y p o p h y s i a l  hormone  a t n e u t r a l pH c o u l d be due t o e i t h e r  of a con-  taminating peptidase active  i n t h e n e u t r a l pH r e g i o n , o r  to the d e - a c t i v a t i o n of the molecule d i s u l f i d e interchange.  t h e presence  as a r e s u l t o f  One p i t u i t a r y p e p t i d a s e has been  d e s c r i b e d as a contaminant o f b o v i n e , o v i n e , and human p i t u i t a r y gonadotrophins. active  However, t h i s p e p t i d a s e i s  a t pH 4 ( 9 2 ) . While t h e presence  cannot be r u l e d  o u t w i t h o u t e x p e r i m e n t a l e v i d e n c e , i t can  be c o n s i d e r e d u n l i k e l y the hormones.  of p e p t i d a s e ( s )  a t t h i s stage of p u r i f i c a t i o n o f  Disulfide  n e u t r a l pH (86).  i n t e r c h a n g e i s known t o occur a t  The r e s u l t o f d i s u l f i d e i n t e r c h a n g e can  be t h e f o r m a t i o n o f a b i o l o g i c a l l y i n a c t i v e  dimer ( 9 0 ) .  The dimer f o r m a t i o n would r e s u l t i n a decrease  i n biological  activity. I n p i l o t experiments  on s e p a r a t i o n o f t h e salmon  hormones a g r a d i e n t t o pH 7.5 was used f o r t h e e l u t i o n o f 8-arg o x y t o c i n .  The o x y t o c i c a c t i v i t y a s s o c i a t e d w i t h  t h i s peak, however, was a t l e a s t t w i c e as g r e a t as t h e a c t i v i t y a s s o c i a t e d w i t h t h e 4-ser, 8 - i l e u peak. t a k e n i n these experiments  Care was  n o t t o l e a v e t h e hormone a t  n e u t r a l c o n d i t i o n s any l o n g e r than was a b s o l u t e l y n e c e s s a r y .  124 Thus, a s s a y i n g of the e f f l u e n t f r a c t i o n s took p l a c e immed i a t e l y upon e l u t i o n , and the hormone c o n t a i n i n g f r a c t i o n s were p o o l e d The  and a c i d i f i e d immediately  afterwards.  second e x p l a n a t i o n f o r the h i g h 8-arg o x y t o c i n  t o 4-ser, 8 - i l e u o x y t o c i n r a t i o o b t a i n e d i n the e x p e r i m e n t s i s t h a t t h e r e was mone.  I f the n e u r o p h y s i n  present  l o s s of the l e s s b a s i c h o r -  or a n e u r o p h y s i n - l i k e complex  does e x i s t i n salmon, and i f the b i n d i n g o f 4-ser, 8 - i l e u o x y t o c i n t o t h a t c a r r i e r m o l e c u l e i s s t r o n g e r than t h a t of 8-arg o x y t o c i n , i t c o u l d be envisaged technique  t h a t the  extraction  d i d not complete d i s s o l u t i o n of the complex.  There  i s some e v i d e n c e of a p r e f e r e n t i a l b i n d i n g of o x y t o c i n t o neurophysin  i n weak (0.25%) a c e t i c a c i d s o l u t i o n a t 2°C  A l t e r n a t i v e l y , the l e s s b a s i c hormone may b i l i t y i n c o l d aqueous a c e t i c The  have a low  (84).  solu-  acid.  t h i r d e x p l a n a t i o n i s the p o s s i b i l i t y of a  v a r i a t i o n i n the r a t i o s of n e u r o h y p o p h y s i a l  seasonal  hormones as  observed by Sawyer and P i c k f o r d i n Fundulus h e t e r o c l i t u s  (50).  These a u t h o r s r e p o r t a d i s a p p e a r a n c e of 4-ser, 8 - i l e u oxyt o c i n from the p i t u i t a r y glands of female f i s h d u r i n g  the  r e p r o d u c t i v e p e r i o d i n June, and the r e t u r n of t h i s hormone a f t e r completion  of the r e p r o d u c t i v e p e r i o d .  t i o n s were based on the p h a r m a c o l o g i c a l crude p i t u i t a r y e x t r a c t s .  The  observa-  c h a r a c t e r i s t i c s of  125 I n the p r e s e n t  study p i t u i t a r y g l a n d s were  obtained  from spawning salmon and c o n s e q u e n t l y the r e s u l t s may  be  i n t e r p r e t e d as c o n f i r m i n g the s t u d i e s on F. h e t e r o c l i t u s . The  r e p r o d u c t i v e stages of the f i s h e s used f o r  of hormones mentioned a t the b e g i n n i n g were not r e p o r t e d .  separations  of t h i s s e c t i o n  I n the s t u d i e s on P a c i f i c salmon, f o u r  p r e p a r a t i v e s c a l e (100 gm)  e x t r a c t s , three using  pituitar-  i e s c o l l e c t e d from males and the f o u r t h from f e m a l e s , showed no d i f f e r e n c e i n hormone c o n t e n t of the g l a n d s ,  the  r e l a t i v e p r o p o r t i o n s of the two hormones, or i n t h e i r chemical  8.  composition.  Evolutionary  Considerations  E v i d e n c e e x i s t s t h a t 3-phe, 8-arg o x y t o c i n p r e s s i n ) i n mammals may  be s y n t h e s i z e d by the  pathway of p r o t e i n s y n t h e s i s the p e p t i d e i s coded by DNA  (93).  (vaso-  conventional  Hence the s t r u c t u r e of  base t r i p l e t s .  This evidence  has been e x t r a p o l a t e d t o o t h e r v e r t e b r a t e s and has l e d t o s p e c u l a t i o n on e v o l u t i o n of n e u r o h y p o p h y s i a l hormones. There appears t o be g e n e r a l agreement w i t h the concept t h a t the two hormones e v o l v e d  from one  a n c e s t r a l molecule.  i d e n t i t y of t h i s m o l e c u l e i s c o n s i d e r e d  uncertain  The  by  Acher (17), Sawyer (34) , and Munsick (26), w h i l e V l i e g e n t h a r t and V e r s t e e g The  c o n s i d e r i t t o be 8-arg o x y t o c i n  (94).  a n c e s t r a l m o l e c u l e concept stems from the f a c t t h a t  126 o n l y one hormone has been found i n c y c l o s t o m e s .  Chemical  i d e n t i f i c a t i o n of the c y c l o s t o m e hormone has not been r e p o r t e d i n the l i t e r a t u r e ; i t was  tentatively identified  8-arg o x y t o c i n on the b a s i s of p h a r m a c o l o g i c a l (17),  (40).  as  studies (6),  Acher proposes gene d u p l i c a t i o n , o c c u r i n g  be-  tween c y c l o s t o m e s and the o t h e r f i s h e s , as the o r i g i n  of  two s e p a r a t e hormones ( 6 ) . V l i e g e n t h a r t and V e r s t e e g p o s t u l a t e s i n g l e p o i n t m u t a t i o n s l e a d i n g t o amino a c i d s u b s t i t u t i o n s i n p o s i t i o n s 3, 4, and  8 (94).  I n a l a t e r paper, they d i s c u s s  codons i n v o l v e d i n these m u t a t i o n s (95). phenylalanine  interchange  Isoleucine-  i n p o s i t i o n 3 can be a c h i e v e d  one base change, t h a t i s , UUU ((Table XVII) .  Phenylalanine  (or UUC)  t o AUU  (or  by  AUC)  i s o n l y found i n 3-phe, 8-arg  o x y t o c i n and 3-phe, 8 - l y s o x y t o c i n i n mammals. other neurohypophysial  the  In a l l  hormones found i n n a t u r e i s o l e u c i n e  occupies p o s i t i o n three. 3-Phe, 8 - l y s o x y t o c i n c o u l d have a r i s e n by one  base  change i n the gene f o r 3-phe, 8-arg o x y t o c i n , i . e . , AGA AGG)  t o AAA  (or AAG).  8-Arg o x y t o c i n c o u l d have g i v e n  (or rise  t o 3-phe, 8-arg o x y t o c i n by one base change i n v o l v i n g the codon f o r amino a c i d 3, and the l a t t e r c o u l d a l s o have, i n t u r n , g i v e n r i s e t o 3-phe, 8 - l y s o x y t o c i n by one base change i n the codon of amino a c i d 8.  127 T A B L E  XVII  The G e n e t i c Code*  2nd 1st  U  C  A  G  3rd  phe  ser  tyr  cys  U  phe  ser  tyr  cys  C  ser  **  **  A  ser  **  try  G  leu  pro  his  arg  U  leu  pro  his  arg  C  leu  pro  gin  arg  A  leu  pro  gin  arg  G  ileu  thr  asn  ser  U  ileu  thr  asn  ser  C  ileu  thr  lys  arg  A  met  thr  lys  arg  G  val  ala  asp  gly  U  val  ala  asp  gly  C  val  ala  glu  gly  A  val  ala  glu  gly  G  u leu leu  c  A  G  *  adapted from C r i c k  .**. t e r m i n a t i o n  (96)  128 The o x y t o c i n t o 8 - i l e u o x y t o c i n t r a n s i t i o n would i n v o l v e o n l y one base change i n t h e l e u c i n e o r i s o l e u c i n e codon (Table  XVII).  In a d d i t i o n t o the 8-arginine  or 8-lysine  contain-  i n g hormones, t h e a n c e s t r a l m o l e c u l e a l s o would have t o give r i s e t o a peptide 8.  series with i l e u or l e u i n p o s i t i o n  Acher r e g a r d s 8 - i l e u o x y t o c i n r a t h e r t h a n o x y t o c i n t o  be a more p r i m i t i v e m o l e c u l e , preceded by 4 - s e r , 8 - i l e u oxytocin  ( 6 ) . Munsick c o n s i d e r s  t h a t o x y t o c i n and 8 - i l e u  o x y t o c i n may have o r i g i n a t e d a t about t h e same t i m e and t h a t t h e l a t t e r gave r i s e t o 4 - s e r , 8 - i l e u o x y t o c i n ( 2 6 ) . Sawyer c o n s i d e r s  o x y t o c i n t o be e i t h e r a v e r y a n c i e n t mole-  c u l e o r one t h a t has a r i s e n s e v e r a l times i n d e p e n d e n t l y i n evolutionary l i n e s (34). The same a r g i n i n e t r i p l e t  (AGA) w h i c h g i v e s r i s e i n  subsequent mammali an e v o l u t i o n t o t h e l y s i n e t r i p l e t (AAA) f o r t h e amino a c i d p o s i t i o n 8, can a l s o become a codon f o r i s o l e u c i n e (AUA) w i t h o n l y one base change  (Table  XVII).  On t h i s b a s i s 8 - i l e u o x y t o c i n i s more l i k e l y t o be o l d e r than o x y t o c i n , because l e u c i n e s u b s t i t u t i o n would have r e q u i r e d two base changes. The a r g i n i n e codon (CGX) w i t h one base change can become codons f o r l e u c i n e  (CUX), (Table X V I I ) .  b a s i s o x y t o c i n can be c o n s i d e r e d  On t h i s  o l d e r t h a n 8 - i l e u oxy-  t o c i n , s i n c e two base changes a r e r e q u i r e d t o produce t h e latter.  A The a r g i n i n e codon (CG ) can y i e l d a g l u t a m i n e p  129  codon (CA.) w i t h one base change.  Glutamine i s found xn  p o s i t i o n 8 o f 4-ser, 8-gln o x y t o c i n  (elasmobranchs). A However, a l y s i n e codon cannot be d e r i v e d from CG^ on t h e b a s i s o f one base change. T h i s suggests t h a t an i n t e r A and AG^, A c o u l d have o c c u r r e d change o f t h e a r g i n i n e codons CG^, G  G  d u r i n g t h e e v o l u t i o n o f t h e genes f o r t h e n e u r o h y p o p h y s i a l hormones. P o s i t i o n four i s occupied oxytocin branchs) .  by s e r i n e i n 4-ser, 8 - i l e u  ( t e l e o s t s ) and i n 4-ser, 8-gln o x y t o c i n In a l l other n a t u r a l l y occuring  hormones g l u t a m i n e i s found i n p o s i t i o n 4.  (elasmo-  neurohypophysial Serine-glutamine  s u b s t i t u t i o n r e q u i r e s two base changes (Table X V I I ) .  One  route i s v i a a 4-proline intermediate, the other i s v i a a nonsense t r i p l e t .  The presence o f p r o l i n e would c o n f e r  new  s t e r i c p r o p e r t i e s onto t h e r i n g o f t h e m o l e c u l e , and p r o found changes i n b i o l o g i c a l a c t i v i t y and o t h e r of t h e hormone c o u l d be e x p e c t e d .  properties  The second p o s s i b l e  r o u t e i s v i a t h e nonsense t r i p l e t UA^ .  The nonsense  G  t r i p l e t c o u l d , however, g i v e r i s e by one base change t o A o r t o g l u t a m i n e (CA^) A . Such a m u t a t i o n e i t h e r s e r i n e (UC_) G  <J  c o u l d a l s o account f o r t h e absence o f one hormone i n c y c l o stomes. A t t h i s s t a g e we have t o c o n c l u d e t h a t much more information i s r e q u i r e d f o r a f u l l understanding  of the  i n t e r r e l a t i o n s h i p and e v o l u t i o n of t h e genes f o r neurohypo-  130 p h y s i a l hormones throughout the v e r t e b r a t e s . i n v e s t i g a t i o n has not uncovered any new  The  present  structures to  help  e l u c i d a t e t h i s problem. The  e v o l u t i o n a r y r e l a t i o n s of the t e l e o s t s are o u t -  l i n e d i n F i g u r e 18, which a l s o shows the groups of t e l e o s t s f o r which the s t r u c t u r e s of the n e u r o h y p o p h y s i a l hormones have been e s t a b l i s h e d c h e m i c a l l y . mon,  r e p r e s e n t a t i v e s of the Gadiformes and  have been s t u d i e d and and  I n a d d i t i o n t o the  8-arg o x y t o c i n .  sal-  Cypriniformes  found t o have 4-ser, 8 - i l e u o x y t o c i n Because of the p o s i t i o n of the salmon-  o i d s i n the e v o l u t i o n a r y t r e e of the t e l e o s t s , i t suggests t h a t t h e s e two hormones are w i d e l y c h a r a c t e r i s t i c of teleosts.  I t a l s o i n d i c a t e s t h a t the s e a r c h  for  the  new  s t r u c t u r e s s h o u l d be d i r e c t e d a t o t h e r l i n e s of e v o l u t i o n .  131  LEPTOLEPIMORPHA  F i g u r e 18:  Family tree of t e l e o s t orders ( a c c o r d i n g t o Romer (37))  Arrows i n d i c a t e t e l e o s t groups f o r which c h e m i c a l i d e n t i f i c a t i o n s o f n e u r o h y p o p h y s i a l hormones a r e available. (a) Gadiformes: ( 1 0 ) , ( 4 2 ) , ( 4 8 ) , ( 4 9 ) , ( 5 1 ) . (b) C y p r i n i f o r m e s : ( 4 6 ) . (c) Salmoniformes: present study.  132 LITERATURE CITED  Van Dyke, H.B., K. Adamsons, J r . , and S.L. E n g e l . A s p e c t s o f b i o c h e m i s t r y and p h y s i o l o g y o f t h e n e u r o h y p o p h y s i a l hormones. I n "Recent P r o g r e s s i n Hormone Research," v o l . X I (ed. G. P i n c u s ) , New York, Academic P r e s s , 1-42 (1955). G r o l l m a n , A. 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