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Isolation and characterization of salmon ultimobranchial calcitonin O'Dor, Ronald Keith 1971

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T H E 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 O F S A L M O N U L T I M O B R A N C H I A L C A L C I T O N I N BY RONALD K E I T H O ' D O R A . B . , U N I V E R S I T Y OF C A L I F O R N I A , B E R K E L E Y * 1967 A T H E S I S S U B M I T T E D IN P A R T I A L F U L F I L M E N T OF T H E R E Q U I R E M E N T S FOR T H E D E G R E E OF DOCTOR OF P H I L O S O P H Y IN T H E D E P A R T M E N T OF P H Y S I O L O G Y WE A C C E P T T H I S T H E S I S AS C O N F O R M I N G TO THE R E Q U I R E D STANDARD SuPERVISOR E X T E R N A L E X A M I N E R T H E U N I V E R S I T Y OF B R I T I S H C O L U M B I A F E B R U A R Y , 1971 In presenting th i s thes i s in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make i t f ree l y ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th i s thes i s for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t ion of th i s thes is f o r f inanc ia l gain sha l l not be allowed without my wr i t ten permission. R O N A L D K E I T H O ' D O R Department of P H Y S I Q L O G Y The Univers i ty of B r i t i s h Columbia Vancouver 8, Canada Date B E B R U A R Y 16, 1971 A B S T R A C T A L T H O U G H T H E F I R S T P R E P A R A T I O N S O F T H E H Y P O C A L C E M I C H O R M O N E , C A L C I T O N I N ( C T ) , W E R E E X T R A C T E D F R O M R A T T H Y R O I D G L A N D S , H I S T O L O G I C A L E V I D E N C E S H O W E D T H A T T H E " C " C E L L S W H I C H P R O D U C E T H E H O R M O N E A R E N O T R E S T R I C T E D T O T H I S G L A N D , B U T A L S O O C C U R I N T H E P A R A T H Y R O I D A N D T H Y M U S T I S S U E S O F M A M M A L S . T H E S E C E L L S A R I S E E M B R Y O L O G I C A L L Y F R O M T H E L A S T B R A N C H I A L P O U C H A N D I N N O N - M A M M A L S T H E Y F O R M A S E P A R A T E U L T I M O B R A N C H I A L G L A N D W H I C H A L S O C O N T A I N S H Y P O C A L C E M I C A C T I V I T Y . T H E W O R K D E S C R I B E D I N T H I S T H E S I S P R O V I D E S E V I D E N C E T H A T T H I S A C T I V I T Y R E S U L T S F R O M P O L Y P E P T I D E S S T R U C T U R A L L Y S I M I L A R T O T H O S E I S O L A T E O F R O M M A M M A L I A N T H Y R O I D T I S S U E S A N D . E X P L O R E S T H E R E L A T I O N S H I P B E T W E E N T H E S T R U C T U R A L A N D F U N C T I O N A L D I F F E R E N C E S O F T H E TWO T Y P E S O F C T . A S U R V E Y O F F O U R M A M M A L I A N T H Y R O I D T I S S U E S ( H U M A N , B O V I N E , P O R C I N E A N D M U R I N E ) A N O F O U R N O N - M A M M A L I A N U L T I M O -B R A N C H I A L T I S S U E S ( T U R K E Y , C H I C K E N , S A L M O N A N D D O G F I S H ) D E M O N S T R A T E D T H A T T H E S E T I S S U E S C O N T A I N E D H Y P O C A L C E M I C P O L Y P E P T I D E S W I T H M O L E C U L A R W E I G H T S O F A B 0 U T 4 0 0 0 A S D E T E R -M I N E D B Y G E L F I L T R A T I O N . W H E N E X T R A C T S W E R E P R E P A R E D U S I N G A N O R G A N I C S O L V E N T M I X T U R E D E V E L O P E D F O R T H E T H Y R O I D C T ' S T H E U L T I M O B R A N C H I A L T I S S U E S Y I E L D E D M O R E H Y P O C A L C E M I C A C T I V I T Y O N A F R E S H W E I G H T B A S I S A N D T H E F I N A L P R O D U C T H A D A H I G H E R S P E C I F I C A C T I V I T Y . S A L M O N U L T I M 0 B R A N C H I A L T I S S U E W A S C O L L E C T E D O N A L A R G E S C A L E A N D E X T R A C T E D T O P R O V I D E I I M A T E R I A L FOR C H E M I C A L C H A R A C T E R I Z A T I O N . A S E R I E S OF T H R E E GEL F I L T R A T I O N S T A G E S ON S E P H A O E X G - 5 0 A L T E R N A T I N G W ITH TWO I O N - E X C H A N G E C H R O M A T O G R A P H Y S T A G E S ON S E - S E P H A D E X C - 2 5 AT TWO P H ' S P R O V I D E D A 3 0 0 , 0 0 0 FOLD P U R I F I C A T I O N AND Y I E L D E D 1 5 MG OF P U R E S A L M O N C T . A M I N O A C I D A N A L Y S I S AND P A R T I A L C H A R A C T E R I Z A T I O N OF T R Y P T I C P E P T I D E S I N D I C A T E D THAT THE U L T I M O B R A N C H I AL HORMONE WAS A 3 2 A M I N O A C I O P O L Y P E P T I D E W ITH A D I S U L F I D E B R I D G E A F THE C - T E R M I N U S . A L T H O U G H T H E S E F E A T U R E S ARE A L S O COMMON TO A L L THE M A M M A L I A N C T ' S , T H E R E ARE A NUMBER OF U N I Q U E F E A T U R E S IN THE S A L M O N C T S T R U C T U R E . T H E S E S T R U C T U R A L D I F F E R E N C E S WERE A L S O R E F L E C T E D IN THE B I O L O G I C A L A C T I V I T Y OF THE H O R M O N E . S A L M O N C T WAS N E A R L Y 5 0 T I M E S MORE A C T I V E THAN HUMAN C T I N T H E S T A N D A R D B I O A S S A Y AND THE R E S P O N S E TO THE S A L M O N HORMONE WAS P R O L O N G E D . T E S T S IN P L A S M A BOTH J_N V I VP AND JI_N VI TRO I N D I C A T E D THAT S A L M O N C T WAS MUCH MORE S T A B L E THAN THE ' T H Y R O I D C T ' S S U G G E S T I N G A P O S S I B L E R E A S O N FOR I T S G R E A T E R P O T E N C Y . A S U R V E Y OF COHN F R A C T I O N S FROM HUMAN P L A S M A SHOWED THAT F R A C T I O N S M'l-O, I V - 1 I V - 4 C O N T A I N E D E N Z Y M E S C A P A B L E OF R A P I D L Y D E G R A D I N G P O R C I N E C T . IN F U R T H E R S T U D I E S ON F R A C T I O N IV—1 A S E L E C T I V E " C A L C I -T O N I N A S E " WAS P U R I F I E D BY C H R O M A T O G R A P H Y ON D E A E - S E P H A D E X , S E P H A D E X G - 2 0 0 AND C M - S E P H A D E X . T H I S E N Z Y M E R A P I D L Y I N A C -T I V A T E D P O R C I N E C T , BUT HAD NO S I G N I F I C A N T E F F E C T ON S A L M O N OR HUMAN C T . S Y N T H E T I C P O R C I N E C T WAS D I G E S T E D W I T H THE E N Z Y M E AND THE R E S U L T A N T P E P T I D E S WERE I S O L A T E O AND I D E N -T I F I E D . T H E N A T U R E OF T H E S E P E P T I D E S I N D I C A T E D THAT THE I I I ENZYME WAS A PEPT IOASE WITH A S P E C I F I C I T Y FOR THE CARBOXYL S IDE OF ARGIN INE RES IDUES . S IM ILAR D IGEST IONS OF PURE NATIVE SALMON CT PRODUCED NO P E P T I D E S PROVIDING AT L E A S T A PART IAL EXPLANAT ION FOR THE GREATER S T A B I L I T Y OF TH IS HORMONE. THESE EXPERIMENTS ALSO SHOWED THAT THE ENZYME WOULD NOT S P L I T AT ALL ARGININE RES IDUES AND WOULD NOT CLEAVE BONDS ASSOC IATED WITH OTHER BAS IC RES IDUES . THE DATA IND I -CATED THAT THE ENZYME HAD A MOLECULAR WEIGHT OF ABOUT 3 0 , 0 0 0 AND PROBABLY WAS DERIVED FROM A PRECURSOR WITH A MOLECULAR WEIGHT OF ABOUT 1 0 0 , 0 0 0 . TESTS OF THE ACTION OF THE ENZYME ON F IBRINOGEN SHOWED THAT IT WAS NOT THROMBIN. COMPARISON OF A V A I L A B L E DATA WITH THAT FOR OTHER PLASMA ENZYMES IND I -CATED S I M I L A R I T I E S TO THE K A L L I K R E I N FAMILY OF ENZYMES, BUT THE " C A L C I TON INASE" DOES NOT APPEAR TO BE IDENTICAL WITH ANY OF THE WELL STUD IES MEMBERS OF TH IS GROUP. IV TABLE OF CONTENTS PAGE ABSTRACT i i L IST OF T A B L E S . . . i x L I S T OF I L L U S T R A T I O N S x ACKNOWLEDGMENTS . x i i i CHAPTER I. INTRODUCTION 1 I I . MOLECULAR WEIGHT OF C A L C I T O N I N S EXTRACTED FROM ULTIMOBRANCHIAL AND THYROID T I S S U E S 6 A. INTRODUCTION. . . . 6 B. EXTRACTION. . . . . . . . . 8 1 . METHODS . 8 A. ISOLATION AND PREPARATION OF GLANDS 8 B. EXTRACTION PROCEDURES 11 c. BIOLOGICAL ASSAY PROCEDURE 1 3 2. RESULTS AND D I S C U S S I O N . 16 C. C A L I B R A T I O N OF SEPHADEX COLUMNS 2 0 1 . METHODS 20 2. RESULTS AND D I S C U S S I O N 22 D. ESTIMATION OF MOLECULAR W'EIGHTS OF HYPOCAL-CEMIC FACTORS 2 3 1 . METHODS 2 3 2. RESULTS AND D I S C U S S I O N . 2 4 I I I . ISOLATION AND CHARACTERIZATION OF SALMON C A L C I -TONIN . . . . 2 9 A. INTRODUCTION. . 2 9 B. P R E L I M I N A R Y P U R I F I C A T I O N S 2 9 v C H A P T E R P A G E I I I . ( C O N T i N U E D ) 1 . E X T R A C T I O N 2 9 2 . S T A G E 1 C H R O M A T O G R A P H Y 3 0 3 . S T A G E 2 C H R O M A T O G R A P H Y . . . . . . . . . 3 1 4 . S T A G E 3 C H R O M A T O G R A P H Y 3 1 5 . S U M M A R Y OF P U R I F I C A T I O N 3 3 C. P R E P A R A T I O N O F P U R E S A L M O N C A L C I T O N I N . . . 3 3 1 . E X T R A C T I O N . . . 3 3 2 . S T A G E 1 C H R O M A T O G R A P H Y 3 4 3 . S T A G E 2 C H R O M A T O G R A P H Y . . . . . . . . . 3 5 4 . S T A G E 3 C H R O M A T O G R A P H Y 3 7 5 . S T A G E 4 C H R O M A T O G R A P H Y . 3 7 6. S T A G E 5 C H R O M A T O G R A P H Y . . . . . . . . . 3 8 7 . S U M M A R Y OF P U R I F I C A T I O N S T A G E S 3 9 8 . C R I T E R I A OF H O M O G E N E I T Y . 4 0 D. C H A R A C T E R I Z A T I O N OF T H E WHOLE M O L E C U L E . . . 4 1 1 . M E T H O D S . . . . . . . 4 1 A. A M I N O A C I D A N A L Y S I S . 4 1 B . E N D G R O U P A N A L Y S I S . . 4 3 2 . R E S U L T S A N D D I S C U S S I O N . . . . . . . . . 4 3 E . P R E P A R A T I O N ANO I S O L A T I O N OF T R Y P T I C P E P T I D E S 4 5 1 . ME T H O D S 4 5 A. P E R F O R M I C A C I D O X I D A T I O N 4 5 B. A M I N O A C I D A N A L Y S I S 4 6 c . D I G E S T I O N W I T H T R Y P S I N 4 6 V I CHAPTER PAGE I I . (CONT iNUED ) D. HIGH VOLTAGE ELECTROPHORESIS. . . . 47 E. PAPER CHROMATOGRAPHY 48 2. RESULTS AND DISCUSSION 48 F. CHARACTERIZATION OF TRYPTIC P E P T I D E S . . . . 50 1. METHODS . . 50 A. AMINO ACID A N A L Y S I S 5 0 B. DETERMINATION OF AMIDE GROUPS . . . 5 0 c . END GROUP A N A L Y S I S . 51 2. RESULTS AND D I S C U S S I O N . . 51 G. SEQUENCE OF SALMON C A L C I T O N I N COMPARED TO KNOWN SEQUENCES OF MAMMALIAN CALCITONINS,, . 54 IV. STRUCTURE-FUNCTION R E L A T I O N S H I P S IN C A L C I T O N I N S 59 A. INTRODUCTION 59 B. ASSAYS FOR I N A C T I V A T I N G ENZYMES . . . . . . 71 C. P A R T I A L P U R I F I C A T I O N OF A S E L E C T I V E " C A L C I -TONIN ASE" 74 1. S E L E C T I O N OF STARTING MATERIAL 74 2. P R E L I M I N A R Y P U R I F I C A T I O N EXPERIMENTS. . 76 3. PREPARATION OF A HIGHLY P U R I F I E D " C A L C I -TONINASE" . . 79 4. SUMMARY OF P U R I F I C A T I O N 8 2 5. TESTS OF PURITY 83 D. S E L E C T I V E I NACT I VATION OF PORCINE CT BY "CALC I TON INASE" . . . . . . . . . . . . . . 84 E. P E P T I D E S FROM C A L C I T O N I N S DIGESTED WITH " C A L C I T O N I N A S E " . . . . . . . . . . . . . . 85 1. ME THODS 85 V I I C H A P T E R P A G E IV. ( C O N T i N U E O ) A . D I G E S T I O N W I T H E N Z Y M E 85 B . H I G H V O L T A G E E L E C T R O P H O R E S I S . . . . 86 c. A M I N O A C I D A N A L Y S I S 86 o. END G R O U P A N A L Y S I S . . 86 2. R E S U L T S AND D I S C U S S I O N 86 F. C O M P A R I S O N O F " C A L C I T O N I N A S E " TO T H R O M B I N . 89 G. G E N E R A L D I S C U S S I O N 90 B I B L I O G R A P H Y . . 96 A P P E N D I C E S A. S T A T I S T I C A L M E T H O D S FOR C A L C U L A T I O N O F L O G D O S E R E S P O N S E C U R V E S BY C O M P U T E R 107 B. A S I M P L E D E S I G N FOR A L A R G E , I N E X P E N S I V E S E P H A -D E X COLUMN. . . . . . 110 C T E C H N I Q U E S FOR H I G H V O L T A G E P A P E R E L E C T R O -P H O R E S is 113 C. A M I N O A C I D A N A L Y S I S . . . . . . . . . 117 E. P R E P A R A T I O N AND I D E N T I F I C A T I O N O F D A N S Y L DER I V A T I V E S . . 128 F. P O L Y A C R i L AM i DE G E L E L E C T R O P H O R E S I S . *' 131 G. S I N G L E L E T T E R N O T A T I O N FOR A M I N O A C I D S . . . . . 133 V I M L I S T OF T A B L E S T A B L E F A C I N G P A G E I . S P E C I F I C B I O L O G I C A L A C T I V I T I E S O F T H Y R O I D AND U L T I M O B R A N C I AL E X T R A C T S . 1 7 I I . B l O L O G I C A L A C T I V I T Y Y I E L D S FROM T H Y R O I D AND U L T I M O B R A N C I A L T I S S U E S 1 8 I I I . M O L E C U L A R W E I G H T E S T I M A T E S FOR C A L C I T O N I N S FROM E L U T I O N V O L U M E S ON A S E P H A D E X G - 5 0 C O L U M N 2 5 I V . A M I N O A C I D A N A L Y S E S O F F R A C T I O N S FROM S T A G E 5 C H R O M A T O G R A P H Y 4 3 V. A M I N O A C I D C O M P O S I T I O N S O F F I V E C A L C I T O N I N S . . 4 4 V I . A M I N O A C I D A N A L Y S E S O F T R Y P T I C P E P T I D E S FROM S A L M O N CT . . . 51 V I I . NET C H A R G E S O F S A L M O N CT T R Y P T I C P E P T I D E S FROM E L E C T R O P H O R E T I C M O B I L I T I E S AND M O L E C U L A R W E I G H T S 52 V I I I . D I S T R I B U T I O N O F 1 2 5 l - C T AND 1 2 5 I - P T H IN R A T T I S S U E S A F T E R I N T E R V E N O U S I N J E C T I O N 6 4 I X . I N A C T I V A T I O N O F P O R C I N E CT O U R I N G I N C U B A T I O N S W I T H HUMAN P L A S M A F R A C T I O N S . 76 I X L I S T O F I L L U S T R A T I O N S F I G U R E F A C ( N G P A G E 1. L O C A T I O N OF A V I A N U L T I M O B R A N C H I A L G L A N D S . . . . 9 2. (A) C O M P A R I S O N OF R E G R E S S I O N S OF LOG DOSE ON R E S P O N S E FOR T U R K E Y UL T I M 0 B R A N C H I A L E X T R A C T S FROM A C E T O N E AND D I C H L O R O E T H A N E D R I E D G L A N D S 16 ( B ) C O M P A R I S O N OF R E G R E S S I O N S OF LOG DOSE ON R E S P O N S E FOR D O G F I S H U L T I M 0 B R A N C H I AL E X T R A C T S P R E P A R E D U S I N G F O R M I C A C I D OR T C A . 16 3. (A) R E G R E S S I O N OF L O G DOSE ON R E S P O N S E FOR HOUSE S T A N D A R D . . . . . 17 ( B) R E G R E S S I O N OF L O G DOSE ON R E S P O N S E FOR E I G H T E X T R A C T S FROM T H Y R O I D AND U L T I M 0 B R A N C H I A L T I S S U E S . . . . . . . . 17 4. C A L I B R A T I O N C U R V E S FOR S E P H A D E X G-50 AND G-75, I N C L U D I N G E L U T I O N V O L U M E S OF H Y P O C A L C E M I A A C T I -V I T Y FROM E X T R A C T S . . 22 5. E L U T I O N P R O F I L E S OF T H Y R O I D E X T R A C T S ON S E P H A D E X G-50 . 25 6. E L U T I O N P R O F I L E S O F U L T I M O B R A N C H I A L E X T R A C T S ON S E P H A D E X G-50 25 7. C O M P A R I S O N OF E L U T I O N P R O F I L E S ON S E P H A D E X G-50 OF TWO B O V I N E T H Y R O I D E X T R A C T S P R E P A R E D BY D I F F E R E N T METHODS 26 8. E L U T I O N P R O F I L E OF S A L M O N U L T I M O B R A N C H I A L S O L V E N T E X T R A C T ON S E P H A D E X G-50* P R E L I M I N A R Y S T A G E 1. . 30 9. E L U T I O N P R O F I L E OF S T A G E 1 P R O D U C T ON S E - S E P H A D E X C-25* P R E L I M I N A R Y S T A G E 2 31 10. E L U T I O N P R O F I L E OF S T A G E 2 P R O D U C T ON B I O - G E L P-10 P R E L I M I N A R Y - S T A G E 3 • 32 11. SUMMARY OF P R E L I M I N A R Y P U R I F I C A T I O N OF S A L M O N C T 33 12. E L U T I O N P R O F I L E OF S A L M O N U L T I M O B R A N C H I A L A C I D E X T R A C T ON S E P H A D E X G-50t S T A G E 1. . . . . . . . 35 13. E L U T I O N P R O F I L E OF S T A G E 1 P R O D U C T ON S E - S E P H A -DEX C-25: S T A G E 2. . . . . . . 36 x FAC ING F IGURE PAGE . 1 4 . ELUT ION PROF ILE OF STAGE 2 PRODUCT ON SEPHADEX G - 5 0 : STAGE 3 37 1 5 . ELUT ION PROF ILE OF STAGE 3 PRODUCT ON SE-S E P H A -DEX C - 2 5 : STAGE 4 . . 38 1 6 . ELUT ION PROF ILE OF STAGE 4 PRODUCT ON SEPHADEX G - 5 0 * STAGE 5 . 38 1 7 . SUMMARY OF P U R I F I C A T I O N OF SALMON C T 39 1 8 . REGRESS ION OF LOG DOSE ON RESPONSE FOR STAGE 1 , 3 AND 5 PRODUCTS . . . 39 19. THIN LAYER CHROMATOGRAPHY OF STAGE 5 PRODUCT ON S I L I CA G E L . . " • 4 0 20. OUTL INE OF PROCEDURES USED TO ISOLATE TRYPT IC P E P T I D E S FROM SALMON C T . . ... 49 21. PH 6 . 5 ELECTROPHORETOGRAM OF SALMON CT TRYPT IC P E P T I D E S 49 2 2 . PH 1 . 9 ELECTROPHORETOGRAM OF REGION A - 2 . . . . . 49 2 3 . PAPER CHROMATOGRAM OF REGION A-1 49 2 4 . PH 1 . 9 ELECTROPHORETOGRAM OF REGION C - 1 . . . . . 49 2 5 . LOGARITHMIC PLOT OF ELECTROPHORET IC M O B I L I T Y , ( A T PH 6 . 5 , R E L A T I V E TO ASPART IC AC ID ) VERSUS MOLE-CULAR WEIGHTS FOR P E P T I D E S 5 0 2 6 . LOGARITHMIC PLOT OF ELECTROPHORET IC MOBIL ITY (AT PH 1 . 9 * R E L A T I V E TO S E R I N E ) VERSUS MOLECULAR WEIGHTS FOR P E P T I D E S 5 0 2 7 . SALMON C T TRYPT IC P E P T I D E S ALIGNED WITH HUMAN C T 53 2 8 . ALIGNMENT OF SALMON C T WITH PORCINE C T , BOVINE C T AND HUM AN C T 54 2 9 . PO S S I B L E CONFIGURATION OF SALMON C T AS DETERMINED FROM A MOLECULAR MODEL, 58 30. PO S S I B L E CONFIGURATION OF PORCINE C T AS DETER -MINED FROM A MOLECULAR MODEL '. 58 31. PO S S I B L E CONFIGURATION OF HUMAN C T AS DETERMINED FROM A MOLECULAR MOOEL . . . . . . 58 1 XI FAC I NG F I G U R E P A G E 3 2 . D I S A P P E A R A N C E O F " I N T A C T " C A L C I T O N I N A F T E R I N J E C T I O N O F 131 L - P O R C I N E C T 6 0 3 3 . A R E A R E S P O N S E C U R V E S FOR V A R Y I N G D O S E S O F HUMAN C T I N R A T S . ., 6 7 3 4 . P L O T S OF L O G D O S E S V E R S U S A R E A R E S P O N S E FOR S E V E R A L C A L C I T O N I N S 6J 3 5 . I N A C T I V A T I O N O F C A L C I T O N I N S FROM S E V E R A L S P E C I E S D U R I N G I N C U B A T I O N IN S E R U M S FROM S E V E R A L S P E C I E S . 6 7 3 6 . C O M P A R I S O N O F IN V I T R O I N A C T I V A T I O N O F S A L M O N AND C H I C K E N C T IN R A T S E R U M TO T H E I R D I S A P P E A R A N C E R A T E S IN NORMAL AND N E P H R E C T O M I Z E D R A T S . . . . . 6 8 3 7 . E L U T I O N P R O F I L E O F AN E X T R A C T O F HUMAN COHN F R A C T I O N I V — 1 ON S E P H A D E X G - 2 0 0 7 7 3 8 . E L U T I O N P R O F I L E O F S E P H A D E X G - 2 0 0 P R O D U C T ON C M -S E P H A D E X C - 2 5 8 0 3 9 . E L U T I O N P R O F I L E O F C M - S E P H A O E X P R O D U C T ON S E P H A -D E X G - 2 0 0 8 1 4 0 . SUMMARY O F P U R I F I C A T I O N P R O C E D U R E U S E D TO P R E P A R E " C A L C I TON I N A S E " 8 2 4 1 . P H 1 . 9 E L E C T R O P H O R E T O G R A M O F P E P T I D E S FROM P O R C I N E C T P R O D U C E D BY " C A L C I T 0 N I N A S E " 8 7 4 2 . C O M P U T E R P R I N T O U T AND P L O T FROM L I N E A R R E G R E S S I O N PROGRAM . . . 1 1 0 4 3 . END P I E C E S FOR 1 0 CM C O L U M N — D I M E N S I O N S 1 1 1 4 4 . 1 0 x 1 5 0 CM G E L F I L T R A T I O N C O L U M N IN S T A N D . . . . 1 1 2 4 5 . IMPROVED P H O T O T R A N S I S T O R D E T E C T O R FOR A M I N O A C I D A N A L Y S E R C I R C U I T D I A G R A M 1 2 3 46. P H O T O T R A N S i S T O R - L O G A M P L 1 F i E R D E T E C T O R FOR A M I N O A C I D A N A L Y S E R — C I R C U I T D I A G R A M . . . . . . . . . . 1 2 6 4 7 . Ml A G R A T I ON O F D A N S Y L D E R I V A T I V E S O F A M I N O A C I D S ON P O L Y A M I D E L A Y E R S IN V A R I O U S S O L V E N T S 1 3 0 X I I ACKNOWLEDGEMENTS I WOULD L I K E TO T H A N K MY S U P E R V I S O R , DR. C O . P A R K E S , FOR H I S C O N T I N U A L H E L P AND G U I D A N C E D U R I N G T H E E X P E R I M E N T A L WORK AND FOR H I S V A L I A N T E F F O R T S TO P R E V E N T T H I S T H E S I S FROM B E C O M I N G A N O V E L . I WOULD A L S O L I K E TO T H A N K DR. D.H. C O P P FOR H I S I N T E R E S T AND E N T H U S I A S M , AND DR. J.C. BROWN FOR H I S A D V I C E AND E N C O U R A G E M E N T . I AM G R A T E F U L TO T H E O T H E R M E M B E R S O F T H E F A C U L T Y O F T H E D E P A R T M E N T O F P H Y S I O L O G Y FOR T H E I R I N S T R U C T I O N D U R I N G MY G R A D U A T E S T U D I E S AND TO T H E M E M B E R S O F T H E S T A F F FOR T H E I R A S S I S T A N C E . S P E C I A L T H A N K S A R E D U E TO DR. K . J . S T E V E N S O N FOR H I S M E T H O O I C A L A D V I C E , TO MR. M.A. COOK F O R H I S C R I T I C A L P R E S E N C E , AND TO MRS. F R A N C I S NEWSOME, MRS. J A N E C O R D Y - S I M P S O N AND MRS. K A T H Y P E R R Y FOR T H E I R A S S I S T A N C E W I T H B I O A S S A Y S . I AM P E R M A N E N T L Y I N D E B T E D TO MR. K U R T H E N Z E AND MR. R A L P H A S S I N N A FOR T H E I R I N V A L U A B L E A I D IN T H E P R E P A R A T I O N OF TH E I L L U S T R A T I O N S IN T H I S T H E S I S AND FOR O T H E R M A T E R I A L C O N T R I B U T I O N S TOO N U M E R O U S "TO M E N T I O N . F I N A L L Y , I WOULD L I K E TO T H A N K MY W I F E , J A N E T , FOR H E R P A T I E N C E AND H E R E F F O R T S I N P R E P A R I N G AND T Y P I N G T H I S T H E S I S . F I N A N C I A L A S S I S T A N C E FROM T H E M E D I C A L R E S E A R C H C O U N C I L IN T H E FORM OF A S T U D E N T S H I P I S G R A T E F U L L Y A C K N O W L E D G E D . X I I I I . I N T R O D U C T I O N THE F I R S T C L E A R E V I D E N C E FOR E N D O C R I N E C O N T R O L O F T H E C A L C I U M C O N C E N T R A T I O N I N P L A S M A C A M E FROM T H E WORK O F M A C C A L L U M AND V O E G T L I N IN 1 9 0 9 (71) W H I C H L I N K E D T H E T E T A N Y F O L L O W I N G P A R A T H Y R O I D E C T O M Y TO A F A L L IN B L O O D C A L C I U M . IT WAS 1 6 Y E A R S B E F O R E T H E E N D O C R I N E F U N C T I O N O F T H E P A R A T H Y -R O I D S WAS C O N F I R M E D BY E X T R A C T I O N O F AN A C T I V E P A R A T H Y R O I D HORMONE ( P T H ) BY C O L L I P I N 1 9 2 5 ( 2 3 ) AND A F U R T H E R 3 4 Y E A R S U N T I L T H E HORMONE WAS I S O L A T E D BY A U R B A C H ( 6 ) AND R A S M U S S E N AND C R A I G ( 1 0 1 ) IN 1 9 5 9 . A L T H O U G H A F A I R L Y C L E A R P I C T U R E O F T H E F U N C T I O N O F T H E HORMONE HAS E M E R G E 0 ( 4 2 , 9 3 * 5 » 1 2 0 ) T H E C H E M I S T R Y O F T H E HORMONE HAS P R O V E D E X T R E M E L Y D I F F I C U L T , AND O N L Y T H E A M I N O A C I D C O M P O S I T I O N O F P O R C I N E P T H ( 7 0 ) AND A P A R T I A L S E Q U E N C E O F B O V I N E P T H ( 9 5 ) A R E A V A I L A B L E AT P R E S E N T . THE R A T E AT W H I C H K N O W L E D G E OF T H E S E C O N D C A L C I U M R E G U -L A T I N G HORMONE, C A L C I T O N I N ( C T ) , I S A C C U M U L A T I N G I S V E R Y MUCH G R E A T E R . A L T H O U G H I T WAS O N L Y R E C O G N I Z E D BY C O P P E T A L ( 3 0 , 2 7 ) I N 1 9 6 1 , S Y N T H E T I C HORMONE WAS P R E P A R E D IN 1 9 6 8 . TWO Y E A R S A F T E R T H E R E C O G N I T I O N O F C T , T H E P R E P A R A T I O N O F A C T I V E E X T R A C T S FROM RAT T H Y R O I D BY H L R S C H E_T A L _ ( 5 6 ) L E D A NUMBER OF L A B O R A T O R I E S TO B E G I N WORK ON I S O L A T I O N O F T H E HORMONE FROM M A M M A L I A N T H Y R O I D T I S S U E ( 1 2 7 » 9 9 » 9 8 , 6 2 , 4 1 T 9 ) - T H I S WORK R E S U L T E D IN T H E S E Q U E N C I N G ( 8 , 9 6 , 8 2 ) AND S Y N T H E S I S ( 5 0 , 1 0 7 ) OF T H E HORMONE FROM P O R C I N E T H Y R O I D - 2 -T I S S U E AND EVENTUALLY TO SEQUENCES FOR HUMAN C T (81) AND BOVINE C T (17). WHILE THE WORK ON THE CHEMISTRY OF THE MAMMALIAN HORMONE WAS IN PROGRESS, STUDIES ON O R I G I N , CONTROL AND FUNCTION PROCEDED APACE AND CONSIDERABLE INFORMATION ON THESE P R O P E R T I E S OF THE HORMONE ARE NOW A V A I L A B L E ( 5 7 , 1 0 2 , 1 2 6 , 1 2 5 ) . STUDIES ON THE CELL OF ORIGIN FOR C T LED TO EVIDENCE THAT C T WAS PRESENT IN NON-MAMMALS L A C K I N G P T H , AND IT IS NOW APPARENT THAT THIS "SECOND" HORMONE I S P H Y L 0 G E N E T I C A L L Y MORE PR I M A T I V E THAN P T H . C T MAY WELL HAVE DEVELOPED IN EARLY MARINE VERTE-BRATES AS A MEANS OF M A I N T A I N I N G CALCIUM CONCENTRATIONS IN THE BODY F L U I D S AT L E V E L S LOWER THAN THE SURROUNDING SEAWATER ( 2 5 ) , WHILE P T H ACTS P R I M A R I L Y TO MAINTAIN HIGH L E V E L S OF C I R C U L A T I N G CALCIUM IN LAND VERTEBRATES FACED WITH AN E N V I -RONMENT HIGH IN PHOSPHATES AND R E L A T I V E L Y LOW IN CALCIUM- ( 2 4 ) • THE INTERACTION OF THESE TWO HORMONES I S PROBABLY RESPON-S I B L E FOR THE CLOSE REGULATION OF PLASMA CALCIUM L E V E L S IN MAN WHERE THE OIURNAL FLUCTUATIONS ARE GENERALLY L E S S THAN ± 5e/o ( 2 1 ) . INCREASES IN PLASMA CALCIUM L E V E L S OF THIS MAG-NITUDE ( 5 $ ) HAVE BEEN SHOWN TO RESULT IN S I G N I F I C A N T R I S E S IN THE SECRETION RATE OF P T H ( 9 4 ) AND IN ITS C I R C U L A T I N G LEVEL ( 4 ) . S I M I L A R L Y , DECREASES IN PLASMA CALCIUM L E V E L S RESULT IN AN INCREASE IN BOTH THE SECRETION ( 2 0 ) AND THE C I R C U L A T I N G L E V E L OF C A L C I T O N I N ( 4 ) . AN INCREASE IN P T H IN TURN ACTS TO RELEASE- CALCIUM STORED IN B 0 N E ( 1 0 0 ) » TO INCREASE CALCIUM ABSORPTION IN THE GUT ( 3 3 ) * TUBULAR REABSORPTI ON OF CALCIUM IN THE KIDNEY ( 9 0 ) ANO TO INCREASE URINARY EXCRETION OF P H O S P H A T E ( 2 3 ) . A L L O F T H E S E A C T I O N S T E N D TO I N C R E A S E P L A S M A C A L C I U M L E V E L S ANO LOWER P L A S M A P H O S P H A T E L E V E L S . AN I N C R E A S E I N C A L C I T O N I N ON T H E O T H E R HAND A P P A R E N T L Y A C T S P R I M A R I L Y TO I N H I B I T B O N E R E S O R P T I O N ( 2 , 1 0 0 ) R E S U L T I N G I N A D E C R E A S E IN P L A S M A C A L C I U M . T H E T I M E C O U R S E O F T H E R E S P O N S E TO PTH I S R A T H E R SLOW AND I T I S G E N E R A L L Y N E A R L Y AN HOUR B E F O R E T H E E F F E C T S O F I T S A D M I N I S T R A T I O N ON P L A S M A C A L C I U M L E V E L S CAN B E O B S E R V E D . T H E M A X I M A L E F F E C T MAY NOT O C C U R FOR S E V E R A L H O URS AND MAY P E R S I S T FOR 2 4 HOURS OR MORE ( 2 4 ) . IN C O N T R A S T C A L C I T O N I N A C T S R A P I D L Y ANO T H E M A X I M A L R E S P O N S E MAY O C C U R IN L E S S T H A N ONE HOUR W I T H A R E T U R N TO NORMAL IN A M A T T E R O F A FEW HOURS ( 3 1 ) . T H E P I C T U R E O F C A L C I U M H O M E O S T A S I S T H A T T H I S S U G G E S T S I S ONE O F E X T R E M E L Y F I N E C O N T R O L B A S E O ON TWO N E G A T I V E F E E D B A C K S Y S T E M S A C T I N G IN O P P O S I T I O N T O . M A I N T A I N P L A S M A C A L C I U M A T A P R E C I S E S E T P O I N T . O N E C O M P O N E N T A C T S O V E R T H E L O N G TERM TO R A I S E P L A S M A C A L C I U M AND T H E S E C O N D A C T S R A P I D L Y TO LOWER P L A S M A C A L C I U M A G A I N S T T H E B A C K G R O U N D O F T H E S L O W E R C O M P O N E N T T H U S A C H I E V I N G T H E P R E C I S E C O N T R O L O B S E R V E D IN T H E I N T A C T A N I M A L . AS E L E G A N T A S T H I S S Y S T E M I S T H E R E I S S T I L L SOME D O U B T A B O U T I T S V A L I D I T Y A S I T HAS P R O V E D O I F F I C U L T TO D E M O N S T R A T E A P H Y S I O L O G I C A L N E E D FOR C A L C I T O N I N . U N T I L R E C E N T L Y T H E PTH S Y S T E M HAD P R O V E D C A P A B L E O F M A I N T A I N I N G P L A S M A C A L C I U M L E V E L S U N D E R A L L S T R E S S E S E X C E P T S U C H U N N A T U R A L P R O C E D U R E S AS I N F U S I O N O F C A L C I U M AND P E R I T O N E A L L A V A G E S W I T H H I G H C A L C I U M S O L U T I O N S ( 1 1 4 ) . GRAY AND MUNSON ( 4 6 ) H A V E NOW SHOWN T H A T T H Y R O I D E C T O M I Z E D R A T S W I T H F U N C T I O N I N G P A R A T H Y R O I D S MAY SHOW S I G N I F I C A N T P L A S M A - 4 -C A L C I U M E L E V A T I O N F O L L O W I N G H I G H C A L C I U M M E A L S . T H U S T H E R E A R E O B V I O U S P A R A L L E L S W I T H T H E I N S U L I N - G L U C A G 0 N S Y S T E M . HOW-E V E R , T H I S S T I L L A P P E A R S TO BE A R E L A T I V E L Y M I N O R R O L E , AND T H E F A C T T H A T C T CAN BE D E T E C T E D IN S I G N I F I C A N T Q U A N T I T I E S I N A N I M A L S W I T H NORMAL P L A S M A C A L C I U M L E V E L S ( 4 ) S U G G E S T S T H A T I T MAY H A V E A D D I T I O N A L I M P O R T A N C E . AMONG T H E P O S S I B I L I T I E S S U G G E S T E D H A V E B E E N A R O L E I N I N C R E A S I N G B O N E D E P O S I T I O N I N F R A C T U R E H E A L I N G I N A D U L T S ( 3 5 ) AND I N T H E F O R M A T I O N O F NEW B O N E I N U T E R O ANO I N E A R L Y L I F E ( 4 3 ) . T H | S S E C O N D P O S S I -B I L I T Y I S S U P P O R T E D BY T H E O B S E R V A T I O N S T H A T T H E R E S P O N S E TO C T I S V E R Y A G E D E P E N D E N T AND T H A T Y O U N G A N I M A L S H A V E A MUCH G R E A T E R S E N S I T I V I T Y TO C T T H A N O L D ( 3 1 ) . T H I S CAN B E R E A D I L Y E X P L A I N E D I N T E R M S O F C A L C I T O N I N ' S I N H I B I T I O N O F B O N E R E S O R P -T I O N , B U T T H I S D O E S NOT D E T R A C T FROM I T S P O S S I B L E R O L E . T H E R E I S A L S O E V I D E N C E T H A T C T I N F L U E N C E S T H E R A T E O F C A L C I U M T R A N S -P O R T A C R O S S T H E M E M B R A N E S O F V A R I O U S C E L L S ( 1 2 , 1 0 4 ) S U G G E S T I N G T H A T I T MAY P L A Y A R O L E I N T H E C O N T R O L O F I N T R A C E L L U L A R AS W E L L A S E X T R A C E L L U L A R C A L C I U M L E V E L S . E L U C I D A T I O N O F T H E R O L E S O F T H E S E E N D O C R I N E S Y S T E M S I N C A L C I U M M E T A B O L I S M AND H O M E O S T A S I S C L E A R L Y R E Q U I R E S C H A R A C T E R I Z A T I O N O F T H E I M P O R T A N C E O F T H E S E AND O T H E R P O S S I B L E A C T I O N S O F C T . T H E A N A L Y S I S O F T H E P H Y L O G E N E T I C O R I G I N O F V A R I O U S H O R M O N E S HAS P R O V I D E D C O N S I D E R A B L E I N S I G H T I N T O T H E I R F U N C T I O N ( 4 5 » 1 1 1 ) » B U T L I T T L E I N F O R M A T I O N A L O N G T H E S E L I N E S WAS A V A I L -A B L E FOR C T . S U C H A N A L Y S I S S H O U L D I N D I C A T E HOW T H E HORMONE F U N C T I O N S I N O T H E R S P E C I E S ANO T H E I M P O R T A N C E O F S U C H F U N C T I O N S IN MAMMALS C O U L D T H E N B E A S S E S S E D . IN 1 9 6 7 P E A R S E AND C A R V A L H E I R A ( 9 1 ) SHOWED T H A T T H E P A R A F O L L I C U L A R OR "C" C E L L S O F T H E M A M M A L I A N T H Y R O I O A R I S E E M B R Y O L O G I C A L L Y FROM T H E T E R M I N A L B R A N C H I A L P O U C H W H I C H IN LOWER V E R T E B R A T E S F O R M S A S E P A R A T E G L A N D , T H E U L T I M O B R A N C H I A L . IN T H E S A M E Y E A R CoPP E T A L ( 2 8 , 2 9 * 3 2 ) AND T A U B E R ( 1 2 4 ) E X T R A C T E D P O T E N T H Y P O C A L C E M I C F A C T O R S FROM T H E U L T I M O B R A N C H I A L G L A N O S O F S E V E R A L N O N - M A M M A L I A N V E R T E B R A T E S , I N C L U D I N G T H E C H I C K E N AND T H E D O G F I S H , B U T T H E R E L A T I O N S H I P B E T W E E N T H E S E U L T I M O B R A N C H I A L F A C T O R S AND T H E M A M M A L I A N C A L C I T O N I N S WAS E S S E N T I A L L Y UNKNOWN. T H E WORK D E S C R I B E D I N T H E P R E S E N T S T U D Y B E G A N A T T H I S P O I N T , AND WAS A I M E D A T A C H E M I C A L C H A R A C T E R I Z A T I O N O F T H E U L T I M O B R A N C H I A L F A C T O R S TO E S T A B L I S H T H E I R R E L A T I O N S H I P TO T H E M A M M A L I A N C A L C I T O N I N S . T H E B E S T A P P R O A C H A P P E A R E D TO BE A S U R V E Y O F C E R T A I N B A S I C F E A T U R E S O F T H E H Y P O C A L C E M I C F A C T O R S FROM A N U M B E R O F S P E C I E S , F O L L O W E D BY A D E T A I L E D S T U D Y O F O N E O F T H E U L T I M O B R A N C H I A L F A C T O R S TO A L L O W C O M P A R I S O N O F I T S S T R U C T U R E TO T H A T O F P O R C I N E C T . A T T H E T I M E T H I S WORK WAS B E G U N T H E A M I N O A C I D S E Q U E N C E O F T H E P O R C I N E M O L E C U L E WAS S T I L L UNKNOWN, B U T I N T E N S I V E S T U D I E S WERE UNDERWAY I N A N U M B E R O F L A B O R A T O R I E S S U G G E S T I N G T H A T T H I S I N F O R M A T I O N WOULD SOON 8 E C 0 M E A V A I L A B L E . T H E D I R E C T I O N O F A N Y F U R T H E R S T U D I E S D E P E N D E D ON T H E O U T C O M E O F T H I S C O M P A R A T I V E S E Q U E N C E WORK, AND W H E T H E R T H E R E WERE S I G N I F I C A N T D I F F E R E N C E S OR S I M I L A R I T I E S B E T W E E N T H E U L T I M O -B R A N C H I A L AND T H Y R O I D C A L C I T O N I N S . T H E F I N A L C H A P T E R D E S C R I B E S A S E R I E S O F E X P E R I M E N T S D E S I G N E D TO C L A R I F Y T H E R E L A T I O N S H I P O F T H E S T R U C T U R A L D I F F E R E N C E S F O U N D TO T H E D I F F E R E N C E S I N B I O L O G I C A L A C T I V I T Y O B S E R V E D . I I . M O L E C U L A R W E I G H T S O F C A L C I T O N I N S E X T R A C T E D F R O M U L T I M O - . B R A N C H I A L A N D T H Y R O I D T I S S U E S . * A . I N T R O O U C T I ON. T H E WORK O F C O P P ( 2 8 , 2 9 ) AND T A U B E R ( 1 2 4 ) HAD I N D I C A T E D T H A T T H E U L T I M O B R A N C H I A L B O D I E S O F C E R T A I N LOWER V E R T E B R A T E S C O N T A I N E D H Y P O C A L C E M I C A C T I V I T Y S I M I L A R TO T H A T O F C T FROM T H E M A M M A L I A N T H Y R O I D , B U T F U R T H E R E V I D E N C E WAS N E C E S S A R Y TO C O N F I R M T H A T T H E S E F A C T O R S WERE C H E M I C A L L Y S I M I L A R TO T H E M A M M A L I A N H O R M O N E S . T H E R E WAS C O N S I D E R A B L E I N F O R M A T I O N S U G G E S T I N G T H A T P O R C I N E C T HAD A M O L E C U L A R W E I G H T O F A B O U T 3000 (49» 9 9 ) AND I T WAS F E L T T H A T C O M P A R I S O N O F T H E M O L E C U L A R W E I G H T S O F U L T I M O B R A N C H I A L F A C T O R S AND T H Y R O I O C A L C I T O N I N S WOULD P R O V I D E S U C H E V I D E N C E . T H I S T Y P E O F C O M P A R I S O N C O U L D B E MADE F A I R L Y E A S I L Y BY C H R O M A T O G R A P H Y O F E X T R A C T S FROM T H E V A R I O U S G L A N D S ON A S E P H A D E X C O L U M N W H I C H HAD B E E N C A L I B R A T E D W I T H M A R K E R S O F KNOWN M O L E C U L A R W E I G H T S ( 3 ) U S I N G T H E B I O A S S A Y F O R H Y P O C A L C E M I C A C T I V I T Y TO D E T E R M I N E T H E E L U T I O N V O L U M E O F T H E A C T I V E M O L E C U L E S . P R E L I M I N A R Y C H R O M A T O G R A P H Y O F E X T R A C T S O F P O R C I N E T H Y R O I D AND D O G F I S H AND C H I C K E N U L T I M O B R A N C H I A L G L A N D S ON S E P H A D E X G - 7 5 ( 3 2 ) HAD SHOWN T H A T A L L O F T H E ! A C T I V E M O L E C U L E S WERE E L U T E D V E R Y N E A R T H E T O T A L V O L U M E ( V T ) O F T H E C O L U M N , AND S I N C E T H E R E L A T I O N S H I P B E T W E E N E L U T I O N V O L U M E AND T H E L O G O F T H E M O L E C U L A R W E I G H T B E C O M E S N O N - L I N E A R N E A R ( 3 ) T H E L E S S P O R O U S S E P H A D E X G - 5 0 WAS C H O S E N F O R M O L E C U L A R W E I G H T * P A R T O F T H E WORK D E S C R I B E D I N T H I S C H A P T E R WAS P R E V I O U S L Y P U B L I S H E D ( 8 6 ) . -6-E S T I M A T I O N . U N F O R T U N A T E L Y T H E E L U T I O N V O L U M E - M O L E C U L A R W E I G H T R E L A T I O N S H I P FOR T H I S S E P H A O E X HAD NOT B E E N D E S C R I B E O A D E Q U A T E L Y IN T H E L I T E R A T U R E AND T H E N U M B E R O F M A R K E R S A V A I L -A B L E I N T H E P R O P E R W E I G H T R A N G E WAS L I M I T E D . I T WAS T H E R E F O R E N E C E S S A R Y TO C H E C K T H E S M A L L M A R K E R S W H I C H WERE A V A I L A B L E A G A I N S T O T H E R L A R G E R M A R K E R S ON T H E B E T T E R C H A R A C T E R I Z E D S E P H A D E X G-75 TO V E R I F Y T H A T T H E Y WOULD Y I E L D T H E D E S I R E D L I N E A R R E L A T I O N S H I P . T H E M O L E C U L A R W E I G H T S T U D I E S R E Q U I R E D A C T I V E E X T R A C T S FROM T H E T H Y R O I D AND U L T I M O B R A N C H I A L G L A N D S O F A V A R I E T Y O F S P E C I E S , ANO P R E P A R A T I O N O F T H E S E E X T R A C T S P R O V I D E D AN O P P O R T U N I T Y TO E V A L U A T E E X T R A C T I O N P R O C E D U R E S AND D E T E R M I N E T H E P O T E N T I A L S O F V A R I O U S U L T I M O B R A N C H I A L T I S S U E S A S P O S S I B L E S O U R C E S O F C T FOR F U R T H E R D E T A I L E D S T U D I E S . T H E E A R L I E R WORK ON U L T I M O B R A N C H I A L T I S S U E S HAD B E E N B A S E D ON A C I D E X T R A C T I O N S , A P R O C E D U R E W H I C H I S U S E F U L ON A S M A L L S C A L E B E C A U S E O F I T S S I M P L I C I T Y B U T W H I C H P R O D U C E S A N U M B E R O F P R O B L E M S WHEN S C A L E D U P . R A S M U S S E N EJR_ AL. ( 1 0 3) D I S C U S S E D S U C H P R O B L E M S AS P A R T I A L H Y D R O L Y S I S AND I N C O M P L E T E D I S S O C I A T I O N O F P E P T I D E S FROM T H E T I S S U E IN T H E I R R E P O R T ON T H E E X T R A C T I O N O F P T H , AND T H E S E S A M E P R O B L E M S A P P L Y TO T H E E X T R A C T I O N O F C T . AN E V E N G R E A T E R DRAWBACK TO U S E ON A L A R G E S C A L E I S T H A T S U C H E X T R A C T I O N S Y I E L D L A R G E V O L U M E S O F A Q U E O U S S O L U T I O N S W H I C H A R E D I F F I C U L T TO H A N D L E AND S I N C E T H E M E T H O D I S R E L A T I V E L Y U N S E -L E C T I V E , G E N E R A L L Y C O N T A I N N U M E R O U S P R O T E I N C O M P O N E N T S , G U D M U N D S S O N E__T _AL_ ( 4 8 ) HAD D E V E L O P E D A S O L V E N T E X T R A C T I O N P R O C E D U R E FOR P O R C I N E C T W H I C H P R O D U C E D A H I G H L Y P U R I F I E D -8-P R O D U C T I N A C O N V E N I E N T FORM, AND P R E L I M I N A R Y WORK ( 3 2 ) I N D I C A T E D T H A T I T WOULD A L S O BE S U I T A B L E FOR U L T I M 0 B R A N C H I A L M A T E R I A L S . TH I S R E L A T I V E L Y S E L E C T I V E P R O C E D U R E HAD T H E A D D E D A D V A N T A G E O F P R O V I D I N G F U R T H E R E V I D E N C E O F T H E S I M I L A R I T Y O F T H E C A L C I T O N I N S FROM V A R I O U S S O U R C E S . S E L E C T I O N O F T H E S P E C I E S TO B E S T U D I E D WAS B A S E D P R I M A R I L Y ON T H E A V A I L A B I L I T Y O F A N I M A L S , T H E E A S E OF G L A N D R E M O V A L ANO T H E S P E C I F I C A C T I V I T Y O F T H E G L A N D S AS S E E N I N A C I D E X T R A C T I O N S S I N C E O N E O F T H E S P E C I E S WOULD E V E N T U A L L Y BE S E L E C T E D FOR F U R T H E R S T U D Y AND L A R G E Q U A N T I T I E S O F S T A R T I N G M A T E R I A L WOULO B E N E E D E D . AN E F F O R T WAS A L S O MADE TO S E L E C T A F A I R L Y B R O A D S P E C T R U M O F S P E C I E S SO T H A T T H E I N F O R M A T I O N O B T A I N E O WOULD BE G E N E R A L L Y A P P L I C A B L E . B . E X T R A C T I O N S . 1. M E T H O D S . A. I S O L A T I O N AND P R E P A R A T I O N O F G L A N D S . THE I N I T I A L S T E P P R I O R TO HORMONE E X T R A C T I O N WAS T H E C O L L E C T I O N O F S U F F I C I E N T Q U A N T I T I E S O F T H Y R O I D AND U L T I M O -B R A N C H I A L T I S S U E FROM T H E V A R I O U S S P E C I E S TO Y I E L D A D E Q U A T E P R O D U C T FOR S T U D Y . T H E TWO A V I A N S P E C I E S S T U D I E D , T U R K E Y ( M E L E A G R A G A L L A P A V O ) AND D O M E S T I C FOWL ( G A L L U S D O M E S T I C U S ) A R E B O T H R A I S E D C O M M E R C I A L L Y AND T H E G L A N D S C O U L D BE C O L L E C T E D AT T H E P R O C E S S I N G P L A N T AS T H E Y FORM A P A R T O F T H E V I S C E R A W H I C H AR E N O R M A L L Y D I S C A R D E D . T H E U L T I M 0 B R A N C H I A L G L A N D S IN B O T H B I R D S A R E B I L A T E R I A L AND L I E A L O N G T H E COMMON C A R O T I D A R T E R I E S N E A R T H E O R I G I N O F T H E S U B C L A V I A N A R T E R Y . T H E I R R E L A T I O N S H I P TO T H E TWO P A R A T H Y R O I O G L A N D S ANO T H E T H Y R O I O I S Esophagus Jugular Vein Carotid Thyroids Parathyroids Ultimobranchials Heart F I G U R E 1. L O C A T I O N O F A V I A N U L T I M O B R A N C H I A L G L A N D S . - 9 -I N D I C A T E D I N F I G U RE 1. T H E G L A N D S WERE S E P A R A T E D FROM S U R R O U N O I N G T I S S U E ANO F R O Z E N ON ORY I C E . T H E S A L M O N G L A N D S WERE A L S O C O L L E C T E D AT A C O M M E R C I A L P R O C E S S I N G P L A N T . WHEN T H E H E A D S O F T H E F I S H A R E R E M O V E D T H E T R A N S V E R S E S E P T U M I S E X P O S E D A S T H E P O S T E R I O R E N D O F T H E H E A D P O R T I O N A L L O W I N G R E L A T I V E L Y E A S Y I S O L A T I O N O F T H E G L A N D U L A R M A T E R I A L , W H I C H F O R M S A D I F F U S E B A N D O F T I S S U E ON T H E A N T E R I O R S U R F A C E O F T H E S E P T U M . T H E M A J O R I T Y O F T H E G L A N O U L A R T I S S U E L I E S B E T W E E N T H E D I A P H R A G M AND T H E S I N U S V E N O S U S S L I G H T L Y V E N T R A L TO T H E E S O P H A G U S . B E C A U S E O F T H E D I F F U S E N A T U R E O F T H E G L A N D T H E E N T I R E S E P T U M WAS R E M O V E D , F R O Z E N AND E X T R A C T E D . MOST O F T H E S A L M O N P R O C E S S E O WERE CHUM, S O C K E Y E OR C O H O ( O N C E R H Y N C H U S K E T A , K I S U T C H OR N E R K A ) AND NO E F F O R T WAS MAOE TO D I F F E R E N T I A T E B E T W E E N T H E S E S P E C I E S . D O G F I S H ( S Q U A L U S S U C K L E Y I ) A R E COMMON I N L O C A L W A T E R S AND WERE C A U G H T AND P R O C E S S E O C O M M E R C I A L L Y F O R A T I M E . D U R I N G T H I S P E R I O D G L A N O S WERE C O L L E C T E D FROM T H E C A R C A S S E S O F P R O C E S S E D F I S H , B U T WHEN T H E F E D E R A L S U B S I D Y FOR T H I S T Y P E O F F I S H I N G WAS T E R M I N A T E D I T B E C A M E N E C E S S A R Y TO C O L L E C T G L A N O S FROM WHOLE F I S H P A C K E D IN I C E AND D E L I V E R E D TO T H E L A B O R A T O R Y . T H I S S I T U A T I O N WAS L E S S T H A N S A T I S F A C T O R Y , B U T S I N C E S Q U A L U S WAS T H E MOST P R I M A T I V E G E N U S SHOWN TO P R O D U C E H Y P O C A L C E M I C F A C T O R S I T WAS E S S E N T I A L T H A T I T BE S T U D I E D . T H E U L T I M O -B R A N C H I A L G L A N D C O N S I S T S O F N U M E R O U S V A S C U L A R F O L L I C L E S L O C A T E D B E N E A T H T H E F L O O R O F T H E P H A R Y N X ON T H E L E F T S I D E A T T H E J U N C T U R E W I T H T H E E S O P H A G U S . I T I S P O S I T I O N E D B E T W E E N T H E P H A R Y N X AND T H E P E R I C A R D I U M I N T H E T R I A N G L E F O R M E D B Y T H E - 1 0 -B A S I B R A N C H I A L AND C E R A T 0 B R A N C H I A L C A R T I L A G E S AND T H E C 0 R A C 0 -B R A N C H I A L M U S C L E . A S W I T H T H E S A L M O N I S O L A T I O N I T I S D I F F I -C U L T AND L A R G E A M O U N T S O F C O N N E C T I V E T I S S U E A R E P R E S E N T I N T H E M A T E R I A L E X T R A C T E D . B E C A U S E O F T H E H I G H P R O T E O L Y T I C E N Z Y M E A C T I V I T Y IN T H E T H Y R O I D I T WAS I M P E R A T I V E T H A T T H E S E G L A N D S BE F R O Z E N IMME-D I A T E L Y A F T E R R E M O V A L . FOR T H I S R E A S O N T H E S A M P L E O F HUMAN T H Y R O I D E X T R A C T WAS O B T A I N E D D U R I N G A P A R T I A L T H Y R O I D E C T O M Y ON A P A T I E N T AT T H E V A N C O U V E R G E N E R A L H O S P I T A L AND F R O Z E N ON DRY I C E . T H E R A T T H Y R O I D S WERE F R O Z E N ON DRY I C E I M M E D I A T E L Y A F T E R R E M O V A L FROM T H E A N I M A L . P O R C I N E AND B O V I N E E X T R A C T S WERE P R E P A R E D FROM C O M M E R C I A L A C E T O N E D R I E D , OE FA T T E 0 T H Y R O I O POWOERS P U R C H A S E O FROM W I L S O N L A B O R A T O R I E S , C H I C A G O , I L L I N O I S . I N A L L C A S E S WHERE F R E S H OR F R O Z E N G L A N D S WERE U S E D T H E T I S S U E S WERE H O M O G E N I Z E D , D R I E D ANO D E F A T T E D P R I O R TO E X T R A C T I O N . TWO M E T H O D S WERE U S E D F O R T H I S . T H E F I R S T M E T H O D WAS A S T A N D A R D A C E T O N E D R Y I N G P R O C E O U R E I N W H I C H G L A N D S WERE H 0 M 0 G E N I Z E 0 I N A W A R I N G B L E N D E R I N C O L O (4° C ) A C E T O N E . T H E H O M O G E N A T E WAS T H E N C E N T R I F U G E D AND T H E S U P E R N A T E D I S C A R D E D . T H E P R E C I P I T A T E WAS WASHED W I T H F R E S H , C O L D A C E T O N E ., AND R E C E N T R I F U G E D . T H I S W A S H I N G P R O C E D U R E WAS R E P E A T E D A N U M B E R O F T I M E S U N T I L T H E A D D I T I O N O F WATER TO T H E S U P E R N A T E NO L O N G E R C A U S E D C L O U D I N G , I N D I C A T I N G T H E A B S E N C E O F L I P I D FROM T H E A C E T O N E . T H E R E M A I N I N G M A T E R I A L , WHEN D R I E D I N A S T R E A M O F A I R I N A B U C H N E R F U N N E L , P R O D U C E D A L I G H T , B U F F -C O L O R E D POWDER R E A D Y FOR E X T R A C T I O N . - 1 1 -A C E T O N E D R Y I N G R E Q U I R E D L A R G E A M O U N T S O F T I M E AND M A T E R I A L S , AND WAS NOT R E A L L Y S U I T A B L E FOR L A R G E S C A L E E X T R A C -T I O N S . T H I S WAS E S P E C I A L L Y T R U E FOR T H E A V I A N G L A N O S W H I C H WERE E M B E D D E D IN A D I P O S E T I S S U E T H A T C O U L D O N L Y B E R E M O V E D W I T H G R E A T D I F F I C U L T Y . O T H E R M E T H O D S WERE E X A M I N E D , AND T H E M OST A T T R A C T I V E O F T H E S E A P P E A R E D TO BE T H A T O F E . L E V I N (69)» W H I C H U S E D 1 , 2 - 0 I C H L O R O E T H A N E TO E X T R A C T T H E L I P I D . A F T E R T H E G L A N D S HAD B E E N H O M O G E N I Z E D IN T H E S O L V E N T , T H E M I X T U R E WAS D I S T I L L E D U N D E R R E D U C E O P R E S S U R E AND A L O W - B O I L I N G , S O L V E N T - W A T E R A Z E O T R O P E R E M O V E D . T H E R E M A I N I N G S O L V E N T C O N T A I N I N G T H E L I P I D WAS R E M O V E D FROM T H E P E L L E T - L I K E G L A N -D U L A R M A T E R I A L BY F I L T R A T I O N AND R E D I S T I L L E D A L O N G W I T H T H E I M M I S I B L E S O L V E N T L A Y E R FROM T H E A Z E O T R O P I C M I X T U R E TO R E C O V E R T H E S O L V E N T . T H E P E L L E T S WERE WASHED W I T H S O L V E N T ON T H E F I L T E R F U N N E L AND A I R D R I E D . B. E X T R A C T I O N P R O C E D U R E S . THE D R I E D , D E F A T T E D POWDERS WERE E X T R A C T E D , U S I N G M O D I -F I C A T I O N S O F ; T H E M E T H O D D E S C R I B E D BY G U D M U N D S S O N E_T AL_ ( 4 8 ) . IN T H E I N I T I A L E X T R A C T I O N AND P R E C I P I T A T I O N S T A G E W H I C H WAS U S E D IN A L L E X P E R I M E N T S , 1 0 0 G O F POWDER I S S T I R R E D G E N T L Y FOR 1 2 HOURS A T ROOM T E M P E R A T U R E IN 1.5 L O F B U T A N O L : A C E T I C A C I D : WATER ( 7 5 : 7.5* 2 1 ) AND T H E N F I L T E R E D . THE R E S I D U E WAS S T I R R E D A G A I N IN 7 5 0 ML O F S O L V E N T F O R 4 H O U R S AND F I L T E R E D , T H E F I L T R A T E FROM T H E F I R S T TWO S T E P S WAS P O O L E D W I T H 3 0 0 ML O F S O L V E N T FROM A T H I R D WASH ANO C O O L E O TO - 1 0 ° C . F L V E V O L U M E S - 1 2 -O F A C E T O N E P R E V I O U S L Y C O O L E D TO - 1 0 ° C ( A P P R O X I M A T E L Y -1 2 . 5 L ) WAS S T I R R E D I N T O T H E P O O L E D F I L T R A T E AND T H E M I X T U R E A L L O W E D TO S T A N D FOR 4 8 H O U R S - A T - 1 0 ° C. T H E M A J O R I T Y O F T H E L I Q U I D C O U L D T H E N BE A S P I R A T E D O F F AND D I S C A R D E D . T H E R E M A I N I N G S L U R R Y WAS C E N T R I F U G E D AT - 1 0 ° C AND T H E S U P E R N A T E D I S C A R D E D . THE P R E C I P I T A T E WAS WASHED T W I C E IN A B O U T 2 L O F - 1 0 ° C A C E T O N E AND D R I E D I N T H E C E N T R I F U G E B O T T L E S U N D E R A S T R E A M O F N I T R O G E N . T H E R E S U L T A N T P R E C I P I T A T E C O U L D T H E N BE T R E A T E D IN S E V E R A L A L T E R N A T E WAYS. T H E P R O C E D U R E OF G U O M U N D S S O N EJT A L ( 4 8 ) WAS F O L L O W E D ON T H E B O V I N E AND P O R C I N E T H Y R O I D P O W D E R S . T H E D R I E D P R E C I P I T A T E WAS E X T R A C T E D W I T H 2 5 0 ML O F 70°/O E T H A N O L , P H 5.5» FOR 2 HOURS AND C E N T R I F U G E D . T H E P R E C I P I T A T E WAS T H E N R E S U S -P E N D E O I N 1 2 5 ML AND T H E P R O C E S S R E P E A T E D . T H E TWO S U P E R N A T E S WERE P O O L E D AND C O N C E N T R A T E D I N V A C U O TO A V O L U M E O F A P P R O X I -M A T E L Y 1 5 0 M L . T R I C H L O R O A C E T I C A C I D ( T C A ) , 1 0 0 $ w/v, WAS T H E N A D D E D TO A C O N C E N T R A T I O N O F 1 2 G / 1 0 0 ML, AND T H E M I X T U R E A L L O W E D TO S T A N D FOR 12 HOURS AT 4 ° C T H E P R E C I P I T A T E WAS WASHED W I T H 1 0 $ TCA (w/v) AND R E S U S P E N D E D IN 50 ML O F 0.5 M A C E T I C A C I D . T H E S U S P E N S I O N WAS T H E N A D D E D TO AN E Q U A L V O L U M E O F I R A - 4 0 0 A N I O N E X C H A N G E R E S I N , 2 0 - 4 0 M E S H , A C E T A T E FORM*, TO R E M O V E T H E TCA. WHEN T H E S O L U T I O N HAD C L E A R E D T H E S L U R R Y WAS F I L T E R E D AND T H E R E S I N WASHED W I T H 1 0 ML O F 0.5 M A C E T I C A C I D . T H E F I L T R A T E WAS L Y O P H I L I Z E D TO Y I E L D T H E F I N A L E X T R A C T . G U D M U N D S S O N U S E D A C E T O N E S E T H E R W A S H E S TO R E M O V E TCA. * T H E R E S I N WAS R E G E N E R A T E D U S I N G 3 W A S H E S I N 5 V O L U M E S O F 1 M S O D I U M H Y D R O X I D E , F O L L O W E D BY 3 D I S T I L L E O WATER W A S H E S . I T WAS T H E N C O N V E R T E D TO T H E A C E T A T E FORM BY 3 W A S H E S I N 5 M A C E T I C A C I D AND P R E P A R E D F O R TCA A B S O R P T I O N BY 3 WATER W A S H E S . - 1 3 -T H I S S A M E P R O C E D U R E WAS U S E O ON S M A L L E R Q U A N T I T I E S O F D R I E D - D E F A T T E D P OWOERS FROM D O G F I S H AND C H I C K E N U L T I M O B R A N C H I A L G L A N D S . T H E M E T H O D WAS E F F E C T I V E B U T T H E L A T E R S T E P S WERE T E C H N I C A L L Y D I F F I C U L T ON S M A L L E R Q U A N T I T I E S AND Y I E L D S WERE P O O R . T H E N E C E S S I T Y F O R W O R K I N G ON A S M A L L E R S C A L E L E D TO T H E F O L L O W I N G M O D I F I C A T I O N S . T H E A C E T O N E P R E C I P I T A T E WAS WASHED T W I C E W I T H C O L D ( 0 ° C ) A C E T O N E AND MOST OF T H E A C E T O N E R E M O V E D IN A S T R E A M O F A I R . T H E M O I S T POWDER WAS T H E N E X T R A C T E D W I T H T E N V O L U M E S O F 0.1 M F O R M I C A C I D AT ROOM T E M P E R A T U R E FOR 4 H O URS AND T H E S U P E R N A T E C O L L E C T E D . TWO F U R T H E R E X T R A C T I O N S U S I N G F I V E V O L U M E S AND TWO V O L U M E S O F 0.1 M F O R M I C A C I D F O R 2 H OURS AND 0.5 H O U R S R E S P E C T I V E L Y WERE P E R F O R M E D AND T H E T H R E E S U P E R N A T E S P O O L E D . L Y O P H I L I Z A T I O N Y I E L D E D T A N P OWOERS W H I C H D I S S O L V E D R E A D I L Y IN T H E B U F F E R S U S E O FOR C O L U M N C H R O M A T O G R A P H Y . U N L E S S S P E C I F I E D T H I S P R O C E D U R E WAS U S E D ON A L L P R E P A R A T I O N S D E S C R I B E D . c. B I O L O G I C A L A S S A Y P R O C E O U R E . T H E B I O L O G I C A L A C T I V I T Y O F T H E V A R I O U S E X T R A C T S WAS D E T E R M I N E D BY A M O D I F I C A T I O N O F T H E M E T H O D O F KUMAR E T A L (66) U S I N G 80-90 G, M A L E L O N G - E V A N S R A T S . M A T E R I A L S TO B E A S S A Y E D WERE S U I T A B L Y D I L U T E D IN AN I N J E C T I O N V E H I C L E O F 0.1% B O V I N E S E R U M A L B U M I N , I N 0.1 M S O D I U M A C E T A T E B U F F E R AT P H 4.5, AND I N J E C T E D I N T O T H E T A I L V E I N . B L O O D S A M P L E S WERE C O L L E C T E D FROM T H E T A I L V E I N 60 M I N U T E S A F T E R I N J E C T I O N ANO D U P L I C A T E P L A S M A C A L C I U M D E T E R M I N A T I O N S O B T A I N E D B Y A - 1 4 -M E T H O D S I M I L A R TO T H A T O F K E P N E R ANO H E R C U L E S ( 6 4 ) W H I C H HAD B E E N A U T O M A T E D U S I N G A T E C H N I C O N A U T O A N A L Y S E R ANO A T U R N E R F L O U R O M E T E R E Q U I P T : W I T H A FLOW C E L L A S D E S C R I B E D BY NEWSOME ( 8 3 ) . T H E M E A S U R E D P L A S M A C A L C I U M L E V E L S WERE T H E N C O M P A R E D W I T H T H O S E O F A S I M I L A R G R O U P O F C O N T R O L R A T S I N J E C T E D W I T H V E H I C L E O N L Y . T H E D I F F E R E N C E B E T W E E N T H E MEAN P L A S M A C A L C I U M L E V E L IN MG/O O F T H E C O N T R O L G R O U P AND T H E P L A S M A C A L C I U M L E V E L OF E A C H E X P E R I M E N T A L BLOOD S A M P L E WAS C A L C U L A T E D AND C A L L E D T H E R E S P O N S E . U S I N G T H I S R E S P O N S E AS T H E D E P E N D E N T V A R I A B L E ANO T H E L O G O F T H E D O S E A S . T H E I N D E P E N D E N T V A R I A B L E I T WAS P O S S I B L E TO C A L C U L A T E A S A M P L E R E G R E S S I O N O F Y ON X FOR E A C H S E R I E S OF D I L U T I O N A S D E S C R I B E D BY S N E D E C O R ( 1 1 6 ) • T H E S E C A L C U L A T I O N S P R O D U C E D T H E F A M I L Y O F L O G D O S E - R E S P O N S E C U R V E S SHOWN I N F I G U R E 4 W H I C H WERE U S E D IN E V A L U A T I N G T H E B I O A S S A Y S . C O M P A R I S O N O F T H E S E C U R V E S TO A C U R V E E S T A B L I S H E D F O R T H E H O U S E S T A N D A R D ( P O R C I N E C T , A L 0 8 3 1 , ARMOUR P H A R M A C E U T I C A L C O . ) AND C H E C K E D A G A I N S T T H E M R C R E S E A R C H S T A N D A R D A , ( W H I C H D E F I N E S T H E M R C U N I T ) Y I E L D E D V A L U E S FOR T H E A C T I V I T Y O F E A C H S A M P L E I N M R C M i L L I U N i T S / M G ( M U / M G ) . B E C A U S E O F V A R I A T I O N S I N T H E S L O P E S O F T H E C U R V E S ANO T H E I N H E R E N T I N A C C U R A C I E S O F T H E B I O A S S A Y S Y S T E M , I T I S P R E F E R A B L E TO E X P R E S S T H E A C T I V I T Y O F A P R E P A R A T I O N AS A R A N G E O F P O S S I B L E V A L U E S R A T H E R T H A N A T T E M P T I N G TO A S S I G N A S I N G L E A C T I V I T Y . THE R A N G E S U S E D WERE B A S E D ON T H E 0 . 9 5 C O N F I D E N C E L I M I T S O F T H E E S T I M A T E D R E S P O N S E (9) AND WERE C A L C U L A T E D A S F O L L O W S . 1 . R E S P O N S E S ( Y ) O F 1 . 0 AND 2 . 0 M G $ C A L C I U M WERE S E L E C T E D AS R E P R E S E N T A T I V E V A L U E S . 2. T H E L O G D O S E ( X ) W H I C H C O R R E S P O N D E D TO AN E S T I M A T E D Y ( ? ) O F 1.0 WAS N O T E D . 3. T H E S T A N D A R D E R R O R O F T H E E S T I M A T E D Y WAS T H E N C A L -C U L A T E D A S I 9 5— S Y = S Y X V1/N +.xV2 4. T H E 0.95 C O N F I D E N C E L I M I T S O F T H E P O P U L A T I O N MEAN (jiy) FOR T H E G I V E N X WAS T H E N V - T _ 0 5 S ? < M < Y + T > Q 5 S ? WHERE T ^ I S D E T E R M I N E D FROM T H E D E G R E E S O F .05 F R E E D O M ( N - 2 ) . ( 1 1 6 ) 5. T H E S E TWO V A L U E S WERE T H E N T R A N S P O S E D TO T H E R E S P O N S E A X I S O F T H E S T A N D A R D C U R V E AND TWO C O R R E S P O N D I N G A C T I V I T I E S I N M i l R E A D . 6. D I V I S I O N O F T H E S E A C T I V I T I E S BY T H E L O G D O S E O B T A I N E D I N S T E P 2 A B O V E Y I E L D S A R A N G E O F A C T I V I T Y I N MU/MG R E P R E S E N T I N G T H E 0.95 C O N F I D E N C E L I M I T S AT . T H I S P O I N T , I F T H E S T A N D A R D C U R V E I S A S S U M E D TO C O N T A I N NO E R R O R . T H I S I S A R E A S O N A B L E A S S U M P T I O N S I N C E T H I S C U R V E I S T H E B A S I S F O R A L L A C T I V I T Y E S T I M A T E S U S E D IN T H E L A B O R A T O R Y AND T H E S E E S T I M A T E S A R E U S E D P R I M A R I L Y FOR C O M P A R I S O N . 7. A R E P E T I T I O N O F S T E P S 2 T H R O U G H 6 U S I N G Y = 2.0 G I V E S AN A D D I T I O N A L R A N G E O F A C T I V I T I E S . T H E F I N A L R A N G E E X P R E S S E D I S T H E E X T R E M E V A L U E S O B T A I N E D FROM T H E TWO C A L C U L A T I O N S . MOST O F T H E S T A T I S T I C A L C A L C U L A T I O N S A B O V E WERE R O U T I N E L Y P E R F O R M E O BY C O M P U T E R . T H E PROGRAM U S E D FOR T H I S C O N T A I N E D ('A) Activity of Turkey Extracts 2.0 1.0 I I I I ~l 1 — I — I I I 11 (B) Activity of Dog Fish Extracts to c & n £ 2.0H 1.0-I I I I I I I I I 1 1 1 — I — I — I I I I 0.1 0.2 0.4 0.6 1.0 2.0 4.0 6.0 10.0 Log Dose (jug/80gm Rat) F I G U R E 2. ( A) C O M P A R I S O N OF R E G R E S S I O N S OF L O G DOSE ON R E S P O N S E FOR T U R K E Y U L T I M O B R A N C H I A L E X T R A C T S FROM A C E T O N E AND D I C H L0R0 E T H A N E D R I E D G L A N D S . • - A C E T O N E , O - D I C H L O R O E T H A N E ( B ) C O M P A R I S O N OF R E G R E S S I O N S OF LOG DOSE ON R E S P O N S E FOR D O G F I S H U L T I M O B R A N C H I A L E X T R A C T S P R E P A R E D U S I N G F O R M I C A C I D OR T C A . • — T C A P R E C I P I T A T I O N , A - F O R M I C A C I D E X T R A C T I O N - 1 6 -M O D I F I C A T I O N S BY T H E A U T H O R AN 0 I T S D E T A I L S A R E I N C L U D E D IN A P P E N D I X A . 2 . R E S U L T S AND D I S C U S S I O N . F I G U R E 2 ( A ) SHOWS T H E L O G D O S E - R E S P O N S E C U R V E S FOR TWO S A M P L E S O F T U R K E Y U L T I M O B R A N C H I A L E X T R A C T S . T H E E X T R A C T I O N S WERE C A R R I E D OUT' IN P A R A L L E L E X C E P T F O R T H E I N I T I A L D R Y I N G S T A G E . T H E B I O L O G I C A L A C T I V I T Y O F T H E A C E T O N E D R I E D M A T E R I A L R A N G E S FROM 5 4 0 TO 8 4 0 ML)/MG W H I L E T H A T O F T H E D I C H L O R O E T H A N E O R I E O M A T E R I A L WAS SOMEWHAT L O W E R , R A N G I N G FROM 4 2 0 - 5 3 0 MU/MG. T H E , LOWER B I O L O G I C A L A C T I V I T Y O F T H E 0 I C H L O R O E T H A N E M A T E R I A L WAS C O N S I D E R E D A M A J O R D I S A D V A N T A G E AT T H I S S T A G E AND A L T H O U G H T H E P R O B L E M D I D NOT S E E M I N S U R M O U N T A B L E , A C O M P L E T E I N V E S T I -G A T I O N WAS NOT C A R R I E D O U T . O N E A T T E M P T A T R E D U C I N G H E A T O E N A T U R A T I ON BY U S I N G A D I C H L O R O M E T H A N E A Z E O T R O P E W I T H A L O W E R B O I L I N G P O I N T P R O D U C E D I N C O N C L U S I V E R E S U L T S . T H E M A T E R I A L O B T A I N E D FROM T H I S P R O C E D U R E HAD E X C E L L E N T P H Y S I C A L P R O P E R T I E S AND A P P E A R E D TO B E F R E E O F L I P I D A F T E R A S I N G L E O P E R A T I O N . F U R T H E R WORK M I G H T W E L L P R O V E I T TO BE A V A L U A B L E T E C H N I Q U E P A R T I C U L A R L Y FOR F A T T Y S T A R T I N G M A T E R I A L S . F I G U R E 2 ( B ) SHOWS T H E L O G D O S E - R E S P O N S E C U R V E S F O R TWO S A M P L E S O F D O G F I S H U L T I M O B R A N C H I A L E X T R A C T S . T H E E X T R A C T I O N S WERE C A R R I E D O U T IN P A R A L L E L U S I N G T H E A C E T O N E D R Y I N G T E C H N I Q U E ANO T H E S O L V E N T E X T R A C T I O N O F G U D M U N O S S O N EJT_ AJ. ( 4 8 ) U P T O T H E S T A G E O F T C A P R E C I P I T A T I O N . O N E S A M P L E WAS T H E N P R E C I P I T A T E D W I T H T C A W H I L E T H E S E C O N D WAS E X T R A C T E D W I T H 0.1 M F O R M I C A C I D A S D E S C R I B E D E A R L I E R . T H E B I O L O G I C A L A C T I V I T Y O F T H E T C A POWDER ( 3 6 0 0 - 4 6 0 0 MU/MG) WAS MORE T H A N D O U B L E T H A T O F T H E TABLE I S P E C I F I C B I O L O G I C A L A C T I V I T I E S O F E X T R A C T S T H Y R O I O AND U L T 1 M O B R A N C 1 A L S O U R C E A C T I v I T Y R A N G E ( M U / M G ) C E N T E R O F R A N G E ( M U / M G ) R E L A T I V E P O T E N C Y ( P O R C I N E = 1 ) HUMAN T H Y R O I D 5 5 0.02 B O V I N E T H Y R O I O * 2 0 - 2 5 2 2 . 5 0.1 M U R I N E T H Y R O I D 1 0 0 - 1 5 0 1 2 5 0.5 P O R C I N E T H Y R O I D * 2 1 0 - 2 8 0 2 4 5 1 T U R K E Y UB 5 4 0 - 8 4 0 6 9 0 3 D O G F I S H UB 8 0 0 - 1 3 0 0 1 0 5 0 4 S A L M O N UB 2 5 0 0 - 4 3 0 0 3 4 0 0 1 4 C H I C K E N UB* 1 3 , 0 0 0 - 1 7 , 0 0 0 1 5 , 0 0 0 6 0 • I N C L U D E S TCA P R E C I P I T A T I O N S T E P . o Log Dose(mU Std /80gm Rat) Loo Dose(/jg/80gm Rat) FIGURE 3. ( A ) REGRESSION OF LOG DOSE ON RESPONSE FOR HOUSE STANDARD. (ARMOUR PORCINE C T , A L 0 8 3 1 ) ( B ) REGRESSIONS OF LOG DOSE ON RESPONSE FOR EIGHT EXTRACTS FROM THYROID AND ULTIMOBRANCHIAL T I S S U E S . CALCULATED A C T I V I T Y RANGES ARE SHOWN IN TABLE I . - 1 7 -F O R M I C A C I D POWDER ( 8 0 0 - 1 3 0 0 ) , B U T B E C A U S E O F T H E S I M P L I C I T Y O F T H E P R O C E D U R E T O T A L R E C O V E R Y WAS MUCH G R E A T E R W I T H T H E F O R M I C A C I D E X T R A C T I O N . E X P R E S S E D I N T E R M S O F M R C M I L L l U N I T S P E R GRAM O F F R E S H T I S S U E (MU/G T I S S U E ) T H E A C T I V I T I E S WERE A S F O L L O W S : T C A POWDER 1 0 0 0 - 1 3 0 0 MU/G T I S S U E F O R M I C A C I D POWDER 4 5 0 0 - 7 3 0 0 MU/G T I S S U E ON T H E B A S I S O F T H I S L O S S O F A C T I V I T Y I T WAS D E C I D E D T H A T T H E A D D I T I O N A L C O M P L I C A T I O N S O F T H E T C A P R E C I P I T A T I O N C O U L D NOT B E J U S T I F I E D , P A R T I C U L A R L Y . S I N C E T H E N E X T S T A G E O F P U R I F I C A T I O N WAS TO BE A G E L F I L T R A T I O N S T E P W H I C H WOULO R E M O V E T H E LOW M O L E C U L A R W E I G H T C O N T A M I N A N T S E Q U A L L Y A S W E L L AS T H E T C A S T E P . F I G U R E 3 ( B ) SHOWS A S E R I E S O F L O G D O S E - R E S P O N S E C U R V E S F O R B I O A S S A Y S ON E X T R A C T S FROM S E V E N S P E C I E S P L O T T E D ON T H E S A M E A X I S . T H E C U R V E I N F I G U R E 3 ( A ) I S T H E H O U S E S T A N D A R D W H I C H I S U S E D TO C A L C U L A T E T H E B I O L O G I C A L A C T I V I T Y O F A P R E P A R A T I O N IN M L)/M G. T H E A C T I V I T Y R A N G E S F O R T H E E X T R A C T S A N D T H E I R R E L A T I V E P O T E N C I E S A R E S U M M A R I Z E D I N T A B L E 1. T H E V A L U E I N C L U O E D F O R T H E HUMAN M A T E R I A L I N T H E T A B L E I S AN E X T R E M E L Y R O U G H E S T I M A T E B A S E D ON A S I N G L E P O I N T . B E T T E R D A T A C O U L D NOT B E O B T A I N E D B E C A U S E O F T H E LOW Y I E L D ANO L I M I T E D S T A R T I N G M A T E R I A L . IT I S O F SOME I N T E R E S T , H O W E V E R , FO R C O M P A R I S O N AND I T I S T Y P I C A L O F T H E Y I E L D FROM HUMAN T H Y R O I D . T H E C H I C K E N , P O R C I N E AND B O V I N E E X T R A C T S WERE P R E P A R E D U S I N G T H E T C A P R E C I P I T A T I O N P R O C E D U R E AND T H E R E F O R E P R O B A B L Y H A V E H I G H E R S P E C I F I C A C T I V I T I E S T H A N WOULD H A V E B E E N O B T A I N E D HAD F O R M I C A C I D E X T R A C T I O N B E E N U S E D . TABLE I I B I O L O G I C A L A C T I V I T Y Y I E L D S FROM T H Y R O I D AND U L T I M O B R A N C H I A L TI S S U E S S O U R C E C E N T E R MG E X T R A C T MU O F R A N G E G T I S S U E G T I S S U E ( M U / M G ) * HUMAN T H Y R O I D 5 1 .0 5 B O V I N E T H Y R O I D 2 2 . 5 ( 0 . 5 ) * * 11 M U R I N E T H Y R O I O 1 2 5 5.2 6 5 0 P O R C I N E T H Y R O I D 2 4 5 ( 0 . 7 ) * * 1 7 0 T U R K E Y UB 6 9 0 3.3 2 3 0 0 D O G F I S H UB 1 0 5 0 5.6 5 9 0 0 S A L M O N UB 3 4 0 0 1.3 4 4 0 0 C H I C K E N UB 1 5 , 0 0 0 0.76 1 1 , 5 0 0 * S E E T A B L E I * * E S T I M A T E A S S U M I N G D R Y , D E F A T T E D W E I G H T E Q U A L S 8 $ O F F R E S H W E I G H T . -18-S P E C I F I C A C T I V I T I E S O F C R U D E E X T R A C T S S U C H AS T H E S E A R E G E N E R A L L Y U S E F U L F OR C A L C U L A T I O N O F HORMONE C O N T E N T O F T H E F R E S H G L A N D S OR AS AN I N D E X O F T H E D E G R E E O F P U R I F I C A T I O N A C H I E V E D I N L A T E R S T A G E S . T H E S E C A L C U L A T I O N S W I L L B E D I S -C U S S E D L A T E R . T H E Y C A N A L S O P R O V I D E AN E S T I M A T E O F T H E P U R I T Y O F T H E M A T E R I A L AND O F T H E E X P E C T E O Y I E L D S O F P U R E HORMONE I F T H E A B S O L U T E S P E C I F I C A C T I V I T Y O F T H E M O L E C U L E S A R E C O M P A R A B L E . ON T H I S B A S I S , K N O W I N G T H A T P U R E P O R C I N E CT HAD A S P E C I F I C A C T I V I T Y O F 2 0 0 - 2 5 0 U/MG, T H E S A L M O N E X T R A C T S H O U L O C O N T A I N 1 TO 2<fo HORMONE. As W I L L B E SHOWN L A T E R , T H I S I S R A T H E R AN O V E R - E S T I M A T E . T A B L E II SHOWS T H E C A L C U L A T E D A C T I V I T I E S O F T H E F R E S H T I S S U E S U S E D I N T H E E X T R A C T S . B O V I N E AND P O R C I N E V A L U E S A R E E S T I M A T E S B A S E D ON T H E A S S U M P T I O N T H A T T H E D R Y , D E F A T T E D P O W D E R S R E P R E S E N T A P P R O X I M A T E L Y 8$ O F T H E F R E S H W E I G H T . T H E R E L A T I V E A C T I V I T I E S O F T H E T I S S U E S FROM T H E E I G H T S P E C I E S A R E C O M P A R A B L E W I T H T H O S E O B T A I N E D IN A C I D E X T R A C T I O N ( 3 2 ) T H O U G H T H E A B S O L U T E V A L U E S A R E G E N E R A L L Y L O W E R . T H E R E A R E TWO P O S S I B L E E X P L A N A T I O N S F OR T H I S F A C T . F l R S T T H E S O L V E N T E X T R A C T I O N M E T H O D I S MORE C O M P L E X AND T H E R E A R E N U M E R O U S O P P O R T U N I T I E S F O R S I G N I F I C A N T L O S S E S TO O C C U R , WHERE A S A S I M P L E A C I D E X T R A C T I O N I N V O L V E S A L M O S T NO L O S S E S D UE TO H A N D L I N G . S E C O N D , T H E P R I M A R Y P U R P O S E O F T H E S E E X T R A C T I O N S WAS TO E S T A B L I S H T H E F E A S A B I L I T Y O F L A R G E R S C A L E E X T R A C T I O N S , AND T H E E M P H A S I S WAS P L A C E D ON M I N I M I Z I N G T H E T I M E S P E N T I N C O L L E C T I N G T H E G L A N D S . T H U S NO G R E A T E F F O R T WAS MADE TO R E M O V E A L L N 0 N - G L A N D U L A R T I S S U E AND T H E S T A R T I N G M A T E R I A L S - 1 9 -WE RE G E N E R A L L Y MUCH C R U D E R T H A N T H O S E U S E D TO E S T I M A T E A C T U A L A C T I V I T Y L E V E L S IN G L A N D S . T H E M A J O R V A L U E O F T H E D A T A , H O W E V E R , I S I T S U S E F U L N E S S IN E S T I M A T I N G T H E AMOUNT O F S T A R T I N G M A T E R I A L N E C E S S A R Y TO P R O V I D E S U F F I C I E N T HORMONE F O R S T R U C T U A L WORK. A L L O F T H E U L T I M O B R A N C H I A L T I S S U E A P P E A R E D TO B E E X C E L L E N T S O U R C E S O F HORMONE S I N C E T H E Y C O N T A I N E D FROM 1 0 TO 5 0 T I M E S T H E A C T I V I T Y O F T H E P O R C I N E T H Y R O I D . T H E C H I C K E N U L T I M 0 B R A N C H I A L S WERE P A R T I C U L A R L Y A T T R A C T I V E S I N C E T H E Y WERE 2 TO 3 T I M E S MORE A C T I V E T H A N E I T H E R T H E D O G F I S H OR S A L M O N G L A N D S . T H I S C O U L D B E D U E , AT L E A S T I N P A R T , TO T H E D I F F U S E N A T U R E O F T H E G L A N D U L A R T I S S U E I N T H E TWO F I S H A S O P P O S E D TO T H E D I S C R E T E G L A N O W H I C H E X I S T S I N T H E C H I C K E N . T H I S E X P L A N A T I O N : D O E S : NOT H O L D , H O W E V E R , I N T H E T U R K E Y W H I C H HAS A D I S C R E T E G L A N D AND A C O N S I D E R A B L Y L O W E R A C T I V I T Y P E R U N I T W E I G H T . ON T H E B A S I S O F T H E E X P E R I M E N T S O U T L I N E D IN T H I S C H A P T E R I T WAS 0 E C I D E D T H A T A L L F O U R O F T H E U L T I M O B R A N C H I A L S O U R C E S C O N T A I N E D S U F F I C I E N T Q U A N T I T I E S O F HORMONE TO A L L O W P U R I F I C A T I O N AND I S O L A T I O N , ANO T H A T T H E S O L V E N T E X T R A C T I O N P R O C E D U R E F O L L O W E D BY A F O R M I C A C I D E X T R A C T I O N WAS A S U I T A B L E M E T H O D F O R P R E P A R I N G T H E S E H O R M O N E S . T H E F I N A L D E C I S I O N TO C A R R Y ON W I T H T H E S A L M O N HORMONE WAS T H E R E F O R E B A S E D ON O T H E R F A C T O R S . T H E D O G F I S H WAS E L I M I N A T E D B E C A U S E O F T H E R E L A T I V E D I F F I C U L T Y IN A C Q U I R I N G S T A R T I N G M A T E R I A L AND T H E A V I A N S O U R C E S WERE P A S S E D O V E R B E C A U S E T H E L A R G E A M O U N T S O F F A T T Y T I S S U E A S S O C I A T E D W I T H T H E G L A N D S MADE T H E A C E T O N E D R Y I N G P R O C E O U R E E X P E N S I V E AND T I M E C O N S U M I N G . T H I S L E F T T H E S A L M O N A S T H E -20-S P E C I E S O F C H O I C E . THE S A L M O N HAD T H E S E C O N D H I G H E S T A C T I V I T Y P E R W E I G H T O F T I S S U E AND T H E S E C O N D H I G H E S T S P E C I F I C A C T I V I T Y IN T H E E X T R A C T , ( T H E H I G H E R A C T I V I T Y O F T H E C H I C K E N E X T R A C T MIGHT-BE E X P L A I NED BY T H E T C A P R E C I P I T A T I O N S T E P W H I C H WAS I N C L U D E D IN T H E C H I C K E N E X T R A C T I O N . ) . T H E T I S S U E WAS E A S Y TO WORK W I T H , AND C O U L D BE O B T A I N E D I N L A R G E Q U A N T I T I E S ON T H E P R O D U C T I O N L I N E O F A L O C A L C A N N E R Y . T H U S I T M E T A L L T H E R E Q U I R E M E N T S FOR A S T A R T I N G M A T E R I A L FOR L A R G E S C A L E E X T R A C T I ON. A S S U M I N G AN A C T I V I T Y A P P R O X I M A T E L Y E Q U A L TO T H A T O F T H E P U R E P O R C I N E HORMONE (200-250 U/MG) I T WAS C A L C U L A T E D T H A T 1 KG O F S A L M O N U L T I M 0 B R A N C H I A L T I S S U E S H O U L D C O N T A I N A B O U T 20 MG O F H O R M O N E . A L L O W I N G F O R A 50$ L O S S D U R I N G P U R I F I C A T I O N T H I S M E A N T T H A T 2 KG O F T I S S U E WOULD B E N E E D E D TO P R O V I D E T H E 10 TO 20 MG O F P U R E HORMONE N E C E S S A R Y F O R S T R U C T U R A L WORK. A L T H O U G H T H I S R E P R E S E N T E D A B O U T 10 T O N S O F S A L M O N , I T WAS S T I L L A G R O S S U N D E R E S T I M A T I O N . C . C A L I S R A T i O N O F S E P H A D E X C O L U M N S . 1. M E T H O D S . T H E P R O C E D U R E U S E D I N P R E P A R I N G T H E C A L I B R A T E D S E P H A D E X G-75 C O L U M N WAS E S S E N T I A L L Y T H A T D E S C R I B E D BY ANDREWS ( 3 ) . A 1.2 x 80 CM C O L U M N O F S E P H D E X G-75 WAS P R E P A R E D U S I N G 0.1 M F O R M I C A C I 0 AS AN E L U A N T . A P P R O X I M A T E L Y 1 MG O F T H E M A R K E R P R O T E I N WAS T H E N D I S S O L V E D I N 0.5 ML O F 2 M U R E A I N 0.1 M F O R M I C A C I D . T H E U R E A I N S U R E D T H A T T H E P R O T E I N WOULD D I S S O L V E ANO I N C R E A S E D T H E D E N S I T Y O F T H E S O L U T I O N A L L O W I N G I T TO B E - 2 1 -L A Y E R E D B E N E A T H T H E E L U A N T ON T H E T O P O F T H E C O L U M N . O N C E T H E S A M P L E WAS A P P L I E D E L U A N T WAS A L L O W E D TO FLOW T H R O U G H T H E C O L U M N U N D E R G R A V I T Y ) AND 3 ML F R A C T I O N S O F E L U A T E WERE C O L L E C T E D . T H E A B S O R B A N C E O F E A C H F R A C T I O N A T 2 8 0 NM WAS M E A S U R E D W I T H A B A U S C H AND LOMB S P E C T R O N I C 6 0 0 S P E C T R O P H O T O -M E T E R ANO WHEN P L O T T E D A G A I N S T T H E T O T A L V O L U M E C O L L E C T E D I T P R O D U C E D A T Y P I C A L E L U T I O N P R O F I L E C O N T A I N I N G A S I N G L E P E A K . T H E C E N T E R O F T H I S P E A K WAS D E S I G N A T E D A S T H E E L U T I O N V O L U M E ( V E ) FOR T H A T P R O T E I N ANO T H I S V A L U E WAS P L O T T E D A G A I N S T T H E L O G O F T H E M O L E C U L A R W E I G H T OF T H E P R O T E I N . ( S E E F I G U R E 4 ) . , T H E M A R K E R S U S E O WERE GAMMA G L O B U L I N ( 1 6 0 , 0 0 0 M . W . ) , M Y O G L O B I N ( 1 7 * 8 0 0 M.W.) C Y T O C H R O M E C ( 1 2 , 4 0 0 M . W . ) , G L U C A G O N ( 3 4 8 5 M . W . ) , B A C I T R A C I N ( 1 4 1 1 M . W . ) , I N S U L I N ( 5 7 2 2 M.W.) AND ACTH ( 4 5 4 2 M.W.-). T H E F I R S T F O U R M A R K E R S HAO B E E N U S E D BY ANDREWS AND WERE C O N S I D E R E D A C H E C K ON T H E O T H E R T H R E E . T H E F I R S T F I V E M A R K E R S WERE O B T A I N E D FROM MANN R E S E A R C H L A B O R A T O R I E S , I N C . NEW YORK. T H E I N S U L I N WAS R E C R Y S T A L I Z E D B O V I N E I N S U L I N , G R A D E B FROM C A L B I O C H E M , LOW A N G E L E S . THE ACTH WAS C H R O M A T O G R A P H I C A L L Y P U R E P O R C I N E ACTH FROM S I G M A C H E M I C A L CO., S T . L O U I S . T H E C A L I B R A T I O N P R O C E D U R E FOR T H E S E P H A D E X G - 5 0 C O L U M N WAS I O E N T I C A L TO T H A T D E S C R I B E D FOR T H E G - 7 5 C O L U M N E X C E P T FOR T H E S E P H A D E X U S E D , T H E C O L U M N S I Z E ( 1 . 5 x 8 0 C M ) AND T H E M A R K E R S . T H E S A M E LOW M O L E C U L A R W E I G H T M A R K E R S WERE U S E D ( B A C I T R A C I N , G L U C A G O N , ACTH AND I N S U L I N ) , B U T B O V I N E S E R U M A L B U M IN ( 6 5 , 0 0 0 M.W.) WAS U S E D TO I N D I C A T E T H E V O I D V O L U M E 1000 I 2000 I M.W. 5000 9000 -I 1 I--105 100 90 80 • Vr 70 H VE (ml) 60 SO 40 Bacitracin B a c i t r a c i n ( I 4 I I ) 1000 Glucagon \(3685) ACTH\(4542) lnsulin\°(5722) Sephadex G-75 Calibration Glucagon\(3485) Porcine\(3600) Bovine Mminor) Sephadex G-50 Calibration Cytochrome\( 12400) L Chicken, Human, Rat » Bovine (major), Salmon A C T H ( 4 5 4 2 ) \ D o g f i s h J u r k e y • Insulin (5722) Myoglobin\(l7800) BSA(65000) 120 110 100 90 (ml) - eo 70 60 50 r-Globulin( 160000) 10000 M.W. 100000 F I G U R E 4 . C A L I B R A T I O N C U R V E S F O R S E P H A D E X G - 5 0 A N D G - 7 5 , I N C L U D I N G E L U T I O N V O L U M E S O F H Y P O C A L C E M I C A C T I V I T Y F R O M E X T R A C T S . G - 7 5 C O L U M N , 1 . 2 X 8 0 E L U A N T , 0.1 M F O R M I C A C I D ; T E M P E R A T U R E , F L O W R A T E 5 M L / H R ; F R A C T I O N S I Z E , 3 . 0 M L . G - 5 0 C O L U M N , 1 . 5 x 8 0 C M ; O T H E R C O N D I T I O N S T H E S A M E . eg; 4 ° C ; o> G-75 M A R K E R S ; • , G-50 M A R K E R S ; A , H Y P O C A L C E M I C A C T I V I T Y . - 2 2 -( V ) OF T H I S C O L U M N . T H E A L B U M I N WAS G R A D E A, C R Y S T A L L I N E 0 FROM CAL B I O C H E M , L O S A N G E L E S . 2 . R E S U L T S AND D I S C U S S I O N . THE R E S U L T S O F T H I S C A L I B R A T I O N A R E SHOWN I N F I G U R E 4 WHERE T H E E L U T I O N V O L U M E S ' ( V ) O F E A C H M A R K E R A R E P L O T T E D V E R S U S T H E L O G O F T H E M O L E C U L A R W E I G H T O F T H A T M A R K E R . T H E L I N E A R I T Y O F T H I S R E L A T I O N S H I P B E T W E E N M.W. 3 0 0 0 ANO 3 0 , 0 0 0 H A S B E E N R E P O R T E D P R E V I O U S L Y AND G L U C A G O N , C Y T O C H R O M E C AND M Y O G L O B I N H A V E B E E N SHOWN TO F I T T H E L I N E W E L L ~ ( 3 ) » IN T H I S E X P E R I M E N T T H E S E R E S U L T S WERE R E P R O D U C E D AND ACTH WAS A L S O SHOWN TO L I E A L O N G T H E L I N E . I N S U L I N , H O W E V E R , D I D N O T B E H A V E A S P R E D I C T E D AND HAD A V £ SOMEWHAT L A R G E R T H A N E X P E C T E D . ON T H E G - 7 5 C O L U M N I N S U L I N HAD AN A P P A R E N T M O L E C U L A R W E I G H T O F S L I G H T L Y B E L O W 5 0 0 0 A V A L U E SOME 1 0 - 1 5 $ B E L O W I T S R E A L M O L E C U L A R W E I G H T O F 5 7 2 2 . TH.IS A N O M A L O U S B E H A V I O R P R O B A B L Y R E S U L T S FROM T H E F A C T T H A T G E L F I L T R A T I O N M E T H O D S A C T U A L L Y M E A S U R E M O L E C U L A R S I Z E W H I C H I S I N F L U E N C E D BY F A C T O R S O T H E R T H A N M O L E C U L A R W E I G H T A S W I L L B E D I S C U S S E D I N A L A T E R C H A P T E R . F I G U R E 4 A L S O SHOWS T H E C A L I B R A T I O N O F T H E G - 5 0 C O L U M N . A G A I N T H E R E I S A L I N E A R R E L A T I O N S H I P B E T W E E N T H E L O G O F T H E M O L E C U L A R W E I G H T ANO T H E E L U T I O N V O L U M E , T H O U G H W I T H G - 5 0 T H E R A N G E I S MUCH S M A L L E R E X T E N D I N G O N L Y FROM A B O U T 3 0 0 0 TO 9 0 0 0 M.W. A S E X P E C T E D , H O W E V E R , T H E R E S O L U T I O N I N T H I S R A N G E WAS G R E A T E R T H A N WAS S E E N W I T H G - 7 5 . I N S U L I N B E H A V E D MUCH A S I T HAD ON G - 7 5 . WHEN I T S R E A L M O L E C U L A R W E I G H T WAS P L O T T E D V E R S U S I T S E L U T I O N V O L U M E T H E P O I N T L A Y TO T H E R I G H T O F T H E L I N E E S T A B L I S H E D . B Y T H E O T H E R M A R K E R S . IF I T S A P P A R E N T - 2 3 -M O L E C U L A R W E I G H T A S S E E N ON G - 7 5 WAS U S E D I N S T E A D , : T H E R E S U L T A N T P O I N T L A Y V E R Y N E A R T H E L I N E . IN T H E A B S E N C E O F O T H E R M A R K E R S IN T H I S W E I G H T R A N G E I T S E E M E D J U S T I F I A B L E TO U S E T H I S A P P A R E N T M O L E C U L A R W E I G H T TO P R O V I D E A P O I N T AT T H E LOWER E N D O F T H E L I N E . T H E R E L I A B I L I T Y O F T H E U P P E R E N D O F T H E L I N E WAS E N H A N C E D WHEN T H E M O L E C U L A R W E I G H T O F P O R C I N E CT, W H I C H HAD O R I G I N A L L Y B E E N C O N S I D E R E D AN UNKNOWN, WAS C O N F I R M E D BY T H E S E Q U E N C E WORK O F B E L L ( 9 ) AND P O T T S ( 9 6 ) . 0. E S T i M A T i O N O F M O L E C U L A R W E I G H T S O F H Y P Q C A L C E M I C F A C T O R S . 1. M E T H O D S . T O O B T A I N E S T I M A T E S O F T H E M O L E C U L A R W E I G H T S OF T H E A C T I V E F A C T O R S IN T H E E X T R A C T S , S A M P L E S O F E A C H E X T R A C T WERE A P P L I E D TO T H E C A L I B R A T E D G - 5 0 C O L U M N I N A MANNER S I M I L A R TO T H A T U S E D FOR T H E M A R K E R S . T H E S A M P L E S WERE A P P L I E D IN 0.1 M F O R M I C A C I D MAOE 8 M IN U R E A . T H E S E S A M P L E S WERE E L U T E D AND T H E A B S O R B A N C E AT 2 8 0 NM O F E A C H 3 ML F R A C T I O N D E T E R M I N E D . R E C O R D S O F T H E E L U T I O N P R O F I L E S A R E SHOWN IN F I G U R E S 5 AND 6. THE E L U T I O N V O L U M E O F T H E A C T I V E M O L E C U L E S WAS D E T E R -M I N E D BY A B I O A S S A Y P R 0 C E 0 U R E S I M I L A R TO T H A T D E S C R I B E D P R E V I O U S L Y . T H E A S S A Y WAS U S E D O N L Y A S A Q U A L I T A T I V E M E T H O O TO I N D I C A T E P R E S E N C E OR A B S E N C E O F H Y P O C A L C E M I C F A C T O R S ; HOW-E V E R , AND NO A T T E M P T WAS MADE TO M E A S U R E T H E A C T U A L AMOUNT O F A C T I V I T Y P R E S E N T . R O U T I N E L Y T H E C O L U M N F R A C T I O N S WERE S U R V E Y E D BY T A K I N G 0.1 ML A L I Q U O T S FROM E V E R Y S E C O N D F R A C T I O N B E T W E E N VQ AND V T ( 5 0 TO 1 5 0 M L ) ANO D I L U T I N G T H E M W I T H 0.2 ML O F S O D I U M A C E T A T E , BSA V E H I C L E . E A C H 0.3 ML S A M P L E WAS T H E N - 2 4 -I N J E C T E O I N T O A R A T AND T H E R A T S P L A S M A C A L C I U M IN M G $ D E T E R -M I N E D A F T E R O N E HOUR. T H E D I F F E R E N C E ( CA, M G $ ) B E T W E E N T H E T E S T R A T ' S P L A S M A C A L C I U M ANO T H E MEAN P L A S M A C A L C I U M O F A G R O U P O F C O N T R O L R A T S I N J E C T E D W I T H V E H I C L E O N L Y WAS T H E N U S E D AS AN I N D E X O F H Y P O C A L C E M I C A C T I V I T Y . A N Y D R O P I N P L A S M A C A L C I U M W H I C H E X C E E D E D 1 M G $ WAS C O N S I D E R E D I N D I C A T I V E O F A H Y P O C A L C E M I C F A C T O R S I N C E V A R I A T I O N S O F T H I S M A G N I T U D E A R E A L M O S T N E V E R S E E N I N V E H I C L E I N J E C T E D R A T S . I F A N Y A M B I G U I T I E S WERE S E E N T H E S U R V E Y WAS R E P E A T E D S A M P L I N G FROM E V E R Y F R A C T I O N . B E C A U S E O F T H E LOW A C T I V I T Y IN T H E HUMAN T H Y R O I D E X T R A C T L A R G E R S A M P L E S WERE R E Q U I R E D FOR I N J E C T I O N . 0.4 ML A L I Q U O T S WERE T A K E N AND 0.1 ML O F A C O N C E N T R A T E D V E H I C L E A D D E D ( 0 . 4 M S O D I U M A C E T A T E P H 4.5 C O N T A I N I N G 0 . 4 $ B O V I N E S E R U M A L B U M I N . ) G E N E R A L L Y T H E A C T I V E R E G I O N O F A N Y C O L U M N WAS B E T W E E N 3 AND 5 F R A C T I O N S W I D E * AND S I N C E T H E A S S A Y U S E D WAS O N L Y Q U A L I T A T I V E I T WAS I M P O S S I B L E TO KNOW W H I C H F R A C T I O N WAS M A X I M A L L Y A C T I V E . F O R T H I S R E A S O N T H E E L U T I O N V O L U M E SHOWN FOR E A C H HORMONE I S T H A T V O L U M E W H I C H C O R R E S P O N D S TO T H E C E N T E R O F T H E A C T I V E R E G I O N . ONE A D D I T I O N A L S A M P L E O F B O V I N E T H Y R O I O E X T R A C T WAS C H R O M A T O G R A P H E D ON T H E C O L U M N . ( S E E F I G U R E 7 ) . T H I S S A M P L E WAS P R E P A R E O U S I N G T H E F O R M I C A C I D E X T R A C T I O N P R O C E D U R E R A T H E R T H A N T H E T C A P R E C I P I T A T I O N . 2. R E S U L T S AND D I S C U S S I O N . ONCE T H E G - 5 0 C O L U M N HAD B E E N C A L I B R A T E D I T WAS P O S S I B L E TO E L U T E S A M P L E S O F T H E V A R I O U S E X T R A C T S FROM T H E C O L U M N AND TO D E T E R M I N E T H E E L U T I O N V O L U M E O F T H E A C T I V E M O L E C U L E S I N TABLE I I I M O L E C U L A R W E I G H T E S T I M A T E S FOR C A L C I T O N I N S FROM E L U T I O N V O L U M E S ON A S E P H A D E X G-50 COLUMN S P E C I E S E L U T I O N E S T I M A T E D V O L U M E ( M L ) . M O L E C U L A R W E I G H T P O R C 1 N E 1 0 8 3 6 0 0 B O V 1 N E 1 1 0 5 3 7 0 0 B O V I N E I I 8 7 4 5 0 0 M U R I N E 9 0 4 3 0 0 HUMAN 9 0 4 3 0 0 C H I C K E N 9 0 4 3 0 0 S A L M O N 8 7 4 5 0 0 T U R K E Y 8 4 4 6 0 0 D O G F I S H 8 4 4 6 0 0 (A) Porcine Thyroid Extract (B) Bovine Thyroid Extract O oo CM 0.20-1 0.15-0.10-< 0.03-(C) Humon Thyroid Extract c O CO CM 0.20 0.IS 0.10 -4 0.09 3.0 5? 2.0 6 - i .oh O 0 o < + 1 . 0 so (0) Murine Thyroid Extract 3.0 -2.0 -- i . o o + 1.0 lll.ll 100 ISO o - I -SO .y/v' " I. i 1 — 100 ISO Ve (ml) F I G U R E 5 . E L U T I O N P R O F I L E S ( A 2 8 0 NM) OF T H Y R O I D E X T R A C T S ON S E P H D E X G - 5 0 . C O N D I T I O N S AS I N D I C A T E D I N F I G U R E 4 . A C A I S T H E DROP IN P L A S M A C A L C I U M A S S E E N I N T H E B I O A S S A Y , (A) Chicken Ultimobranchial Extract 0.20 E c O a> 0.10-Gl 0.05-0 J 3.0 2.0 £ -i.o 5 ° < + ..0 L i nl. (C) Salmon Ultimobranchial Extract 0.20n o.is 6 c o.io o co CM - o.osH o J 3 30 9 2.0 6 — 1.0 a O 0 -M.0»-Li —r— 90 (B) Turkey Ultimobranchial Extract 3.0 2.0 -i.o o + I.0L. J l M \ J ' VA (D) Dog Fish Ultimobranchial Extract 3.0 2.0 -i.o o + 1.0 nil il TpTTJIT too ISO o 80 ft 100 ISO Ve (ml) F I G U R E 6 . E L U T I O N P R O F I L E S ( A 2 8 0 NM) O F U L T I M O B R A N C H I A L E X T R A C T S ON S E P H A D E X G-50. C O N D I T I O N S A S I N D I C A T E D IN F I G U R E 5. - 2 5 -E A C H O F T H E E X T R A C T S U S I N G T H E B I O A S S A Y . T H E E L U T I O N P R O F I L E S AND B I O A S S A Y S O F T H E E L U A N T F R A C T I O N S FROM T H E T H Y R O I O E X T R A C T S A R E SHOWN I N F L G U R E 5 AND T H O S E O F T H E U L T I M O B R A N C H I A L E X T R A C T S I N F I G U R E 6. T H E E L U T I O N V O L U M E S O F T H E H O R M O N E S A R E SUMMAR-I Z E D I N T A B L E I I I . T H I S T A B L E A L S O SHOWS T H E M O L E C U L A R W E I G H T E S T I M A T E S F O R E A C H HORMONE A S C A L C U L A T E D FROM T H E C A L I B R A T I O N C U R V E I N -FlGURE 4 WHERE T H E P O S I T I O N S O F T H E H O R M O N E S A R E I N D I C A T E D ON T H E G-50 C A L I B R A T I O N C U R V E . T H E E S T I M A T E D M O L E C U L A R WE I G H T S O F T H E M O L E C U L E S V A R I E D FROM A B O U T 3 6 0 0 TO 4 6 0 0 W I T H T H E M A J O R I T Y N E A R E R T H E H I G H F I G U R E . T H E B O V I N E E X T R A C T , HOWEVER* WAS U N U S U A L I N T H A T I T Y I E L D E D TWO A C T I V E R E G I O N S , ONE W I T H A M O L E C U L A R W E I G H T O F 3 7 0 0 AND A S E C O N D W I T H A W E I G H T O F 4 5 0 0 . T H E R E A S O N F O R T H E P R E S E N C E O F TWO A C T I V E R E G I O N S I N T H E B O V I N E E L U A T E I S NOT C L E A R . S I N C E T H I S WORK WAS C O M P L E T E D B R E W E R n A L ( 1 7 ) HAS I S O L A T E D AND C H A R A C T E R I Z E D B O V I N E T H Y R O I D CT AND H I S P U B L I S H E D WORK TO D A T E M A K E S NO M E N T I O N O F A N Y S I M I L A R O B S E R V A T I O N . HE D I D F I N D TWO A C T I V E M O L E C U L E S D U R I N G I O N - E X C H A N G E C H R O M A T O G R A P H Y , B U T T H E S E D I F F E R E D O N L Y I N C H A R G E D U E TO T H E O X I D A T I O N O F M E T H I O N I N E , A D I F F E R E N C E W H I C H WOULD P R O B A B L Y B E U N D E T E C T A B L E BY G E L F I L T R A T I O N . T H I S S T I L L D O E S NOT R U L E O U T T H E P O S S I B I L I T Y T H A T A N O T H E R B O V I N E CT O F S L I G H T L Y D I F F E R E N T S T R U C T U R E M I G H T E X I S T * AND P O S S I B L Y HAS B E E N O V E R L O O K E D OR U N R E P O R T E D BY B R E W E R , J U S T A S K E U T M A N E T AJ. ( 6 5 ) H A V E SHOWN T H E P R E S E N C E O F A M I N O R C O M P O N E N T IN S A L M O N U L T I M O B R A N C H I A L E X T R A C T W H I C H WAS O V E R L O O K E D I N T H E P R E S E N T S T U D Y . A S E C O N D E X P L A N A T I O N C O U L D B E T H E F O R M A T I O N Bovine Thyroid Extract ; (A) TCA (B) Formic Acid o. 15 -o co 0.20-1 3.0 2.0 - 1 . 0 O + 1.0 0.10 -0 .05 -r I p r r -•••••• \ T 50 I 100 150 3.0r-2.0 -- 1.0 -O + I . 0 L T t, till? lllfl, K *\ A T O 50 Ve (ml) i 100 I 150 F I G U R E 7. C O M P A R I S O N O F E L U T I O N P R O F I L E S ON G-50 O F TWO B O V I N E T H Y R O I D E X T R A C T S P R E P A R E D BY D I F F E R E N T M E T H O D S . C O N D I T I O N S AS I N D I C A T E D I N F I G U R E 5* T C A P R E C I P I T A T I O N AND F O R M I C A C I D E X T R A C T I O N P R O C E D U R E S A R E D E S C R I B E D IN T H E T E X T . -26-O F A D I M E R S U C H A S T H A T R E P O R T E D BY N E H E R EJT AL. ( 8 0 ) I N E X T R A C T S O F HUMAN T H Y R O I D M E D U L L A R Y C A R C I N O M A T I S S U E . T H I S O O E S NOT S E E M TOO L I K E L Y , H O W E V E R , S I N C E T H E A C I D C O N D I T I O N S M A I N T A I N E D T H R O U G H O U T T H I S E X T R A C T I O N AND C H R O M A T O G R A P H Y S H O U L D H A V E P R E V E N T E D T H E D I S U L F I D E I N T E R C H A N G E N E C E S S A R Y FOR T H E F O R M A T I O N O F S U C H A D I M E R . A L S O T H E A P P A R E N T M O L E -C U L A R W E I G H T O F T H E L A R G E R A C T I V E S P E C I E S IN T H E B O V I N E E X T R A C T WAS MUCH S M A L L E R T H A N WOULD H A V E B E E N E X P E C T E D FOR S U C H A D I M E R . T H E O N L Y O T H E R E X P L A N A T I O N W H I C H S E E M S R E A S O N -A B L E I S T H E E X I S T E N C E O F AN I N T E R A C T I O N B E T W E E N P A R T O F T H E B O V I N E HORMONE AND SOME O T H E R S M A L L M O L E C U L E C A U S I N G T H E HORMONE TO B E H A V E A S T H O U G H I T WERE L A R G E R . B E C A U S E O F T H I S D E V I A T I O N FROM T H E P A T T E R N O F T H E O T H E R E X T R A C T S , I T WAS D E C I D E D T H A T F U R T H E R E X P E R I M E N T S S H O U L D B E C A R R I E D O U T TO V E R I F Y T H E P R E S E N C E O F T H E TWO A C T I V E C O M P O N E N T S . A NEW E X T R A C T WAS P R E P A R E D FROM B O V I N E T H Y R O I D POWDER U S I N G T H E F O R M I C A C I D E X T R A C T I O N S T E P R A T H E R T H A N T H E T C A P R E C I P I T A T I O N , AND T H E E X T R A C T WAS C H R O M A T 0 G R A P H E D ON T H E C A L I B R A T E D G - 5 0 C O L U M N . T H E R E S U L T A N T E L U T I O N P R O F I L E AND B I O A S S A Y D A T A A S SHOWN I N F I G U R E 7 ( B ) C O N F I R M E D T H E R E S U L T S S E E N P R E V I O U S L Y . T H I S E X P E R I M E N T A L S O S E R V E D AS A C H E C K ON T H E R E P R O D U C I B I L I T Y O F T H E D A T A O B T A I N E D FROM T H E G - 5 0 C O L U M N D U R I N G T H E P E R I O D O F I T S U S E , S I N C E T H E B O V I N E T C A E X T R A C T WAS T H E S E C O N D UNKNOWN E L U T E O AND T H E B O V I N E F O R M I C A C I D E X T R A C T WAS T H E L A S T . BY C O M P A R I N G F I G U R E 7 ( A ) TO 7 ( B ) I T CAN B E S E E N T H A T T H E E L U T I O N V O L U M E S O F T H E A C T I V E C O M P O N E N T S V A R Y BY A B O U T ± 3 ML ( T H E V O L U M E O F T H E F R A C T I O N S C O L L E C T E D ) A F T E R A B O U T - 2 7 -6 M O N T H S OF C O L U M N U S A G E . IN T H E R E G I O N O F T H E UNKNOWNS T H I S R E P R E S E N T S A V A R I A T I O N IN M O L E C U L A R W E I G H T O F A B O U T ± 1 5 0 . I T S H O U L D BE N O T E D T H A T T H I S V A R I A T I O N A P P L I E S O N L Y TO R E P R O D U C I B I L I T Y AND NOT TO T H E A C C U R A C Y O F T H E E S T I M A T E S S I N C E MANY O T H E R F A C T O R S I N F L U E N C E A C C U R A C Y ( E G . D I F F E R E N C E S I N M O L E C U L A R S H A P E A S D I S C U S S E D FOR I N S U L I N . ) ONE A D D I T I O N A L F A C T W H I C H I S I L L U S T R A T E D BY T H E E L U T I O N P R O F I L E S O F F I G U R E S 7 ( A ) AND 7 ( B ) I S T H E D I F F E R E N C E B E T W E E N T H E T C A P R E C I P I T A T E D E X T R A C T S AND T H O S E P R E P A R E D W I T H F O R M I C A C I D . WITH T H E T C A E X T R A C T T H E P E A K AT T H E V Q I S R E L A T I V E L Y L A R G E AND T H A T AT V^ . I S R E L A T I V E L Y S M A L L . T H I S I S TO B E E X P E C T E D S I N C E T C A I S G E N E R A L L Y U S E D TO P R E C I P I T A T E L A R G E P R O T E I N S , AND T H E S A M E P A T T E R N CAN BE S E E N IN T H E O T H E R T C A E X T R A C T S . T H E R E V E R S E I S T R U E O F T H E F O R M I C A C I D E X T R A C T S , B U T IN B O T H C A S E S T H E A B S O R B A N C E AT 2 8 0 NM IN T H E R E G I O N O F T H E A C T I V E M O L E C U L E S I S Q U I T E LOW I N D I C A T I N G T H A T S E P H A D E X G-50 C H R O M A T O G R A P H Y S H O U L O P R O V I D E AN E X C E L L E N T P U R I F I C A T I O N O F E I T H E R T Y P E O F E X T R A C T . T H U S I F S E P H A D E X C H R O M A T O G R A P H Y WERE TO B E T H E N E X T S T A G E O F P U R I F I C A T I O N T H E R E WAS L I T T L E OR NO A D V A N T A G E I N U S I N G T H E MORE C O M P L E X T C A P R E C I P I T A T I O N P R O C E D U R E . T H I S WAS P A R T I C U L A R L Y T R U E O F T H E U L T I M O B R A N C H I A L M A T E R I A L S W H I C H G E N E R A L L Y HAD R E L A T I V E L Y S M A L L P E A K S A T V T I N A N Y C A S E . , T H E R E S U L T S O F T H I S S E R I E S O F E X P E R I M E N T S I N D I C A T E D T H A T T H E H Y P O C A L C E M I C F A C T O R S E X T R A C T E D FROM T H E U L T I M O B R A N C H I A L G L A N D S O F T H E F O U R LOWER V E R T E B R A T E S S T U D I E D WERE I N F A C T P O L Y P E P T I D E S S I M I L A R IN S I Z E TO T H E C A L C I T O N I N S I S O L A T E D FROM M A M M A L I A N T H Y R O I D , AND T H A T CT WAS A W E L L E X T A B L I S H E D HORMONE A R I S I N G A T A V E R Y E A R L Y S T A G E O F V E R T E B R A T E E V O L U T I O N . T H E R E WAS AN A P P A R E N T V A R I A T I O N I N M O L E C U L A R W E I G H T S FROM A B O U T 3 6 0 0 TO 4 6 0 0 , T H O U G H I T B E C A M E C L E A R W I T H F U R T H E R WORK T H A T T H E A C T U A L V A R I A T I O N WAS P R O B A B L Y MUCH L E S S T H A N T H I S . T H E R E A S O N S F O R T H I S A P P A R E N T V A R I A T I O N W I L L B E E X P L O R E D I N C H A P T E R I I I . I I I . I S O L A T I O N A N D C H A R A C T E R I Z A T I O N O F S A L M O N C A L C I T O N I N . * A . I N T R O D U C T I O N . T H E S T U D I E S D E S C R I B E D I N T H E P R E C E E D I N G C H A P T E R SHOWED T H E F E A S I B I L I T Y O F L A R G E S C A L E P U R I F I C A T I O N S O F S A L M O N U L T I M O B R A N C H I A L C T , AND WORK WAS B E G U N TO A L L O W D E T E R M I N A T I O N O F I T S P R I M A R Y S T R U C T U R E . T H E E A R L I E R WORK HAD SHOWN T H E V A L U E O F S E P H A D E X G-50 A S A P U R I F I C A T I O N S T E P AND F U R T H E R P U R I F I C A T I O N S B A S E D ON T H E C H A R G E C H A R A C T E R I S T I C S O F T H E M O L E C U L E WERE A N T I C I P A T E D . O N C E P U R E HORMONE WAS O B T A I N E D AND I T S A M I N O A C I D C O M P O S I T I O N D E T E R M I N E D , W E L L E S T A B L I S H E D P R O C E D U R E S FOR T H E P R O D U C T I O N AND S E Q U E N C I N G O F O V E R L A P P I N G P E P T I D E F R A G M E N T S S U C H A S T H O S E U S E D E A R L I E R IN S E Q U E N C E S T U D I E S ON P O R C I N E C T ( 9 6 , 1 0 , 8 3 ) AND HUMAN C T ( 8 2 ) C O U L D BE U S E D TO E L U C I D A T E T H E P R I M A R Y S T R U C T U R E O F S A L M O N C T . K N O W I N G T H I S S T R U C T U R E AND T H E B I O L O G I C A L A C T I V I T I E S O F T H E HORMONE I T WAS R E A S O N A B L E TO A S S U M E T H A T C E R T A I N C H A R A C T E R -I S T I C S ANO P R O P E R T I E S O F T H E C A L C I T O N I N S AND O F T H E S P E C I E S P R O D U C I N G T H E M WOULD B E C L A R I F I E D . B. P R E L i M I N A R Y E X P E R I M E N T S . 1. E X T R A C T i O N S . T H E E X T R A C T F O R T H E P I L O T P U R I F I C A T I O N WAS P R E P A R E O BY T H E S O L V E N T E X T R A C T I O N P R O C E D U R E D E S C R I B E D IN C H A P T E R I I . T H E S T A R T I N G M A T E R I A L WAS 5 9 0 G O F S E P T A L T I S S U E FROM CHUM, S O C K E Y E AND COHO S A L M O N W H I C H HAD B E E N F R O Z E N I M M E D I A T E L Y * P A R T O F T H E WORK D E S C R I B E D IN T H I S C H A P T E R WAS P R E V I O U S L Y P U B L I S H E D ( 8 7 ) . - 2 9 -F I G U R E 8 . E L U T I O N P R O F I L E O F S A L M O N U L T I M O B R A N C H I A L S O L V E N T E X T R A C T ON S E P H A D E X G-50: P R E L I M I N A R Y S T A G E 1. COLUMN, 5 x 1 5 0 CM; E L U A N T , 0.1 M F O R M I C A C I D ; T E M P E R A T U R E , 4 ° C; FLOW R A T E , 1 2 M L / H R ; F R A C T I O N S I Z E , 1 5 . 0 M L . - 3 0 -A F T E R C O L L E C T I O N . A C E T O N E D R Y I N G R E D U C E D T H E W E I G H T O F T H E T I S S U E TO 8 5 G. S O L V E N T E X T R A C T I O N O F T H E DRY POWDER Y I E L D E D 2 . 4 G O F E X T R A C T W I T H A S P E C I F I C A C T I V I T Y O F 3 M R C U/MG. 2 . S T A G E 1 C H R O M A T O G R A P H Y . T H E F I R S T G E L F I L T R A T I O N S T A G E WAS C A R R I E D O U T ON A 5 X 1 3 0 CM C O L U M N O F S E P H A D E X G - 5 0 AT 4 ° C U S I N G A 0 . 1 M F O R M I C A C I D E L U A N T , AND C O L L E C T I N G 1 5 ML F R A C T I O N S AT A R A T E O F 1 2 M L / H R . T H E A B S O R B A N C E A T 2 8 0 NM WAS D E T E R M I N E D FOR T H E E L U A T E F R A C T I O N S A S AN I N D E X O F P R O T E I N C O N C E N T R A T I O N AND T H E Y WERE S U R V E Y E D F O R B I O L O G I C A L A C T I V I T Y AS D E S C R I B E D I N C H A P T E R I I , U S I N G 0 . 3 ML I N J E C T I O N S O F S U I T A B L Y D I L U T E D E L U A T E . T H E 2 . 4 G O F E X T R A C T , WAS D I S S O L V E D I N 2 0 ML O F 0 . 1 M F O R M I C A C I D AND A P P L I E D TO T H E C O L U M N I N TWO 1 0 ML V O L U M E S . T H E V O F E T H E C T WAS T A K E N A S T H E C E N T E R O F T H E B I O L O G I C A L L Y A C T I V E R E G I O N AND U S E D TO C A L C U L A T E A V A L U E FOR K . AN I N D E X O F D M O L E C U L A R W E I G H T W H I C H I S I N D E P E N D E N T O F C O L U M N S I Z E AND I S E Q U A L TO ( V E - V Q ) / ( V - J - V ^ ) WHERE T H E V O L U M E T E R M S H A V E T H E D E F I N I T I O N S U S E O I N C H A P T E R I I . T H E E L U T I O N P R O F I L E AND A C T I V I T Y S U R V E Y FOR T H E S A M P L E S WERE S I M I L A R TO T H O S E S E E N IN C H A P T E R I I A S SHOWN I N F I G U R E 8 . T H E A C T I V E R E G I O N S O F T H E E L U A T E S , F R A C T I O N S 1 1 8 TO 1 4 5 , WERE P O O L E D AND L Y O P H I L I Z E D TO Y I E L D 1 3 0 MG O F S L I G H T L Y O F F - W H I T E POWDER W I T H A S P E C I F I C A C T I V I T Y O F 4 0 M R C U/MG. D E T A I L S O F R E C O V E R Y AND D E G R E E O F P U R I F I C A T I O N A R E I N C L U D E D I N T H E S U M M A R Y . T H E K O F T H E A C T I V E R E G I O N WAS A P P R O X I M A T E L Y 0 . 6 . D 0 100 2 0 0 3 0 0 4 0 0 Elution Volume (ml) F I G U R E 9 . E L U T I O N P R O F I L E O F S T A G E 1 P R O D U C T ON S E - S E P H A Q E X C - 2 5 : P R E L I M I N A R Y S T A G E 2 . COLUMN, 1 . 2 x 2 0 CM; E L U A N T , L I N E A R G R A D I E N T O F P H 3 . 4 AMMONIUM F O R M A T E FROM 0 . 2 M TO 0 . 5 M ; T E M P E R A T U R E , 4 ° C; FLOW R A T E , 1 0 M L / H R . - 3 1 -3 . S T A G E 2 C H R O M A T O G R A P H Y . A 1 . 2 X 2 0 CM C O L U M N O F S E - S E P H A O E X C - 2 5 W A S P R E P A R E D A N D E Q U I L I B R A T E D A T 4 ° C W I T H 0 . 2 M A M M O N I U M F O R M A T E B U F F E R A T P H 3 . 4 . 9 5 M G O F S T A G E 1 P O W D E R W A S D I S S O L V E D I N T H E S T A R T I N G B U F F E R A N D A P P L I E D T O T H E C O L U M N . A P T E R W A S H I N G W I T H 5 0 M L O F S T A R T I N G B U F F E R , T H E M A T E R I A L R E M A I N I N G O N T H E C O L U M N W A S E L U T E D W I T H A L I N E A R G R A D I E N T O F A M M O N I U M F O R M A T E B U F F E R F R O M 0 . 2 M ( P H 3 . 4 ; C O N D U C T I V I T Y , 8 . 0 M M H O S ; V O L U M E , 1 5 0 M L ) . T O 0 . 5 M ( P H 3 . 4 , C O N D U C T i v i T Y , 1 8 . 0 M M H O S ; V O L U M E , 1 5 0 M L ) . T H E E L U A T E W A S C O L L E C T E D A T A R A T E O F 1 0 M L / H R A N D A B S O R B A N C E A T 2 8 0 N M A N D B I O L O G I C A L A C T I V I T Y F O L L O W E D A S B E F O R E . T H E C O N D U C T I V I T Y O F E A C H F R A C T I O N W A S A L S O D E T E R M I N E D U S I N G A R A D I O M E T E R M O O E L C O M 2 0 C O N D U C T I V I T Y M E T E R . T H E E L U T I O N P R O F I L E , C O N D U C T I V I T Y A N D B I O L O G I C A L A C T I V I T Y O F T H E E L U A T E F R A C T I O N S A R E S H O W N I N F L G U R E 9 . T H E B I O L O -G I C A L A C T I V I T Y C A M E O F F T H E C O L U M N I N A B R O A D P E A K C E N T E R E D O N A C O N D U C T I V I T Y O F A B O U T 1 2 M M H O S . T H E E N T I R E A C T I V E R E G I O N F R O M F R A C T I O N 1 5 T O 2 8 W A S P O O L E D A N D L Y O P H I L I Z E D . A S T H E A M M O N I U M F O R M A T E B U F F E R W A S N O T S U F F I C I E N T L Y V O L A T I L E TO A L L O W C O M P L E T E D E S A L T I N G B Y L Y 0 P H I L I Z A T I O N , S P E C I F I C A C T I V I T Y W A S N O T D E T E R M I N E D A T T H I S S T A G E . 4 . S T A G E 3 C H R O M A T O G R A P H Y . B L O - G E L P - 1 0 , A P O L Y A C R I L A M I O E G E L W I T H P R O P E R T I E S S I M I L A R TO S E P H A D E X G - 5 0 , W A S U S E D T O D E S A L T A N D F U R T H E R P U R I F Y T H E S T A G E 2 P R O D U C T . A 1 . 2 X 2 0 0 CM C O L U M N W A S 0.15 w 2 0 | 0 20 40 60 80 Fraction Number F I G U R E 10. E L U T I O N P R O F I L E O F S T A G E 2 P R O O U C T O N B I O - G E L P-10: P R E L I M I N A R Y S T A G E 3. C O L U M N , 1.2 x 200 C M ; E L U A N T , 0.4 M A C E T I C A C I D ; T E M P E R A T U R E , 4 C; F L O W R A T E , 6 M L / M R ; F R A C T I O N S I Z E , 3.4 M L . P R E P A R E D I N 0.4 M A C E T I C A C I D TO P R O V I D E M A X I M U M R E S O L U T I O N A N D T H E P O O L E D A C T I V I T Y F R O M S T A G E 2 W A S A P P L I E D TO T H E C O L U M N I N 1 M L O F E L U A N T M A D E 4 M I N U R E A . T H E E L U A T E W A S C O L L E C T E D I N 3.4 M L F R A C T I O N S A T A R A T E O F 6 M L / H R . As S E E N I N F I G U R E 1 0 T H E B I O L O G I C A L A C T I V I T Y WAS A S S O -C I A T E D W I T H T H E TWO L A R G E , U N R E S O L V E D P E A K S B U T A D E T A I L E D B I O A S S A Y O F T H E R E G I O N S H O W E D T H A T O N L Y T H E B R O A D S H O U L D E R O N T H E L E A D I N G S I D E W A S A C T I V E . F R A C T I O N N U M B E R 4 6 A T T H E C E N T E R O F T H E S H O U L D E R W A S L Y O P H I L I Z E D A N D A N A L I Q U O T O X I -D I Z E D W I T H P E R F O R M I C A C I D A S D E S C R I B E D I N S E C T I O N E . S A M P L E S O F B O T H O X I D I Z E D A N D U N O X I D I Z E D M A T E R I A L W E R E H Y O R O L Y S E D A N D A M I N O A C I D A N A L Y S E S P E R F O R M E D . T H E M O L E R A T I O S O F S E V E R A L A M I N O A C I D S W E R E F A R B E L O W U N I T Y I N D I C A T I N G T H A T T H E P E A K C O U L D N O T C O N T A I N A S I N G L E M O L E C U L E O F T H E E X P E C T E D M O L E C U L A R W E I G H T . T H E Q U A N T I T I E S O F C Y S T I N E A N D C Y S T E I C A C I D W E R E Q U I T E L O W A N D S I N C E TWO R E S I D U E S O F - j p - C Y S T I N E W E R E E X P E C T E D I N T H E N A T I V E M O L E C U L E I T W A S C L E A R T H A T T H E H O R M O N E WAS O N L Y A M I N O R C O M P O N E N T O F T H E P E A K . F R A C T I O N 4 6 C O N -T A I N E D A P P R O X I M A T E L Y 1.5 M G O F L Y O P H I L I Z E D M A T E R I A L ( 0 . 4 5 M G / M L ) R E P R E S E N T I N G 0 . 3 3 M M O L E S ' OF P U R E M A T E R I A L W I T H A M O L E -C U L A R W E I G H T O F 4 5 0 0 . AS T H E F R A C T I O N C O N T A I N E D O N L Y 0.1 JiMOLES O F - j y - C Y S T I N E , E Q U I V A L E N T T O 0.05 _ M M ° L E S O F H O R M O N E , I T W A S E S T I M A T E D T H A T T H E P E A K C O N T A I N E D A B O U T 1 5 $ H O R M O N E . T H E S P E C I F I C A C T I V I T Y O F T H E M A T E R I A L W A S A S S A Y E D A S A B O U T 7 0 0 MRC U/MG S U G G E S T I N G T H A T T H E P U R E H O R M O N E M I G H T H A V E A S P E C I F I C A C T I V I T Y O F N E A R L Y 5 0 0 0 MRC U/MG, A N D I N D I C A T I N G T H A T M U C H L A R G E R Q U A N T I T I E S O F T I S S U E W O U L D B E R E Q U I R E D T O Y I E L D -FIGURE 11 S U M M A R Y O F P R E L I M I N A R Y P U R I F I C A T I O N O F S A L M O N CT P R O C E D U R E S P E C I F I C A P P R O X . W E I G H T A C T I V I T Y A C T I V I T Y (MRC U/MG) (MRC U) S E P T A L T I S S U E D R Y A N D D E F A T W I T H A C E T O N E A C E T O N E P O W D E R 5 9 0 G 8 5 G E X T R A C T W I T H B u O H s H A c s H 20 F I L T R A T E R E S I D U E P R E C I P I T A T E W I T H 5 V O L O F A C E T O N E A T - 2 0 C R E S I D U E P R E C I P I T A T E ( D I S C A R D E D ) E X T R A C T W I T H 0.1M F O R M i c A C I D S U P E R N A T E * R E S I D U E ( D I S C A R D E D ) L Y O P H I L I Z E D F I N A L E X T R A C T 2 4 0 0 M G M O L E C U L A R S I E V I N G O N S E P H A D E X G-50 S T A G E 1 A C T I V I T Y 1 3 0 M G 4 0 9 5 M G , I O N E X C H A N G E O N S E - S E P H A D E X , P H 3.4 S T A G E 2 A C T I V I T Y M O L E C U L A R S I E V I N G ON B I O - G E L P - 1 0 S T A G E 3 A C T I V I T Y 8.3 M G 4 0 0 7 0 0 0 5 2 0 0 3 7 0 0 - 3 3 -S U F F I C I E N T HORMONE FOR A N A L Y S I S . . T H E R E M A I N D E R O F T H E A C T I V E R E G I O N , F R A C T I O N S 4 1 - 5 0 , WAS P O O L E D AND L Y O P H I L I Z E D Y I E L D I N G 3.8 MG O F POWDER. THE T O T A L Y I E L O O F A C T I V I T Y A T T H I S S T A G E WAS A B O U T 3 7 0 0 MRC U FROM AN I N I T I A L S A M P L E C O N T A I N I N G 3 8 0 0 MRC U. TH I S M A T E R I A L WAS U S E D I N A S E R I E S O F T E S T C O L U M N S TO D E T E R M I N E T H E B E S T C O N D I T I O N S FOR F U R T H E R P U R I F I C A T I O N . E V E N T U A L L Y C H R O M A T O -G R A P H Y ON AN S E - S E P H A D E X C O L U M N E Q U I L I B R A T E D A T A H I G H E R P H P R O V E D M O S T E F F E C T I V E . 5. SUMMARY O F P U R I F I C A T I O N . F I G U R E 11 S U M M A R I Z E S T H E M A J O R S T A G E S U S E D I N T H E P R E -L I M I N A R Y E X P E R I M E N T S . B I O L O G I C A L A C T I V I T Y WAS NOT A S S A Y E D D U R I N G T H E I N I T I A L E X T R A C T I O N , B U T AT T H E F I N A L S T A G E T H E E X T R A C T HAD AN A C T I V I T Y O F A B O U T 3 U/MG FOR A T O T A L O F A B O U T 7 0 0 0 U. A F T E R S E P H A D E X C H R O M A T O G R A P H Y A B O U T 7 5 $ O F T H E A C T I V I T Y WAS R E C O V E R E D . 3 8 0 0 U WERE A P P L I E D TO T H E I O N E X C H A N G E C O L U M N AND 3 7 0 0 U R E C O V E R E D A F T E R T H E F I N A L G E L F I L T R A T I O N S T A G E F O R A R E C O V E R Y O F 9 7 $ . T H E O V E R A L L R E C O V E R Y FROM T H E E X T R A C T TO T H E 1 5 $ P U R E P R O D U C T O F S T A G E 3 WAS T H E R E -F O R E A B O U T 7 3 $ . T H E M A T E R I A L A T T H I S S T A G E R E P R E S E N T E D A 5 0 , 0 0 0 F O L D P U R I F I C A T I O N O V E R T H E F R E S H G L A N D S AND R E Q U I R E D A F U R T H E R S E V E N - F O L D P U R I F I C A T I O N . C. P R E P A R A T I ON O F P U R E S A L M O N C A L C I T O N I N . 1. E X T R A C T I O N . T H E P R E L I M I N A R Y E X P E R I M E N T S MADE I T C L E A R T H A T MUCH L A R G E R Q U A N T I T I E S O F G L A N O S WOULD H A V E TO B E P R O C E S S E D AND A S C O L L E C T I O N T E C H N I Q U E S I M P R O V E D TO T H E P O I N T WHERE O V E R 2 0 0 P O U N D S O F S E P T A L T I S S U E WERE C O L L E C T E D FROM 5 0 0 T O N S O F S A L M O N I N A S E A S O N j L A B O R A T O R Y S C A L E D R Y I N G AND E X T R A C T I O N WERE NO L O N G E R F E A S I B L E . Q U A N T I T I E S O F F R O Z E N S E P T A L T I S S U E WERE S H I P P E D TO C A N A D A P A C K E R S L T D . , T O R O N T O FOR S O L V E N T E X T R A C T I O N AND TO ARMOUR P H A R M A C E U T I C A L Co., K A N K A K E E , I L L . F O R E X T R A C T I O N BY A M E T H O D T H E Y HAD D E V E L O P E D FOR P O R C I N E CT ( 1 5 ) . T H E S T A R T I N G M A T E R I A L W H I C H E V E N T U A L L Y Y I E L D E D P U R E S A L M O N C A L C I T O N I N I N S I G N I F I C A N T Q U A N T I T Y WAS AN E X T R A C T P R E P A R E D BY ARMOUR. T H I S M A T E R I A L H A S B E E N R E P O R T E D TO H A V E A S P E C I F I C A C T I V I T Y O F 2 3 0 MRC U/MG ( 6 5 ) ; H O W E V E R , WHEN A S S A Y E D AS D E S C R I B E D I N C H A P T E R I I A SOMEWHAT LOWER S P E C I F I C A C T I V I T Y ( 1 5 0 - 2 0 0 MRC U/MG) WAS O B T A I N E D . 5 0 KG O F S E P T A L T I S S U E Y I E L D E D 4 G O F T H I S M A T E R I A L A T O T A L O F B E T W E E N 6 0 0 , 0 0 0 AND 9 0 0 , 0 0 0 MRC U N I T S . 2 . S T A G E 1 C H R O M A T O G R A P H Y . T H E P R O C E D U R E D E S C R I B E D I N T H E P R E L I M I N A R Y E X P E R I M E N T WAS S C A L E D U P TO H A N D L E T H E E N T I R E 4 G S A M P L E ON A S I N G L E C O L U M N . T H E S T A R T I N G M A T E R I A L WAS D I S S O L V E D I N 4 0 ML O F 2 M U R E A I N 0 . 1 M F O R M I C A C I D AND A P P L I E D TO A 1 0 X 1 5 0 CM C O L U M N O F S E P H A D E X G - 5 0 . T H E C O N S T R U C T I O N O F T H I S C O L U M N I S D E S C R I B E D I N A P P E N D I X B . T H E S A M P L E WAS E L U T E O W I T H 0 . 1 :M F O R M I C A C I D AT 4°C AND C O L L E C T E D A T A R A T E O F 2 5 0 M L / H R . A B S O R B A N C E AT 2 8 0 NM AND B I O L O G I C A L A C T I V I T Y O F T H E S A M P L E S WERE F O L L O W E D A S D E S C R I B E D P R E V I O U S L Y W I T H S A M P L E S S U I T A B L Y D I L U T E D P R I O R TO I N J E C T I O N . S P E C I F I C F I G U R E 1 2 . E L U T I O N P R O F I L E O F S A L M O N U L T I M O B R A N C H I A L A C I D E X T R A C T ON S E P H A D E X G-50S S T A G E 1. C O L U M N , 1 0 X 1 5 0 C M ; E L U A N T , 0.1 M F O R M I C A C I D ; T E M P E R A T U R E , 4 ° C ; F L O W R A T E , 2 5 0 M L / H R . A C T I V I T Y WAS D E T E R M I N E D I N T H E B I O A S S A Y D E S C R I B E D I N C H A P T E R LL ON AN A L I Q U O T O F T H E P O O L E D S A M P L E W H I C H WAS L A T E R L Y O -P H I L I Z E D AND W E I G H E D . T H E E L U T I O N P R O F I L E AND A C T I V I T Y S U R V E Y O F T H E C O L U M N I S SHOWN I N F I G U R E 1 2 . THE P R O F I L E D I F F E R E D M A R K E D L Y FROM T H A T S E E N FOR T H E S O L V E N T E X T R A C T S U S E D P R E V I O U S L Y , B U T V A R I E O O N L Y S L I G H T L Y FROM T H A T S E E N BY K E U T M A N N EJT _A_L_ ( 6 5 ) I N A S I M I L A R E X P E R I M E N T . T H E KQ V A L U E O F T H E B I O L O G I C A L A C T I V I T Y ( 0 . 6 ) , H O W E V E R , WAS N E A R L Y I D E N T I C A L TO T H A T S E E N J N 0 T H*E R E X P E R I M E N T S ON T H E S A L M O N HORMONE. T H E A C T I V E R E G I O N E X T E N D E D FROM AN E L U T I O N V O L U M E O F 8.3 L TO 9.6 L , B U T TO A V O I D C O N T A M I N A T I O N FROM T H E L A R G E L E A D I N G P E A K O N L Y T H E R E G I O N FROM 8.5 TO 9.5 L WAS P O O L E D . A F T E R L Y O P H I L I Z A T I O N T H I S P O O L Y I E L D E D 3 5 0 MG O F M A T E R I A L W I T H A S P E C I F I C A C T I V I T Y O F A B O U T 6 5 0 MRC U/MG ( S E E F I G U R E 1 8 ) . T H E Low R E C O V E R Y ON T H I S C O L U M N ( 3 0 $ TO 4 0 $ ) WAS D U E P R I M A R I L Y TO T H E NARROW R E G I O N P O O L E D . D E T A I L S ON R E C O V E R Y AND D E G R E E O F P U R I F I C A T I O N A R E G I V E N I N T H E SUMMARY. 3. S T A G E 2 C H R O M A T O G R A P H Y . T H E C O L U M N U S E D I N T H I S S T A G E WAS S I M I L A R TO T H A T D E S C R I B E D IN T H E P R E L I M I N A R Y E X P E R I M E N T T H O U G H S L I G H T L Y -L A R G E R TO H A N D L E T H E G R E A T E R Q U A N T I T Y O F M A T E R I A L . A 2.0 X 2 0 CM C O L U M N O F S E - S E P H A D E X C-25 WAS E Q U I L I B R A T E D AT 4 ° C W I T H 0.2 M AMMONIUM F O R M A T E AT PH 3.4. T H E 3 5 0 MG O F M A T E R I A L FROM S T A G E 1 WAS A P P L I E D TO T H E C O L U M N IN 1 0 ML O F S T A R T I N G B U F F E R AND E L U T E D W I T H 4 0 0 ML O F S T A R T I N G B U F F E R „ 3.0 N Fraction Number F I G U R E 13. E L U T I O N P R O F I L E O F S T A G E 1 P R O D U C T O N S E - S E P H A D E X 025* S T A G E 2. C O L U M N , 2 x 20 C M ; E L U A N T , L I N E A R G R A D I E N T O F P H 3.4 A M M O N I U M F O R M A T E F R O M 0.2 M TO 0.6 M, F O L L O W E D B Y 1 M A M M O N I U M H Y D R O X I D E ; T E M P E R A T U R E 4° C; F L O W R A T E , 20 M L / H R ; F R A C T I O N S I Z E , 12 M L . - 3 6 -F O L L O W E D BY A L I N E A R G R A D I E N T O F pH 3.4 AMMONIUM F O R M A T E B U F F E R FROM 0.2 M ( C O N D U C T I V I TY 8..0 MMHOS i V O L U M E » 5 0 0 ML ) TO 0.6 M ( C O N D U C T I V I T Y , 24.0,MMHOS; V O L U M E , 5 0 0 M L ) . A F T E R T H E G R A D I E N T WAS C O M P L E T E D T H E C O L U M N WAS WASHED W I T H 5 0 0 ML O F 1 M AMMONIUM H Y D R O X I D E TO E N S U R E R E C O V E R Y O F A L L A P P L I E D M A T E R I A L S . T H E E L U A T E WAS C O L L E C T E D I N 1 2 ML F R A C T I O N S AT A R A T E O F 2 0 M L / H R . A B S O R B A N C E A T 2 8 0 NM, C O N D U C T I V I T Y AND B I O L O G I C A L A C T I V I T Y WERE F O L L O W E D AS P R E V I O U S L Y D E S C R I B E D . F I G U R E 1 3 SHOWS T H E R E S U L T S O F T H E S T A G E 2 E L U T I O N . ONLY ONE M A J O R P E A K WAS E L U T E D D U R I N G T H E G R A D I E N T A L T H O U G H T H I S P E A K A P P E A R E D TO C O N T A I N A T L E A S T TWO C O M P O N E N T S . T H E C O N D U C T I V I T Y A T T H E P E A K WAS S L I G H T L Y H I G H E R ( 1 6 MMHOS V S . 1 2 MMHOS) T H A N S E E N P R E V I O U S L Y , B U T T H E S H O U L D E R O F T H E P E A K C O R R E S P O N D E D W E L L TO T H E M A J O R R E G I O N O F B I O L O G I C A L A C T I V I T Y , A S E C O N D M I N O R R E G I O N O F B I O L O G I C A L A C T I V I T Y E L U T E D P R I O R TO T H E P E A K WAS NOT I N V E S T I G A T E D F U R T H E R . IN R E T R O S P E C T I T I S L I K E L Y T H A T T H I S R E G I O N MAY H A V E R E P R E S E N T E D T H E M I N O R C O M P O N E N T O E S C R I B E O BY K E U T M A N N E T A L ( 6 5 ) , AND MAY I N D I C A T E T H A T T H E D I F F E R E N T S P E C I E S O F S A L M O N FROM W H I C H T H E G L A N D S WERE C O L L E C T E O DO, I N F A C T , H A V E C A L C I T O N I N S O F S L I G H T L Y D I F F E R E N T S T R U C T U R E S . TO M I N I M I Z E C O N T A M I N A T I O N A NARROW R E G I O N O F M A X I MUM A C T I V I T Y WAS P O O L E D ( F R A C T I O N S 8 4 TO 9 8 ) ANO L Y O P H I L I Z E D . S P E C I F I C A C T I V I T Y C O U L D NOT B E D E T E R M I N E D B E C A U S E O F R E S I D U A L S A L T IN T H E L Y O P H I L I Z E D P R O D U C T , B U T T H E , D E G R E E O F P U R I F I C A T I O N D I D NOT A P P E A R TO B E G R E A T S I N C E O N L Y M I N O R C O N T A M I N A N T P E A K S WERE R E M O V E D . C U R E 14. E L U T I O N P R O F I L E O F S T A G E 2 P R O D U C T O N S E P H A D E X G-50: S T A G E 3. C O L U M N , 1.2 x 2 2 0 C M ; E L U A N T 0.1 M F O R M I C A C I D ; T E M P E R A T U R E , 4° C; F L O W R A T E , 6 M L / H R ; F R A C T I O N S I Z E , 3.4 M L . - 3 7 -4 . S T A G E 3 C H R O M A T O G R A P H Y . T H I S S T A G E WAS E S S E N T I A L L Y A D E - S A L T I N G S T E P , B U T A L O N G , H I G H R E S O L U T I O N C O L U M N WAS U S E D TO R E M O V E C O N T A M I N A N T S L E F T BY T H E F I R S T L A R G E G E L F I L T R A T I O N C O L U M N . A 1 . 2 X 2 2 0 CM C O L U M N O F S E P H A D E X G - 5 0 WAS P R E P A R E D A T 4 ° C I N 0 . 1 M F O R M I C A C I D . T H E S T A G E 2 P R O D U C T WAS D I S S O L V E D IN 7 ML O F 8 M U R E A I N 0 . 1 M F O R M I C A C I D AND L A Y E R E D ONTO T H E T O P O F T H E C O L U M N . T H E E L U A T E WAS C O L L E C T E D I N 3 . 4 ML F R A C T I O N S AT A R A T E O F 6 M L / H R . As SHOWN I N F I G U R E 1 4 , T H E B I O L O G I C A L A C T I V I T Y WAS C O N F I N E D TO A S I N G L E M A J O R P E A K W I T H A K Q O F 0 . 6 . T H I S P E A K WAS S L I G H T L Y S K E W E D , B U T L A T E R R E S U L T S I N D I C A T E D T H A T T H I S A S Y M M E T R Y WAS A C H A R A C T E R I S T I C O F T H E C O L U M N U S E D . F R A C T I O N S . 4 8 TO 6 3 WERE P O O L E D AND A F T E R L Y O P H I L I Z A T I O N T H E P O O L Y I E L D E D 5 5 MG O F M A T E R I A L W I T H A S P E C I F I C A C T I V I T Y O F A B O U T 1 2 0 0 MRC U/MG ( S E E F I G U R E 1 8 ) F O R A T O T A L R E C O V E R Y O F 6 5 , 0 0 0 MRC U . 5 . S T A G E 4 C H R O M A T O G R A P H Y . FOR T H E S E C O N D IO N E X C H A N G E S T A G E A 1 . 2 x 2 0 CM C O L U M N OP S E - S E P H A D E X C - 2 5 E Q U I L I B R A T E D A T 4 ° C W I T H 0 . 1 M AMMONIUM A C E T A T E A T PH 4 . 5 WAS P R E P A R E D . T H E 5 5 MG S A M P L E O F S T A G E 3 M A T E R I A L WAS D I S S O L V E D I N 3 ML O F S T A R T I N G B U F F E R ANO A L L O W E D TO FLOW ONTO T H E C O L U M N . A F T E R T H E C O L U M N HAD B E E N WASHEO W I T H 4 0 0 ML O F 0 . 1 M AMMONIUM A C E T A T E , A L I N E A R G R A D I E N T O F P H 4 . 5 AMMONIUM F O R M A T E B U F F E R FROM 0 . 1 M ( C O N D U C T I V I T Y , 3 . 5 MMHOS; V O L U M E , 3 5 0 M L ) TO 0 . 5 M ( C O N D U C T I V I T Y , 1 6 . 0 MMHOS; 9$ 2.0 0 2 0 4 0 ' 6 0 8 0 100 120 Fraction Number F I G U R E 1 5 . E L U T I O N P R O F I L E O F S T A G E 3 P R O D U C T O N S E - S E P H A D E X C - 2 5 * S T A G E 4 . C O L U M N , 1 . 2 x 2 0 C M ; E L U A N T , L I N E A R G R A D I E N T S O F P H 4 . 5 A M M O N I U M A C E T A T E F R O M 0 . 1 M T O 0 . 2 M A N D F R O M 0 . 2 M T O 0 . 5 M; T E M P E R A T U R E 4 ° C ; F L O W R A T E , 1 2 M L / H R ; F R A C T I O N S I Z E , 9 M L . 0 20 40 60 80 Fraction Number F I G U R E 1 6 ; E L U T I O N P R O F I L E O F S T A G E 4 P R O D U C T O N S E P H A D E X G-50: S T A G E 5. C O N D I T I O N S I D E N T I C A L TO F I G U R E 14 - 3 8 -VOLUME, 3 5 0 ML) WAS BEGUN. WHEN THE F I R S T PEAK BEGAN TO APPEAR IN THE E L U A T E THE 0.5 M BUFFER WAS DISCONNECTED AND E L U T I O N CONTINUED WITH 2 5 0 ML OF BUFFER AT THE E S T A B L I S H E D CONCEN-R T R A T I O N . WHEN THE ABSORBANCE HAD RETURNED TO ZERO A NEW GRADIENT WAS BEGUN WITH PH 4.5 AMMONIUM A C E T A T E BUFFER FROM 0.2 M (C O N D U C T I V I T Y , 5.6 MMHOS; VOLUME, 3 0 0 ML) TO 0.5 M ( C O N D U C T I V I T Y , 1 6 . 0 MMHOS: VOLUME, 3 0 0 M L ) . TH I S GRADIENT WAS USED UNT IL A NEW PEAK B E G A N , AT WHICH TIME THE PROCEDURE D E S C R I B E D ABOVE WAS R E P E A T E D . THE E L U A T E WAS C O L L E C T E D IN 9 ML F R A C T I O N S AT A RATE OF 1 2 M L / H R . F IGURE 1 5 SHOWS THE E L U T I O N P R O F I L E , CONDUCTIV ITY GRADIENT AND B I O L O G I C A L A C T I V I T Y S U R V E Y . THREE WELL DEF INED PEAKS WERE E L U T E D FROM THE COLUMN, ONLY ONE OF WHICH WAS A S S O C I A T E D WITH ANY B I O L O G I C A L ACT I V I T Y . T H I S PEAK WAS SYME T R I C A L AND SHOWED NO INDICAT ION OF CONTAMINAT ION. FR A C T I O N S 1 0 9 TO 121 WERE POOLED AND L Y O P H I L I Z E D . 6. STAGE 5 CHROMATOGRAPHY. THE PROCEDURES AND EQUIPMENT USED IN THE STAGE 5 CHROMATOGRAPHY WERE IDENT ICAL IN ALL R E S P E C T S TO THOSE D E S C R I B E D FOR STAGE 3. THE SAMPLE USED WAS THE L Y O P H I L I Z E D POOL FROM ST A G E 4 D I S S O L V E D IN 1 ML OF 0.1 M FORMIC A C I D , B L O L O G I CAL ACT I V ITY WAS MEASURED IN ASSAYS ON AN ALIQUOT FROM FRACT ION 56 AS P R E V I O U S L Y D E S C R I B E D , AND S P E C I F I C A C T I V I T Y C A L C U L A T E D FROM THE WEIGHT OF A L Y O P H I L I Z E D ALIQUOT OF THIS F R A C T I O N . THE ELUT ION P R O F I L E SHOWN IN F IGURE 16 FOR THE F INAL P U R I F I C A T I O N STAGE CONTAINED A S INGLE MAJOR PEAK AND A SMALL F IGURE 1 7 SUMMARY OF P U R I F I C A T I O N OF SALMON CT PROCEDURE WEIGHT S P E C I F I C A P P R O X . A C T I V I T Y A C T I V I T Y (MRC U/MG) (MRC U ) S E P T A L T I S S U E 5 0 KG A C I D E X T R A C T I O N BY ARMOUR C O . • F INAL EXTRACT 4 G 1 5 0 - 2 3 0 7 5 0 , 0 0 0 MOLECULAR S I E V I N G ON SEPHADEX G - 5 0 v — S T A G E 1 A C T I V I T Y 3 5 0 MG 6 5 0 2 3 0 , 0 0 0 I ON EXCHANGE ON S E - S E P H A D E X , PH 3 . 4 STAGE 2 A C T I V I T Y - - -MOLECULAR S I E V I N G ON SEPHADEX G - 5 0 V S T A G E 3 A C T I V I T Y 5 5 MG 1 2 0 0 6 5 , 0 0 0 ION EXCHANGE ON S E - S E P H A D E X , PH 4 . 5 STAGE 4 A C T I V I T Y - - -MOLECULAR S I E V I N G ON SEPHADEX G - 5 0 • S T A G E 5 A C T I V I T Y 1 5 MG 4 5 0 0 6 5 , 0 0 0 F IGURE 1 8 . REGRESSIONS OF LOG DOSE ON RESPONSE FOR STAGE 1, 3 AND 5 PRODUCTS . CA L C U L A T I O N S AS DESCRIBED FOR F IGURE 3 . o - STAGE T , V - STAGE 3» a - STAGE 5. i - 3 9 -PEAK AT V T INDICAT ING THAT THE STAGE 5 MATERIAL WAS E S S E N T I A L L Y F R E E OF PROTE IN CONTAMINANTS. THE S L I G H T ASYMMETRY WAS A C H A R A C T E R I S T I C OF THE COLUMN U S E D , AS THE T E S T S FOR HOMOGENEITY D E S C R I B E D IN S E C T I O N G SHOWEO THAT ONLY A S INGLE P E P T I D E WAS PRESENT IN THE P E A K . THE B I O L O G I C A L ACTI V ITY C0RRESPONDED WELL TO THE PEAK AND VARIED WITH THE CONCENTRATION IN EACH F R A C T I O N . THE R E S U L T S OF THE B IOASSAY ON THE MATERIAL IN FRACTION 56 ARE SHOWN IN F IGURE 1 8 . ON THE B A S I S OF T H I S ASSAY AND THE P E P T I D E CONCENTRATION IN THE F R A C T I O N , 0.69 MG/ML, THE ABSOLUTE S P E C I F I C A C T I V I T Y OF PURE SALMON C T WAS C A L C U L A T E D TO BE CLOSE TO 4 5 0 0 M R C L)/MG NEARLY 2 5 T IMES THE S P E C I F I C A C T I V I T Y OF PURE PORCINE C T . THE Y I E L D AT T H I S STAGE WAS 1 4 . 5 MG OF PURE HORMONE, A TOTAL OF 6 5 , 0 0 0 M R C U R E P R E S E N T I N G E S S E N T I A L L Y 1 0 0 $ OF THE MATERIAL A P P L I E D IN ST A G E 4. 7. SUMMARY OF PU R I F I C A T I O N SC H E D U L E . F IGURE 1 7 SUMMARIZES THE S T A G E S USED IN THE P R E P A R A T I O N OF PURE SALMON C T . THE RESULTS OF B IOASSAYS AT STAGES 1,3 AND 5 ARE SHOWN IN F|GURE 1 8 0 THE RECOVERY AT EACH STAGE IS ALSO RECORDED IN F IGURE 1 7 . ALTHOUGH HIGH L O S S E S OCCURED DURING THE F I R S T THREE S T A G E S , PROBABLY DUE IN PART TO AN A C T I V E MINOR COMPONENT WHICH WAS REMOVED, THE R E C O V E R I E S IN THE TWO F INAL S T A G E S WERE E S S E N T I A L L Y 1 0 0 $ . THE OVERALL RECOVERY WAS ABOUT 1 0 $ , BUT A 3 0 0 , 0 0 0 FOLD P U R I F I C A T I O N WAS A C H I E V E D FROM S E P T A L T I S S U E WITH AN E S T I M A T E D S P E C I F I C A C T I V I T Y OF 1 5 M R C L)/G TO PURE HORMONE WITH AN A C T I V I T Y OF 4 5 0 0 M R C U/MG. (a) C l -4-5 8 5 6 5 4 5 2 0 + H— 5 0 ' 4 8 0 C 2 (b) 10 K 10 A 2 0 K F I G U R E 1 9 . T H I N L A Y E R C H R O M A T O G R A P H Y O F S T A G E 5 P R O D U C T ON S I L I C A G E L . S O L V E N T S Y S T E M ; B U T A N O L , A C E T I C A C I D , W A T E R , P Y R I D I N E ( 1 5 : 1 0 : 1 2 : 3 ) . O-PAQ P O S I T I V E , © - PAQ A N D N I N H Y D R I N P O S I T I V E . ( A ) 1 0 M L S A M P L E S O F A L T E R N A T E F R A C T I O N S F R O M 4 8 T O 5 8 . C-1, 1 N M O L E A R G . C-2, 0.1 N M O L E A R G . ( B ) 1 0 A N D 2 0 ML S A M P L E S F R O M F R A C T I O N 5 4 . -40-8. CR I T E R I A OF HOMOGENEITY . S I N C E ALL S T A G E S OF P U R I F I C A T I O N HAD INVOLVED S E P A R A T I O N S BASED ON E I T H E R CHARGE OR MOLECULAR S I Z E A S E P A R A T I O N BASED ON D I F F E R E N T I A L S O L U B I L I T I E S SEEMED MOST L I K E L Y TO DETECT ANY CONTAMINANT. TH IN LAYER CHROMATOGRAPHY IN A SYSTEM USED ON PORCINE CT (15) WAS CHOSEN AS THE BEST P R O C E D U R E . TH IN LAYER CHROMATOGRAMS WERE RUN IN AN ASCENDING SOLVENT SYSTEM ON EASTMAN PRECOATED 20 x 20 C M , 6061, S I L I C A GEL P L A T E S (D I S T I L L A T I O N PRODUCTS IN D U S T R I E S , ROCHESTER N.Y.) A C T I V A T E D AT 110°C FOR 30 M I N U T E S . A B U T A N O L , P Y R I D I N E , WATER, A C E T I C AC ID (I5t10t12t3) BUFFER SYSTEM WAS U S E D . 10 U.L AL IQUOTS FROM F R A C T I O N S 50, 52, 54, 56, 58 WERE A P P L I E D TO THE ORIGIN AND ALLOWED TO MIGRATE FOR A PERIOD OF 5 HOURS. THE P L A T E S WERE THEN SPRAYED WITH PHENANTHRENEQUI NONE ( P A Q ) R E A G E N T . AF T E R DRYING AND OBSERVATION UNDER UV L I G H T TO DETECT ARGIN INE CONTAINING SPOTS THE P L A T E S WERE SPRAYED A SECOND TIME WITH A S P E C I A L NINHYDRIN REAGENT DESIGNED TO FOLLOW THE P A Q . PR E P A R A T I O N OF THESE REAGENTS IS D E S C R I B E D IN APPENDIX C. IN A SECOND E X P E R I M E N T AL IQUOTS OF VARIOUS S I Z E S WERE TAKEN FROM FRACT ION 54 AND TREATED IN A S IMILAR MANNER. ONE CHANNEL WAS RETAINED U N D E V E L O P E D , HOWEVER, AND THE P E P T I D E CONTAINING REGION E L U T E D WITH V E H I C L E AND B I O A S S A Y E D . TR A C I N G S OF THE THIN LAYER CHROMATOGRAMS ARE SHOWN IN F IGURE 19(A). ONLY FRACT IONS 52, 54, AND 56 CONTAINED S U F F I C I E N T MATERIAL FOR D E T E C T I O N BY BOTH R E A G E N T S . THE P A Q REAGENT PRODUCED D I S T I N C T FLOURESCENT SPOTS IN ALL BUT TWO F R A C T I O N S AND ON T E S T SAMPLES CONTAINING 0.1 AND 1.0 NM OLE - 41 -OF A R G I N I N E . THE 1 NMOLE SAMPLE OF ARGININE WAS F A I N T L Y NINHYORIN P O S I T I V E . IN THE FRACT IONS 5 0 TO 5 6 ONLY A S INGLE SPOT COULD BE DETECTED ANO IN EACH CASE THE SPOT HAD AN R OF ABOUT 0 . 5 WITH THE MORE CONCENTRATED SAMPLES BE ING S L I G H T L Y R E T A R D E D . THUS IT APPEARED THAT THE PEAK CONTAINED ONLY A S I N G L E COMPONENT. TO CONFIRM THIS A SECOND EXPERIMENT WAS DONE (F I G U R E 19B) USING TWICE AS MUCH SAMPLE WITH S IMILAR R E S U L T S . B IOASSAY OF AN UNDEVELOPED SAMPLE FROM THE SECOND CHROMATOGRAM CONFIRMED THAT THIS COMPONENT WAS B I O L O G I C A L L Y A C T I V E . AMINO ACID A N A L Y S E S PERFORMED ON ALTERNATE FRACT IONS BETWEEN NUMBERS 5 0 AND 5 6 AS DESCRIBED IN S E C T I O N E FURTHER CONFIRMED THE HOMOGENEITY OF THE A C T I V E P E A K . THE RESULTS OF THESE A N A L Y S E S AS SHOWN IN TABLE I V INDICATE NO S I G N I F I C A N T D I F F E R E N C E BETWEEN THE MOLE RATIOS OF THE AMINO ACIDS IN THE VARIOUS F R A C T I O N S . ON THIS B A S I S IT WAS CONCLUDED THAT THE MATERIAL IN FRACT IONS 4 9 ~ 5 7 REPRESENTED PURE SALMON C T IN S U F F I C I E N T QUANTITY TO ALLOW COMPLETE CHEMICAL C H A R A C T E R I -Z A T I O N . D. CH A R A C T E R I Z A T I O N OF THE WHOLE M O L E C U L E . 1 . ME T H O D S . A . AMINO A c I O CO M P O S I T I O N . AMINO ACID A N A L Y S E S WERE CARRIED OUT ON FRACTIONS 5 0 , 5 2 , 5 4 , AND 5 6 OF THE ST A G E 5 E L U A T E . 1 0 0 ML AL IQUOTS OF THESE FRACT IONS WERE L Y O P H I L I Z E D AND HYDROLYSED IN 5 . 7 M HYDROCHLORIC ACID FOR 1 7 HOURS AT 1 1 0 ° C . THE HYDROLYSED S A M P L E S W E R E D R I E D J_N V A C U O A N D A I R O X I D I Z E D T O I N S U R E C O M P L E T E R E C O V E R Y O F - ^ - - C Y S T I N E . T H E D E T A I L E D M E T H O D O L O G Y I S D E S C R I B E D I N A P P E N D I X D . S A M P L E S E Q U I V A L E N T T O 4 0 J4.L O F T H E O R I G I N A L A L I Q U O T S W E R E T H E N S U B J E C T E D T O A U T O M A T E D A M I N O A C I D A N A L Y S I S B Y T H E M E T H O D O F S P A C K M A N E_T AJ. (119) O N A B I O C H R O M MO D E L 2 0 0 A N A L Y S E R W H I C H H A D B E E N M O D I F I E D W I T H A W H E A T S T O N E B R I D G E - P H O T O T R A N S I S T O R D E T E C T I O N C I R C U I T A N D S P E C I A L L O N G P A T H L E N G T H F L O W C E L L S ' T O A L L O W Q U A N T I T A T I O N O F A M I N O A C I D S A T L E V E L S DOWN T O 2 N M O L E S . * T H E S E P R O C E D U R E S A L L O W E D D E T E R M I N A T I O N O F A L L A M I N O A C I D S E X C E P T G L U T A M I N E A N D A S P A R A G I N E W H I C H A R E D E A M I DA T E D B Y A C I D H Y O R O L . Y S E S . ( I D E N T I F I-* C A T I O N O F T H E S E R E S I D U E S W I L L B E D I S C U S S E D L A T E R ) A N D T R Y P T O P H A N W H I C H I S D E S T R O Y E D B Y A C I D H Y D R O L Y S I S . D E T E R M I N A T I O N O F T R Y P T O P H A N WAS B A S E D ON M E A S U R E M E N T S O F T H E M O L A R E X T I N C T I O N C O E F F I C I E N T S O F T H E W H O L E M O L E C U L E A T 2 8 0 A N D 2 3 0 NM A N O ON T H E R E S U L T S ON A T E S T W I T H E H R L I C H R E A G E N T W H I C H I S S P E C I F I C F O R T R Y P T O P H A N . M I L L I G R A M E X T I N C -T I O N C O E F F I C I E N T S ( A B S O R B A N C E IN A 1 CM P A T H L E N G T H O F A S O L U T I O N C O N T A I N I N G 1 M G / M L O F T H E P R O T E I N ) W E R E C A L C U L A T E D F R O M T H E M E A S U R E D A B S O R B A N C E O F F R A C T I O N 56 I N A 1 CM C E L L A T 2 3 0 A N D 2 8 0 NM I N A B A U S C H A N D L O M B S P E C T R O N I C 6 0 0 S P E C T R O -P H O T O M E T E R A T P H 2 IN 0.1 M F O R M I C A C I D A G A I N S T A B L A N K O F T H E S A M E B U F F E R . S A M P L E S O F T H E F R A C T I O N W E R E L A T E R L Y O -* E X T E N S I V E M O D I F I C A T I O N O F T H I S M A C H I N E O C C U R E D O U R I N G T H E C O U R S E O F T H E S E S T U D I E S A N D T H E A N A L Y S E S P R E S E N T E D I N V A R I O U S S E C T I O N S O F T H I S T H E S I S W E R E P E R F O R M E D A T D I F F E R E N T S T A G E S O F T H I S M O D I F I C A T I O N . A P P E N D I X D C O N T A I N S A D E S C R I P T I O N O F T H E E V O L U T I O N O F T H E A M I N O A C I D A N A L Y S E R , A N D T H E D E T A I L S IN T H E T E X T A R E P R E S E N T E D O N L Y T O A L L O W T H E R E A D E R T O I D E N T I F Y T H E S T A G E A T W H I C H A P A R T I C U L A R A N A L Y S I S WAS P E R F O R M E D . T A B L E I V A M I N O A C I O A N A L Y S E S O F F R A C T I O N S F R O M S T A G E 5 C H R O M A T O G R A P H Y 5 0 ~ 5 2 : " 5 4 R 5D" A M I N O A C I D S N A N O - M O L E * N A N O - M O L E N A N O - M O L E N A N O - M O L E M O L E S R A T I O M O L E S R A T I O M O L E S R A T I O M O L E S R A T I O A S P A R T I C A C I O 5 .1 2 . 0 8 . 7 2 . 0 1 5 . 1 2 . 0 1 8 . 0 2 . 0 T H R E O N 1 N E 1 2 . 8 5 . 1 2 2 . 4 5.1 3 7 . 8 5 . 0 4 5 . 0 5 . 0 S E R 1 N E 1 0 . 0 4 . 0 1 7 . 4 4 . 0 2 4 . 6 3 . 3 3 7 . 0 4 . 1 G L U T A M I C A C I D 7 . 6 3 . 0 1 2 . 9 3 . 0 2 2 . 7 3 . 0 2 6 . 6 3 . 0 P R O L 1 N E 6 . 2 2 . 5 9 . 3 2 . 1 1 3 . 4 1 . 8 1 7 . 3 1 . 9 G L Y C 1 N E 8 . 8 3 . 5 1 4 . 1 3 . 2 2 3 . 2 3 . 0 2 8 . 8 3 . 2 A L A N I N E 0 0 0 0 0 0 0 0 C Y S T I N E 4 . 6 1 . 8 9 . 3 2 . 1 1 2 . 5 1 . 7 1 7 . 0 1 . 9 V A L 1 N E 2 . 7 1 . 1 4 . 8 1.1 6 . 5 0 . 9 8 . 0 0 . 9 M E T H 1 ON 1NE 0 0 0 0 0 0 0 0 1 S O L E U C 1 N E 0 0 0 0 0 0 0 0 L E U C 1 N E 1 3 . 0 5 . 2 2 0 . 2 4 . 7 3 8 . 5 5 . 1 4 8 . 7 5 . 4 T Y R O S 1 N E 2 . 4 1 . 0 3 . 8 0 . 9 6 . 5 0 . 9 7 . 4 0 . 8 P H E N Y L A L A N 1NE 0 0 0 0 0 0 0 0 S E P A R A T E A N A L Y S E S ON S H O R T C O L U M N L Y S I N E 7 . 0 2 . 0 1 6 . 3 2 . 0 H i S T 101NE 3 . 7 1 . 0 9 . 1 1 . 1 A R G I N I N E 4 . 0 1 . 1 8 . 1 1 . 0 A M M O N i A 1 9 . 0 5 . 3 4 4 . 4 5 . 5 * W l T H R E F E R E N C E TO A S P A R T I C Ac ID OR L Y S I N E . - 4 3 -P H I L I Z E D A N O W E I G H E D TO D E T E R M I N E P R O T E I N C O N C E N T R A T I O N . MO L A R E X T I N C T I O N C O E F F I C I E N T S W E R E C A L C U L A T E D F R O M T H E S E V A L U E S A N O T H E M O L E C U L A R W E I G H T O F T H E P O L Y P E P T I D E A S D E T E R M I N E D F R O M T H E A M I N O A C I D . A N A L Y S E S * , T H E E H R L I C H R E A G E N T U S E WAS A 0 . 5 $ (W / V ) S O L U T I O N 0 F IP -D I M E T H Y L AM I N 0 B E N Z A L D E H Y 0 E ( F l S H E R S C I E N T I F | C Co.) I N 9 5 $ E T H A N O L C O N T A I N I N G 2 $ ( v / v ) C O N C E N T R A T E D H Y D R O C H L O R I C A C I D ( 6 8 ) . T H I S WAS S P R A Y E D O N T O A S I L I C A G E L T H I N L A Y E R P L A T E W H I C H H A D B E E N S P O T T E D W I T H 1 0 yUL O F F R A C T I O N 5 6 . B . EN D G R O U P A N A L Y S I S . TH E N - T E R M I N A L A M I N O A C I D I N T H E N A T I V E M O L E C U L E WAS D E T E R M I N E D B Y T H E D A N S Y L M E T H O D O F G R A Y A N D H A R T L E Y ( 4 7 ) . A O N E N M O L E S A M P L E O F T H E H O R M O N E WAS D I S S O L V E D I N 0.2 M S O D I U M B I C A R B O N A T E A N O T R E A T E O W I T H D A N S Y L C H L O R I D E I N A C E T O N E . H Y O R O L Y S I S O F T H E S A M P L E I N 5.7 M H Y D R O C H L O R I C A C I D WAS U S E D T O R E L E A S E T H E D A N S Y L D E R I V A T I V E O F T H E N - T E R M I N A L A M I N O A C I D W H I C H WAS T H E N I D E N T I F I E D B Y P O L Y A M I D E L A Y E R C H R O M A T O -G R A P H Y A S D E S C R I B E D B Y W O O D S A N D W A N G ( 1 3 2 ) A N D M O D I F I E D B Y H A R T L E Y ( 5 2 ) . D E T A I L S O F T H E S E M E T H O D S A R E I N C L U D E D I N A P P E N D I X E. 2. R E S U L T S A N D D I S C U S S I O N . T H E R E S U L T S O F T H E A M I N O A C I D A N A L Y S E S O F T H E F O U R F R A C T I O N S A C R O S S T H E A C T I V E P E A K A R E S H O W N I N T A B L E I V . No S I G N I F I C A N T D I F F E R E N C E C O U L D B E S E E N I N T H E M O L E R A T I O S O F T H E V A R I O U S S A M P L E S D E S P I T E T H E O B V I O U S D I F F E R E N C E S IN T H E A B S O L U T E Q U A N T I T I E S A N A L Y S E D . T H E A V E R A G E V A L U E S F O R T H E TABLE V AMINO A C I D COMPOSITIONS OF F I V E C A L C I T O N I N S AMINO A C I D S SALMON CT* HUMAN CT (80) PORC1NE CT (96) BOV1NE C T ( 1 7 ) OV 1 NE CT(91 A S P A R T I C ACID 2 3 4 4 4 THREONINE 5 5 2 2 2 SER1NE 4 1 4 4 4 GLUTAMIC ACID 3 2 1 1 1 PROL1NE 2 2 2 2 2 G L Y C I N E 3 4 3 3 3 ALAN I NE 0 2 1 1 1 •g- CYST i NE 2 2 2 2 2 V A L I N E 1 1 1 1 1 METH1 ON 1NE 0 1 1 1 1 1SOLEUC1NE 0 1 0 0 0 L E U C I N E 5 2 3 3 3 TYROSINE 1 1 1 2 3 PHENYLALAN 1NE 0 3 3 2 1 L Y S 1 N E 2 1 0 1 1 H I S T I D I N E 1 1 1 1 1 A R G I N I N E 1 0 2 1 1 AMMON1 A 5 5 5 4 4 TRYPTOPHAN 0** 0 1 1 1 * MOLE INTEGERS BASED ON AVERAGES FROM EIGHT ANALYSES. **FROM A O Q ~ AND NEGATIVE E H R L I C H ' S TEST. 280 NM - 4 4 -M O L E R A T I O S B A S E D ON A T O T A L O F E I G H T A N A L Y S E S A N O A R E V E R Y C L O S E T O T H E I N T E G R A L V A L U E S A S S H O W N I N T A B L E V . AS T H E Y I E L D S O F A L L A M I N O A C I D S W E R E G O O D A N D C O N S I S T E N T F R O M S A M P L E T O S A M P L E H Y D R O L Y S E S F O R V A R Y I N G T I M E I N T E R V A L S W E R E N O T P E R F O R M E D . , TH E A B S O R B A N C E S A T 2 8 0 NM ( 0 . 2 9 2 ) A N D 2 3 0 NM ( 1 . 3 9 ) F O R A S O L U T I O N C O N T A I N I N G 0.69 MG O F P E P T I D E P E R ML. I N D I C A T E D M I L L I G R A M E X T I N C T I O N C O E F F I C E N T S O F 0.45 A T 2 8 0 NM A N D 2.0 A T 2 3 0 N M . B A S E D ON A M O L E C U L A R W E I G H T O F 3 4 2 7 F R O M A M I N O A C I D A N A L Y S I S T H I S S O L U T I O N WAS 2.0 X 10""^ M I N H O R M O N E , A N D T H E M O L A R E X T I N C T I O N C O E F F I C I E N T S O F T H E P O L Y P E P T I D E W E R E 1 5 0 0 A T 2 8 0 NM A N D 7 0 0 0 A T 2 3 0 N M . T H I S M O L A R E X T I N C T I O N C O E F F I C I E N T A T 2 8 0 NM WAS A P P R O X I M A T E L Y E Q U A L T O T H A T O F T Y R O S I N E U N D E R S I M I L A R C O N D I T I O N S I NO I C A T I N G T H A T T H E S I N G L E T Y R O S I N E F O U N D B Y A M I N O A C I D A N A L Y S I S WAS T H E O N L Y A R O M A T I C R E S I O U E P R E S E N T IN T H E M O L E C U L E . T H E T E S T W I T H E H R L I C H R E A G E N T C O N F I R M E D T H E A B S E N C E O F T R Y P T O P H A N . T A B L E V T H E R E F O R E R E P R E S E N T S T H E C O M P L E T E C O M P O S I T I O N O F S A L M O N CT A S C O M P A R E D T O T H E C O M P O S I T I O N S O F P O R C I N E , B O V I N E , O V I N E , A N D H U M A N C A L C I T O N I N S . I D E N T I F I C A T I O N O F T H E N-T E R M I N A L R E S I D U E O F T H E S A L M O N M O L E C U L E A S C Y S T I N E C O N F I R M E D T H A T A L L F I V E H O R M O N E S C O N S I S T O F A 32 A M I N O A C I D C H A I N W I T H A D I S U L F I D E R I N G A T T H E N— T E R M I N U S A N D TWO P R O L I N E R E S I D U E S , B U T D R A M A T I C D I F F E R E N C E S E X I S T E D F O R M O S T O F T H E O T H E R R E S I -D U E S . TH E S A L M O N M O L E C U L E L A C K E D F I V E O F T H E E I G H T E E N D E T E C T A B L E R E S I D U E S , C O N T A I N I N G N O A L A N I N E , I S O L E U C I N E , M E T H I O N I N E , P H E N Y L A L A N I N E OR T R Y P T O P H A N . I T S L O W C O N T E N T O F - 4 5 -A R O M A T I C A N D H Y D R O P H O B I C R E S I D U E S A N D T H E P R E S E N C E O F T H R E E A D D I T I O N A L R E S I D U E S C O N T A I N I N G H Y D R O X Y L G R O U P S ( N I N E V E R S U S S I X F O R P O R C I N E ) P R O B A B L Y A C C O U N T S F O R T H E H I G H E R A P P A R E N T M O L E C U L A R W E I G H T O F S A L M O N C T O B S E R V E D ON S E P H A D E X G - 5 0 . M O L E C U L E S O F L O W A R O M A T I C A N D H Y D R O P H O B I C C O N T E N T A R E R E T A R D E D L E S S ON S E P H A D E X T H A N T H O S E W I T H H I G H C O N T E N T S ( 6 0 ) A N D T H E L A R G E R H Y D R A T E D R A D I U S O F A H Y O R O P H | L I C M O L E C U L E C O U L D R E I N F O R C E T H I S T E N D E N C Y ( 1 7 ) . A T O T A L O F F I V E A C I D I C R E S J D U E S W E R E F O U N D A S IN T H E O T H E R C A L C I T O N I N S , B U T T H E F I V E A M M O N I A R E S I D U E S I N D I C A T E D T H A T M O S T O F T H E S E W E R E O R I G I N A L L Y A M I D A T E D , A C C O U N T I N G F O R T H E S M A L L C H A N G E I N C H A R G E W H I C H R E S U L T E D F R O M I N C R E A S I N G T H E B U F F E R P H F R O M 3 . 4 T O 4 . 5 ON T H E S E C O N D S E - S E P H A 0 E X C O L U M N . T H E B I N D I N G O F T H E H O R M O N E T O T H I S R E S I N WAS D U E T O T H E N U M B E R O F B A S I C R E S I D U E S P R E S E N T ( T W O L Y S I N E S , O N E A R G I N I N E A N D O N E H I S T I D I N E ) W H I C H W A S G R E A T E R T H A N I N A N Y O F T H E O T H E R C A L C I T O N I N S C H A R A C T E R I Z E D . E. P R E P A R A T I ON A N D I S O L A T I ON O F T R Y P T I C P E P T I D E S . T H E P R E S E N C E O F TWO L Y S I N E S A N D O N E A R G I N I N E I N T H E M O L E C U L E S U G G E S T E D T H A T D I G E S T I O N W I T H T R Y P S I N S H O U L D P R O V I D E F O U R P E P T I D E S O F C O N V E N I E N T S I Z E F O R S E Q U E N C E D E T E R M I N A T I O N B Y E D M A N D E G R A D A T I O N S . T H E M O L E C U L E WAS F I R S T O X I D I Z E D W I T H P E R F O R M I C A C I D T O A V O I D D I S U L F I D E I N T E R C H A N G E A N D O T H E R P R O B L E M S W I T H C Y S T E I N E W H I C H H A D B E E N E N C O U N T E R E D D U R I N G S E Q U E N C E WORK ON P O R C I N E C T ( 8 , 9 ) . 1. M E T H O D S . A . P E R F O R M I C A C I D O X I O A T I O N . -46-PERFORMIC ACID WAS PREPARED BY ADDING 0.5 ML OF 30$ HYDROGEN PEROXIDE TO 9.5 ML OF 90$ FORMIC ACID AND ALLOWING THE MIXTURE TO STAND AT ROOM TEMPERATURE FOR FOUR HOURS. IT 0 WAS THEN COOLED TO 0 C ANO 1 ML ADDED TO 10 MG OF POOLED STAGE 5 MATERIAL IN A VESSEL PRECOOLEO TO 0°C. THE MIXTURE WAS SHAKEN, ALLOWED TO STANO FOR 3 HOURS AT 0° C.AND LYO-P H I L I Z E D . THE RESIDUE WAS REOISSOLVED IN 2 ML D I S T I L L E D WATER AND R E L Y O P H I L I Z E D . T H I S LAST STEP WAS REPEATED AND A 10 UX SAMPLE WAS TAKEN FOR AMINO ACID A N A L Y S I S . B. AMINO A C I D A N A L Y S I S . P R E P A R A T I O N FOR A N A L Y S I S OF THE O X I D I Z E D MATERIAL WAS CARRIED OUT AS PREVIOUSLY DESCRIBED, BUT WITHOUT THE A I R OXIDATION S T E P . THE ANALYSES WERE DONE AS BEFORE, BUT USING A SINGLE COLUMN, THREE BUFFER SYSTEM (38) WHICH AL L 0 WE 0 DETERMINATION OF ALL RESIDUES ON A SINGLE SAMPLE. D E T A I L S OF THIS SYSTEM ARE DESCRIBED IN APPENDIX D. c. D I G E S T I O N WITH T R Y P S I N . T R Y P S I N ( WORTHINGTON, T R 6 E A ) WAS DISSOLVED TO A CONCENTRATION OF 10 MG/ML IN 1 M M HYDROCHLORIC ACID WHICH WAS 1 M M IN CALCIUM CHLORIDE. 20 yLL OF THIS STOCK SOLUTION WAS ADDED TO 10 MG OF O X I D I Z E D MATERIAL IN 1 ML OF 0.5$ (w/v) AMMONIUM BICARBONATE BUFFER AT PH 8.0 TO GIVE AN ENZYME TO SUBSTRATE RATIO OF 1:50 (w/w). AFTER INCUBATION AT 37° C FOR 4 HOURS THE SAMPLE WAS L Y O P H I L I Z E D ANO REDISSOLVEO IN 3 ML OF 0.1 M FORMIC ACID AND STORED FROZEN. -47-D. H IGH VOLTAGE E L E C T R O P H O R E S I S . THE I N I T I A L S E P A R A T I O N OF THE P E P T I D E S WAS BY HIGH VOLTAGE E L E C T R O P H O R E S I S ( H V E ) AT PH 6.5 AND PH 1.9 IN THE SYSTEM D E S C R I B E D IN APPENOIX C. FOR EXPERIMENTS AT PH 6.5f 300 ^IL SAMPLES OF THE T R Y P T I C D I G E S T SOLUTION CONTAINING APPROXIMATELY 0.3 M M 0 F P E P T I D E WERE A P P L I E D TO 3 CM OF THE ORIGIN L I N E 32 CM FROM THE CATHODE END OF A 57 CM SHEET OF WHATMAN 3 MM PAPER AND FLANKED WITH 5 }XL SPOTS OF TWO S T A N -OARO AMINO ACID SOLUT IONS TO T E S T D E V E L O P I N G REAGENTS ANO ALLOW C A L C U L A T I O N OF R E L A T I V E M O B I L I T I E S (SEE F IGURE 21). V I S I B L E DYE MARKERS WERE ALSO EMPLOYED AS DESCRIBED IN AP P E N D I X C. AF T E R SATURAT ION WITH PH 6.5 BUFFER THE SHEET WAS P L A C E D IN THE E L E C T R O P H O R E S I S TANK AND RUN AT 4000 V AND APPROXIMATELY 5 M I L L I A M P E R E S PER CENT IMETER OF PAPER WIDTH UNT IL THE MARKERS REACHED A PREDETERMINED P O I N T . THE ELECTROPHORETOGRAM WAS THEN REMOVED AND ALLOWED TO DRY. A 5 X 57 CM S T R I P WAS REMOVED FROM THE CENTER OF THE S H E E T SUCH THAT It CONTAINED APPROXIMATELY 75$ OF THE A P P L I E D S A M P L E , AND THE REMAINOER OF THE SHEET OEVELOPED WITH E I T H E R PAQ R E A G E N T , CADM IUM-NINHYORIN OR BOTH AS DESCRIBED IN APPENDIX C. THE SEGMENTS OF THE UNDEVELOPED S T R I P CONTAINING P E P T I D E S WERE CUT OUT ANO SAVED FOR FURTHER P U R I F I C A T I O N OR E L U T I O N . EL E C T R O P H O R E S I S AT PH 1.9 WAS PERFORMED ON THE E X C I Z E D , UNDEVELOPED S T R I P S FROM THE PH 6.5 ELECTROPHORE TOGRAMS AND FROM PAPER CHROMATOGRAMS BY SEWING THE S T R I P S ONTO NEW S H E E T S OF WHATMAN 3 MM ON AN ORIGIN L I N E 10 CM FROM THE ANODE AND - 4 8 -R E M O V I N G T H E A R E A O F T H E NEW S H E E T B E N E A T H T H E S E W E D I N t S T R I P . E L E C T R O P H O R E S I S WAS T H E N P E R F O R M E D A S P R E V I O U S L Y D E S C R I B E D A N D T H E E D G E S T R I P S O F T H E R E S U L T A N T E L E C T R O -P H O R E T O G R A M S DR I E D A N D D E V E L O P E D . S E G M E N T S O F T H E U N D E -V E L O P E D C E N T E R S T R I P W H I C H C O N T A I N E D P E P T I O E S W E R E C U T O U T A N D E L U T E D W I T H 0.5 M A C E T I C A C I D ( 5 8 ) . E . P A P E R C H R O M A T O G R A P H Y . P E P T I D E S W H I C H C O U L D N O T B E S E P A R A T E D B Y E L E C T R O P H O R E S I S A L O N E W E R E S U B J E C T E D T O D E C E N D I N G P A P E R C H R O M A T O G R A P H Y I N A S I N G L E P H A S E S Y S T E M C O N S I S T I N G O F B U T A N O L , A C E T I C A C I D , P Y R I O I N E A N D W A T E R ( 3 0 S 6 S 2 0 S 2 4 ) ( 1 3 1 ) . U N D E V E L O P E D S T R I P S F R O M E L E C T R O P H O R E T O G R A M S W E R E S E W E D ON A T T H E O R I G I N , 7 CM F R O M T H E T O P O F 1 5 X 4 6 CM S H E E T S O F W H A T M A N 3 M M P A P E R . T H E S E S H E E T S W E R E C U T F R O M S T A N D A R D 4 6 x 5 7 CM S H E E T S S U C H T H A T T H E S O L V E N T F L O W W A S P E R P E N D I C U L A R T O T H E G R A I N O F T H E P A P E R . UN D E R T H E S E C O N D I T I O N S T H E S O L V E N T F R O N T R E Q U I R E D A B O U T 1 4 H O U R S T O R E A C H T H E B O T T O M O F T H E P A P E R . A F T E R D R Y I N G T H E C H R O M A T 0 G R A M S W E R E D I V I D E D A N D T H E E D G E S T R I P S D E V E L O P E D W I T H T H E R E A G E N T S U S E D F O R T H E E L E C T R O P H O R E T O G R A M S . T H E U N D E V E L O P E D S E G M E N T S C O N T A I N I N G P E P T I D E S W E R E C U T O U T F O R E L U T I O N OR F U R T H E R P U R I F I C A T I O N . 2. R E S U L T S A N D D I S C U S S I O N . T H E A M I N O A C I D A N A L Y S I S O F T H E O X I D I Z E D S A M P L E WAS S I M I L A R T O T H A T O F T H E N A T I V E H O R M O N E E X C E P T F O R T H E L O S S O F T H E TWO - G — C Y S T I N E R E S I D U E S A N O P A R T I A L C O N V E R S I O N O F T Y R O S I N E T O C H L O R O T Y R O S I N E ( 1 2 8 ) . C Y S T E I C A C I D WAS P R E S E N T FIGURE 20 O U T L I N E OF PROCEDURES USED TO ISOLATE T R Y P T I C P E P T I D E S FROM SALMON CT. SEPARATION PROCEDURES P E P T I D E S ELUTED HVE AT PH~5T3" A - 1 , A-2, A-3, A-4 -•HVE AT PH 1 .9 B-1 CHROMATOGRAPHY C- 1 , C-2 HVE AT PH 1.9 D - l , D-2 — -•ST-1 A -•ST-1 -•ST-4A + ST-3 -•ST-4 >> ST-2 : = = = = = = z i O K OH n @R MG n U l\ u fi YEL/g| A - l FR|P -0-O.@ — 1 o DNP '+ SCT Y E L Q A - 3 (ST-1) Y E LO A - 4 (ST-IA) o O D OG o O C A FIGURE 21. PH 6 . 5 ELECTROPHORETOGRAM OF SALMON C T T R Y P T I C P E P T I D E S . COMPOSITE OF SEVERAL RUNS. O- N I N -HYDRIN P O S I T I V E , P O S I T I V E , © - P A Q AND NINHYDRIN P O S I T I V E . SINGLE LETTER DESIGNATIONS USED FOR AMINO A C I D S . OTHER A B B R E V I A T I O N S TA , TAURINE; C A , C Y S T E I C A C I D ; MG , METHYL GREEN; D N P , « - D N P - L Y S i N E ; O G , ORANGE G ; Y E L , YELLOW NINHYDRIN COLOR; F R , FAST RED; Y R , YELLOW CHANGING TO RED. SINGLE LETTER DESIGNATIONS (E G . A-3) INDICATE P E P T I D E CONTAINING REGIONS. TWO LETTER DESIGNATIONS ( E G . ST-1) INDICATE ISOLATED P E P T I D E S . DOTTED L I N E IS NEUTRAL. O K O H 0°« 0 o v O L § M O E O Y 8?„ OT: O D 0 YR 0-DNP| o (ST-4A) H 1 h O C A F I G U R E 22. P H 1.9 E L E C T R O P H O R E T O G R A M OF R E G I O N A-2. O T H E R D A T A AS D E S C R I B E D I N F I G U R E 21, F I G U R E 2 3 . P A P E R C H R O M A T O G R A M O F R E G I O N A-1 IN B U T A N O L , A C E T I C A C I D , P Y R I D I N E , W A T E R S Y S T E M . ( 3 0 ? 6T 2 0 : 2 4 ) . O T H E R D A T A A S O E S C R I B E D IN F I G U R E 21 D O T T E D L I N E I S S O L V E N T F R O N T . I F I G U R E 2 4 . PH 1.9 ELECTROPHORETOGRAM OF REGION C-1. DATA AS DESCRIBED IN F l G U R E 21. O T H E R - 4 9 -WITH A MOLE RATIO OF 1 , 8 IND ICAT ING THE E X P E C T E D OXIDATION HAD OCCURED AND THAT THE MATERIAL WAS S U I T A B L E FOR D IGEST ION WITH T R Y P S I N . THE P E P T I D E S PRODUCED BY T H I S D IGEST ION WERE SEPARATED BY A S E R I E S OF E L E C T R O P H O R E T I C AND CHROMATOGRAPHIC STAGES AS SHOWN IN F I G U R E S 2 1 THRU 2 4 . THE P E P T I D E MAPS SHOWN IN THESE F I G U R E S ARE COMPOSITES BASED ON MORE THAN ONE EXPERIMENT S I N C E ALL OF THE DATA PRESENTED COULD NOT BE OBTAINED FROM A S I N G L E S A M P L E . THE S I N G L E L E T T E R , S I N G L E - N U M B E R D E S I G -NATIONS REFER TO P E P T I D E CONTAINING REGIONS WHILE THE DES IGNAT IONS IN P A R E N T H E S I S ARE USED ONLY FOR REGIONS C O N -T A I N I N G A S I N G L E COMPONENT. THE I N I T I A L SEPARAT ION BY H V E AT PH 6 . 5 SHOWN IN FIGURE 2 1 Y I E L D E D FOUR NINHYORIN P O S I T I V E REGIONS AND TWO MINOR SPOTS SEEN ONLY WITH P A Q WHICH WERE NOT I N V E S T I G A T E D FURTHER. TWO OF THE FOUR MAJOR REGIONS WERE SHOWN TO BE ISOLATEO P E P T I D E S ( S T - 1 AND S T - 1 A ) , BUT THE OTHER TWO CONTAINED MORE THAN ONE COMPONENT AND WERE S U B J E C T E D TO FURTHER P U R I F I C A T I O N AS OUTLINED IN F IGURE 2 0 . TWO MINOR CONTAMINANTS WERE SEPARATED FROM A S I N G L E MAJOR P E P T I D E ( S T - 4 A ) IN REGION A - 2 BY H V E AT PH 1 . 9 AS SHOWN IN F IGURE 2 2 , AND REGION A - 1 WAS S U B J E C T E D TO PAPER CHROMATOGRAPHY FOR FURTHER P U R I F I C A T I O N . TH IS CHROMATOGRAPHY Y I E L D E D TWO N I N -HYDRIN P O S I T I V E REGIONS AS SHOWN IN F IGURE 2 3 , BUT ONLY ONE OF THESE WAS HOMOGENEOUS ( S T - 3 ) . THE SECOND REGION ( C - 1 ) WAS FURTHER P U R I F I E D BY H V E AT PH 1 . 9 AS SHOWN IN FIGURE 2 4 TO Y I E L D TWO ISOLATED P E P T I D E S , S T - 2 AND S T - 4 . THUS THE T R Y P T I C D IGEST WAS SHOWN TO CONTAIN S IX MAJOR P E P T I D E S RATHER 1 1.5 2 2.5 3 4 5 6 7 8 9 10 1.5 2 2.5 3 MOLECULAR WEIGHT x 1 0 OF ELECTROPHORETIC M O B I L I T Y VE TO A S P A R T I C A C I D ) VERSUS FOR P E P T I D E S . ALIGNMENTS T CHARGES AS INDICATED. (88) FIGURE 25. LOGARITHMIC PLOT (AT PH 6.5 RELATI MOLECULAR WEIGHT CORRESPOND TO UNI I o x >-CD o < _) tr 6 7 8 9 10 MOLECULAR WEIGHT X 10 FIGURE 2 6 . LOGARITHMIC PLOT OF ELECTROPHORETIC MO B I L I T Y (AT PH 1.9 R E L A T I V E TO S E R I N E ) VERSUS MOLECULAR WEIGHT FOR P E P T I D E S . ALIGNMENTS CORRESPOND TO UNIT CHARGES AS INDICATED. ( 8 8 ) - 5 0 -T HA N T H E E X P E C T E D F O U R . T H E S E W E R E REC0VERE0 B Y E L U T I O N F R O M T H E P A P E R S T R I P S I N Q U A N T I T I E S A D E Q U A T E T O A L L O W C H A R -A C T E R I Z A T I O N A S D E S C R I B E D I N T H E N E X T S E C T I O N . F. C H A R A C T E R i Z A T I O N O F T R Y P T I C P E P T I P E S . A L I Q U O T S O F THE E L U T E O P E P T I D E S WERE T A K E N FOR A M I N O A C I D A N A L Y S I S T O D E T E R M I N E T H E I R C O M P O S I T I O N ANO THE M O B I L I T I E S O F THE P E P T I O E S D U R I N G H V E WERE C O M P A R E D T O T H E S E C O M P O S I T I O N S T O C A L C U L A T E NET C H A R G E AND D E T E R M I N E W H E T H E R OR NOT THE A C I D I C R E S I D U E S WERE A M l D A T E D . N — T E R M INAL A N A L Y S E S WERE C A R R I E D OUT TO O B T A I N DATA ON THE O R D E R O F T H E P E P T I O E S I N T H E W H O L E M O L E C U L E . 1. M E T H O D S . A . AM I N O A C I D A N A L Y S I S . P R E P A R A T I O N F O R A N A L Y S I S W A S C A R R I E D O U T A S D E S C R I B E D P R E V I O U S L Y , BUT W I T H O U T AIR O X I D A T I O N . THE A N A L Y S E S WERE P E R F O R M E D U S I N G A S I N G L E C O L U M N - T W O B U F F E R S Y S T E M AND A 6 MM D I A M E T E R C O L U M N T O I N C R E A S E S E N S I T I V I T Y . THE D E T E C T I O N S Y S T E M U S E D WAS A LOG A M P L I F | E R - P H O T 0 T R A N S I S T O R C I R C U I T A S D E S C R I B E D I N A P P E N D I X D . B . D E T E R M I N A T I O N O F AM I D E G R O U P S . 0FF0RD ( 8 8 ) HAS S H O W N THAT P E P T I D E S O F KNOWN E L E C T R O -P H O R E T I C M O B I L I T Y R E L A T I V E T O A S P A R T I C A C I D ( U ) A T PH 6.5 A S P OR TO S E R I N E (j-lg^ R ) A T PH 1 . 9 ANO K N O W N M O L E C U L A R W E I G H T F A L L I N T O C E R T A I N P O P U L A T I O N S W H E N P L O T T E D G R A P H I C A L L Y A S S H O W N I N F I G U R E S 2 5 A N D 2 6 . P E P T I D E S W H I C H L I E N E A R A G I V E N TABLE VI AM I N O A C I D A N A L Y S E S O F T R Y P T I C P E P T I D E S F R O M S A L M O N CT AM I N O Ac ios ST-1 MO L E R A T I O S S T - 1 A ST-2 ST-3 ST-4 ST-4A C Y S T E I C A C I D 1.9 1.7 0 0 0 0 A S P A R T I C A C I D 1.0 1.0 0 0 1.0 1.0 T H R E O N I N E 0.8 0.8 0 0.8 2.9 2.8 S E R I N E 1.8 1.7 0.8 0 1.1 0.8 G L U T A M I c Ac 1D 0 0 2.1 1.2 0 0 P R O L I N E 0 0 0 1.1 1.1 1 .0 GLYC1NE 1.1 1.0 0 0 2.0 2.1 A L A N i N E 0 0 0 0 0 0 V A L 1 N E 1.0 1.1 0 0 0 0 ME T H I O N I N E 0 0 0 0 0 0 1 S O L E U C 1 N E 0 0 0 0 0 0 L E U C I N E 1.7 1.7 1.8 1.0 0 0 T Y R O S I N E 0 0 0 1* 0 0 P H E N Y L A L A N i N E 0 0 0 0 0 0 L Y S I N E 0.9 0.8 1.0 0 0 0 H 1 S T 1 D 1 N E 0 0 0.8 0 0 0 ARG1N1NE 0 0 0 1.0 0 0 $ Y I E L D * * 5 . 5 $ 1 0 $ 7 5 $ 3 5 $ 7 0 $ 1 0 $ * D E T E R M I N E D A S C H L O R O T Y R O S I N E * * A P P R O X I M A T I O N S -51-L I N E ON T H E S E G R A P H S H A V E B E E N S H O W N T O H A V E T H E N E T C H A R G E I N D I C A T E D F O R T H A T L I N E A T T H E G I V E N P H . T H E R E L A T I V E M O B I L -I T I E S O F T H E T R Y P T I C P E P T I D E S W E R E C A L C U L A T E D F R O M T H E A V A I L -A B L E D A T A I N T H E P R E C E D I N G , S E C T I O N A N D C O M P A R E D T O T H E M O L E -C U L A R W E I G H T S C A L C U L A T E D F R O M T H E A M I N O A C I D C O M P O S I T I O N D A T A T O G I V E T H E N E T C H A R G E O F T H E P E P T I D E . K N O W I N G T H E N E T C H A R G E A N D T H E C O M P O S I T I O N A S D E T E R M I N E D B Y AM I N O A C I D A N A L Y S I S O F H Y D R O L Y S E D S A M P L E S I T WAS G E N E R A L L Y P 0 S S f B L E T O D E T E R M I N E W H E T H E R OR N O T T H E C A R B O X Y L G R O U P S P R E S E N T H A D B E E N I N T H E A M I D E F O R M P R I O R TO H Y D R O L Y S I S . c EN D GR O U P A N A L Y S I S . T H E N - T E R M I N A L R E S I D U E S O F T H E P E P T I D E S W E R E D E T E R M I N E D B Y DA N S Y L A T I O N O F 0.7 N M O L E S A M P L E S O F T H E P E P T I D E S A N D S U B S E Q U E N T I D E N T I F I C A T I O N A S D E S C R I B E D I N A P P E N O I X E . T H E C — T E R M I N A L R E S I D U E S O F 4 O F T H E S I X M A J O R P E P T I D E S C O U L D B E D E T E R M I N E D F R O M T H E I R C O M P O S I T I O N S A N D K N O W N S P E C I F I C I T Y O F T R Y P S I N F O R T H E C A R B O X Y L S I D E O F L Y S I N E A N D A R G I N I N E R E S I D U E S . 2. R E S U L T S A N D D I S C U S S I O N . T A B L E V I S H O W S T H E A M I N O A C I D A N A L Y S E S O F T H E S I X T R Y P T I C P E P T I D E S A N D T H E A P P R O X I M A T E Y I E L D S O F E A C H P E P T I . D E . T H E Y I E L D F I G U R E S A R E O N L Y R O U G H E S T I M A T E S S I N C E T H E P E R -C E N T A G E L O S T ON T H E D E V L O P E D E D G E S T R I P S V A R I E S I N A N I N D E T E R M I N A N T M A N N E R * B U T T H E Y W E R E U S E F U L I N D E T E R M I N I N G T H E S O U R C E O F T H E TWO E X T R A P E P T I D E S . As S H O W N I N T H E T A B L E T H E Y I E L D S O F P E P T I D E S ST-1A A N D ST-4A W E R E Q U I T E L O W A N D T H E I R A M I N O A C I D A N A L Y S E S W E R E I D E N T I C A L T O T H O S E O F ST-1 TABLE VI I NET CHARGES OF SALMON CT TR Y P T I C P E P T I D E S FROM E L E C T R O -PHORET IC MO B I L I T I E S AND MOLECULAR WEIGHTS ( 8 8 ) PE P T 1 O E S ASP ( PH 6.5) SER (PH 1.9) MOLECULAR WE 1GHT NET CHARGE ST-1 0 . 3 0 1 2 2 0 -1 ST-1 A 0 . 5 9 1 2 2 0 -2 ST-2 - 0 . 4 1 1.31 8 5 4 +1.5 +3 S T - 3 - 0 . 4 1 7 7 7 +1 ST-4 - 0 . 4 4 0.52 7 3 4 +1 +1 ST-4 A 0 0.*47 7 3 4 0 +1 -52-A N D S T - 4 . TH E M O B I L I T I E S O F T H E M I N O R P E P T I D E S W E R E Q U I T E D I F F E R E N T F R O M T H O S E O F T H E M A J O R P E P T I O E S , H O W E V E R , A N O A S S H O W N I N T A B L E V I I , T H E Y E A C H A P P E A R E O T O H A V E O N E M O R E N E G A T I V E C H A R G E T H A N T H E C O R R E S P O N D I N G M A J O R P E P T I D E . . T H E C A L C U L A T E D N E T C H A R G E S O F T H E M A J O R P E P T I D E S I N D I C A T E D T H A T T H E Y E A C H C O N T A I N E D A S P A R A G I N E A N D T H E A D D I T I O N A L N E G A T I V E C H A R G E ON T H E M I N O R P E P T I D E S S U G G E S T E D T H A T T H E Y C O N T A I N E D A S P A R T I C A C I D . IT T H U S A P P E A R E D L I K E L Y T H A T A P A R T I A L D E A M I D A T I O N H A D O C C U R E D D U R I N G T H E P E R F O R M I C A C I D O X I D A T I O N A N D T H A T T H E N A T I V E M O L E C U L E H A D C O N T A I N E D A S P A R A G I N E R A T H E R T H A N A S P A R T I C A C I D . T H E C O M P O S I T I O N O F S T - 1 I N D I C A T E D T H A T I T WAS T H E N — T E R M I N A L P E P T I D E S I N C E I T C O N T A I N E D B O T H C Y S T E I C A C I O R E S I D U E S A N D I D E N T I F I C A T I O N O F D A N S Y L - C Y S T E I C A C I D A F T E R D A N S Y L A T I O N C O N F I R M E D T H I S . S T - 4 C O N T A I N E D N E I T H E R L Y S I N E N O R A R G I N I N E A N D T H E R E F O R E C O U L D O N L Y H A V E B E E N T H E C - T E R - , M I N A L P E P T I D E . T H E F A C T T H A T I T S M O B I L I T Y I N D I C A T E D T H A T I T C A R R I E D A N E T +1 C H A R G E I N S P I T E O F T H E A B S E N C E O F B A S I C R E S I D U E S S U G G E S T E D T H A T I T S C - T E R M I N U S WAS B L O C K E D — A F U R T H E R I N D I C A T I O N T H A T I T WAS T H E C - T E R M I N A L P E P T I D E . T H E P R E S E N C E O F P R O L I N E IN T H I S P E P T I O E S U G G E S T E D T H A T T H I S 8 L 0 C K E D C - T E R M I N A L R E S I D U E WAS P R O B A B L Y P R O L Y L A M I D E , A S S E E N I N T H E O T H E R C A L C I T O N I N S , B U T T H I S WAS N O T C O N F I R M E D . T H E N — T E R M I N A L R E S I D U E O F T H I S P E P T I D E WAS T H R E O N I N E . P E P T I D E S T - 3 H A D AN N — T E R M I N A L L E U C I N E A N D A C - T E R M I N A L A R G I N I N E , A N D I T S N E T C H A R G E A T P H 6.5 W A S +1 I N D I C A T I N G T H A T I T C O N T A I N E D G L U T A M I N E R A T H E R T H A N G L U T A M I C A C I D . P E P T I D E 1 0 1 5 2 0 2 5 3 0 F I G U R E 2 7 . S A L M O N CT T R Y P T I C P E P T I D E S A L I G N E D W I T H H U M A N CT. ! • P O S S I B L E H O M O L O G I E S , '•}/•/•/: P O S S I B L E C O N S E R V A T I V E S U B S T I T U T I O N S . S I N G L E L E T T E R N O T A T I O N U S E D F O R AM I NO A C I O S . - 5 3 -S T - 2 H A D A N N— T E R M IN A L L E U C I N E A S W E L L A N D A C — T E RM IN A L L Y S I N E , B U T 8ECAUSE O F T H E H I S T I D I N E P R E S E N T T H E M O B I L I T Y D A T A A T P H 6.5 D I D N O T F I T A N Y O F T H E L I N E S W E L L ( 8 8 ) . I T D I D H O W E V E R C A R R Y A N E T P O S I T I V E C H A R G E I N D I C A T I N G T H A T A T L E A S T O N E O F T H E TWO G L U T A M I C A C I D R E S I D U E S H A D B E E N A M I D A T E D , A N O T H E F A C T T H A T O N L Y F I V E A M M O N I A R E S I D U E S W E R E S E E N I N T H E A N A L Y S I S O F T H E W H O L E M O L E C U L E S U G G E S T E D T H A T O N L Y O N E WAS A M I D A T E D S I N C E TWO A S P A R A G I N E S , TWO G L U T A M I N E S A N D A C — T E R M I N A L A M I D E W O U L D A C C O U N T F O R A L L O B S E R V E D A M M O N I A . T H U S A L L O F T H E A V A I L A B L E D A T A WAS C O N S I S T E N T W I T H T H E S T R U C T U R E I N D I C A T E D I N F I G U R E 2 7 W H I C H S U M M A R I Z E S T H E I N F O R M A T I O N O B T A I N E D F R O M T R Y P T I C D I G E S T I O N O F S A L M O N C A L C I T O N I N . T H I S I N F O R M A T I O N D O E S N O T A L L O W O R D E R I N G O F T H E TWO C E N T R A L P E P T I D E S , B U T W H E N C O M P A R E D T O T H E K N O W N S E Q U E N C E S O F H U M A N C T O N L Y T H E O R D E R S H O W N I N F I G U R E 2 8 A L L O W S A N Y S I G N I F I C A N T H O M O L O G Y B E T W E E N T H E M O L E C U L E S I N T H I S R E G I O N . E V E N IN T H I S A R R A N G E M E N T , H O W E V E R , H O M O L O G Y WAS P O S S I B L E A T O N L Y F O U R P O I N T S B E T W E E N T H E H U M A N A N D S A L M O N M O L E C U L E S I N T H E C E N T R A L R E G I O N A N D N O S I G N I F I C A N T H O M O L O G Y B E T W E E N S A L M O N C T A N D P O R C I N E C T WAS P O S S I B L E IN E I T H E R A R R A N G E -M E N T , I T S E E M E D C L E A R , E V E N W I T H O U T F U R T H E R S E Q U E N C E D A T A , T H A T T H E TWO E N D S O F T H E S A L M O N M O L E C U L E W E R E S I M I L A R T O T H O S E O F T H E O T H E R C A L C I T O N I N S , B U T T H A T T H E C E N T R A L R E G I O N S D I F F E R E D E X T E N S I V E L Y . P O R C I N E BoV I N E S A L M O N HU M A N C-S-hW S-N-: i-S-T-i-V-t-S-A-Ls -T- i -V- : t ' i -S -A - : :¥ - W- R - N - t > N- N-:£r H- R-iP. ti-s-T-C-V-L:-G4&-I Ls-T-i-M-r'L-G-T-: 4-W-K-D-:-T Q-D-: i - N - N - : Y - H - R -r S-G-M-G-f:-G-P-E-T-P-NH2 r^S-G-M-G-iF>G-P-E-T-P-NH2 ;^.N-K-:F'-H-T-.^ fiUT-N-T-G- S-G-T-P- NH2 -P-Q-T-A-.:j:-Gr-V-G-A-P-NH2 1 0 1 5 2 0 2 5 3 0 F I G U R E 2 8 . A L I G N M E N T O F S A L M O N CT ( 8 4 ) W I T H P O R C I N E CT ( 9 6 ) , B O V I N E CT ( 1 7 ) , A N O H U M A N CT ( 8 0 ) . V : Y C O N S I S T E N T HY0R0PH0B I C R E S I D U E S , , O T H E R H O M O -L O G I E S A N D C O N S E R V A T I V E S U B S T I T U T I O N S , j>Xv! U N I Q U E C H A R G E P L A C E M E N T I N S A L M O N CT. S I N G L E L E T T E R N O T A T I O N U S E D F O R A M I N O A C I D S . - 5 4 -G . SE Q U E N C E O F S A L M O N C A L C I T O N I N C O M P A R E D T O KNOWN  S E Q U E N C E S O F M A M M A L I A N C A L C I T O N I N S . WH I L E T H E WORK ON T H E T R Y P T I C P E P T I D E S W A S B E I N G C A R R I E O O U T T H E C O M P L E T E S E Q U E N C E O F T H E S A L M O N H O R M O N E WAS A N N O U N C E D B Y NI A L L E_T AJL ( 8 4 ) B A S E D ON A U T O M A T E D S E Q U E N T I A L E D M A N D E G R A D A T I O N S . IT WAS T H U S F U T I L E T O C O N T I N U E I N D E -P E N D E N T S E Q U E N C E W O R K , P A R T I C U L A R L Y S I N C E B O T H S T A R T I N G M A T E R I A L S H A D B E E N O B T A I N E D F R O M T H E S A M E S O U R C E A N D S I N C E T H E WORK C O M P L E T E O C O N F I R M E D T H E I R R E P O R T . T H E S E Q U E N C E O F T H E S A L M O N H O R M O N E I S S H O W N I N F L G U R E 2 8 A L I G N E D W I T H T H E M A M M A L I A N H O R M O N E S O F K N O W N S T R U C T U R E . C O N S I D E R A 8 L E I N F O R -M A T I O N ON T H E R E L A T I O N S H I P O F S T R U C T U R E T O B I O L O G I C A L A C T I V I T Y CAN B E O B T A I N E D B Y A N A L Y S I S O F T H E S E S E Q U E N C E S , . , T H E i t N— T E R M I N A L A N D C - T E R M I N A L E N D S O F T H E M O L E C U L E S A R E V E R Y S I M I L A R . T H R E E O F T H E F O U R H O R M O N E S A R E I D E N T I C A L F O R T H E F I R S T 9 R E S I D U E S A N O T H E H U M A N H O R M O N E V A R I E S O N L Y A T P O S I T I O N S 2 A N D 8 . T H E 1 - 7 D I S U L F I D E B R I D G E I S C O M M O N T O A L L F O U R , A N D M O S T E V I D E N C E I N D I C A T E S T H A T I T I S E S S E N T I A L F O R B I O L O G I C A L A C T I V I T Y ( 1 6 , 1 2 3 ) . B E L L E T AJL ( 1 0 ) H A V E R E P O R T E D , H O W E V E R , T H A T W H E N R E D U C E D W I T H M E R C A P T O E T H A N O L T H E H O R M O N E R E T A I N S B I O L O G I C A L A C T I V I T Y , B U T T H E Y O F F E R N O E V I D E N C E T H A T R E D U C T I O N A C T U A L L Y O C C U R E O , A N D I T I S P O S S I B L E T H A T T H E B R I D G E WAS R E F O R M E D D U R I N G T H E B I O A S S A Y . T H E C - T E R M I N A L R E S I D U E S O F T H E F O U R M O L E C U L E S A R E A L S O I D E N T I C A L ( P R O L Y L A M I D E ) , A N D E V I D E N C E F R O M T H E S Y N T H E T I C WORK O F G U T T M A N N E_T AJL ( 5 0 ) A N D S l E B E R JET A L ( 1 1 . 5 ) I N D I C A T E S T H A T E V E N R E M O V A L O F T H E C - T E R M I N A L A M I D E R E S U L T S IN A 9 7 $ L O S S O F B I O L O G I C A L A C T I V I T Y . T H E R E S I D U E S I N P O S I T I O N S 2 8 , 3 0 - 5 5 -A N O 31 A L S O S H O W C O N S I D E R A B L E H O M O L O G Y B E T W E E N T H E F O U R M O L E C U L E S . IN T H E C E N T R A L R E G I O N O F T H E M O L E C U L E S T H E S I M I L A R I T I E S A R E M O R E S U B T L E . O N L Y O N E H O M O L O G Y E X I S T S H E R E B E T W E E N T H E S A L M O N A N O U N G U L A T E H O R M O N E S — T H E L E U C I N E I N P O S I T I O N 1 6 . P O S I T I O N S 1 0 , 1 4 , 1 8 , 2 1 , 2 3 , A N D 2 5 A R E T H E S A M E I N T H E S A L M O N A N O H U M A N M O L E C U L E S , H O W E V E R . M O R E I N T E R E S T I N G T H A N T H E H O M O L O G I E S H O W E V E R , A R E T H E C O N S E R V A T I V E S U B S T I T U T I O N S . T H E H Y D R O P H O B I C R E S I D U E S S U C H A S P H E N Y L A L A N I N E , T Y R O S I N E , A N D L E U C I N E O C C U R W I T H M A R K E D R E G U L A R I T Y I N P O S I T I O N S 4, 9, 1 2 , 1 6 , 1 9 A N D 2 2 . T H E S E H Y D R O P H O B I C R E S I D U E S M A Y C O N T R I B U T E T O T H E S T A B L I L I Z A T I ON O F A T E R T I A R Y S T R U C T U R E O F T H E T Y P E F R E Q U E N T L Y S E E N I N L A R G E R G L O B U L A R P R O T E I N S ( 7 2 ) . T H I S U N I M O L E C U L A R M I C E L L U L A R C O N F O R M A T I O N I N D I L U T E A Q U E O U S S O L U T I O N S W O U L D B E M A I N T A I N E D B Y V A N D E R ' W A A L S F O R C E S B E T W E E N T H E H Y D R O P H O B I C G R O U P S I N T E R N A L L Y A N D B Y H Y D R O G E N B O N D I N G T O T H E H Y D R A T I O N S P H E R E E X T E R N A L L Y . T H E WORK O F BR E W E R A N D E O E L H O C H ( 1 4 ) ON T H E P O R C I N E M O L E C U L E S U P P O R T S S U C H A C O N C L U S I O N S I N C E H E S H O W S T H A T T H E R A N D O M C O I L F O R M A T I O N P R E D O M I N A T E S I N A Q U E O U S S O L U T I O N , A N O T H A T T H I S R A N D O M N E S S I S R E A R R A N G E D I N T O A N A - H E L I C A L C O N F O R M A T I O N I N 2 - C H L O R O E T H A N O L . H E S U G G E S T S T H A T T H I S T Y P E O F R E A R R A N G E M E N T M A Y B E I M P O R T A N T A T T H E R E C E P T O R S I T E ON T H E C E L L U L A R M E M B R A N E W H E R E T H E W A T E R C O N C E N T R A T I O N I S S I G N I F I C A N T L Y R E D U C E D . I T I S L I K E L Y T H A T T H E R E G U L A R L Y P L A C E D H Y D R O P H O B I C R E S I D U E S W O U L D P L A Y A M A J O R R O L E I N I N D U C I N G S U C H A C O N F O R M A T I O N A L C H A N G E . T H E O N L Y E V I D E N C E C O N F I R M I N G T H E I M P O R T A N C E - 5 6 -O F T H E S E R E S I D U E S F O R B I O L O G I C A L A C T I V I T Y I S F U R T H E R WORK B Y B R E W E R ( 1 3 ) W H I C H S H O W S T H A T O X I D A T I O N O F T H E T Y R O S I N E IN P O R C I N E C A L C I T O N I N W I T H T Y R O S I N A S E R E S U L T S IN A 9 0 $ L O S S O F A C T I V I T Y . O X I D A T I O N O F T H E M E T H I O N I N E IN P O S I T I O N 8 O F H U M A N C T S I G N I F I C A N T L Y R E D U C E S B I O L O G I C A L A C T I V I T Y ( 8 0 ) , A N D T H I S P O S I T I O N I S A L S O H Y D R O P H O B I C IN A L L F O U R M O L E C U L E S S I N C E B O V I N E , P O R C I N E A N D S A L M O N C A L C I T O N I N S H A V E V A L I N E R E S I D U E S H E R E . IN C O N T R A S T T H E M E T H I O N I N E I N P O S I T I O N 2 5 IN T H E U N G U L A T E C A L C I T O N I N S M A Y B E O X I D I Z E D W I T H O U T A C T I V I T Y L O S S ( 1 6 ) A N D C O R R E S P O N D S TO H Y O R O P H I L I C T H R E O N I N E R E S I D U E S IN T H E S A L M O N A N D H U M A N M O L E C U L E S . T H E O N L Y O T H E R S I G N I F I -C A N T C O N S E R V A T I V E S U B S T I T U T I O N S A R E T H E T H R E E A C I D I C R E S I D U E S IN H U M A N , S A L M O N A N O B O V I N E C A L C I T O N I N S A T P O S I T I O N 1 5 B U T T H E P O R C I N E M O L E C U L E C O N T A I N S A N A S P A R A G I N E H E R E I N D I C A T I N G T H A T A C H A R G E D G R O U P I S N O T E S S E N T I A L F O R A C T I V I T Y . A S T H E O T H E R C H A R G E D R E S I D U E S A R E S C A T T E R E D T H R O U G H O U T T H E C E N T R A L P O R T I O N O F T H E M O L E C U L E S I T I S U N L I K E L Y T H A T T H E I R P O S I T I O N S A R E C R I T I C A L F O R A C T I V I T Y . T H E S I M I L A R I T I E S B E T W E E N T H E F O U R M O L E C U L E S D I S C U S S E D G I V E AN I N D I C A T I O N O F W H A T S T R U C T U R A L C H A R A C E R I S T I C S A R E R E Q U I R E D F O R B I O L O G I C A L A C T I V I T Y , B U T O F F E R L I T T L E I N F O R M M A T I ON ON T H E R E A S O N S F O R T H E M U C H H I G H E R B I O L O G I C A L A C T I V I T Y O F S A L M O N C T . P O R C I N E C T ( 1 5 ) , B O V I N E C T ( 1 7 ) A N D H U M A N C T ( 8 0 ) A L L H A V E A C T I V I T I E S B E T W E E N 100 A N O 3 0 0 M R C U / M G W H E N A S S A Y E D IN T H E R A T W H I L E S A L M O N C T H A S A C O R R E S P O N D I N G A C T I V I T Y B E T W E E N 2 5 0 0 A N D 4 5 0 0 M R C U/MG ( 6 5 , 8 7 ) , A N D W H E N A S S A Y E D I N T H E M O U S E ( 8 9 ) T H E S A L M O N - 5 7 -H O R M O N E H A S M O R E T H A N 2 5 0 T I M E S T H E A C T I V I T Y O F P O R C I N E C T (65). AN E X A M I N A T I O N O F T H E D I F F E R E N C E S B E T W E E N S A L M O N C T A N D T H E M A M M A L I A N H O R M O N E S C O U L D E S T A B L I S H S O M E S T R U C T U R A L B A S I S F O R I T S H I G H E R B I O L O G I C A L A C T I V I T Y . ON T H I S B A S I S P O S I T I O N S W H I C H S H O W S I M I L A R I T I E S C A N B E E L I M I N A T E D A N D O N L Y T H O S E P O S I T I O N S ON T H E S A L M O N M O L E C U L E W H I C H A R E N E I T H E R H O M O L O G O U S N O R C O N S E R V A T I V E L Y S U B S T I T U T E D A R E O F I N T E R E S T . T H I S L E A V E S O N L Y S E V E N P O S I T I O N S : 1 0 , 1 7 , 2 0 , 2 4 , 2 6 , 2 7 , A N D 2 9 . FO U R O F T H E S E S U B S T I T U T I O N S I N V O L V E C H A R G E D O F P O T E N T I A L L Y C H A R G E D R E S I D U E S A N D T H E E Q U A L L Y S P A C E D L Y S I N E , H I S T I D I N E A N D A R G I N I N E R E S I D U E S IN P 0 S I T I 0 N S 1 0 , 1 7 • A N D 2 4 C O U L D B E H I G H L Y S I G N I F I C A N T IN B I N D I N G T H E M O L E C U L E T O I T S R E C E P T O R S I T E . T H E F O U R T H C H A R G E D I F F E R E N C E I S IN P O S I T I O N 2 0 W H E R E T H E G L U T A M I N E IN T H E S A L M O N M O L E C U L E I S R E P L A C E D B Y A H I S T I D I N E I T\ T H E M A M M A L I A N M O L E C U L E S . TH E L A S T T H R E E P O S I T I O N S O F D I F F E R E N C E O C C U R N E A R T H E C— T E R M IN A L E N D IN A R E G I O N W H E R E T H E S A L M O N M O L E C U L E I S H I G H L Y H Y D R O P H I L I C . IN C O N T R A S T T H E T H R E E M A M M A L I A N M O L E C U L E S C O N T A I N H Y D R O P H O B I C R E S I D U E S I N P O S I T I O N 2 7 . I F T H E M I C E L L U L A R S T R U C T U R E O I S C U S S E O E A R L I E R D O E S E X I S T T H I S H Y D R O P H O B I C P O S I T I O N W O U L D B E E X P E C T E D T O B E I N V O L V E D I N T H E M A M M A L I A N H O R M O N E S , B U T T H E C - T E R M I N A L E N D O F T H E S A L M O N M O L E C U L E W O U L D B E F R E E R E S U L T I N G I N A M A R K E D D I F F E R E N C E I N C O N F O R M A T I O N . T H U S T H E R E G I O N B E T W E E N T H E P R O L I N E A T P O S I T I O N 2 3 A N D T H E C - T E R M I N U S O F T H E S A L M O N M O L E C U L E W O U L D S E E M W E L L S U I T E D T O F O R AN A - H E L I C A L S T R U C T U R E IN A Q U E O U S S O L U T I O N . BR E W E R E T A L ( 1 7 ) H A V E IN F A C T S U G G E S T E D T H A T F I G U R E 29» A P O S S I B L E C O N F I G U R A T I O N O F S A L M O N C T A S D E T E R M I N E D F R O M A M O L E C U L A R M O D E L . U P P E R P H O T O G R A P H , O P E N C H A I N . L O W E R P H O T O G R A P H , P O S S I B L E C L O S E D C O N F O R M A T I O N E M P H A S I Z I N G H Y D R O P H O B I C B O N D I N G . H Y D R O P H O B I C R E S I D U E S A R E S H O W N I N B L A C K . F I G U R E 3 0 . A P O S S I B L E C O N F I G U R A T I O N O F P O R C I N E C T A S D E T E R M I N E D F R O M A M O L E C U L A R M O D E L . O T H E R D A T A A S A B O V E . F I G U R E 3 1 A P O S S I B L E C O N F I G U R A T I O N O F D E T E R M I N E D F R O M A M O L E C U L A R D A T A A S A B O V E . H U M A N C T A S M O D E L . O T H E R -=58-T H E A P P A R E N T L Y H I G H M O L E C U L A R W E I G H T O F T H E S A L M O N H O R M O N E O U R I N G G E L F I L T R A T I O N M A Y R E S U L T F R O M S U C H D I F F E R E N C E S I N T E R T I A R Y S T R U C T U R E . TO T E S T T H E V A L I D I T Y O F T H I S C O N J E C T U R E ON C O N F O R M A T I O N A L D I F F E R E N C E S T H E P O R C I N E , H U M A N A N D S A L M O N M O L E C U L E S W E R E C O N S T R U C T E D U S I N G S P A C E F I L L I N G M O D E L S ( L . A N D E -ED M U N D M O L E C U L A R M O D E L S , ED M U N D S C I E N T I F I C C O . , B A R R I N G T O N , N . J . ) . T H E S E M O D E L S A R E S H O W N IN F I G U R E S 2 9 , 30, A N D 31 I N O P E N C O N F I G U R A T I O N S ( A ) A N D IN P O S S I B L E R A N D O M C O I L C O N F O R M A T I O N S ( B ) W H I C H E M P H A S I Z E H Y D R O P H O B I C B O N D I N G . T H E H Y D R O P H O B I C G R O U P S A R E B L A C K F O R C O N T R A S T A N D I T C A N B E S E E N T H A T E V E N I N T H E S E S M A L L M O L E C U L E S I T I S P O S S I B L E T O F O L D T H E H Y D R O P H O B I C S I D E C H A I N S I N T O C O M P A C T A P O L A R R E G I O N S . A C T U A L L Y M A N Y C O N F O R M A T I O N S A R E P O S S I B L E , B U T T H E O N E S S H O W N A L L O W A H I G H D E G R E E O F S I D E C H A I N H Y D R O G E N B O N D I N G A N D I O N I C B O N D I N G B E T W E E N C H A R G E D G R O U P S . T H E A B S E N C E O F H Y D R O P H O B I C G R O U P S N E A R T H E C - T E R M I N U S O F S A L M O N C T I S C L E A R L Y S H O W N A N D T H E L O N G H Y D R O P H I L I C T A I L I S C L E A R L Y D I F F E R E N T F R O M P O R C I N E C T A N D H U M A N C T . T H E T A I L I S N O T S H O W N I N I T S P O S S I B L E O R — H E L I C A L S T R U C T U R E S I N C E O N L Y E I G H T R E S I O U E S A R E P R E S E N T B E T W E E N P R O L I N E S S U G G E S T I N G T H A T S U C H A S T R U C T U R E W O U L D H A V E R E L A T I V E L Y L I T T L E S T A B I L I T Y ( 1 9 ) . C H A R G E A N D C O N F O R M A T I O N A L D I F F E R E N C E S S U C H A S T H E S E W O U L D P R O B A B L Y I N F L U E N C E T H E I N T E R A C T I O N B E T W E E N T H E H O R M O N E S A N O T H E I R R E C E P T O R S , B U T A S I T S E E M E D L I K E L Y T H A T A N U M B E R O F O T H E R F A C T O R S W E R E I N V O L V E D D E T E R M I N I N G T H E R E L A T I V E B I O L O G I C A L A C T I V I T I E S O F T H E H O R M O N E S F U R T H E R I N V E S T I G A T I O N S W E R E U N D E R T A K E N T O C L A R I F Y T H E S T R U C T U R E - F U N C T I 0N R E L A T I O N -S H I P S . ' I V . S T R U C T U R E - F U N C T I O N R E L A T I O N S H I P S I N C A L C I T O N I N S . A . I N T R O D U C T I ON IN T H E P R E C E D I N G C H A P T E R A N U M B E R O F P O S S I B L E C O N F O R -M A T I O N A L V A R I A T I O N S IN T H E C A L C I T O N I N S W E R E I N D I C A T E D , A N D I T WAS S U G G E S T E D T H A T T H E S E D I F F E R E N C E S P L A Y E O A R O L E I N T H E A C T I O N S O F T H E H O R M O N E S A T T H E R E C E P T O R S I N T H E T A R G E T O R G A N S . WH I L E S U C H A C T I O N ON T H E T A R G E T O R G A N S I S T H E U L T I M A T E R E Q U I R E M E N T F O R A H O R M O N E , I T I S O N L Y O N E O F M A N Y F U N C T I O N S A H O R M O N E M O L E C U L E M U S T P E R F O R M . A L L D E F I N I T I O N S O F H O R M O N E S I N C L U D E T H E C O N C E P T O F R E L E A S E A T A D I S T A L S I T E F O L L O W E D B Y T R A N S P O R T W I T H I N T H E C I R C U L A T I O N T O T H E T A R G E T O R G A N S . I N V E S T I G A T I O N O F T H E B E H A V I O R O F T H E H O R M O N E D U R I N G T H I S T R A N S P O R T S T A G E I N V O L V E S T H E D E F I N I T I O N O F T H E D Y N A M I C S O F S E C R E T I O N A N D R E M O V A L W H E R E B Y T H E C I R C U L A T I N G L E V E L O F H O R M O N E I S C O N T R O L L E D IN T H E N O R M A L A N I M A L A N D I N T H E R E S P O N S E O F T H E A N I M A L T O E X O G E N O U S H O R M O N E . T H I S C H A P T E R W I L L B E C O N F I N E D T O T H E F A C T O R S I N V O L V E D I N T H E R E M O V A L O F C T F R O M C I R C U L A T I O N R A T H E R T H A N T H E D Y N A M I C S O F I T S S E C R E T I O N . U N D E R S T A N D I N G O F T H E S E F A C T O R S R E Q U I R E S I N F O R -M A T I O N ON B O T H T H E C I R C U L A T I N G L I F E O F T H E H O R M O N E A N D T H E R O U T E S B Y W H I C H I T I S R E M O V E D F R O M T H E C I R C U L A T I O N . S U C H R E M O V A L S H O U L D B E E S P E C I A L L Y C R I T I C A L F O R A H O R M O N E L I K E C T W H I C H IN M A M M A L S , I S F A S T A C T I N G (31) A N D R E S P O N D S T O R A P I D C H A N G E S I N P L A S M A C A L C I U M L E V E L S (46). T H E M A I N T E N A N C E O F A L O W C I R C U L A T I N G H O R M O N E L E V E L W O U L D B E E S S E N T I A L T O E F F E C T I V E E N D O C R I N E F U N C T I O N B E C A U S E T H E R E L E A S E O F NEW H O R M O N E W O U L D H A V E L I T T L E E F F E C T ON AN A L R E A D Y H I G H -59-Time (min.) F I G U R E 3 2 . D I S A P P E A R A N C E O F " I N T A C T " C A L C I T O N I N A F T E R I N J E C T I O N O F 051 I ) - P O R C I N E CT (130). - 6 0 -C I R C U L A T I N G L E V E L A N D T H E R E F O R E E X E R T L I T T L E C O N T R O L . TH I S I S C E R T A I N L Y T R U E I N M A M M A L S W H E R E CT L E V E L S A R E U S U A L L Y L O W A N O T H E R E S P O N S E T O A C H A L L E N G E I S A R A P I D R E L E A S E O F A P U L S E O F H O R M O N E F O L L O W E D B Y A S T E A D Y R E L E A S E A T A L O W E R R A T E ( 4 ) . E A R L Y S T U D I E S ON S A L M O N CT S H O W E D T H A T I T H A D A P R O L O N G E D A C T I O N I N M A M M A L S A N D WAS I N A C T I V A T E D A T A M U C H L O W E R R A T E I N I N V I T R O I N C U B A T I O N S W I T H P L A S M A ( 2 6 ) . T H E S E R E S U L T S S U G G E S T E D T H A T T H E E N D O C R I N E S Y S T E M M I G H T F U N C T I O N D I F F E R E N T L Y IN T H E S A L M O N A N D T H A T T H E S A L M O N H O R M O N E M I G H T B E U S E F U L C L I N I C A L L Y . T H E Y A L S O S U G G E S T E D A P O S S I B L E E X P L A N A T I O N F O R T H E H I G H S P E C I F I C A C T I V I T Y O F S A L M O N CT, A N O T H I S S T U D Y WAS U N D E R T A K E N T O E L U C I D A T E T H E R E L A T I O N S H I P S B E T W E E N T H E K N O W N S T R U C T U R A L D I F F E R E N C E S O F T H E C A L C I T O N I N S A N O T H E I R B I O L O G I C A L A C T I V I T I E S . ; TH E B E S T D A T A O N T H E C I R C U L A T I N G H A L F - L I F E O F CT C O M E S F R O M T H E S T U D I E S O F WE S T EJ_ AL^ (1 3 0 ) ON T H E C L E A R A N C E O F E X O G E N O U S P O R C I N E CT F R O M T H E P I G . T H E H O R M O N E C O N C E N T R A T I O N D E C R E A S E S A S S H O W N I N F I G U R E 3 2 , W H I C H I S A T W O - C O M P O N E N T , E X P O N E N T I A L C U R V E S H O W I N G T H E D E C L I N E IN R A D I O A C T I V I T Y A S S O C I A T E D W I T H 0 ^ l ) - P O R C I N E CT I N P L A S M A T A K E N A T I N T E R -V A L S A F T E R AN I N J E C T I O N O F L A B E L L E O CT. A S I M I L A R D E C L I N E WAS S E E N IN B I O L O G I C A L A C T I V I T Y I N E X P E R I M E N T S U S I N G L A R G E D O S E S O F U N L A B E L L E D P O R C I N E CT. T H E A U T H O R S U S E D T H E M O D E L S H O W N B E L O W T O A N A L Y S E T H E K I N E T I C S O F T H E D I S A P P E A R A N C E I N T H E E X P E R I M E N T S H O W N I N F I G U R E 3 2 , A N D S U G G E S T E D T H A T T H E R A P I D C O M P O N E N T ( T i = 5 M I N ) R E P R E S E N T S T H E E Q U I L I B R A T I O N O F T H E TWO C O M P A R T M E N T S W H I L E T H E S L O W E R C O M P O N E N T - 6 1 -(T^•= 37 M I N ) R E P R E S E N T S T H E A C T U A L R A T E O F D E G R A D A T I O N O F 2 T H E H O R M O N E . Input X21 —-p X1,2 T H E I R A N A L Y S I S A L S O I N D I C A T E D V O L U M E S O F 9 $ A N D 1 6 $ O F B O D Y W E I G H T F O R C O M P A R T M E N T S 1 A N D 2 R E S P E C T I V E L Y . W H I L E T H I S M O D E L S Y S T E M C A N P R O D U C E T H E P A T T E R N S E E N IN F I G U R E 3 2 , T H E R E A R E A N U M B E R O F O T H E R M O D E L S W H I C H C O U L D E Q U A L L Y W E L L P R O D U C E T H I S R E L A T I V E L Y C O M M O N T Y P E O F C U R V E ( 1 0 9 , 1 1 7 ) , A N O A V A R I E T Y O F D A T A I M P L Y T H A T T H I S M O D E L I S AN O V E R -S I M P L I F I C A T I O N . TH E A U T H O R S T H E M S E L V E S S U G G E S T T H A T T I S S U E I N A C T I V A T I O N I S A M A J O R F A C T O R IN T H E D E C L I N E O F A C T I V I T Y , B U T T H I S M O D E L F A I L S T O A L L O W F O R I N A C T I V A T I O N O U T S I D E O F C O M P A R T M E N T 1. A C O M P L E T E M O D E L M U S T A L S O I N D I C A T E T H E R O U T E S O F D I S A P P E A R A N C E A N D A L L O W A N A L Y S I S O F T H E I R R E L A T I V E I M P O R T A N C E . TH E C U R R E N T L Y A V A I L A B L E D A T A F O R CT DO N O T A L L 6 W S U C H A N A N A L Y S I S , B U T T H E Y DO A T , L E A S T P R O V I D E A B A S I S F O R S U G G E S T I N G A M O R E C O M P R E H E N S I V E . M O D E L . S T U D I E S O F O T H E R P O L Y P E P T I D E H O R M O N E S S U C H A S O X Y T O C I N ( 5 3 , 5 5 ) , ADH ( 3 9 , 1 2 9 ) A N D I N S U L I N ( 6 3 , 1 0 6 ) H A V E P R O V I D E D A F A I R L Y C L E A R P I C T U R E O F T H E F A C T O R S I N V O L V E D IN T H E R E M O V A L O F T H I S T Y P E O F H O R M O N E F R O M - 6 2 -T H E C I R C U L A T I O N , A N D T H E R O L E O F M A N Y O F T H E S E F A C T O R S IN R E M O V I N G C T H A S B E E N I N V E S T I G A T E . T H E O V E R A L L P I C T U R E I S Q U I T E C O M P L E X , B U T I T C A N B E B R O K E N DOWN I N T O T H E F I V E M A J O R F A C T O R S L I S T E D B E L O W . 1. E Q U I L I B R A T I O N O F F R E E H O R M O N E IN T H E P L A S M A W I T H H O R M O N E 8 0 U N 0 T O P L A S M A P R O T E I N S . 2. R E N A L E X C R E T I O N O F H O R M O N E . 3. U P T A K E O F H O R M O N E B Y T A R G E T O R G A N S A N D O T H E R O R G A N S . 4. I N A C T I V A T I O N O F H O R M O N E B Y T A R G E T O R G A N S A N D O T H E R O R G A N S . 5. I N A C T I V A T I O N O F H O R M O N E IN P L A S M A . TH E F I R S T F A C T O R WAS N O T C O N S I D E R E D I N T H E M O D E L O F WE S T E_T M . A L T H O U G H C O N S I D E R A B L E E V I D E N C E E X I S T S F O R T H E B I N D I N G O F C T T O P L A S M A P R O T E I N S ( 6 7 , 1 2 1 ) . T H E R E I S , H O W E V E R , NO C L E A R E V I D E N C E T H A T T H E S E P R O T E I N S A R E S P E C I F I C C A R R I E R S . HO R M O N E B O U N D T O L A R G E P R O T E I N S W O U L D B E H A V E Q U I T E D I F F E R E N T L Y F R O M F R E E H O R M O N E S I N C E I T W O U L D P R O B A B L Y B E P R O T E C T E D F R O M D I F F U S I O N O U T O F T H E P L A S M A , R E N A L E X C R E T I O N A N D I N A C T I V A T I O N B Y P L A S M A E N Z Y M E S . T H E P R E S E N C E O F S U C H P R O T E I N S C O U L D IN F A C T E X P L A I N C E R T A I N A N O M A L I E S IN T H E R E S U L T S O F WE S T EJT_ A_L W H I C H I N D I C A T E D T H A T T H E V O L U M E O F C O M P A R T M E N T 1 WAS N E A R L Y D O U B L E T H E P L A S M A V O L U M E A S D E T E R - ' M I N E D F R O M T H E V O L U M E O F D I S T R I B U T I O N F O R (^  31 I ) - P O R C I N E A L B U M I N (5$ B O D Y W E I G H T ) . T H E IR S T U D Y A L S O S H O W E D T H A T T H E A P P A R E N T V O L U M E O F T H I S C O M P A R T M E N T I N C R E A S E D A S T H E D O S E O F I N J E C T E D C A L C I T O N I N I N C R E A S E D . T H E S E R E S U L T S W O U L D B E - 6 3 -E X P E C T E D I F C O M P A R T M E N T 1 C O N T A I N E D TWO C O M P O N E N T S , O N E B O U N D A N D O N E F R E E , A N D I F P L A S M A S A M P L I N G A C T U A L L Y M E A S U R E D B O T H C O M P A R T M E N T S . W H I L E T H E R E I S N O C L E A R E V I O E N C E F O R T H I S F R O M T R A C E R S T U D I E S , WORK B Y L_E G GA T E JET AL . ( 6 7 ) I N D I C A T E S T H A T B O T H B O U N D A N O F R E E C T C A N B E D E T E C T E D B Y B I O A S S A Y . IF T H I S S I T U A T I O N D O E S E X I S T H I G H E R D O S E S O F C T W H I C H I N C R E A S E T H E L E V E L O F F R E E H O R M O N E W O U L D S H I F T I N C R E A S I N G A M O U N T S O F H O R M O N E O N T O B I N D I N G P R O T E I N S A N D T H E A P P A R E N T S I Z E O F T H E C O M P A R T M E N T W O U L D I N C R E A S E . T H E S E B I N D I N G P R O T E I N S W O U L D C E R T A I N L Y P L A Y A M A J O R R O L E I N R E T A I N I N G C T I N T H E P L A S M A , A N D S T R U C T U A L D I F F E R E N C E S B E T W E E N C A L C I T O N I N S C O U L D I N F L U -E N C E T H E D E G R E E O F B I N D I N G . D E T A I L E D I N F O R M A T I O N O F T H E R O L E O F R E N A L E X C R E T I O N I N T H E R E M O V A L O F C T F R O M P L A S M A I S N O T A V A I L A B L E , H O W E V E R , S E V E R A L R E P O R T S S U G G E S T T H A T I T I S R E L A T I V E L Y M I N O R . N E E R E_T M . ( 7 9 ) H A V E R E P O R T E D T H A T T H E R A 0 I 0 - I MM U N 0 A S S A Y A B L E C T I N T H E U R I N E F O L L O W I N G A N I N F U S I O N O F P O R C I N E C T I N T O A P A T I E N T W I T H P A G E T ' S D I S E A S E A C C O U N T E D F O R O N L Y 0 . 1 $ O F T H E T O T A L I N F U S E D . W E S T E T A L ( 1 3 0 ) R E P O R T E D S O M E W H A T H I G H E R V A L U E S F O R T C A P R E C I P I T A B L E L A B E L L E D C T IN U R I N E A F T E R I N J E C T I O N S O F ( 1 5 1 I ) - P O R C I N E C T R A N G I N G F R O M 2 . 5 T O 1 4 $ 0F T H E T O T A L I N J E C T I O N . T H E I R R E S U L T S I N O I C A T E D T H A T A T L E A S T P A R T O F T H I S M A T E R I A L WAS S T I L L B I O L O G I C A L L Y A C T I V E . T H U S E X C R E T I O N I S A R O U T E O F R E M O V A L , B U T I T S R E L A T I V E I M P O R T A N C E I S U N C L E A R . TH E Q U E S T I O N O F U P T A K E B Y V A R I O U S O R G A N S A P P E A R S TO B E C O N S I D E R A B L Y M O R E I M P O R T A N T , A N D M O R E C O M P L E X . U P T A K E B Y T A R G E T O R G A N S M U S T O C C U R I F T H E H O R M O N E I S T O C A U S E A R E S P O N S E , TABLE V I I I 125 1 25 D I S T R I B U T I O N O F I-CT A N D ^ l-PTH I N RA T T I S S U E S A F T E R I N T E R V E N O U S I N J E C T I O N . (36) T i S S U E PTH CT % P E R G T I S S U E G T I S S U E io P E R CT io I N T O T A L T I S S U E M A S S PTH $ I N T O T A L T I S S U E M A S S L I V E R 6.4 3.2 1 3.9 7.0 S K E L E T A L M U S C L E * * 1 3.0 14.7 B L O O D 1.0 1.5 6.5 10.1 BO N E 0.3 0.6 4.4 7.5 K I O N E Y 3.2 2 2 . 6 2.6 18.0 * NO T R E P O R T E D . -64-A N D T H E C O N S E Q U E N C E S O F T H I S U P T A K E A R E Q U I T E D I F F E R E N T F R O M T H O S E O F U P T A K E B Y O T H E R O R G A N S . T H E H O R M O N E T A K E N U P B Y T H E T A R G E T O R G A N S C A U S E S T H E P H Y S I O L O G I C A L R E S P O N S E W H I L E T H A T T A K E N U P B Y O T H E R O R G A N S I S A T B E S T S T O R E D A N D I S M O S T L I K E L Y C O M P L E T E L Y R E M O V E D F R O M A N Y P O S S I B L E E F F E C T ON T H E R E S P O N S E . T H E R E S U L T S O F D E L U I S E EJT AJ_ (36) S H O W N I N T A B L E V I I I I N D I -C A T E T H E D I S T R I B U T I O N O F R A D I O A C T I V I T Y IN V A R I O U S T I S S U E S 1 0 / 1 2 S v , 1 2 5 . M I N U T E S A F T E R IV I N J E C T I O N O F ( ^ l ) - P O R C I N E C T A N D ( I ) -P T H I N T O R A T S . T H E S E R E S U L T S C O N F I R M T H E R A P I D D I S A P P E A R A N C E O F C T F R O M T H E P L A S M A A N D I N D I C A T E T H A T M U C H O F T H E H O R M O N E M O V E S I N T O V A R I O U S T I S S U E S . T H E A M O U N T P R E S E N T I N B O N E , T H O U G H T TO B E T H E P R I M A R Y T A R G E T O R G A N F O R C T , S E E M S S M A L L ; B U T I N T E R M S O F T H E A M O U N T O F L I V I N G T I S S U E I T I S A C T U A L L Y Q U I T E A L A R G E P R O P O R T I O N O F T H E T O T A L . T H E L I V E R A L S O C O N -T A I N S A D I S P R O P O R T I O N A T E L Y L A R G E A M O U N T O F C T A N D A S M A L L A M O U N T O F P T H S U G G E S T I N G S O M E D E G R E E O F S E L E C T I V E U P T A K E B Y T H I S O R G A N . A L T H O U G H T H I S WORK E S T A B L I S H E S T H A T C T is R A P I D L Y T A K E N U P B Y V A R I O U S T I S S U E S , T H E R E I S NO I N D I C A T I O N W H E T H E R T H E H O R M O N E ' S B I O L O G I C A L A C T I V I T Y I S T H E N E N D E D OR I F I T I S M E R E L Y S T O R E D P E N D I N G L A T E R R E L E A S E . IF T H E C T IN T H E S E T I S S U E S I S R A P I D L Y I N A C T I V A T E D T H E M O V E M E N T B E T W E E N P L A S M A A N D T I S S U E W I L L B E E S S E N T I A L L Y U N I D I R E C T I 0 N A L , S E R V I N G O N L Y T O M A I N T A I N L O W C I R C U L A T I N G L E V E L S . I F , ON T H E O T H E R H A N D , I T I S N O T D E S T R O Y E D - , T H E C T IN T H E T I S S U E S C O U L D R E T U R N T O T H E P L A S M A W H E N T H E C I R C U L A T I N G L E V E L WAS R E D U C E D A N D T H E R E S P O N S E TO A S I N G L E D O S E OR P U L S E W O U L D B E P R O L O N G E D . -65-F U R T H E R WORK B Y D E L U I S E EJ_ AJL ( 3 7 ) I N D I C A T E S T H E I N A C T I V A T I O N O F P O R C I N E C T B Y V A R I O U S T I S S U E S L I C E S A N D H O M O G E N A T E S F R O M R A T S , P A R T I C U L A R L Y F R O M T H E L I V E R , I S R A P I D . O T H E R W O R K E R S H A V E A L S O S E E N S I M I L A R R A P I D I N A C T I V A T I O N O F P O R C I N E C T IN T I S S U E H O M O G E N A T E S ( 4 4 , 7 5 ) W H I C H S U P P O R T T H E I D E A T H A T T H I S T Y P E O F I N A C T I V A T I O N I S I M P O R T A N T IN T H E D I S T R U C -T I O N O F C T . A S E C O N D T Y P E O F I N A C T I V A T I O N O C C U R S I N T H E P L A S M A , T A S H J I A N A N D M U N S O N ( 1 2 1 ) F I S T S H O W E D T H I S D U R I N G I N C U B A T I O N S O F P O R C I N E C T I N H U M A N S E R U M . WE S T E_T AJ_ (1 3 0 ) H A V E R E P O R T E D A T I F O R P O R C I N E C T IN P O R C I N E P L A S M A IN V I T R O A T 3 7 ° C O F 2 1.5 - 2.0 H O U R . I F A S I M I L A R R A T E O F I N A C T I V A T I O N O C C U R S IN V I V O I T C O U L D A C C O U N T F O R O N L Y A B O U T 1 0 $ O F T H E T O T A L I N A C T I V A T I O N S E E N IN T H E S L O W C O M P O N E N T O F F I G U R E 3 2 . W H I L E T H I S I S Q U A N T I T A T I V E L Y O N L Y A R E L A T I V E L Y S H A L L F R A C T I O N O F T O T A L I N A C T I V A T I O N I T M A Y H A V E A D I S P R O P O R T I 0N A T E E F F E C T ON T H E R E S P O N S E S I N C E I T S A C T I O N E X T E N D S F R O M T H E I N S T A N T O F R E L E A S E OR I N J E C T I O N T O T H E T I M E O F U P T A K E B Y T H E T A R G E T O R G A N S A N D A L L O F T H E H O R M O N E I T E F F E C T S I S P O T E N T I A L L Y A C T I V E . HO R M O N E T A K E N U P B Y O T H E R O R G A N S M A Y NO L O N G E R B E A C T I V E E V E N T H O U G H I T H A S N O T Y E T B E E N D E S T R O Y E D . T H E M O D E L S H O W N ON T H E N E X T P A G E P R O V I D E S F O R T H E E Q U I L I B R A T I O N A N D I N A C T I V A T I O N D I S C U S S E D A N D C A N S T I L L F I T T H E T Y P E O F D O U B L E E X P O N E N T I A L C U R V E S E E N IN F I G U R E 3 2 . IN T H I S M O D E L C O M P A R T M E N T 1 R E P R E S E N T S F R E E H O R M O N E IN T H E P L A S M A , W H I L E C O M P A R T M E N T 3 R E P R E S E N T S H O R M O N E B O U N D T O P L A S M A P R O T E I N S . A 3 F 1 A N D \ ^ R E P R E S E N T T H E E Q U I L I B R I U M B E T W E E N - 6 6 -B O U N D A N D F R E E H O R M O N E , A N D H O R M O N E IN C O M P A R T M E N T 3 I S A S S U M E D TO B E I M M U N E T O D I R E C T I N A C T I V A T I O N OR E X C R E T I O N . C O M P A R T M E N T 2 I N C L U D E S A L L E X T R A P L A S M A S P A C E S A N D X 0 \ »I A N D ^1 f2 A R E T H E F R A C T L 0 N A L T U R N O V E R R A T E S B E T W E E N T H E S E C O M P A R T M E N T S . \ N I N C L U D E S DI S T R U C T I 0N IN P L A S M A A N D U, 1 E X C R E T I O N , W H I L E X n R E P R E S E N T S T H E C O M B I N E D D I S T R U C T I O N R A T E S F O R A L L O F T H E E X T R A P L A S M A T I S S U E S . C L E A R L Y T H E D A T A A V A I L A B L E D O E S N O T P E R M I T A N A L Y S I S O F A L L O F T H E S E C O M P O N E N T S , B U T T H E M O D E L I S U S E F U L IN D E T E R M I N I N G T H E I M P O R T A N C E O F T H E V A R I O U S F A C T O R S I N V O L V E O . T H I S I S P A R T I C U L A R L Y V A L U A B L E S I N C E T H E R E I S C O N S I D E R A B L E ' E V I D E N C E T H A T T H E R E L A T I V E I M P O R T A N C E O F T H E S E F A C T O R S V A R I E S F R O M S P E C I E S T O S P E C I E S A N D M U S T B E A L L O W E D F O R IN C O M P A R A T I V E S T U D I E S . T H E V A R I A T I O N S IN T H E P E R C E N T O F A T O T A L D O S E E X C R E T E D M E N T I O N E D E A R L I E R I S AN E X A M P L E O F T H I S . A N U M B E R O F S U C H C O M P A R A T I V E I N A C T I V A T I O N S T U D I E S H A V E B E E N O O N E IN T H I S D E P A R T M E N T I N D I C A T I N G T H A T S T R U C T U R A L D I F F E R E N C E S M A Y B E C O R R E A L A T E D W I T H V A R I A T I O N S IN I N A C T I -V A T I O N R A T E . E A R L Y WORK ( 2 6 ) S U G G E S T E O A R E L A T I O N S H I P B E T W E E N T H E M A G N I T U D E O F T H E A R E A R E S P O N S E S (31 ) A N D T H E F I G U R E 3 3 . A R E A R E S P O N S E C U R V E S F O R V A R Y I N G D O S E S O F H U M A N C T IN R A T S ( 8 5 ) . O - V E H I C L E O N L Y , • - 2 0 M R C U , X - 100 M R C U , A - 4 0 0 M R C U . 36 i6o Dose (MRC mU) 2S6 sod G U R E 3 4 . P L O T S O F L O G D O S E V E R S U S A R E A R E S P O N S E F C A L C I T O N I N S F R O M S E V E R A L S P E C I E S ( 8 5 ) . O - H U M A N C T , A - B O V I N E C T ( 3 1 - ) » D ~ P O R C I N E C T , » - C O D C T , A — S A L M O N C T , • - C H I C K E N C T . 0 4 8 12 16 ~20 24 28 Time ( h r ) F I G U R E 35. I N A C T I V A T I O N O F C A L C I T O N I N S F R O M S E V E R A L S P E C I E S D U R I N G I N C U B A T I O N IN S E R U M S F R O M S E V E R A L S P E C I E S (85). ( A) RA T S E R U M . P O R C I N E CT, A - H U M A N CT, • - S A L M O N CT, T - C H I C K E N CT. ( B) P O R C I N E CT I N : A - P O R C I N E S E R U M , • -C H I C K E N S E R U M , • - S A L M O N S E R U M . S A L M O N CT I N : A - P O R C I N E S E R U M , O - C H I C K E N S E R U M , S A L M O N S E R U M . (c) HUMAN S E R U M . • - H U M A N CT, • - P O R C I N E CT. RA T S E R U M . • - H U M A N CT, v- P O R C I N E CT. - 6 7 -IN V I T R O I N A C T I V A T I O N R A T E S O F T H E H O R M O N E S I N C U B A T E D IN P L A S M A . MO R E R E C E N T E X P E R I M E N T S (85) H A V E C O N F I R M E D A N D E X T E N D E D T H E S E F I N D I N G . T H E T A B L E B E L O W P R E S E N T S A T Y P I C A L S E T O F A R E A R E S P O N S E S F O R H U M A N C T IN T H E R A T C A L C U L A T E D F R O M T H E D A T A IN F I G U R E " 3 3 . DO S E ( M R C M U ) A R E A ( M G $ - H O U R ) 2 0 3 . 5 1 0 0 6.8 4 0 0 13.3 WHEN T H E A R E A R E S P O N S E S A R E P L O T T E D A G A I N S T T H E L O G O F T H E D O S E A L I N E A R R E L A T I O N S H I P I S S E E N A S S H O W N IN F I G U R E 3 4 . T H I S F I G U R E A L S O S H O W S T H E P L O T S F O R S I M I L A R D A T A . F O R T W O . O T H E R T H Y R O I D C A L C I T O N I N S A N D F O R T H R E E U L T I M O B R A N C H I A L C A L C I T O N I N S . T H E S L O P E S O F T H E S E L I N E S I N D I C A T E D T H A T A C L E A R D I C H O T O M Y E X I S T E D B E T W E E N T H E S E TWO T Y P E S O F C T A N D T H A T T H E R E S P O N S E TO T H E U L T I M O B R A N C H I A L C A L C I T O N I N S , H A D A M A R K E D L Y D I F F E R E N T T I M E C O U R S E . WHEN T H E S E C A L C I T O N I N S W E R E I N C U B A T E D W I T H S E R U M F R O M V A R I O U S S P E C I E S A T 3 7 A N D T H E R A T E O F I N A C T I V A T I O N D E T E R -M I N E D B Y B I O A S S A Y , A F A I R L Y C L E A R P A T T E R N E M E R G E D W I T H T H E M A M M A L I A N C A L C I T O N I N S A S A G R O U P B E I N G M O R E R A P I D L Y I N A C T I -V A T E D T H A N T H E U L T I M O B R A N C H I A L C A L C I T O N I N S A S S H O W N IN F I G U R E 3 5 ( A ) . AS S H O W N IN F I G U R E 3 5 ( B ) A N O ( C ) T H I S R E L A T I O N S H I P WAS R E L A T I V E L Y C O N S T A N T R E G A R D L E S S O F T H E P L A S M A U S E D A S T H E I N A C T I V A T I N G M E D I U M , S U G G E S T I N G T H A T S O M E F U N D A M E N T A L S T R U C T U R A L D I F F E R E N C E B E T W E E N T H E TWO T Y P E S O F C A L C I T O N I N S WAS R E S P O N S I B L E F O R D I F F E R E N T I A L I N A C T I V A T I O N 20-T Time (hr) 24 26 F I G U R E 3 6 . C O M P A R I S O N O F IN V I T R O I N A C T I V A T I O N O F S A L M O N A N D C H I C K E N C T IN R A T S E R U M TO T H E I R D I S A P P E A R -A N C E R A T E S IN N O R M A L A N D N E P H R E C T O M I Z E D R A T S ( 8 5 ) . ( A ) C H I C K E N C T • - R A T S E R U M , A - N E P H R E C -T O M I Z E D R A T S , • - N O R M A L R A T S . ( B ) S A L M O N CT • - R A T S E R U M , A - N E P H R E C -T O M I Z E D R A T S , O - N O R M A L R A T S . - 6 8 -R A T E S IN P L A S M A . T H U S T H E S T A B I L I T Y O F A C T I N V I T R Q IN S E R U M WAS A F A I R L Y G O O D I N D E X O F T H E E X P E C T E D T I M E C O U R S E F O R T H E I N V I V O R E S P O N S E T O T H A T C T . T H I S D I D N O T M E A N T H A T T H E R E WAS A D I R E C T C A U S E A N D E F F E C T R E L A T I 0 N S H I P , HOWE V E R . TH E R E S U L T S O F D E L U I S E J IT AJ_ ( 3 7 ) I N D I C A T E D T H A T T H E I N A C T I V A T I O N O F S A L M O N C T B Y L I V E R H O M O G E N A T E S 1 WAS A L S O M U C H S L O W E R T H A N T H E I N A C T I V A T I O N O F P O R C I N E C T , S U G G E S T I N G T H A T T H E M E C H A N I S M F O R I N A C T I V A T I O N IN T H E L I V E R M I G H T B E S I M I L A R TO T H A T I N P L A S M A . P E R H A P S T H E S A M E E N Z Y M E S W E R E R E S P O N S I B L E IN B O T H C A S E S S I N C E T H E L I V E R I S K N O W N TO B E T H E S O U R C E O F M A N Y P L A S M A E N Z Y M E S . AN A D D I T I O N A L P O S S I B I L I T Y W H I C H H A D T O B E C O N S I D E R E D W A S T H A T T H E D E C R E A S E D I N A C T I V A T I O N O F T H E U L T I M O B R A N C H I A L C A L C I T O N I N S R E S U L T E D F R O M I N C R E A S E O B I N D I N G TO P R O T E C T I N G P R O T E I N S R A T H E R T H A N F R O M A D E C R E A S E D R A T E O F E N Z Y M A T I C D E G R A D A T I O N . T H E D I S A P P E A R A N C E R A T E S O F B I O L O G I C A L A C T I V I T Y F O R TWO C A L C I T O N I N S IN J_N V I V O E X P E R I M E N T S I N N O R M A L A N O N E P H R E C T O M I Z E D R A T S A R E S H O W N IN F L G U R E 36 C O M P A R E D T O T H E D I S A P P E A R A N C E R A T E S R E S U L T I N G F R O M IN V I T R O I N C U B A T I O N S I N R A T S E R U M . T H E V A R I A T I O N S IN T H E IN V I V O D I S A P P E A R A N C E R A T E S W E R E N O T S I G N I F I C A N T A N D S H O W E D NO C O R R E L A T I O N W I T H T H E A R E A R E S P O N S E D A T A . IT S H O U L D B E N O T E D T H A T T H E D O S E S G I V E N W E R E N O T L A R G E E N O U G H TO A L L O W M E A S U R E M E N T O F T H E S E C O N D E X P O N E N T I A L C O M P O N E N T , S I N C E C I R C U L A T I N G H O R M O N E L E V E L S D R O P P E D T O U N M E A S U R A B L E L E V E L S B E F O R E T H I S P A R T O F T H E C U R V E W O U L D B E R E A C H E D . W E S T JE_J_ _AL_ ( 1 3 0 ) R E P O R T E D S I M I L A R R E S U L T S I N E X P E R I M E N T S U S I N G R E L A T I V E L Y L O W D O S E S -69-W H E R E H O R M O N E L E V E L S W E R E D E T E R M I N E D B Y B I O A S S A Y . T H E S E C U R V E S T H E R E F O R E G I V E NO I N F O R M A T I O N A B O U T I N A C T I V A T I O N R A T E S , B U T R A T H E R I N D I C A T E T H A T N E I T H E R T H E D I F F U S I O N R A T E S F R O M C O M P A R T M E N T 1 T O C O M P A R T M E N T 2 N O R R E N A L E X C R E T I O N R A T E S F O R T H E S E C A L C I T O N I N S D I F F E R S I G N I F I C A N T L Y . T H I S S U G G E S T S T H A T T H E B I N D I N G O F T H E C A L C I T O N I N S T O P L A S M A P R O T E I N S I S N O T A M A J O R F A C T O R IN D E T E R M I N I N G T H E A R E A R E S P O N S E S . A L L OF T H E DATA DISCUSSED ABOVE P O I N T E D T O W A R D S T H E P R O T E O L Y T I C E N Z Y M E S I N P L A S M A A S A M A J O R F A C T O R IN D E T E R -M I N I N G T H E T I M E . C O U R S E O F T H E P H Y S I O L O G I C A L R E S P O N S E : T O S T R U C T U A L L Y D I F F E R E N T C A L C I T O N I N S . T H E P O S S I B I L I T Y S T I L L E X I S T E D T H A T T H E P L A S M A E N Z Y M E S W E R E N O T D I R E C T L Y R E S P O N -S I B L E , B U T I T S E E M E D C L E A R . T H A T T H E R A T E O F I N A C T I V A T I O N B Y T H E S E E N Z Y M E S WAS I N D I C A T I V E O F T H E O V E R A L L R A T E O F I N A C T I -V A T I O N . TH E R O L E O F C A R R I E R P R O T E I N S WAS A L S O U N C L E A R , B U T I T A P P E A R E D T H A T A B E T T E R K N O W L E D G E O F T H E C H A R A C T E R I S T I C S O F T H E D E G R A D I N G E N Z Y M E S W O U L D B E R E Q U I R E D B E F O R E T H I S P R O B L E M C O U L D B E S T U D I E D . IT WAS T H E R E F O R E E S S E N T I A L T O S T U D Y T H E S E E N Z Y M E S IN G R E A T E R D E T A I L T O O B T A I N A C L E A R E R P I C T U R E O F HOW V A R I A T I O N S I N H O R M O N E S T R U C T U R E I N F L U E N C E D T H E R E S P O N S E T O T H E H O R M O N E S . T H I S T Y P E O F I N F O R M A T I O N I S P A R T I C U L A R L Y I M P O R T A N T S I N C E WORK B Y N E E R J£_T AJ_ (79) H A S S H O W N T H A T T H E R E S P O N S E D I F F E R E N C E S O C C U R E D I N MAN A S W E L L A S T H E R A T A N D T H A T T H E M O R E S T A B L E S A L M O N CT I S P O T E N T I A L L Y M U C H M O R E V A L U A B L E T H E R A P E U T I C A L L Y T H A N I S P O R C I N E CT. S I N C E O N E O F T H E M A J O R -70-V A L U E S O E C O M P A R A T I V E O A T A I S I T S A P P L I C A T I O N T O F I N D I N G O R D E S I G N I N G M O R E S T A B L E A N O M O R E A C T I V E H O R M O N E S F O R C L I N I C A L A P P L I C A T I O N , H U M A N P L A S M A W A S U S E D I N T H E S E S T U D I E S . T H E W O R K O F T A S H J I A N A N D V O E L K E L ( 1 2 2 ) O N T H E H U M A N P L A S M A I N A C T I V A T O R S P R O V E D V A L U A B L E I N D E S I G N I N G E A R L Y E X P E R I M E N T S . A L A R G E N U M B E R O F P R O T E O L Y T I C E N Z Y M E S A R E K N O W N T O E X I S T I N H U M A N P L A S M A A N D S E R U M , A N D A N U M B E R O F T H E M C O U L D A C T O N T H E C A L C I T O N I N S . T H E C H A R A C T E R I Z A T I O N O F T H E R O L E S O F A L L T H E S E E N Z Y M E S I N T H E I N A C T I V A T I O N O F T H E C A L C I T O N I N S W A S B E Y O N D T H E S C O P E O F T H I S S T U D Y A N D W O R K W A S C O N F I N E D T O E N Z Y M E S W H I C H A C T E D A S M A J O R P A T H W A Y S F O R D E G R A D A T I O N A N D W H I C H A P P E A R E D T O D I F F E R E N T I A T E B E T W E E N C A L C I T O N I N S W I T H K N O W N S T R U C T U R A L D I F F E R E N C E S . A M O N G T H E E N Z Y M E S O F K N O W N S P E C I F I C I T I E S W H I C H M I G H T B E E X P E C T E D T O F I T T H I S D E S C R I P T I O N W E R E T H O S E W H I C H C L E A V E D A T A R O M A T I C R E S I D U E S S U C H A S P E P S I N , C O N V E R T I N G E N Z Y M E A N D P 0 S S I B L Y C H Y M 0 T R Y P S I N ; A L L O F W H I C H A R E K N O W N T O O C C U R I N P L A S M A T O A T L E A S T A L I M I T E D E X T E N T A N D W O U L O B E M U C H M O R E D A M A G I N G T O T H E M O R E A R O M A T I C M A M M A L I A N C A L C I T O N I N S T H A N T O T H E S A L M O N H O R M O N E . A S E C O N D G R O U P O F E N Z Y M E S L I K E L Y T O B E I N V O L V E D I N D E G R A D I N G T H E C A L C I T O N I N S W E R E T H O S E W I T H S P E C I F I C I T I E S F O R B A S I C R E S I D U E S S U C H A S T R Y P S I N , T H E P L A S M A - K A L L I K R E I N S T H E E N Z Y M E S F R O M P R O T H R O M B I N ( A U T 0 P R O T H R O M B I N C A N O T H R O M B I N ) A N O T H E P L A S M I N T Y P E E N Z Y M E S . A N O T H E R R E A C T I O N W H I C H C O U L D O C C U R , A D D I N G E V E N M O R E C O N F U S I O N T O T H E P I C T U R E , I S R E D U C T I O N , B Y G L U T A T H I O N E A N D O T H E R R E D U C T I V E A G E N T S A N D D I S U L F I D E I N T E R C H A N G E W I T H O T H E R P L A S M A P R O T E I N S . T H E R E D U C T I V E I N A C T I V A T I O N R E A C T I O N S M A Y O C C U R A T 0 I F F E R E N T R A T E _I_N V I V P A N D _J_N V I T R O S I N C E C O N T I N U A L R E N E W A L O F T H E R E O U C T I O N A G E N T W O U L D B E P O S S I B L E IN v i vo. TH E P R O B L E M O F D I S U L F I D E I N T E R C H A N G E H A S B E E N P A R T I C U L A R L Y P R E V A L E N T W I T H H U M A N C T W H I C H A P P E A R S TO D I M E R I Z E R E A 6 I L Y ( 8 0 ) . T H E WORK D E S C R I B E D IN T H I S C H A P T E R P A R T I A L L Y C H A R A C T E R I Z E S T H E R O L E S O F S O M E O F T H E S E F A C T O R S IN T H E D I F F E R E N T I A L I N A C T I -V A T I O N O F T H E V A R I O U S C A L C I T O N I N S , A N D P R O V I D E S A S T A R T I N G P O I N T F O R F U R T H E R S T U O I E S ON T H E D E T A I L S O F T H I S U N O O U B T A B L Y C O M P L E X P R O B L E M . T H E F I R S T S T E P I N T H I S T Y P E O F S T U D Y I S T O E S T A B L I S H P R O C E D U R E S F O R A S S A Y I N G T H E I N A C T I V A T I N G P R O P E R T I E S O F T H E V A R I O U S P L A S M A C O M P O N E N T S . T H E P R O B L E M S I N V O L V E D IN S U C H AN A S S A Y A N D T H E P R O C E D U R E S W H I C H W E R E T R I E D A N D E V E N T U A L L Y A D O P T E D A R E D E S C R I B E D I N T H E N E X T S E C T I O N . B. A S S A Y P R O C E D U R E S . IT WAS D E C I D E D T H A T O N L Y T H E B I O A S S A Y C O U L D P R O V I D E U N E Q U I V O C A L D A T A ON I N A C T I V A T I O N S I N C E P R O C E D U R E S S U C H A S 1 3 1 T C A P R E C I P I T A T I O N O F I L A B E L L E D C T A N D R A D I O I M M U N O A S S A Y S C A N N O T B E E X P E C T E D T O C O R R E S P O N D TO T H E B I O A S S A Y U N D E R T H E C O N D I T I O N S O F T H E E X P E R I M E N T S . F O R E X A M P L E T H E S P E C I F I C I T I E S O F T H E R A D I O I M M U N O A S S A Y S F O R C A L C I T O N I N S T U D I E D H A V E B E E N S H O W N TO B E F O R O N L Y O N E P O R T I O N O F T H E M O L E C U L E ( 1 8 , 3 4 , 4 0 ) . C L E A R L Y T H I S P R O C E D U R E C O U L D N O T D I S T I N G U I S H B E T W E E N I N A C T I V E F R A G M E N T S A N D T H E A C T I V E W H O L E M O L E C U L E . T H U S T H E P R O B L E M S O F T H E I N A C T I V A T I O N A S S A Y W E R E C O M P O U N D E D B Y T H E I N H E R E N T V A R I A B I L I T Y O F T H E B I O A S S A Y . IN T H E E A R L Y S T A G E S O F T H E E X P E R I M E N T S A T T E M P T S W E R E M A D E TO D E V E L O P Q U A N T I T A T I V E A S S A Y P R O C E D U R E S W H I C H W O U L D A L L O W D E T E R M I N A T I O N O F T H E T O T A L I N A C T I V A T O R P R E S E N T IN A S A M P L E A N D P E R M I T E S T I M A T I O N O F R E C O V E R Y IN S U B S E Q U E N T P U R I -F I C A T I O N S T A G E S . T H E S E M E T W I T H S O M E S U C C E S S A N D I T WAS P O S S I B L E T O M E A S U R E R A T E C O N S T A N T S A N O E S T I M A T E ' M A X I M U M V E L O C I T I E S F O R S O M E O F T H E S T A R T I N G M A T E R I A L S . T H E P R O C E D U R E S I N V O L V E O W E R E Q U I T E C O M P L E X H O W E V E R , A N O R E Q U I R E D V E R Y E X T E N S I V E U S E O F B I O A S S A Y S S I N C E N U M E R O U S I N C U B A T I O N S W E R E N E C E S S A R Y W I T H F R E Q U E N T S A M P L E S F O R B I O A S S A Y . I T B E C A M E E V I D E N T T H A T T H I S ; P R O C E D U R E WAS N O T S U I T A B L E F O R S U R V E Y I N G C O L U M N E L U A T E S B E C A U S E O F I T S C O M P L E X I T Y A N D B E C A U S E O F T H E I N S T A B I L I T Y O F T H E C A L C I T O N I N S A T L O W C O N C E N T R A T I O N I N B U F F E R S O L U T I O N S . A L D R E D A N O S C H L U E T E R ( 1 ) H A V E C H A R A C T E R I Z E D T H I S T Y P E O F I N A C T I V A T I O N IN A N U M B E R O F S I T U A T I O N S . B R I E F L Y , T H E I R R E S U L T S I N D I C A T E D T H A T I N A C T I V A T I O N O C C U R R E D R A P I D L Y A B O V E PH 5 IN B U F F E R S O L U T I O N S A N D T H A T T H E I N A C T I V A T I O N C O U L D B E R E T A R D E D B Y T H E P R E S E N C E O F A V A R I E T Y O F O T H E R P R O T E I N S IN T H E S O L U T I O N . IT WAS F O U N D , F O R E X A M P L E , T H A T W H I L E I N C U B A T I O N O F C T IN S E R U M OR IN S O D I U M A C E T A T E B U F F E R C A U S E D C O M P L E T E I N A C T I V A T I O N IN 4 H O U R S A T 3 7 ° C , T H E I N A C T I -V A T I O N WAS I N S I G N I F I C A N T A T S E R U M D I L U T I O N S O F 1 11OO T O 1 : 1 0 0 0 . TH U S S M A L L Q U A N T I T I E S O F P R O T E I N S W E R E P R O T E C T I V E . T H I S T Y P E O F P H E N O M E N O N O C C U R E D I N T H E P R E S E N T S T U D Y A N D L E D TO S I T U A T I O N S W H E R E C O L U M N E L U A T E F R A C T I O N S C O N T A I N I N G L I T T L E OR NO P R O T E I N A P P E A R E D T O C O N T A I N M O R E I N A C T I V A T O R T H A N T H E S T A R T I N G M A T E R I A L A C L E A R L Y U N S A T I S F A C T O R Y R E S U L T . E V E N T U A L L Y A S E M I - Q U A N T I T A T I V E A S S A Y F O R I N A C T I V A T O R WAS - 7 3 -D E V E L O P E D W H I C H WAS N O T S E N S I T I V E T O V A R I A T I O N S IN P R O T E I N C O N C E N T R A T I O N . IT S E E M E D L I K E L Y T H A T A M A J O R F A C T O R IN T H I S B U F F E R I N A C T I V A T I O N WAS A D S O R P T I O N O F T H E CT T O T H E W A L L S O F T H E I N C U B A T I O N V E S S E L S , A N D A S E R I E S O F T E S T S W E R E C A R R I E D O U T T O S E E I F T H I S A D S O R P T I O N C O U L D B E B L O C K E D B Y M O D I F Y I N G T H E V E S S E L W A L L S . S l N C E A D D I T I O N O F E X T R A N E O U S P R O T E C T I V E P R O T E I N S S E E M E D L I K E L Y TO I N T E R F E R W I T H T H E E N Z Y M A T I C I N A C T I V A T I O N W H I C H WAS B E I N G M E A S U R E D T H I S A P P R O A C H WAS N O T S U I T A B L E F O R T H E P R E S E N T P U R P O S E S . S I L I C O N I Z E D G L A S S A N D P L A S T I C V E S S E L S P R O V E D TO C A U S E A M O R E R A P I D I N A C T I V A T I O N T H A N G L A S S , A N O A C I D W A S H E D G L A S S WAS E Q U A L L Y U N S U I T A B L E . IN F A C T , A C I D W A S H E D G L A S S I N A C T I V A T E D A T A G R E A T E R R A T E T H A N D E T E R G E N T W A S H E D G L A S S S U G G E S T I N G T H A T T H E G L A S S S U R -F A C E M I G H T B E A C T I N G A S A C A T I O N E X C H A N G E R , A N D G L A S S V E S S E L S W A S H E D I N 2 M S O D I U M H Y D R O X I D E P R I O R TO A D I S T I L L E D W A T E R R I N S E W E R E T R I E D . CT C O U L D B E I N C U B A T E D A T 3 7 ° C IN pH 7.4- T R I S - H C L B U F F E R F O R P E R I O D S U P , T O TWO H O U R S IN T H E S E V E S S E L S W I T H L I T T L E OR NO I N A C T I V A T I O N . IF T H E I N C U B A T I O N S W E R E E X T E N D E D F O R L O N G E R P E R I O D S A S L O W I N A C T I V A T I O N O C C U R E O P O S S I B L Y R E S U L T I N G F R O M D I S U L F I D E I N T E R C H A N G E A T T H I S H I G H P H . S I N C E I N C U B A T I O N O F CT W I T H T H E M A J O R I N A C T I V A T O R F R A C T I O N S R E S U L T E D IN M A R K E D R E D U C T I O N I N A C T I V I T Y IN A 30 M I N U T E P E R I O D A T 3 7 ° C A N D PH 7 . 4 , I T WAS P O S S I B L E TO U S E T H I S R E L A T I V E L Y S I M P L E P R O C E D U R E T O I N D I C A T E W H I C H F R A C -T I O N S C O N T A I N E D S I G N I F I C A N T I N A C T I V A T O R A L T H O U G H N O D E T A I L E D R E C O R D O F Q U A N T I T A T I V E Y I E L D S WAS P O S S I B L E . IN G E N E R A L , - 7 4 -A L I Q U O T S OF A S O L U T I O N C O N T A I N I N G 2.5 M R C U/ML OF P O R C I N E C T (ARMOUR P H A R M A C E U T I C A L . Co . A L 0 8 3 1 ) I N PH 7.4, 0.1 M T R I S - H C L WERE M I X E D WITH AN EQUAL VOLUME OF V A R I O U S COLUMN E L U A T E F R A C T I O N S A D J U S T E D TO PH 7.4 WITH T RIS B A S E AND I N C U B A T E D FOR ONE HOUR AT 3 7 ° C S A M P L E S , T A K E N AT ZERO T I M E AND ONE HOUR WERE S U I T A B L Y D I L U T E D AND B I O A S S A Y E O AS D E S C R I B E D IN C H A P T E R I I . Two D I L U T I O N S OF E A C H S A M P L E WERE A S S A Y E D WITH THE SECOND U S U A L L Y A TWO FOLD D I L U T I O N OF THE F I R S T . FOUR RATS W E R E U S E D F O R E A C H D I L U T I O N A N D T H E R E M A I N I N G B I O L O G I C A L A C T I V I T Y C A L C U L A T E D FROM THE A V E R A G E R E S P O N S E S OF E A C H GROUP.. THE ZERO T I M E A C T I V I T Y WAS T A K E N AS 1 0 0 $ AND THE P E R C E N T A G E OF B I O L O G I C A L A C T I V I T Y REMOVED IN ONE HOUR USED AS AN INDEX OF THE I N A C T I V A T O R P R E S E N T . S L I G H T V A R I A T I O N S OF T H I S PROCEDURE WERE USED FOR V A R I O U S A S S A Y S AND THE D E T A I L S W I L L BE I N C L U D E D IN THE D E S C R I P T I O N S OF THE P U R I F I C A T I O N S T A G E S . C . PART I AL PUR I F I CAT I ON OF JA S E L E C T I VE " C A L C I TON I N A S E " . 1 . S E L E C T i O N QF S T A R T i N G M A T E R I A L . THE WORK OF T A S H J I A N AND V O E L K E L ( 1 2 2 ) SHOWED THE P R E S E N C E OF P O R C I N E C T I N A C T I V A T I N G F A C T O R S I N HUMAN AND RAT SERUMS AND P A R T I A L L Y C H A R A C T E R I Z E D T H E S E F A C T O R S . T H E WORK D E S C R I B E D I N THE I N T R O D U C T I O N TO T H I S C H A P T E R I N D I C A T E D THAT T H E S E F A C T O R S WERE NOT P A R T I C U L A R L Y E F F E C T I V E IN I N A C T I V A T I N G SALMON C T AND THAT T H I S F A I L U R E TO I N A C T I V A T E THE SALMON HORMONE P R O B A B L Y C O N T R I B U T E D S I G N I F I C A N T L Y TO THE ENHANCED P H Y S I O L O G I C A L E F F E C T OF T H I S HORMONE. FURTHER C H A R A C T E R I -Z A T I O N OF THESE FACTORS COULD A I D IN D E F I N I N G THE S T R U C T U R A L D I F F E R E N C E S IN THE SALMON M O L E C U L E R E S P O N S I B L E FOR T H I S - 7 5 -R E S I S T A N C E TO I N A C T I V A T I O N . SUCH C H A R A C T E R I Z A T I O N RE QUI RE 0 THAT THE I N A C T I V A T I N G E N Z Y M E S BE O B T A I N E D I N R E L A T I V E L Y PURE FORM SO THAT ONLY ONE T Y P E OF I N A C T I V A T I O N OCCURED IN A G I V E N I N C U B A T I O N A L L O W I N G I D E N T I F I C A T I O N OF THE R E A C T I O N PRODUCTS TO D E T E R M I N E THE S P E C I F I C I T Y OF THE E N Z Y M E . B E C A U S E OF THE VAST NUMBER OF COMPONENTS P R E S E N T IN SERUM AND THE R E L A T I V E L Y L A R G E Q U A N T I T Y OF S T A R T I N G M A T E R I A L WHICH WOULD BE NEEDED TO Y I E L D S I G N I F I C A N T P U R I F I E D PRODUCT C O M M E R C I A L L Y P R E P A R E D COHN P L A S M A F R A C T I O N S WERE USED AS S T A R T I N G M A T E R I A L . IT WAS F I R S T N E C E S S A R Y , HOWEVER, TO DEMONSTRATE THAT UN C L O T T E D P L A S M A AND SERUM HAD A S I M I L A R I N A C T I V A T I N G A B I L I T I E S S I N C E THE P O S S I B I L I TY EX I S T E D THAT THE C L O T T I N G E N Z Y M E S WERE R E S P O N S I B L E FOR THE I N A C T I V A T I O N . TO T E S T T H I S , S A M P L E S OF R A B B I T SERUM AND H E P A R I N I Z E D R A B B I T P L A S M A WERE P R E P A R E D C O N T A I N I N G 20 MRC U/ML OF P O R C I N E CT AND I N C U B A T E D AT 3 7 °C FOR 75 M I N . A L I Q U O T S WERE T A K E N FROM THE I N C U B A T I O N S A M P L E S AT ZERO T I M E AND 75 M I N . , AND WERE B I O A S S A Y E D AS P R E V I O U S L Y D E S C R I B E D . THE MEAN P L A S M A C A L C I U M L E V E L OF THE TEN RATS I N J E C T E D WITH BOTH ZERO T I M E S A M P L E S WAS 8.5 ± 0 . 1 MG$ ( ± S . E . M . ) . A F T E R THE 75 M I N I N C U B A T I O N THE MEAN P L A S M A C A L C I U M L E V E L OF F I V E RATS I N J E C T E D WITH THE SERUM S A M P L E WAS 9.2 ± 0 . 1 MG$. THE C O R R E S P O N D I N G V A L U E FOR THE P L A S M A S A M P L E WAS 9.3 ± 0 . 1 MG$. BOTH V A L U E S WERE S I G N I F I C A N T L Y LOWER THAN THE ZERO T I M E L E V E L (P<.005) , BUT THERE WAS NO S I G N I F I C A N T D I F F E R E N C E BETWEEN THE TWO. V A L U E S , I N D I C A T I N G THAT THERE WAS L I T T L E D I F F E R E N C E IN THE AMOUNT OF I N A C T I V A T O R P R E S E N T . TABLE IX INACTIVATION OF PORCINE C T DURING INCUBATIONS WITH HUMAN PLASMA FRACTIONS INCUBAT I ON SOLUTI ON PLASMA CALCIUM LEVELS +S.E.M. (MG$) ZERO TIME 3 HOURS BUFFER ONLY LYOPHILiZEO PLASMA COHN I COHN I I COHN I I 1-0 COHN I I I COHN IV-1 COHN IV-4 COHN V 1 0 . 2 + 0.1 (5)* 8.4 + 0.1 (5) 9.1 + 0.1 ( 2 ) 8.3 + 0.1 ( 2 ) 8.3 + 0.1 ( 2 ) 10.1 + 0.1 ( 2 ) 8.5 + 0.1 ( 2 ) 10.4 + 0.1 ( 2 ) 10.7 + Q.1 ( 2 ) 8.8 + 0.1 ( 2 ) * NUMBER OF ANIMALS INJECTED - 7 6 -COHN F R A C T I O N S FROM HUMAN P L A S M A ( N U T R I T I O N A L B I O C H E M -I C A L CORP., C L E V E L A N D , O H I O ) AS L I S T E D IN T A B L E I X WERE ADDED TO PH 7 . 4 , 0 . 1 M T R i S - H C L B U F F E R IN C O N C E N T R A T I O N S A P P R O X I M A T I N G THOSE WHICH E X I S T IN P L A S M A ( S E E 9 2 ) , AND S H A K E N FOR 3 0 M I N . ANY I N S O L U B L E R E S I D U E WAS REMOVED BY C E N T R I F U G A T I O N AND THE S U P E R N A T E S USED FOR I N C U B A T I O N S WITH P O R C I N E C T . THE S A M P L E S WERE MADE UP TO A C O N C E N T R A T I O N OF ' 0 2 0 M R C MU/ML, AND I N C U B A T E D AT 3 7 C . B I O A S S A Y S WERE C A R R I E D OUT AT ZERO T I M E AND A F T E R 3 HOURS OF I N C U B A T I O N , AS THE P L A S M A C A L C I U M S OF THE RATS I N J E C T E D AT ZERO T I M E D I D NOT VARY S I G N I F I C A N T L Y THE DATA WAS P O O L E D . THE AMOUNT OF C T I N THE RED I S S O L V E D L Y O P H I L I Z E D P L A S M A S A M P L E A F T E R 3 HOURS WAS REDUCED MARKEDLY AND NO M E A S U R A B L E C T R E M A I N E D IN THREE OF THE S E V E N COHN F R A C T I O N S , I N D I C A T I N G THAT I N A C T I V A T O R S WERE P R E S E N T IN MORE THAN ONE F R A C T I O N E I T H E R B E C A U S E MORE THAN ONE E N Z Y M E WAS I N V O L V E D OR B E C A U S E A S I N G L E ENZYME WAS P R E S E N T IN MORE THAN ONE F R A C T I O N . F U R T H E R T E S T S ON D I L U T E D S A M P L E S OF T H E S E THREE F R A C T I O N S I N D I C A T E D THAT F R A C T I O N I V - 1 WAS S L I G H T L Y MORE POTENT IN I N A C T I V A T I N G P0RC I NE C T AND T H I S F R A C T I O N WAS CHOSEN FOR FURTHER WORK. 2 . P R E L I M I N A R Y P U R I F I C A T I O N E X P E R I M E N T S . D U R I N G THE E X P E R I M E N T S I N THE P R E V I O U S S E C T I O N I T WAS O B S E R V E D THAT A L A R G E P E R C E N T A G E OF THE M A T E R I A L I N F R A C T I O N I V - 1 WAS I N S O L U B L E IN THE PH 7 . 4 B U F F E R . T H U S A C O N S I D E R A B L E P U R I F I C A T I O N COULD BE A C H I E V E D BY E X T R A C T I N G THE F R A C T I O N WITH T H I S B U F F E R . IN AN I N I T I A L E X P E R I M E N T 1 G OF F R A C T I O N I V - 1 WAS E X T R A C T E D FOUR T I M E S WITH 1 0 ML A L I Q U O T S OF B U F F E R . F I G U R E 3 7 . E L U T I O N P R O F I L E OF AN E X T R A C T OF HUMAN COHN F R A C T I O N IV-1 ON S E P H A O E X G-200. COLUMN, 2.5 x 9 0 CM; E L U A N T , PH 7.4, 0.1 M T R I S - H C L B U F F E R ; T E M P E R A T U R E , 4 ° C; FLOW R A T E , 2 0 CM/HR; F R A C T I O N S I Z E , 6.8 ML. C/OCT REMOVED D E T E R M I N E D IN I N A C T I -V A T I O N A S S A Y AS D E S C R I B E D IN T E X T . - 7 7 -A F T E R A D O I T I O N OF E A C H 1 0 ML A L I Q U O T , THE M I X T U R E WAS S H A K E N FOR 3 0 M I N AND THEN C E N T R I F U G E D AND THE S U P E R N A T E S S A V E D . AN A L I Q U O T OF EACH S U P E R N A T E WAS THEN I N C U B A T E D WITH P O R C I N E C T AS P R E V I O U S L Y D E S C R I B E D TO D E T E R M I N E I T S I N A C T I -V A T I O N A C T I V I T Y . THE I N A C T I V A T I O N RATE DROPPED MARKEDLY I N THE L A S T TWO S A M P L E S AND I T WAS D E C I D E D THAT ONLY THE F I R S T TWO S A M P L E S SHOULD BE POOLED FOR F U R T H E R P U R I F I C A T I O N . A 3 ML S A M P L E OF THE POOLED M A T E R I A L WAS T A K E N FOR CHROMATOGRAPHY AT 4 ° C ON A 2.5 X 9 0 CM S E P H A D E X A D J U S T A B L E COLUMN C O N T A I N I N G S E P H A D E X G-200 E Q U I L I B R A T E D I N 7.4, 0.1 M T R I S - H C L B U F F E R . THE E L U A T E WAS C O L L E C T E D IN 6.8 ML F R A C T I O N S AT A RATE OF 2 0 M L / H R AND THE A B S O R B A N C E AT 2 8 0 NM FOLLOWED AS AN I N D E X OF P R O T E I N . THE S A M P L E WAS A P P L I E O TO THE BOTTOM OF THE COLUMN THROUGH A THREE-WAY V A L V E ANO E L U A N T FLOW WAS UPWARDS TO P R E V E N T THE S E P H A D E X FROM P A C K I N G . THE E L U T I O N P R O F I L E FOR THE COLUMN I S SHOWN IN F I G U R E 3 7 . THE COLUMN E L U A T E WAS D I V I D E D INTO F I V E POOLS AS I N D I C A T E D IN THE F I G U R E ANO E A C H POOL A S S A Y E D FOR I N A C T I VAT0R AS D E S C R I B E D IN S E C T I O N B E X C E P T THAT THE CT S O L U T I O N C O N T A I N E D ONLY 4 0 MRC MU/ML SO THAT THE F I N A L I N C U B A T I O N M I X T U R E C O N T A I N E D 2 0 MRC MU/ML. THE P E R C E N T OF CT A C T I V I T Y REMOVED IN ONE HOUR I S SHOWN IN THE F I G U R E ABOVE THE E L U T I O N P R O F I L E . THE R E S U L T S OF T H I S E X P E R I M E N T WERE I N MANY WAYS S I M I L A R TO THOSE OF T A S H J I A N AND V O E L K E L ( 1 2 2 ) ON WHOLE HUMAN SERUM. TWO R E G I O N S WERE O B S E R V E O TO C O N T A I N I N A C T I V A T O R S IN E A C H C A S E AND THE M O L E C U L A R WEIGHTS OF THE I N A C T I V A T O R S WERE S I M I L A R . THE L A R G E R COMPONENT HAD A K D L E S S THAN 0.3 IN - 7 8 -EACH C A S E WHILE THE S M A L L E R COMPONENTS HAD KQ'S G R E A T E R THAN 0.7. T H E S E K O * S I N D I C A T E M O L E C U L A R WEIGHTS G R E A T E R THAN 1 0 0 , 0 0 0 AND L E S S THAN 3 0 , 0 0 0 R E S P E C T I V E L Y . T H I S DATA CANNOT B E T A K E N TO I N D I C A T E THAT THE SAME COMPONENTS WERE R E S P O N S I B L E FOR I N A C T I V A T I O N IN BOTH C A S E S , BUT T H I S P O S S I B I L I T Y CANNOT BE RULED OUT. POOL B WAS I N V E S T I G A T E D F I R S T S I N C E I T A P P E A R E D TO C O N T A I N MORE I N A C T I V A T I N G A C T I V I T Y THAN THE OTHER P O O L S . THE E N T I R E R E M A I N I N G VOLUME OF POOL B WAS ALLOWED TO FLOW ONTO A 2 X 2 0 CM COLUMN OF D E A E - S E P H A D E X WHICH HAD B E E N E Q U I L I B R A T E D WITH P H 7.4, 0.1 M T R I S - H C L B U F F E R AND THE E L U A T E C O L L E C T E D IN 6.8 ML F R A C T I O N S AT A RATE OF 1 0 ML/HR. A L I N E A R G R A D I E N T OF T R I S - H C L B U F F E R FROM 0.1 M ( P H 7.4; C O N D U C T I V I T Y , 3.5 MMHOS; VOLUME, 50 M L ) TO 0.5 M ( P H 7.4; C O N D U C T I V I T Y , 1 6 . 0 MMHOS; VOLUME, 5 0 M L ) WAS THEN A P P L I E D TO THE COLUMN. UNDER THE C O N D I T I O N S OF T H I S COLUMN NONE OF THE I N A C T I V A T I N G A C T I V I T Y R E M A I N E D ON THE RESIN:- IN THE I N I T I A L B U F F E R , BUT ABOUT 5 0 $ OF THE M A T E R I A L A B S O R B I N G AT 2 8 0 NM WAS ADSORBED AND S U B S E Q U E N T L Y REMOVED AS A BROAD P E A K BY THE G R A D I E N T . T H I S R E S U L T S U G G E S T E D THAT D E A E - S E P H A D E X COULD BE USED IN A BULK A D S O R P T I O N S T E P F O L L O W I N G THE E X T R A C T I O N TO REMOVE A NUMBER OF C O N T A M I N A N T S . THE I N A C T I V A T I N G M A T E R I A L FROM THE I N I T I A L E L U A T E F R A C T I O N S OF T H I S COLUMN WERE POOLED AND USED IN A S E R I E S OF E X P E R I M E N T S TO D E F I N E C O N D I T I O N S FOR A D S O R P T I O N OF THE A C T I V E COMPONENT TO C M - S E P H A D E X FOR FURTHER P U R I F I C A T I O N . THE C O N D I T I O N S WHICH PROVED MOST S U C C E S S F U L WERE USED IN S U B S E Q U E N T E X P E R I M E N T S AND ARE D E S C R I B E D I N THE I PUR.T F I CAT I ON S C H E D U L E IN THE NEXT S E C T I O N . 3. P R E P A R A T I O N OF H I G H L Y P U R I F I E D " C A L C I T O N I N A S E " . A 5 G S A M P L E OF COHN F R A C T I O N IV-1 WAS M I X E D WITH 3 0 ML OF PH 7.4, 0.1 M T R I S - H C L B U F F E R AND S H A K E N FOR ONE HOUR AT ROOM T E M P E R A T U R E . IT WAS THEN C E N T R I F U G E D TO Y I E L D A C L E A R S U P E R N A T E WHICH WAS REMOVED AND S A V E D . THE R E S I D U E WAS R E S U S P E N D E D IN ANOTHER 3 0 ML OF B U F F E R AND S H A K E N A G A I N . T H I S P R O C E S S OF R E S U S P E N S I O N AND C E N T R I F U G A T I ON WAS R E P E A T E D A TOTAL OF THREE T I M E S Y I E L D I N G 8 0 ML OF POOLED S U P E R N A T E S . THE POOLED S U P E R N A T E S WERE ADDED TO 2 0 G OF D E A E - S E P H A D E X WHICH HAD B E E N P R E V I O U S L Y E Q U I L I B R A T E D WITH PH 7.4» 0,1 M T R I S - H C L B U F F E R AND THE M I X T U R E WAS S T I R R E D FOR TWO HOURS AT ROOM T E M P E R A T U R E . THE M I X T U R E WAS THEN F I L T E R E D AND THE S E P H A D E X WASHED TWICE WITH 5 0 ML OF PH 7.4» 0.1 M T R I S - H C L B U F F E R . T H I S WAS FOLLOWED BY TWO WASHES WITH 5 0 ML OF D I S T I L L E D WATER, AND A L L OF THE F I L T R A T E S WERE P O O L E D . THE TOTAL F I L T R A T E VOLUME OF ABOUT 3 0 0 ML WAS REDUCED TO 1 0 ML BY L Y O P H I L I Z A T I O N , AND T H I S 1 0 ML E X T R A C T USED FOR FURTHER P U R I F I C A T I O N BY COLUMN CHROMATOGRAPHY. THE E N T I R E 1 0 ML S A M P L E WAS A P P L I E D TO A S E P H A D E X G-200 COLUMN I D E N T I C A L TO THAT D E S C R I B E D IN THE P R E C E D I N G S E C T I ON E X C E P T THAT 0.1 M F O R M I C A C I D WAS USED AS AN E L U A N T . THE H I G H M O L E C U L A R WEIGHT COMPONENTS CAME OFF I N A S I N G L E BROAD P E A K WHICH WAS WELL S E P A R A T E D FROM THE S M A L L E R COMPONENTS ANO THE S A L T , AND THE COLUMN F R A C T I O N S WERE D I V I D E D INTO TWO —- lOOn 0 80 160 240 Fraction Number F I G U R E 3 8 . E L U T I O N P R O F I L E OF S E P H A D E X G-200 PRODUCT ON CM-SEPHADEX C-25. COLUMN, 2 x 2 0 CM; E L U A N T , L I N E A R G R A D I E N T S OF PH 5.0 AMMONIUM A C E T A T E FROM 0.1 M TO 0.5 M AND FROM 0.5 M TO 1.0 M; T E M P E R A T U R E , 4 ° C; FLOW R A T E , 2 0 M L / H R ; F R A C T I O N S I Z E , 6.8 ML. - 8 0 -P 0 0 L S . THE POOL CONTAINING THE LARGE COMPONENTS ( F R A C T I O N S 2 0 THROUGH 4 5 , SEE FIGURE 3 7 ) HAD A TOTAL VOLUME OF 1 7 0 ML. WHEN THIS SOLUTION WAS AOJUSTED TO PH 5 . 0 WITH AMMONIUM HYDROXIDE FOR A P P L I C A T I O N TO THE NEXT COLUMN THE SOLUTION CLOUDED. THE SUSPENDED COLLOID WAS NOT REMOVED BUT THE ENTIRE SAMPLE INCLUDING THE SUSPENDED COLLOID WAS A P P L I E D TO A 2 X 2 0 CM COLUMN OF C M - S E P H A D E X , C - 2 5 E Q U I L I B R A T E D WITH 0 . 1 M AMMONIUM ACETATE BUFFER AT PH 5 . 0 . THE COLUMN WAS WASHED WITH 2 5 0 ML OF 0 . 1 M AMMONIUM ACETATE BUFFER AT PH 5 . 0 AND THE MATERIAL WHICH REMAINED ADSORBED ON THE COLUMN WAS ELUTED WITH A L I N E A R GRADIENT OF AMMONIUM ACETATE BUFFER FROM 0 . 1 M (PH 5 . 0 ; CONDUCTIVITY, 3 . 5 MMHOS; VOLUME, 2 5 0 ML) TO 0 . 5 M ( P H 5 . 0 ; CONDUCTIVITY, 1 6 . 0 MMHOS; VOLUME, 2 5 0 M L ) . A SECOND LINEAR GRADIENT OF AMMONIUM ACETATE BUFFER FROM 0 . 5 M (PH 5 . 0 ; CONDUCTIVITY, 1 6 . 0 MMHOS; VOLUME, 2 5 0 ML) TO 1 . 0 M (PH 5 . 0 ; CONDUCTIVITY, 2 6 . 0 MMHOS; VOLUME, 2 5 0 ML) WAS EMPLOYED TO INSURE THAT ALL COMPONENTS WERE REMOVED. ELUATE WAS COLLECTED IN 6 . 8 ML FRACTIONS AT A RATE CF 2 0 M L / HR . THE RESULTS FROM THIS COLUMN ARE SHOWN IN FIGURE 3 8 . THE ELUATE FRACTIONS WERE POOLED IN THE S I X REGIONS I N D I -CATED AND THE C T I N A C T I V A T I N G A C T I V I T Y ASSESSED AS PREVIOUSLY DESCRIBED. 2 . 0 MRC U OF PORCINE C T WERE ADDED TO EACH SAMPLE TO A FINAL CONCENTRATION OF 1 . 0 MRC L)/ML, AND THE BIOLOGICAL A C T I V I T Y REMAINING AFTER 3 0 MIN OF INCUBATION AT 3 7 ° C DETERMINED. THE PERCENTAGE A C T I V I T Y REMOVED IS SHOWN IN THE FIGURE FOR EACH POOL. POOL H CONTAINED THE MAJOR J K L M 0.4 i Fraction Number F I G U R E 39. E L U T I O N P R O F I L E OF CM-SEPHADEX PRODUCT ON S E P H A D E X G-200. E L U A N T , 0.05 M A C E T I C A C I D ; OTHER C O N D I T I O N S AS I N D I C A T E D I N F l G U R E 37. - 8 1 -INACTIVATOR ACITIVITY ANO WAS SELECTED FOR FURTHER PURIFI-CATION. THE ADJACENT POOLS G AND I ALSO CONTAINED SOME INACTIVATOR, BUT IT SEEMED LIKELY THAT THIS WAS DUE PRI-MARILY TO OVERLAP OF THE CENTRAL PEAK INTO THE ADJACENT POOLS. POOL H CONTAINED 1 2 0 ML OF ELUATE AND WAS LYOPHILIZED TO ORYNESS TO REMOVE EXCESS WATER AND SALT. THE DRY SAMPLE WAS RED I SSOLVED IN 1 0 ML OF DISTILLED WATER AND 5 ML OF THIS WAS APPLIED TO A COLUMN OF SEPHADEX G - 2 0 0 AS PREVIOUSLY DESCRIBED BUT USING 0 . 0 5 M ACETIC ACID AS AN ELUANT. THE ELUATE WAS COLLECTED IN 6 . 8 ML FRACTIONS AT A RATE OF 2 0 ML/HR. THE ELUATE FRACTIONS WERE DIVIDED INTO FOUR POOLS AND THEIR INACTIVATOR ACTIVITY ASSESSED AS DESCRIBED FOR THE PREVIOUS COLUMN. As SHOWN IN FIGURE 39 THE COLUMN PROFILE AND INACTIVATOR ASSAY RESULTS WERE RATHER UNEXPECTED. THE SAMPLE, WHICH HAD ORIGINALLY CONTAINED ONLY MATERIAL OF RELATIVELY HIGH MOLECULAR WEIGHT WAS COMPRISED PRIMARILY OF MATERIAL OF RELATIVELY LOW MOLECULAR WEIGHT, AND POOL M CONTAINED MOST OF THE INACTIVATOR. THE TWO POOLS, J AND K, WHERE INACTI-VATOR WAS EXPECTED ACTUALLY CONTAINED LITTLE PROTEIN AND ONLY A SMALL FRACTION OF THE INACTIVATOR, SUGGESTING THAT THE LOW MOLECULAR WEIGHT INACTIVATOR WAS DERIVED FROM THE LARGER COMPONENT AND PERHAPS REPRESENTED THE ONLY ACTIVE ENZYME INVOLVED IN INACTIVATION. AN OBVIOUS ANALOGY FOR THIS TYPE OF RELATIONSHIP EXISTS IN THE PR0THR0MBIN-THR0MBIN SYSTEM WHERE IT HAS BEEN SHOWN THAT THE ACTIVE ENZYME CAN BE RELEASED FIGURE 4 0 SUMMARY OF PURIFICATION PROCEDURE FOR "CALCITONINASE" COHN FRACTION IV-1 (5G) RESIDUE (DiSCARDED ) EXTRACTED WITH PH 7.4, 0.1 M TRIS HYDROCHLORIDE 'SUPERNATE F l LTRATE' CONTAMINANTS ABSORBED ON DEAE-SEPHADEX IN PH 7.4, 0.1M TRIS ^RESIDUE (DiSCARDED) LYOPHILIZED TO 1 0 ML VOLUME FINAL EXTRACT MOLECULAR SIEVING ON SEPHADEX G-200 IN 0.1M FORMIC ACID POOLED HIGH MOLECULAR WEIGHT COMPONENTS ION EXCHANGE ON CM-SEPHAOEX, PH 5.0 POOL H LYOPHILIZED 5 0 $ * ^ 5 0 $ SAVED MOLECULAR SIEVING ON SEPHADEX G-200 IN 0.05 M ACETIC ACID POOL M FINAL PRODUCT ( 1 6 . 5 MG) - 8 2 -F R O M T H E P R E C U R S O R B Y I N C U B A T I O N I N A 2 5 $ (w/v) S O D I U M C I T R A T E S O L U T I O N ( 1 1 1 ) . P E R H A P S A S I M I L A R T Y P E R E L E A S E O C C U R E D I N T H E C O N C E N T R A T E D A M M O N I U M A C E T A T E S O L U T I O N W H I C H R E S U L T E D F R O M L Y O P H I L I Z A T I O N O F P O O L H . E L U C I D A T I O N O F T H E R E L A T I O N S H I P S I N V O L V E D I N T H I S T Y P E O F S Y S T E M W A S B E Y O N D T H E S C O P E O F T H I S S T U D Y A N D I T W A S L I K E L Y T H A T F U R T H E R P U R I F I -C A T I O N W O U L D P R O V E D I F F I C U L T S I N C E A N U M B E R O F S P O N T A N E O U S L Y G E N E R A T E D I N A C T I V E F R A G M E N T S C O U L D B E P R O D U C E D . IT W A S D E C I D E D T H E R E F O R E T H A T P O O L M R E P R E S E N T E D M A T E R I A L O F S U F F I C I E N T P U R I T Y F O R U S E I N C O M P A R A T I V E S T U D I E S . T H E D E G R E E O F P U R I T Y A N D S O M E C H A R A C T E R I S T I C S O F T H E E N Z Y M E W E R E E X A M I N E D B U T N O F U R T H E R P U R I F I C A T I O N W A S A T T E M P T E D . P O O L M C O N T A I N E D 1 1 0 M L A N D L Y O P H I L I Z E D S A M P L E S O F T H I S P O O L Y I E L D E D 0 . 1 5 M G O F P R O T E I N P E R M L . T H I S P O O L T H U S C O N T A I N E D 1 6 . 5 M G O F M A T E R I A L P R E P A R E D F R O M T H E E Q U I V A L E N T O F 2 . 5 G O F C O H N F R A C T I O N I V - 1 . 4 . S U M M A R Y O F P U R I F I C A T I O N . F I G U R E 40 O U T L I N E S T H E S T A G E S O F P U R I F I C A T I O N U S E D T O P R E P A R E T H E ; M A T E R I A L I N P O O L M W H I C H W A S U S E D F O R A L L F U R T H E R S T U D I E S . T H E 1 6 . 5 M G O F M A T E R I A L F R O M 2 . 5 G O F C O H N F R A C T I O N I V - 1 R E P R E S E N T S 1 50 F O L D R E D U C T I O N I N W E I G H T O V E R T H E C O H N F R A C T I O N U S E D A S A S T A R T I N G M A T E R I A L , B U T S I N C E T H I S F R A C T I O N R E P R E S E N T S O N L Y 6 $ O F T H E T O T A L P L A S M A P R O T E I N T H E F I N A L P R O D U C T R E P R E S E N T S A 2 5 0 0 F O L D R E D U C T I O N I N W E I G H T F R O M T O T A L P L A S M A P R O T E I N . T H I S I S N O T N E C E S S A R I L Y E Q U I V A L E N T T O T H E O E G R E E O F P U R I F I C A I O N , B U T I T I S T H E B E S T -83-E S T I M A T E A V A I L A B L E S I N C E NO Q U A N T I T A T I V E MEASUREMENT OF I N A C T I V A T O R WAS P O S S I B L E . 5. T E S T S OF PURI TY. S A M P L E S OF POOL M WERE S U B J E C T E D TO E L E C T R O P H O R E S I S ON C E L L U L O S E A C E T A T E S T R I P S AND ON P O L Y A C R I L A M IDE G E L S TO D E T E R M I N E THE NUMBER OF COMPONENTS P R E S E N T . E L E C T R O P H O R E S I S ON C E L L U L O S E A C E T A T E S T R I P S WAS C A R R I E D OUT U S I N G A GELMAN #51170 CHAMBER AND S E R A P H O R E M L S T R I P S FROM THE GELMAN INSTR U M E N T CO., ANN ARBOR, M I C H I G A N . THE B U F F E R WAS S T A N -DARD H I G H R E S O L U T I O N B A R B I T O N E B U F F E R AT PH 8.6 FROM THE SAME S O U R C E . . S A M P L E S C O N T A I N I N G 25 >XG 0 F P R O T E I N I N 5 RL WERE A P P L I E D NEAR THE CATHODE AND ALLOWED TO M I G R A T E FOR 75 M I N AT 360 V O L T S . T H E S T R I P S WERE THEN D E V E L O P E D I N A S O L U T I O N C O N T A I N I N G 0.2$ (w/v) P O N C E A U S S.TAIN IN 5$ (w/ V ) T C A AND D E S T A I N E D WITH 3 WASHES I N 5$ A C E T I C A C I D . HUMAN P L A S M A S A M P L E S WERE RUN S I M U L T A N E O U S L Y AS C O N T R O L S . P O L Y A C R I L A M IDE GEL E L C T R O P H O R E S I S WAS C A R R I E D OUT AS D E S C R I B E D IN A P P E N O I X F. A F T E R THE G E L S HAO B E E N E Q U I L I -B R A T E D WITH 0.01 M A C E T I C A C I D A 10 j^L S A M P L E C O N T A I N I N G 2 NG/^L OF POOL M M A T E R I A L IN 1 M SUCROSE I N 0.002 M A C E T I C A C I D WAS A P P L I E D TO THE TOP OF THE GEL AND THE A P P L I C A T I O N ZONE S H A R P E N E D FOR 25 M I N IN A 32 VOLT F I E L D . E L E C T R O -P H O R E S E S WAS THEN C A R R I E D OUT AT 320 V O L T S FOR 70 M I N U T E S AND THE G E L S WERE REMOVED FROM THE T U B E S FOR S T A I N I N G AS ' D E S C R I B E D IN THE A P P E N D I X . TWO D I S T I N C T COMPONENTS WERE S E P A R A T E D BY E L E C T R O -- 8 4 -P H O R E S I S ON C E L L U L O S E A C E T A T E S T R I P S . THE MAJOR BAND M I G R A T E D WITH THE 01 2"-GL 0 B UL I N S WHILE THE MINOR BAND HAD A H I G H E R M O B I L I T Y A P P R O X I M A T E L Y EQUAL TO A L B U M I N . T H E S E P A R A T I O N ON THE P O L Y A C R I L A M IOE G E L S R E V E A L E D 5 MINOR BANDS OF H I G H M O B I L I T Y AND A MAJOR BAND OF R E L A T I V E L Y LOW M O B I L I T Y . T H I S C O N F I R M E D THAT POOL M C O N T A I N E D A NUMBER OF COMPONENTS AND THAT I S O L A T I O N OF THE I N A C T I V A T I N G E N Z Y M E WOULD R E Q U I R E FURTHER P U R I F I C A T I O N S T A G E S . T H E P U R I F I C A T I O N A C H I E V E D WAS ADEQUATE, HOWEVER, TO ALLOW A N A L Y S I S OF S E V E R A L C H A R A C T E R -I S T I C S OF THE E N Z Y M E . D * S E L E C T i V E I N A C T i V A T i O N OF P O R C I N E C A L C I T O N I N BY " C A L C I TON I N A S E " . I N C U B A T I O N OF THE THREE C A L C I T O N I N S WITH THE POOL M M A T E R I A L WAS C A R R I E D OUT IN THE MANNER S I M I L A R TO THAT D E S C R I B E D FOR A S S A Y I N G I N A C T I V A T O R . THE I N C U B A T I O N M I X T U R E S C O N T A I N E D 75 J^G/ML OF POOL M M A T E R I A L AND A P P R O X I M A T E L Y 1 0 |IG/ML OF C T IN 0.1 M T R I S - A C E T A T E B U F F E R AT PH 7.4. THE M A T E R I A L S USED WERE PURE N A T I V E SALMON C T AS D E S C R I B E D I N C H A P T E R I I I , S Y N T H E T I C HUMAN C T FROM C I B A L T D . , B A S L E , AND P O R C I N E C T ( A L 0 8 3 1 ) FROM ARMOUR P H A R M A C E U T I C A L CO. K A N K A K E E , I L L I N O I S . THE TWO PURE M A T E R I A L S WERE WEIGHED D I R E C T L Y , BUT THE WEIGHT OF P O R C I N E C T WAS B A S E D ON A B I O L O G I C A L A C T I V I T Y OF 2 5 0 U/MG. A L I Q U O T S OF E A C H OF THE THREE M I X T U R E S WERE S U I T A B L Y D I L U T E D AND B I O A S S A Y E D P R I O R TO I N C U B A T I O N . A F T E R I N C U B A T I O N AT 3 7 ° C FOR ONE HOUR A L I Q U O T S WERE S I M I L A R L Y D I L U T E D AND B I O A S S A Y E D . THE P L A S M A C A L C I U M L E V E L S A F T E R I N J E C T I O N OF P O R C I N E C T - 8 5 -WERE 7 . 9 6 ± 0 . 0 4 M0/a AT ZERO TIME AND 9.11 ±-0.08 MG$ AFTER ONE HOUR OF INCUBATION. T H I S WAS A S I G N I F I C A N T DIFFERENCE (P < 0 . 0 0 1 ) AND REPRESENTED A LOSS OF 54$ OF THE CT. THE HUMAN AND SALMON C A L C I T O N I N S SHOWED NO S I G N I F I C A N T LOSS OF BIOLOGICAL A C T I V I T Y AFTER INCUBATION THUS CONFIRMING THAT THE ENZYME ACTED S E L E C T I V E L Y ON PORCINE C T . E. P E P T I P E S FROM C A L C I T O N I N S DIGESTED WITH " C A L C I T O N I N A S E . " i. METHODS. A. D I G E S T I O N WITH ENZYME. TWO PROCEOURES WERE USEP FOR D I G E S T I O N S . THE F I R S T WAS A MODIFICATION OF THE I N A C T I V A T I O N ASSAY PROCEDURE, AND CONSISTED OF D I S S O L V I N G THE C T SUBSTRATE IN THE POOL M SOLUTION ( 0 . 1 5 MG PROTEIN PER ML IN 0 . 0 5 M A C E T I C A C I D ) TO A CONCENTRATION OF 5 M G/M L. THE PH OF THE SOLUTION WAS THEN ADJUSTED TO PH 7 . 5 BY ADDING 0 .1 ML OF 2 . 0 M TRIS BASE SOLUTION PER 1 ML OF ENZYME SOLUTION. THE RESULTANT T RIS-ACETATE BUFFER SOLUTION WAS I NCUBATEO OVER-NIGHT (17 HR) AT 37° C AND ANALYSED FOR P E P T I D E S . T H I S PROCEDURE WAS USED FOR DIGESTION OF SYNTHETIC PORCINE CT S U P P L I E D BY ClBA LTD., BASLE AND FOR PURE NATIVE SALMON C T . THE SECOND DIGESTION PROCEDURE WAS A MODIFICATION OF THAT USED BY MUTT _E_T AJ. (78) FOR DIGESTION OF S E C R E T I N WITH THROMBIN. IN THIS PROCEDURE 1 MG OF CT WAS DISSOLVED IN 0 . 5 ML OF 1 . 0 $ AQUEOUS AMMONIUM BICARBONATE AND 10 JW- 0 F A N ENZYME STOCK SOLUTION ADOED EVERY 2 HOURS. THE STOCK SOLUTION CONTAINED 2 MG/ML OF POOL M MATERIAL IN 1$ AQUEOUS AMMONIUM -86-8 I CARBONATE. AFTER INCUBATION FOR 6 HOURS AT ROOM TEMPER-ATURE (21° C ) THE SOLUTION WAS LYOPHILIZED AND REDISSOLVED IN DISTILLED WATER. THE SOLUTION WAS THEN PLACED IN BOILING WATER FOR 6 MINUTES AND RELY0PHILIZE0. THIS PROCEDURE WAS USED FOR A SECOND DIGESTION OF PURE NATIVE SALMON C T . B. HIGH VOLTAGE ELECTROPHORESIS. THE DIGEST PRODUCTS WERE SPOTTED ON 3 CM OF THE ORIGIN LINE ON SHEETS OF WHATMAN 3 M M PAPER AND SUBJECTED TO ELECTROPHORESIS AT PH 1.9 AS DESCRIBED IN CHAPTER III. EDGE STRIPS WERE DEVELOPED WITH PAQ AND ACID NINHYDRIN REAGENTS. UNDEVELOPED AREAS CONTAINING PEPTIDES WERE CUT OUT ANO ELUTED AS PREVIOUSLY DESCRIBED. c. AMINO ACID ANALYSIS. ALIQUOTS OF THE ELUTED PEPTIDES WERE HYOROLYSED AND AMINO ACID ANALYSES CARRIED OUT IN THE SAME MANNER USED FOR THE TRYPTIC PEPTIDES IN CHAPTER III. D. END GROUP ANALYSIS. N— TERM IN A L RESIDUES OF THE PEPTIDES WERE DETERMINED BY DANS Y L A T I 0 N AS DESCRIBED FOR THE TRYPTIC PEPTIDES IN CHAPTER I I I . 2. RESULTS AND DISCUSSION. DURING THE INCUBATIONS A CONSIDERABLE PROPORTION OF THE MATERIAL IN SOLUTION PRECIPITATED OUT. THIS PRECIPITATION BEGAN AS SOON AS THE PH WAS RAISED AND INCREASED DURING THE INCUBATION PERIOD. SINCE ANY FRAGMENTS PRODUCED WOULD OR OH 0 s°0 MG J O A / I Ov O L 8? PM £ P ® 3 0 T YO : - ; OE P-2 O DNP QP-I T A O C A FIGURE 41 • PH 1 . 9 ELECTROPHORETOGRAM OF PEPT IDES FROM PORCINE CT PRODUCED BY 11 C A L C I T ON I N A S E " . OTHER DATA AS INDICATED IN F IGURE 2 1 . -87-TEND TO BE MORE S O L U B L E THAN THE WHOLE M O L E C U L E THE D I G E S T S WERE C E N T R I F U G E D TO REMOVE THE P R E C I P I T A T E AND THE S U P E R -N A T E S ONLY WERE A N A L Y S E D FOR P E P T I D E S . A T T E M P T S TO R E D I S S O L V E THE P R E C I P I T A T E S IN D I L U T E A C I D MET WITH ONLY L I M I T E D S U C C E S S I N D I C A T I N G THAT THE P R E C I P I T A T I O N P R O C E S S WAS NOT C O M P L E T E L Y R E V E R S I B L E , AND S U G G E S T I N G THAT THE HORMONES WERE UN D E R G O I N G D I S U L F I D E I N T E R C H A N G E IN THESE C O N C E N T R A T E D S O L U T I O N S R E S U L T I N G IN P O L Y M E R I Z A T I O N . T H E S U P E R N A T E S OF THE P O R C I N E C T D I G E S T S C O N T A I N E D A N A L Y S A B L E Q U A N T I T I E S OF P E P T I D E F R A G M E N T S , I N D I C A T I N G THAT THE I N A C T I V A T I N G ENZYME I N POOL M WAS IN FACT A P E P T I D A S E . F I G U R E 41 SHOWS THE P A T T E R N OF P E P T I D E S PRODUCED BY E L E C T R O P H O R E S I S OF THE S U P E R N A T E AT PH 1.9. F L V E WELL I S O L A T E D P E P T I D E S WERE D E T E C T E D , BUT ONLY FOUR WERE P R E S E N T IN S U F F I C I E N T Q U A N T I T Y FOR FURTHER A N A L Y S I S . A MINO A C I D A N A L Y S I S OF P E P T I D E P-1 I N D I C A T E D I T S C O M P O S I T I O N WAS THR, S E R , G L U , P R 0 2 » G L Y ^ , MET, PHE ^ . A N D I T S N — TERM I N A L GROUP WAS P H E N Y L A L A N I N E . P E P T I D E P-2 C O N T A I N E D A S P , THR, S E R , , 3 A L A , i-CYS^, V A L , L E U ^ , TYR AND ARG WITH AN N — TERM I NAL C Y S T I N E . THE C O M P O S I T I O N OF P-3 W,AS A S P ^ , THR, S E R , G L U , P R 0 o , G L Y ^ , MET, L E U , P H E , , H I S , ARG ANO THE N — TERM I N A L R E S I D U E WAS A S P A R T I C A C I D . P E P T I D E P-4 A L S O HAD AN N — TERM I N A L A S P A R T I C A C I D , BUT C O N T A I N E D ONLY A S P ^ , L E U , P H E , H I S AND ARG. C O M P A R I S O N OF THESE C O M P O S I T I O N S TO THE SEQUENCE OF P O R C I N E C A L C I T O N I N SHOWN IN F l G U R E 29 P E R M I T S I D E N T I F I C A T I O N OF THE P E P T I D E S I N V O L V E D . P - 1 , P-2 AND P-3 ARE THE THREE P E P T I O E S PROOUCED BY S P L I T S AT THE C - T E R M I N A L - 8 8 -S I O E OF THE TWO A R G I N I N E R E S I D U E S IN THE M O L E C U L E WITH P-1 AS THE C — TERM IN A L P E P T I D E , P-2 AS THE N — TERM IN A L P E P T I D E AND P-4 AS THE S M A L L E R C E N T R A L P E P T I D E . THE FOURTH P E P T I D E , P-3, R E S U L T E D FROM AN I N C O M P L E T E S P L I T AT THE SECOND A R G I N I N E AND C O N T A I N S BOTH THE C E N T R A L AND C — TERM IN A L P E P T I D E S . T H E S E R E S U L T S I N D I C A T E D THAT THE ENZYME IN POOL M HAD A S P E C I F I C I T Y FOR P E P T I D E BONDS ON THE C A R B O X Y L S I D E OF A R G I N I N E R E S I D U E S , BUT THAT OTHER F A C T O R S WERE I N V O L V E D IN THE S P E C I F I C I T Y S I N C E E V E N A F T E R 17 HOURS OF I N C U B A T I O N AT 37° C A R E L A T I V E L Y L A R G E AMOUNT OF P E P T I D E P-3 R E M A I N E D . C L E A R L Y THE A R G - A S N BONO WAS S P L I T MORE R E A O I L Y THAN THE A R G - P H E , AND THE S P E C I F I C I T Y WAS NOT EQUAL FOR A L L A R G I N I N E BONDS. TH E R E S U L T S OF THE D I G E S T I O N S OF SALMON C T P R O V I D E D F URTHER E V I D E N C E THAT NOT A L L A R G I N I N E BONDS WERE S U S C E P -T A B L E , S I N C E NO P E P T I D E S WERE D E T E C T E D IN THE S U P E R N A T E S OF E I T H E R OF THE D I G E S T S OF T H I S M A T E R I A L . IF THE A R G I N I N E IN THE SALMON M O L E C U L E HAD B E E N S U S C E P T A B L E THE C— TE RMIN A L P E P T I D E PRODUCED WOULD HAVE B E E N R E A D I L Y I D E N T I F I A B L E S I N C E IT WOULD HAVE B E E N I D E N T I C A L WITH ONE OF THE T R Y P T I C P E P T I D E S P R E V I O U S L Y I S O L A T E D . THE P R E S E N C E OF A P R O L I N E ON THE N— TERM IN A L S I D E OF THE A R G I N I N E IN SALMON C T MAY HAVE C O N T R I B U T E D TO THE R E S I S T A N C E OF THE BOND TO THE E N Z Y M E , S I N C E T H I S R E S I D U E CAN BLOCK THE A C T I O N OF P E P T I -D A S E S WHEN IT OCCURS NEAR A NORMALLY S U S C E P T A B L E BOND ( 6 8 ) . THE F A I L U R E OF THE ENZYME TO S P L I T THE SALMON M O L E C U L E A L S O - 8 9 -S U G G E S T E O THAT THE S P E C I F I C I T Y WAS ONLY FOR A R G I N I N E AND NOT A L L B A S I C R E S I D U E S S I N C E THE M O L E C U L E A L S O C O N T A I N S TWO L Y S I N E S . T H I S S P E C I F I C I T Y WAS C L E A R L Y C O M P A T A B L E WITH THE I N A C T I V A T I N G C H A R A C T E R I S T I C S OF THE E N Z Y M E S I N C E HUMAN C T C O N T A I N S NO A R G I N I N E AND ONLY P O R C I N E C T C O N T A I N S A R G I N I N E R E S I D U E S S U S C E P T A B L E TO A T T A C K . T H I S S P E C I F I C I T Y A L S O S U G G E S T E D THE P O S S I B I L I T Y THAT THE " C A L C I TON I N A S E " MIGHT BE THROMBIN WHICH I S KNOWN TO A T T A C K C E R T A I N T Y P E S OF P E P T I D E BONDS ON THE C A R B O X Y L S I D E OF A R G I N I N E ( 1 1 , 7 7 , 7 8 ) . T H I S P 0 S S I B I L I T Y WAS R E I N F O R C E D BY THE B E H A V I O R OF THE ENZYME D U R I N G THE P U R I F I C A T I O N S T A G E S WHERE AN A C T I V E M A T E R I A L WITH M O L E C U L A R WEIGHT AROUND 3 0 , 0 0 0 WAS R E L E A S E D FROM A MUCH L A R G E R P R E C U R S O R M O L E C U L E . THE E X P E R I M E N T S IN THE NEXT S E C T I O N WERE D E S I G N E D TO T E S T T H I S P O S S I B I L I T Y . F. COMPAR I SON OF " C A L C I TON I NASE 1 1 TO THROMB I N. S I N C E THE NATURAL S U B S T R A T E FOR THROMBIN I S F I B R I N O G E N AN E X P E R I M E N T WAS D E S I G N E D TO COMPARE THE A C T I O N OF T H R O M B I N ANO THE " C A L C I TON I N A S E " ON T H I S S U B S T R A T E . TWO 1 ML S A M P L E S C O N T A I N I N G 2 . 5 MG COHN F R A C T I O N 1 WHICH I S 5 0 TO 6 0 $ F I B R I N O G E N D I S S O L V E D IN 0 . 1 M AMMONIUM B I C A R B O N A T E WERE P R E P A R E D AND 4 0 JUL OF THE E N Z Y M E STOCK S O L U T I O N D E S C R I B E D ON P A G E 8 5 ADDED TO ONE S A M P L E . A F T E R I N C U B A T I O N FOR TWO HOURS AT ROOM T E M P E R A T U R E THERE WAS NO E V I D E N C E OF C L O T T I N G IN E I T H E R S A M P L E AND 1 0 U N I T S OF AN IMPURE B O V I N E THROMBIN P R E P A R A T I O N FROM U P J O H N C O . K A L A M A Z O O , M I C H I G A N WAS ADDED - 9 0 -T0 THE CONTROL S A M P L E R E S U L T I N G I N A R A P I D CLOT FORMATJ ON IN L E S S THAN 1 0 M I N . A F T E R THREE HOURS TOTAL I N C U B A T I O N THERE WAS NO S I G N OF CLOT F O R M A T I O N IN THE " C A L C I TON I N A S E " S A M P L E ANO 2 5 0 /(L OF POOL M S O L U T I O N WAS ADDED. A F T E R A F U R T H E R TWO HOURS OF I N C U B A T I O N THERE WAS S T I L L NO CLOT AND 1 0 U N I T S OF THE B O V I N E THROMBIN WAS ADDED. TH I S C A U S E D A R A P I D C L O T T I N G S I M I L A R TO THAT S E E N IN THE CONTROL. S U B S E Q U E N T T E S T S OF THE POOL M S O L U T I O N C O N F I R M E D THAT IT S T I L L R E T A I N E D I T S P O R C I N E C T I N A C T I V A T I N G P R O P E R T I E S . T H I S F A I L U R E OF A C T I V E " C A L C I T O N I N A S E " TO C A U S E C L O T T I N G I N D I C A T E D THAT IT C O N T A I N E D NO S I G N I F I C A N T THROMBIN A C T I V I T Y . THE FACT THAT THE F I B R I N O G E N S O L U T I O N WAS S T I L L C L O T T E D BY THROMBIN A F T E R A P R I O R 5 HOUR I N C U B A T I O N WITH THE ENZYME S U G G E S T E D FURTHER THAT THE " C A L C I TON I N A S E " HAD NO P L A S M I N A C T I V I T Y S I N C E P R E - I N C U B A T I ON OF F I B R I N O G E N WITH P L A S M I N R E T A R D S OR P R E V E N T S CLOT F O R M A T I O N BY THROMBIN ( 1 1 ) . IT I S T H E R E F O R E U N L I K E L Y THAT T H E " C A L C I T 0 N I N A S E " I N POOL M C O R R E S -PONDED TO E I T H E R OF THESE E N Z Y M E S . G. GENERAL D I S C U S S I O N . IN THE I N T R O D U C T I O N TO T H I S C H A P T E R THE R E S P O N S E S OF S E V E R A L S P E C I E S OF MAMMALS TO C A L C I T O N I N S FROM A V A R I E T Y OF OTHER S P E C I E S WERE D I S C U S S E D AND E V I O E N C E WAS P R E S E N T E D THAT THE V A R I A T I O N S IN THE R E S P O N S E TO T H E S E S T R U C T U R A L L Y D I F F E R E N T C A L C I T O N I N S WERE DUE AT L E A S T IN PART TO D I F F E R -E N C E S IN THE RATE AT WHICH THEY WERE I N A C T I V A T E D . FROM A P H Y S I O L O G I C A L S T A N D P O I N T T H E S E O B S E R V A T I O N S DO NOT A P P E A R , . ' - 9 1 -PARTICULARLY VALUABLE SINCE THE POSSIBILITY OF A MAN SECRETING SALMON C T IS QUITE REMOTE, BUT THEY MAY BE USEFUL IN UNDERSTANDING THE ROLE OF INACTIVATION IN THE OVERALL CONTROL SYSTEM. A DETAILED KNOWLEDGE OF THE ROUTES AND MECHANISMS INVOLVED IN THE INACTIVATION OF THE HORMONE IS ALSO IMPORTANT, IF C T IS TO BE USED THERAPEUTICALLY IN MAN. IN STUDIES ON A PATIENT WITH PAGET'S DISEASE NEER ET AL (79) DESCRIBED THE USE OF OVER 2 0 0 MG OF PORCINE C T DURING AN 11 WEEK COURSE OF TREATMENT. IN STUDIES ON A PATIENT WITH A PARATHYROID CARCINOMA IN THE SAME WORK THE^  AUTHORS USE 8 0 MG OF PORCINE C T TO ACHIEVE A 1.5 MG$ DROP IN THE PLASMA CALCIUM LEVEL. AN INFUSION OF 0 . 6 MG OF SALMON C T IN THE SAME PATIENT PRODUCED A DROP IN PLASMA CALCIUM OF NEARLY 2 . 5 MG$. BECAUSE, IN TERMS OF M R C U, THIS DOSE WAS ONLY 2 0 $ OF THE PORCINE C T USED, SALMON C T PROMISES TO BE A MORE EFFICIENT MEANS OF CONTROLLING BONE RESORPTION THAN PORCINE C T . THE REASONS FOR SUCH VARIATIONS IN THE MAGNITUOE OF RESPONSE TO THE DIFFERENT CALCITONINS MUST ULTIMATELY DEPEND ON THE STRUCTURAL DIFFERENCES BETWEEN THE HORMONE MOLECULES. THE EXPERIMENTAL WORK IN THIS CHAPTER DESCRIBES AN ENZYME FROM HUMAN PLASMA WHICH RAPIDLY INACTIVATES PORCINE C T BY SPLITTING TWO PEPTIDE BONDS WHICH ARE ABSENT IN THE SALMON MOLECULE. THIS INDICATES A STRUCTURAL DIFFERENCE WHICH CONTROLS THE INACTIVATION RATE OF THE TWO HORMONES, AND PROVIDES A MECHANISM WHICH COULD EXPLAIN WHY SALMON'" C T HAS A MUCH GREATER POTENCY IN MAN THAN -92-PORCINE C T . THE CONCLUSION ONE WOULD LIKE TO DRAW FROM THIS IS THAT CALCITONINS WHICH DO NOT HAVE LABILE ARGININE BONDS ARE MORE STABLE ANO THEREFORE HAVE A GREATER B I 0 -LOBICAL ACTIVITY. UNFORTUNATELY THE PROBLEM IS MUCH MORE COMPLEX THAN THIS FOR HUMAN C T WHICH CONTAINS NO ARGININE HAS A LOWER B I OLOGICAL ACT I V I TY THAN PORCINE C T IN BOTH THE STANOARD BIOASSAY AND THE AREA RESPONSE ASSAY. THE SITUATION IS SIMILAR FOR BOVINE C T WHICH HAS A LYSINE IN T H E POSITION O F T H E M O S T L A B I L E A R G I N I N E B O N D IN T H E P O R C I N E MOLECULE AND HAS THE SAME ACTIVITY IN THE STANDARO BIOASSAY. FIGURE 34 SHOWS THAT THE AREA RESPONSE TO LOW DOSES OF BOVINE C T IS ALSO SLIGHTLY LOWER THAN PORCINE, BUT THE SLOPE OF THE LINE IS GREATER. THUS THIS PLASMA ENZYME WHICH SELECTIVELY INACTIVATES PORCINE.CT CAN ONLY BE CONSIDERED AS AN INDICATION OF THE TYPE OF PHENOMENA WHICH MAY BE INVOLVED IN DETERMINING THE RELATIONSHIP BETWEEN THE STRUCTURE AND FUNCTION OF CALCITONINS AND IS NOT A COMPLETE ANSWER. THIS ENZYME DOES NOT ADEQUATELY ACCOUNT FOR INACTIVATION WITHIN THE PLASMA MUCH LESS THE OVERALL RELATIONSHIP BETWEEN STRUCTURE AND FUNCTION. A COMPARISON OF THE INACTIVATION RATES OF SALMON C T AND HUMAN C T ( F l G U R E 35 (A)) NEITHER OF WHICH WERE INACTIVATED BY THIS ENZYME INDICATES THAT OTHER MODES OF INACTIVATION MUST EXIST FOR THE HUMAN HORMONE. IN THE INTRODUCTION TO THIS CHAPTER A NUMBER OF ENZYMES WERE MENTIONED WHICH SEEMED LIKELY TO DIFFERENTIATE 8ETWEEN THE MAMMALIAN AND SALMON CALCITONINS AND ANY ONE OF -93-THESE OR P E R H A P S MANY OTHER E N Z Y M E S COULD BE I N V O L V E D . THE ENZYME WHICH WAS S T U D I E D IN T H I S C H A P T E R DOES NOT A P P E A R TO BE ANY OF THE E N Z Y M E S S U G G E S T E D E A R L I E R . THE S P E C I F I C I T Y SUG-G E S T S A P L A S M IN OR K A L L I K R E IN T Y P E E N Z Y M E , BUT I T S MO L E C U L A R S I Z E AND CHARGE C H A R A C T E R I S T I C S DO NOT C O I N C I D E WITH THOSE OF THE B E S T S T U D I E D E X A M P L E S OF T H E S E E N Z Y M E S ( 1 0 8 , 1 3 5 ) , HOWEVER, THE " C A L C I TON I N A S E " MAY B E L O N G TO ONE OF THESE E N Z Y M E F A M I L I E S , IN ANY CASE I T HAS THUS FAR PROVED I M P O S S I B L E TO R E L A T E THE " C A L C I TON I N A S E " TO ANY OTHER WELL C H A R A C G E R I ZED P L A S M A E N Z Y M E , THE O B S E R V A T I O N THAT AT L E A S T TWO OTHER COHN F R A C T I O N S C O N T A I N E D P O T E N T C T I N A C T I V A T I N G F A C T O R S S U G G E S T S THAT THERE MAY BE MANY OTHER " C A L C I TON I N A S E S " WHICH MAY OR MAY NOT CORRESPOND TO KNOWN P L A S M A E N Z Y M E S . E V E N A C O M P L E T E C H A R A C T E R I Z A T I O N OF A L L THE P L A S M A " C A L C I TORI I N A S E S " AND E V A L U A T I O N OF T H E I R R E L A T I V E E F F E C T S W I L L NOT F U L L Y E X P L A I N HOW S T R U C T U R E I N F L U E N C E S THE COURSE OF THE R E S P O N S E S I N C E THERE ARE MANY OTHER F A C T O R S I N V O L V E D . I F , FOR E X A M P L E , A " S U P E R " C T WERE D E S I G N E D FOR T H E R A P Y WHICH WAS R E S I S T A N T TO A T T A C K BY A L L P L A S M A E N Z Y M E S , I T WOULD S T I L L 8E C L E A R E D FROM THE P L A S M A AND A V A R I E T Y OF FA C T O R S WOULD I N F L U E N C E THE RATE OF C L E A R A N C E . L O O K I N G AT THE THREE COMPARTMENT MOOEL D E S C R I B E D E A R L I E R I T I S E V I D E N T THAT ANY D E C R E A S E IN THE RATE OF I N A C T I V A T I O N I N THE P L A S M A MUST R E S U L T I N AN I N C R E A S E IN THE A C T I V E C T MOVING INTO COMPARTMENT 2 AND IN THE C T E L I M I N A T E D BY RENAL E X C R E T I O N . THE M A G N I T U D E OF T H I S I N C R E A S E WOULD BE D E P E N D E N T ON THE DEGREE OF B I N D I N G OF THE C T TO P L A S M A P R O T E I N S . I F THE - 9 4 -B I N D I N G I S VERY STRONG MOST OF THE CT W I L L R E M A I N IN COMPARTMENT 3 WHERE I T I S U N A V A I L A B L E FOR E X C R E T I O N ANO CANNOT MOVE INTO COMPARTMENT 2. THUS IT M I G H T L A S T A LONG T I M E AND BE E S S E N T I A L L Y U S E L E S S S I N C E THE R E S P O N S E OCCURS ONLY A F T E R THE HORMONE R E A C H E S THE TARGET ORGAN I N COMPARTMENT 2. IF ON THE OTHER HAND B I N D I N G I S WEAK » THE CT W I L L MOVE R A P I D L Y INTO COMPARTMENT 2 AND A F A I R L Y L A R G E P R O P O R T I O N WILL A P P E A R IN THE U R I N E . HOW T H I S WOULD E F F E C T THE R E S P O N S E D E P E N D S ON WHAT H A P P E N S IN COMPARTMENT 2. I F , AS I S S U G G E S T E D IN THE WORK OF DE L U I S E E_T _AL (36), A L A R G E P E R C E N T A G E OF THE CT I S T A K E N UP BY THE L I V E R THE D U R A T I O N OF THE R E S P O N S E W I L L DEPEND ON WHETHER IT I S I N A C T I V A T E D ANO ON WHETHER THE U P T A K E I S R E V E R S I B L E . DE L U I S E ET AL (37) A L S O SHOWED THAT C A L C I T O N I N S R E S I S T A N T TO P L A S M A I N A C T I V A T I O N ARE R E S I S T A N T TO L I V E R I N A C T I V A T I O N . IF UNDER THESE C O N D I T I O N S THE L I V E R U P T A K E WERE R E V E R S I B L E CT COULD SLOWLY RETURN TO THE P L A S M A AND BE E X C R E T E O OR R E A C H THE TARGET ORGAN AND PROLONG THE R E S P O N S E . THE F I N A L FACTOR I N F L U E N C I N G THE E F F E C T I V E N E S S OF T H I S " S U P E R 1 1 CT I S I T S A C T I O N ON THE TARGET ORGAN, AND SOME OF THE S T R U C T U R A L C H A R A C T E R I S T I C S WHICH M I G H T E F F E C T T H I S A C T I O N WERE D I S C U S S E D IN THE P R E C E E D I N G C H A P T E R . E V A L U A T I N G T H E S E S T R U C T U R A L C H A R A C T E R I S T I C S R E Q U I R E S E L I M I N A T I O N OF I NACT I VAT I ON AS AN I N F L U E N C E BY USTNG E I T H E R M O L E C U L E S WHICH ARE NOT I N A C T I V A T E D OR A S Y S T E M WHICH HAS NO I N A C T I -V A T O R S . E V E N IN J_N V I T R O S T U D I E S ON BONE E X P L A N T S ( 1 0 5 ) I N A C T I V A T I O N I S AN IMPORTANT FACTOR S I NCE' R E P E A TED S M A L L -95-DOSES OF PORCINE C T WERE MORE EFFECTIVE IN SUPPRESSING ACTIVE RESORPTION THAN AN EQUIVALENT SINGLE DOSE. FURTHER MORE STABLE ULTIMOBRANCHIAL CALCITONINS HAD A MUCH GREATER AND MORE PROLONGED EFFECT. WHILE THIS INACTIVATION MAY HAVE RESULTED FROM THE SMALL PERCENTAGE OF SERUM USED IN THEIR INCUBATION MEDIUM, LYSOZOMAL ENZYMES RELEASED BY OSTEOCLASTIC CELLS WOULD UNDOUBTEDLY BE CAPABLE OF INACTI-VATING THE MORE LABILE CALCITONINS. THIS TYPE OF INACTI-VATION, WHICH WAS NOT DISCUSSED EARLIER, MAY BE CRITICAL IN DETERMINING THE EFFECTIVENESS OF A C T AFTER IT HAS REACHED THE RECEPTOR. THE PRESENCE OF SUCH INACTIVATORS IN THE TAR-GET ORGANS OF OTHER HORMONES HAS BEEN DESCRIBED (55). IT THUS MAY PROVE EXTREMELY DIFF|CULT T 0 SEPARATE ACTION ON THE RECEPTOR FROM INACTIVATION AS FACTORS WHICH INFLUENCE THE POTENCY OF A GIVEN MOLECULE, ANO MODIFICATIONS WHICH RESULT IN A DECRESED STABILITY CAN BE EXPECTED TO CAUSE PROFOUND CHANGES IN THE POTENCY OF THE MOLECULE WHETHER OR NOT THEY INFLUENCE ACTION AT THE RECEPTOR SITE. 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AND WANG, K. S E P A R A T I O N OF D A N S Y L - A M I N O A C I D BY P O L Y A M I D E L A Y E R CHROMATOGRAPHY. B I O C H I M . B I O P H Y S . A C T A , J _ 3 3 , 3 6 6 ( 1 9 6 7 ) . 1 3 3 . YAMADA, S. AND I T A N O , H.A. P H E N A N T H R E N Q U I N O N E AS AN A N A L Y T I C A L REAGENT FOR A R G I N I N E AND OTHER MONOSUB-S T I T U T E D G U A N I D I N E S . B I O C H I M . B I O P H Y S . A C T A , 1 5 0 , 1 3 4 . A M B L E R , R.P. THE AMINO A C I D S E Q U E N C E OF PSEUDOMONAS CYTOCHROME C - 5 5 1 . B I O C H E M . J . 89, 3 4 9 ( 1 9 6 3 ) . 1 3 5 . C O L E M A N , R.W., M A T T L E R , L. AND S H E R R Y , S. S T U D I E S ON THE P R E K A L L I K R E IN ( K A L L I KE IN0GEN ) - K A L L I K R E E N ENZYME S Y S T E M OF HUMAN P L A S M A I . I S O L A T I O N ANO P U R I F I C A T I O N OF P L A S M A K A L L I K R E E N S . J . C L I N . I N V E S T . 4 8 , 1 1 ( 1 9 6 9 ) . A P P E N D I C E S A . STAT I S T I C A L METHODS FOR C A L C U L A T I O N OF LOG D O S E - R E S P O N S E  C U R V E S BY COMPUTER* C A L C U L A T I O N OF R E G R E S S I O N L I N E FOR A G I V E N B I O A S S A Y R E Q U I R E D THE F O L L O W I N G DATA: 1. THE DOSE OF C T G I V E N IN MG, 2. THE WEIGHT OF THE RAT I N J E C T E D , 3. THE P L A S M A C A L C I U M L E V E L MEASURED IN MG $ I N THAT RAT A F T E R 1 HOUR. IN P R A C T I C E THE DOSE G I V E N WAS C A L C U L A T E D ON THE B A S I S OF A 0.3 ML I N J E C T I O N FOR AN 8 0 G RAT AND THE DOSE WAS C O R R E C T E D FOR RAT WEIGHT BY A D J U S T I N G I N J E C T I O N VOLUME. T H I S A V O I D E D THE P R O B L E M OF R E C O R D I N G RAT WEIGHT, BUT THE PROGRAM WAS D E S I G N E D TO C O M P E N S A T E FOR V A R I A T I O N S I N WEIGHT I F A CONSTANT DOSE WAS USED FOR E A C H A N I M A L . E A C H S E T QF S A M P L E DAATA MUST A L S O I N C L U D E DATA FOR R A T S I N J E C T E D WITH V E H I C L E ONLY AS A CONTROL. FROM T H I S DATA C A L C U L A T I O N S WERE C A R R I E D OUT AS SHOWN IN THE P R I N T O U T S H E E T S I N C L U D E D AT THE END OF THE A P P E N D I X . THE MEAN V E H I C L E R E S P O N S E WAS C A L C U L A T E D F I R S T ( V R E S A V ) AS THE SUM OF THE P L A S M A C A L C I U M S OF V E H I C L E I N J E C T E D R A T S D I V I D E D BY THE NUMBER OF V E H I C L E RATS (DUM/FNV) I N L I N E 18. THE R E S P O N S E ( R E S P ) WAS THEN C A L C U L A T E D AS THE D I F F E R E N C E BETWEEN V R E S A V AND THE MEASUREO P L A S M A C A L C I U M ( C A ) FOR E A C H S A M P L E I N J E C T E D R A T . THE LOG DOSE ( N D O S E ) WAS THEN ' i C A L C U L A T E D AS THE L O G J Q OF I N J E C T E D DOSE D I V I D E D BY THE RAT WEIGHT ( R D O S E / W E I G H T ) . T H I S DATA WAS THEN P L A C E O I N THE - 1 0 7 --108-EXECUT ION ARRAY ANO THE TOTALS CALCULATED (TRES AND TND). THE MEAN VALUES (MRES AND MND) WERE DETERMINED BY D IV I S ION BY THE NUMBER OF EXPERIMENTAL SAMPLES INCLUDED (FNE) AND WERE PLACED IN THE ARRAY. FROM THESE VALUES IT WAS POSS IBLE TO CALCULATE DEVI AT IONS IN DOSES (X) AS THE D I FFERENCE BETWEEN THE LOG OF EACH DOSE (NDOSE) AND THE MEAN (MND) AND THE DEV IAT IONS IN RESPONSE (Y) AS THE D IFFERENCE BETWEEN THE RESPONSE (RESP) AND THE MEAN (MRES). TH E S E WERE ALSO PLACED IN THE ARRAY TOGETHER WITH THE CALCULATED SQUARES OF X AND Y. THE XSQ AND YSQ TERMS WERE THEN SUMMED AND D ISPLAYED BELOW THE COLUMNS IN THE ARRAY. S IM ILAR PROCEDURES WERE THEN CARRIED OUT FOR THE PRODUCT XY. THE VALUES OBTAINED TO THIS POINT ALLOWED CALCULAT ION OF DATA NECESSARY FOR DETERMINATION OF THE POINT E S T I M A T E S OF THE PARAMETERS OF THE REGRESS ION POPULAT IONS AND OF THEIR INTERVAL EST IMATES BY THE METHODS DESCRIBED BY SNEDECOR (115). THE SAMPLE REGRESS ION C O E F F I C I E N T ( B ) OF Y ON X FOR THE EQUATION Y • BX IS EQUAL TO THE SUM OF THE XY TERMS D IV IDED BY THE SUM OF THE XSQ TERMS ( B = SXY/SUM XSQ) AS SHOWN IN L I N E 49. THE EST IMATED RESPONSES , YC, WERE THEN THEN CALCULATED IN L I N E 57 BY THE FORMULA YC = BX + MRES, WHERE B IS THE SAMPLE REGRESSION C O E F F I C E N T , X IS THE D E V I -ATION OF A GIVEN DOSE FROM THE MEAN DOSE, AND MRES IS MEAN RESPONSE. THE EST IMATED RESPONSES FOR THE GIVEN DOSES WERE THCN FLACED IN THE ARRAY AND USED TO PLOT BOTH THE COMPUTER GRAPH AND THE F IGURES IN THE T E X T . MOST OF THE REMAINING CALCULAT IONS IN THE PROGRAM WERE -109-AIMED AT A CALCULATION OF "T" TO TEST THE HYPOTHESIS THAT THE REGRESSION COEFFICENT FOR THE POPULATION IS ZERO. THIS WAS REALLY OF LITTLE VALUE SINCE IT WOULD IMPLY THAT THERE WAS NO RESPONSE OR THAT ALL RESPONSES WERE EQUIVALENT, AND IT IS UNLIKELY THAT SUCH DATA WOULD EVER BE TESTED. IT IS ALSO POSSIBLE TO USE THE TEST TO COMPARE THE SAMPLE POPULATION REGRESSION COEFFICIENT TO ANOTHER REGRESSION COEFFICIENT (E.G. THAT OF THE STANDARD CURVE), BUT THIS WAS NOT DONE SINCE THE VARIATIONS IN THE SOURCES OF MATERIAL BEING TESTED MADE SUCH COMPARISONS OF LITTLE VALUE. THESE CALCU-LATIONS ALSO SUPPLIED PART OF THE OATA USED TO DETERMINE CONFIDENCE LIMITS OF ESTIMATED RESPONSES WHICH WERE THEN USED TO CALCULATE THE ACTIVITY RANGES IN TABLE 1. S l N C E ( B - g ) / S B FOLLOWS THE T-0 I STRI BUTI ON WHEN g IS THE POPULATION REGRESSION COEFFICIENT AND SB IS THE STANDARD DEVIATION OF THE SAMPLE REGRESSION COEFFICIENT, IN THE NULL HYPOTHESIS WHERE 3lIS ZERO T IS EQUAL TO B/SB. SB IS THE SQUARE ROOT OF VARIANCE OF THE SAMPLE REGRESSION COEFFICIENT WHICH"IS EQUAL TO SYXSQ/SUMXSQ. SUMXSQ i s IN THE ARRAY AND SYXSQ IS THE MEAN SQUARE DEVIATION FROM REGRESSION AND IS EQUAL TO THE SUM OF THE SQUARES OF THE DEVIATIONS FROM THE REGRESSION ( R E S P - Y C ) OR SDYXSQ DIVIDED BY THE DEGREES OF FREEDOM ( S D Y X S Q / ( N - 2 ) ) . THE DEGREES OF FREEDOM ARE EQUAL TO THE NUMBER OF SAMPLES N MINUS 2 SINCE TWO AVERAGES WERE USED IN CALCULATING THE DEVIATIONS. THE VALUE OF SDYXSQ MAY BE CALCULATED FROM THE AVAILABLE DATA BY THE FORMULA SDYXSQ = SUMYSQ - SXYSQ/SUMXSQ WHERE SXYSQ IS EQUAL TO FIGURE 4 2 . COMPUTER PRINT OUT AND PLOT FROM LINEAR REGRESSION PROGRAM (NEXT 4 PAGES) f FORTRAN IV G COMPILER MA IN 01-15-70 15:01:51 PAGF 0001 C CASS1 MAIN PROGRAMME C PROGRAMME TC COMPUTE THE LINEAR REGRESSION OF LOGCCSE/RESPONSE C PROGRAMMER PETER GRAYSTDNE PHYSIOLGGY DEPT. UBC FEBRUARY 1968 s C MCDIFIEU RY RON CDCR JANUARY 1970 / / C REFERENCE CHAPTER 6. SNEDECOR , STA T I ST I C AL METHODS t I CWA STATE COL PRESS \ C C NV IS NUMBER OF VEHICLE SAMPLES. NT IS NUMBER OF TOTAL SAMPLES INC NV r . • • - - •• C COLS 79,80 CF RUN IDENTIFIER CARD SETS THE X AXIS CRIGIN 0CC1 CALL PLCTS 0002 REAL NOOSE,MNC.MKfS 0003 INTEGER DATE, STD COCA 0 I MENS I CN P.DOSE (100 I, HEIGHT 1100 ),CA( 100), TITLE 1 15 I.NDOSEI IOC) 0005 DIMENSION XI 100),Y<100),XSO(100),YSQI100),XY(1001,DYX(100I.DYXSQI1 COO),YC(100),RESP(100).DEVI100I.DEVSCI100) 0006 1 READ(5,10> DATE,NV,NT,TITLE, STO 0C07 10 F0RMAT(1X,I6,1X,I3,1X,I3.1X,15A4,2X,I2( ccoa IF(OATE.LT.0IGC TC 999 occs WRI TE(6,1010ATE.NV.NT,TITLE 0010 READI5.20) (ROOSE(I).HEIGHT!I),CA(11,1 = 1,NT) 001 1 20 FORMAT!1X.F8.3,1X.F8.3,1X.F6.2I 0012 IF(NV.EO.OIGO TO 25 0013 FNV=NV 0C14 DUM=0. 001 5 00 30 1 = 1,NV • . . . 0016 DUM = OUMt-CA(I) CC17 30 CONTINUE 0018 VPESAV=OUM/FNV 0019 GO TO 35 0C2C 25 VRESAV=0. 002 1 35 NVP = NV+1 : 0022 TPES=0. 0023 TNO=0. 0C24 no 40 l=NVP,NT 0025 NCCSE(II = AL0G10IR0OSem/HEIGHT(II*1000.) 0026 RESP(I)=VRESAV-CA(I) C THE ARRAYS NOOSE AND RESP NOH CONTAIN THE LOG DOSE AND RESPONSE DATA 0C27 TNO=TND + NOOSE ( I ) 0C28 TRES = TPES + RESP( I) 0029 40 CONTINUE 003C FNE=NT-NV 0C31 MNC=T NO/FNE 0032 MRES=TRES/FNE C MND CONTAINS MEAN CF LOGOCSE AND MRES CONTAINS MEAN OF RESPONSE 0033 SUMXSO=0. 0034 SUI-YSC-O. 0035 SUMXY=0. 0036 DO 50 I=NVP,NT 0037 XI I)=NOOSE(Il-MND , . 0038 XSOII)=X(I)*X(I) 0039 Y(I)=RESP(I l-MRES 0040 SUMXSQ=SUMXSC*XSQU 1 004 1 XY(I 1=XI I 1»Y( I 1 0042 YSC(I) = YI I l*Y( I ) 0043 SUMYSQ=SUMYSQ+YSQ(I) J o FORTRAN IV G COMPILER MAIN 01-15-70 15:01:51 PAGE 0002 0044 SUM XY=SLMXY + XYt I) 0C45 SXYSO=SUMXY*SUMXY C SOYXSQ IS SUM OF RESP<Il-YCI I) 0C46 ' 0047 RMSQ=SORT(SUMXSQ) oc48 50 CONTINUE C SAMPLE REGRESSION COEFFICIENT OF Y ON X IS B (Y=BX) ,. .. . 00*9 B=SUMXY/SUMXSO CC5C DCF =FNE-2. C MEAN SCIIARE OEVIATICN FROM REGRESSION IS SYXSQ 0051 SYXSC=50YXS0/C0F . C SAMPLE STANDARD OEVI AT ICN FROM REGRESSION IS SYX 0052 SYX=SORTISYXSQ) C SAMPLE STANDARD DEVIATION OF THE REGRESSION COEFFICIENT IS SB 0053 SruSYX/RMSQ 0C54 T=B/SR 0055 SLAM=SQRTISUMXY/FNEl/B 0056 DO 7C I=NVP,NT 0057 YC( I )=B*X( I KMRES 0058 70 C YC CONTINUE CONTAINS ESTIMATED RESPONSES 0C59 WRITEI6.100) 0C6C 100 FGRPATI//3X,109H RAWDOSE WEIGHT CA RDG LOGDCSE RESP C LD DVN RES OVN LD SC RES SQ LD*RES EST RESP) 0061 WRITE(6,110)1RC0SE!I I.WEIGHT!I),CA(I).ND0S6(Il,RESPU),X<II,Yll),X. C S Q I I ) , Y S Q ( I ) , X Y ( I ) , Y C ( I ) , I = NVP,NTI ..... 0062 110 FORMAT( 1X.11F10.4) 0063 WP. ITE (6, 120 ) 0C64 120 F0PMAT(1X,3HSUM) 0065 WRITE(6.130)TNO.TRES,SUMXSQ,SUMYSO,SUMXY 0066 130 FCPMAT(31X,2F10.4,2CX,3F10.4/I 0067 WRITEI6,140) 0068 140 FORMAT!IX,4HMEAN) 0069 WRITE16,150)MND,MKES 0070 150 FORMAT! 31X.2F1C.4//) 0071 WRITE 16,160)B 0072 160 FORMAT!IX,55HSAMPLE REGRESSION COEFFICIENT OF RESPONSE ON LOGDOSE CIS.F10.4//) . 0073 WPITF(6,170ISYXSQ 0074 170 FORMAT!IX,40HMEAN SQUARE DEVIATION FRCM REGRESSION IS, F10.4//I 0C75 WR ITEI6.180ISYX 0C76 180 FORMAT!IX,44HSAMPLE STANDARD DEVIATION FROM REGRESSION IS.F10.4//I 0077 WRITE(6,190)SB 0078 190 FORMAT 1IX,5BHSAMPLE STANOARO DEVIATION CF THE REGRESS1CN COEFFICIE C NT IS.F10.4//I 0C79 WP.ITE(6,2C0IT,DCF 0080 200 FORMAT!IX,13H.T,TEST, T IS,F10.4,13H DOF (N-2) IS.F10.4//I 0081 WP.ITEI6.220) SLAM 0082 220 FORMAT!IX,10H LAMBDA IS.F8.4//) 0083 CALL REGPL!NDCSE,RESP,NVP.NT,TITLE,DATE,YC,B,STD,T,SLAM 1 0081 WP.ITE(6,55) 0085 55 FORMAT(1H1) 0C86 GO TO 1 0087 999 CONTINUE 0088 CALL PLOTND EXECUTION TERMINATED © tRUN -LOAC»+«NFL+»BPLOT 9=-P EXECUTION BEGINS 171167 6 28 PORC RAWCOSE 0.0060 WE IGHT 80.0000 RAT CA ROG B.4000 4fcK KUMAR LOGOOSE - 1 . 1249 RESP 1.8333 LO OVN -0.2133 RES DVN 0.0523 0.1023 -C.94 77 -C.7477 -1.1477 -1.0477 -0.0477 LO SO 0.0455 0.C455 0 .0455 0.0455 0.0455 0.0455 0.0078-; RES SO U.U027 O.C105 0.8982 0.5591 1.3173 1.0977 O.C023 LD*.RES -0.0112 EST RESP 1.2116 0.C060 0.C06C 0.C060-0. CC60-0.0060 o.cceo 80.0000 80.CO0O 80.0000 80.0000 80.0000 80.0000 3500 4000 2000 6000 5000 8.5000 -1, -1, -1, -1, -1. -1. 1249 1249 1249 1249 1249 ooco 1.8833 0.8333 1.0333 0.6333 0.7333 1.7333 -0.2133 -0.2133 -0.2133 -0.2133 -0.2133 -0.CR84 -0.0218 0.2022 0.1595 0.2449 0.2235 0.C042 . 1.2116 1.2116 1.2116 1.2116 1.2116 1.5451 1.7353 1.9333 1.633,3 1. 2333 1 .2333 1.9333 -0.0884 -0.C884 -0.C884 0.C877 C.C877 0.0877 -0.0477 0.1523 -0.1477 -0.5477 -0.5477 0.1523 O.COfiO -0.0080-o.coeo-0.0120• 0.C12C-0.C120-80.00CO 80.0000 80.0000 80.0000 80.0000 80.0000 8.5000 8.3000 8.6000 9.0000 9.0000 8.3000 0000 0000 0000 8239 8239 8239 0 .00 IH • 0.0078\ 0.0078) 0.0077 " 0.C077 0.0077 0.0077 0.0077 0.0077 0.0341 0.0341 0.0341 0.0023 0.0232 0.0218 "0.3000 0.3000 0..0232 0.0232 0.3050 0.3050 1.3277 0.C636 0.063b 0.0U42 -0.0135 0.0131 -0.0480 -0.0480 0.0134 T4~yr~ 5451 5451 0151 0151 0151 0.0120-0.0120-0.0120-0.0150-0.0150. 0.0150-80.0000 BO.0000 80.0000 BO.0000 80.0000 BO.OOCO 8.3000 7.9000 7.9000 7.3000 8.20C0 8.2000 -0.8239 -0.B239 -0.B239 -0.7270 -0.7270 -0.7270 1.9333 2.3333 2.3333 2.9333 2.0333 2.0333 0.C877 0.0877 0.0877 0.1846 0. 1846 0.1846 0.152 3 0.5523 0.5523 1.1523 0.2523 0.2523 U.0134 0.0484 0.0484 0.2127 0.0466 0.0466 2.0151 2.0151 2.C151 2.2738 2.273* 2.273B 0.4523 0.6523 0.7523 0.0341 0.0341 0.0341 0.5549 0.204b 0.4255 0.5659 7.8424 0.083b 0.1204 0.1389 1.4813 2.2733 2.2738 2.2738 0.C150, 0.0150-0.0150 80.0000 80.0000 80.0000 8.0000 7.8000 7.7000 -0.7270 -0.7270 -0.7270 2.2333 2.4333 2.5333 6.1846 0.1846 0.1846 S U M -20.0550 39.1831 MEAN - 0 . 9 1 1 6 1.7811 SAMPLE REGRESSION COEFFICIENT OF RESPONSE CN LOGDOSE IS 2 .6692 MEAN SCUARE DEVIATION FROM REGRESSION IS 0 . 1 9 4 4 SAMPLE STANOARO OEVIATICN FROM REGRESSION IS 0 . 4 4 0 9 SAMPLE STANOARO DEVIATION OF THE REGRESSION COEFFICIENT IS 0.5919 .T.TEST, T IS 4.5095 DOF (N-21 IS 2C.0000 LAMBOA IS 0 . 0 9 7 2 ; i ' -JJ | i i l l ! M ; i 4 4 n~ • t j i • 1 ; • : i i . : " ••'"; i 111 - i j j - r ! T u . ! j ' 1 1 ; . : J : i ± + r r _ - • i r -p I -T' X ! i i 4- _:_L.1..L i" T i : r _ 1 | ) T ~pr " ; ' . ~ i ! 1 ! 1 • ! ' < i ; T i"i~r - : i t t < t I f • ! ,~'~\ " i ; i M i -. ; • ' ' * I • ; 1 ' } i t 1 t : - 4+ 1 1 i I | i • i i i i i_ ! t ; • i i ' 1 1 M l ! :ir i i ! 1 T j i • • ; '-i-i ' 1 1 ; i 1 1 1 i l l ! ! ! 1-4 "1~n?.i~ X i ' 1 | 1 i M . i . i j i ' i 1 1 | | • M < X p j in. XJl i 1 _ 1 * i ' : i i \ .' i " 1 1 1 , M ; < 1 1 . . . ' r i • ' i i i • i . i • i 41-' I I I i I I I . 1 i i • t i J i t ' i ! i i i ' 1 1 i 1 ! 1 I M I : , : a l "i" I I ' M i > i i | I 1 1 1 t 1 1 ! i r r r ' M 1 J j . ; ;.. I I I ! _ i i i T l - •  I i ' i '. 1 MM" ; i • 1 i i i i 1 I ' ' : i T!3T 44 _ i ! - 1 M i 1 1 1 1 i M M 1 1 1 i : ' i - 1 " 1 ! I 1 l -n • i ' • • i • I ' l l i 1 i . i : : M M i i i , 4- ; l,i , M M i • - , i ' 1 l _ ; - l - t - t -i i i • i : 1 1 ! I ' l l M i ' ~M.~M' j 4+ r i i i | i I : i i i T - f - h T i i • i ! • I t . ' j . _1_ 1 i_i ' i i i . ' T ' T i " 1 | 1 1 1 i i !. ! . - t i i ' - ' < ; "!~ 1 1 j • i M ' 1 ! 1 | TJ > ' i i i .• . i • : 1 : i • . i [ ' 1 1 i i i ; 1 cn • i . ! i f 1 i l l ' 1 f 1 1 i i 1 1 ; i ; ! | 1 1 ; i | ! M i - i i i i 1 ' M i l ' i i i i \ . • • • i i | 1 1 1 ! 1 . , 1 1 1 j i i { ! , i ; i i i i -: • •; 1 i » 1 ! 1 1 1 . ' I • ; 1 i i , t . r _ i - i i — n •. i . ! ' it M • i '. 1 l : . i -• t • . - i , •i Ll. i. I.I ! 1 M M . 1 • 1 • 1 M • 1 M J l > . i i I'M M M 1 i 1 i 1 1 ' * •1 i , : >_ .:. : M ! 1 | ! ! I i I _ • i 1 • 1 : I ' M in ru l ! i ' i 1 ! U ~ L T i . 1 1 - . . i . • . i : i M T M i 1 1 1 . i M * , i • . . . i i L-l-l 1 ..1. , i . 1 i ! I ' l l M j i • , i t I ' l l 1 " i t ! ! M I I t ' ' ! m _ : ; i . ; i ! ; • i i I 1 ! 1 i i • 1 1 : i t 1 : M j ! L) 1. I | ! • • 1 1 I 1 1 , M i i i ; i i - ; ! i , i i ' : M 1 i 1 : i i i • 1 ; ' 1 • 1 I , 1 M M 1 ! 1 1 I i i ! i : • • 1 ' , 1 i 1 M i 1 ' I 1 i 1 1 . • 1 1 : i - H~r 1 1 1 ! | | • , • i i i ; I 1 1 , • ! I i i . < M 1 1 • ; • • ! 1 i : ' i I I I ' ! [ ' 1 \ | I 1 1 ' 1 ' ! I . 1 . . i i > i ' i 1 I J 1 1 i ' 1 I • 1 : . i ' M i 1 J-J _ j T : i 1 1 1 l_ 1 1 l 1 1 i M | I I I . M i ' M M i 1 ' i I i i i I 1 I . M | I I I 1 1 ' I I I | i : ' ! i M M | ' I -[ 1 i i i : i : ' M _ • M 1 M 1 1 I.M 1 i r • , in i • M • : i l ; 1 1 i ' i i 1 • n i i i . j j M J | 1 j 1 ' 1 • 1 : • i i - 1 1 ' • • • 1 i .j ; ; i i i • i . ! ' M l U-J_ | I 1 .. _i ! .!_ | | 1 t i l l t i l : 1 ] l i M l ! i r M -14-' 1 1 ' • : ' i • 1 . i. 1 . i m | 1 | 1 ! | 1 M i l i j M M M M 1 I i I j M 1 , . i 1 ! ! i ! M M M M 1 j i 1 i | I i l l ! ! 1 . ! M i r i 1 1 . 1 • ! 1 ! ! 1 i 1 i i ! • •- ' 1 ; | l • i i : i M J . J : • 1 i i ' 1 i ! i M ' | | i i i i i , ! i i 1 1 1 i ' 1 1 M M i M 1 M i l I ' M 1 1 1 i l i t 1 i 1 M M M M I ! i M M ' 1 M M ii. !. i 1 1 : in 1 ] : , : 1 1 " ' J | 1 < i • ' ' ,11 ' , , | „ I i 1 | j M l ] I ' M f I i M , 1 IT" 1 1 • x-X ~X^:~4 i X i i i . i ' | l • 1 . i , - ! : ; i 11 1 ; 1 1 j i x M M M I ' M 1 1 1 — ; - X r j i t ! _i._J_ + i p I ' M 1 1 M i M M IL | | • I T M l , 1 ' 1 1 . ; 1 1 i V.' I i 1 i | ! t i" i 1 : ' i | i i • • ! i ' i i 1 i i : -uU- M l ; : . : ' ! J : -T+rr* 4- -4+ i 1 1 i i ! i M i I I I ! 4-j-f 1 -p M ; , - r r r r i i ! : : i • 1 1 . ; 1 _J_L iH-i-1 i •i-r- 1 -~j~r T'l •;• i • i j I 1 "T ; i ; 1 1 : • 1 i , M i : Ir' : ! ; " S : , , • • ~* 'i '•" i X . , K X T\ , > i i 1 ; . . i : : j i M i l liii i 1 M . i 1 V I i r M I I I ! 1 ! i i i i i i i . ' i 1 • , i 1 1 M 1 J!M i 1 i : M M i ! f M i M M j , M l 1 | M | • • • \ • n i OJ _ M M I 1 ! I W i i i ; TT-H 3 n P T T rr x f l : 3gr ex X3 i r i f H -ro-l=M "IE -tr tr :CT> ±CD. -ICE J_LJJ. i n -H4 m 4 1 •at T T - P r 7 T H ' 'SI. •on -H-i •n-L0: - M S - ' - 4-I--0 1N33 b c l mi •P4-T 4 T M f : M ± : m t-r4+ •rrr XDXl ±H--110-S U M X Y SQUARED AS SHOWN IN L I N E 4 5 . T MAY THEN BE C A L C U L A T E D AS I N D I C A T E D P R E V I O U S L Y . S Y X S Q AS C A L C U L A T E D ABOVE I S THE SQUARE OF S Y X ( L I N E 5 2 ) THE S A M P L E STANDARD D E V I A T I O N FROM R E G R E S S I O N . THE V A L U E S FOR S Y X , N AND S U M X S Q WERE USED TO C A L C U L A T E THE STANDARD ERROR FOR THE E S T I M A T E D Y OF A S E L E C T E D X AS SHOWN IN THE TEXT I N C H A P T E R 1. FURTHER C A L C U L A T I O N S AS SHOWN ALLOWED D E T E R M I N A T I O N OF C O N F I D E N C E L I M I T S AND RANGES AS I N D I C A T E D I N THAT C H A P T E R . B. S I M P L E P E S I GN FOR A. LARGE, I N E X P E N S I V E - S E P H A D E X COLUMN . S E P H A O E X CHROMATOGRAPHY I S A V A L U A B L E T E C H N I Q U E FOR P R O T E I N P U R I F I C A T I O N , BUT IT HAS C E R T A I N L I M I T A T I O N S WHEN USED ON A P R E P A R A T I V E S C A L E , P A R T I C U L A R L Y I F A H I G H DEGREE OF S E P A R A T I O N OF COMPONENTS I S R E Q U I R E D . UNDER SUCH C O N D I T I O N S THE ZONE OF S A M P L E A P P L I C A T I O N SHOULD BE ONLY 1 TO 2 $ OF COLUMN L E N G T H . FOR E X A M P L E , A 2 . 5 X 100 CM COLUMN HAS A . C R O S S - S E C T I 0NAL AREA OF ABOUT 5 CM, A L L O W I N G A MAXIMUM S A M P L E VOLUME OF ABOUT 10 ML. THE AMOUNT OF P R O T E I N W H I C H CAN BE P R O C E S S E D ON SUCH A COLUMN MAY BE L I M I T E D BY S O L U B I L I T Y OR, I F S O L U B I L I T Y I S H I G H , BY THE H I G H V I S C O S I T Y OF THE S A M P L E WHICH L E A D S TO BROAD, SKEWED P E A K S . ONE S O L U T I O N I S TO P R O C E S S L A R G E S A M P L E S BY R E P E T E T I V E CHROMA-TOGRAPHY, BUT IN A D D I T I O N TO M U L T I P L Y I N G THE L A B O R ANO T I M E R E Q U I R E D T H I S M U L T I P L I E S THE P O T E N T I A L FOR A C C I D E N T S AND ERRORS/AND I N C R E A S E S L O S S E S ON V E S S E L W A L L S , E T C . THE B E S T METHOD FOR H A N D L I N G L A R G E R S A M P L E S I S TO glass fiber disc screen -1/8 in.dia. 1/8 in. pipe thread -2 in.- 1/4H-F l G U R E 43 END-PIECES FOR 10 CM COLUMN-VIEWS SHOWN. DISCS MACHINED SHEET. -DIMENSIONS. TWO FROM 1 IN LUC ITE INCREASE THE CROSS-SECTIONAL AREA OF THE COLUMN, BUT THIS CAN BE.QUITE EXPENSIVE SINCE IT REQUIRES MUCH GREATER QUANTITIES OF GEL AND SPECIALLY DESIGNED COLUMNS. THE COST OF SUCH COLUMNS APPEARS TO INCREASE EXPONENTIALLY WITH DIAMETER, AND COMMERCIALLY AVAILABLE 10 CM COLUMNS COST WELL OVER $1000. SUCH EXPENSE CAN BE AVOIDED BY CONSTRUCTING THE REQUIRED COLUMNS FROM PARTS AVAILABLE FROM NON-SCIENTIFIC SOURCES. THE COLUMN USED FOR THE PURIFICATION OF SALMON C T IN CHAPTER III WAS 10 x 150 CM AND WAS CONSTRUCTED FOR LESS THAN $100. THE CONSTRUCTION REQUIRED THE MANUFACTURE OF ONLY TWO IDENTICAL END PIECES AND PRODUCED SEPARATIONS EQUIVALENT TO SMALLER COMMERCIAL COLUMNS ON SAMPLES AS LARGE AS 150 ML. THE BASIC COLUMN WAS A 5 FT. SECTION OF 4 IN.DIAMETER PYREX GLASS PIPE WITH PREBEADED ENDS ORDERED FROM CORNING GLASS WORK, CORNING, N.Y. THESE PIPE SECTIONS, DESIGNED FOR CONSTRUCTING ACID PROOF DRAINS, CAN BE ORDERED IN A VARIETY OF SIZES AND ARE NORMALLY CONNECTEO TOGETHER WITH TEFLON LINED RUBBER JUNCTIONS (CORNING PART #4009). Two END PIECES DESIGNED AS SHOWN IN F l G U R E 43 WERE MACHINED FROM 1 IN. L.UC ITE SHEETS BY FIRST CUTTING A ROUGH 5 IN. CIRCLE ANO THEN DRILLING A CENTER HOLE OF THE DIAMETER REQUIRED FOR ELUANT FLOW. THIS HOLE WAS FURTHER ENLARGED ON ONE SIDE AND THREADED TO TAKE A STANDARD NYLON PIPE THREAD TO HOSE CONNECTOR. A SECTION OF 1/8 IN PIPE WAS THEN TIGHTENED INTO PLACE AND USED TO CHUCK THE PLASTIC PIECE INTO THE LATHE WHERE IT WAS TURNED TO THE DIMENSIONS FIGURE 44. 10 x 150 CM GEL FILTRATION COLUMN IN STAND. -112-SHOWN. CIRCULAR PIECES OF NYLON WINDOW SCREEN AND GLASS FIBER PAPER ( G F P - 2 , APPLIED SCIENTIFIC LABORATORIES, INC., STATE COLLEGE, PA.) WERE THEN CUT TO THE DIMENSIONS SHOWN AND PLACED ON THE END PIECES. WHEN PLACED IN THE STANDARD JUNCTION FITTINGS THE"INNER LIP OF THE FITTING PROVIDED A GOOO SEAL AGAINST THE GLASS FIBER DISC PREVENTING GEL LEAKAGE AND THE NYLON SCREEN ALLOWED ELUANT FLOW FROM THE ENTIRE CROSS-SECTION TOWARD THE OUTLET HOLE. WITH THE BOTTOM END PLATE IN PLACE THE COLUMN WAS MOUNTED ON A LABORATORY STOOL AS SHOWN IN FLGURE 44 WITH A DEXION (WESTERN FABRICATORS, BURNABY, B . C . ) FRAME TO SUPPORT THE UPPER PART OF THE COLUMN. THE COLUMN WAS PLACEO IN A COLD ROOM AT 4 ° C TO MAINTAIN CONSTANT TEMPERATURE AND PARTIALLY FILLED WITH ELUANT WHICH WAS FLUSHED BACK AND FORTH THROUGH THE OUTLET HOLE TO REMOVE AIR BUBBLES FROM BENEATH THE GLASS FIBER DISK. SEPHADEX G - 5 0 WAS ADDED TO THE COLUMN IN STAGES UNTIL THE ENTIRE COLUMN INCLUDING THE UPPER FITTING WAS PACKED WITH GEL. THE SECOND END PLATE WITH NYLON SCREEN AND GLASS FIBER DISCS WAS PRESSED INTO THE FITTING AND TIGHTENED UNTIL A GOOD SEAL WAS OBTAINED. THIS PROVIDED A 150 CM COLUMN ENTIRELY FILLED WITH GEL SO THAT EITHER NORMAL DOWNWARD OR REVERSE FLOW COULD BE USED. A THREE WAY VALVE IN THE INPUT LINE ALLOWED THE SAMPLE TO FLOW DIRECTLY ONTO THE COLUMN. As SHOWN IN FIGURE 12 V Q OF THE COLUMN WAS 4 L AND V T 14 L. EXCELLENT SEPARATION WAS ACHIEVED AND THE STABILITY WAS SUCH THAT THE COLUMN SELDOM REQUIRED REPOURING. -113-C. T E C H N I Q U E S FOR H iGH V O L T A G E P A P E R E L E C T R O P H O R E S I S . 1. A P P A R A T U S H I G H V O L T A G E E L E C T R O P H O R E S I S WAS C A R R I E D OUT IN THE M L C H L V E R T I C A L S T R I P A P P A R A T U S (74, 75) M O D I F I E D AS D E S C R I B E D BY R Y L E EJ_ AJ. (110), THE MONOGRAPH BY . L E G G E T T - B A I L E Y C O N T A I N S A D E T A I L E D D I S C U S S I O N OF T H I S T E C H N I Q U E ( 6 8 ) . THE A P P A R A T U S USED WERE C O N S T R U C T E D IN A L L - G L A S S SHANOON CHROMATANKS (22. 5'I'N. x 9IN. X 22.5 I N . D E E P ) FROM C O N S O L I D A T E D L A B O R A T O R I E S (WESTON, O N T A R I O ) . T H E S E WERE F I T T E D WITH G L A S S RACKS TO SUPPORT THE U P P E R G L A S S B U F F E R TROUGHS, G L A S S RODS TO P R E V E N T THE P A P E R C O N T A C T I N G THE WALLS AND L U C I T E L I D S S U P P O R T I N G THE G L A S S C O O L I N G C O I L S . P L A T I N U M E L E C T R O D E S I N G L A S S E L E C T R O D E HOLDERS WERE P L A C E D I N THE U P P E R B U F F E R TROUGHS ( C A T H O D E ) AND S I M I L A R E L E C T R O D E S P L A C E D TWO C E N T I M E T E R S FROM THE BOTTOM OF THE TANKS WHICH C O N T A I N E D 8 U F F E R TO A D E P T H OF A P P O X I M A T E L Y FOUR C E N T I M E T E R S . BOTH E L E C T R O D E S WERE A T T A C H E D TO CONNECTORS MOUNTED E X T E R N A L L Y ON THE CHROMOTANKS. A C A N A D I A N R E S E A R C H I N S T I T U T E (DON M I L L S , O N T A R I O ) MODEL E P S K - 2 0 0 D.C. POWER S U P P L Y WAS USED TO M A I N T A I N CONSTANT V O L T A G E S UP TO 5 K V AT 200 MA. THE E N T I R E S Y S T E M WAS P L A C E D IN A FUME HOOD TO REMOVE T O X I C V A P O R S . THE HOOD WAS E Q U I P E D WITH L U C I T E OOORS CONNECTED TO AN I N T E R L O C K S A F E T Y S Y S T E M A L L O W I N G THE OPERATOR TO VIEW THE P A P E R D U R I N G O P E R A T I O N WHILE M I N I M I Z I N G THE E L E C T R I C A L HAZARD. THE TWO V O L A T I L E B U F F E R S Y S T E M S USED I N THE P R E S E N T - 1 1 4 -STUDY WERE FIRST DESCRIBED BY AMBLER ( 1 3 4 ) . THE BUFFERS AND COOLANTS USED WERE AS FOLLOWS! PH 1.9 BUFFER: 2$ FORMIC ACID (V/V) 8$ ACET I C AC 1:0 (v/v ) IN DEIONIZED WATER COOLANT: VARSOL PH 6.5 BUFFER: 879 ML DEIONIZED WATER 100 ML PYRIDINE 3 ML GLACIAL ACETIC ACID COOLANT: 92$ TOLUENE, 8$ PYRIDINE (BY VOLUME) ALL REAGENTS WERE FISHER CERTIFIED GRADE. VARSOL WAS PURCHASED FROM THE IMPERIAL OIL CO. 2. STANDARDS AND MARKERS. TWO AMINO ACID STANDARD MIXTURES WERE PREPARE D MCON— TAINING 2 NM OL E S PER U.L OF EACH OF THE ACIDS LISTED. S-1 LYSINE, ARGININE, VALINE, LEUCINE, METHIONINE, PROLINE, PHENYLALANINE, TYR0SINE, CYSTE I C ACID. S-2 HLSTIDINE, GLYC I NE , . ALAN I NE , SERINE, ISOLEUCINE, THREONINE, GLUTAMIC ACID, ASPARTIC ACID, TAURINE. 5 /J.L OF EACH MIXTURE WAS ROUTINELY APPLIED TO THE ORIGIN LINE ON SPOTS OUTSIDE THE SAMPLE AREA. IN THE PH 6.5 BUFFER ONLY THE THREE BASIC AND THREE ACIDIC ACIDS MOVE AS INDICATED IN FIGURE 21. IN THE PH 1.9 BUFFER THE ACIDS ARE FOUNO IN THE ORDER LISTED MOVING DOWN FROM THE CATHODE AS SHOWN IN FLGURE 22. THESE STANDARDS INDICATE WHETHER THE DETECTION REAGENTS - 1 1 5 -ARE WORKING AS E X P E C T E D AND ARE USED IN C A L C U L A T I N G R E L A T I V E M O B I L I T I E S . IN A D D I T I O N TO THE AMINO A C I D M I X T U R E S E A C H E L E C T R O -PHORETOGRAM I S S P O T T E D WITH V I S I B L E MARKER D Y E S TO I N D I C A T E ; THE C O N D I T I O N S D U R I N G THE RUN AND TO DETERM I NE: THE L E N G T H OF THE RUN. FOR RUNS AT PH 6.5 THREE MARKERS ARE U S E D : METHYL GREEN ( C . I . NO. 4 2 5 9 0 , F l S H E R C E R T I F I E D B I O L O G I C A L S T A I N ) ORANGE G ( C . I . NO. 1 6 2 3 0 , F I S H E R C E R T I F I E S B I O L O G I C A L S T A I N ) AND - D I N I T R O P H E N Y L - L Y S I NE ( C A L B I O C H E M , LOS A N G E L E S ) . CURRENT I S NORMALLY A P P L I E D TO THESE RUNS U N T I L THE METHYL GREEN HAS RUN TO A MARK 2 0 CM FROM THE O R I G I N . THE T - D I N I T R O -P H E N Y L - L Y S I N E I S N E U T R A L AT T H I S PH AND MOVES ONLY BY E L E C -TRO E N D 0 S M 0 S I S, AND I T S L O C A T I O N ON THE E L E C T R O P H O R E T O G R A M I S USED TO I N D I C A T E THE N E U T R A L L I N E FOR C A L C U L A T I O N S OF R E L A T I V E M O B I L I T Y . FOR RUNS AT PH 1.9 ONLY METHYL GREEN: ANO. « - D I N I T R O P H E N Y L - L Y S I N E MARKERS ARE U S E D , AND THE METHYL GREEN I S ALLOWED TO T R A V E L 3 0 CM FROM THE O R I G I N . T H E T A U R I N E IN THE AMINO A C I D STANDARD I S N E U T R A L AT T H I S PH ANO I S USED TO I N D I C A T E THE N E U T R A L L I N E . 3. D E T E C T I O N R E A G E N T S . THE CADM I UM-N I NHYDR I N R E A G E N T OF H E I L M A N E_T AL ( 5 4 ) WAS. USED WHEN MAXIMUM S E N S I T I V I T Y (1 TO 5 NMOLES/CM ) WAS R E Q U I R E D OR WHEN I N F O R M A T I O N ON THE , N— TERM IN A L AMINO A C I D WAS N E E D E D . T H I S R E A G E N T WAS P R E P A R E D BY M I X I N G 1 5 ML OF A STOCK S O L U T I O N C O N T A I N I N G 5 G CADMIUM A C E T A T E ( F L S H E R C E R T I F I E D R E A G E N T ) IN 2 5 0 ML OF G L A C I A L A C E T I C A C I D ANO -116-500 ML D I S T I L L E D WATER WITH 100 ML OF 0.5$ (W/V) N I N H Y D R I N ( P I E R C E C H E M I C A L CO.) IN A C E T O N E ( F I S H E R REAGENT G R A D E ) . P A P E R S WERE D I P P E D IN T H I S S O L U T I O N AND ALLOWED TO A I R DRY U N T I L THE ACET O N E WAS REMOVED. THE P A P E R WAS THEN P L A C E O I N AN OVEN AT 60° C FOR 20 M I N U T E S FOR COLOR D E V E L O P M E N T . IN GENE R A L P E P T I D E S 'HAVING THE N - T E R M I N A L AMINO A C I D S L I S T E D BELOW HAVE B E E N SHOWN TO G I V E THE COLORS I N D I C A T E D WITH T H I S REAGENTo YELLOW ( S T A B L E 1 WEEK) G L Y , S E R , THR, C Y S , P R O , C Y S T E I C RED ORANGE S E R , H I S SLOW RED ( O V E R N I T E ) ( L E , VAL "FAST RED L E U , L Y S , ARG, A S P , G L U , TYR, P H E , MET, M E T SO2, T R P , ALA T H E S E COLOR D I F F E R E N C E S ARE U S E F U L IN P R E D I C T I N G THE P R O C E D U R E S NEEDED TO I D E N T I F Y THE D A N S Y L - D E R I V A T I V E S OF THE P E P T I D E S . B E C A U S E OF I T S H I G H S E N S I T I V I T Y FOR A R G I N I N E P E P T I D E S (0.1 NMOLES/C M ^ ) AND THE EX T R A I N F O R M A T I O N I T P R O V I D E D , THE P A Q R E A G E N T WAS R O U T I N E L Y USED I N C O M B I N A T I O N WITH A S P E C I A L N I N H Y D R I N R E A G E N T FOR P E P T I D E D E T E C T I O N . THE R E A G E N T S USED WERE D E S C R I B E D BY YAMADA AND I..TANO (133). THE P A Q R E A G E N T WAS P R E P A R E D BY M I X I N G EQUAL VOLUMES OF A S O L U T I O N C O N T A I N I N G 0.02$ (W/V) P H E N A N T H R E N Q U I NONE ( F L S H E R S C I E N T I F I C , CO., H I G H E S T P U R I T Y ) IN ANHYDROUS ETHANOL AND 10$ w/v SOOIUM H Y O R O X I D E I N 60$ E T H A N O L . THE P A P E R S WERE D I P P E D I N T H I S M I X T U R E AND ALLOWED TO A I R DRY 20 M I N U T E S - 1 1 7 -PRIOR TO EXAMINATION UNDER A LONGWAVE UV LAMP. AFTER AN ADDIT IONAL 2 0 MINUTES DRYING THE PAPER COULD BE DEVELOPED WITH AN ACID NINHYDRIN REAGENT MADE BY MIXING 2 0 ML OF A SOLUTION CONTAINING 1 G OF CADMIUM ACETATE IN 1 5 0 ML OF GLAC IAL A C E T I C ACID AND 50 ML OF D I S T I L L E D WATER WITH 8 0 ML OF 0.5 i (w/v) NINHYDRIN IN ACETONE . IN TH IS REAGENT ALL P E P T I D E S PRODUCED A V I O L E T - R E D COLOR ON DRYING AT 6 0 ° C FOR 2 0 M INUTES. D. AM 1 NO Ac 1o ANALYS i s , 1. HYDROLYS IS . HYDROLYSES WERE CARRIED OUT E S S E N T I A L L Y AS DESCRIBED BY MOORE AND ST E I N ( 7 7 ) . AL IQUOTS OF SOLUTIONS CONTAINING 2 TO 2 0 NMOLES OF PROTEIN WERE L Y O P H I L I Z E D IN 1 2 X 7 5 MM PYREX CULTURE TUBES AND RED ISSOLVED IN 0.5 ML OF 5.7 M HYDROCHLORIC AC ID . T H E . T U B E S WERE THEN HEATED NEAR THE TOP WITH A F I N E , HOT GAS-OXYGEN FLAME AND DRAWN OUT TO Y I ELD A NARROW (1MM) , HEAVY WALLED NECK. THE CONTENTS WERE THEN FROZEN IN A DRY I C E - A C E T O N E MIXTURE TO PREVENT BUBBLING AND THE TUBES EVACUATED TO ABOUT 1 0 " ^ TORR BEFORE SEAL ING THE NECK WITH A MODERATELY HOT GAS-OXYGEN FLAME . HYDROLYSIS ON THE SEALED TUBES WAS THEN ROUTINELY CARRIED OUT FOR 1 7 HOURS AT 1 1 0 ° C IN A TEMP-BLOK FROM L A B - L L N E INSTRUMENTS, MELROSE PARK, IL L I N O I S. AFTER HYDROLYSIS THE TUBES WERE OPENED AND DRIED IN A VACUUM DESS ICATOR OVER SODIUM HYOROXIOE. THE HYDROCHLORIC ACID USED WAS CONSTANT BO IL ING AZEOTROPE O I S T I L L E D FROM B A K E R AND ADAMSON REAGENT GRADE HYDROCHLORIC - 1 1 8 -A c i D FROM A L L I E D C H E M I C A L S CANADA, L T D . D I L U T E D WITH D I S T I L L E D , DEIONI2ED WATER. THE T U B E S USED FOR H Y D R O L Y S I S WERE WASHED IN STRONG D E T E R G E N T AND R I N S E D FOUR T I M E S WITH HOT TAP WATER FOLLOWED BY FOUR R I N S E S WITH D I S T I L L E D WATER. THEY WERE THEN ALLOWED.TO STAND O V E R N I T E IN 1 M H Y D R O C H L O R I C A C I D , R I N S E D ONCE MORE IN THE SAME A C I D AND D R I E D D I R E C T L Y FROM THE A C I D R I N S E IN A 6 0 ° C OVEN. 2. A I R O X I D A T I O N OF C Y S T E I N E . D R I E D , H Y D R O L Y S E D S A M P L E S C O N T A I N I N G C Y S T E I N E WERE D I S S O L V E D IN 2 0 0 /J.L OF 0.1 M P H O S P H A T E B U F F E R AT PH 6.5 AND ALLOWED TO STAND OPEN TO THE A I R FOR FOUR HOURS TO I N S U R E C O M P L E T E R E C O V E R Y AS -G * —CYSTINE. THE S A M P L E S WERE THEN A D J U S T E D TO P H 2 WITH 5 JU- 0 F ^ M H Y D R O C H L O R I C A C I D FOR A P P L I C A T I O N TO THE A N A L Y S E R COLUMN. 3. AUTOMATED A N A L Y S I S . THE F I R S T D E T A I L E D REPORT OF AN AUTOMATEO S Y S T E M FOR AMINO A C I D A N A L Y S I S CAME FROM S P A C K M A N JIT_ AJ_ IN 1 9 5 8 ( 1 1 8 ) AND A L T H O U G H ' S E V E R A L A L T E R N A T E S Y S T E M S HAVE S I N C E B E E N R E P O R T E D THE O R I G I N A L PROCEDURE I S S T I L L THE B A S I S OF THE M A J O R I T Y OF AMINO A C I D A N A L Y S E S P E R F O R M E D . THERE ARE NOW A NUMBER OF C O M M E R C I A L I N S T R U M E N T S ON THE MARKET WHICH USE T H I S B A S I C P R O C E D U R E , AND T H E S E I N S T R U M E N T S VARY P R I M A R I L Y IN THE D E S I G N AND V A R I E T Y OF A N C I L L A R Y E Q U I P M E N T A V A I L A B L E . T H E I R COMMON F E A T U R E S I N C L U D E S E P A R A T I O N OF AMINO A C I D S BY E L U T I O N FROM S U L F O N A T E D P O L Y S T Y R E N E R E S I N S , S I M I L A R TO DOWEX 50 X 8, BY A S E R I E S OF C I T R A T E B U F F E R S FOLLOWED BY - 1 1 9 -D E T E C T I O N AND Q U A N T I T A T I O N B A S E D ON THE R E A C T I O N OF THE E L U T E D AMINO A C I D S WITH A N I N H Y D R I N R E A G E N T . THE AMOUNT OF COLORED R E A C T I O N PRODUCT I S MEASURED BY A PHOTOMETER AND RECORDED. T H E S E P R O C E D U R E S ARE C A R R I E D OUT M O N L I N E 1 ' IN A C O N T I N U O U S L Y FLOWING S Y S T E M WHICH R E Q U I R E S A B U F F E R PUMP TO MOVE THE E L U A N T THROUGH THE COLUMN AND A N I N H Y D R I N PUMP TO M I X A CONSTANT P R O P O R T I O N OF T H I S R E A G E N T WITH THE E L U A T E . T H I S M I X T U R E I S THEN P A S S E D THROUGH A C O I L OF NARROW BORE T E F L O N T U B I N G I N A' B O I L I N G WATER BATH TO A C C E L E R A T E THE R E A C T I O N AND THENCE TO A S E R I E S OF FLOW C E L L S I N THE PHOTO-METER WHERE THE A B S O R B A N C E AT 5 7 0 NM I S RECORDED FOR THE AMINO A C I D S AND AT 4 4 0 NM FOR THE I M I N O A C I D S . THE ORDER AND DEGREE OF S E P A R A T I O N OF THE AMINO A C I D S IN THE E L U A T E CAN BE V A R I E D BY CHANGES IN THE T E M P E R A T U R E OF THE COLUMNS AND THE PH AND I O N I C S T R E N G T H OF THE B U F F E R S U S E D . MOST C O M M E R C I A L M A C H I N E S HAVE PROGRAMMED S Y S T E M S FOR S W I T C H I N G B U F F E R S AND T E M P E R A T U R E AND C O N T R O L L I N G N I N H Y D R I N FLOW, THE RECORDER AND THE R E G E N E R A T I O N C Y C L E FOR THE COLUMN. THE F L E X I B I L I T Y OF THESE PROGRAMMING S Y S T E M S I S ONE OF THE MAJOR V A R I A B L E S IN THE C O M M E R C I A L I N S T R U M E N T S . THE SECOND MAJOR V A R I A B L E I S S E N S I T I V I T Y . MOST COMMERCIAL I N S T R U M E N T S ARE R O U T I N E L Y USEO FOR Q U A N T I T A T I O N IN THE RANGE OF 5 0 TO 2 5 0 NANOMOLES OF AMINO A C I D , AND ALTHOUGH THEY HAVE R E P O R T E D D E T E C T I O N L I M I T S V A R Y I N G FROM 1 TO 1 0 NMOLES T H E S E L I M I T S ARE OF L I T T L E S I G N I F I C A N C E S I N C E AT THESE L E V E L S IT I S P O S S I B L E ONLY TO DETECT THE AMINO A C I D S NOT TO QUANT I T ATE THEM. A T H I R D V A R I A B L E WHICH HAS PROVED Q U I T E C R I T I C A L I S -120-R E L I A B I L I T Y S I N C E I N C R E A S E D S E N S I T I V I T Y I S OF L I T T L E USE I F THE S Y S T E M R E Q U I R E S F I V E S A M P L E S TO A C H I E V E ONE GOOD A N A L Y S I S . THE WORK D E S C R I B E D IN T H I S T H E S I S AND OTHER WORK UNDER-WAY IN T H I S L A B O R A T O R Y R E Q U I R E D A H I G H L Y S E N S I T I V E PROCEDURE FOR AMINO A C I D A N A L Y S I S . S I N C E THE P R O T E I N S UNDER STUDY WERE A V A I L A B L E IN ONLY Q U I T E L I M I T E D Q U A N T I T I E S . A M INO A C I D A N A L Y S I S I S THE HEART OF A L L P R O T E I N S T U D I E S AND I F THE A N A L Y S E S ARE C A R R I E O OUT AT THE L E V E L S R E Q U I R E D BY THE C O M M E R C I A L L Y A V A I L A B L E E Q U I P M E N T IT I S THE L I M I T I N G FACTOR I N D E T E R M I N I N G THE Q U A N T I T I E S OF P R O T E I N WHICH MUST BE A V A I L A B L E FOR STUDY. THE A D V A N C E S IN OTHER A R E A S OF P R O T E I N C H E M I S T R Y HAVE FAR O U T S T R I P E D THOSE IN AMINO A C I D A N A L Y S I S . As AN E X A M P L E OF T H I S THE P R E S E N T USE OF THE D A NSYL-EDMAN METHOD (52) ALLOWS S E Q U E N C E D E T E R M I N A T I O N ON P E P T I D E S U S I N G L E S S THAN H A L F THE M A T E R I A L N E C E S S A R Y FOR A S I N G L E A N A L Y S I S AT THE 50 NMOLE L E V E L . C L E A R L Y R O U T I N E Q U A N T I T A T I O N AT OR BELOW THE 1 NMOLE L E V E L WAS E S S E N T I A L TO B R I N G T H I S PROCEDURE IN L I N E WITH THE OTHER A V A I L A B L E T E C H N I Q U E S . To A C H I E V E T H I S , M O D I F I C A T I O N S WERE C A R R I E O OUT ON A NEWLY D E V E L O P E D C O M M E R C I A L M A C H I N E , THE BlOCHROM 200, WHICH HAD THE H I G H E S T A V A I L A B L E S E N S I T I V I T Y AND THE D E S I R E D F L E X I B I L I T Y I N PROGRAMMING. THE B A S E S OF T H I S H I G H S E N S I T I V I T Y WERE LONG P A T H L E N G T H FLOW C E L L S (14 MM V E R S U S THE MORE COMMON 7 MM) AND A H I G H DEGREE OF A M P L I F I C A T I O N IN THE RECORDER C I R C U I T (5.5MV F U L L S C A L E D E F L E C T I O N ) . E A R L Y E X P E R I M E N T S AT LOW L E V E L S I N D I C A T E D THAT T H I S S Y S T E M WAS - 1 2 1 -I N A D E QU A T E S I N C E THE S E N S I T I V I T Y WAS S T I L L NOT GREAT ENOUGH ANO THE S I G N A L TO N O I S E R A T I O OF THE S Y S T E M WAS POOR. R E L A T I V E L Y S M A L L P E A K S WERE N E A R L Y H I D D E N IN THE RANDOM F L U C T U A T I O N S IN THE B A S E L I N E . A MAJOR PART OF T H I S P R O B L E M WAS THE I N H E R E N T N O I S E IN THE P H 0 T O M U L T I P L I E R T U B E S USED IN THE PHOTOMETER WHICH WAS G R E A T L Y A M P L I F I E D BY THE RECORDER, T O OVERCOME T H I S P R O B L E M A D E T E C T I O N C I R C U I T WAS D E S I G N E D B A S E D ON A NEWLY D E V E L O P E D P H 0 T 0 T R A N S I S T 0 R ( F P T 1OO, FA I R -C H I L D S E M I C O N D U C T O R , MOUNTAIN V I E W , C A L I F O R N I A ) WHICH WAS USED AS ONE S I D E OF A WHEATSTONE B R I D G E C I R C U I T . V A R I A T I O N OF THE R E S I S T A N C E ON THE O P P O S I T E S I D E OF THE B R I O G E ALLOWED A D J U S T M E N T OF THE OUTPUT TO SET THE 8 A S E L I N E , WHILE D R A I N I N G PART OF T H I S OUTPUT O F F TO GROUND THROUGH A SECOND V A R I A B L E R E S I S T A N C E ALLOWED CONTROL OF THE G A I N IN THE C I R C U I T , D.C. CURRENT WAS S U P P L I E D BY MERCURY C E L L S WHICH HAVE AN E X T R E M E L Y S T A B L E OUTPUT. T H E N O I S E L E V E L IN T H E S E C I R C U I T S WAS U N D E T E C T A B L E ON THE RECORDER AND THE H I G H G A I N THEY P R O V I D E D A L L OWE 0 R E D U C T I O N OF THE G A I N (55MV F U L L S C A L E ) IN THE N O I S I E R RECORDER A M P L I F I E R . T H R E E S E P A R A T E C I R C U I T S WERE USED TO MEASURE THE A B S O R B A N C E AT 5 7 0 NM WITH A 14 MM FLOW C E L L , AT 5 7 0 NM WITH A 7 MM FLOW C E L L ANO AT 4 4 0 NM WITH A U MM F L O V C E L L . THE A N A L Y S E S IN T A B L E I V WERE C A R R I E D OUT U S I N G T H I S S Y S T E M AND A C C E P T A B L E Q U A N T I T A T I O N WAS P O S S I B L E AT THE 2 T 0 3 NMOLE L E V E L . As I N D I C A T E D IN THE T A B L E THE STANDARD TWO COLUMN S Y S T E M WAS USED. THE A C I D I C AND N E U T R A L AMINO A C I D S WERE S E P A R A T E D ON A 5 2 X 0.9 CM COLUMN O F . B | 0 - R A D - 1 2 2 -A M I N E X A-6 R E S I N A T 5 2 ° C U S I N G 0.2 N S O D I U M C I T R A T E B U F F E R S A T PH 3.25 A N O PH 4.40 P R E P A R E D I N D I S T I L L E D , D E I O N I Z E D W A T E R A S D E S C R I B E D B E L O W . PH 3.25 4.40 S O D I U M C I T R A T E D I H Y D R A T E ( A M I N O A C I D A N A L Y S I S G R A D E 7 8 . 4 G 7 8 . 4 G B I O - R A D L A B O R A T O R I E S , R I C H M O N D , C A L I F O R N I A ) C O N C . H Y D R O C H L O R I C A C I D ( B A K E R - A D A M S O N R E A G E N T , 4 9 . 4 M L 3 0 . 0 M L A L L I E D C H E M I C A L S C A N A D A , L T D . ) O C T A N O I C A C I D ( A . A . A . G R A D E , B I O - R A D . ) 0.5 M L 0.5 M L F I N A L V O L U M E 4.0 L 4.0 L TH E PH 3.25 B U F F E R WAS P U M P E D A T A R A T E O F 7 0 M L / H R F O R 6 0 M I N . A N D P H 4.40 B U F F E R F O R AN A D D I T I O N A L 1 0 0 M I N . , F O R A T O T A L R U N T I M E O F 2 H O U R S A N D 4 0 M I N U T E S . R E G E N E R A T I O N W I T H 0.2 M S O D I U M H Y D R O X I D E A N D R E - E Q U I L I B R A T I 0N R E Q U I R E D AN A D D I T I O N A L 7 0 M I N U T E S . B A S I C A M I N O A C I D S W E R E S E P A R A T E D O N A 14 x 0.9 CM C O L U M N O F B I O - R A D A M I N E X A-5 R E S I N A T 5 2 ° C U S I N G A 0.35 N S O D I U M C I T R A T E B U F F E R A T PH 5.28 P R E P A R E D A S D E S C R I B E D B E L O W . PH 5.28 S O D I U M C I T R A T E D I H Y D R A T E ( A . A . A . G R A D E , B I O - R A D ) 1 3 7 . 2 G C O N C . H Y O R O C H L O R I C ACI0 ( B & A R E A G E N T ) 2 6 M L O C T A N O I C A C I D ( A . A . A . G R A D E , B | 0 - R A D ) 0.5 M L F I N A L V O L U M E 1 4 . 0 L E L U T I O N O F T H E S E A C I D S A T 70 M L / H R R E Q U I R E D 7 0 M I N A N D A T O T A L C Y C L E T I M E O F 1 1 0 M I N . N I N H Y D R I N R E A G E N T P R E P A R E D A S I N D I C A T E D B E L O W W A S P U M P E D A T A R A T E O F 3 5 M L / H R A N D M I X E D W I T H T H E E L U A T E S F O R 15K' 10K 20K FFTIOO He 5MF Recorder 6.8K< 10K — FPT100 Recorder 5MF n e A A A - t 20K 20K , AAA—± Recorder 180a AAA—, IN4004 r IN4004 AAA— I 180a F I G U R E 45 IM P R O V E D P H O T O T R A N S I S T O R D E T E C T O R F O R A M I N O A C I D A N A L Y S E R — C I R C U I T O I A G R A M . - 1 2 3 -D E T E C T I ON. N I N H Y D R I N REAGENT t • METHYL C E L L O S O L V E 3 L ( S E Q U A N A L G R A D E , P I E R C E C H E M I C A L CO., • --Ro:G^ |i|0»-O!ifeysiiL4,N:O I s) • • • 4 M SODIUM A C E T A T E . ( F I S H E R C E R T I F I E D ) 1 L N I N H Y D R I N ( R E A G E N T GRADE, P I E R C E C H E M . ) 8 0 G STANNOUS C H L O R I D E D I H Y D R A T E ( A . A . A . G R A D E , B I O - R A D ) 1 . 6 0 G O X Y G E N WAS REMOVED BY B U B B L I N G THE M I X T U R E WITH N I T R O G E N ( L GRADE, C A N A D I A N L I Q U I D A L R L T D . ) FOR 3 0 M I N AND THE R E A G E N T WAS STORED UNDER N I T R O G E N . UNDER THESE C O N D I T I O N S A C O M P L E T E A N A L Y S I S R E Q U I R E D A M I N I M U M OF 5 NMOLES OF P R O T E I N AND A TOTAL OF ALMOST 6 HOUR. THUS WITH C A R E F U L P L A N N I N G AND F U L L USE OF AUTOMATED P R O C E D U R E S TWO A N A L Y S E S COULD BE C A R R I E D OUT IN A NORMAL WORKING DAY. A L T H O U G H T H I S S Y S T E M WAS A MARKED IMPROVEMENT OVER THE O R I G I N A L THE M A T E R I A L R E Q U I R E M E N T S WERE S T I L L TOO H I G H , AND THE N O I S E L E V E L IN THE B A S E L I N E WAS U N S A T I S F A C T O R Y WHEN MAXIMUM G A I N WAS U S E D , P A R T I C U L A R L Y ON THE 4 4 0 CHANNEL S I N C E A B S O R B A N C E OF THE I M I N O A C I D PRODUCT I S L E S S THAN THAT OF THE AMINO A C I D S . T H E P H O T O T R A N S I S T O R C I R C U I T HAD PROVED TO BE MUCH Q U I E T E R THAN THE R E S T OF THE S Y S T E M AND IT WAS T H E R E F O R E D E C I D E D TO I N C R E A S E THE G A I N IN T H I S C I R C U I T BY R E D E S I G N AS SHOWN IN F I G U R E 4 5 AND TO CUT DOWN THE N O I S E WHICH WAS NOW KNOWN TO COME P R I M A R I L Y FROM THE H Y D R A U L I C S Y S T E M . H A M I L T 0 N ( 5 1 ) HAD P R E S E N T E D A D E T A I L E D D I S C U S S I O N OF THE D I F F I C U L T I E S E N C O U N T E R E D IN H I G H S E N S I T I V I T Y A N A L Y S I S - 1 2 4 -AND EFFORTS WERE MADE TO CORRECT MANY OF THE FAULTS HE HAD POINTED OUT. THE TWO BASIC CAUSES OF NOISE IN THE BASELINE ARE RHYTHMICAL FLUCTUATIONS DUE TO MINUTE VARIATIONS IN PUMPING RATES AND RANDOM FLUCTUATIONS DUE TO PARTICULATE MATTER AND BUBBLES IN THE FLUIDS. RHYTHMICAL FLUCTUATIONS WERE ELIMINATED BY CAREFULLY MATCHING THE SPEED OF THE BUFFER AND NINHYDRIN PUMPS, BUT THE PROBLEM OF PARTICULATE MATTER WAS MORE COMPLEX. IMPROVED PROCEDURES FOR FILTRATION OF BUFFERS AND NINHYDRIN REAGENT HELPED AND IT WAS POSSIBLE TO REDUCE THE TIME IN THE REACTION BATH FROM 1 5 TO 5 MIN WITHOUT EFFECTING COLOR.DEVELOPMENT (117) BY SHORTENING THE COIL. THIS HAD THE EFFECT OF REDUCING BUBBLE FORMATION AND PRECIPITATION. THESE CHANGES REDUCED BUT DID NOT ELIMINATE THE PROBLEM SO THAT MORE BASIC CHANGES WERE REQUIRED. THE PHOTOMETER WAS REDESIGNED TO MINIMIZE THE IMPORTANCE OF THE HYDRAULIC FLUCTUATIONS BY DECREASING THE LENGTH OF THE FLOW CELLS TO 7 MM AND COMPENSATING THIS REDUCTION BY INCREASED GAIN IN THE QUIET ELECTRONIC SYSTEMS. FURTHER IMPROVEMENTS WERE OBTAINED BY REDUCING THE NUMBER OF FLOW CELLS TO TWO AND DIVIDING THE OUTPUT OF THE 5 7 0 CHANNEL ELECTRICALLY TO PROVIDE A SUPPRESSED CHANNEL. THIS REDUCED MIXING VOLUME AND INCREASED PEAK HEIGHT RELATIVE TO BASE LINE VARIATIONS. A CHANGE IN THE CON-FIGURATION OF THE PHOTOMETER SET UP ALLOWED BOTH FLOW CELLS TO BC ILLUMINATED FROM A SINGLE LAMP RATHER THAN THE THREE LAMPS USED ORIGINALLY. THIS REDUCED THE POWER REQUIRE-MENTS FOR THE VOLTAGE STABILIZER SUPPLYING THE LAMPS TO ABOUT - 1 2 5 -O N E F O U R T H O F I T S R A T E D C A P A C I T Y A N D E L I M I N A T E D L A M P N O I S E W H I C H H A D O C C U R E D P R E V I O U S L Y D U R I N G S W I T C H I N G O P E R A T I O N S . IT A L S O E L I M I N A T E D R A N D O M V A R I A T I O N S V E T W E E N C H A N N E L S D U E T O I N S T A B I L I T I E S IN T H E L A M P S . A N A D D I T I O N A L C H A N C E W H I C H H A L V E D T H E M A T E R I A L R E Q U I R E ' M E N T S WAS T H E A D O P T I O N O F AN A C C E L E R A T E D S I N G L E C O L U M N S Y S T E M S I M I L A R T O T H A T O F D E V E N Y I ( 3 8 ) W H I C H A L L O W E D D E T E R M I N A T I O N O F A L L A M I N O A C I D S IN A S I N G L E R U N . T H I S P R O C E D U R E R E Q U I R E D T H E U S E O F A T H I R D B U F F E R W I T H I N C R E A S E D I O N I C S T R E N G T H ( 0 . 8 N IN S O D I U M I O N S ) P R E P A R E D A S S H O W N B E L O W A N D E L E V A T I O N O F O P E R A T I N G T E M P E R A T U R E TO 5 8 ° C. T H E PH 6.55 S O D I U M H Y D R O X I D E ( F I S H E R C E R T I F I E D R E A G E N T ) 1 6 0 G C I T R I C A C I D ( A . A . A . G R A D E , B I O - R A D ) 2 5 9 c O C T A N O I C A C I D ( A . A . A . G R A D E , B I O - R A D ) 0.5 M . F I N A L V O L U M E 4 L R E D U C E D V I S C O S I T Y A T T H I S E L E V A T E D . T E M P E R A T U R E A L L O W E D T H E P U M P I N G R A T E S T O B E I N C R E A S E D T O 8 0 M L / H R F O R B U F F E R A N D 4 0 M L / H R F O R N I N H Y D R I N . T H I S S Y S T E M WAS U S E D F O R T H E A N A L Y S E S O F T H E O X I D I Z E D S A M P L E O F S A L M O N C T A N O A L L O W E D C O M P L E T E Q U A N T I T A T I V E A M I N O A C I D A N A L Y S I S O N A S L I T T L E A S 2 N M O L E S O F P R O T E I N . T H E T I M E R E Q U I R E D F O R A C O M P L E T E A N A L Y S I S WAS R E D U C E D TO L E S S T H A N 5 H O U R S ( P H 3 . 2 5 , 50 M I N ; PH 4 . 4 0 , 6 0 M I N ; PH 6.45, 9 5 M I N ; R E C Y C L E , 7 0 M I N ) . T H E S E M O D I F I C A T I O N S A L L O W E D R O U T I N E O P E R A T I O N A T T H E 5 N M O L E L E V E L , A 50 F O L D I N C R E A S E IN S E N S I T I V I T Y , B U T I T W A S S T I L L N O T P O S S I B L E TO WORK A T T H E 1 N M O L E L E V E L . - 1 2 6 -B A S E L I N E NOISE FROM THE HYDRAULIC SYSTEM WAS THE MAJOR PROBLEM SINCE THE S O L I D STATE ELECTRONICS WERE E S S E N T I A L L Y NOISE FREE AND COULD PROVIDE ALMOST UNLIMITED G A I N . L I N E A R A M P L I F I C A T I O N OF THE OUTPUT SIGNAL HOWEVER, MAGNIFIED THE NOISE AS WELL AS THE SIGNAL DUE TO THE NINHYDRIN PRODUCTS. C A L C U L A T I O N S OF AMINO ACID CONCENTRATIONS IN THE ELUATE OF THIS SYSTEM ARE BASED ON THE BEER-LAMBERT LAW ( L / L 0 = E ~ U C X ) WHICH STATES THAT THE RATIO OF THE I N T E N S I T Y OF TRANSMITTED LIGHT ( L ) TO THAT OF INCIDENT LIGHT ( L Q ) IS A LOGARITHMIC FUNCTION OF THE ABSORPTION COEFFI CENT (JJT) OF THE DISSOLVED COMPOUND, THE CONCENTRATION OF THAT COMPOUND (c) AND THE PATHLENGTH (x). WHEN JJt, l Q AND X ARE CONSTANT, I , THE QUANTITY MEASURED, IS A LOG FUNCTION OF THE CONCEN-TRATION, IN THIS SYSTEM A CONCENTRATION CHANGE FROM 0 T01 NMOLE AND FROM 1 5 TO 2 0 NMOLES BOTH RESULT IN A RECORDER DEFLECTION WHICH IS 1 0 $ 0F FULL S C A L E . THUS BUBBLES AND P A R T I C L E S CAUSE LARGE FLUCTUATIONS IN THE B A S E L I N E , BUT LARGE CHANGES IN CONCENTRATION SOMETIMES HAVE ONLY A RELA-. T I V E L Y SMALL E F F E C T . IT IS THEREFORE P O S S I B L E TO IMPROVE THE SIGNAL TO NOISE RATIO OF THE SYSTEM BY USING AN A M P L I F I E R WITH AN OUTPUT WHICH IS A LOGARITHMIC FUNCTION OF ITS INPUT. THE USE OF SUCH A L O G - A M P L I F I E R MEANS THAT PEAK HEIGHT BECOMES DIRECTLY PROPORTIONAL TO CONCENTRATION AND THAT SMALL V A R I A T I O N S NEAR THE B A S E L I N E ARE NO LONGER EXAGGERATED. FIGURE 46 SHOWS THE C I R C U I T DIAGRAM OF A L O G - A M P L I F I E R DESIGNED BY TECHCAL ELECTRONIC S E R V I C E S , VANCOUVER, B . C . FOR USE WITH THE PHOT 0 -TRANSIST 0R DETECTION SYSTEM. - 1 2 7 -FURTHER IMPROVEMENTS MADE TO THE SYSTEM AT THIS TIME INCLUDED REDUCTION OF COLUMN DIAMETER.TO 0 . 6 CM WHICH REDUCED ELUTION VOLUME BY HALF, EFFECTIVELY DOUBLING SAMPLE CONCENT TRATION IN THE ELUATE; AND THE USE OF A TWO.BUFFER-SINGLE COLUMN ELUTION SYSTEM. THE FIRST BUFFER WAS THE SAME AS THAT PREVIOUSLY USED BUT THE P H 4 , 4 0 BUFFER WAS MADE UP TO 0 , 8 N IN SODIUM IONS BY THE ADDITION OF 3 5 . 1 G/L OF SODIUM CHLORIDE ( F l S H E R REAGENT G R A D E ) . IT HAD BEEN OBSERVED EARLIER THAT BOTH SODIUM CITRATE AND CITRIC ACID APPARENTLY CONTAINED MINOR CONTAMINANTS WHICH WERE NINHYDRIN POSITIVE. THIS MEANT THAT INCREASING IONIC STRENGTH WITH CITRATE BUFFERS INCREASED THE BACKGROUND COLOR IN THE PHOTOMETER AND IN THE HIGH SENSITIVITY SYSTEM THIS RESULTED IN A MARKED SHIFT IN BASELINE DURING THE THIRD BUFFER WHICH WAS AN INCON-V E N I E N C E . THIS WAS ELIMINATED BY MAINTAINING THE CONCEN-TRATION OF CITRATE IN BOTH BUFFERS CONSTANT ANO INCREASING IONIC STRENGTH WITH SODIUM CHLORIDE WHICH CAUSED NO CHANGE IN BACKGROUND COLOR. THIS SYSTEM WAS USED FOR THE ANALYSES ON THE SALMON C T TRYPTIC PEPTIDES (TABLE V l ) AND FOR PORCINE C T PEPTIDES (CHAPTER I V ) , AND IS RUN ROUTINELY AT THE 5 NMOLE LEVEL. PUMPING RATES OF 5 0 ML/HR FOR BUFFER AND 2 5 ML/HR FOR NIN-HYDRIN ALLOW COMPLETE AMINO ACID ANALYSES IN SLIGHTLY OVER 4 HR ON SAMPLES OF LESS THAN 1 NMOLE WHEN THE FULL GAIN OF THE LOG-AMPLIFIER IS USED. THE DECREASED-ANALYSIS TIME ALSO MEANS THAT THE AUTOMATED SYSTEM ALLOWS COMPLETION OF THREE ANALYSES DURING A NORMAL DAY. A FURTHER ADVANTAGE OF THE - 1 2 8 -S Y S T E M I S T H A T T H E L I N E A R R E L A T I O N S H I P B E T W E E N P E A K H E I G H T A N D C O N C E N T R A T I O N A L L O W S D I R E C T D E T E R M I N A T I O N O F C O N C E N -T R A T I O N F R O M M E A S U R E M E N T S O F P E A K H E I G H T . V A R I A T I O N IN P E A K H E I G H T B E T W E E N A M I N O A C I D S C A N B E C O M P E N S A T E D F O R B Y T H E U S E O F C O R R E C T I O N F A C T O R S A N D T H E T E D I O U S P R O C E S S O F A R E A C A L C U L A T I O N S A N D T H E I N A C C U R A C I E S O F I N T E G R A T I N G U N I T S A R E E L I M I N A T E D . C O M P A R I S O N S O F T H E A C C U R A C Y O F A R E A A N D H E I G H T M E A S U R E M E N T S IN T H I S S Y S T E M H A V E S H O W N NO S I G N I F I C A N T V A R I A T I O N B E T W E E N T H E TWO M E T H O D S . E . P R E P A R A T I ON A N D I D E N T I F I C A T I O N O F D A N S Y L P E R I V A T T V E S . N— T E R M IN A L A M I N O A C I D S W E R E D E T E R M I N E D B Y A M O D I F I C A T I O N O F T H E D A N S Y L P R O C E D U R E O F G R E Y A N D H A R T L E Y ( 4 7 ) . A N A L I Q U O T O F T H E S A M P L E T O B E A N A L Y S E D C O N T A I N I N G A P P R O X I M A T E L Y 1 N M O L E O F P E P T I D E WAS P L A C E D IN A 4 X 5 0 MM P Y R E X C U L T U R E T U B E W H I C H H A D B E E N C L E A N E D B Y T H E S A M E M E T H O D S O E S C R I B E D F O R T H E H Y D R O L Y S I S T U B E S . T H E S A M P L E WAS D R I E D IN V A C U U O A N D R E D I S S O L V E D IN 1 0 JU.L O F 0 . 2 M S O D I U M B I C A R B O N A T E ( B A K E R C H E M I C A L , R E A G E N T G R A D E ) IN O E I O N I Z E D ; D I S T I L L E D W A T E R . 1 0 J J J - O F D A N S Y L C H L O R I D E ( 1 - 0 I M E T H Y L A M I N 0-N A P H T H A L E N E - 5 ~ S U L F O N Y L C H L O R I D E , C A L B L O C H E M , L O S A N G E L E S ) IN F L S H E R R E A G E N T G R A D E A C E T O N E ( 1 M G / M L ) WAS T H E N A D D E D A N D T H E T U B E S E A L E D W I T H P A R A F I L M . T H E S A M P L E WAS T H E N I N C U B A T E D A T 3 7 ° C F O R 2 H O U R S A N D D R I E D IN A V A C U U M D E S I C C A T O R O V E R S U L F U R I C A C I D . A F T E R D R Y I N G 5 0 JUL O F C O N S T A N T B O I L I N G H Y D R O C H L O R I C A C I D WAS A D D E D A N D T H E T U B E D R A W N O U T A N D F L A M E S E A L E D . A F T E R F O U R H O U R S O F H Y D R O L Y S I S A T 1 0 5 ° C T H E T U B E W A S O P E N E D - 1 2 9 -A N D D R I E D IN A V A C U U M D E S S I C A T O R O V E R S O D I U M H Y D R O X I D E . T H E R E S I D U E WAS T H E N D I S S O L V E D IN 1 0 JAL O F A N A C E T O N E - G L A C I A L A C E T I C A C I D M I X T U R E ( 3 * 2 ) F O R A P P L I C A T I O N T O T H E P O L Y A M I D E P L A T E S U S E D F O R S E P A R A T I O N . TH E S Y S T E M U S E D F O R I D E N T I F Y I N G T H E D A N S Y L D E R I V A T I V E S P R O D U C E D WAS S I M I L A R T O T H A T O F W O O D S A N D W A N G ( 1 3 2 ) A S M O D I F I E D B Y H A R T L E Y ( 5 2 ) . T H E B A S I C P R O C E D U R E I S T W O -D I M E N S I O N A L , T H I N - L A Y E R C H R O M A T O G R A P H Y ON P O L Y A M I D E P L A T E S ( C H E N G - C H I N T R A D I N G CO . L T D . T A I P E I , T A I W A N ) U S I N G F O U R S O L V E N T S Y S T E M S W H I C H - A L L O W P O S I T I V E I D E N T I F I C A T I O N O F A L L N A T U R A L A M I N O A C I D D E R I V A T I V E S F R O M A S I N G L E S A M P L E . T H E O R I G I N A L 1 5 CM S Q U A R E P L A T E S A R E C U T I N T O N I N E 5 CM S Q U A R E P L A T E S W H I C H A R E U S E D T O R U N T H E S A M P L E S . TH I S R E D U C E S B O T H R U N N I N G T I M E A N O C O S T , T H O U G H S I N C E P L A T E S C A N B E W A S H E D A N D R E U S E D T H E C O S T F A C T O R I S N O T R E A L L Y S I G N I F I C A N T . 0.5 M L 0 F A S T A N D A R D S O L U T I O N C O N T A I N I N G 6 0 P I C O M O L E / ^ L L O F E A C H D A N S Y L A M I N O A C I D I S A P P L I E D T O O N E S I D E O F T H E P L A T E 0.5 CM I N F R O M T H E S I D E S IN O N E C O R N E R , A N D A S I M I L A R S A M P L E O F T H E U N K N O W N C O N T A I N I N G 1 0 TO 5 0 P M O L E S I S A P P L I E D T O T H E O P P O S I T E S I D E A T T H E . S A M E P O I N T . T H E F O U R S O L V E N T S Y S T E M S U S E D A R E L I S T E D B E L O W . 1. 1 . 5 ^ A Q U E O U S F O R M I C A C I D 2. B E N Z E N E , A C E T I C A C I D , 9*1 3. N - H E X A N E , N - B U T A N O L , A C E T I C A C I D , 3*3*1 4. 0.1 M A M M O N I U M H Y D R O X I D E , E T H A N O L , 9*1 TH E B A S I C T W O - D I M E N S I O N A L R U N C O N S I S T S O F S O L V E N T ( 1 ) IN D I M E N S I O N ( 1 ) F O R 3 T O 5 M I N U T E S , F O L L O W E D B Y S O L V E N T ( 2 ) IN D I M E N S I O N ( 2 ) F O R 5 T O 7 M I N U T E S . E X T R E M E C A R E M U S T B E T A K E N TO D R Y T H E P L A T E T H R O U G H L Y A F T E R S O L V E N T ( 1 ) . BY •P •V NH BH BK# M A0 OH aH R BH M« ' BK« G # # T # * K W # NY OY«E »S * H •D _Q±L ( A ) -M, t2. NH OH (B) t2, t3. NH >aH »R OH (C)-»-1, t2, t1 (0) t2, t 3 , t4. NH UK OH (E) t2, t4. F I G U R E 47. M I A G R A T I O N O F D A N S Y L D E R I V A T I V E S O F A M I N O A C I O S O N P O L Y A M I O E L A Y E R S I N V A R I O U S S O L V E N T S . S O L V E N T S A R E D E S C R I B E D I N T H E T E X T A N O W E R E R U N I N T H E D I M E N S I O N S I N D I C A T E D , S I N G L E L E T T E R N O T A T I O N F O R T H E P A R E N T A M I N O A C I D S A R E U S E D F O R M O S T D E R I V A T I V E S . A D D I T I O N A L A B B R E V I A T I O N S I N C L U D E ; OH, D N A S Y L H Y D R O X I D E ; NH, D A N S Y L A M I N E ; BY, B I S - D N S - T Y R ; BH, Bis - D N S-His; BK, B i S - D N S - L Y S ; C A , D N S - C Y S T E I C A C I D ; NY, N - D N S - T Y R ; OY, O - D N S - T Y R ; aH, a -DNS-Hls; eK, e - D N S - L Y S . -1 3 0 -F l G U R E 47 ( A ) S H O W S T H E P O S I T I O N S O F T H E F L O U R E S C E N T S P O T S V I S I B L E A F T E R T H E S E TWO S T A G E S . T H E D A N S Y L D E R I V A T I V E S O F P R O L I N E , I S O L E U C I N E , L E U C I N E , V A L I N E , P H E N Y L A L A N I N E , G L Y C I N E , M E T H I O N I N E , T R Y P T O P H A N A N D A L A N I N E C A N B E P O S I -T I V E L Y I D E N T I F I E D A T T H I S P O I N T . T H E D I - D A N S Y L O E R I A T I V E S O F T Y R O S I N E , L Y S I N E A N D H I S T I D I N E C A N A L S O B E I D E N T I F I E D , A S C A N D A N S Y L A M I N E A N D D A N S Y L H Y D R O X I D E . IF I D E N T I F I C A T I O N C A N N O T BE M A D E A T T H I S S T A G E S O L V E N T ( 3 ) M A Y B E R U N IN D I M E N S I O N ( 2 ) F O R A B O U T 8 M I N U T E S TO S E P A R A T E T H E D E R I V A -T I V E S O F G L U T A M I C A C I D F R O M A S P A R T I C A C I D : T H R E O N I N E F R O M S E R I N E ? A R G I N I N E F R O M E - L Y S I N E A N D 0 1 — H I S T I D I N E ; A N D C Y S T I N E , O C - T Y R O S I N E A N D 0 - T Y R O S I N E F R O M D A N S Y L H Y D R O X I D E . 7*HE P O S I -T I O N S O F T H E S P O T S A T T H I S S T A G E A R E S H O W N IN F l G U R E 4 7 ( B ) . AT T H I S S T A G E T H E C Y S T E I C A C I D D E R I V A T I V E M A Y S T I L L BE H I D D E N B E N E A T H T H E D A N S Y L H Y D R O X I D E S T R E A K I F T H E S T R E A K I S L A R G E A N D T H E E - L Y S I N E A N D OC— H I S T I D I N E D E R I V A T I V E S A R E U N R E S O L V E D . D A N S Y L C Y S T E I C A C I D M A Y B E S E P A R A T E D F R O M D A N S Y L H Y D R O X I D E B Y R U N N I N G S O L V E N T ( 1 ) IN D I M E N S I O N ( 2 ) F O R 5 T O 5 M I N U T E S ( F I G U R E 4 7 C ) . TH E D E R I V A T I V E S O F E - L Y S I N E A N D CC— H I S T I D I N E M A Y B E S E P A R A T E D B Y R U N N I N G S O L V E N T ( 4 ) I N D I M E N S I O N ( 2 ) F O R 3 T O 5 M I N U T E S ( F I G U R E 4 7 D ) . CC— H I S T I D I N E , E - L Y S I N E A N O A R G I N I N E D E R I V A T I V E S M A Y A L S O B E S E P A R A T E D B Y R U N N I N G S O L V E N T ( 4 ) I M M E D I A T E L Y A F T E R S O L V E N T ( 2 ) , A N D I F T H E S E A R E T H E O N L Y E X P E C T E O D E R I V A T I V E S T H I S P R O C E D U R E I S P R E F E R A B L E S I N C E I T G I V E S A B E T T E R S E P A R A T I O N A S S H O W N IN F l G U R E 4 7 ( E ) . T H E P L A T E S M A Y BE W A S H E D F O R R E U S E BY S T A N D I N G O V E R N I T E IN S O L V E N T (1) A N D W A S H I N G T W I C E IN A M I X T U R E O F 1 M A M M O N I U M - 1 3 1 -H Y D R O X I D E A N D A C E T O N E , 1:1. THE S T A N D A R D D A N S Y L A M I N O A C I D S U S E D WERE P R E P A R E D FROM T H E A M I N O AC I D S ( C A . L B I O C H E M , Los A N G E L E S ) B Y T H E F O L L O W I N G P R O C E D U R E . 6.5 /J.MOLES O F AN A M I N O A C I D WERE D I S S O L V E D I N 1 ML O F 0.1 M S O D I U M B I C A R B O N A T E . S O L U T I O N ANO M I X E D W I T H 1 ML O F D A N S Y L C H L O R I D E I N A C E T O N E ( 6 M G / M L ) . T H E M I X T U R E WAS A L L O W E D TO S T A N D O V E R N I T E A N D T H E S O D I U M B I C A R B O N A T E P R E C I P I T A T E D W I T H 8 ML O F A C E T O N E . T H E S T O C K S O L U T I O N S T H U S C O N T A I N E D 6 5 0 P M O L E S O F D E R I V A T I V E P E R J 4.L. AN A L I Q U O T O F T H E H I S T I D I N E S A M P L E WHICH C O N T A I N E D P R I M A R I L Y T H E B I S - . H I S T I D I N E D E R I V A T I V E WAS H Y D R O L Y S E D IN 5.7 M H Y D R O C H L O R I C A C I D F O R 1 7 H O U R S TO P R O O U C E T H E CC-H I, S T I 6 I NE D E R I V A T I V E ( 4 7 ) . S - D A N S Y L - L Y S I N E WAS P U R C H A S E D FROM C A L B l O C H E M , L O S A N G E L E S A N D M A D E U P TO A S I M I L A R C O N C E N T R A T I O N I N A C E T O N E . T H E A B O V E S O L U T I O N S WERE M I X E D I N E Q U A L V O L U M E TO P R O D U C E T H E S T A N D A R D S O L U T I O N U S E D I N T H I N - L A Y E R C H R O M A T O G R A P H Y . F. P O L Y A C R I L A M I DE GEL E L E C T R O P H O R E S I S . P O L Y A C R I L A M I DE G E L E L E C T R O P H O R E S I S WAS C A R R I E D O UT U S I N G T H E M E T H O D O F J O H N S ( 6 1 ) . T H E G E L S WERE P R E P A R E D I N 5 X 7 5 MM G L A S S T U B E S U S I N G 1 ML OF A S O L U T I O N C O N T A I N I N G 1 0 ML O F MONOMER S T O C K (409$ w/v A C R I L A M I OE » 0 . 6 $ w/v N , N 1 - M E T H Y L E N E B I S A C R I L A M I D E I N D I S T I L L E D W A T E R ) , 1 0 ML O F C A T A L Y S T 1 ( 0 . 5 $ v/v N , N , N ' N ' - T E T R A M E T H Y L E N E D I A M I N E I N 4.6 N A C E T I C A C I O ) A N D 6 ML O F C A T A L Y S T 2 ( 0 . 6 $ w/V AMMO-N I U M P E R S U L F A T E ) F O R E A C H T U B E . O R G A N I C R E A G E N T S WERE P U R C H A S E D FROM E A S T M A N O R G A N I C C H E M I C A L S , R O C H E S T E R , N.Y., -1 32-A N D A M M O N I U M P E R S U L F A T E WAS F l S H E R R E A G E N T G R A D E . T H E G E L S O L U T I O N WAS D E G A S S E O F O R 3 0 M I N U T E S IN A V A C U U M O E S S I C A T O R B E F O R E B E I N G P L A C E D IN T H E T U B E S . D I S T I L L E D W A T E R WAS L A Y E R E D C A R E F U L L Y ON T H E T O P O F T H E G E L W I T H AN I N F U S I O N P U M P TO E L I M I N A T E T H E M I N I S C U S , A N D T H E G E L S A L L O W E D T O P O L Y M E R I Z E . E L E C T R O P H O R E S I S WAS C A R R I E D O U T IN A S H A N O O N M O D E L 1 2 7 3 4 A P P A R A T U S ( C O L A B C A N A D A L T D . , W E S T O N , O N T A R I O ) . G E L S W E R E P L A C E D IN T H E A P P A R A T U S S O T H A T S A M P L E M I G R A T I O N WAS T O W A R D T H E C A T H O D E A N D T H E G E L S W E R E E Q U I L I B R A T E D W I T H 0 . 0 1 M A C E T I C A C I D F O R 3 H O U R S A T 3 2 V O L T S . A F T E R T H E E Q U I L I B R A T I O N B U F F E R WAS R E P L A C E D W I T H F R E S H B U F F E R 2 0 JA.G O F P R O T E I N D I S S O L V E D IN 1 0 JJ.L O F 1 M S U C R O S E IN 0 . 0 0 2 M A C E T I C A C I D WAS A P P L I E D T O T H E T O P O F T H E G E L A N D T H E A P P L I C A T I O N Z O N E S H A R P E N E D IN A 3 2 V O L T F I E L D F O R 2 5 M I N . E L E C T R O P H O R E S I S WAS-'-, T H E N C A R R I E D O U T A T 3 2 0 v F O R 7 0 M I N A N D T H E T U B E S R E M O V E D . T H E T U B E S W E R E T H E N C R A C K E D I N A V I S E A N D T H E G E L S P L A C E D IN 1 2 . 5 $ TCA F O R 3 0 M I N T O F I X T H E P R O T E I N F O R S T A I N I N G . T H E S T A I N U S E D WAS 0 . 0 5 $ (w/v) C O O M A S S I E B R I L L I A N T B L U E R 2 5 0 ( C O L A B L A B O R A T O R I E S , I N C . , C H I C A G O H E I G H T S , I L L I N O I S ) IN 1 2 . 5 $ TCA ( 2 2 ) . A F T E R 1 H O U R I N T H E S T A I N I N G S O L U T I O N T H E G E L S W E R E O E S T A I N E D O V E R N I T E l N 1 2 . 5 $ TCA. -1 33-G. S i N G L E L E T T E R N O T A T I O N F O R AM I N O A C I D S . (59) A - A L A N i N E B - A S P A R T I C A C I D OR A S P A R A G I N E C - C Y S T E I N E D - A S P A R T I C A C I D E' - G L U T A M I C A C I D F - P H E N Y L A L A N 1 N E G - G L Y C 1N E H — H I S T I D I N E I - I S O L E U C I N E K - L Y S 1 N E L - L E U C I N E M - M E T H I O N I N E N — A S P A R A G I N E P - P R O L I N E Q - G L U T A M I N E R - A R G I N I N E S - S E R I N E T - T H R E O N I N E V - V A L I N E W- T R Y P T O P H A N X - UN K N O W N OR " O T H E R " Y - T Y R O S I N E Z - G L U T A M I C A C I D OR G L U T A M I N E 

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