"Science, Faculty of"@en . "Zoology, Department of"@en . "DSpace"@en . "UBCV"@en . "Wilson, Nadine"@en . "2011-06-30T17:52:56Z"@en . "1968"@en . "Doctor of Philosophy - PhD"@en . "University of British Columbia"@en . "The neurohypophysial hormones of Pacific chinook salmon (Oncorhynchus tschawytscha) were purified and the amino acid sequence of both hormones determined.\r\nThe extraction and purification procedure was developed in an effort to maximize the yields of pure hormones.\r\nPituitary glands were extracted at 4\u00B0C using 0.2 M acetic acid. Purification consisted of gel filtration, ultrafiltration, and ion exchange. Gel filtration on Sephadex G-15 columns was used to separate neurohypophysial peptides from high molecular weight material. Separation of the two hormones was accomplished on one of three cation exchangers: carboxymethylcellulose, phosphocellulose or sulfoethyl-Sephadex. The hormones were eluted from cation exchange columns using a sodium or ammonium ion concentration gradient at constant pH; pH 5 was used for chromatography on carboxymethylcellulose and on phosphocellulose; pH 2.45 was used for chromatography on sulfoethyl-Sephadex.\r\nThe two hormones were purified further by rechromatography of individual hormones on another cation exchange medium. The material obtained by rechromatography was pure as determined by amino acid analyses. The yields of pure hormones at the end of purification procedure was 48% of the starting material. The specific activities of the two purified hormones were 145 and 229 oxytocic units per milligram for Hormone I and Hormone II respectively.\r\nThe amino acid sequence of Hormone I was determined by a combination of three methods: subtractive Edman degradation, partial acid hydrolysis, and Dansyl-Edman technique. The amino acid sequence of Hormone I was found to be that of 4-serine, 8-isoleucine oxytocin.\r\nThe amino acid sequence of Hormone II was determined by the Edman subtractive method, the Dansyl-Edman technique, and the mobility of the C-terminal residue on high voltage electrophoresis. The amino acid sequence of Hormone II was found to be that of 8-arginine oxytocin.\r\nThe two neurohypophysial hormones described from salmon have amino acid sequences identical with those described from four species of Gadiformes and one species of Cypriniformes by other workers. The position of Salmoniformes on the evolutionary tree of teleost fishes suggests that these structures are characteristic of a wide range of teleosts."@en . "https://circle.library.ubc.ca/rest/handle/2429/35833?expand=metadata"@en . "I S O L A T I O N AND AMINO A C I D SEQUENCE OF NEUROHYPOPHYSIAL HORMONES OF P A C I F I C CHINOOK SALMON ( O n c o r h y n c h u s t s c h a w y t s c h a ) b y N a d i n e W i l s o n B . A . , U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1955 A T H E S I S SUBMITTED IN P A R T I A L F U L F I L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF D o c t o r o f P h i l o s o p h y i n t h e D e p a r t m e n t o f Z o o l o g y We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE U N I V E R S I T Y OF B R I T I S H COLUMBIA A u g u s t , 1968 Nadine Wilson 1969 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced deg ree a t the U n i v e r s i t y o f B r i t i s h Co l umb i a , I ag r ee t h a t t he L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and S t u d y . I f u r t h e r ag ree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y pu r po se s may be g r a n t e d by the Head o f my Department o r by h.iis r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l no t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Depar tment o f The U n i v e r s i t y o f B r i t i s h Co lumb ia Vancouve r 8, Canada August 9, 1968 ABSTRACT The neurohypophysial hormones of P a c i f i c chinook salmon (Oncorhynchus tschawytscha) were p u r i f i e d and the amino a c i d sequence of both hormones 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-veloped i n an e f f o r t to maximize the y i e l d s of pure hor-mones. P i t u i t a r y glands were e x t r a c t e d at 4\u00C2\u00B0C using 0.2 M a c e t i c a c i d . 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. Gel f i l t r a t i o n on Sephadex G-15 columns was used to separate neurohypophysial peptides from high molecular weight m a t e r i a l . Separation of the two hormones was accomplished on one of three c a t i o n exchangers: carboxymethylcellulose, phosphocellulose or sulfoethyl-Sephadex. The hormones were e l u t e d from c a t i o n exchange columns using a sodium or ammonium i o n concentra-t i o n g r adient at constant pH; pH 5 was used f o r chromato-graphy on carboxymethylcellulose and on phosphocellulose; 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 rechroma-tography 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 obtained by rechromatography was pure as determined by amino a c i d analyses. The y i e l d s of pure hormones at the end of p u r i f i c a t i o n procedure was 48% of the 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 of the 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 per 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 of Hormone I was determined by a combination of three methods: s u b t r a c t i v e Edman degradation, 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 of Hormone I was found to be t h a t of 4-serine, 8 - i s o l e u c i n e o x y t o c i n . The amino a c i d sequence of Hormone I I was determined by the Edman s u b t r a c t i v e method, the Dansyl-Edman tech-nique, and the m o b i l i t y of the C-terminal residue on high voltage e l e c t r o p h o r e s i s . The amino a c i d sequence of Hormone I I was found to be tha t of 8-arginine o x y t o c i n . The two neurohypophysial hormones described from salmon have amino a c i d sequences i d e n t i c a l w i t h those described from four species of Gadiformes and one species of Cypriniformes by other workers. The p o s i t i o n of Sa'lmoniformes' on the e v o l u t i o n a r y t r e e of t e l e o s t f i s h e s suggests t h a t these s t r u c t u r e s are c h a r a c t e r i s t i c of a wide range of t e l e o s t s . i v T A B L E OF C O N T E N T S I . INTRODUCTION 1. Background 1 2. C h a r a c t e r i z a t i o n of neurohypophysial hormones . 3 3. Neurohypophysial hormones of ve r t e b r a t e s other than f i s h e s . 4 4. Neurohypophysial hormones of f i s h e s other than t e l e o s t s 6 5. Neurohypophysial hormones of t e l e o s t f i s h e s . . 8 6. Present study: neurohypophysial hormones i n Oncorhynchus tschawytscha 10 I I . 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. B i o l o g i c a l assays 14 3. A n a l y t i c a l methods i . Measurement of p r o t e i n concentration . . 17 : : i i . S p e c i f i c c o n d u c t i v i t y measurements . . . 18 4. E x t r a c t i o n of 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 at 4\u00C2\u00B0C 20 i i . E x t r a c t i o n at 100\u00C2\u00B0C i n 0.25% a c e t i c a c i d of acetone powder prepared from frozen p i t u i t a r i e s 21 i i i . A d d i t i o n a l e x t r a c t i o n methods 22 5. Concentration of crude and p a r t i a l l y p u r i f i e d e x t r a c t s 23 6. U l t r a f i l t r a t i o n 24 V 7. Gel f i l t r a t i o n i . P r e p a r a t i o n of the g e l and column packing 26 i i . Loading and e l u t i o n of g e l f i l t r a t i o n columns 27 8. Ion exchange chromatography i . P r e p a r a t i o n of exchangers and column packing . . . . . . . 28 i i . B u f f e r s used i n c a t i o n exchange. . . . 31 i i i . Loading and e l u t i o n of c a t i o n exchange columns 31 i v . Repeated use of the same column. . . . 32 9. Performic a c i d o x i d a t i o n 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 analyses i . Procedure . 36 i i . Q u a n t i t a t i o n of r e s u l t s 37 12. P a r t i a l a c i d h y d r o l y s i s 38 13. High voltage e l e c t r o p h o r e s i s 38 14. Edman degradation 40 15. S u b t r a c t i v e Edman method i n amino a c i d sequence determination 42 16. Dansylation method i n amino a c i d sequence determination i . D a nsylation r e a c t i o n and h y d r o l y s i s . . 43 i i . I d e n t i f i c a t i o n of dansyl amino acids by t h i n l a y e r chromatography 44 I I I . RESULTS A. S t a b i l i t y of o x y t o c i c 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 of Oncorhynchus t schawytscha 1. S t a b i l i t y of o x y t o c i c a c t i v i t y during storage of glands at -196\u00C2\u00B0C 42 v i 2. S t a b i l i t y of 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 e x t r a c t s 49 B. E x t r a c t i o n of neurohypophysial hormones from salmon p i t u i t a r i e s 51 C. P u r i f i c a t i o n of neurohypophysial hormones of salmon 51 1. Gel f i l t r a t i o n of crude salmon p i t u i t a r y e x t r a c t s 54 2. D e - s a l t i n g of neurohypophysial hormones . 61 i . D e - s a l t i n g on Sephadex G-10 61 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 . . . . 62 3. Separation of neurohypophysial hormones of salmon by i o n exchange chromatography 64 4. p u r i f i c a t i o n of salmon neurohypophysial Hormone I by i o n exchange chroma-tography 74 5. P u r i f i c a t i o n of salmon neurohypophysial Hormone I I by i o n exchange chroma-tography 77 D. Amino a c i d sequence of neurohypophysial hormones of Oneorhynchus tschawytscha . . . 84 1. Amino a c i d sequence of Hormone I i . Determination of N-terminal pentapeptide 84 i i . C h a r a c t e r i z a t i o n of C-terminal t e t r a p e p t i d e 89 2. Amino a c i d sequence of Hormone I I i . Determination of N-terminal pentapeptide 92 i i . Determination of C-terminal t e t r a p e p t i d e 94 3. Reaction y i e l d s encountered i n the course of amino a c i d sequence work. . . 97 v i i IV. DISCUSSION 1. Neurohypophysial hormone content of salmon p i t u i t a r i e s 100 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 103 3. E x t r a c t i o n of neurohypophysial hormones. . . . 104 4. P u r i f i c a t i o n of neurohypophysial hormones. . . 106 5. S t r u c t u r a l a n a l y s i s of neurohypophysial hormones of t e l e o s t s 113 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 . . . . 113 i i . Amino a c i d sequence st u d i e s of the l e s s b a s i c t e l e o s t hormone 113 i i i . Amino a c i d sequence st u d i e s of the more b a s i c t e l e o s t hormone . . . . . . . 117 i v . S t r u c t u r e of the 0. tschawytscha neurohypophysial hormones 118 v. Comments on the a n a l y t i c a l methods . . . 119 6. S p e c i f i c a c t i v i t i e s of salmon neurohypo-p h y s i a l hormones 119 7. R e l a t i v e proportions of neurohypophysial hormones i n t e l e o s t s 120 8. E v o l u t i o n a r y c o n s i d e r a t i o n s 125 LITERATURE CITED . 132 v i i i L I S T O F T A B L E S I . Munsick's b u f f e r e d t i s s u e bath s o l u t i o n 16 I I . 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 tschawytscha c o l l e c t e d at three d i f f e r e n t l o c a t i o n s 48 I I I . E x t r a c t i o n of 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 of 0. tschawytscha 52-53 IV. 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. tschawytscha using D i a f l o UM-2 membranes 65 V. 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. tschawytscha using D i a f l o UM-3 membranes 66-67 VI. Ion exchange chromatography of neurohypophysial hormones of 0. tschawytscha: s p e c i f i c conduc-t i v i t i e s of e l u t e d hormones 73 V I I . Amino a c i d composition (residues per mole) of neurohypophysial Hormone I of 0. tschawytscha. . 79 V I I I . Amino a c i d composition (residues per mole) of neurohypophysial Hormone I I of 0.tschawytscha. . 83 IX. The amino a c i d sequence of N-terminal t e t r a -peptide of Hormone I of 0. tschawytscha 85 X. 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 degradation c y c l e s 87 XI. 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 of amino acids 6 and 7 of Hormone I 90 X I I . The amino a c i d sequence of N-terminal penta-peptide of Hormone I I of 0. tschawytscha 93 X I I I . 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 of amino acids 6, 7 and 8 of Hormone I I 95 i x XIV. Comparison of neurohypophysial hormone contents of t e l e o s t p i t u i t a r i e s 102 XV. Comparison of y i e l d s obtained at 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 XVI. Comparison of q u a n t i t a t i v e amino a c i d analyses of t e l e o s t neurohypophysial hormones 114 XVII. The Genetic Code 127 X L I S T O F I L L U S T R A T I O N S FIGURE 1. Neurohypophysial hormones of ve r t e b r a t e s 2 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 three d i f f e r e n t methods 19 3. Loss of 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 at n e u t r a l and s l i g h t l y b a s i c pH at 25\u00C2\u00B0C 50 4. P u r i f i c a t i o n sequence of larg e s c a l e salmon p i t u i t a r y e x t r a c t s 55 5. Gel f i l t r a t i o n of 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 6. Comparison of g e l f i l t r a t i o n on Sephadex G-25 and Sephadex G-15 57 7. Gel f i l t r a t i o n of crude salmon p i t u i t a r y ex-t r a c t using a tandem s e r i e s of Sephadex G-15. . . 59 8. Rechromatography of 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 of equal bed volumes and d i f f e r e n t dimensions. . . . 60 9. D e - s a l t i n g of ox y t o c i n on Sephadex G-10 63 10. Separation of salmon neurohypophysial hormones on Whatman CM-32 69 11. Separation of salmon neurohypophysial hormones on S e l e c t a c e l phosphocellulose 70 12. Separation of salmon neurohypophysial hormones on SE-Sephadex. 72 13. Rechromatography of Hormone I on SE-Sephadex. . . 76 14. Rechromatography of Hormone I on Whatman CM-22. . 78 15. Rechromatography of Hormone I I on Whatman CM-22 . 82 x i 16. I d e n t i f i c a t i o n of amino a c i d 7 of Hormone I as DNS-proline 91 17. I d e n t i f i c a t i o n of amino a c i d 7 of Hormone I I as DNS-proline 96 18. Family t r e e of t e l e o s t orders 131 x i i A B B R E V I A T I O N S U S E D A.a. - amino a c i d a l a - a l a n i n e arg - a r g i n i n e asn - asparagine asp - a s p a r t i c a c i d cys-cys - c y s t i n e CyS03H - c y s t e i c a c i d g i n - glutamine g l u - glutamic a c i d g l y - g l y c i n e gly-NR^ - glycinamide h i s - h i s t i d i n e i l e u - i s o l e u c i n e l e u - l e u c i n e l y s - l y s i n e met - methionine phe - phenylalanine pro - p r o l i n e ser - s e r i n e t r p - tryptophan t y r - t y r o s i n e t h r - threonine v a l - v a l i n e A o o n - absorbance at 280 nm* 2 80 nm CM - carboxymethyl C-terminal - carboxyterminal DNS-dansyl 1-dimethylamino-naphthalene-5-s u l f o n y l -DEAE - di e t h y l a m i n o e t h y l mho - 1/ohm ymole, ym - micromole nm - nanometer N-terminal - amino-terminal p . s . i . - pounds per square inc h PTC-peptide - phenylthiocarbamyl peptide SE - s u l f o e t h y l TCA - t r i c h l o r o a c e t i c a c i d U C A G u r i d i n e c y t o s i n e adenosine guanosine one absorbance u n i t i s that amount of substance which when d i s s o l v e d i n 1 ml of solve n t has an absorbance of 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 wish to express my thanks to Dr. Michael Smith f o r h i s enthusiasm, 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 the sharing of h i s knowledge which I have enjoyed throughout the past three years. P a r t i c u l a r thanks are due to Miss 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 techniques, and to Dr. G.H. Dixon of the Department of Biochemistry, U n i v e r s i t y of B r i t i s h Columbia, and Dr. John Black, now w i t h the U n i v e r s i t y of Oregon Medical School f o r much help and advice. I am indebted to Dr. C. Finnegan of the Depart-ment of 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 throughout my graduate 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 to Mr. S. F a l l e r t and the s t a f f of Green River Hatchery, Washington, f o r t h e i r co-operation during the c o l -l e c t i o n of p i t u i t a r y glands. I would a l s o l i k e to thank Mr. K. Henze of the Department 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. Wylie and my other colleagues 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 of the glands and f o r many other instances of help and encouragement. The research work was done i n i t i a l l y i n the Vancouver Laboratory of the F i s h e r i e s Research Board of Canada, and x i v l a t t e r l y i n the Department of Biochemistry at t h i s u n i v e r -s i t y . F i n a n c i a l support was provided by the Medical Research C o u n c i l of Canada through a grant to Dr. Michael Smith. L a s t , I would l i k e to acknowledge the encouragement, help and patience of my husband, S t u a r t , throughout the pe r i o d of my graduate work. To my c h i l d r e n Gregory, Katya, and Duncan I . INTRODUCTION (1) Background Hormones having o x y t o c i c , pressor 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 the neurohypophyses of repre-s e n t a t i v e s of a l l c l a s s e s of v e r t e b r a t e s . The neurohypo-physis i s defined as the anatomical s t r u c t u r e c o n s i s t i n g of hypothalamic n u c l e i of neurons, t h e i r axons passing down the i n f u n d i b u l a r s t a l k , and the i n f u n d i b u l a r process or pars nervosa of the p i t u i t a r y gland (1). The b i o s y n t h e s i s of the neurohypophysial hormones i s thought to occur i n the c e l l bodies of neurons which comprise the hypothalamic n u c l e i (2). In mammals n u c l e i supraopticus and paraven-t r i c u l a r i s have been described and, i n f i s h e s , nucleus praeopticus (3). The hormones are transported 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 or p r o t e i n s by axoplasmic flow along the infundibulum f o r storage i n , and l a t e r r e l e a s e from, the pars nervosa (4). These peptide hormones co n t a i n a chain of nine amino a c i d s , which i n general s t r u c t u r e f o l l o w a uniform p a t t e r n (Figure 1). They have a d i s u l f i d e bridge between c y s t e i n e residues at p o s i t i o n 1 (the amino end of the peptide chain) 2 1 2 3 4 5 6 7 (a) O - O \u00E2\u0080\u0094 t 8 9 O - \u00E2\u0080\u00A2 1 2 3 4 5 6 7 8 9 (b) \u00E2\u0080\u00A2 - \u00E2\u0080\u00A2 - I L E U - G L N - # - * - # - L E U - # 1 2 3 4 5 6 7 8 9 (c) T \u00E2\u0080\u0094 PHE -- G L N - * - \u00E2\u0080\u00A2 -ARG- \u00E2\u0080\u00A2 (d) 1 2 3 4 5 6 7 8 9 T \u00E2\u0080\u00A2 PHE-- GLN-#-Y~ -\u00E2\u0080\u00A2- LYS--\u00E2\u0080\u00A2 1 2 3 4 5 6 7 8 9 (e) T \u00E2\u0080\u0094 ILEU - G L N - # - ^ --\u00E2\u0080\u00A2-\u00E2\u0080\u00A2ARG 1 2 3 4 5 6 7 8 9 ( f ) y-0 - l LEU -GLN\u00E2\u0080\u0094#\u00E2\u0080\u0094 ^ - \u00C2\u00BB - I L E U - + 1 2 3 4 5 6 7 8 9 (0) \u00E2\u0080\u00A2 - \u00E2\u0080\u00A2 - I L E U - S E R - # - \u00C2\u00AB - # - I L E U - # ^ - \u00E2\u0080\u00A2 - I L E U - S E R-# J-1 2 3 4 5 6 7 8 9 (h) y- \u00E2\u0080\u00A2 - I L E U - S E R - \u00C2\u00BB - ^ - \u00C2\u00BB - G L N - \u00C2\u00BB Figure 1: Neurohypophysial hormones of vert e b r a t e s F i l l e d c i r c l e s : p o s i t i o n s occupied by i d e n t i c a l amino acids throughout v e r t e b r a t e s (1 - 1/2 Cys; 2 - Tyr; 5 - Asn; 6 -1/2 Cys; 7 - Pro; 9 - Gly-NH 2)\u00E2\u0080\u00A2 Empty c i r c l e s : p o s i t i o n s of amino a c i d s u b s t i t u t i o n s . (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 ox y t o c i n (arginine v a s o p r e s s i n ) ; (d) 3-phe, 8-lys 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 (arginine v a s o t o c i n ) ; (f) 8 - i l e u o x y t o c i n (mesotocin); (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-ser, 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 , asparagine, p r o l i n e , and glycinamide 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 . V a r i a t i o n s 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 Hormones Neurohypophysial hormones are o f t e n c h a r a c t e r i z e d on the b a s i s of t h e i r pharmacological a c t i v i t i e s . Compari-son of the a c t i v i t y r a t i o s i n a number of b i o l o g i c a l assays (pharmacological p r o f i l e ) of an unknown hormone w i t h the a c t i v i t i e s of known peptides has been used to p r e d i c t the chemical i d e n t i t y of the unknown wi t h considerable pre-c i s i o n (5). This procedure i s o f t e n used because the s e n s i t i v i t y of the b i o l o g i c a l assays and t h e i r s p e c i f i c i t y allows the examination of very small amounts of impure hormones. However, pharmacological s t u d i e s can give errone-ous or ambiguous r e s u l t s (5). The determination of the amino a c i d content of a hormone and the d e f i n i t i o n of the sequence i n which the amino acids are arranged i s conse-quently the most fundamental approach to c h a r a c t e r i z a t i o n . This has been attempted l e s s o f t e n than pharmacological 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 completely pure form. 4 S t u d i e s by e i t h e r p h a r m a c o l o g i c a l o r c h e m i c a l m e t h o d s h a v e l e d t o t h e c o n c l u s i o n t h a t i n e a c h v e r t e b r a t e s p e c i e s t h e r e a r e two n e u r o h y p o p h y s i a l h o r m o n e s . P o s s i b l e e x c e p t i o n s t o t h i s g e n e r a l i z a t i o n a r e t h e c y c l o s t o m e s i n w h i c h o n l y one a c t i v e p r i n c i p l e h a s b e e n d e t e c t e d (6) and e l a s m o b r a n c h s i n w h i c h t h e p r e s e n c e o f t h r e e n e u r o h y p o p h y -s i a l h ormones 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 t i m e ( 7 ) . (3) Ne u r o h yp ophy s i a1 Hormones i n V e r t e b r a t e s O t h e r t h a n F i s h e s \u00E2\u0080\u00A2 The f i r s t s t r u c t u r a l a n a l y s i s o f n e u r o h y p o p h y s i a l h ormones was done b y du V i g n e a u d ' s g r o u p on 3-phe, 8 - a r g 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 ) ( 8 ) a n d o n o x y t o c i n (9) o f ox p i t u i t a r i e s . S t u d i e s on f o u r o t h e r mammals f o l l o w e d c l o s e l y ( s h e e p , h o r s e , man, and w h a l e ) and r e v e a l e d two s u b s t a n c e s i n e a c h s p e c i e s w i t h t h e amino a c i d c o m p o s i t i o n o f o x y t o c i n a n d 3-phe, 8 - a r g o x y t o c i n ( 1 0 ) . A p h a r m a c o -l o g i c a l s u r v e y o f r e p r e s e n t a t i v e s o f m o s t f a m i l i e s o f mammals by F e r g u s o n a nd H e l l e r c o n f i r m e d t h e w i d e d i s t r i b u t i o n o f t h e s e hormones ( 1 1 ) . One e x c e p t i o n f o u n d i s 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 ) f r o m h og p i t u i t a r i e s w h e r e i t r e p l a c e s 3-phe, 8 - a r g o x y t o c i n ( 1 2 ) . The two p e p t i d e s d i f f e r i n one amino a c i d i n p o s i t i o n 8. The s u r v e y o f F e r g u s o n a nd H e l l e r i n d i c a t e d t h e p r e s e n c e o f 3-phe, 8 - l y s o x y t o c i n i n o r d e r S u i f o r m e s ( 1 1 ) . 5 Oxytocin has been found i n b i r d s (13), (14), but 3-phe, 8-arg o x y t o c i n i s replaced by 8-arg o x y t o c i n ( a r g i n i n e vasotocin) (10, (15). A s y n t h e t i c p r e p a r a t i o n of 8-arginine o x y t o c i n was reported before the hormone was found i n nature (16). I t subsequently has been found i n p i t u i t a r i e s of represen-t a t i v e s of 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 exception of mammals (17). In mammals, however, 8-arginine o x y t o c i n has been reported from the p i n e a l gland of beef on the ba s i s of chromatographic and pharmacological evidence (18). Sub-sequently 8-lys o x y t o c i n was found i n the p i n e a l bodies of pigs (19). I t i s d i f f i c u l t to 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 to 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 not s u c c e s s f u l (20). According to the data a v a i l a b l e at present, p i t u i -t a r y 8-arginine o x y t o c i n appears to have remained unchanged throughout 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 other hormone, on the c o n t r a r y , has been subject to e v o l u t i o n a r y change. Oxytocin has been chemically i d e n t i f i e d from chicken (13) and pharmacologically from turkey (21) and two other b i r d s (14) . The p o s s i b l e existence of o x y t o c i n i n lower v e r t e b r a t e s ( r e p t i l e s , amphibians, f i s h e s ) i s a disputed s u b j e c t . Another neurohypophysial homologue, 8 - i l e u o x y t o c i n (mesotocin) has been chemically character-i z e d i n s e v e r a l amphibian species (22), (23), (24), (25). However, whi l e Acher described 8 - i l e u o x y t o c i n from Rana esculenta i n chemical terms (25), Munsick i d e n t i f i e d o x y t o c i n pharmacologically 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 the ba s i s of amino a c i d a n a l y s i s from some r e p t i l i a n species (27), (28), yet a recent r e p o r t on cobra, Naja naja, suggests the presence of both o x y t o c i n and 8 - i l e u o x y t o c i n (29). (4) Neurohypophysial Hormones of Fishes Other than Teleosts In cyclostomes only one neurohypophysial hormone, 8-arg o x y t o c i n , has been i n d i c a t e d pharmacologically (6), (17) . The hormone, 4-ser, 8-gln o x y t o c i n (glumitocin) was c h a r a c t e r i z e d chemically by Acher 1s group i n 1965 from elasmobranch f i s h e s (30), and subsequently found by the 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 of 4-ser, 8-gln o x y t o c i n was confirmed by snyth e s i s by K l i e g e r (33). Acher 1s work i n d i c a t e s the presence of small amounts of the second hormone, 8-arg o x y t o c i n , i n elasmobranchs. The presence of o x y t o c i c p r i n c i p l e s other than 4-ser, 8-gln o x y t o c i n has not yet been excluded f o r some elasmobranchs (7), (34). The study of Sawyer on Hydrolagus c o l l i e i f a i l e d to 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 present together 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 to 8-arg o x y t o c i n , both 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 phar-m a c o l o g i c a l l y . 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 species of A f r i c a n l u n g f i s h (Protopterus aethiopicus) 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 (23). P i c k e r i n g and McWatters, however, reported o x y t o c i n from a South American specie s , L e p i d o s i r e n paradoxa (35). Sawyer and van Dyke a l s o found o x y t o c i n i n the A f r i c a n P. aethiopicus (36). Sawyer's specimens were c o l l e c t e d from a d i f f e r e n t lake than were those of H e l l e r . Sawyer i s a l s o reported to have found 8-i l e u o x y t o c i n i n another batch of glands from the 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 neurohypophysial hormones of Dipnoi were on a pharmacological b a s i s . Sawyer and van Dyke i n d i c a t e the presence of 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 the p r i m i t i v e b i c h i r , Polypterus (36). The c l a s s i f i c a t i o n of Polypterus adopted by d i f f e r e n t authors v a r i e s . Romer c l a s s i f i e d Polypterus w i t h the Chondrostei (37); Co l b e r t a l s o placed P o l y p t e r i n i w i t h i n the Chondrostei (38) ; N i k o l s k i i included them i n the A c t i n o p t e r y g i i , but separated them from Chondrostei (39); F o l l e t and H e l l e r separate them from the A c t i n o p t e r y g i i as B r a c h i o p t e r y g i i (40). 8 Present knowledge of the neurohypophysial hormones of the other Chondrostei r e l i e s e x c l u s i v e l y on the pharmaco-l o g i c a l data of F o l l e t and H e l l e r (40). Their s t u d i e s detected only one neurohypophysial hormone i n the p a d d l e f i s h , Polyodon spathula. On the other hand, they found two hor-mones i n three Acipenser species examined. One was t e n t a -t i v e l y i d e n t i f i e d as 8-arginine o x y t o c i n , a l s o present i n the p a d d l e f i s h . The second hormone remained u n i d e n t i f i e d since i t represented only a small 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 included 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 presence, i n a d d i t i o n to 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 chromato-g r a p h i c a l l y or pharmacologically from the t e l e o s t hormone, 4-ser, 8 - i l e u o x y t o c i n ( i s o t o c i n , i c h t h y o t o c i n ) ( 4 0 ) . (5) Neurohypophys1a1 Hormones of Teleost Fishes In t e l e o s t f i s h e s examined up to present two hormones have been found: 4-ser, 8 - i l e u o x y t o c i n , and 8-arg oxy-t o c i n . The 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 the 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 , P o l l a c h i u s v i r e h s . The f i r s t chemical data on the s t r u c t u r e of t h i s new hormone were published by Acher e t 'al. i n 1962 (42) . This group studied three marine species 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 Merluccius merluccius and b i b cod Gadus lusc u s . The s t r u c t u r e of 4-ser, 8 - i l e u o x y t o c i n was confirmed s h o r t l y a f t e r Acher's r e p o r t by the independent syntheses of Guttman (4) and J o h l (44). 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 the s y n t h e t i c standard (45). 8-Arg oxy 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 chemically i n the above studi e s of neurohypophysial hormones of marine Gadidae. In 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 reported from a f r e s h water t e l e o s t , Cyprinus c a r p i o (Family Cyprinidae) by Acher's group. The i d e n t i f i c a t i o n of both hormones was 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 to be as ubiquitous i n 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 exception of mammals (17) . S h o r t l y a f t e r t h i s peptide was f i r s t synthesized i n the l a b o r a t o r y (16), i t was i d e n t i f i e d 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) . The f i r s t amino a c i d analyses of 8-arg ox y t o c i n from t e l e o s t f i s h were reported i n 1961 from Gadus luscus by Acher (48) and by Rasmussen from Urophycis tenuis (49) , (50). Two s p e c i e s , P o l l a c h i u s v i r e n s and Merluccius merluccius have been used as sources f o r amino a c i d sequence studi e s reported by Acher (51). 10 Thus i t can be s a i d t h a t f i v e species of t e l e o s t f i s h have so f a r been examined on a chemical b a s i s w i t h respect to neurohypophysial hormones. These species are: P o l l a c h l u s v i r e n s , Merluccius merluccius, Gadus lu s c u s , Cyprinus c a r p i o and Urophycis tenuis (one hormone only i d e n t i f i e d from the l a t t e r species) as w e l l as Scombridae species r e f e r r e d to by Acher i n 1967 (32) . The pharmaco-l o g i c a l survey 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 species 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 , Cyprinus 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 of 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 survey are based on bioassays of f r a c t i o n s obtained by paper chromatography of p i t u i t a r y e x t r a c t s (40). In t h e i r survey of neurohypophysial 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 acologically, 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). In another survey, on bony f i s h e s and cyclostomes, the same group of workers pharmacologically 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) Present Study: Neurohypophys1a1 Hormones i n 'Oneorhync'h'us tschawytscha The p o s i t i o n of salmonid 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 salmoniform order 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 considers Salmoniformes as a p r i m i t i v e stock at the base of the e v o l u t i o n a r y l i n e 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 neurohypophysial hormones have been stud i e d p r e v i o u s l y (Gadidae, Cyprinidae and Scombridae) are p o s i t i o n e d by Romer moderately w e l l along the branches 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). Because of t h i s the c h a r a c t e r i z a t i o n of neurohypophysial hormones i n salmonids may be considered an important step 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 present 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 neurohypophysial hormones of the P a c i f i c chinook salmon, Oncorhynchus tschawytscha. Deter-mination of the s t r u c t u r e by chemical r a t h e r than pharmaco-l 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 , the method i s p o t e n t i a l l y l e s s ambiguous. Exhaustive chemical s t u d i e s of t e l e o s t neurohypophysial hormones have not been found i n the l i t e r a t u r e . This a l s o made i t d e s i -r a b l e to do a chemical study i n the present i n v e s t i g a t i o n . The study of amino a c i d composition and sequence of the two neurohypophysial hormones which have been i s o l a t e d i n the course of t h i s work from salmon was p o s s i b l e f o r two major reasons. F i r s t , a l a r g e number of p i t u i t a r y glands was a v a i l a b l e because of the s l a u g h t e r i n g of s e x u a l l y mature f i s h which accompanies hatchery 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 possible by such recent advances i n peptide sequencing methods as the Dansyl method of Gray and Hartley (54) and by the increased s e n s i t i v i t y of automatic amino acid analyzers which became available i n the course of the l a s t decade. I t was thus possible to determine the sequence of amino acids i n one to two micromoles of neurohypophysial hormones, which i n terms of weight represents one to two milligrams of p u r i f i e d material. The determination of amino acid sequence requires material of at least 90% pur i t y . In the present study an extraction and p u r i f i c a t i o n procedure on the preparative scale was developed with the aim of minimizing losses of material at each successive step of processing. Extraction methods, molecular exclusion, u l t r a f i l t r a t i o n and cation exchange techniques were extensively examined leading to a r e l a t i v e l y simple procedure for i s o l a t i o n of pure hormones i n good y i e l d s . 13 I I . EXPERIMENTAL PROCEDURES (1) C o l l e c t i o n of P i t u i t a r y Glands P i t u i t a r y glands of spawning Oncorhynchus tschawytscha were c o l l e c t e d at h a t c h e r i e s i n Washington State. The glands 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 during 1963, 1964, and 1965 at three l o c a t i o n s : Green River State Hatchery (near Auburn) and Spring Creek and L i t t l e White Federal Hatcheries (near Bingen). In 1966 and 1967 glands were c o l l e c t e d only at Green R i v e r . The c o l l e c t i o n took place i n September and October, the spawning season i n those areas. The f i s h were k i l l e d by hatchery workers and gonadal products removed. The s l a u g h t e r i n g techniques differed'.: from one hatchery to another. At Green River f i s h were k i l l e d by severance of the s p i n a l chord at the l e v e l of the operculum. At Spring 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. Using an e l e c t r i c borer, a c y l i n d r i c a l core was r e -moved from the head of the f i s h i n such a manner as to i n -clude the p i t u i t a r y glands. The d e t a i l s of the procedure have been described by Tsuyuki, Schmidt and Smith (53). Male and female cores were kept s e p a r a t e l y . The i n t a c t 14 glands were removed from i s o l a t e d cores using a pointed l a b o r a t o r y s p a t u l a . The glands were placed 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 kept 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 at Vancouver, B.C. In t h i s manner the glands were frozen at most w i t h i n two hours a f t e r the death of the f i s h . The length of storage 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 the s i t e and date of c o l l e c t i o n . In the e a r l i e r years (1963 to 1965) the glands remained i n s o l i d carbon d i o x i d e up to f i v e days, and upon a r r i v a l at the l a b o r a t o r y were stored i n l i q u i d n i t r o g e n (-196\u00C2\u00B0C). In subsequent years (1966 and 1967), the glands remained i n s o l i d carbon d i o x i d e f o r a maximum p e r i o d of twelve hours. Upon a r r i v a l i n Vancouver these glands were stored i n a -80\u00C2\u00B0C f r e e z e r (Revco, U l t r a Low Temperature). The number of glands c o l l e c t e d v a r i e d from year to year. About 7,800 glands were c o l l e c t e d i n 1966 and 3,900 glands i n 1967. This represented approximately 780 and 390 grams of t i s s u e , r e s p e c t i v e l y . (2) B i o l o g i c a l Assays The assays, based on c o n t r a c t i o n of r a t uterus i n presence of o x y t o c i c substances, were performed according to Holton (55) on v i r g i n Wistar l a b o r a t o r y r a t s , weighing from 180 to 230 gm. The stage of the estrous c y c l e was de-termined by microscopic examination of an unstained v a g i n a l smear, and only animals i n pro-estrus or estrus were used f o r the experiments (5). Rats were k i l l e d w i t h an overdose of ether, whole u t e r i excised and one horn used f o r the assay. The t i s s u e was suspended i n the buffered 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 of the u t e r i n e horn was anchored to a g l a s s a i r lead at the bottom of the t i s s u e bath, while the ovary was attached by means of a thread t o the arm of a kymograph (C.F. Palmer L t d . ) . The l e v e r arm of the kymograph was e i t h e r not weighted, or was weighted w i t h a piece of p l a s t i c i n e weighing up to 0.5 gm. The temperature of the jacketed g l a s s t i s s u e bath (volume 10 ml) was maintained constant at 35\u00C2\u00B0C by a c i r c u l a t i n g water pump (Haake, Model F ) . At the end of each c o n t r a c t i o n the e n t i r e s o l u t i o n was removed through an o u t l e t at the bottom of the t i s s u e bath using the s u c t i o n of a water a s p i r a t o r . The uterus was r i n s e d immediately w i t h Munsick's s o l u t i o n and then r e -suspended i n the same s o l u t i o n i n pr e p a r a t i o n f o r the next bioassay. Munsick's s o l u t i o n was dispensed from a p l a s t i c squeeze b o t t l e kept i n a water bath at 35\u00C2\u00B0C. The pH of both the t i s s u e bath and the stock Munsick's s o l u t i o n was maintained at 7.4 - 7.5 by constant bubbling of 5% CO2 i n a i r mixture. This a l s o served to mix the s o l u t i o n s i n the t i s s u e bath. Syntocinon-10 (Sandoz Pharmaceuticals) was used as a standard. 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 I Munsick's Buffered Tissue Bath S o l u t i o n (56) A. Stock s o l u t i o n : NaCl 120.67 gm (18 L) NaHCO-j 46.62 gm KCl 8.275 gm 0.02% phenol red 270 ml B. Phosphate b u f f e r , stock: (a) 22.714 gm Na 2HP0 4 d i l u t e d i n hot d i s t i l l e d water to 1,000 ml (b) 5.520 gm Nal^PO^ d i l u t e d as above (a) and (b) were mixed i n approximately equal q u a n t i t i e s u n t i l pH of 7.4 was obtained and the 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 ml of A For the sake of convenience the stock s o l u t i o n A was o f t e n made more concentrated ( i n three i n s t e a d 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 to 988 ml as re q u i r e d . 10 ml of B 1 ml of 0.5 M CaCl 500 mg glucose 17 Units of o x y t o c i n per ml was d i l u t e d to 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 to monitor f r a c t i o n a t i o n of molecular e x c l u s i o n and i o n ex-change 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 . Q u a n t i t a t i v e assays, however, were performed on a l l pooled 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 . Q u a n t i t a t i v e assays: four p o i n t assays were per-formed according to the method of Holton (55). When the l i m i t of the standard e r r o r was to be c a l c u l a t e d , f i v e to ten groups of four doses were used f o r each unknown. For a quick check of the 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 separations were to be defined) only one to three groups of four assays were performed. For assays of the o x y t o c i c e f f e c t of hormone prepar-a t i o n s i n the presence of magnesium i o n (57), magnesium c h l o r i d e was added to Munsick's s o l u t i o n i n order to make i t 0.5 mM w i t h respect to magnesium i o n . In a l l other r e s -pects, the assay was performed i n the i d e n t i c a l manner as the assay without 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 concentration was measured by the F o l i n Lowry method (58) and by absorbance at 280 nm. Absorbance at 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 of the same column measured by these three methods. I t can be seen th a t the e l u t i o n p r o f i l e based on 260 nm readings i s sharper, but otherwise 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 obtained 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 e l u t e d 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 not 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 of the sample f o r measurements. The amount of 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 at the s t a r t of the i s o l a t i o n procedure, but increases r a p i d l y as the p u r i f i c a t i o n progresses. The spectrophotometers used f o r measurements of absorbance i n the course of t h i s i n v e s t i g a t i o n were: Beckman models DU, DK and DB, and Unicam SP 820, s e r i e s 2. The l a t t e r instrument was used w i t h and without an expansion s c a l e . ( i i ) S p e c i f i c c o n d u c t i v i t y measurements S a l t concentration was monitored 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 purpose. The c e l l constant of the e l e c -trode was 0.5586. Thus the conductance measurements ob-tai n e d by d i r e c t reading were d i v i d e d by t h i s number to ob t a i n s p e c i f i c c o n d u c t i v i t y values. 19 A. B. C. 14. On Fraction No. Figure 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 three d i f f e r e n t methods\"! Loading sample: 10 ml, 600 mg Folin-Lowry peptide; 6 80 A ' - U -280 nm; 8 3 0 A\" U-260 nm* B i o 9 e l p \" 2 ' A. Folin-Lowry peptide, absorbance at 660 nm B. Absorbance at 280 nm C. Absorbance at 260 nm 20 (4) E x t r a c t i o n of Oxytocic 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 at 4\u00C2\u00B0C The 0.2 M a c e t i c a c i d and the glassware to be used f o r the e x t r a c t i o n were precooled. The e x t r a c t i o n was c a r r i e d out i n a c o l d room (temperature approximately 4\u00C2\u00B0C). Frozen glands (100 gm) were weighed q u i c k l y and then placed i n t o 0.2 M a c e t i c a c i d (1,000 ml, pH 2.6). The t i s s u e was di s r u p t e d immediately i n a Waring blender at maximum speed u n t i l the suspension was of even consistency. This re q u i r e d 0.5 to 1.0 minutes. Further d i s r u p t i o n of the t i s s u e was c a r r i e d out using an e l e c t r i c homogenizer of Potter-Elvehjem type (TRI-R STIR-R, Model S-63, s e t t i n g 5) f o r approximately one minute. The t i s s u e homogenate was then c e n t r i f u g e d at 48,200 g f o r 30 min at 0\u00C2\u00B0C, using a S e r v a l l RC 2B c e n t r i f u g e and SS-34 head. The supernatant l i q u i d was decanted and kept on i c e . The sediment was resuspended i n 100 - 200 ml of c o l d 0.2 M a c e t i c a c i d and the suspension c e n t r i f u g e d at 48,000 g f o r 45 to 60 min at 0\u00C2\u00B0C, using the S e r v a l l RC 2B c e n t r i f u g e and SS-34 head. The two supernatant s o l u t i o n s were pooled to y i e l d the 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 bioassay. An attempt to prepare the crude e x t r a c t by c e n t r i -f u g a t i o n at 27,300 g f o r 45 minutes at 0\u00C2\u00B0C, using the 21 S e r v a l l RC 2B c e n t r i f u g e and GSA head was not s u c c e s s f u l because the r e s u l t i n g crude e x t r a c t was opaque and req u i r e d r e - c e n t r i f u g i n g . The supernatant s o l u t i o n obtained by r e - e x t r a c t i o n of the sediment was bioassayed on s e v e r a l occasions, and found to c o n t a i n from 5.9 to 17.2% of the t o t a l a c t i v i t y of the e x t r a c t . A t h i r d e x t r a c t i o n of sediment d i d not y i e l d d e t e c t a b l e o x y t o c i c a c t i v i t y . ( i i ) E x t r a c t i o n at 100\u00C2\u00B0C i n 0.25% a c e t i c a c i d of acetone powder prepared from frozen p i t u i t a r i e s The acetone was d r i e d over calcium 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 of de s s i c a n t . The acetone was then cooled i n an i c e bath and the frozen p i t u i t a r i e s placed i n the acetone. Fol l o w i n g t h i s , the acetone was replaced three times at h a l f hour i n t e r v a l s , and once again s i x hours a f t e r the t h i r d change. The glands were then l e f t i n acetone at 4\u00C2\u00B0C f o r an a d d i t i o n a l 18 hours. At the end of t h i s p e r i o d the acetone was decanted and the glands 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 . Dry glands were homogenized i n 0.25% a c e t i c a c i d at 25\u00C2\u00B0C using 10 ml of e x t r a c t a n t per gm of wet t i s s u e . An e l e c t r i c homogenizer, 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 of the t i s -sue. The t i s s u e homogenate was then placed i n a b o i l i n g water bath f o r 3 minutes, cooled on i c e and c e n t r i f u g e d at 48,200 g f o r 30 min at 0\u00C2\u00B0C, using the S e r v a l l RC 2B c e n t r i -fuge and an SS-34 head. The supernatant l i q u i d was decanted and kept on i c e . The sediment was resuspended i n 20% of the o r i g i n a l e x t r a c t a n t volume, and the 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 supernatant s o l u t i o n s were pooled to y i e l d the 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. This e x t r a c t i o n procedure was intended to approximate the method reported by Kamm (59) and to compare the y i e l d obtained by Kamm's procedure w i t h the e x t r a c t i o n procedure developed f o r salmon p i t u i t a r i e s ( i ) . ( i i i ) 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 co n s i s t e d of v a r i a t i o n s on the two procedures described above. V a r i a t i o n s on the f i r s t e x t r a c t i o n procedure were as f o l l o w s : (a) Lengthening the time of e x t r a c t i o n . In these experiments the 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 p e r i o d of e x t r a c t i o n a t 4\u00C2\u00B0C or at 25\u00C2\u00B0C. (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 the e x t r a c t i o n of the 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 inconvenient to handle because the excess a c i d had to be removed by l y o p h y l i z a t i o n . This could only be done a f t e r d i l u t i o n w i t h water to approxi-mately 1 M a c e t i c a c i d . Such d i l u t i o n produced lar 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 consequently consumed a great d e a l 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 r e q u i r e d the removal of excess a c i d by l y o p h y l i z a t i o n , but the d i l u t i o n r e q u i r e d f o r t h i s process was two-fold r a t h e r than approxi-mately s i x t e e n - f o l d as i n the case of g l a c i a l a c e t i c a c i d . (c) Omission of the Potter-Elvehjem homogenized step from the e x t r a c t i o n procedure, the d i s r u p t i o n being c a r r i e d out using Waring blender only. (d) Use of 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 i n v o l v e d f r e e z e - d r y i n g of whole frozen glands. L y o p h y l i z a t i o n f a c i l i -t a t e d the t i s s u e d i s r u p t i o n step. The only v a r i a n t on e x t r a c t i o n procedure ( i i ) con-s i s t e d i n omission of the acetone powder step and subsequent e x t r a c t i o n i n b o i l i n g water bath f o r 5 minutes r a t h e r than 3 minutes. The timing began when the temperature of the homogenate reached a plateau at approximately 92\u00C2\u00B0C. The e x t r a c t i o n procedures used i n the course of t h i s study and the y i e l d s of o x y t o c i c a c t i v i t y obtained are summarized i n Table I I I , p. 52-53. (5) Concentration of the Crude and P a r t i a l l y P u r i f i e d E x t r a c t s Crude e x t r a c t s d i d not have to be concentrated i n large s c a l e (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 (cf. Figure 7). P r i o r to t h i s , crude e x t r a c t was concentrated to remove excess a c i d and to reduce the volume o f the sample f o r f u r t h e r p u r i f i c a t i o n s t e p s . C o n c e n t r a t i o n o f 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 , be -cause p r e c i p i t a t i o n o f m a t e r i a l took p l a c e above the membrane and s u b s t a n t i a l l y reduced the f low r a t e . U l t r a f i l t r a t i o n o f p a r t i a l l y p u r i f i e d e x t r a c t s i n o r d e r to c o n c e n t r a t e and to d e - s a l t them was c a r r i e d out r o u t i n e l y . U l t r a f i l t r a t i o n i s d i s c u s s e d i n s e c t i o n 6 of t h i s c h a p t e r . C o n c e n t r a t i o n and removal o f excess a c i d by l y o p h y l i -z a t i o n was done r e p e a t e d l y i n the course o f i s o l a t i o n p r o -c e d u r e . A Thermovac I n d u s t r i e s C o r p . f r e e z e d r y e r and r o u n d -bottom l y o p h y l i z a t i o n f l a s k was used f o r t h i s p u r p o s e . Losses o f hormonal a c t i v i t y were not observed i n the course o f t h i s l y o p h y l i z a t i o n p r o c e d u r e . I t was e s s e n t i a l t o the r e c o v e r y of the hormones, however, t h a t a l l o f the d r y m a t e r i a l i n the f l a s k be brought i n t o s o l u t i o n by r e p e a t e d r i n s i n g o f the f l a s k . (6) U l t r a f i l t r a t i o n The D i a - f l o u l t r a f i l t r a t i o n apparatus i s d e s i g n e d to c a r r y out f a s t s e l e c t i v e f i l t r a t i o n o f aqueous s o l u t i o n s . The h i g h r a t e i s a c h i e v e d 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 to the chamber c o n t a i n i n g the sample , and the s e l e c t i v i t y i s i n t r o d u c e d by membranes o f v a r y i n g pore s i z e s through which the s o l u t i o n i s f i l t e r e d . Three types of membranes were a v a i l a b l e : UM-1, UM-2, and UM-3, w i t h molecular weight c u t - o f f ranges of 10,000, 1,000, and 350, r e s p e c t i v e l y . The molecular weights of neurohypophysial hormones are i n the 1,000 range, while the molecular weight of 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 to concentrate a s o l u t i o n of neurohypophysial hormones. The u l t r a f i l t r a t i o n apparatus was used according to the d i r e c t i o n s of the manufacturer (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. Pressure a p p l i e d was 80 p . s . i . and flow r a t e s ranging from 1.2 to 2.0 ml/min were obtained. By v a r y i n g the pressure a p p l i e d and the concen-t r a t i o n of the s o l u t i o n i n the chamber, i t was observed that the flow r a t e appeared to depend on the conce n t r a t i o n of the s o l u t e i n the chamber r a t h e r than on the pressure a p p l i e d to tha t chamber. In order to minimize l o s s e s of hormone during u l t r a -f i l t r a t i o n , a compromise was reached between lowering of 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 concentrating the sample above the u l t r a f i l -t r a t i o n membrane and thereby reducing the s a l t content of the sample by a f a c t o r dependent on the r a t i o between the s t a r t i n g volume and t h a t of the f i n a l concentrate (10 to 15 ml). The t o t a l s a l t content was thereby reduced, but u s u a l l y not s u f f i c i e n t l y to permit c a t i o n exchange chromatography. The s o l u t i o n was adjusted l a t e r to the d e s i r e d conductance f o r l oading the 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 of the g e l and column packing Two types of g e l f i l t r a t i o n media were used i n the course of these experiments: c r o s s l i n k e d dextran (Sephadex, Pharmacia) and c r o s s l i n k e d polyacrylamide ( 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 respect to s w e l l i n g and i n packing of the column. The treatment d i f f e r e d s l i g h t l y only at one p o i n t . More care had to be e x e r c i s e d i n sus-pending the dry B i o g e l because i t tended to form l a r g e aggregates. Consequently the suspension had to be s t i r r e d v i g o r o u s l y and the dry g e l added slowly. The s w e l l i n g u s u a l l y was allowed to take place over-n i g h t or f o r twenty-four hours, and f i n i n g of the g e l was accomplished during t h a t time. The s w e l l i n g and packing was done i n the same m o l a r i t y of a c e t i c a c i d i n which the column was to be run. O c c a s i o n a l l y the Sephadex g e l was swollen by the f a s t method recommended by the manufacturer; f o r one hour i n a 60\u00C2\u00B0C bath (61). Small columns ( i . e . 200 mm x 12 mm diameter) were packed by pouring the 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 the pouring began and the o u t l e t was opened s l i g h t l y when the bed was packed to a depth of 2 0 to 30 mm. Larger columns (25 to 50 mm i n diameter) were packed using a funnel i n which the g e l sus-pension was continuously s t i r r e d . A l a r g e (500 ml) g l a s s funnel w i t h a short wide stem was i n s e r t e d i n t o a rubber stopper f i t t e d to the top of the column. An e l e c t r i c r o t a r y blade s t i r r e r (Gerald T. H e l l e r Company, GT 21) was used to s t i r the suspension i n the f u n n e l . The funnel was kept f i l l e d w i t h g e l s l u r r y . When the column bed reached the d e s i r e d height the funnel was removed and a p e r l o n d i s c placed on top of the g e l bed. The column was washed wi t h two bed volumes of eluant and was ready to use. The packing of a l l columns was c a r r i e d out at 25\u00C2\u00B0C. Blue Dextran (Pharmacia) s o l u t i o n was run through newly packed Sephadex columns to monitor the q u a l i t y of packing. Blue Dextran could not be used w i t h B i o g e l , since i t was r e t a i n e d by the g e l . Sephadex G-10 Palso tended to spread the Blue Dextran band. On G-15, however, Blue Dextran behaved as described by the manufacturer (62). ( i i ) Loading and e l u t i o n of g e l f i l t r a t i o n columns Experiments were g e n e r a l l y c a r r i e d out at 25\u00C2\u00B0C. 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 preparations were loaded i n the appropriate volume and then washed i n t o ti^gelswifn3 0..5 to,* li0jv0.i/ ml of b u f f e r . 28 (10 ml was used f o r 450 mm x 50 mm diameter columns). The column was then e l u t e d w i t h the 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 mechanical 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 t i m e r ) . In order to process a larg e volume of e x t r a c t (approximately 1 l i t e r ) , a Sephadex G-15 column (450 mm x 50 mm diameter) was used repeatedly to f r a c t i o n a t e 225 ml batches of the e x t r a c t (tandem g e l f i l t r a t i o n ) . When t h i s technique was employed a f r e s h sample of e x t r a c t was app l i e d to the column as soon as the b i o l o g i c a l l y a c t i v e m a t e r i a l 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 run was between 20 and 25 hours. The absorbance of f r a c t i o n s at 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 pooled and e i t h e r processed f u r t h e r immediately or stored at 5\u00C2\u00B0C. Packed Sephadex columns were stored at 25\u00C2\u00B0C w i t h 0.02% sodium azide a f t e r washing w i t h s e v e r a l bed volumes of b u f f e r . (8) Ion Exchange Chromatography (i) P r e p a r a t i o n of the exchangers and column packing P r e c y c l i n g of Whatman CM-32 and of Whatman CM-22 was c a r r i e d out according to the d i r e c t i o n s of the manufacturers (63). U s u a l l y the amount precy c l e d was about 30% more than r e q u i r e d by the column. The dry exchanger was s t i r r e d i n t o 0.5 N sodium hydroxide (15 ml per gm of exchanger) and l e f t f o r 30 minutes. At the end of t h i s p e r i o d the l i q u i d was decanted and the exchanger washed wi t h d i s t i l l e d water i n a 500 ml Buchner funnel under s u c t i o n u n t i l the washings were at pH 8. The exchanger was then s t i r r e d i n t o 0.5 N hydro-c h l o r i c a c i d (15 ml per gm of exchanger) and again l e f t f o r 30 minutes. The l i q u i d was decanted and the exchanger washed on a Buchner funnel u n t i l the 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 treatment was repeated once. E q u i l i -b r a t i o n w i t h the appropriate b u f f e r was c a r r i e d out at 2M i o n i c strength and the c o r r e c t pH value (pH 5). I t was found convenient to c a r r y out t h i s step i n a 500 ml graduated c y l i n d e r . The exchanger was s t i r r e d w i t h the b u f f e r and allowed to s e t t l e f o r 10 minutes. At 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 r e f i l l e d 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 times and again allowed to s e t t l e . This step was repeated 7 to 8 times. The conduc-tance and pH of the 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 the s t a r t i n g b u f f e r and allowed to s e t t l e f o r 30 minutes. The l i q u i d was removed by s u c t i o n and the step repeated once. Most of the removal of f i n e p a r t i c l e s was accomplished during the e q u i l i b r a t i o n step. A t h i c k s l u r r y of the prepared ex-changer (approximately 20% w/v) was degassed using a water 30 pump. I t was found to be more convenient to prepare a t h i c k s l u r r y and pour i t i n t o the column i n one pass, because the flow r a t e of t h i s exchanger i s low and packing of the column would take too long when a t h i n s l u r r y was used. For the same reason i t was found d e s i r a b l e to have the s t a r t i n g b u f f e r to a depth of only 20 to 30 mm at the bottom of the column p r i o r to pouring. The exchanger was allowed to s e t t l e to a height of about 20 mm before the o u t l e t was opened. The column was u s u a l l y packed i n the l a t e a f t e r -noon and l e f t to wash wi t h s t a r t i n g b u f f e r overnight. By morning complete e q u i l i b r a t i o n was achieved w i t h regard to both c o n d u c t i v i t y and pH. Phosphocellulose ( S e l e c t a c e l ) d i d not r e q u i r e s p e c i a l p r e c y c l i n g . 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 the column i n the s t a r t i n g 0.002 M) b u f f e r . Because of the f i b r o u s nature of the exchanger i t was packed i n a t h i n s l u r r y . The column was e q u i l i b r a t e d overnight w i t h the s t a r t i n g b u f f e r . SE-Sephadex C-25 (Pharmacia) was precy c l e d according to the d i r e c t i o n s of the manufacturer (64). The dry ex-changer (30 gm) swelled to a volume of 300 ml while soaking i n water. No appreciable shrinkage was n o t i c e d as the column was packed. The exchanger was allowed to s w e l l i n water f o r at 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 funnel w i t h 500 ml of 0.5 N sodium hydroxide followed w i t h 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. E q u i l i b r a t i o n was c a r r i e d out i n 2 M b u f f e r of d e s i r e d pH. Several changes of b u f f e r were re 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 overnight as i n the p r e p a r a t i o n of phosphocellulose columns. A l l c a t i o n 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 at 25\u00C2\u00B0C. ( i i ) B u f f e r s used i n c a t i o n exchange Sodium acetate, ammonium acetate, and ammonium formate b u f f e r s were made by t i t r a t i o n of the a c i d at d e s i r e d molar-i t y w i t h the appropriate base to the re q u i r e d pH. A Radiometer 26 pH meter was used f o r the t i t r a t i o n . A 2 M stock s o l u t i o n was f r e s h l y made before use and lower i o n i c s trength b u f f e r s obtained by d i l u t i o n . In ammonium formate b u f f e r s , the pH had to be adjusted w i t h formic a c i d f o l l o w i n g d i l u t i o n . ( i i i ) Loading and e l u t i o n of c a t i o n exchange columns Loading and e l u t i o n were conducted at 25\u00C2\u00B0C. The f r a c t i o n s were c o l l e c t e d using the G i l s o n f r a c t i o n c o l l e c t o r . U s u a l l y the sample was loaded onto the column from a r e s e r -v o i r and the c o l l e c t i o n of 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 of the loading sample was adjusted by d i l u t i o n w i t h d i s t i l l e d water to make i t i l o w e r than the c o n d u c t i v i t y needed to e l u t e Hormone I . A f t e r a l l of the sample p e r c o l a t e d i n t o the column bed, the w a l l s of the column above the 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 of s t a r t i n g b u f f e r and the r i n s i n g s allowed to pass i n t o the bed. The column was washed wi t h s t a r t i n g b u f f e r u n t i l the u l t r a v i o l e t absorbance of the eluate de-creased. 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 out using a gradient forming system w i t h equal volumes of appropriate b u f f e r s i n a mixing chamber and i n a r e s e r v o i r of equal dimensions (65). In most cases, bioassay and the 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 the column. The gra d i e n t was checked by measuring the conduc-tance of the f r a c t i o n s . (iv) Repeated use of the same c a t i o n column Unless i t could be re-used immediately, the exchanger was discarded a f t e r use. On a few occasions a l l three ex-changers were re-used w i t h i n a day or two of the f i r s t run. 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 the column wi t h high i o n i c strength (1 - 2 M) b u f f e r followed by a two to three l i t e r wash wi t h the s t a r t i n g b u f f e r . SE-Sephadex, phosphocellulose, and Whatman CM-22 could be re-used without re-packing. Whatman CM-32 req u i r e d re-packing because of decreasing flow r a t e . Phosphocellulose i n wet form was never kept longer 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 A c i d O x i d a t i o n Cystine and c y s t e i n e residues were converted to c y s t e i c a c i d residues by performic o x i d a t i o n (66). C y s t e i c a c i d gives b e t t e r q u a n t i t a t i v e data than c y s t e i n e on the automatic amino a c i d analyser. 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 bridge i n neurohypophysial hormones thus f a c i l i t a t i n g sequence deter-mination . The sample of peptide intended f o r o x i d a t i o n , the f r e s h l y prepared performic 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 copied i n i c e to 0\u00C2\u00B0C. Performic a c i d was added to the peptide sample, the t e s t tube stoppered w i t h a ground gla s s stopper and the r e a c t i o n allowed to proceed at 0\u00C2\u00B0C f o r two to three hours. Excess reagents were removed by l y o p h y l i z a t i o n . The f i r s t l y o p h y l i z a t i o n was c a r r i e d out u n t i l no peroxide could be detected 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 oxidant was used, i . e . 0.2 ml of performic a c i d to 0.05 ymoles of peptide. Performic a c i d was prepared by mixing 88% formic a c i d and 30% hydrogen peroxide i n 9 : 1 proportions and al l o w i n g the mixture to stand at room temperature f o r one hour. A mixture of formic a c i d to peroxide (9.5 : 0.5) warmed f o r f i v e minutes i n a 50\u00C2\u00B0C bath has a l s o been used and found s a t i s f a c t o r y . (10) A c i d H y d r o l y s i s of Peptides T o t a l h y d r o l y s i s of the sample was re q u i r e d i n three types of experiments: (1) amino a c i d a n a l y s i s of the whole molecule; (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 of amino a c i d sequence determination; and (3) 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 of N-terminal residues of whole or p a r t i a l l y degraded hormones. The sample to be analyzed was placed 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 pentoxide and sodium hydroxide. Kimax c u l t u r e tubes, 10 x 75 mm, were used f o r t o t a l hydrolyses and f o r s u b t r a c t i v e Edmans. Hy d r o l y s i s of dansylated peptides was c a r r i e d out i n the 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 (20 u l f o r 0.01 ymole of peptide) was added to the d r i e d sample and the t e s t tube p u l l e d out i n a hot flame to form a 1 - 2 mm diameter neck i n the middle. The hot g l a s s was cooled i n a stream of a i r or at room temperature, and the contents frozen i n an acetone and s o l i d carbon d i o x i d e bath. The mouth of the tube was then attached to an o i l vacuum pump and the tube evacuated. The s o l u t i o n was allowed to melt and evacuation continued u n t i l bubbling ceased. When bubbling was too in t e n s e , as was the case w i t h the hydro-l y z a t e s of dansylated peptides, i t was c o n t r o l l e d by repeated f r e e z i n g and thawing of the sample. The tube was sealed under vacuum using a small flame. 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 temper-ature heating block (Temp-Blok Module Heater, Lab-Line Instruments) which was covered w i t h s e v e r a l thicknesses of aluminium f o i l w i t h a hole f o r a thermometer stem. The h y d r o l y s i s was allowed to proceed f o r 16 hours at 105\u00C2\u00B0C, unless otherwise i n d i c a t e d i n the t e x t . F o l l o w i n g h y d r o l y s i s , the sealed tube was opened and the h y d r o c h l o r i c a c i d evaporated under vacuum over sodium hydroxide p e l l e t s . D e s s i c a t i o n was allowed to proceed longer than would be warranted f o r the appearance of the sample i n order t o remove a l l traces of 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 , the sample was l e f t under vacuum f o r 1.5 hours. The dry sample was then ready f o r the next step; e i t h e r amino a c i d a n a l y s i s or t h i n l a y e r chromatography. I f the next step was not done immediately the sample was stored at -20\u00C2\u00B0C. (11) Amino A c i d Analyses On occasion during t h i s i n v e s t i g a t i o n , three models of amino a c i d analyzer were used. They were a Beckman Model 120, a Technicon analyzer, and a Beckman Model 120 C, equipped wi t h an i n t e g r a t o r . The bulk of t o t a l hydrblyzates and a l l of the s u b t r a c t i v e Edman r e s u l t s were analyzed on the Beckman Model 120 C, and the experimental procedure described i s f o r t h i s model. (i ) Procedure The sample c o n t a i n i n g 0.01 to 0.03 umoles of peptide was d i s s o l v e d i n 100 or 200 y l of pH 2.2 sodium c i t r a t e b u f f e r . When only the long column was to be used, as i n sequencing of Hormone I , the sample was d i s s o l v e d i n 100 y l and the t o t a l amount a p p l i e d to the column. When the short column was to be run i n a d d i t i o n to the long column, the sample was d i s s o l v e d i n 200 y l of b u f f e r and 100 y l of s o l u t i o n was ap p l i e d to each column. The long column on Beckman 120 C i s used f o r r e s o l u t i o n of a c i d i c and n e u t r a l amino a c i d s ; the short column i s used f o r r e s o l u t i o n of b a s i c r e s i d u e s . The run was performed according to the d i r e c t i o n s of the manufacturer (67) , (68) . The sample was ap p l i e d using n i t r o g e n pressure and the w a l l s of the column r i n s e d twice w i t h the s t a r t i n g b u f f e r (pH 3.52 or pH 5.18). The space above the r e s i n was f i l l e d to the top of the column wi t h the s t a r t i n g b u f f e r , the lead from the appropriate pump attached 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 acids was c a r r i e d out w i t h a pH 5.18 sodium c i t r a t e b u f f e r and the time r e q u i r e d was 50 minutes. The e l u t i o n of a c i d i c and n e u t r a l amino acids 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 of the l a t t e r group. The b u f f e r change was from the s t a r t i n g b u f f e r to pH 4.25 b u f f e r and was set to begin a u t o m a t i c a l l y 25 to 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 length of the column used. 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 long column run was 125 to 130 minutes. ( i i ) 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 values f o r each amino a c i d were e i t h e r read o f f the i n t e g r a t o r tape, or c a l c u l a t e d by m u l t i p l y i n g the net height of each peak by i t s width. Q u a n t i t a t i o n f o r p r o l i n e was obtained from the 440 nm recorder t r a c i n g , while the 570 nm l i n e was used f o r i n t e g r a t i o n of a l l other amino a c i d s . The numerical value obtained above was d i v i d e d by the constants obtained from a standard amino a c i d run to give values f o r the amounts of amino acids i n micromoles. In order to transpose the number of micromoles i n t o residues per mole, a l l values f o r amino acids found i n approximately equal amounts i n the molecule were averaged and the average was equated w i t h one residue per mole. 38 To express the s p e c i f i c a c t i v i t y of p u r i f i e d hor-mones i n terms of o x y t o c i c u n i t s per m i l l i g r a m hormone, the t o t a l number of o x y t o c i c u n i t s contained 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 the average value of micromoles obtained from t h i s a n a l y s i s and then d i v i d e d by the number of m i l l i g r a m s equal to one micromole of the hormone (Tables V I I and V I I I ) . (12) P a r t i a l A c i d H y d r o l y s i s The method of p a r t i a l a c i d h y d r o l y s i s described by CM. Tsung and Fraenkel-Conrat (69) was followed. This method i s intended to 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 of aspar-t i c a c i d (and asparaginyl) r e s i d u e s . The peptide 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 to a f i n a l c oncentration of 1 mg/ml and hydrolyzed at 105\u00C2\u00B0C i n a sealed evacuated tube. The h a l f time f o r h y d r o l y s i s of a s p a r t i c a c i d r e s i -dues i s reported to be 5.5 hours and of asparagine residues 11 hours. In c o n t r o l experiments i t was found th a t 22 hour h y d r o l y s i s of 3-phe, 8-lys o x y t o c i n r e s u l t e d i n s i x fragments i n a d d i t i o n to a s p a r t i c a c i d . For Hormone I , an 18 hour h y d r o l y s i s was used. (13) High Voltage E l e c t r o p h o r e s i s High voltage e l e c t r o p h o r e s i s was used to monitor the p u r i t y of hormone preparations and to i d e n t i f y peptide f r a g -ments obtained 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 or as a r e s u l t of Edman degradation c y c l e s . The experiments were c a r r i e d out on Whatman 3 MM paper at e i t h e r pH 6.5 ( p y r i -dine : a c e t i c a c i d : water; 25 : 1 : 225) or pH 3.6 (py r i d i n e : a c e t i c a c i d : water; 1 : 10 : 289) . E i t h e r a f l a t - p l a t e apparatus (Savant Instruments) or a v e r t i c a l l i q u i d cooled apparatus (Michl-Ryle type) equipped w i t h a high v o l t a g e D.C. power supply (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 separations. Reference amino acids (6.5 umoles/ml i n 0.1 M sodium bicarbonate) were each a p p l i e d at the o r i g i n i n a streak 5 mm long. Peptide 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 streak 20 - 25 mm long at the o r i g i n i n such a manner th a t about one f i f t h of the peptide band could be cut o f f and st a i n e d along w i t h amino a c i d markers, wh i l e the remaining peptide area(s) could 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 , the o r i g i n was d r i e d i n a stream of warm a i r , and the paper wetted w i t h b u f f e r using appropriate precautions to prevent movement or d i f f u s i o n of substances at the o r i g i n . F o l l o w i n g the 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 , the paper was d r i e d and the edge bearing the amino a c i d markers and some of the unknown was cut 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 to use from 1% n i n h y d r i n i n acetone, 15 mis, and 85 mis of a s o l u t i o n of cadmium acetate (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 ml). 40 Areas marked f o r e l u t i o n on the unstained s t r i p were cut out and the e l u t i o n c a r r i e d out using the method of Heppel (70) . The v e r t i c a l M i c h l - R y l e type high voltage apparatus was found to be p r e f e r a b l e to the f l a t p l a t e type i n peptide work because considerable spreading was observed w i t h the l a t t e r apparatus. (14) 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 of two steps: formation of a phenylthiocarbamyl peptide (PTC-peptide) and s p l i t t i n g o f f of the N-terminal 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 shortening of the peptide chain by one amino a c i d r e s i d u e . Edman i n 1950 described the a p p l i c a t i o n of t h i s r e a c t i o n to the determination of amino a c i d sequence i n p r o t e i n s (71). The method used i n the present work was tha t of Black, Kauffman and Dixon (72) w i t h some m o d i f i c a t i o n s . The peptide (0.1 ymole) was d i s s o l v e d i n 200 y l of 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 graduated 15 ml c e n t r i f u g e tube equipped w i t h a g l a s s stopper. 25 y l of N-ethyl morpholine ( d i s t i l l e d ) and 10 y l of phenyliso-. thiocyanate ( d i s t i l l e d ) were added, the tube flushed w i t h n i t r o g e n , stoppered, and the coupling r e a c t i o n allowed to proceed f o r 3 hours at 37\u00C2\u00B0C. 41 At the end of t h i s time, 2 drops of de- i o n i z e d water were added and the mixture e x t r a c t e d w i t h an equal volume of benzene to remove excess reagents. I n i t i a l l y , the benzene e x t r a c t i o n step was repeated three times, but lo s s e s of PTC-peptide were observed, and the benzene e x t r a c t i o n step was t h e r e a f t e r only done once on each sample. Benzene ex-t r a c t i o n could be omitted a l t o g e t h e r when only the s u b t r a c t i v e Edman method was used i n amino a c i d sequence determination. The aqueous phase was d r i e d i n a vacuum d e s s i c a t o r overnight. T r i f l u o r o a c e t i c a c i d (0.1 ml) was added to the dry residue and the cleavage r e a c t i o n allowed to proceed at 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 at 50\u00C2\u00B0C f o r 30 minutes or at 25\u00C2\u00B0C f o r 90 minutes. The dry residue was i n i t i a l l y d i s s o l v e d i n water and ex t r a c t e d three times with 1 ml of b u t y l acetate. Because los s e s of peptide m a t e r i a l were observed to occur, the ex-t r a c t i o n w i t h b u t y l acetate was performed once on the dry residue i n subsequent experiments using 0.5 ml of b u t y l acetate. Excess b u t y l acetate was removed i n a vacuum d e s i c -c a t o r and the residue r e d i s s o l v e d i n 5'0%=&pyridine ( d i s t i l l e d ) i n water. (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 of the r e s i d u a l peptide at the end of each de-gradation step described i n s e c t i o n 14 of t h i s chapter. A s e r i e s of amino a c i d analyses was thus obtained showing a g r a d u a l l y decreasing peptide chain as the successive amino acids were removed from the N-terminus by the Edman degrada-t i o n r e a c t i o n . The next Edman degradation c y c l e was not begun u n t i l the r e s u l t s of the previous c y c l e were obtained from amino a c i d a n a l y s i s . I t was found t h a t the sequence of four to f i v e N-terminal residues could be defined by the s u b t r a c t i v e Edman method. (16) Dansylation Method i n Amino A c i d Sequence Determination While the 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 to e s t a b l i s h which amino a c i d was removed, and hence which was the 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 of each successive N-terminus as i t becomes exposed f o l l o w i n g an Edman degradation c y c l e (54). Dansyl c h l o r i d e (l-dimethylaminonaphthalene-5-sulfonyl c h l o r i d e ) i s reacted w i t h an a l i q u o t of the peptide f o l l o w i n g each Edman degradation c y c l e to form a dansylated peptide (dansyl c h l o r i d e r e a c t s not only with alpha amino groups, but a l s o w i t h the e p s i l o n amino group of l y s i n e and w i t h the phenolic hydroxy1 groups). Dansylated peptide i s then hydrolyzed and dansylated amino acids i d e n t i f i e d . The method followed i n the present work i s approximately t h a t described by Black, Kauffman and Dixon (72). (i ) D a nsylation 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 of peptide was r e -moved from the pyridine-water s o l u t i o n of the peptide at the end of each Edman degradation c y c l e ( s e c t i o n 1'A of t h i s c h a p t e r ) . The a l i q u o t was placed 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 pentoxide and sodium hydroxide. 20 y l of sodium bicarbonate was added and the peptide d r i e d again. 10 y l of de- i o n i z e d water was added to the dry peptide and the pH of the r e s u l t i n g s o l u -t i o n checked w i t h :pir:; ': paper. I f the pH was l e s s than 8, the step was repeated. 10 y l of dansyl c h l o r i d e (3 mg/ml i n acetone) was added to the aqueous s o l u t i o n of peptide, the tube sealed w i t h P a r a f i l m , and the r e a c t i o n allowed to pro-ceed at 37\u00C2\u00B0C for.1.5 to 3 hours, or u n t i l the yellow colour 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 hydrolyzed 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 twice) f o r 16 hours at 105\u00C2\u00B0C a f t e r s e a l i n g the tube under vacuum, as described i n s e c t i o n 9 of t h i s chapter. A sh o r t e r h y d r o l y s i s time (6 hours) was used where a dansyl-p r o l i n e residue was suspected, since d a n s y l - p r o l i n e i s destroyed by prolonged a c i d h y d r o l y s i s . ( i i ) I d e n t i f i c a t i o n of dansyl amino acids by t h i n l a y e r chromatography Dansyl amino a c i d standards were prepared by the method of Boulton and Bush (73) and checked against s t a n -dards purchased from Calbiochem. Both sets of standards were used. The commercially obtained standards were f r e e from dansyl s u l f o n i c a c i d . The l a t t e r provided a u s e f u l reference compound i n some t h i n l a y e r systems, while i n others i t was found to obscure the 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 valu 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 of dansyl amino acids (72): 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 : water; 80 : 20). Systems A and B separated most of the dansyl d e r i v a t i v e s of amino acids present i n neurohypophysi hormones, whi l e system C was used to improve the separation of DNS-aspartic and DNS-cysteic a c i d s . Because i t i s b a s i c System B enhanced fluorescence and was an e x c e l l e n t medium to b r i n g out weak fluorescence of some peptide d e r i v a t i v e s . 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 of water) using the g l a s s a p p l i c a t o r rod according to the d i r e c t i o n s of the manufacturer (74). A f t e r d r y i n g s u f f i c i e n t l y to tu 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 to an 85\u00C2\u00B0C oven f o r one hour and then stored i n a d e s s i c a t o r over calcium c h l o r i d e . P r i o r to s p o t t i n g , the p l a t e was d i v i d e d i n t o 7 - 1 0 mm channels using a sharp object to mark the coating on the p l a t e . The dry sample of dansylated peptide hydrolyzate was d i s s o l v e d i n 1 H ammonium hydroxide (5 y l ) and spotted repeatedly u n t i l a l l fluorescence was t r a n s f e r r e d from the t e s t tube as observed i n the U.V. viewer (Chromato-Vue, U l t r a v i o l e t Products L t d . , San G a b r i e l , C a l i f . ) . The standard s o l u t i o n s were such t h a t one to two a p p l i c a t i o n s y i e l d e d a spot of s u i t a b l e i n t e n s i t y . Small compact spots improved sepa r a t i o n . For t h i s reason, a t h i n c a p i l l a r y tube (1 mm i n outside diameter) was used f o r a p p l i c a t i o n , and the spot d r i e d immediately i n a current of 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 the solvent system. A f r e s h s o l v e n t mixture was used f o r each run. 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 spotted and chromatographed simultaneously i n two s o l v e n t systems. When the amount of m a t e r i a l was l i m i t e d , the same p l a t e was re-run 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 p e r i o d . When the spots to be i d e n t i f i e d were slow running i n the f i r s t s o lvent system, the 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 spots were f a s t running i n the f i r s t system, i t was found convenient to reverse the p l a t e and to do the second chromatography i n the opposite d i r e c t i o n . Chromato-graphy i n system A r e q u i r e d approximately 1 hour; i n B, 2-1/2 hours; and i n C, 3-1/2 hours. Fol l o w i n g chromatography, the p l a t e s were d r i e d and photographed. The photography was done using a P o l a r o i d Model 160 camera equipped w i t h a copy lens and U.V. f i l t e r ( T i f f e n Photar, U.V., 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 aperture 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 source ( U l t r a v i o l e t Products, San G a b r i e l , C a l i f . ) . 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 propanol : ammonia to 45 seconds f o r the p l a t e s run i n chloroform : 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 f l u o r e s c e n c e . The development of the photograph was allowed to proceed f o r 10 seconds. 47 I I I . RESULTS A. 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 t h e 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 s c h a w y t s c h a (1) 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 D u r i n g S t o r a g e o f Glands a t -196\u00C2\u00B0C 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 g l a n d s s t o r e d i n l i q u i d n i t r o g e n (-196\u00C2\u00B0C) f o r p e r i o d s r a n g i n g from two t o t w e n t y - s i x months was su r v e y e d i n o r d e r t o dete r m i n e which group o f g l a n d s would g i v e t h e b e s t y i e l d o f n e u r o h y p o p h y s i a l hormones. I n t h e same ex p e r i m e n t a comparison o f g l a n d s 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 were made. A p p r o x i -m a t e l y one gram o f each 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 manner, u s i n g 2.5 ml o f 0.25% a c e t i c a c i d p e r gram and f o l -l o w i n g t h e p r o c e d u r e 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 method o f H o l t o n (55). The time i n t e r v a l between t h e e x t r a c t i o n o f t h e g l a n d s and t h e performance o f the b i o a s s a y was i d e n t i c a l . 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 o b s e r v e d were m i n o r , c o n s i d e r i n g t h a t t h e average e r r o r o f t h e b i o a s s a y was 10%. A l l p r e p a r a t i v e e x t r a c t s were from p i t u i t a r i e s c o l l e c t e d a t Green R i v e r H a t c h e r y . O x y t o c i c a c t i v i t y s u r -48 T A B L E I I 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 tschawytscha c o l l e c t e d at Three D i f f e r e n t Locations S i t e of C o l l e c t i o n Months Stored at -196\u00C2\u00B0C S p e c i f i c A c t i v i t y L i m i t of S e x Oxytocic u n i t s E r r o r of per A.U. 2 8 0 ^ Bioassay L i t t l e White 26 L i t t l e White 14 L i t t l e White 2 Spring Creek 2 Green Ri v e r 2 Green Ri v e r 2 female female female female female male 0.0437 0.0471 0.0463 0.0582 0.0593 0.0512 6.0 10.9 7.7 12.3 14.3 8.1 Green Ri v e r Hatchery ( S t a t e ) , near Auburn, Wash. Spring Creek and L i t t l e White Hatcheries (Federal), near Bingen, Wash. 49 v i v e d extended c o l d storage of the i n t a c t glands. This method of p r e s e r v a t i o n at -196\u00C2\u00B0C, or a l t e r n a t i v e l y , at -80\u00C2\u00B0C, was adopted as the standard procedure. (2) S t a b i l i t y of Oxytocic A c t i v i t y 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 E x t r a c t s Figure 3 demonstrates the l o s s of o x y t o c i c a c t i v i t y 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 condi-t i o n s . I t can be seen th 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 at pH 7.6 (50% of i n i t i a l value found a f t e r 80 minutes), while at pH 8.1 o x y t o c i c a c t i v i t y of a crude e x t r a c t i s l o s t at a higher r a t e . In the course of long term storage (21 days) of 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 at 4\u00C2\u00B0C losses up to 68% of the 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. Storage i n 0.05 M a c e t i c a c i d under the same c o n d i t i o n s r e -s u l t e d i n lo s s e s of o x y t o c i c a c t i v i t y up to 42%. Losses of a c t i v i t y were not observed when the crude p i t u i t a r y e x t r a c t s were stored i n 0.2 M a c e t i c a c i d at 4\u00C2\u00B0C f o r 21 days. S i m i l a r l o s s e s were not observed i n c o n t r o l experiments using Parke-Davis o x y t o c i n . A p a r t i a l l y p u r i f i e d e x t r a c t ( b i o l o g i c a l l y a c t i v e e f -f l u e n t of 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 of oxy-t o c i c a c t i v i t y was stored f o r two months at -20\u00C2\u00B0C as a l y o p h y l i z e d powder. The bioassay f o l l o w i n g t h i s p e r i o d of 50 Each p o i n t on the graph represents one four p o i n t assay on r a t uterus without magnesium (55). storage showed tha t 530-57 u n i t s of o x y t o c i c a c t i v i t y sur-v i v e d the storage p e r i o d . B. E x t r a c t i o n of Neurohypophysial Hormones from Salmon P i t u i t a r i e s The e f f e c t on the y i e l d of o x y t o c i c a c t i v i t y of v a r y i n g s e v e r a l of the parameters i n the e x t r a c t i o n pro-cedure are recorded i n Table I I I . Factors which were i n -v e s t i g a t e d i n c l u d e d the temperature of e x t r a c t i o n , pH, conc e n t r a t i o n of e x t r a c t a n t , the r e l a t i v e amounts of sol v e n t and t i s s u e , the s t a t e of the t i s s u e (frozen, l y o p h y l i z e d , or acetone d r i e d glands) and the method of d i s r u p t i n g the t i s s u e . Factors which l e d to increased y i e l d of o x y t o c i c a c t i v i t y i n c l u d e d adequate d i s i n t e g r a t i o n of t i s s u e , low temperature, low pH and adequate amounts of e x t r a c t i n g s o l -vent. The concent r a t i o n of a c e t i c a c i d above 0.2 M, the s t a t e of the t i s s u e (frozen, l y o p h y l i z e d or acetone dried) were r e l a t i v e l y unimportant i n o b t a i n i n g optimum y i e l d s . The procedure subsequently adopted f o r e x t r a c t i o n of hormones in v o l v e d d i s r u p t i o n of t i s s u e i n 0.2 M a c e t i c a c i d at 4\u00C2\u00B0C. C. P u r i f i c a t i o n of Neurohypophysial Hormones of Salmon Twenty four p i t u i t a r y e x t r a c t s were made i n the course of t h i s work. The f i r s t twenty e x t r a c t s served to T A B L E E x t r a c t i o n of Oxytocic A c t i v i t y State of t i s s u e Method of d i s r u p t i o n * E x t r a c t a n t Volume of ex t r a c t a n t E x t r a c t i o n (ml) per gm Temp. Duration wet t i s s u e wt.(\u00C2\u00B0C) Frozen Potter- -Elvehjem 0.25% HOAc 2 .5 100 5 min. Acetone powder Potter- -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 hr. Lyophyl-i z e d Potter- -Elvehjem 2M HOAc . 10 .0 25 24 hr. Lyophyl-i z e d Potter- -Elvehjem 2 M HOAc - 10 .0 4 24 hr. 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 r .0 4 5 min. * Potter-Elvehjem g l a s s t i s s u e homogenizer. TRI-R STIR-R (model S-63) e l e c t r i c t i s s u e homogenizer, s e t t i n g 5. Waring blender, f u l l speed. from t h e _ P i t u i t a r i e s of Oncorhynchus tschawytscha Crude e x t r a c t . ^ obtained Absorbance Oxytocic u n i t s per r a t i o nm 280/260 A- U'280nm G m w e t w t ' L i m i t of e r r o r of bioassay 0.67 0.81 0.0593 0.0765 1.8 2.5 6.0 13.0 0.84 _ 0.1040 4.5 10.2 0.83 0.0360 0.144 6.3 9.5 8.6 7.3 0.81 0.93 0.88 0.87 0.90 0.148 0.076 0.010 0.079 0.098 8.5 8.2 1.4 10.0 7.3 11.0 6.2 12.9 10.6 9.3 develop optimal 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 steps, and u t i l i z e d from one to ten grams of t i s s u e each. The l a s t four e x t r a c t s were done on pr 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 of t i s s u e , and provided pure hormones f o r the analyses of amino a c i d composition and sequence. The flow chart i l l u s t r a t e d i n Figure 4 summarizes the p u r i f i c a t i o n schedules developed f o r larg e s c a l e e x t r a c t s . (1) Gel F i l t r a t i o n of Crude Salmon P i t u i t a r y E x t r a c t s Two types of 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 the neurohypophysial hormones of salmon: B i o g e l and Sephadex. Only Sephadex G-15 was used i n pr e p a r a t i v e work. Figure 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 ob-ta i n e d using B i o g e l P-2 and 0.2 M a c e t i c a c i d as the e l u t i n g b u f f e r . Spreading of 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 . S i m i l a r spreading of s y n t h e t i c o x y t o c i n took place under i d e n t i c a l c o n d i t i o n s . A comparison of Sephadex G-25 and G-15 i s shown i n Figure 6. A spreading of hormonal a c t i v i t y , s i m i l a r to th a t observed on B i o g e l P-2, d i d not take place although a low i o n i c s t r e n g t h b u f f e r was used. 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 than t h a t obtained on G-25 columns. This observation-was made c o n s i s t e n t l y , since Sephadex G-25 columns were used i n p u r i f y i n g the f i r s t ten 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 tschawytscha, frozen p i t u i t a r i e s , 100 g I PRECIPITATE r e - e x t r a c t e d FILTRATE 0.2 M HOAc, c o l d , 10:1 v/w TISSUE HOMOGENATE 48,200 g, 30 min. i : HORMONE I I ( u l t r a f i l t r a t i o n ; l y o p h y l i z a t i o n ) SE-Sephadex C-25 or Whatman CM-32 CRUDE EXTRACT (1 L) Sephadex G-15 5 tandem runs ACTIVE EFFLUENT (1 L) UM-2 or UM-3 u l t r a f i l t r a t i o n s ( l y o p h y l i z a t i o n ) CONCENTRATE one of: Whatman CM-32 SE-Sephadex G-25 Phosphocellulose HORMONE I I HORMONE I U l t r a f i l t r a t i o n ; l y o p h y l i z a t i o n HORMONE I I S p e c . a c t i v i t y : * 1.25-1.45 x 10' 2.09-2.29 x 10' Figure 4: P u r i f i c a t i o n Sequence of Large Scale Salmon P i t u i t a r y E x t r a c t * Oxytocic u n i t s per mg Procedure shown i n brackets was not always employed. 56 3.00-1 2 . 5 0 -2 . 0 0 -1.50-E c O CO OJ < 1.00-0.50 -Salt Hormone 1 10 \u00E2\u0080\u0094i\u00E2\u0080\u0094 20 I 30 I\u00E2\u0080\u0094 40 Fraction No. \u00E2\u0080\u0094 i 50 Figure 5: Gel f i l t r a t i o n of 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. Coliamn 320 mm x 30 mm diameter; eluent 0.2 M a c e t i c a c i d ; flow r a t e 1.0 ml/min; f r a c t i o n volume 5.0 ml. Loading sample: 5.0 ml; 94.5 A.U. 2 g Q ; 15.9JI1.2 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 3.4 f o l d ; recovery of 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_ G-25 B. G-15 2.50 -\u00C2\u00A3 2 .00-0 10 20 30 40 50 0 10 20 30 40 50 Fract ion No. Fract ion No. Figure 6: Comparison of g e l f i l t r a t i o n on Sephadex G-25 and Sephadex G-15. Columns 550 mm x 10 mm diameter; eluent 0.002 M a c e t i c a c i d ; flow 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). Loading sample (A and B): 0.8 ml; 51.0 A.U.o D n ; 0.7610.051 oxytocxc u n i t s . 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); recovery of 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 at 280 nm; cross-hatched: hormone co 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 . 58 l a t e r experiments, but the m o l a r i t y of e l u t i n g b u f f e r was increased to 0.2 M. This was done i n order to preserve maximum o x y t o c i c a c t i v i t y of the e x t r a c t , as discussed i n s e c t i o n 2 of t h i s chapter. Further adaptation of the Sephadex G-15 g e l f i l t r a -t i o n i n v o l v e d using the same column repeatedly (tandem separations) to allo w r a p i d processing of lar g e volumes of e x t r a c t (Figure 7). This permitted g e l f i l t r a t i o n immediately 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 the need f o r con c e n t r a t i o n . The e l u t i o n of hormones co i n c i d e d w i t h the 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 o x y t o c i c 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 wit h 0.2 M a c e t i c a c i d as eluent ranged from 90 to 100%. However, i n one larg e s c a l e experiment, the s a l t peak was used i n s t e a d of the bioassay to l o c a t e the hormones, and the recovery of the i n i t i a l o x y t o c i c a c t i -v i t y was 78%. Rechromatography of 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 from Sephadex G-15 on Sephadex G-10 or 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 afforded at t h i s stage by G-10 columns was comparable wi t h t h a t of G-15 columns. Ah increase i n length of the column had no e f f e c t on p u r i f i -c a t i o n obtained by t h i s rechromatography (Figure 8). A two^fold p u r i f i c a t i o n at t h i s e a r l y stage of the i s o l a t i o n sequence was regarded to be of l i t t l e value to the o v e r a l l I \u00E2\u0080\u00A2 . . _ _ . . _ Fraction No. _ ' Figure 7: Gel f i l t r a t i o n of crude salmon p i t u i t a r y e x t r a c t using a tandem s e r i e s of Sephadex G-15. Column 450 mm x 50 mm diameter; eluent 0.2 M a c e t i c a c i d ; flow r a t e 4 ml/min; f r a c t i o n volume 20 ml. T o t a l loading sample: 1050 ml; 12060 A . U . 2 8 q 954-103 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 4.4 f o l d ; recovery of 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 at 280 nm; dotted l i n e : s p e c i f i c c o n d u c t i v i t y ; bars: 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 . Ul 60 E c O CO CM 8.00-1 6 . 0 0 -4.00 -2 . 0 0 -0-15 . 2.5x92 Salt Hormone 1 -T\u00E2\u0080\u0094 20 T\" ft T 30 4 0 Fraction No. \u00E2\u0080\u0094 i 90 Bl 6 - 1 5 . 5x44 salt I 1 Hormone I I T 1\u00E2\u0080\u0094 10 20 30 40 Fraction No. T\" -1 90 Figure 8: Rechroma tog raphy of 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 of equal g e l bed volumes and d i f f e r e n t dimensions. A. Column 920 mm x 25 mm diameter; eluent 0.2 M a c e t i c a c i d ; flow r a t e 0.6 ml/min; f r a c t i o n volume 10.0 ml. Loading s o l u t i o n : 12.0 ml; 723 A.U.280 11111' 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 ; recovery of o x y t o c i c a c t i v i t y 93%. B. Column 440 mm x 50 mm diameter; eluent 0.2 M a c e t i c a c i d ; flow r a t e 4.0 ml/min; f r a c t i o n volume 20.0 ml. Loading s o l u t i o n : 41 ml; 800 A.U. 2go 1)1X1 > 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 ; recovery of o x y t o c i c a c t i v i t y 94%. 61 p u r i f i c a t i o n of the e x t r a c t . On the other hand, every a d d i t i o n a l step included i n the 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 of v a l u a b l e m a t e r i a l . For t h i s reason rechromatography of the a c t i v e eluent from the f i r s t g e l f i l t r a t i o n step 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 of Neurohypophysial 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 necessary p r e l i m i n a r y to i o n exchange chroma-tography. I t was observed during g e l f i l t r a t i o n experiments on salmon p i t u i t a r y e x t r a c t s ( c f. s e c t i o n 4(a) of t h i s chapter) t h a t the s a l t peak c o i n c i d e d w i t h the peak of oxy-t 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 of d e - s a l t i n g were examined i n the course 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 -f i l t r a t i o n . ( i ) D e - s a l t i n g on Sephadex G-10 Commercial preparations of o x y t o c i n (Sigma, synthe-t i c powder, Grade IV) and bovine albumen (Armour, c r y s t a l i n e ) were used f o r these experiments. 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 place of Blue Dextran (Pharmacia), because the l a t t e r e x h i b i t s a high degree of spreading on Sephadex G-10 g e l . A Sephadex G-10 ( l * l m x 10 mm diameter) column pro-vides adequate separation of ox y t o c i n and sodium c h l o r i d e (Figure 9,A). However, sodium acetate and ammonium acetate could not be separated from o x y t o c i n i n the same system (Figure 9,B). Attempts were made to ob t a i n separation by var y i n g temperature, flow r a t e , and the composition of the e l u t i n g b u f f e r . The flow r a t e was v a r i e d from 0.5 to 13.7 ml/min, the temperature from 9.5\u00C2\u00B0C to 73.0\u00C2\u00B0C, and the 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 the concentration of a c e t i c a c i d from 0.002 M to 0.200 M and by a d d i t i o n of 20% methyl a l c o h o l . None of the c o n d i t i o n s t r i e d , s i n g l y or i n combination, achieved the d e s i r e d s e p a r a t i o n . ( 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 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 of p i t u i t a r y e x t r a c t s of salmon. The b i o l o g i c a l l y a c t i v e eluent from Sephadex G-15 was subjected to u l t r a -f i l t r a t i o n p r i o r to the separation of hormones on a c a t i o n exchanger. Fol l o w i n g t h i s s eparation and before rechroma-tography of 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 to which the 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 at each step was deter-mined by the i o n i c s t r e n g t h at which the d e s i r e d substance was expected t o be e l u t e d from the i o n exchange column. -|2.00 2 \u00C2\u00B0 30 40 90 60 70 20 30 40 SO 60 70 Fraction No. Fraction No. Figure 9: D e - s a l t i n g of oxy t o c i n on Sephadex G-10. Column 1.1 m x 10 mm diameter; eluent 0.002 M a c e t i c a c i d ; flow r a t e 0.3 ml/min; f r a c t i o n volume 1.0 ml. Peal; 1 ( s o l i d l i n e ) bovine 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 IV; peak 3 (dotted l i n e ) - s a l t ? Left-hand 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 acetate. 64 Tables IV and V i l l u s t r a t e the re c o v e r i e s of m a t e r i a l f o l l o w i n g u l t r a f i l t r a t i o n . The r e s u l t s obtained appeared to be independent of the contents or of the volume of the sample, and subject to i n d i v i d u a l p e c u l i a r i t i e s of the mem-branes used. The re c o v e r i e s ranged from the very good to the very poor. In former cases, a considerable concomitant p u r i f i c a t i o n of the sample was observed as can be seen from the absorbance measurements. I t can be seen from these r e s u l t s t h a t two types of losses of m a t e r i a l were exper-ienced: passing of the hormones i n t o the f i l t r a t e and complete l o s s of the hormones. The second type of l o s s could not be explained since the d e t a i l s of membrane s t r u c -ture are not reported by the manufacturers (60). Some experiments w i t h UM-1 membranes, d i r e c t e d at removing high molecular weight contaminants were l a r g e l y negative. Sandoz \"Syntocinon\" was passed q u a n t i t a t i v e l y i n t o the u l t r a f i l t r a t e , as was expected from the molecular we i g h t s c u t - o f f range of t h i s type of membrane (10,000) and the molecular weight of 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 of salmon p i t u i t a r i e s , on the other hand, was not passed by the UM-1 membranes. (3) Separation of Neur ohypophy s i a1 Hormones of Salmon by Ion Exchange Chromatpgraphy Foll 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 , the two neurohypophysial hormones of salmon were separated by i o n T A B L E IV 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 Oncorhynchus tschawytscha Using D i a f l o UM-2 Membranes Volume of sample (ml) 100 (Syntocinon) 70 105 % 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 i n f i l t r a t e 97.5 92.0 107.8 n i l 7.2 n i l % of i n i t i a l absorbance @ 280 nm i n concentrate i n f i l t r a t e 70.5 (b) (a) 26.8 10.5 225 300 100 41.2 80.4 83.8 13.7 15.7 12.5 44.6 49.4 72.8 50.4 54.4 30.4 (a) absorbance too low f o r measurement (b) not t e s t e d : 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 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, wi t h the exception of Syntocinon, were p u r i f i e d by passage through a Sephadex G-15 column. Pressure of 80 p . s . i . was used i n a l l experiments. T A B L E V 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 Oncorhynchus tschawytscha Using D i a f l o UM-3 Membranes Loading S o l u t i o n Contents Sandoz 'Syntocinon' pH 4.8 Sandoz 'Syntocinon 1 pH 2.1 Sephadex G-15 e f f l u e n t U l t r a f i l t r a t i o n concentrate of Sephadex G-15 e f f l u e n t Sephadex G-15 e f f l u e n t Sephadex G-15 e f f l u e n t Hormone I a f t e r i o n exchange sepa r a t i o n Volume, mli 160 160 600 % 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 i t i a l absorbance @ 280 nm concentrate f i l t r a t e concentrate f i l t r a t e 250 1335 1050 70 22.8 25.6 77.8 89.1 83.0 103.0 90.5 23.0 23.2 20.6 12.6 7.9 n i l 5.8 (a) (a) 92.0 80.3 66.8 65.2 (a) (a) 10.6 18.8 55.0 45.5 28.0 (continued) T A B L E V (continued) Loading S o l u t i o n Contents % 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 i t i a l absorbance \u00C2\u00A7 280 nm Volume, mfc concentrate f i l t r a t e concentrate f i l t r a t e Hormone I a f t e r i o n exchange sepa r a t i o n Hormone I I a f t e r i o n exchange separation Hormone I I a f t e r i o n exchange separation Hormone I I a f t e r i o n exchange rechromatography 57 600 250 350 56.4 103.0 32.4 96.0 5.4 n i l 68.4 15.8 50.0 31.0 (a) (a) 55.0 (a) (a) (a) (a) absorbance too low f o r measurement. (b) not t e s t e d : 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. 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. 68 exchange chromatography ( c f . Figure 4). Three types of c a t i o n exchangers were used f o r t h i s purpose: Whatman carboxymethylcellulose (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 phosphocelluose and S u l f o e t h y l Sephadex (SE-Sephadex, CM-25). The i o n exchange media were f i r s t standardized w i t h known s y n t h e t i c neurohypophysial hormones (Sigma o x y t o c i n , Grade IV or Sandox Syntocinon, and N.B.C. 3-phe, 8-arg o x y t o c i n ) . The neurohypophysial hormones of salmon were e l u t e d from these c a t i o n exchangers at the i o n i c strengths corresponding to those at 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 at a given pH the charge borne by the salmon neurohypophysial hormones were i d e n t i c a l w i t h the 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 the separation of hormones on Whatman CM-32 and on phosphocellulose was achieved using an e l u t i o n method reported by Sawyer (45). The method i n v o l v e s the e l u t i o n of the l e s s b a s i c hormone using 0.02 M ammonium acetate b u f f e r , pH 5, followed by a gradient to 0.2 M b u f f e r of pH 7.5 to e l u t e the more b a s i c hormone. Subsequently t h i s method was s u b s t i t u t e d by a continuous s a l t gradient from 0.002 M to 0.200 M sodium or ammonium acetate at constant pH (pH 5) f o r e l u t i o n of 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 phosphocellulose, using a continuous s a l t g r adient at pH of 5, are i l l u s t r a -ted i n Figures 10 and 11 r e s p e c t i v e l y . 69 Loading and A A \u00E2\u0080\u00A2 B IM N a O A c I H H H Fraction No. Figure 10: Separation of salmon neurohypophysial hormones on Whatman CM-3 2. Column 390 mm x 12 mm diameter; flow r a t e 0.6 ml/min; f r a c t i o n volume 5.0 ml; sodium acetate, pH 5. Buf f e r A (to f r a c t i o n 30) 0.196 mmho, 0.002 M. Buf f e r B (A \u00E2\u0080\u0094 B gr a d i e n t , f r a c t i o n s 31 \u00E2\u0080\u0094 200) 14.8 mmho, 0.200 M. Loading s o l u t i o n : 42 ml; s p e c i f i c a c t i v i t y (oxytocic u n i t s per A.U^so 1 1 1 1 1) 1*4. Hormone I , s p e c i f i c a c t i v i t y 25.8; Hormone I I , s p e c i f i c a c t i v i t y 65.0. Recovery of o x y t o c i c a c t i v i t y : 98%. L i g h t black l i n e : absorbance at 280 nm; heavy black 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 fo u r - p o i n t assay); 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 ' B ^ I M NH.OA^ GO 100 ISO 200 Fraction No. Figure 11: Separation of salmon neurohypophysial hormones on S e l e c t a c e l phosphoce1lulose. Column 420 mm x 12 mm diameter; flow r a t e 1.7 ml/min; f r a c t i o n volume 5.0 ml; ammonium acetate, pH 5. Buf f e r A (to f r a c t i o n 30) 0.59 mmho, approx. 0.004 M; Buf f e r B (A \u00E2\u0080\u0094 B gr a d i e n t , f r a c t i o n s 31 \u00E2\u0080\u0094 200) 9.3 mmho, 0.200 M. Loading s o l u t i o n : 97 ml; s p e c i f i c a c t i v i t y (oxytocic u n i t s per A.U.280 1*6. Hormone I , s p e c i f i c a c t i v i t y 8.9; Hormone I I , s p e c i f i c a c t i v i t y 93.0. Recovery of o x y t o c i c a c t i v i t y : 88%. L i g h t black l i n e : absorbance at 280 nm; heavy black 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 assay). 71 C h r o m a t o g r a p h y o n S E - S e p h a d e x a t l o w pH ( 2 . 4 5 ) was d e v e l o p e d i n t h e c o u r s e o f t h i s work i n a n e f f o r t t o p u r i f y Hormone I . S u b s e q u e n t l y t h e s y s t e m was s c a l e d up a n d a p p l i e d t o t h e s e p a r a t i o n o f t h e h o r m o n e s . A n e l u e n t p r o -f i l e o f a l a r g e s c a l e s e p a r a t i o n on S E - S e p h a d e x i s shown i n F i g u r e 1 2 . W h i l e t h e e l u t i o n o f Hormone I g i v e s a s y m -m e t r i c a l n a r r o w p e a k , t h e e l u t i o n o f Hormone I I r e s u l t s i n a b r o a d b a n d . I n a s u b s e q u e n t e x p e r i m e n t t h e e l u t i o n o f Hormone I I was d o n e i n a s t e p w i s e m a n n e r w i t h 2 M ammonium a c e t a t e a t pH 5 , a n d o x y t o c i c a c t i v i t y was e l u t e d i n t h r e e f r a c t i o n s . T h e c h o i c e o f c a t i o n e x c h a n g e r t o be u s e d f o r t h e i n i t i a l s e p a r a t i o n o f h o r m o n e s was g o v e r n e d by t h e p u r p o s e f o r w h i c h t h e s e p a r a t e d h o r m o n e s w e r e i n t e n d e d . T o w a r d t h e e n d o f t h i s i n v e s t i g a t i o n a d d i t i o n a l amounts o f Hormone I w e r e r e q u i r e d f o r s t r u c t u r a l s t u d i e s , a n d s e p a r a t i o n o n t h e s u l f o e t h y l c o l u m n p r o v i d e d a f a s t m e t h o d f o r o b t a i n i n g t h i s h o r m o n e . T a b l e V I i l l u s t r a t e s t h e i o n i c s t r e n g t h s a t w h i c h t h e h o r m o n e s w e r e e l u t e d f r o m t h e t h r e e c a t i o n e x c h a n g e m e d i a u s e d . T h e r e s u l t s i n d i c a t e t h a t Hormone I was b e s t r e t a i n e d b y S E - S e p h a d e x a t a pH o f 2 . 4 5 , a n d s u c c e s s i v e l y l e s s w e l l b y S E - S e p h a d e x a t pH 5 , b y p h o s p h o c e l l u l o s e , a n d b y Whatman C M - 3 2 . I n c r e a s e i n l e n g t h o f Whatman C M - 3 2 c o l u m n s a n d a c h a n g e f r o m s o d i u m t o ammonium a c e t a t e b u f f e r d i d n o t a p p e a r t o a f f e c t t h e i o n i c s t r e n g t h a t w h i c h Hormone Loading A A i H H B B H i i O 10 SO 100 150 200 250 Fraction No. Figure 12: Separation of salmon neurohypophysial hormones on SE-Sephadex. Column 430 mm x 25 mm diameter; flow r a t e 0.7 ml/min; f r a c t i o n volume 10 ml; ammonium formate pH 2.45. Bu f f e r A (to f r a c t i o n 40) 1.75 mmho, 0.085 M. Bu f f e r B (A \u00E2\u0080\u0094 B g r a d i e n t , f r a c t i o n s 41 \u00E2\u0080\u0094 220; B, f r a c t i o n s 221 \u00E2\u0080\u0094 250) 22.0 mmho,0.2 M. Loading s o l u t i o n : 130 ml; s p e c i f i c a c t i v i t y (oxytocic u n i t s per A.U^SO 1\u00E2\u0084\u00A2) 0.42. Hormone I , s p e c i f i c a c t i v i t y 2.7; Hormone I I , s p e c i f i c a c t i v i t y 3.3. Recovery of o x y t o c i c a c t i v i t y : 92%. L i g h t black l i n e : absorbance at 280 nm; heavy black 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 assay) . n j T A B L E VI Ion Exchange Chromatography of Neurohypophysial Hormones of Oncorhynchus tschawytscha: 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 Cation Exchanger Column length x diameter mm E l u t i n g b u f f e r used S a l t pH S p e c i f i c c o n d u c t i v i t y Hormone I % i n i t i a l o x y t o c i c Hormone I I a c t i v i t y recovered Whatman CM-32 90 X 12 sodium acetate 5.0-7 R 0.8 - 1. 7 8.2 - 9. 2 87 Whatman CM-32 90 X 12 sodium acetate / . -> 5.0 0.8 - 1. 0 7.1 -11. 2 70 Whatman CM-32 190 X 12 ammon. acetate 5.0 0.8 - 1. 9 89 Whatman CM-32 390 X 12 ammon. acetate 5.0 0.9 - 1. 0 5.7 - 6. 7 98 Phosphocellulose 420 X 12 ammon. acetate 5.0 1.3 - 2. 0 8.0 - 8. 8 88 SE-Sephadex 120 X 12 ammon. acetate 5.0 2.2 - 2. 5 10.7 -11. 2 77 SE-Sephadex 120 X 12 ammon. formate 2.45 3.7 = 4. 0 68 SE-Sephadex 430 X 25 ammon. formate 2.45 9.6 -11. 1 20.0 92 Mixing chamber/reservoir volumes used were as f o l l o w s : 250 : 250 cc f o r 90 x 12 columns; 450 : 450 cc f o r 420 x 12 columns; 950 : 950 cc f o r 420 x 25 columns. Ammonium and sodium acetate gradients were from 0.002 M to 0.2 M; ammonium formate from 0.085 M to 2.0 M. A pH gradient was a p p l i e d only i n the f i r s t experiment i n the Table. 74 I was e l u t e d . The r e c o v e r i e s of i n i t i a l o x y t o c i c a c t i v i t y d i d not appear to vary 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 to 98 percent. At t h i s stage of the p u r i f i c a t i o n , the extent 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' chemical i d e n t i t y . The p o t e n t i a t i o n values ranged from 2.1 to 3.8 f o r Hormone I and from 1.5 to 2.5 f o r Hormone I I . I t was not found p o s s i b l e to p r e d i c t the i d e n t i t y of the hormones on the b a s i s of these r e s u l t s and 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 other 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 to t h a t of Hormone I was c o n s i s t e n t l y observed to l i e between 2.0 and 3.0 i n each of the c a t i o n exchange separations of the two hormones. (4) P u r i f i c a t i o n of Salmon Neurohypophysial Hormone I_ by Ion Exchange Chromatography In order to determine the sequence of amino acids w i t h i n the molecule i t was necessary to prepare a s u f f i c i e n t amount of the salmon hormone of 90%, or g r e a t e r , p u r i t y . 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 the hormone pre p a r a t i o n was used as a c r i t e r i o n of p u r i t y . An amino a c i d a n a l y s i s of the Hormone I was 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 (cf . preceeding s e c t i o n ) , because n e i t h e r the s p e c i f i c a c t i v i t y of the hormone at 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 warranted an a n a l y s i s . Rechromatography of Hormone I on Whatman CM-32 or on phosphocellulose 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 analyses. A b e t t e r p u r i f i c a t i o n was obtained by rechromatography on SE-Sephadex at pH 5, yet the l e v e l of contaminating amino acids was s t i l l too high to attempt sequence s t u d i e s : g l u , l y s , a l a , v a l , l e u , phe, h i s , and arg were present i n amounts ranging from 0.1 to 0.4 residues per molecule. A r e d u c t i o n i n pH of 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 prepar-a t i o n of Hormone I . Figure 13 i l l u s t r a t e s t h i s rechroma-tography step. The absorbance readings corresponding to the 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, could 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 of the p u r i f i e d Hormone I . The s p e c i f i c a c t i v i t y was c a l c u l a t e d using the data obtained 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 (c f . Experimental Procedures). Further s t u d i e s on the i s o l a t i o n of Hormone I showed tha t the sequence i n which the chromatographies were con-ducted at pH 5 and pH 2.45 r e s p e c t i v e l y was not r e l e v a n t to 76 B to tube 185 E E 8.0- i 6 . 0 -4.0 - -0.2 O 3 \"D C o o E c O CO CM 2.0 -4-0.1 < 30 Hormone J_ x x\u00E2\u0080\u0094 x\u00E2\u0080\u0094 Lx-60 Fract ion No. $ t LX\u00E2\u0080\u0094X\u00E2\u0080\u0094X\u00E2\u0080\u0094X\u00E2\u0080\u0094 \u00E2\u0080\u00A2x - i \u00E2\u0080\u0094 90 2 *9< \u00E2\u0080\u0094r- $ "Thesis/Dissertation"@en . "10.14288/1.0104257"@en . "eng"@en . "Zoology"@en . "Vancouver : University of British Columbia Library"@en . "University of British Columbia"@en . "For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use."@en . "Graduate"@en . "Isolation and amino acid sequence of neurohypophysial hormones or Pacific chinook salmon (Oncorhynchus tschawytscha)."@en . "Text"@en . "http://hdl.handle.net/2429/35833"@en .