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

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THE  ISOLATION  SALMON  AND  CHARACTERIZATION  ULTIMOBRANCHIAL  OF  CALCITONIN  BY  RONALD A.B.,  A  UNIVERSITY  THESIS THE  OF  KEITH  CALIFORNIA,  SUBMITTED  IN  REQUIREMENTS DOCTOR  IN  THE  O'DOR  PARTIAL FOR  OF  THE  BERKELEY*  1967  FULFILMENT  OF  DEGREE  OF  PHILOSOPHY  DEPARTMENT  OF  PHYSIOLOGY  WE  ACCEPT  TO  THE  THIS  REQUIRED  THESIS  AS  CONFORMING  STANDARD  SuPERVISOR  EXTERNAL  THE  UNIVERSITY  OF  EXAMINER  BRITISH  FEBRUARY,  1971  COLUMBIA  In p r e s e n t i n g t h i s  thesis  in p a r t i a l  fulfilment of  the requirements f o r  an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e I  f u r t h e r agree t h a t p e r m i s s i o n  for  I agree  that  r e f e r e n c e and study.  f o r e x t e n s i v e copying o f t h i s  thesis  f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s of  representatives.  this  thesis  It  is understood that copying o r p u b l i c a t i o n  f o r f i n a n c i a l gain s h a l l  not be allowed without my  written permission.  RONALD  Department  of  PHYS IQLOGY  The U n i v e r s i t y o f B r i t i s h Vancouver 8, Canada  Date  BEBRUARY  16,  1971  Columbia  K E I T H  O ' D O R  ABSTRACT  ALTHOUGH HORMONE, GLANDS, WHICH BUT  THE  F I R S T  C A L C I T O N I N  ( C T ) , WERE  H I S T O L O G I C A L  EVIDENCE  PRODUCE  ALSO  THE  OCCUR  HORMONE  IN  THE  MAMMALS.  THESE  C E L L S  BRANCHIAL  POUCH  AND  U L T IM O B R A N C H IA L A C T I V I T Y . E V I D E N C E  THE THAT  STRUCTURALLY THYROID  P R E P A R A T I O N S  GLAND WORK  THIS  STRUCTURAL  AND  WHICH  ALSO  A C T I V I T Y  IN  RAT  THYROID  THE  "C"  C E L L S  TO  AND  T I S S U E S  THYMUS  THEY  THIS  AND.EXPLORES  A  OF  THE  LAST  SEPARATE  PROVIDES  P O L Y P E P T I D E S  FROM  MAMMALIAN  R E L A T I O N S H I P  D I F F E R E N C E S  GLAND,  HYPOCALCEMIC  T H E S I S  ISOLATEO THE  FORM  FROM  THIS  FROM  CONTAINS  RESULTS  THOSE  FUNCTIONAL  FROM  R E S T R I C T E D  NON-MAMMALS  TO  THAT  HYPOCALCEMIC  EMBRYOLOGICALLY  D E S C R I B E D  S I M I L A R  T I S S U E S  NOT  PARATHYROID  IN  THE  EXTRACTED SHOWED  ARE  ARISE  OF  OF  THE  BETWEEN TWO  THE  T Y P E S  OF  CT. A  SURVEY  BOVINE,  OF  PORCINE  BRANCHIAL  FOUR AND  T I S S U E S  MAMMALIAN  MURINE)  ( T U R K E Y ,  THAT  THESE  P O L Y P E P T I D E S  WITH  MOLECULAR  MINED  F I L T R A T I O N .  AN  GEL  ORGANIC  THE  SOLVENT  A C T I V I T Y  ON  A  S P E C I F I C  HIGHER  WAS  A  COLLECTED  FRESH  ON  T I S S U E S WEIGHT  LARGE II  OF  B A S I S  SALMON AND  FOR MORE  AND  AND  THE  ULTIMO-  D O G F I S H )  HYPOCALCEMIC  AB0UT  EXTRACTS  Y I E L D E D  (HUMAN,  NON-MAMMALIAN  CONTAINED  DEVELOPED  SCALE  T I S S U E S  SALMON  WEIGHTS  WHEN  A C T I V I T Y . A  FOUR  T I S S U E S  MIXTURE  ULTIMOBRANCHIAL  ANO  C H I C K E N ,  DEMONSTRATED  BY  THYROID  WERE THE  4 0 0 0  AS  DETER-  PREPARED  U S I N G  THYROID  CT' S  HYPOCALCEMIC FINAL  PRODUCT  U L T I M 0 B R A N C H I AL EXTRACTED  TO  HAD  T I S S U E  PROVIDE  MATERIAL  FOR  CHEMICAL  GEL  FILTRATION  TWO  ION-EXCHANGE  AT  TWO  15  MG  PH'S OF  STAGES  U L T I M O B R A N C H I AL A  DISULFIDE  ARE OF  ALSO  THE 50 AND IN WAS  BRIDGE  THE  MORE  MUCH  FROM  HUMAN  ENZYME TIFIED.  WAS  THE  THAN  CT.  AND  PLASMA  SHOWED  ON BY  OF  BUT  SALMON  THE  PORCINE  THAT  THESE III  NUMBER  THAT  TESTS  SALMON  SURVEY  A  SELECTIVE  ON  COHN  M'l-O,  DEGRADING  CT  A  OF  I V-1  PORCINE "CALCI-  DEAE-SEPHADEX,  ENZYME  RAPIDLY  EFFECT  ON  WAS  DIGESTED  WITH  WERE  ISOLATEO  AND  PEPTIDES  NEARLY  BIOASSAY  SUGGESTING A  SIGNIFICANT  IN  C T WAS  FRACTIONS  IV—1  THIS  PEPTIDES  A  PROLONGED.  CT' S  RAPIDLY  CT  ARE  STANDARD  WAS  CHROMATOGRAPHY  NO  WITH  FEATURES  REFLECTED  INDICATED  FRACTION  HAD  THE  POLYPEPTIDE  ALSO  POTENCY.  CM-SEPHADEX.  OF  IN  'THYROID  CAPABLE  RESULTANT  NATURE  THE  PARTIAL  STRUCTURE.  WERE  CT  YIELDED  ALTHOUGH THESE  HORMONE  GREATER  SYNTHETIC  THE  THE  THAN  PURIFIED  CT,  HUMAN  WITH  THAT  C T ' S , THERE  CT  A N D JI_N V I TRO  ITS  ACIO  HORMONE.  SALMON  STUDIES  AND  THE  THREE  AND  AND  INDICATED  MAMMALIAN  OF  OF  SE-SEPHADEX C - 2 5  ANALYSIS  3 2 AMINO  SALMON  SERIES  PURIFICATION  ACID  DIFFERENCES  ENZYMES  G-200  A  ON  C-TERMINUS.  THE  THE  FOR  PORCINE  HUMAN  TO  REASON  TONINASE"  OR  IN  STABLE  FURTHER  TIVATED  ALL  ACTIVITY  I V-4 CONTAINED  SEPHADEX  THE  B O T H J_N V I V P  FRACTIONS  IN  AF  FOLD  PEPTIDES  WAS  A  G - 5 0 ALTERNATING  STAGES  AMINO  TRYPTIC  ACTIVE  MORE  POSSIBLE  CT.  TO  RESPONSE  PLASMA  CT.  STRUCTURAL  BIOLOGICAL TIMES  OF  FEATURES  THESE  SEPHAOEX  300,000  HORMONE  COMMON  UNIQUE  A  SALMON  CHARACTERIZATION  ON  CHROMATOGRAPHY  PROVIDED  PURE  CHARACTERIZATION.  INDICATED  THAT  INACSALMON THE IDENTHE  ENZYME SIDE  WAS  A PEPTIOASE  OF A R G I N I N E  NATIVE  SALMON  PARTIAL  WITH  RESIDUES.  C T PRODUCED  EXPLANATION  FOR  SIMILAR  THE  THESE  EXPERIMENTS  WOULD  NOT  SPLIT  AT  BONDS  ASSOCIATED  CATED  THAT  AND  THE  PROBABLY  WEIGHT  OF  WITH  ENZYME  WAS  ABOUT  OF  AVAILABLE  CATED  DATA  HAD  " C A L C I TON I N A S E "  ANY  OF  TO  FROM  RESIDUES  OF NOT  THAT  FOR  OTHER  APPEAR  MEMBERS  IV  OF  THE  ACTION  FAMILY TO BE  THIS  LEAST A  THIS ENZYME NOT  CLEAVE  DATA  OF ABOUT  INDI30,000  A MOLECULAR OF  THROMBIN.  KALLIKREIN  AT  OF  WITH  PLASMA  PURE  WOULD  WEIGHT  THE  WAS  NOT  AND  CARBOXYL  OF  THE  RESIDUES.  IT  DOES  THAT  A PRECURSOR  TESTS  THE  STUDIES  STABILITY  SHOWED  BASIC  THE  PROVIDING  A MOLECULAR  THAT  WITH  THE  WELL  OTHER  100,000.  SIMILARITIES  THE  ARGININE  DERIVED  ON F I B R I N O G E N SHOWED  GREATER ALSO  FOR  DIGESTIONS  NO P E P T I D E S  HORMONE.  ALL  A SPECIFICITY  THE  ENZYME  COMPARISON ENZYMES  INDI-  OF E N Z Y M E S ,  IDENTICAL  GROUP.  WITH  BUT  TABLE  OF  CONTENTS PAGE i i  ABSTRACT .  L I S T OF  TABLES  LIST  ILLUSTRATIONS  OF  .  .  i x x  .  ACKNOWLEDGMENTS  xiii  CHAPTER I. II.  INTRODUCTION MOLECULAR  1  WEIGHT  ULTIMOBRANCHIAL  AND  A.  INTRODUCTION.  B.  EXTRACTION. 1.  2. C.  D.  CALCITONINS EXTRACTED THYROID  TISSUES  6 6  . . . . .  . . .  METHODS  8 .  A.  ISOLATION  B.  EXTRACTION  c.  BIOLOGICAL ASSAY  RESULTS  FROM  . . .  AND  C A L I B R A T I O N OF 1.  III.  OF  AND  PREPARATION  OF  GLANDS  PROCEDURES PROCEDURE  13 16  COLUMNS  20  METHODS  20  2. R E S U L T S AND D I S C U S S I O N E S T I M A T I O N OF M O L E C U L A R W'EIGHTS OF CEMIC FACTORS 1 .  METHODS  2.  RESULTS  ISOLATION TONIN .  AND  8 11  DISCUSSION. SEPHADEX  8  22 HYPOCAL23 23  AND  DISCUSSION.  C H A R A C T E R I Z A T I O N OF  A.  INTRODUCTION.  .  B.  PRELIMINARY PURIFICATIONS  24 SALMON  CALCI. . .  29 29  v  29  CHAPTER III.  PAGE  (CONTiNUED  C.  D.  1.  EXTRACT ION  29  2.  STAGE  1  CHROMATOGRAPHY  30  3.  STAGE  2  CHROMATOGRAPHY.  4.  STAGE  3  CHROMATOGRAPHY  31  5.  SUMMARY  OF  PURIFICATION  33  PREPARATION  OF  PURE  SALMON  .  .  .  .  .  .  CALCITONIN  .  .  31  . . .  .  33  1.  EXTRACTION.  2.  STAGE  1  CHROMATOGRAPHY  3.  STAGE  2  CHROMATOGRAPHY.  4.  STAGE  3  CHROMATOGRAPHY  37  5.  STAGE  4  CHROMATOGRAPHY.  37  6.  STAGE  5  CHROMATOGRAPHY.  .  7.  SUMMARY  OF  STAGES  39  8.  CRITERIA  .  40  2.  .  OF  HOMOGENEITY OF  THE  .  .  WHOLE  .  .  .  .  .  .  .  .  .  .  .  MOLECULE.  35  .  .  38  .  AMINO  B.  END  ACID  GROUP  41 41  ANALYSIS ANALYSIS.  RESULTS  AND  DISCUSSION.  PREPARATION  ANO  ISOLATION  1 .  .  M E T H O D S . . . . . . . A.  33 34  PURIFICATION  CHARACTERIZATION 1.  E.  )  OF  . .  41 43  .  .  .  .  TRYPTIC  ME T H O D S  .  .  .  .  PEPTIDES  43 45 45  A.  PERFORMIC  B.  AMINO  c.  DIGESTION  ACID  VI  ACID  OXIDATION  ANALYSIS WITH  TRYPSIN  45 46 46  CHAPTER I I.  PAGE (CONTiNUED )  2. F.  HIGH  E.  PAPER  RESULTS  2.  ELECTROPHORESIS.  AND  OF  AMINO  ACID  B.  DETERMINATION  c.  END  AND  OF  OF  .  51  IN C A L C I T O N I N S  59 59  I N A C T I V A T I N G ENZYMES  1.  S E L E C T I O N OF  2.  PRELIMINARY  3.  P R E P A R A T I O N OF TONINASE" . .  TESTS  TO  RELATIONSHIPS  P A R T I A L P U R I F I C A T I O N OF TONIN ASE"  E.  51  54  ASSAYS  5.  50  CALCITONINS,, .  B.  SUMMARY  . . .  MAMMALIAN  C.  4.  GROUPS  C A L C I T O N I N COMPARED  INTRODUCTION  SELECTIVE  AMIDE  ANALYSIS.  A.  D.  50  DISCUSSION.  SEQUENCES  FOR  50  ANALYSIS  SALMON  STRUCTURE-FUNCTION  48  50  GROUP  OF  48  TRYPTIC PEPTIDES. . . .  A.  SEQUENCE  47  DISCUSSION  .  RESULTS  . . .  CHROMATOGRAPHY  METHODS .  KNOWN IV.  VOLTAGE  CHARACTERIZATION 1.  G.  D.  OF  OF  A  . . . . . .  SELECTIVE  71  "CALCI74  STARTING  MATERIAL  74  PURIFICATION EXPERIMENTS. A HIGHLY  PURIFIED  .  "CALCI79  PURIFICATION  82  PURITY  I NACT I V A T I O N  76  83 OF  PORCINE  CT  BY  " C A L C I TON I N A S E " . . . . . . . . . . . . . .  84  P E P T I D E S FROM C A L C I T O N I N S D I G E S T E D WITH "CALCITONINASE" . . . . . . . . . . . . . .  85  1.  85  ME THODS VII  CHAPTER IV.  PAGE  (CONTiNUEO )  2.  A.  DIGESTION  B.  HIGH  c.  AMINO  o.  END  RESULTS  F.  COMPARISON  G.  GENERAL  WITH  VOLTAGE ACID  GROUP AND OF  ENZYME  85  ELECTROPHORESIS.  . . .  86  ANALYSIS  86  ANALYSIS.  .  86  DISCUSSION  86  " C A L C I T O N IN A S E "  TO  THROMBIN  .  DISCUSSION  89 90  BIBLIOGRAPHY.  .  96  APPENDICES A.  B.  STATISTICAL  CURVES  BY  A  DESIGN  FOR  SIMPLE  DEX C  METHODS  RESPONSE  FOR  CALCULATION  OF  A  LARGE,  INEXPENSIVE .  FOR  DOSE 107  COLUMN.  TECHNIQUES  LOG  COMPUTER  HIGH  VOLTAGE  PAPER  SEPHA.  .  .  .  110  ELECTRO-  P H O R E S is C.  AMINO  E.  PREPARATION DER  ACID  113 ANALYSIS.. AND  .  IDENTIFICATION  OF  .  .  .  .  P O L Y A C R i L AM i D E  G.  SINGLE  LETTER  .  . GEL  ELECTROPHORESIS.  NOTATION  V I M  FOR  AMINO  117  DANSYL  I VAT I VES  F.  .  .  *'  ACIDS.  128 131  .  .  .  .  133  LIST  OF  TABLES  TABLE  FACING PAGE  I.  SPECIFIC  BIOLOGICAL  U L T I M O B R A N C I AL I I.  BlOLOGICAL  EXTRACTS  ACT IV ITY  U L T IMOBRANC I AL III.  MOLECULAR FROM  IV.  AMINO  ACID  OF  THYROID  AND  .  17  YIELDS  FROM  THYROID  AND  TISSUES  WEIGHT  ELUTION  ACTIVITIES  18  ESTIMATES  VOLUMES  ANALYSES  ON OF  A  FOR  CALCITONINS  SEPHADEXG-50  FRACTIONS  FROM  COLUMN STAGE  5  CHROMATOGRAPHY  43  V.  AMINO  ACID  COMPOSITIONS  VI.  AMINO  ACID  ANALYSES  VII.  VIII.  TISSUES IX.  OF  OF  FIVE  TRYPTIC  CALCITONINS.. PEPTIDES  OF  AFTER  INACTIVATION WITH  HUMAN  1  2 5  l-CT  AND  INTERVENOUS OF  PORCINE  PLASMA  CT  FRACTIONS  IX  1 2 5  I-PTH  IN  .  51 52  RAT  INJECTION OURING  44  FROM  SALMON CT . . . NET CHARGES OF SALMON CT TRYPTIC PEPTIDES FROM ELECTROPHORETIC M O B I L I T I E S AND MOLECULAR WEIGHTS DISTRIBUTION  25  64 INCUBATIONS 76  LIST  OF  ILLUSTRATIONS  FIGURE 1.  LOCATION  2.  (A)  OF  AVIAN  COMPARISON  OF  R E S P O N S E FOR FROM A C E T O N E (B)  3.  (A)  (B)  OF  LOG  DOSE  ON 16  C O M P A R I S O N OF R E G R E S S I O N S O F L O G D O S E 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  REGRESSION  OF  STANDARD  .  .  LOG . .  R E G R E S S I O N OF E X T R A C T S FROM  DOSE  ON  RESPONSE  FOR  HOUSE  .  17  L O G D O S E ON T H Y R O I D AND  R E S P O N S E FOR E I G H T ULTIM0BRANCHIAL  . . . . . . .  17  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-50 AND G-75, I N C L U D I N G E L U T I O N VOLUMES 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 . .  5.  ELUTION  6.  ELUTION PROFILES S E P H A D E X G-50  7.  REGRESSIONS  GLANDS.  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 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  TISSUES. 4.  ULT I MOBRANCHIAL  FAC(NG PAGE . . . 9  PROFILES  OF  THYROID  EXTRACTS  ON  22  SEPHADEX  . 25  G-50 OFULTIMOBRANCHIAL  EXTRACTS  ON 25  C O M P A R I S O N OF E L U T I O N O F TWO B O V I N E T H Y R O I D D I F F E R E N T METHODS  PROFILES EXTRACTS  G-50 26  8.  ELUTION EXTRACT  P R O F I L E OF S A L M O N ON S E P H A D E X G-50*  9.  E L U T I O N P R O F I L E OF S T A G E C-25* P R E L I M I N A R Y S T A G E  10.  E L U T I O N P R O F I L E OF S T A G E PRELIMINARY-STAGE 3  11.  SUMMARY  OF  12.  ELUTION EXTRACT  P R O F I L E OF SALMON ON S E P H A D E X G-50t  13.  ELUTION PROFILE D E X C-25: S T A G E  PRELIMINARY  OF  ON S E P H A D E X P R E P A R E D BY  ULTIMOBRANCHIAL SOLVENT PRELIMINARY S T A G E 1. . 1 PRODUCT  ON  SE-SEPHADEX  PRODUCT  ON  BIO-GEL  2  2  PURIFICATION  STAGE  SALMON  ULTIMOBRANCHIAL S T A G E 1. . . . 1  PRODUCT  2. . . . . . . x  OF  ON  30 31  P-10 • 32 CT  33  ACID  . . . . 35  SE-SEPHA-  36  FACING PAGE  FIGURE .14.  ELUTION  ELUTION DEX  16.  17. 18.  19.  AND  21.  OF S T A G E  3 PRODUCT  ON  SE-SEPHA-  STAGE 4 . .  REGRESSION  THIN  ON S E P H A D E X 37  38  OF S T A G E 4  OF LOG DOSE  5 PRODUCTS LAYER  PRODUCT  ON S E P H A D E X 38  OF P U R I F I C A T I O N  SUMMARY  S I L I CA 20.  PROFILE  C-25:  ELUTION PROFILE G - 5 0 * STAGE 5 .  3  OF S T A G E 2 PRODUCT  STAGE 3  G-50: 15.  PROFILE  .  .  OF  SALMON  CT  ON R E S P O N S E  39  FOR  STAGE 1 ,  .  39  CHROMATOGRAPHY  OF S T A G E  5 PRODUCT  ON  GEL..  O U T L I N E OF P R O C E D U R E S USED TO P E P T I D E S FROM SALMON C T . .  ISOLATE  PH 6 . 5 E L E C T R O P H O R E T O G R A M OF SALMON  "•  4 0  ...  49  TRYPTIC  TRYPTIC  CT  PEPTIDES  49  22.  PH  23.  PAPER  24.  PH  . . . .  49  25.  L O G A R I T H M I C P L O T OF E L E C T R O P H O R E T I C M O B I L I T Y , ( A T PH 6 . 5 , R E L A T I V E TO A S P A R T I C A C I D ) V E R S U S M O L E CULAR WEIGHTS FOR P E P T I D E S  5 0  L O G A R I T H M I C P L O T OF E L E C T R O P H O R E T I C M O B I L I T Y PH 1 . 9 * R E L A T I V E TO S E R I N E ) V E R S U S M O L E C U L A R WEIGHTS FOR P E P T I D E S  5 0  26.  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 CHROMATOGRAM  OF R E G I O N  A-1  27.  SALMON C T T R Y P T I C  28.  A L I G N M E N T OF SALMON AND HUM AN C T  29.  P O S S I B L E C O N F I G U R A T I O N OF SALMON FROM A M O L E C U L A R M O D E L ,  30.  31.  C T WITH  ALIGNED  (AT  HUMAN  C T , BOVINE  C T AS  CT CT  53  54  DETERMINED 58  OF P O R C I N E MODEL '.  POSSIBLE CONFIGURATION FROM A M O L E C U L A R MOOEL  OF HUMAN  XI  C-1.  WITH  PORCINE  POSSIBLE CONFIGURATION MINED FROM A M O L E C U L A R  1  49 49  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  PEPTIDES  A-2. . . . .  C T AS  DETER58  C T AS .  DETERMINED . . . . .  58  FAC FIGURE 32.  DISAPPEARANCE INJECTION  33.  AREA CT  34.  RATS.  PLOTS  OF  LOG  40.  DOSES  OF  VARYING  VERSUS  AREA  DOSES  OF  HUMAN  RESPONSE  FOR 6J  CALCITONINS  COMPARISON  OF  IN  IN  RAT  CT  IN  ELUTION  NORMAL  IV—1  ELUTION  SERUMS  VITRO  TO  SEVERAL  SPECIES  SEVERAL  SPECIES.  THEIR  AN  EXTRACT  OF  SALMON  AND  OF  RATS  HUMAN  .  .  .  .  .  7 7  G-200  PRODUCT  ON  CM-  C - 2 5  8 0  PROFILE  OF  CM-SEPHAOEX  PRODUCT  ON  SEPHA-  G-200  SUMMARY  81  OF  PURIFICATION  PROCEDURE  USED  TO  PREPARE  " C A L C I TON IN A S E " 4 1 .  42.  P H  1.9  CT  PRODUCED  8 2  ELECTROPHORETOGRAM  COMPUTER  BY  OF  END  PRINT  OUT  AND  PLOT  44.  1 0  45.  x  1 5 0  IMPROVED  GEL  CM  ON  OF  POLYAMIDE  LINEAR  REGRESSION  .  1 1 0  COLUMN  DETECTOR  IN  111  STAND.  FOR  .  AMINO  .  .  DANSYL LAYERS  1 2 3 DETECTOR  DIAGRAM.  .  DERIVATIVES IN  VARIOUS  X I I  1 1 2  ACID  DIAGRAM  ANALYSER—CIRCUIT  Ml A G R A T I ON  PORCINE  COLUMN—DIMENSIONS  FILTRATION  CIRCUIT  .  PHOTOTRANSiSTOR-LOGAMPL1FiER ACID  47.  1 0  PHOTOTRANSISTOR  ANALYSER 46.  FOR  CM  FROM  8 7  FROM  .  PIECES  PEPTIDES  "CAL C IT0N INA SE"  PROGRAM 43.  6 8  COHN  G-200  SEPHADEX  6 7  DISAPPEARANCE  N E P H R E C T O M IZ E D  SEPHADEX OF  FROM FROM  INACTIVATION  SERUM  OF  ON  PROFILE  IN  AND  PROFILE  ELUTION  DEX  FOR  6 0  6 7  INCUBATION  SEPHADEX 39.  CURVES  AFTER  CT  CALCITONINS  FRACTION 38.  CALCITONIN  L-PORCINE  CHICKEN RATES 37.  "INTACT"  .,  INACTIVATION DURING  36.  OF 131  OF  RESPONSE  IN  SEVERAL 35.  I NG  PAGE  . OF  .  FOR  AMINO  .  .  .  AMINO  SOLVENTS  .  .  .  1 2 6  ACIDS 1 3 0  ACKNOWLEDGEMENTS  I FOR  WOULD  AND  FOR  BECOMING  A  HIS  INTEREST  OF  FACULTY  KATHY  RALPH THE  THEIR  FOR  TO  FOR  MR.  HER  OF  AND  OF  TO  D.H.  BROWN  THE  PHYSIOLOGY AND  THESIS  DR.  J.C.  PARKES,  EXPERIMENTAL  THIS  THANK  DR.  STUDIES  TO  M.A.  IN  K.J.  DR. COOK  MRS.  INDEBTED  TO  FOR THE  FROM COPP  FOR H I S  OTHER  MEMBERS  THEIR MEMBERS  OF  THIS  THESIS "TO  LIKE  EFFORTS  PRESENCE, AND  MRS.  BIOASSAYS. KURT  AID  IN  FOR  HENZE  THE  AND  MR.  PREPARATION  OTHER  OF  MATERIAL  MENTION.  TO  IN  HIS CRITICAL  MR.  AND  FOR H I S  CORDY-SIMPSON  WITH TO  STEVENSON  FOR  JANE  INVALUABLE  ASSISTANCE A  TO  GRATEFUL  ASSISTANCE  THEIR  I WOULD  FINANCIAL FORM  DUE  TOO NUMEROUS  FINALLY, AND  GRADUATE  THE  PREVENT  LIKE  AM  NEWSOME,  THEIR  ILLUSTRATIONS  THE  ALSO  TO  DEPARTMENT  ARE  PERMANENTLY  CONTRIBUTIONS  IN  EFFORTS  DURING  C O .  ASSISTANCE.  FRANCIS  ASSINNA  PATIENCE  MY  ADVICE,  MRS.  AM  GUIDANCE  I  DR.  SUPERVISOR,  ENTHUSIASM,  THE  THANKS  PERRY I  AND  OF  FOR  METHOOICAL TO  AND  I WOULD  DURING  SPECIAL  AND  MY  ENCOURAGEMENT.  INSTRUCTION STAFF  HELP  NOVEL.  AND  THE  THANK  HIS VALIANT  ADVICE  THE  TO  HIS CONTINUAL  WORK  FOR  LIKE  THANK  MY  PREPARING  FROM  STUDENTSHIP  XIII  WIFE, AND  THE MEDICAL  IS GRATEFULLY  JANET,  TYPING  FOR  THIS  RESEARCH  HER  THESIS.  COUNCIL  ACKNOWLEDGED.  I.  THE  FIRST  CALCIUM  INTRODUCTION  CLEAR  EVIDENCE  CONCENTRATION  IN PLASMA  MACCALLUM  AND  FOLLOWING  PARATHYROIDECTOMY  WAS  1 6 YEARS  ROIDS  WAS  HORMONE UNTIL  CONFIRMED  ( P T H ) BY  CRAIG  ( 1 0 1 )  THE  FUNCTION  CHEMISTRY  OF  PARTIAL  LATING  RATE  AT  HORMONE,  OF  ISOLATED  NUMBER  OF  HORMONE  BOVINE  WHICH  9 )  -  THIS  SYNTHESIS  WORK  RESULTED  ( 5 0 , 1 0 7 ) OF  OF  BEGIN  WORK  THE  ( 6 ) AND CLEAR  YEARS  RASMUSSEN PICTURE  ( 4 2 ,9 3 * 5»  ON  1 2 0 ) THE AND  P T H ( 7 0 ) AND AT  IS  BY  A  PRESENT.  CALCIUM  PREPARED  REGU-  VERY  COPP  ET AL  IN 1 9 6 8 .  E_T A L _ ( 5 6 )  ISOLATION  OF LED A  OF THE  ( 1 2 7 » 9 9 » 9 8 ,6 2 ,  SEQUENCING FROM  OF  DIFFICULT,  AVAILABLE  HLRSCH  TISSUE  T H E HORMONE  3 4  C T , THE PREPARATION  BY  IT  PARATHY-  FURTHER  RECOGNIZED WAS  TETANY  PARATHYROID  IS ACCUMULATING  ONLY  OF  THE  THE  THE SECOND  THYROID  IN  A  PORCINE  HORMONE  THYROID  OF  EXTREMELY  OF  OF THE  CALCIUM.  ACTIVE  FAIRLY  OF  ( C T ) ,  I T WAS  TO  A  PROVED  KNOWLEDGE  RAT  MAMMALIAN  AN  EMERGE0  THE RECOGNITION FROM  IN BLOOD  P T H ( 9 5 ) ARE  CALCITONIN  LABORATORIES  FROM  HAS  HAS  2 7 ) IN 1 9 6 1 , S Y N T H E T I C  EXTRACTS  LINKED  AURBACH  ALTHOUGH  ( 3 0 ,  ACTIVE  BY  COMPOSITION  ALTHOUGH  AFTER  FALL  I N 1 9 2 5 ( 2 3 ) AND  GREATER.  YEARS  WHICH  OF  MUCH  TWO  A  EXTRACTION  T H E HORMONE  SEQUENCE  THE  TO  CONTROL  T H E WORK  BY  WAS  ACID  FROM  FUNCTION  T H E HORMONE  THE AMINO  CAME  THE ENDOCRINE  IN 1 9 5 9 .  OF  ENDOCRINE  I N 1 9 0 9 (71)  COLLIP  T H E HORMONE  AND  ONLY  VOEGTLIN  BEFORE  FOR  ( 8 ,  PORCINE  4 1 T  9 6 , 8 2 ) AND THYROID  -2TISSUE  AND  EVENTUALLY  SEQUENCES  FOR  HUMAN  C T (81)  WHILE WAS  THE  WORK ON  IN PROGRESS,  PROCEDED  APACE  AND  HORMONE  ARE  ON  THE  CELL  ORIGIN  PRESENT  THE  MAMMALIAN  T H I S "SECOND"  THAN  PTH.  THE  AS  BODY  (25),  C T MAY  RONMENT  FOR  BOVINE  TO  IN LAND  IN PHOSPHATES  FOR  THE  CLOSE  WHERE  THE  OIURNAL  e  ( 5 $ ) HAVE  SECRETION  BEEN  RATE  OF  IT  I S NOW  THAN  THE  THESE  OF  HORMONES PLASMA  FLUCTUATIONS IN PLASMA SHOWN  TO  FACED  ARE  RESULT  P T H ( 9 4 ) AND  IN  SECRETION  ( 2 0 ) AND  RELEASE- C A L C I U M ABSORPTION THE  STORED  I N THE  KIDNEY  (4).  GUT  ( 9 0 ) ANO  AN  INCREASE  IN B 0 N E ( 1 0 0 ) » ( 3 3 ) * TUBULAR TO  INCREASE  AN  OF ENVI-  RESPON-  LEVELS LESS  IN  THAN  THIS  ITS C I R C U L A T I N G  INCREASE  CALCITONIN  SEAWATER  MAG-  I N S I G N I F I C A N T R I S E S IN  IN  OF  IN  IN CALCIUM- ( 2 4 ) •  L E V E L S OF  CALCIUM  LEVEL  WITH  CALCIUM  IN PLASMA  THE  VERTE-  LEVELS  GENERALLY  CALCIUM  PRIMATIVE  I S PROBABLY  S I M I L A R L Y , DECREASES I N BOTH  APPARENT  MARINE  HIGH  R E L A T I V E L Y LOW TWO  STUDIES WAS  SURROUNDING  MAINTAIN  VERTEBRATES AND  PROPERTIES  CONCENTRATIONS  (4). AN  THESE  THAT C T  IN EARLY  CALCIUM  TO  REGULATION  INCREASES  ON  EVIDENCE  DEVELOPED  PRIMARILY  OF  FUNCTION  I S P H Y L 0 G E N E T I C A L L Y MORE  HAVE  HORMONE  (57, 102, 126, 1 2 5 ) .  C T LED  L E V E L S LOWER  CALCIUM  AND  INFORMATION  MAINTAINING  INTERACTION  5 /o ( 2 1 ) .  THE  OF  P T H ACTS  HIGH  THE  NITUDE  O R I G I N , CONTROL  AVAILABLE  HORMONE  F L U I D S AT  CIRCULATING  MAN  NOW  WELL  A MEANS  WHILE  SIBLE  ON  OF  I N NON-MAMMALS L A C K I N G P T H , AND  THAT  BRATES  CHEMISTRY  CONSIDERABLE  THE  OF  THE  STUDIES  OF  IN  AND  (17).  CT  ±  TO  LEVELS THE  INCREASE  R E A B S O R P T I ON URINARY  RESULT  CIRCULATING  I N P T H I N TURN TO  LEVEL  ACTS  TO  CALCIUM OF  EXCRETION  CALCIUM OF  PHOSPHATE CALCIUM IN  (23).  LEVELS  CALCITONIN  INHIBIT  BONE  ANO ON  CALCIUM.  RATHER  SLOW  BE  OF  AND  ITS  IN A  LESS FEW  THIS  THAN  PLASMA  RAPIDLY  LONG  SLOWER  OBSERVED  AS ABOUT  OF  THE  VALIDITY  ALL  CALCIUM  (114). RATS  PROVED  AND  THUS  IT  RESPONSE  IN  PLASMA  ONE  CALCIUM  CALCIUM  CONTRAST OCCUR  MATTER  OF  THAT  BASEO  ON  TWO  TO.MAINTAIN  COMPONENT AND  AGAINST THE  IN  A  CAN  HOMEOSTASIS  OPPOSITION  POINT.  THE  SEVERAL  MAY  IN  CONTROL  IS  LEVELS  (24).  TO  IN  PTH  FOR  NORMAL  FINE  TO  BEFORE  OCCUR  CALCIUM  ACHIEVING  HAS  FOR  MUNSON  IS  THE  THE  PRECISE  THERE  PROVED  OF  UNTIL  MAINTAINING  SUCH  UNNATURAL  LAVAGES  ( 4 6 )  IS  HAVE  PARATHYROIDS  STILL  OIFFICULT  CALCITONIN.  EXCEPT  FUNCTIONING  SET  SYSTEM  PERITONEAL  AND  OF  TO  INCREASE  DECREASE  HOUR  MORE  AN  ACTS  SECOND  BACKGROUND  OF  CONTROL  ANIMAL.  CAPABLE  STRESSES  GRAY  WITH  NEED  RETURN  A  PLASMA  PRIMARILY  CALCIUM  NOT  OR  IN  AN  MAXIMAL  ACTING  PLASMA  THIS AS  MAY  EXTREMELY  RAISE  INTACT  AS  A  ACTS  RESPONSE  PLASMA  HOURS THE  PRECISE TO  ON  PICTURE  OF  LOWER  ELEGANT  HAD  A  TERM TO  ANO  SYSTEMS  AT  PHYSIOLOGICAL  UNDER  ONE  24  THE  NEARLY  EFFECT  WITH  THE  COMPONENT  IN  ITS  SYSTEM  HOUR  IS  CALCIUM  ACTS  A  ONE  FEEDBACK  THE  THE  RAPIDLY  OF  INCREASE  LEVELS.  RESULTING  GENERALLY  FOR  TO  APPARENTLY  COURSE  MAXIMAL  (31).  OVER  HAND  ADMINISTRATION  TEND  PHOSPHATE  ( 2 , 100)  TIME  IS  ACTIONS  PLASMA  OTHER  PERSIST  SUGGESTS  NEGATIVE  IT  ACTS  HOURS  THE  THE  MAY  CALCITONIN  THESE  LOWER  THE  AND  OBSERVED.  HOURS  OF  RESORPTION  PLASMA  EFFECTS  ALL  WITH NOW MAY  TO  SOME  DEMONSTRATE  RECENTLY  PLASMA  SHOWN SHOW  CALCIUM THAT  THE  CALCIUM  PROCEDURES  HIGH  DOUBT  AS  PTH LEVELS  INFUSION  SOLUTIONS  THYRO I DECTOM I ZED  SIGNIFICANT  PLASMA  - 4 CALCIUM ARE  ELEVATION  OBVIOUS  EVER, THE  THIS  FACT  ANIMALS  IT  MAY  FRACTURE IN  BILITY CT  IS  STILL  AGE  TION,  THIS  BUT  TERMS  ACROSS  THE  THAT  IT  PLAY  ENDOCRINE  CLEARLY AND  OTHER  HORMONES  ABLE  FOR  OF  LITTLE  CT.  SUCH  IN  OTHER  MAMMALS  COULD  THAT  THAN  OLD  INFLUENCES OF  IN  IN  THE  CALCIUM  OF  INFORMATION  BE  SHOULD ANO  OF  POSSI-  CAN OF  A  TO  MUCH  BE  READILY  BONE  RESORP-  ROLE.  THERE  CALCIUM  TRANS-  INTRACELLULAR  METABOLISM THE  NEW  ( 1 2 ,1 0 4 ) SUGGESTING  ELUCIDATION  OF  OF  HAVE  OF  IN  OF  THE  AND  AS  ROLES  WELL OF  HOMEOSTASIS  IMPORTANCE  OF  THESE  CT.  CONSIDERABLE  SPECIES  RATE  THAT  RESPONSE  POSSIBLE  CELLS  PHYLOGENETIC  ANALYSIS  THEN  THE  CONTROL  LEVELS.  THE  THIS  ITS  VARIOUS  THE  SECOND  INHIBITION  FROM  DEPOSITION  ANIMALS  ( 3 1 ) .  AND  QUANTITIES  FORMATION  THAT  YOUNG  ROLE,  POSSIBILITIES  BONE  TH|S  HOW-  ( 4 ) SUGGESTS  THE  THE  OBSERVATIONS  ACTIONS  PROVIDED  1 1 1 ) » BUT  FUNCTIONS  ROLE  IN  ( 4 3 ) .  CHARACTERIZATION  ANALYSIS  (45»  LIFE  DETRACT  CT  SYSTEMS  POSSIBLE  HAS  INCREASING  THERE  SYSTEM.  MINOR  LEVELS  AMONG  CALCITONIN'S  NOT  THUS  SIGNIFICANT  ( 3 5 ) AND  AND  CT  CALCIUM  REQUIRES  THE  IN  A  IN  CALCIUM  IN  THE  MEMBRANES  EXTRACELLULAR  THESE  ROLE  MEALS.  RELATIVELY  IMPORTANCE.  TO  THAT  A  DETECTED  EARLY  OF  CALCIUM  INSULIN-GLUCAG0N  BE  PLASMA  BY  DOES  EVIDENCE  MAY  TO  DEPENDENT  PORT  AS  IN  HIGH THE  ADULTS  SENSITIVITY IN  ALSO  A  IN  ANO  EXPLAINED  IS  BEEN  SUPPORTED  VERY  BE  NORMAL  HEALING UTERO  GREATER  CAN  ADDITIONAL  HAVE  IS  WITH  APPEARS  CT  WITH  HAVE  SUGGESTED  BONE  PARALLELS  THAT  IN  FOLLOWING  THE  ASSESSED.  ORIGIN  INSIGHT ALONG  OF  INTO  THESE  INDICATE  IN  THEIR  LINES  HOW  IMPORTANCE  VARIOUS  OF  THE  WAS  AVAIL-  HORMONE  SUCH  1 9 6 7 PEARSE  FUNCTION  AND  FUNCTIONS  CARVALHEIRA OF  THE  MAMMALIAN  BRANCHIAL GLAND, 29*  WHICH  IN  TAUBER  (124)  ULTIMOBRANCHIAL  VERTEBRATES,  INCLUDING  RELATIONSHIP  BETWEEN  MAMMALIAN THE POINT,  THAT  THE  EMBRYOLOGICALLY  LOWER  VERTEBRATES  IN T H E  WORK  AND  WAS  AIMED  ULTIMOBRANCHIAL  GLANOS  SEVERAL  WAS  AT  FACTORS  OF  CHICKEN  ESSENTIALLY IN  A  THE  TO THE  SURVEY  OF  CERTAIN  FEATURES  FROM  NUMBER  OF  A  THE  OF  ULTIMOBRANCHIAL  STRUCTURE  TO  BEGUN  AMINO  THE  UNKNOWN,  THAT  BUT  AVAILABLE. OUTCOME  OF  THIS  SERIES THE  AND OF  OF  THE  WERE  OR  OBSERVED.  A  APPEARED  FOUND  FACTORS  BUT  THE  AND  THE  AT  THIS  THE  TIME  PORCINE  IN  A  THE  ONE  ITS WAS  WAS  NUMBER SOON  DEPENDED  WHETHER  STILL OF 8EC0ME ON  ULTIMO-  DESCRIBES  RELATIONSHIP  DIFFERENCES  THE  THERE  THE  CHAPTER  THE  THE  A  OF  WORK  BETWEEN  FINAL  CLARIFY  OF  WOULD  AND  TO  FACTORS  STUDY  THIS  STUDIES  WORK,  TO  THE  BE  MOLECULE  UNDERWAY  THE  OF  TO  COMPARISON  SIMILARITIES  TO  (28,  AL  RELATIONSHIP  DETAILED  FURTHER  CALCITONINS. DESIGNED  BEGAN  INFORMATION  SEQUENCE  DIFFERENCES  ACTIVITY  OF  ANY  ET  HYPOCALCEMIC  ALLOW  THIS  DIFFERENCES  EXPERIMENTS  STRUCTURAL  BIOLOGICAL  THAT  FACTORS  THEIR  BY  AT  STUDIES  COMPARATIVE  THYROID  TO  CoPP  DOGFISH,  STUDY  THE  CT.  SEQUENCE  DIRECTION  SIGNIFICANT  BRANCHIAL A  ACID  PORCINE  SUGGESTING THE  OF  OF  OF  TERMINAL  UNKNOWN.  APPROACH  FOLLOWED  FACTORS  INTENSIVE  LABORATORIES  WERE  SPECIES,  BEST  THE  SEPARATE  CHARACTERIZATION  ESTABLISH  CALCITONINS.  A  CELLS  NON-MAMMALIAN  THE  PRESENT  CHEMICAL  FROM  "C"  HYPOCALCEMIC  ULTIMOBRANCHIAL  MAMMALIAN  BASIC  AND  OR  FORMS  YEAR  POTENT  THESE  DESCRIBED  SAME  EXTRACTED  THE  CALCITONINS  PARAFOLLICULAR  ARISE  ULT I MOBRANCHIAL.  AND  THE  SHOWED THYROIO  POUCH  THE  32)  FROM  (91)  IN  I I .  MOLECULAR  WEIGHTS  BRANCHIAL  A.  INTROOUCTI  THAT  WORK  THE  ULTIMOBRANCHIAL  CONFIRM  THAT  (49»  WEIGHTS WOULD BE  THESE  THAT 99)  OF  IT  SUCH  GLANDS  ON  OF  FOR  HYPOCALCEMIC  THE  ACTIVE  SINCE OF  THE  LESS *PART  AND  HAD  VERY THE  CHEMICALLY  A  MOLECULAR  THAT  WAS  AND  THIS  CT  NECESSARY TO  WEIGHT OF  THYROIO  OF  CHROMATOGRAPHY  OF  EXTRACTS  MOLECULAR TO  WHICH  DETERMINE  HAD  (3)  WEIGHTS  BEEN  USING  THE  THE  MOLECULAR  CALCITONINS  COMPARISON  COLUMN  TO  ABOUT  THE  OF  SEPHADEX  FROM  INFORMATION  COMPARISON  FACTORS  OF  SIMILAR  HAD  CHROMATOGRAPHY CHICKEN  SHOWN  NEAR  THE  SEPHADEX  THE  WORK  ALL  TOTAL  WEIGHT  OF  ELUTION  EXTRACTS  ULTIMOBRANCHIAL  THAT  RELATIONSHIP  POROUS OF  EVIDENCE  VERTEBRATES  TYPE  ACTIVITY  MOLECULAR  PUBLISHED  THAT  INDICATED  COULD FROM  THE  CALIBRATED THE  BIOASSAY  VOLUME  OF  MOLECULES.  DOGFISH  ELUTED  A  TO  CONSIDERABLE  FELT  BY  KNOWN  PRELIMINARY  (32)  ULTIMO- .  HAD  LOWER  WAS  EVIDENCE.  VARIOUS  MARKERS  FROM  (124)  CERTAIN  SIMILAR  WERE  ULTIMOBRANCHIAL  PROVIDE  OF  FURTHER  CT  WAS  EASILY  G-75  BUT  PORCINE  AND  AND  BODIES  THERE  FAIRLY  AND  TAUBER  FACTORS  MADE  WITH  29)  ACTIVITY  THYROID,  HORMONES.  SUGGESTING  EXTRACTED  T I S S U E S . *  (28,  HYPOCALCEMIC  MAMMALIAN  3000  COPP  OF  MAMMALIAN  C A L C I T O N I N S THYROID  ON.  THE  CONTAINED THE  OF  AND  OF  BETWEEN  WAS  DESCRIBED  IN  ) OF  T  ELUTION  BECOMES  G-50  ( V  VOLUME  THIS  (86).  -6-  FOR  ON  WERE  COLUMN,  AND  AND  THE  LOG  (3)  THE  NEAR  MOLECULAR  CHAPTER  THYROID  SEPHADEX  MOLECULES  THE  NON-LINEAR  CHOSEN  PORCINE  GLANDS  THE ! A CT IVE  VOLUME  OF  WAS  WEIGHT  PREVIOUSLY  ESTIMATION. WEIGHT  RELATIONSHIP  ADEQUATELY ABLE  IN  IN  THE  NECESSARY AGAINST  LINEAR  TO  SEPHAOEX AND  RANGE  SMALL  THE WAS  ON  THAT  HAD  NOT  BEEN  OF  LIMITED.  THE  THEY  VOLUME-MOLECULAR  NUMBER  MARKERS  MARKERS  VERIFY  THE  MOLECULAR  THE  THYROID ANO  OF  EARLIER  WORK  AND  WHICH  WOULD  DESCRIBEO  MARKERS IT  WERE  BETTER  CT  WAS  AVAILTHEREFORE  AVAILABLE  CHARACTERIZED  YIELD  WHEN  PROBLEMS  THE  DESIRED  ULTIMOBRANCHIAL  OF  AS  AN  EVEN  GREATER  RASMUSSEN  PARTIAL  HYDROLYSIS  TISSUE  SAME  LARGE  HANDLE  GENERALLY  AND  TO  (48)  PORCINE  CT  REPORT  AQUEOUS  WHICH  LARGE  METHOD  A  PRODUCED  THE  A  NUMBER  OF  DISSOCIATION EXTRACTION  EXTRACTION IS  OF  THAT  ARE  UNSE-  COMPONENTS,  HIGHLY  CT.  SUCH  WHICH  RELATIVELY  SOLVENT A  SMALL  SOLUTIONS  IS  ON  DISCUSSED  SCALE  PROTEIN A  3)  ON  THE  OF  DEVELOPED  ON  INCOMPLETE  TO  POSSIBLE  BASED  (10  VOLUMES  HAD  AS  EJR_ A L .  A  NUMEROUS  BEEN  USEFUL  ON  CONTAIN  AN  THE  HAD  AND  OF  DETERMINE  PRODUCES  APPLY  THE  AND  TISSUES  USE  SINCE  VARIETY  WHICH  THEIR  PROBLEMS  DRAWBACK  YIELD  IN  IS  BUT  UP.  THE  E__T _AL_  WHICH  A  EXTRACTS  PROVIDED  STUDIES.  TISSUES  SIMPLICITY  SCALED  THESE  FOR  PROCEDURE  ITS  AND  PROCEDURE  A  OF  PROCEDURES  DETAILED  P T H ,  GUDMUNDSSON  EXTRACTION  FURTHER  OF  LECTIVE,  EXTRACTS  FOR  FROM  TO  THESE  ULT IMOBRANCHIAL  PEPTIDES  DIFFICULT  OF  ACTIVE  GLANDS  VARIOUS  OF  EXTRACTIONS  REQUIRED  OF  ON  BECAUSE  STUDIES  ULTIMOBRANCHIAL  EVALUATE  EXTRACTIONS,  PROBLEMS  WEIGHT  PREPARATION TO  SOURCES  SUCH  THE  LARGER  POTENTIALS  SCALE  WEIGHT  CHECK  TO  THIS  ELUTION  RELATIONSHIP.  OPPORTUNITY  ACID  FOR  THE  LITERATURE  PROPER  G-75  SPECIES,  THE  THE  OTHER  SEPHADEX  FROM  UNFORTUNATELY  EXTRACTION PURIFIED  -8PRODUCT  IN  A  INDICATED  THAT  MATERIALS. ADVANTAGE THE  CONVENIENT IT  TO  BE  OF  ANIMALS,  OF  THE  GLANDS  SPECIES LARGE  FROM  AS  WOULD  OF IN  OF MADE  SO  THAT  THE  TO  SUITABLE  EVIDENCE  BASED  BE  FOR  OF  PRIMARILY  MATERIAL  SELECT  A  INFORMATION  BE  BROAD  OBTAINEO  OF  THE  ONE  ACTIVITY  OF  THE  STUDY  AND  NEEDED.  AN  SPECTRUM  WOULD  ADDED  AVAILABILITY  FURTHER  WOULO  FAIRLY  OF  SPECIFIC  SINCE  FOR  THE  SIMILARITY  THE  THE  EXTRACTIONS  SELECTED  HAD  THE  ON  ANO  (32)  U L T I M 0 B R A N C H I AL  SELECTION  REMOVAL  ACID  WORK  PROCEDURE  SOURCES.  STARTING  ALSO  PRELIMINARY  SELECTIVE  GLAND  EVENTUALLY  WAS  SPECIES  WAS  SEEN  QUANTITIES  EFFORT  BE  VARIOUS  EASE  AND  FURTHER  STUDIED  THE  ALSO  RELATIVELY  PROVIDING  CALCITONINS  SPECIES  WOULD  TH I S OF  FORM,  BE  OF  GENERALLY  APPLICABLE.  B.  EXTRACT  1.  METHODS.  A.  ISOLATION THE  BRANCHIAL  AT  BOTH THE  WHICH BOTH  OF  PREPARATION STEP  FROM  STUDY.  GALLAPAVO) RAISED  BIRDS  ARTERIES  ARE  NEAR  RELATIONSHIP  TO  HORMONE  VARIOUS  THE  TWO  AND  DOMESTIC  AS  AVIAN  THEY  ORIGIN  THE  TWO  AND OF  AND  TO  SPECIES  STUDIED,  FORM THE  THE  THYROID  SPECIES  THE  L I E  EXTRACTION  OF  FOWL  AND  DISCARDED.  BILATERIAL THE  GLANDS.  QUANTITIES  THE  PLANT  NORMALLY  OF  TO  COMMERCIALLY  PROCESSING ARE  PRIOR  SUFFICIENT  TISSUE  FOR  (MELEAGRA ARE  AND  INITIAL  COLLECTION  PRODUCT  IONS.  YIELD  (GALLUS  COULD  A  OF  THE  ULTIM0BRANCHIAL ALONG  THE  SUBCLAVIAN  PARATHYROIO  GLANDS  COMMON ARTERY. ANO  THE  ULTIMOADEQUATE TURKEY  DOMESTICUS)  GLANDS PART  WAS  THE  BE  COLLECTED  VISCERA GLANDS  IN  CAROTID THEIR THYROIO  IS  Esophagus  Thyroids  Jugular Vein  Parathyroids  Carotid  Ultimobranchials  Heart  FIGURE  1.  LOCATION  OF  AVIAN  ULTIMOBRANCHIAL  GLANDS.  -9INDICATED  IN  SURROUNOING THE  TISSUE  SALMON  PROCESSING THE  F I G U RE  HEAD  PORTION  GLANDULAR THE  MATERIAL,  GLANOULAR VENOSUS  SLIGHTLY  DIFFUSE  NATURE  FROZEN CHUM, AND  AND  DURING  THE  EASY A  THE  OR  COHO  THE  WAS  MAOE  (SQUALUS AND  PERIOD  FROM  WHOLE  FISH  THIS  SITUATION MOST  GLAND  BENEATH  JUNCTURE  THE  PHARYNX  PACKED WAS  LESS  THE  FLOOR  THE  THE  IT ICE  OF  AND  IT  WAS  FROM  TO  BE  ESOPHAGUS.  PERICARDIUM  IT IN  IS  THE  NERKA ) SPECIES. WATERS  CARCASSES  FOR  THIS  COLLECT  TYPE GLANOS  LABORATORY.  SINCE  SQUALUS  HYPOCALCEMIC THE  VASCULAR ON  OR  THE  BUT  STUDIED.  WERE  TIME.  TO  TO  THE  REMOVED,  THE  PRODUCE  PHARYNX  OF  LOCAL  SUBSIDY  ON  SINUS  THESE  A  THE  THE  THE  FOR  DELIVERED  NUMEROUS THE  IN  NECESSARY  SHOWN  OF  KISUTCH  SATISFACTORY,  THAT OF  FEDERAL  TISSUE  PROCESSEO  COMMON  BECAME  GENUS  CONSISTS  SEPTUM  COLLECTED  THE  THAN  ESSENTIAL  WITH AND  IN  AND  COMMERCIALLY  WERE  WHEN  TERMINATED  PRIMATIVE  WAS  THE  PROCESSEO  BUT  OF  OF  THE  BECAUSE  KETA,  ARE  END  OF  D I F F E R E N T I A T E BETWEEN  SUCKLEYI)  GLANOS  FISH,  WAS  TO  REMOVED  MAJORITY  SALMON  (ONCERHYNCHUS  ARE  BAND  DIAPHRAGM  THE  COMMERCIAL  ISOLATION  MOST  OF  A  POSTERIOR  DIFFUSE  SEPTUM.  BETWEEN  VENTRAL  THE  AT  FISH  EXTRACTED.  FISHING  LOCATED  AS  THE  ENTIRE  OF  BRANCHIAL  OF  FORMS  OF  THE  THIS  IT  EXPOSED  RELATIVELY  LIES  OF  CAUGHT  FACTORS  HEADS  GLAND  PROCESSED  THE  COLLECTED  THE  OF  WAS  ALSO  FROM  ICE.  ESOPHAGUS.  EFFORT  WERE  ORY  SEPARATED  THE  DOGFISH AND  IS  ON  WERE  TO  SOCKEYE  NO  THE  WHICH  SURFACE  TISSUE  WERE  WHEN  SEPTUM  GLANDS  FROZEN  GLANDS  ALLOWING  ANTERIOR  THE  ANO  PLANT.  TRANSVERSE  1.  ULTIMO-  FOLLICLES  THE  LEFT  POSITIONED TRIANGLE  SIDE  AT  BETWEEN  FORMED  BY  THE  -10BASI BRANCH IAL BRANCHIAL CULT THE  AND  MUSCLE.  AND  LARGE  MATERIAL  OF  IT  DIATELY  AFTER  THYROID  EXTRACT  DRY  AFTER  THE  ALL  HOMOGENATE THE  LONGER FROM  STREAM COLORED  BE  D I F F I -  PRESENT  IN  WAS  THEN WAS THIS THE  POWDER  WERE  FRESH  SAMPLE  IMME-  OF  HUMAN  PARTIAL  THYROIDECTOMY  HOSPITAL  AND  FROZEN  ON  WERE  DRY  FROZEN  ICE  PREPARED  POWOERS  OR  DRIED WERE  FROZEN ANO  USED  DRYING  ON  IMMEDIATELY  FROM  PURCHASEO  IN  FOR  COLO  AND  WASHED  FRESH,  WITH  WASHING  WATER  INDICATING REMAINING  BUCHNER FOR  THE  PROCEDURE OF  GLANDS  DEFATTED  PROCEOURE  BLENDER  ADDITION  READY  GENERAL  CENTRIFUGED  THE A  A  THE  FROZEN  THE  COMMERCIAL  FROM  WILSON  ILLINOIS.  CLOUDING,  IN  REASON  DURING  THYROIO  WARING  ACETONE. AIR  THIS  EXTRACTS  METHODS  A  THESE  IN  ANIMAL.  WHERE  ACETONE  IN  CAUSED  OF  THE  BOVINE  TWO  UNTIL  THE  GLANDS  THAT  HOMOGENIZED,  PRECIPITATE  TIMES  C0RAC0-  IS  ACTIVITY  THYROIDS  CHICAGO,  RECENTRIFUGED. OF  IT  ENZYME  FOR  OE FA T T E 0  STANDARD  H0M0GENIZE0  ISOLATION  PROTEOLYTIC  VANCOUVER  CASES  WERE  HIGH  THE  AND  EXTRACTION. A  THE  ARE  OBTAINED  FROM  LABORATORIES,  WAS  SALMON  AND  TISSUE  WAS  RAT  DRIED,  TISSUES  THE  CARTILAGES  CONNECTIVE  IMPERATIVE  AT  PORCINE  IN  OF  REMOVAL.  REMOVAL  ACETONE  THE  WAS  PATIENT ICE.  WITH  AMOUNTS  THYROID  A  A S  AL  EXTRACTED.  BECAUSE  ON  CERAT0BRANCHI  TO  THE  (4°  WAS THE  PRODUCED  EXTRACTION.  WHICH C )  FIRST  METHOD  GLANDS  WERE  ACETONE.  THE  DISCARDED.  A C E T O N E ., A N D REPEATED  A  SUPERNATE  ABSENCE  MATERIAL,  FUNNEL,  THE  THE  TO  SUPERNATE COLD  USED  PRIOR  THIS. IN  WERE  OF  WHEN A  NUMBER NO  LIPID  DRIED  LIGHT,  IN  A  BUFF-  -11ACETONE MATERIALS, TIONS.  DRYING  AND  THIS  WAS  WAS  WERE  EMBEDDED  WITH  GREAT  MOST  ATTRACTIVE  WHICH  GLANDS  WAS  DISTILLED  HAD  CONTAINING  THE  12  ACID:  STIRRED THE OF  SOLVENT  A  AND  THE  LEVIN  LIPID.  (69)»  AFTER  THE  MIXTURE  SOLVENT  PELLET-LIKE  REDISTILLED  LAYER  THE  AZEOTROPIC  WASHED  WHICH  REMOVED  E.  REMAINING THE  EXTRAC-  LOW-BOILING,  AND  WERE  OF  SOLVENT,  FILTRATION FROM  BE  EXAMINED,  THE  AND  WITH  GLAN-  ALONG  WITH  MIXTURE  TO  SOLVENT  ON  THE RECOVER  THE  DRIED.  DEFATTED  POWDERS  WERE  METHOD  DESCRIBED  BY  EXTRACTION  ROOM  (75:  FILTRATE  PRESSURE  SCALE  PROCEDURES.  AT  AGAIN  THE  FROM  EXPERIMENTS,  WATER  IN  AND  GLANOS  ONLY  THAT  EXTRACT  REMOVED  WAS  AVIAN  WERE BE  THE  AIR  OF;THE  HOURS  TO  TIME  LARGE  COULD  REMOVED.  AND  DRIED,  ALL  REDUCEO  PELLETS  INITIAL  IN  TO  OF  FOR  THE  METHODS  APPEARED  THE  EXTRACTION  USED FOR  BY  FOR THAT  HOMOGENIZED  LIPID  SOLVENT  FICATIONS IN  THE  FUNNEL  THE  TRUE  TISSUE  THESE  AMOUNTS  SUITABLE  OTHER  AZEOTROPE  SOLVENT.  B.  OF  BEEN  MATERIAL  FILTER  ADIPOSE  UNDER  SOLVENT-WATER  THE  REALLY  ESPECIALLY  IN  LARGE  1 ,2-0 ICHLOROETHANE  THE  IMMISIBLE  NOT  DIFFICULTY.  USED  DULAR  REQUIRED  7.5*  IN  750  FROM  FROM  A  THE  AND 100  G  OF  ML  AND  OF  THEN  SOLVENT  FIRST  THIRD  POWDER IN  WASH  TWO ANO  USING  GUDMUNDSSON  PRECIPITATION  TEMPERATURE 21)  EXTRACTED,  1.5  STAGE  IS L  STEPS  4  OF  COOLEO  THE  TO  -10°  ACETIC WAS  FILTERED,  WITH C.  WAS  GENTLY  RESIDUE  AND  POOLED  (48).  WHICH  BUTANOL:  HOURS  WAS  AL_  STIRRED  FILTERED. FOR  E_T  MODI-  300  FLVE  ML VOLUMES  -12OF  ACETONE  WAS  STIRRED  TO  STAND  COULD  THE  INTO  FOR  THEN  SLURRY  THE  48  BE  WAS  COOLED  ASPIRATED  CENTRIFUGED  AND  WAS  DRIED  OFF  RESULTANT  ALTERNATE FOLLOWED  THE  AND  THE  THE  MIXTURE  MAJORITY  DISCARDED.  -10°  C  TWICE  AND  THE  IN  ABOUT  CENTRIFUGE  WAYS.  THE  OF  THE  2  L  L)  ALLOWED  THE  LIQUID  REMAINING  SUPERNATE  BOTTLES  PRECIPITATE  WAS  EXTRACTED  FOR  AND  CENTRIFUGED.  HOURS  PENDEO WERE  IN  125  POOLED  150  MATELY ADDED  TO  STAND  A  ML  AND  AND  AND  THE  OF  DISCARDED.  -10°  UNDER  (w/v)  TCA  THE  SUSPENSION  HOURS  AND  AT  THEN  EXCHANGE  RESIN,  TCA.  WHEN  SOLUTION  THE  RESIN  FILTRATE  WAS  GUDMUNDSSON *THE  RESIN  SODIUM  A  C STREAM  USED WAS  AND  TO  FOLLOWED TO  THE  PREPARED  THE  POWDERS.  THE  TO  WAS  THE  A  AND  THE  PRECIPITATE  EQUAL  VOLUME  OF  ACETATE  FORM*,  TO  3  M  TO  WASHES FORM  BY  WAS  3  ALLOWED WITH  IRA-400 REMOVE  THE  FILTERED THE  EXTRACT.  5  TCA.  VOLUMES  WATER  ABSORPTION  THEN  ACID.  ACID.  REMOVE IN  WAS  ACETIC  ACETIC  FINAL  DISTILLEO  ACETATE TCA  THE  WASHES  USING 3  0.5  APPROXI-  WASHED  AN  SLURRY  5.5»  RESUS-  MIXTURE  WAS M  DRIED  PH  w/v,  TO  THE  0.5  WAS  SUPERNATES  OF  ML  OF  OF  TWO  100$  SEVERAL  THEN  VOLUME  (TCA), ML,  ETHANOL,  50  YIELD  FOR  (48)  IN  ML  BY  EJT A L  70°/O  OF  ACID  ETHER  REGENERATED  IN  REPEATED.  CLEARED  10  ACETONES  CONVERTED  ACID  WITH  TREATED  PRECIPITATE  MESH,  HAD  L Y O P H I L IZED  HYDROXIDE,  THEN  ACETIC  WASHED  ADDED  ML  G/100  4 ° C  20-40  ANION  AND  12  RESUSPENDED  WAS  THE  OF  BE  THYROID  VACUO  TRICHLOROACETIC  12  10$  THE  IN  THEN  GUOMUNDSSON  250  WITH  PROCESS  CONCENTRATION  FOR  OF  PORCINE  CONCENTRATED  ML.  COULD  PROCEDURE  BOVINE  2  ON  PRECIPITATE  THE  WAS  C.  AND  WASHED  IN  ( A P P R O X I M A T E L Y -1 2 . 5  FILTRATE  -10°  AT  -10°C  N I TROGEN. THE  TO  TO  POOLED  HOURS-AT  PRECIPITATE  ACETONE OF  PREVIOUSLY  WASHES BY  OF  WASHES. 3  IN  WATER  5  1  M  IT M WASHES.  -13THIS  SAME  PROCEDURE  DRIED-DEFATTED GLANDS.  THE  TECHNICALLY  POWOERS  METHOD  FROM  WAS  DIFFICULT  WAS  USEO  ON  DOGFISH  EFFECTIVE  ON  SMALLER  SMALLER AND  BUT  QUANTITIES  CHICKEN  THE  ULTIMOBRANCHIAL  LATER  QUANTITIES  OF  STEPS  AND  WERE  YIELDS  WERE  POOR. THE  NECESSITY  FOLLOWING TWICE IN  A  TEN  WITH  COLD(0°C)  STREAM  OF  AIR.  VOLUMES  OF  0.1  AND  USING  FIVE  HOURS  WORKING  MODIFICATIONS.  HOURS  2  FOR  THE  AND  ACETONE THE  M  0.5  AND  HOURS  WHICH  DISSOLVED  READILY  CHROMATOGRAPHY.  UNLESS  c.  BIOLOGICAL THE  ASSAY  BY  (66)  80-90  0.1% AT  ANO  BOVINE  PH  WERE  WERE  4.5,  A  DUPLICATE  WAS  ROOM TWO  VOLUMES  OF  THE  0.1  BUFFERS  SPECIFIED  THEN  M  THIS  EXTRACTED  PERFORMED YIELDED FOR  PROCEDURE  WITH  FOR  4  EXTRACTIONS  FORMIC  USEO  THE  REMOVED  TEMPERATURE  LYOPHILIZATION IN  WASHED  ACETONE  FURTHER  WERE  WAS  ACID AND  TAN  FOR  THE POWOERS  COLUMN WAS  USED  ACTIVITY  OF  THE  VARIOUS  MODIFICATION  OF  THE  METHOD  OF  RATS.  MATERIALS  SERUM  COLLECTED  AT  THE  TO  ON  PROCEOURE.  G,  MALE  SUITABLY  AND  OF  LED  DESCRIBED.  DETERMINED  ASSAYED  ACID  SCALE  PRECIPITATE  POWDER  COLLECTED.  POOLED.  BIOLOGICAL  USING  SMALLER  MOST  RESPECTIVELY  SUPERNATES  PREPARATIONS  MOIST  TWO  THREE  ALL  A  ACETONE AND  FORMIC  SUPERNATE  VOLUMES  THE  ON  LONG-EVANS  DILUTED  ALBUMIN,  IN  IN  AN  0.1  M  INJECTED  INTO  THE  FROM  TAIL  VEIN  PLASMA  THE  CALCIUM  EXTRACTS  INJECTION SODIUM  KUMAR  WAS ET TO  VEHICLE  ACETATE  AL BE  OF  BUFFER  TAIL  VEIN.  BLOOD  SAMPLES  60  MINUTES  AFTER  INJECTION  DETERMINATIONS  OBTAINED  BY  A  - 1 4 METHOD BEEN  SIMILAR  TO  AUTOMATED  FLOUROMETER  THAT  USING  OF  A  EQUIPT:  KEPNER  ANO  TECHNICON  WITH  A  HERCULES  ( 6 4 ) WHICH  AUTOANALYSER  FLOW  CELL  AS  ANO  A  HAD  TURNER  DESCRIBED  BY  NEWSOME ( 8 3 ) . THE WITH  THOSE  VEHICLE LEVEL  OF  MEASURED OF  A  ONLY.  IN  EACH  MG/O  PLASMA  SIMILAR THE  OF  THE  CONTROL  BLOOD  THE  RESPONSE.  USING  ANO  THE  THE  POSSIBLE SERIES  OF  TO  OF  SHOWN  IN  STANDARD  CHECKED  THE  MRC MRC  SLOPES  4 OF  YIELDED  PREPARATION  THE  TO  AS  A  RANGES  USED  A  BY  FAMILY  THE  LOG  IN  TO  A L 0 8 3 1 ,  MRC  RESEARCH FOR  THE  OF  ESTIMATED  RESPONSE  1.  RESPONSES  (Y)  1.0  IT  X  WAS  FOR  (1 1 6 ) •  EACH  THESE  THE  CURVES BIOASSAYS. FOR  PHARMACEUTICAL (WHICH  ACTIVITY  OF  EACH  OF  VARIATIONS  INACCURACIES EXPRESS VALUES  THE  CO.) DEFINES  SAMPLE IN  OF  THE  THE  ACTIVITY  RATHER  THE  OF  THAN  ACTIVITY.  BASED (9)  VARIABLE  A,  INHERENT  SINGLE  ON  LEVEL  CALLED  STANDARD  BECAUSE  POSSIBLE  AND  ESTABLISHED  ARMOUR  TO  Y  CALCIUM  DOSE-RESPONSE  CURVE  THE  PREFERABLE  OF  WITH  CALCIUM  VARIABLE  EVALUATING  A  CT,  THE  PLASMA  DEPENDENT  SNEDECOR  OF  USED  CURVES  WERE  OF  AS  REGRESSION  RANGE  ASSIGN  PLASMA  SAMPLE  VALUES  IS  MEAN  CALCULATED  ANO  IT  THE  WAS  RESPONSE  COMPARED  INJECTED  SAMPLE  OF  SYSTEM,  RATS  INDEPENDENT  THESE  CURVES  BETWEEN  THEN  THE  WERE  THE  CONTROL  AS.THE  THE  (PORCINE  WERE  AND  (MU/MG).  THE  OF  DESCRIBED  WHICH  LEVELS  GROUP  MiLLIUNiTS/MG  ATTEMPTING  OF  AS  AGAINST  UNIT)  BIOASSAY A  A  PRODUCED  FIGURE  AND  IN  DOSE  DILUTION  COMPARISON HOUSE  THIS  CALCULATE  CALCULATIONS  GROUP  DIFFERENCE  EXPERIMENTAL  LOG  CALCIUM  ON  AND AND  THE WERE  2.0  MG$  0 . 9 5  CONFIDENCE  CALCULATED CALCIUM  LIMITS  AS  FOLLOWS.  WERE  SELECTED  AS 2.  REPRESENTATIVE THE  Y(?) 3.  LOG OF  THE  DOSE  1.0  WAS  THE (jiy)  Y  ERROR  THESE AXIS  ^  =  YX V / 1  S  GIVEN  OF  0  5  X S  <  ?  VALUES  THE  STANDARD  OF  THESE  IN  IN  REPRESENTING  ESTIMATES ARE A  USED  STEP  IF  USED  AN  RANGE  EXPRESSED  MOST PERFORMEO  Y  THE  T  +  >  Q  THE  THEN  POPULATION  S  5  CAL-  MEAN  ?  DEGREES  IS  OF  IS  THE  THE  AND  TWO  BY  OF  0.95  IS  STEPS  IS  FOR  THE  RESPONSE  DOSE  OF  CONFIDENCE  ACTIVITY LIMITS  ASSUMED  REASONABLE  BASIS  ALL AND  AT  .  TO  ASSUMPTION  ACTIVITY THESE  ESTIMATES  COMPARISON. 2  RANGE THE  LOG  RANGE  CURVE A  TO  CORRESPONDING  THE  A  LABORATORY  FOR  ADDITIONAL  TRANSPOSED  YIELDS  THE  THIS  IN  THEN  STANDARD  PRIMARILY  REPETITION  TWO  OF  FROM  ABOVE  THE  CURVE  GIVES  THE  2  ERROR.  THIS  WAS  5—  ACTIVITIES  OBTAINED  SINCE  <  CURVE  DIVISION  NO  Y  THEN  M  WERE  M i l READ.  CONTAIN  ESTIMATED  (116)  TWO  POINT,  AN  +.xV2  N  WAS  IN  THIS  ESTIMATED 9  DETERMINED  (N-2).  MU/MG  THE  LIMITS  ACT IV IT IE S 6.  7.  IS  .05  FREEDOM 5.  OF  CONFIDENCE  THE  TO  NOTED.  V - T _ T  CORRESPONDED  I  0.95 FOR  WHERE  WHICH  AS S  4.  (X)  STANDARD  CULATED  VALUES.  THROUGH OF  6  USING  ACTIVITIES.  EXTREME  VALUES  Y  =  2.0  THE  FINAL  OBTAINED  FROM  CALCULATIONS.  OF  THE  STATISTICAL  BY  COMPUTER.  THE  CALCULATIONS PROGRAM  USED  ABOVE FOR  WERE  THIS  ROUTINELY  CONTAINED  ('A)  Activity of Turkey Extracts  2.0  1.0 I I  (B)  I  I  ~l  1 — I — I I I 11  Activity of Dog Fish Extracts  to  c  & n  £ 2.0H 1.0I  0.1  0.2  I  I  0.4  I  I  I  0.6  I  I  I  1.0  1  2.0  1  1—I—I—I  4.0  6.0  I  I I  10.0  Log Dose (jug/80gm Rat)  FIGURE  2.  ( A ) C O M P A R I S O N O F R E G R E S S I O N S O F L O G D O S E 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 A N D D I C H L 0 R 0 E T H A N E D R I E D G L A N D S . • - ACETONE, O - DICHLOROETHANE ( B ) C O M P A R I S O N O F R E G R E S S I O N S O F L O G D O S E 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 APRECIPITATION, A - FORMIC ACID EXTRACTION  -16MODIFICATIONS APPENDIX  2.  AND  FIGURE  WERE  OF  THE  RANGES  FROM  OENATURATI  ON  TECHNIQUE  TCA  ACID  AS  TCA  MAJOR NOT  BY  POINT  OF  TCA  INCLUDED  IN  DESCRIBED  POWDER  TO  FREE  MIGHT  WELL  FOR  FATTY  THE  LOG  OF  THIS  SECOND  WAS  EARLIER. MU/MG)  A  OF  HAD  WAS  MORE  A  PHYSICAL  AFTER  IT  TO  A  BE  SINGLE A  VALUABLE  MATERIALS. CURVES THE  0.1  TECHNIQUE U P T O  THE  PRECIPITATED M  FORMIC  ACTIVITY DOUBLE  TWO  EXTRACTIONS  (48)  THEN  WITH  FOR  DRYING  EJT_ AJ.  WAS  THAN  WITH  EXCELLENT  ACETONE  BIOLOGICAL  HEAT  THE  EXTRACTS.  EXTRACTED  INVESTI-  RESULTS.  PROVE  SAMPLE  MU/MG.  ALTHOUGH  REDUCING  LIPID  THE  AND  AZEOTROPE  THANE  MATERIAL  COMPLETE  AT  GUDMUNOSSON  THE  420-530  STAGE  DOSE-RESPONSE  ONE  MATERIAL  D ICHLOROE  FROM  STARTING  USING  DRYING  0 ICHLOROETHANE AT  TWO  EXTRACTIONS  DRIED  THE  RANGING  PROCEDURE  PARALLEL  (3600-4600  OF  INCONCLUSIVE  BE  EXTRACTION  THE  THAT  FOR  INITIAL  ACETONE  DICHLOROMETHANE  THIS  CURVES THE  THE  ATTEMPT  ULTIMOBRANCHIAL  IN  THE  THE  ONE  PRECIPITATION.  WHILE  OF  FOR  INSURMOUNTABLE,  WORK  SHOWS  OUT  SOLVENT  A  FROM  DOGFISH  LOWER,  OUT.  APPEARED  2(B)  WHILE  PRODUCED  PARTICULARLY  OF  DISADVANTAGE  SEEM  USING  FURTHER  CARRIED  WITH  ARE  EXTRACTS.  EXCEPT  ACTIVITY  CARRIED  AND  FIGURE  OF  840  SOMEWHAT  OBTAINED  OPERATION.  STAGE  A  DID  BOILING  THE  TO WAS  NOT  PROPERTIES  ANO  PARALLEL  ML)/MG  PROBLEM  WERE  DETAILS  DOSE-RESPONSE  540  THE  SAMPLES  ITS  LOG  ACTIVITY  CONSIDERED  MATERIAL  IN  BIOLOGICAL  WAS  THE  BIOLOGICAL  WAS  LOWER  AN 0  ULTIMOBRANCHIAL  OUT'  MATERIAL  THE, LOWER  SHOWS  TURKEY  STAGE.  GATION  AUTHOR  DISCUSSION.  2(A)  CARRIED  ORIEO  THE  A.  RESULTS  SAMPLES  BY  OF  THAT  THE OF  THE  TABLE I SPECIFIC  BIOLOGICAL  ACTIVITIES  OF  THYROIO  AND  ULT1MOBRANC1AL  EXTRACTS  SOURCE  ACTIvITY RANGE (MU/MG)  HUMAN BOVINE  THYROIO*  MURINE  THYROID  PORCINE TURKEY DOGFISH SALMON  THYROID* UB UB UB  CHICKEN  •INCLUDES  UB*  TCA  RANGE  (MU/MG)  5  5  THYROID  CENTER OF  20-25  22.5  RELATIVE POTENCY (PORCINE  0.02 0.1  100-150  125  0.5  210-280  245  1  540-840  690  3  800-1300  1050  4  2500-4300  3400  14  15,000  60  13,000-17,000  PRECIPITATION  STEP.  =  1)  o  Log Dose(mU Std /80gm Rat)  Loo Dose(/jg/80gm Rat)  FIGURE  3.  ( A ) R E G R E S S I O N OF LOG DOSE STANDARD. (ARMOUR P O R C I N E  ON R E S P O N S E FOR C T , AL0831)  HOUSE  ( B ) R E G R E S S I O N S OF LOG 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 O B R A N C H I A L TISSUES. C A L C U L A T E D A C T I V I T Y R A N G E S ARE SHOWN I N T A B L E I .  -17FORMIC  ACID  OF  PROCEDURE  THE  FORMIC PER  ACID  GRAM  AS  POWDER  OF  TOTAL  RECOVERY  FRESH  (MU/G  TISSUE  BECAUSE  WAS  EXPRESSED  MUCH IN  OF  SIMPLICITY  GREATER  TERMS  TISSUE)  THE  THE  OF  WITH  THE  MILLlUNITS  MRC  ACTIVITIES  WERE  FOLLOWS:  THE  BASIS  ADDITIONAL  OF  TO  BE  A  MOLECULAR  AXIS.  WHICH  IS  VALUE  DATA  FOR  CURVE  NOT  THE  PREPARED  USING  PROBABLY  HAVE  OBTAINED  ACTIVITY THE  FOR  .SINCE STEP  3(A)  IT  IS  TCA  HIGHER  A  FORMIC  ACID  THE  IS  OF OF  AND  AS  THE  THE  FOR  IN  THE  THE SOME  THE  IN  BOVINE  THE  YIELD  IN T E R E S T , FROM  EXTRACTS  PROCEDURE  STEP.  CURVES ON  OF  THE  AND  ACTIVITIES  THAN  WOULD  EXTRACTION  BEEN  USED.  A  EXTRACTS  TABLE  POINT.  YIELD  LOW  STANDARD  TABLE  LOW  NOT  T C A  PLOTTED HOUSE  THE  PURIFICATION  ACTIVITY  SINGLE OF  OF  DOSE-RESPONSE  RANGES  PRECIPITATION  SPECIFIC  WELL  THAT  COULD  REMOVE  SUMMARIZED  ON  TYPICAL  DECIDED  STAGE  BIOLOGICAL  BECAUSE  PORCINE  AS  IS  MATERIAL  IT  WAS  SPECIES  THE  CHICKEN,  HAD  SEVEN  ACTIVITY  BASED  TISSUE  WOULO  LOG  ARE  MATERIAL.  THE  THE  TISSUE  MU/G  NEXT  POTENCIES  OBTAINED  AND  THE  OF  MU/G  PRECIPITATION  WHICH  FIGURE  HUMAN  IT  EQUALLY  FROM  THE  ESTIMATE BE  TCA  SERIES  CALCULATE  STARTING  COMPARISON  A  IN  M L)/M G .  ROUGH  COULD  OF  EXTRACTS  TO  IN  THYROID.  BEEN  ON  INCLUOED  LIMITED  SHOWS  RELATIVE  EXTREMELY  OF  CONTAMINANTS  3(B)  USED  THEIR  LOSS  FILTRATION  THE  PREPARATION AND  POWDER  PARTICULARLY  GEL  BIOASSAYS  SAME  THIS  WEIGHT  FIGURE FOR  ACID  COMPLICATIONS  JUSTIFIED,  WAS  1000-1300 4500-7300  POWDER  FORMIC  BE  BUT  EXTRACTION.  TCA  ON  (800-1300),  1.  IS  THE  AN  BETTER ANO HOWEVER, HUMAN WERE THEREFORE HAVE  TABLE BIOLOGICAL  ACTIVITY  YIELDS  FROM TI  SOURCE  I I THYROID  CENTER OF  AND  ULT I M O B R A N C H I A L  SSUES  RANGE  MG G  EXTRACT TISSUE  MU G  TISSUE  (MU/MG)*  HUMAN BOVINE  THYROID  MURINE  THYROIO  PORCINE TURKEY  SALMON  **  UB UB  CHICKEN  SEE  THYROID UB  DOGFISH  *  TABLE  FRESH  22.5 125 245  1 .0  5 11  (0.5)** 5.2  650 170  (0.7)**  690  3.3  2300  1050  5.6  5900  3400  1.3  4400  15,000  UB  ESTIMATE OF  5  THYROID  0.76  11,500  I ASSUMING WEIGHT.  DRY,  DEFATTED  WEIGHT  EQUALS  8$  -18SPECIFIC GENERALLY FRESH  ACTIVITIES  USEFUL  GLANDS  ACHIEVED  OR  LATER.  PURITY  OF  HORMONE  A  SPECIFIC  II  TISSUES  USED  ESTIMATES POWDERS  POSSIBLE  ACID  HANDLING.  APPROXIMATELY OF  THOSE  METHOD  VALUES  I S MORE  SECOND,  ESTABLISH  AND  THE EMPHASIS  COLLECTING  OBTAINED  EXTRACTION  TO  OF  OF  THE  THAT  BE  DIS-  OF THE  YIELDS  PURE  MOLECULES  PURE  PORCINE  CT  EXTRACT  As  LATER,  WILL  BE  THAT  8$  SHOWN  OF  COMPLEX  LOSSES  PURPOSE  THE FEASABILITY PLACED  A L L N0N-GLANDULAR  THUS  ON NO  TISSUE  TO  ALMOST  OCCUR,  LOSSES  OF  THESE  MINIMIZING  AND  SCALE  NUMEROUS  WAS  THE STARTING  AS A  DUE TO EXTRACTIONS EXTRACTIONS,  THE TIME  EFFORT  ARE  SOLVENT  WHERE  NO  ARE  (32)  THE  ARE  THE  SPECIES  THERE  FlRST  OF LARGER  GREAT  WEIGHT.  LOWER.  ARE  DEFATTED  EXTRACTION  THERE  FRESH  VALUES  THE EIGHT  FACT. AND  THE  PORCINE  THE FRESH  FROM  FOR T H I S  OF  THE DRY,  ARE GENERALLY  INVOLVES  THE GLANDS.  AND  IN ACID  T H E PRIMARY  WAS  ACTIVITIES  THE TISSUES  FOR S I G N I F I C A N T  WAS  OF THE  U/MG, T H E S A L M O N  BOVINE  THE ASSUMPTION  EXPLANATIONS  OPPORTUNITIES  REMOVE  ON  THE ABSOLUTE  EXTRACTION  HORMONE.  IN THE EXTRACTS.  WITH  ACTIVITY  200-250  WILL  ESTIMATE  KNOWING  ARE  OF PURIFICATION  THE EXPECTEO  THE CALCULATED  ACTIVITIES  COMPARABLE  SIMPLE  SHOWS  BASED  AN  THESE  OVER-ESTIMATE.  REPRESENT  RELATIVE  PROVIDE  AS  CONTENT  CALCULATIONS  BASIS,  2<fo  SUCH  HORMONE  THE DEGREE  SPECIFIC  OF  EXTRACTS OF  THESE  AND OF  THIS  TO  AN  OF  CAN ALSO  ON  1  CONTAIN  TABLE  TWO  STAGES.  ACTIVITY  I S RATHER  THOUGH  INDEX  I F THE ABSOLUTE  HAD  THIS  AN  THE MATERIAL  COMPARABLE.  SHOULO  AS  THEY  ARE  CRUDE  FOR C A L C U L A T I O N  IN LATER  CUSSED  OF  SPENT  MADE  IN  TO  MATERIALS  -19WE R E  GENERALLY  ACTIVITY  LEVELS  THE IN  MAJOR  ESTIMATING  PROVIDE  MUCH IN  VALUE THE  ULTIMOBRANCHIAL  OF  THE  SINCE  PARTICULARLY ACTIVE BE  THAN  DUE,  AT  GLANDULAR GLANO  HOLD,  AND  A  IT  WAS  AND  ON  THE  TO  CHICKEN  TWO  THE  AS  IN  THE  CHICKEN.  HOWEVER,  IN  THE  TURKEY  WHICH  ACTIVITY  PER  LOWER OF  THAT  THE ALL  IN  ACQUIRING  QUANTITIES  THAT  FORMIC THESE  SALMON  DOGFISH  OVER  ANO  WAS  ACID  HORMONE  AND  THE  TIME  AND  CONSUMING.  THE THIS  THE  3  A  OF  THE  THE  THE  IN  TO  A  DISCRETE  CHAPTER  SOURCES  ALLOW  PURIFICATION  PROCEDURE METHOD  DECISION  TO  BASED  OTHER  ON  RELATIVE  THE  CARRY FACTORS.  DIFFICULTY  SOURCES  FATTY  ACETONE LEFT  GLAND  THIS  SUITABLE  AVIAN OF  COULD  THE  DISCRETE  EXTRACTION  OF  MORE  WEIGHT.  HORMONE  FINAL  WERE  EXPLANATION:DOES:  UNIT  WAS  OF  THIS  NATURE TO  THE  ACTIVITY  TIMES  ULTIMOBRANCHIAL  AMOUNTS  MADE  OF  TO  SOURCES  GLANDS.  HAS  THEREFORE  BECAUSE  LARGE  GLANDS  OF  THE  WAS  TO  OUTLINED  EXTRACTION  MATERIAL  THE  THE  SOLVENT  HORMONES.  STARTING  WITH  THE  ELIMINATED  BECAUSE  OF  2  THIS  EXPERIMENTS FOUR  USEFULNESS  ALL  TIMES  OPPOSED  EXISTS  THE  ACTUAL  NECESSARY  WORK.  DIFFUSE  FISH  ITS  ULTIM0BRANCHIALS  SALMON  TO  IS  50  OR  THE  THE  EXPENSIVE  10  DOGFISH  IN  A  ESTIMATE  EXCELLENT  WERE  TISSUE  SUFFICIENT  ASSOCIATED  BE  THEY  PART,  PREPARING  PASSED  TO  SINCE  IN  BY  TO  MATERIAL  STRUCTUAL  FROM  THE  ATTRACTIVE  ISOLATION,  WITH  FOR  CONTAINED  THYROID.  0ECIDED  FOLLOWED FOR  THEY  BASIS  USED  HOWEVER,  STARTING  APPEARED  LEAST  THE  CONTAINED  THOSE  DATA,  OF  HORMONE  CONSIDERABLY ON  THE  AMOUNT  EITHER  WHICH  NOT  OF  TISSUE  PORCINE  THAN  GLANDS.  SUFFICIENT  HORMONE  CRUDER  WERE  TISSUE  DRYING SALMON  PROCEOURE AS  THE  -20SPECIES  OF  THE OF  CHOICE.  SALMON  TISSUE  AND  HAD  THE  THE  SECOND  SECOND  HIGHEST  EXTRACT, ( T H E  HIGHER  ACTIVITY  BE  BY  TCA  E X P L A I NED  INCLUDED TO  WORK  IN  THE  THE  WITH,  CHICKEN  AND  THE  PRODUCTION  THE  REQUIREMENTS  COULD  LINE  OF  FOR  A  HIGHEST  ACTIVITY  SPECIFIC  OF  THE  STEP  EXTRACTION.).  T H E  BE  LARGE  A  LOCAL  STARTING  WHICH  MATERIAL  THE  MIGHT-  WAS  TISSUE  WAS  EASY  QUANTITIES  THUS  CANNERY.  IN  EXTRACT  PRECIPITATION  IN  WEIGHT  ACTIVITY  CHICKEN  OBTAINED  PER  IT  FOR  LARGE  EQUAL  TO  MET  ON ALL  SCALE  E X T R A C T I ON. ASSUMING THE  PURE  THAT  1  ABOUT  OF MG  OF  PROVIDE  THIS 10  STRUCTURAL  WORK.  SALMON,  WAS  IT  CALISRATiON  1.  METHODS.  G-75 A  1.2  x  FORMIC PROTEIN FORMIC ANO  PROCEDURE  COLUMN 80  WAS CM  ACI0 WAS  AS  INCREASED  MEANT  THAT  TO  MG  20  A  GROSS  USED  IN  AN  OF  DISSOLVED  THE  UREA  THE  DENSITY  WAS  A  THAT  50$  TISSUE  HORMONE  OF  CALCULATED  SHOULD  CONTAIN  LOSS  DURING  WOULD  BE  NEEDED  NECESSARY  REPRESENTED  ABOUT  FOR  10  TONS  OF  UNDERESTIMATION.  COLUMNS.  PREPARING THAT  SEPHDEX  ELUANT.  OF  PURE  IT  TISSUE  FOR  KG  THIS  SEPHADEX  COLUMN  2  OF  ESSENTIALLY  THEN  ACID.  ALLOWING  ALTHOUGH  OF  U/MG)  ULTIM0BRANCHI AL  STILL  C.  THE  (200-250  HORMONE.  THE  APPROXIMATELY  HORMONE  SALMON  PURIFICATION TO  ACTIVITY  PORCINE  KG 20  AN  THE  CALIBRATED  DESCRIBED G-75  WAS  0.5  INSURED OF  ML  THAT  THE  OF THE  ANDREWS  PREPARED  APPROXIMATELY IN  BY  1 2  MG M  OF  (3).  USING THE  UREA  PROTEIN  SOLUTION  SEPHADEX  IN  M  MARKER 0.1  WOULD  ALLOWING  0.1  IT  M DISSOLVE TO  BE  -21LAYERED  BENEATH  THE  ELUANT  TOP  WAS  ALLOWED  SAMPLE  WAS  THE  COLUMN  UNDER  GRAVITY)  THE  ABSORBANCE  OF  BAUSCH  LOMB  MEASURED METER  WITH  ANO  PRODUCED  WHEN A  THE  VOLUME  ( V  (SEE  FIGURE  THE  LOG  ACTH  AND  CHEMICAL THE  FIVE  CO.,  IOENTICAL  FOR  THE  ALBUM IN  TOTAL  WAS  ANO  FLOW OF AT  DESIGNATED  AS  VALUE  THE  WAS  THE  ELUTION  PLOTTED  THE  PROTEIN.  GLOBULIN  ( 1 6 0 , 0 0 0  M.W.),  M.W.),  ( 1 4 1 1M.W.),  INSULIN  ( 5 7 2 2  FIRST  FOUR A  CHECK  OBTAINED  YORK. FROM  MARKERS  THE  ON  INSULIN  PURE  THE  PORCINE  BEEN  USED  THREE.  RESEARCH  WAS LOW  M.W.)  OTHER  MANN  FROM  CALBIOCHEM,  HAO  GLUCAGON  RECRYSTALIZED ANGELES.  ACTH  FROM  THE  SIGMA  LOUIS. PROCEDURE  THAT USED,  SAME  LOW  GLUCAGON,  ( 6 5 , 0 0 0  IT  SINGLE  ( 1 2 , 4 0 0  WERE  B  GRADE  ST.  SEPHADEX  WAS  COLLECTED  C  CONSIDERED  I N C . NEW  TO  WERE  6 0 0 SPECTROPHOTO-  VOLUME  OF  THROUGH  2 8 0 NM  A  WEIGHT  ONCE  ELUATE  CONTAINING  THIS  CYTOCHROME  THE  MARKERS  CALIBRATION  (BACITRACIN,  THE  GAMMA  CHROMATOGRAPHICALLY  WAS  MARKERS.  WERE  M.W.)  WERE  INSULIN,  WAS  USEO  M.W.-).  LABORATORIES,  ACTH  SPECTRONIC  MOLECULAR  BACITRACIN  ( 4 5 4 2  FIRST  THE  TO  FRACTION  PROFILE  PROTEIN  COLUMN.  FRACTIONS  EACH  PEAK  THE  ,  ( 1 7 * 8 0 0  ANDREWS  BOVINE  OF  THIS  ML  OF  4 ) .  M.W.),  ( 3 4 8 5  OF  3  AGAINST  ELUTION  THAT  MARKERS  MYOGLOBIN  BY  ) FOR  THE  THE  AND  E  CENTER  AND  AND  PLOTTED  TYPICAL  PEAK.  AGAINST  A  ELUANT  THE  THE  COLLECTED.  APPLIED  ON  M.W.)  FOR  DESCRIBED THE  FOR  COLUMN  MOLECULAR  ACTH WAS  AND  USED  THE THE  SIZE WEIGHT  SEPHADEX  G - 7 5 COLUMN ( 1 . 5x  8 0  MARKERS  INSULIN), TO  G - 5 0  INDICATE  BUT THE  CM) WERE  BOVINE VOID  COLUMN  EXCEPT AND  THE  USED SERUM VOLUME  I  5000  I  105 Bacitracin 100  M.W.  2000  1000 B a c i t r a c i n  Glucagon\(3485) Porcine\(3600) Bovine Mminor)  ACTH\(4542)  Sephadex G-50  70 H  120  100  Calibration L Chicken, Human, Rat » Bovine (major), Salmon ACTH(4542)\  80 •  V  9000 I--  110  lnsulin\°(5722)  Vr E  1  ( I 4 I I )  Glucagon \(3685)  90  -I  Sephadex G-75 Calibration  90 (ml)  D o g f i s h J u r k e y  - eo  (ml) • Insulin (5722)  Cytochrome\( 12400)  60  SO  70  60  Myoglobin\(l7800) BSA(65000)  50  40 r-Globulin( 160000) 1000  F I G U R E  4.  10000  C A L I B R A T I O N I N C L U D I N G A C T I V I T Y  eg;  FROM  4°C;  FLOW  G-50  C O L U M N ,  S A M E  C U R V E S  E L U T I O N  E L U A N T ,  100000  M.W.  FOR  S E P H A D E X  V O L U M E S  E X T R A C T S .  0.1  R A T E  5  1.5  M L / H R ; x  F R A C T I O N  C M ;  M A R K E R S ;  o>  G-75  H Y P O C A L C E M I C  • ,  G-50  A C T I V I T Y .  G-75,  O T H E R  1.2  X  80  T E M P E R A T U R E , 3.0  M L .  C O N D I T I O N S  S I Z E ,  T H E  .  A ,  AND  C O L U M N ,  A C I D ;  80  G-50  H Y P O C A L C E M I C  G-75  F O R M I C  M  OF  M A R K E R S ;  - 2 2 (V  0  )  OF  FROM  2.  THIS  BIOCHEM,  CAL  RESULTS THE  WHERE  AND  LINEARITY  LOG OF  OF  BEEN  AND  MYOGLOBIN  OF  ALSO  SHOWN  TO  BEHAVE  AS  ON  THE  G-75  OF  SLIGHTLY  MOLECULAR  BELOW  THE  MOLECULAR  MOLECULAR FIGURE  4  THERE  A  WEIGHT  THE  IS  9 0 0 0 WAS AS  M.W.  IT  HAD  PLOTTED OF  THE  ON  A,  CRYSTALLINE  FACT  THAN  AS  THE  G - 7 5 . ITS  WHEN  ESTABLISHED.BY  C  WELL~(3)»  HOWEVER,  METHODS  NOT  EXPECTED. WEIGHT  ITS  BEHAVIOR  WAS  DID  THAN  BELOW  IN  ACTH  AND  MOLECULAR  1 0 - 1 5 $  REAL  PROBABLY ACTUALLY  FACTORS  OTHER  DISCUSSED  IN  A  LATER  CHAPTER.  THE  G-50  CALIBRATION  VOLUME  OF  BETWEEN  VOLUME, ONLY  THE  G - 7 5 .  REAL  THE  3 0 , 0 0 0  BY  HOWEVER,  ELUTION  THE  ANO  LARGER  APPARENT  EXTENDING  ITS  PLOTTED  CYTOCHROME  INSULIN,  4  INFLUENCED  BE  WITH  FIGURE  MARKER.  3 0 0 0  LINE  FILTRATION  ELUTION  SEEN  M.W.  THE  RELATIONSHIP  THE  THAT  ANOMALOUS  IS  WILL  SMALLER  WAS  GEL  OF  IN ARE  REPRODUCED  SOME  TH.IS  MARKER  SOMEWHAT  £  AN  VALUE  SHOWS  ANO  V  HAD  WHICH  LINEAR  WERE  A  THAT  SIZE  F I T  HAD  5 7 2 2 .  EACH  SHOWN  GLUCAGON,  LINE.  A  EXPECTED,  VERSUS LINE  OF  MUCH  A S  GREATER  5 0 0 0  ARE  BETWEEN  THE  INSULIN  WEIGHT  MOLECULAR RANGE  GRADE  WEIGHT  TO  RESULTS  AND  ALSO  IS  OF  AND  SHOWN  ALONG  COLUMN  MEASURE  AGAIN  BEEN  PREDICTED  FROM  THAN  PREVIOUSLY  L I E  RESULTS  ' ( V )  MOLECULAR  THESE  WEIGHT  CALIBRATION  RELATIONSHIP  HAVE  EXPERIMENT  THIS  THE  REPORTED  THIS  WAS  ANGELES.  VOLUMES  THIS  HAS  ALBUMIN  DISCUSSION.  ELUTION  THE  THE  LOS  RESULTS  THE  VERSUS  COLUMN.  THE  THOUGH FROM  IN  WEIGHT  THE  LAY  OTHER  MARKERS.  TO IF  THE  G - 5 0 3 0 0 0  THIS  BEHAVED  MOLECULAR POINT  OF  WITH  ABOUT  RESOLUTION INSULIN  LOG  COLUMN.  TO RANGE  MUCH  WAS THE ITS  RIGHT APPARENT  -23MOLECULAR  WEIGHT  RESULTANT  POINT  OTHER  MARKERS  USE  THIS  THE  LOWER  END  OF  LINE  0.  EST i MATiON  1.  METHODS.  FACTORS TO  TO  USED  M  THE  AND  THE  5  THE MINED  AND  THE  THE  8  M  ABSORBANCE OF  AT  THE  SAMPLES  280  OF  POINT THE  OF  UPPER  AN  IN  A  THESE OF  FACTORS.  3  PROFILES  THE  EXTRACT  WERE  SIMILAR  APPLIED  SAMPLES  EACH  ML  (96).  POTTS  OF  MANNER WERE  OF  UNKNOWN,  AND  EACH  TO  AT  WEIGHT  WEIGHTS  SAMPLES  NM  A  HYPQCALCEMIC  MOLECULAR  OF  JUSTIFIABLE  ( 9)  BELL  OF  UREA.  ABSENCE  CONSIDERED  OF  ELUTION  VOLUME  OF  THE  PR0CE0URE  ASSAY  PRESENCE ATTEMPT  WAS  OR WAS  MADE  0.1  AND  V  T  ACETATE,  (50 BSA  SIMILAR  ABSENCE  BY  TAKING  ACTIVE  USED  SURVEYED  SODIUM  BEEN  THE  IN  THE  MOLECULAR  COLUMN  MARKERS.  MAOE  THE  WORK  G-50  IN  RELIABILITY  EXTRACTS,  ROUTINELY  OF  PROVIDE  THE  PRESENT.  VQ  TO  WEIGHTS  ACTIVITY  BETWEEN  WEIGHT  WHEN  OF  CALIBRATED  THE  NO  SEEMED  SEQUENCE  ESTIMATES IN  IT  THE  MOLECULAR  BIOASSAY  INDICATE  EVER,  OF  RANGE  ORIGINALLY  THE  INSTEAD,: THE  IN  WERE  FRACTION  ARE  SHOWN  IN  6.  AND  A  HAD  USED  LINE.  ENHANCED  RECORDS  PREVIOUSLY. TO  WAS  LINE.  WAS  THE  WEIGHT  THE  ELUTION  BY  G-75  MOLECULAR  ACID  DETERMINED.  FIGURE S  THIS  FOR  FORMIC  ELUTED  NEAR  BY  OBTAIN  APPLIED  0.1  VERY  WHICH  CONFIRMED  THAT  LAY  OF  WAS  ACTIVE  ON  IN  END  CT,  T O  SEEN  APPARENT  THE  PORCINE  AS  ML TO  TO THE  ONLY OF  ML)  VEHICLE.  TO AS  THAT A  COLUMN FROM  ANO EACH  WAS  QUALITATIVE  THE  EVERY  DILUTING ML  METHOO  FACTORS;  ACTUAL  FRACTIONS  0.3  DETER-  DESCRIBED  HYPOCALCEMIC  MEASURE  ALIQUOTS 150  MOLECULES  HOW-  AMOUNT  OF  WERE  SECOND THEM SAMPLE  FRACTION  WITH WAS  0.2 THEN  ML  -24INJECTEO MINED TEST  AFTER  USED  OF AS  PLASMA A  ARE  AN  ML  SEEN  THE  M  SERUM  ALBUMIN.)  BETWEEN  3  LARGER  WAS  ONLY  WAS  MAXIMALLY  CENTER  OF  HORMONE THE  CHROMATOGRAPHED PREPAREO  2.  THE  T C A  RESULTS ONCE ELUTE  TO  DETERMINE  IS  LOW  ONLY ANY  OF  FOR  PH  4.5  CONTAINING  THAT  A  AND  VOLUME  INDICATIVE  THE  FROM  HUMAN  INJECTION.  0.4$ OF  SINCE TO  IN  CONCENTRATED  REGION  REASON  THEN  SAMPLING  REQUIRED OF  THE  ANY  COLUMN  ASSAY  WHICH  ELUTION  WHICH  VEHICLE  BOVINE  THE  KNOW  A  I F ANY  IN  ML  OF  MAGNITUDE  RATS.  0.1  THIS  WAS DROP  ACTIVITY  IMPOSSIBLE  THE  THIS  AND  WIDE*  BETWEEN  CALCIUM  REPEATED  ACTIVE  DETER-  CONSIDERED  INJECTED WAS  MG$  USED  FRACTION  VOLUME  CORRESPONDS  TO  REGION.  SAMPLE  OF  ON  THE  COLUMN.  USING  THE  FORMIC  BOVINE (SEE  THYROIO FIGURE  EXTRACT 7).  ACID  EXTRACTION  BEEN  CALIBRATED  WAS  THIS  SAMPLE  PROCEDURE  RATHER  PRECIPITATION.  AND  THE  TO  FOR  WAS  WERE  THE  WAS  ACTIVE  ADDITIONAL  THE  FRACTIONS IT  VEHICLE  M G$  IN  MG$)  PLASMA  VARIATIONS  SAMPLES  GENERALLY  ACTIVE.  EACH  OF  ACETATE  5  1  CA,  ACTIVITY.  SURVEY  TAKEN  QUALITATIVE  FOR  ONE  WERE  AND  WITH  CALCIUM  (  MEAN  VEHICLE  BECAUSE  SODIUM  WAS  THE  SINCE  WERE  (0.4  THAN  FACTOR IN  ADDED  SHOWN  EXCEEDED  SEEN  ALIQUOTS  ANO  INJECTED  NEVER  EXTRACT  PLASMA  HYPOCALCEMIC  WHICH  FRACTION.  THYROID  WAS  OF  RATS  DIFFERENCE  CALCIUM  HYPOCALCEMIC  EVERY  THE T H E  RATS  INDEX  CALCIUM  AND  HOUR.  CONTROL  AMBIGUITIES  THE  RAT  PLASMA  ALMOST  0.4  A  ONE  RAT'S  GROUP  OF  INTO  DISCUSSION.  G-50  SAMPLES THE  OF  COLUMN THE  ELUTION  HAD  VARIOUS VOLUME  EXTRACTS OF  THE  FROM  ACTIVE  IT THE  WAS  POSSIBLE  COLUMN  MOLECULES  AND IN  TABLE MOLECULAR  WEIGHT  ESTIMATES  VOLUMES  SPECIES  ON  A  FOR  II I CALCITONINS  SEPHADEX  G-50  ELUTION VOLUME  (ML).  FROM  ELUTION  COLUMN  ESTIMATED MOLECULAR WEIGHT  108  3600  105  3700  87  4500  MURINE  90  4300  HUMAN  90  4300  CHICKEN  90  4300  SALMON  87  4500  TURKEY  84  4600  DOGFISH  84  4600  PORC1NE BOV1NE  1  BOVINE  II  (A) Porcine Thyroid Extract  (B) Bovine Thyroid Extract  0.20-1  0.15-  O 0.10oo CM < 0.03-  (C) Humon Thyroid Extract 0.20 5?  3.0  3.0 -  2.0  2.0 -  6 -i.oh  0.IS  c O 0.10 -  (0) Murine Thyroid Extract  O  -i.o  0  <  lll.ll  o  o  + 1.0  +1.0  CO CM  4 0.09  so  100  ISO  o  - I  .y/v'  -  i  SO  100  " I. 1 —  ISO  Ve (ml)  FIGURE  5.  ELUTION ON  FIGURE AS  PROFILES  SEPHDEX SEEN  4. IN  G-50. ACA THE  (A280  NM)  OF  CONDITIONS IS  THE  DROP  BIOASSAY,  THYROID AS  IN  EXTRACTS  INDICATED PLASMA  IN  CALCIUM  (A)  Chicken  (B)  Ultimobranchial Extract  0.20  3.0  3.0  2.0  2.0  £ -i.o  5 °  E c  O a> 0.10-  Turkey Ultimobranchial  Li  -i.o  nl.  Jl  o  < + ..0  Extract  + I.0L.  Gl  M  0.05-  \ 0  Salmon  (C)  (D)  Ultimobranchial  c  o  Extract  'VA  Dog Fish Ultimobranchial  0.20n  3 9  2.0  2.0  o.is  — a O  1.0  -i.o  6  6  J  J  Extract  3.0  30  Li  0  nil il  o + 1.0  -M.0»-  TpTTJIT  o.io  co  ft  CM  -  o.osH  o  J  —r—  too  90  ISO  80  o  100  ISO  Ve (ml)  FIGURE  6.  ELUTION  PROFILES  EXTRACTS INDICATED  ON IN  (A280  SEPHADEX FIGURE  NM)  G-50. 5.  OF  ULTIMOBRANCHIAL  CONDITIONS  AS  -25EACH  OF  THE  EXTRACTS  AND  BIOASSAYS  ARE  SHOWN  IN  IN  FIGURE  IZED  IN  WEIGHT  THE  FLGURE  6.  THE  TABLE  HORMONES  ARE  ESTIMATED  THIS  FOR  MOLECULAR WITH  FRACTIONS  TABLE  ON  EXTRACT,  HOWEVER*  WAS  ACTIVE  REGIONS,  ONE  A  SECOND  WITH  THE  ELUATE  BREWER  n  CT  HIS  AND  SIMILAR  TO  (17)  THE  OXIDATION  OUT  RULE  SLIGHTLY  OVERLOOKED (65)  SALMON PRESENT  HAVE  OR  CLEAR.  ISOLATED WORK  STUDY.  A  THE  IN  FROM  THE  OF  THE  CURVE.  HIGH  WEIGHT  SUMMAR-  MOLECULAR  VARIED  THAT  EXTRACTS  ARE  POSITIONS  THE  HE  OF  OF  TWO  THE  FROM  ABOUT  FIGURE.  IT  THE  YIELDED  3700  OF  AND  TWO  AND  A  MAKES  DID  FIND  TWO  GEL  THE  SECOND  BY  BREWER,  PRESENCE  OF  WHICH  EXPLANATION  BOVINE  BOVINE  A WAS  JUST MINOR  OF  ONLY  THIS  AND  BE  DURING IN  CT  WOULD DOES  OF HAS  KEUTMAN  COMPONENT  THE  CHARGE  STILL  POSSIBLY AS  THYROID  ANY  WHICH  OVERLOOKED  COULD  THE  COMPLETED  DIFFERENCE  EXIST*  IN  MOLECULES  DIFFERED  ANOTHER  MIGHT  WAS  MENTION  FILTRATION.  THAT  EXTRACT  A  NO  ACTIVE  THESE  METHIONINE, BY  WORK  REGIONS  CHARACTERIZED  DATE  BUT  ACTIVE  THIS  TO  UNREPORTED  ULTIMOBRANCHIAL  EXTRACTS  MOLECULES  SINCE  STRUCTURE  SHOWN  THE  MOLECULAR  POSSIBILITY  DIFFERENT  THYROIO  CALCULATED  NEARER  PRESENCE  UNDETECTABLE THE  THE  HORMONES  SHOWS  UNUSUAL  CHROMATOGRAPHY,  NOT  AJ.  HAS  OBSERVATION.  BE  ET  THE  NOT  PROFILES  4500.  OF  PUBLISHED  PROBABLY  BEEN  FOR  IS  AL  ION-EXCHANGE DUE  WEIGHT  REASON  BOVINE  WITH  ELUTION  CALIBRATION  THE  MAJORITY  THE  ULTIMOBRANCHIAL  THE  AS  G-50  OF  BOVINE  A  ALSO  WHERE  THE  FROM  THE OF  HORMONE  WE I G H T S  THE  OF  VOLUMES  -FlGURE 4  IN  BIOASSAY.  THOSE  EACH  INDICATED  4600  TO  AND  ELUTION  CURVE  THE  ELUANT 5  I I I .  ESTIMATES  CALIBRATION  3600  OF  USING  IN  IN THE  FORMATION  Bovine Thyroid Extract ; (A)  0.20-1  o. 15  -  3.0 r  TCA  (B)  I  3.0r-  2.0  2.0 -  -1.0  - 1.0 -  O  T t, till? lllfl,  O  prr-  + 1.0  o co  Formic Acid  + I.0  L  0.10 -  0.05K  ••••••  T 50  *\ A  \  I 100  150  T  i 100  50  O  I  150  Ve (ml)  FIGURE  7.  COMPARISON BOVINE  METHODS. TCA  OF  THYROID  ELUTION  CONDITIONS  PRECIPITATION  PROCEDURES  ARE  PROFILES  EXTRACTS AND  AS  IN  ACID THE  G-50 BY  INDICATED  FORMIC  DESCRIBED  ON  PREPARED  OF  TWO  DIFFERENT  IN  FIGURE  EXTRACTION  TEXT.  5*  -26OF  A  DIMER  SUCH  EXTRACTS  OF  OOES  SEEM  NOT  MAINTAINED SHOULD FOR  CULAR  A  ABLE  IS  BOVINE  WAS  DIMER. THE  TO  DEVIATION DECIDED VERIFY  FORMIC AND  FROM  ACID  COLUMN.  THE  AS  IN  THIS  SMALLER ONLY  AND  AS  THE  WAS  FIGURE  ALSO  DATA  OBTAINED  OF  ITS  USE,  SINCE  BY  WOULD  AND  THE  ELUTION  VOLUMES  ±3  ML  VOLUME  THE  SMALL  IT OF  WERE THE  THE  TWO  STEP  ELUTION  AS  THE  BOVINE BOVINE  IN  THE  BEEN  BOVINE  EXPECTED  WHICH  SEEMS PART  OF  EXTRACTS, BE  IT  CARRIED  OUT  COMPONENTS. POWDER THE  A  AND  BIOASSAY  RESULTS  CHECK  ON  COLUMN  EXTRACT  OF  ACTIVE  WAS  IT  FRACTIONS  THE  EXTRACT CAN  COMPONENTS  G-50  DATA PREVIOUSLY.  REPRODUCIBILITY  DURING  ACID  7 ( B )  SEEN  THE  THE  PRECIPITATION,  PROFILE THE  TO  NEW  USING  T C A  THIS  WAS  CALIBRATED  FORMIC  THE  BECAUSE  THE  TCA  REASON-  LARGER.  THAN  G-50  FOR  OF  ON  A  MOLE-  THE  THYROID  7 ( A ) TO  THE  APPARENT  CAUSING  SHOULD  FIGURE  OF  NECESSARY  BETWEEN  OTHER  RATHER  CONDITIONS  MOLECULE  ACTIVE  BOVINE  THIS  CHROMATOGRAPHY  EXPLANATION  OTHER  THE  ACID  THE  HAVE  INTERACTION  SERVED  THE  COMPARING  (THE  AN  FROM  AND  SPECIES  7 ( B ) CONFIRMED  THE  LAST.  ACTIVE  CHROMAT0GRAPHED  OF  ELUTEO  DIMER.  EXPERIMENTS  RESULTANT  THE  ALSO  PATTERN  EXTRACTION  SINCE  TISSUE.  A  OTHER  FROM  CARCINOMA  INTERCHANGE  THOUGH  OF  EJT AL. ( 8 0 ) I N  DISULFIDE  THAN  OF  SOME  PREPARED  EXPERIMENT  UNKNOWN  SUCH  FURTHER  EXTRACT  SHOWN  THE  NEHER  EXTRACTION  LARGER  PRESENCE  WAS  THE  OF  BEHAVE  THE  THIS  EXISTENCE  THAT  EXTRACT  HOWEVER,  THE  HORMONE  HORMONE  LIKELY,  THE  MUCH  BY  MEDULLARY  PREVENTED  OF  REPORTED  THYROID  THROUGHOUT  WEIGHT  SUCH  TOO  FORMATION  EXTRACT  THAT  HUMAN  HAVE  THE  AS  BE  VARY  COLLECTED)  THE  PERIOD  SECOND WAS  THE  SEEN  THAT  BY  ABOUT  AFTER  ABOUT  -276  MONTHS  THIS  A  I T SHOULD  USAGE.  MANY  BE  OTHER  MOLECULAR ONE  PROFILES THE  TCA  ACID.  NOTED AND  SHAPE  AS  AND  AND  THAT  PROTEINS,  AND  TCA AT  SINCE  EXTRACT  V^. I S  TCA THE  IS  GENERALLY  SAME  PATTERN  REVERSE  IN  THE  ACTIVE  CASES  MOLECULES  IS  ABSORBANCE IS  QUITE  CHROMATOGRAPHY  SHOULO  EITHER  EXTRACT.  WERE NO  TYPE  TO  BE  OF THE  ADVANTAGE  PROCEDURE. MATERIALS IN  ANY ,  THE  NEXT  USING  THIS  WAS  WHICH  LOW  THE  OF  SMALL.  HAD  ESTIMATES DIFFERENCES  280  V  IN  IF  SEPHADEX  FORMIC  RELATIVELY  IS  TO  BE LARGE  OTHER  TCA  EXTRACTS,  REGION  OF  SEPHADEX  BUT  THE G-50  PURIFICATION  OF  CHROMATOGRAPHY  PURIFICATION  THERE  TCA OF  RELATIVELY  BETWEEN  IS  ACID  THAT  ELUTION  WITH  THE  THE  EXCELLENT  TRUE  Q  IN  FORMIC  COMPLEX  THE  PRECIPITATE  SEEN  NM  BY  THIS  TO  BE  THE  PARTICULARLY  GENERALLY  THE  AN  MORE  (EG.  PREPARED  INDICATING  THUS  THE  ONLY  WAS  LITTLE  OR  PRECIPITATION THE  U L T I M O B R A N C H I AL  SMALL  PEAKS  AT  V  T  CASE.  THE  RESULTS  HYPOCALCEMIC  GLANDS  AT  OF  DIFFERENCE  AT  USED  OF  PROVIDE  STAGE  IN  PEAK  CAN  TRUE  THE  THOSE  RELATIVELY  ABOUT  INSULIN.)  IS  AND  OF  APPLIES  ILLUSTRATED  7(B)  UNKNOWNS  WEIGHT  VARIATION  FOR  THE  THE  ACCURACY  IS  EXTRACTS  EXTRACTS. THE BOTH  DISCUSSED  7(A)  OF  ACCURACY  INFLUENCE  OF  THE  THIS THE  WHICH  FIGURES  REGION  MOLECULAR  TO  FACT  PRECIPITATED  EXPECTED  IN  THAT  NOT  FACTORS  THE  ADDITIONAL  WITH  LARGE  IN  VARIATION  REPRODUCIBILITY  SINCE IN  COLUMN  REPRESENTS  ±150. TO  OF  OF  THE  POLYPEPTIDES  OF  THIS  FACTORS  FOUR  LOWER  SIMILAR  IN  SERIES  OF  EXTRACTED  EXPERIMENTS FROM  VERTEBRATES SIZE  TO  THE  THE  STUDIED  INDICATED  THAT  ULTIMOBRANCHIAL WERE  CALCITONINS  IN  FACT  ISOLATED  FROM  MAMMALIAN ARISING WAS  3600 THE  AN TO  THYROID,  AT  VERY  APPARENT  4600,  ACTUAL  REASONS  A  AND EARLY  THOUGH  CHAPTER I I I .  THIS  IT  CT  STAGE  VARIATION  VARIATION  FOR  THAT  IN  OF  APPARENT  A  WELL  CLEAR  PROBABLY  EXTABLI SHED  VERTEBRATE  MOLECULAR  BECAME  WAS  WAS  MUCH  VARIATION  EVOLUTION.  WEIGHTS  WITH  WILL  FROM  FURTHER  LESS BE  HORMONE  THAN  THERE  ABOUT  WORK  THAT  THIS.  THE  EXPLORED  IN  I I I .  ISOLATION  A.  STUDIES  DESCRIBED  FEASIBILITY  OF  ULTIMOBRANCHIAL OF  I T S PRIMARY  VALUE  OF  LARGE  C T , AND  WORK  STRUCTURE.  SEPHADEX  SALMON  G-50  AS  A  MOLECULE  ANTICIPATED.  I T S AMINO  PROCEDURES  ACID  FOR  ON  C A L C I T O N I N . *  FRAGMENTS  STUDIES  ON  PORCINE  BE  TO  ELUCIDATE  KNOWING  THIS  HORMONE  I T WAS  ISTICS  ANO  PRODUCING  WOULD  PREL i MINARY  1.  EXTRACTiONS.  THE  STARTING  SOCKEYE *PART  OF  AND  BE  WORK  STEP  HORMONE  AND  DETERMINATION SHOWN  AND  USED  EARLIER  AND  HUMAN  STRUCTURE  ASSUME  THAT  THE CALCITONINS  WAS  WELL  SEQUENCING  THE BIOLOGICAL TO  HAD  SALMON  CHARACTERISTICS PURE  THE PRIMARY  OF  ALLOW  DETERMINED,  THOSE  OF  OF  SHOWED  FURTHER OF THE OBTAINED  ESTABLISHED  OF  OVERLAPPING  IN  SEQUENCE  C T  (82)  COULD  SALMON  C T .  ACTIVITIES CERTAIN AND  THE  OF  OF THE  CHARACTERTHE  SPECIES  CLARIFIED.  EXPERIMENTS.  EXTRACT  SOLVENT  ONCE  AND  TO  PURIFICATION  (96, 10, 83)  PROPERTIES  THE  AS  REASONABLE  B.  THE  SUCH C T  BEGUN  THE CHARGE  STRUCTURE  THEM  WAS  THE PRODUCTION  CHAPTER  PURIFICATIONS  COMPOSITION  PEPTIDE  USED  THE PRECEEDING  THE EARLIER  BASED  WERE  IN  SCALE  PURIFICATIONS  AND  OF  INTRODUCTION. THE  THE  A N D C H A R A C T E R I Z A T I O N  FOR  THE PILOT  EXTRACTION MATERIAL COHO  T H E WORK  PURIFICATION  PROCEDURE WAS  SALMON  590  WHICH  DESCRIBED  PUBLISHED ( 8 7 ) .  -29-  G  DESCRIBED OF  HAD  IN  SEPTAL BEEN  THIS  WAS  PREPAREO  IN CHAPTER TISSUE  FROZEN  CHAPTER  FROM  BY  I I . CHUM,  IMMEDIATELY  WAS  PREVIOUSLY  FIGURE  8.  ELUTION  PROFILE  EXTRACT  ON  COLUMN,  5  x  TEMPERATURE, SIZE,  15.0  OF  SEPHADEX 150 4° ML.  CM; C;  SALMON  ULTIMOBRANCHIAL  G-50:  PRELIMINARY  ELUANT, FLOW  0.1  RATE,  12  M  FORMIC ML/HR;  SOLVENT  STAGE  1.  ACID; FRACTION  - 3 0 AFTER  COLLECTION.  TISSUE  TO  YIELDED  8 5  2.4  G. G  OF  ACETONE  DRYING  REDUCED  SOLVENT  EXTRACTION  EXTRACT  WITH  A  OF  THE  THE  SPECIFIC  WEIGHT  DRY  OF  THE  POWDER  ACTIVITY  OF  3  MRC  U/MG. 2.  STAGE  1  THE 5  X  OF  FIRST  1 3 0 CM  FORMIC 1 2  ELUATE WERE  CHROMATOGRAPHY. GEL  COLUMN  FILTRATION OF  ACID  ELUANT,  ML/HR.  THE  FRACTIONS  SURVEYED  I I ,  USING  2.4  G  AND  APPLIED  OF  ML  INDEX  BIOLOGICAL INJECTIONS  EXTRACT, TO  AN  WAS  THE  WAS  G-50  COLLECTING  ABSORBANCE  FOR  0 . 3  SEPHADEX AND  AS  STAGE  COLUMN  4 °  C  1 5  ML  FRACTIONS  2 8 0 NM  OF  PROTEIN  ACTIVITY  IN  IN  TWO  1 0  A  0.1  M  AT  A  RATE  FOR  THE  CONCENTRATION  AND  THEY  DESCRIBED DILUTED  ML  ML  A  ON  DETERMINED  AS  2 0  USING  WAS  SUITABLY  DISSOLVED  OUT  AT  AT  OF  CARRIED  OF  0.1  IN  CHAPTER  ELUATE.  THE  M  ACID  VOLUMES.  FORMIC THE  V  OF E  THE  CT  REGION  WAS  TAKEN  AND  USED  MOLECULAR EQUAL  TO  E  DEFINITIONS THE WERE THE  ACTIVE  POWDER  WITH  OF D  CALCULATE  IN  AND THE  IS  OF  A  THE  VALUE  BIOLOGICALLY FOR  INDEPENDENT WHERE  THE  K  OF  VOLUME  D  .  AN  ACTIVE  INDEX  OF  COLUMN  SIZE  AND  TERMS  HAVE  THE  IS  CHAPTER I I .  PROFILE THOSE OF  THE  SPECIFIC DEGREE ACTIVE  AND  SEEN  LYOPHILIZED A  CENTER  ) / ( V-J-V^)  USEO  TO  THE  WHICH  REGIONS  AND  K  Q  ELUTION  POOLED  THE  - V  SIMILAR  RECOVERY  TO  WEIGHT ( V  AS  OF  TO  ACTIVITY  IN  CHAPTER  ELUATES, YIELD  ACTIVITY  WAS  I I AS  OF  FOR  4 0  OF MRC  ARE  THE  SHOWN  FRACTIONS  1 3 0 MG  PURIFICATION  REGION  SURVEY  1 1 8  IN TO  SLIGHTLY U/MG.  FIGURE  IN  0 . 6 .  8.  1 4 5 , WERE OFF-WHITE  DETAILS  INCLUDED  APPROXIMATELY  SAMPLES  THE  OF SUMMARY.  0  100  200  Elution  FIGURE  9.  ELUTION C-25:  PROFILE  OF  STAGE  1  STAGE  2.  FROM  LINEAR  0.2  ML/HR.  M  TO  GRADIENT 0.5  M;  400  (ml)  PRELIMINARY  ELUANT, 1 0  Volume  300  OF  PRODUCT  ON  COLUMN, P H 3.4  TEMPERATURE,  SE-SEPHAQEX 1.2  x  2 0  AMMONIUM 4 °  C;  FLOW  CM;  FORMATE RATE,  - 3 1 3.  STAGE A  AND AT  2  1.2  CHROMATOGRAPHY. X  2 0  CM  E Q U I L I B R A T E D PH  3.4.  S T A R T I N G WITH THE  50  95  ML  OF  COLUMN  AT  MG  BUFFER  AND  WAS  1 5 0  ML).TO  VOLUME,  1 5 0  M L ) .  AND  FOLLOWED ALSO  AS  FROM  BEFORE.  POWDER  WAS  PREPARED  FORMATE  DISSOLVED  BUFFER  IN  THE  COLUMN.  APTER  BUFFER,  THE  MATERIAL  REMAINING  M M  A  L I N E A R  (PH  3.4;  (PH  AT  A  WAS  2 8 0  GRADIENT  OF  WASHING  CONDUCTIVITY,  COLLECTED  NM  AND  8.0  A  OF  EACH  MOOEL  COM  MMHOS; MMHOS;  RATE  B I O L O G I C A L  CONDUCTIVITY RADIOMETER  AT  ON  AMMONIUM  CONDUCTiviTY,1 8.0  3.4,  ELUATE  THE  USING  AMMONIUM  WAS  THE  0.2  THE  M  C-25  TO  ABSORBANCE  DETERMINED  0.2  1  WITH  0.5  SE-SEPHAOEX  WITH  A P P L I E D  ELUTED  VOLUME,  OF  STAGE  S T A R T I N G  BUFFER  ML/HR  4 ° C  OF  FORMATE  10  COLUMN  OF  A C T I V I T Y  FRACTION 20  WAS  CONDUCTIVITY  METER. THE OF  THE  GICAL ON  A  E L U T I O N  ELUATE  P R O F I L E ,  FRACTIONS  A C T I V I T Y  CAME  CONDUCTIVITY  OF  REGION  FROM  AS  THE  AMMONIUM  TO  ALLOW  COMPLETE  WAS  NOT  DETERMINED  4.  STAGE  FRACTION  3  P U R I F Y  TO THE  ARE  SHOWN  THE  COLUMN 1 2  MMHOS.  1 5  2 8  WAS  TO  BUFFER  D E S A L T I N G T H I S  BY  A  THE  NOT  9.  THE  BROAD  POOLED  WAS  B I O L O G I C A L  FLGURE  IN  ABOUT  FORMATE  AT  IN  AND  PEAK  ENTIRE AND  A C T I V I T Y  B I O L O CENTERED  A C T I V E  L Y O P H I L I Z E D .  S U F F I C I E N T L Y  L Y 0 P H I L I Z A T I ON ,  V O L A T I L E  S P E C I F I C  STAGE.  CHROMATOGRAPHY.  BLO-GEL S I M I L A R  OFF  CONDUCTIVITY  P - 1 0 ,  SEPHADEX STAGE  2  A  P O L Y A C R I LAM IOE G-50,  WAS  PRODUCT.  USED A  1.2  GEL TO X  WITH  DESALT 2 0 0  CM  P R O P E R T I E S AND COLUMN  FURTHER WAS  A C T I V I T Y  0.15  w  2  0  |  0  20  40  60  Fraction  FIGURE  10.  ELUTION P-10: ELUANT, FLOW  P R O F I L E  Number  OF  STAGE  2  P R E L I M I N A R Y  STAGE  3.  0.4  RATE,  M 6  A C E T I C ML/MR;  80  A C I D ;  PROOUCT  ON  COLUMN,  BIO-GEL 1.2  TEMPERATURE,  FRACTION  S I Z E ,  3.4  x 4 ML.  200 C;  CM;  PREPARED AND  THE  COLUMN  IN  1  COLLECTED As  THE  OF  3.4  SEEN  IN  OF  OF  AMINO  SEVERAL THE  FIGURE  REGION SIDE  MOLECULAR  PERFORM IC  A C I D S NOT  LOW  EXPECTED  IN  CONTAIN  WAS  A  AND  THE  SINCE  APPROXIMATELY  MG/ML)  R E P R E S E N T I N G  CULAR  WEIGHT  JiMOLES IT  WAS  OF  OF  0.33  4500.  -jy-CYSTINE,  ESTIMATED  THAT  THE  A C T I V I T Y  700  MRC  SUGGESTING  MUCH  A C T I V I T Y  LARGER  OF  OF  THE  OF  THE THAT  NEARLY  Q U A N T I T I E S  OF  TO  AND  EXPECTED  C Y S T E I C  THAT  _M °LES M  ABOUT  WAS  THE  HORMONE U/MG,  WOULD  THAT  THE  BE  CON(0.45  WITH ONLY  A  MOLE-  0.1  HORMONE,  15$  ASSAYED  AND  HORMONE  46  OF  ACID  WERE  MATERIAL  MATERIAL  T I S S U E  OF  THE  CONTAINED  CONTAINED  5 0 0 0 MRC  SAMPLES  RATIOS  MATERIAL  0.05  PURE  E.  FRACTION  PURE  O X I -  HYOROLYSED  OF  CLEAR  THE  ALIQUOT  -jp-CYSTINE  PEAK.  ' OF  AT  I N D I C A T I N G  OF  FRACTION  PEAK  MOLE  C Y S T I N E  WAS  46  S E C T I O N  L Y O P H I L I Z E D  EQUIVALENT  S P E C I F I C  S P E C I F I C  THE  MMOLES  AS  THE  U/MG  OF  MG  OF  IT  SHOULDER  MOLECULE  RESIDUES  MOLECULE  BROAD  WERE  UNITY  SINGLE  TWO  1.5  THE  BELOW  A  COMPONENT  TAINED  MATERIAL  ASSO-  D E T A I L E D  AN  IN  WAS  A  NUMBER  AND  WAS  ML/HR.  BUT  THE  THE  ELUATE  A C T I V I T Y  FRACTION  Q U A N T I T I E S  NATIVE  MINOR  FAR  6  PEAKS  DESCRIBED  TO  THE  OF  ONLY  PERFORMED.  WERE  UREA.  RATE  THAT  RESOLUTION  A P P L I E D  B I O L O G I C A L  UNOXIDIZED  THE  IN  L Y O P H I L I Z E D AS  MAXIMUM  WAS  UNRESOLVED  WAS  ANALYSES  2  A  A C T I V E .  ACID  AND  WEIGHT.  QUITE  ONLY  WAS  M  AT  SHOWED  SHOULDER  COULD  4  THE  LARGE,  PROVIDE  STAGE  MADE  10  THE  ACID  FROM  TO  FRACTIONS  TWO  AMINO  PEAK  WERE  ML  O X I D I Z E D  ACID  ELUANT  THE  THE  WITH  BOTH  AND  OF  L E A D I N G  CENTER DIZED  ML  WITH  A C E T I C  A C T I V I T Y  IN  BIOASSAY ON  M  POOLED IN  CIATED  0.4  HORMONE. AS  MIGHT  ABOUT HAVE  I N D I C A T I N G  REQUIRED  TO  A THAT  YIELD-  F I G U R E 11 SUMMARY  OF  PRELIMINARY  P U R I F I C A T I O N  OF  WEIGHT  PROCEDURE  SALMON  S P E C I F I C  APPROX.  ACTIVITY  ACTIVITY  (MRC U / M G ) SEPTAL  TISSUE DRY  AND  WITH ACETONE  590  G  85  G  CT  (MRC  DEFAT  ACETONE  POWDER EXTRACT  WITH  BuOHsHAcsH 0 2  FILTRATE  RESIDUE  P R E C I P I T A T E OF  RESIDUE  WITH  ACETONE  AT  5  -20  VOL C  P R E C I P I T A T E  (DISCARDED) EXTRACT 0.1M  S U P E R N A T E *  WITH  FORMic  ACID  RESIDUE (DISCARDED)  LYOPHILI  ZED  2400 MG FINAL  STAGE  1  MOLECULAR  S I E V I N G  SEPHADEX  G-50  130  MG,  ION  EXCHANGE  SE-SEPHADEX, 2  BIO-GEL 3  PH  MG  40  5200  400  3700  ON  3.4  A C T I V I T Y MOLECULAR  STAGE  ON  ACTIVITY 95  STAGE  7000  EXTRACT  A C T I V I T Y  S I E V I N G  ON  P-10  8.3  MG  U)  -33SUFFICIENT  THE POOLED YIELO AN  HORMONE  REMAINDER  AND OF  IN  ACTIVITY  A  SERIES FOR  GRAPHY  ON  PROVED  MOST  AN  ANALYSIS.  THE  ACTIVE  YIELDING  THIS  STAGE  OF  TEST  FURTHER  SE-SEPHADEX  COLUMN  11  SUMMARIZES  EXPERIMENTS.  THE  INITIAL  HAD  COLUMN  FORE  ABOUT  MAJOR  EXTRACTION,  U  TH I S  STAGES  TOTAL  FROM  MATERIAL THE  AT  WAS  BEST CHROMATO-  A  FOR TO  73$.  A  THE  P R E P A R A T I ON  1.  EXTRACT  OF  U/MG  RECOVERED OF  PURE  MATERIAL  PURIFICATION  SEVEN-FOLD  15$  3  AT  WERE  RECOVERY  THE  THE  U  U  OVER  AT THE  USED  ACTIVITY  BUT  ABOUT  3800  3700  AND  STAGE  C.  LARGER  THE  BIOLOGICAL  RECOVERED.  EXTRACT  FOLD  3 7 0 0 MRC  EQUILIBRATED  CHROMATOGRAPHY  THE  THE  EVENTUALLY  SEPHADEX  FROM  POWDER.  DETERMINE  AFTER  EXCHANGE  THE  U.  OF  WAS  FURTHER  ABOUT  ACTIVITY  ACTIVITY  50,000  WAS  AN  FILTRATION  OF  WAS  HIGHER  PH  EFFECTIVE.  FIGURE  U.  MG  TO  41-50,  FRACTIONS  3.8  PURIFICATION.  PURIFICATION.  EXTRACT  REGION,  COLUMNS  OF  DURING  .  3 8 0 0 MRC  CONTAINING  SUMMARY  LIMINARY  7000  AT  SAMPLE  CONDITIONS  5.  OF  LYOPHILIZED  INITIAL  USED  FOR  WAS  A  ABOUT  APPLIED AFTER  97$.  PRODUCT THIS  OF  STAGE  FRESH  STAGE  75$ TO  PRE-  ASSAYED  TOTAL  THE  THE  THE  NOT  FINAL  FOR  IN  THE  OF  ABOUT  OF  THE  THE  ION  FINAL  GEL  OVERALL STAGE  3  RECOVERY WAS  THERE-  REPRESENTED  GLANDS  AND  A  REQUIRED  PURIFICATION.  PURE  SALMON  CALCITON IN.  ION.  PRELIMINARY  QUANTITIES  OF  EXPERIMENTS GLANOS  WOULD  MADE HAVE  IT TO  CLEAR BE  THAT  MUCH  PROCESSED  AND  A  AS  COLLECTION  200  POUNDS  SALMON  IN  OF  NO  WERE  SHIPPED  LONGER  EXTRACTION  CT  TISSUE  WERE  TO  CANADA  ARMOUR  EXTRACTION  BY  A  THE  LTD.,  OF  THEY  HAD  WHERE 500  FROM  DRYING  AND  TORONTO  TONS  SEPTAL  FOR  Co.,  OVER  TISSUE  SOLVENT  KANKAKEE,  DEVELOPED  OF  EXTRACTION  FROZEN  PHARMACEUTICAL  METHOD  POINT  COLLECTED  SCALE  PACKERS  TO  TO  QUANTITIES  FEASIBLE.  AND  FOR  I L L .  PORCINE  (15). THE  SALMON  STARTING  BY  SPECIFIC  ASSAYED  TISSUE  THE WAS  1  UP  THE  2  IN  COLUMN IS  OF  FORMIC  ML/HR. THE  OF  CHAPTER  MRC  A  WAS  BEEN  (65);  II  WAS  THIS  HAS  U/MG  MRC  U/MG)  REPORTED  SOMEWHAT  A  EXTRACT  HOWEVER,  50  OBTAINED.  MATERIAL  AN  PURE  TOTAL  TO  HAVE  WHEN  LOWER  SPECIFIC  KG  SEPTAL  OF  OF  BETWEEN  UNITS.  DESCRIBED  HANDLE  STARTING 0.1  M  IN  SUITABLY  G-50.  AT  IN  4°C  THE  ENTIRE  MATERIAL  APPENDIX  ACID  WERE  THE  FORMIC  ABSORBANCE  SAMPLES  SAMPLES  TO  SEPHADEX  DESCRIBED  0.1:M  G  QUANTITY  YIELDED  CHROMATOGRAPHY.  COLUMN. UREA  IN  MRC  PROCEDURE  SCALED  M  4  EVENTUALLY  MATERIAL  230  OF  900,000  AND  STAGE  THIS  DESCRIBED  (150-200  WHICH  SIGNIFICANT  ARMOUR.  YIELDED  600,000  IN  ACTIVITY  AS  ACTIVITY  2.  MATERIAL  CALCITONIN  PREPARED A  SEPTAL  IMPROVED  SEASONjLABORATORY  A  WERE  FOR  TECHNIQUES  ACID  WAS AND  THE  PRELIMINARY 4  G  DISSOLVED APPLIED  B.  THE  AND  COLLECTED  SAMPLE  NM  AND  FOLLOWED  AS  DESCRIBED  D I LUTED  PRIOR  TO  AT  A  OF  WAS A  BIOLOGICAL  ON  A  10  ML  CM  COLUMN  ELUTEO RATE  OF  150  X  THIS  WITH  OF  ACTIVITY  PREVIOUSLY  INJECTION.  SINGLE  40  IN  TO  CONSTRUCTION  280  AT  SAMPLE  EXPERIMENT  250 OF  WITH  SPECIFIC  FIGURE  12.  ELUTION  P R O F I L E  EXTRACT  ON  10  X  4°  C;  150  CM;  FLOW  OF  SALMON  SEPHADEX ELUANT,  RATE,  250  ULTIMOBRANCHIAL  G-50S 0.1  STAGE M  ML/HR.  FORMIC  1.  ACID  COLUMN,  ACID;  TEMPERATURE,  ACTIVITY  WAS  LL  ALIQUOT  ON  AN  PHILIZED  AND  THE IS  IN  IN  SEEN  VARIEO A  L  AFTER WITH  SLIGHTLY  DEGREE  3.  STAGE  THE  HOWEVER,  ONLY  TO  THE  WAS THE  K  THE  IN  CHAPTER  LATER  LYO-  NEARLY  OF  ON  NARROW  THIS  REGION ARE  COLUMN  PREVIOUSLY, KEUTMANN OF  THE  FROM  BUT  EJT _A_L_  (65)  B IOLOG I CAL TO  THAT  SEEN  VOLUME  OF  HORMONE. AN  ELUTION  CONTAMINATION 8.5  TO  YIELDED  ABOUT  THE  MARKEDLY  IDENTICAL  FROM  FROM  OF  FROM 9.5  350 MRC  650  COLUMN  L  MG  OF  TO  POOLED. MATERIAL  (SEE 40$)  POOLED.  DETAILS  GIVEN  THE  IN  LARGE  WAS  U/MG  (30$  THE  FIGURE WAS  ON  DUE  RECOVERY  SUMMARY.  CHROMATOGRAPHY.  DESCRIBED  IN  LARGER  HANDLE  COLUMN M  ACTIVITY  BY  VALUE  Q  POOL  PURIFICATION  COLUMN  TO  THIS  SURVEY  USED  SALMON  REGION  RECOVERY  OF  2  WAS  DIFFERED  SEEN  AVOID  L,  SPECIFIC  0.2  THAT  BUT  AND  WITH  EXTRACTS  FROM  ON  PROFILE  9.6  TO  DESCRIBED  WHICH  ACTIVITY  EXTENDED  PRIMARILY  CM  SAMPLE  REGION  Low  20  THE  EXPERIMENT.  PEAK  THE  BIOASSAY  ACTIVE  THE  18).  AND  SOLVENT  LYOPHILIZATION A  THE  POOLED  12.  EXPERIMENTS  TO  LEADING  THE  PROFILE  THE  (0.6),  THE 8.3  FOR  SIMILAR  0 T H*E R  IN  WEIGHED.  FIGURE  ONLY  ACTIVITY JN  OF  ELUTION  SHOWN  THAT  DETERMINED  THE  OF  USED  FROM  STARTING  BUFFER  THIS  STAGE  WAS  SIMILAR  PRELIMINARY  EXPERIMENT  THE  QUANTITY  GREATER  SE-SEPHADEX  AMMONIUM  MATERIAL  IN  STAGE AND  FORMATE 1  WAS  ELUTED  C-25 AT  WAS PH  APPLIED WITH  THOUGH  OF  400  MATERIAL.  THE  THE ML  THAT  SLIGHTLY  EQUILIBRATED  3.4. TO  TO  OF  A AT  350  MG  OF  COLUMN  IN  10  STARTING  -  2.0 4°  X  C  ML  BUFFER  OF  „  3.0  N  Fraction  FIGURE  13.  ELUTION  025*  P R O F I L E STAGE  GRADIENT 0.6  M,  OF  PH  FOLLOWED  TEMPERATURE S I Z E ,  2.  12  ML.  4°  OF  Number  STAGE  COLUMN,  3.4 BY C;  1  PRODUCT  2 x 20  AMMONIUM 1  M  FLOW  FORMATE  AMMONIUM RATE,  CM;  20  ON  SE-SEPHADEX  ELUANT, FROM  0.2  LINEAR M  HYDROXIDE; ML/HR;  FRACTION  TO  -36FOLLOWED  BY  BUFFER  FROM  TO  M  0.6  THE OF  A  0.2  M  WAS  RATE  AND  OF  20  ELUATE  ML/HR.  BIOLOGICAL  pH  3.4  THE TO  WAS  ENSURE  ACTIVITY  WERE  500  IN  280  FOLLOWED  12  ML  NM, AS  OF  500  ML).  WASHED  RECOVERY  COLLECTED AT  WAS  FORMATE  VOLUME »  VOLUME,  COLUMN  ABSORBANCE  AMMONIUM  MMHOS i  8..0  24.0,MMHOS;  HYDROXIDE  THE  OF  ( C O N D U C T I V I TY  COMPLETED  AMMONIUM  MATERIALS. A  M  GRADIENT  (CONDUCTIVITY,  GRADIENT 1  LINEAR  AFTER  WITH ALL  ML )  500  ML  APPLIED  FRACTIONS  AT  CONDUCTIVITY PREVIOUSLY  DESCRIBED. FIGURE ONLY  ONE  THIS  PEAK  13  MAJOR  MMHOS)  SECOND  THE  WAS  THAT  COMPONENT  COLLECTEO  REGION BY  STRUCTURES.  REGION  MAXIMUM  BECAUSE DEGREE MINOR  OF OF  MAY  KEUTMANN  FACT, TO  SALMON  HAVE  MINIMIZE  SPECIFIC  ACTIVITY  PURIFICATION PEAKS  IN  DID  NOT  WERE  OF  AND  MAY  LYOPHILIZED APPEAR  REMOVED.  TO  BE  IS  INDICATE  THE  GLANDS  SLIGHTLY  (FRACTIONS NOT  TO  IT  MINOR  CONTAMINATION  COULD  PRIOR  THE  OF  PEAK  ACTIVITY,  RETROSPECT  WHICH  VS.  THE  ELUTED  (65), FROM  POOLED  THE  IN  THE  MMHOS  BIOLOGICAL  CALCITONINS  WAS  SALT  (16  ACTIVITY  AL  ALTHOUGH  COMPONENTS.  REPRESENTED ET  ELUTION.  SHOULDER  OF  FURTHER.  HAVE  OF  TWO  THE  2  GRADIENT  HIGHER  REGION  ACTIVITY  RESIDUAL  CONTAMINANT  BUT  STAGE  THE  LEAST  BIOLOGICAL  SPECIES IN  THE  SLIGHTLY  MAJOR  OF  OF  DURING AT  INVESTIGATED  DO,  LYOPHILIZED.  WAS  THE  DIFFERENT  ANO  CONTAIN  REGION  DIFFERENT  OF  ELUTED  PREVIOUSLY,  TO  OESCRIBEO  WERE  RESULTS  PEAK  SEEN  THIS  THE  WAS  THE  NOT  THAT  THE  TO  WELL  MINOR  PEAK  LIKELY  AT  THAN  CORRESPONDED A  PEAK  APPEARED  CONDUCTIVITY 12  SHOWS  BE  A  NARROW  84  98)  DETERMINED  PRODUCT, GREAT  TO  BUT  SINCE  THE, ONLY  CURE  14.  ELUTION G-50: 0.1 6  P R O F I L E STAGE  M  FORMIC  ML/HR;  OF  3.  ACID;  F R A C T I O N  STAGE  COLUMN,  2 1.2  PRODUCT x  TEMPERATURE, S I Z E ,  3.4  ML.  220 4°  ON CM; C;  SEPHADEX ELUANT FLOW  RATE,  -374.  STAGE  3  THIS HIGH BY  STAGE  FIRST OF  ACID.  THE  0.1  6  M  LARGE  FORMIC  WAS  TO  AND  SINGLE  TO  REMOVE  DISSOLVED  LAYERED IN  4°  AT  ONTO  STEP,  BUT  A  CONTAMINANTS  COLUMN.  PREPARED WAS  WAS  A  A  1.2  X  C  IN  0.1  THE  ML  OF  TOP  OF  THE  THE  BIOLOGICAL  FRACTIONS  AT  A  CM  M  ML  LONG,  LEFT  220  7  IN  3.4  63  WERE  55  MG  OF  MATERIAL  U/MG  MRC  U.  5.  STAGE  4  FOR  THE  POOLED  AT  MATERIAL FLOW  WITH  FORMIC  8  M  UREA  COLUMN.  RATE  OF  4.5  OF  THE  OF  Q  0.6.  COLUMN  A  ACTIVITY  TOTAL  WAS THIS  INDICATED  THAT  USED.  LYOPHILIZATION  SPECIFIC FOR  ION  C-25 4.5  WAS  THE OF  EXCHANGE  EQUILIBRATED  THIS  POOL  YIELDED  1200  ABOUT  RECOVERY  PEAK  FRACTIONS.  THE  OF  IN  COLUMN.  0.1  M  ML  350  BUFFER ML)  TO  A  1.2  4°  C  WITH  THE OF  AFTER  AMMONIUM  FORMATE  VOLUME,  3  STAGE AT  PREPARED.  DISSOLVED  AMMONIUM  MMHOS;  A  K  A  RESULTS  AFTER  18)  SECOND  PH  WAS  ML  WITH  WITH  ACTIVITY  65,000  OF  CHROMATOGRAPHY.  ONTO  400  LATER  AND  FIGURE  (SEE  SE-SEPHADEX  ACETATE  BUT  PEAK  CHARACTERISTIC  TO  MRC  14,  MAJOR  SKEWED,  48  3.5  PRODUCT  FIGURE  IN  A  SLIGHTLY  ASYMMETRY  PH  WAS  COLLECTED  SHOWN  CONFINED  TO  USED  DE-SALTING  ML/HR. As  OP  A  FILTRATION  G-50  ACID  WAS  WAS  GEL  2  STAGE  ELUATE  ESSENTIALLY  COLUMN  SEPHADEX  COLUMN  THE  WAS  RESOLUTION  THE  IN  CHROMATOGRAPHY.  55  0.1  CM M  SAMPLE  OF  STARTING  BUFFER  ANO  COLUMN  ACETATE, FROM M  0.1  A  HAD  LINEAR M  BEEN  COLUMN AMMONIUM  MG  THE  0.5  20  x  STAGE  3  ALLOWED WASHEO  GRADIENT  OF  (CONDUCTIVITY,  (CONDUCTIVITY,  16.0  MMHOS;  9$  0  2.0  20  40  ' 6 0  Fraction  FIGURE  15.  ELUTION C-25* LINEAR 0.1 4°  M C;  P R O F I L E STAGE 0.2  FLOW  M  STAGE  COLUMN,  GRADIENTS TO  OF  AND  RATE,  100  120  Number  OF  4.  80  12  PH FROM  3  PRODUCT  1.2  4.5 0.2  ML/HR;  x  20  AMMONIUM M  TO  SE-SEPHADEX  ON CM;  0.5  FRACTION  ELUANT,  ACETATE M; S I Z E ,  FROM  TEMPERATURE 9  ML.  0  20  40 Fraction  FIGURE  16;  ELUTION G-50:  PROFILE  STAGE  5.  OF  60  80  Number  STAGE  4  CONDITIONS  PRODUCT  ON  IDENTICAL  SEPHADEX TO  FIGURE  14  -38VOLUME, IN  THE  3 5 0 ML) ELUATE  WAS  THE  BUFFER  0.5 M  CONTINUED  WITH 2 5 0 ML OF  TRATION.  WHEN  GRADIENT 0.2  WAS  THE  BEGUN  WITH  WAS  A NEW  UNTIL  DESCRIBED 9  ML  ABOVE  FRACTIONS FIGURE  GRADIENT PEAKS  AT  ELUTED WITH  SYME T R I C A L  AND  6.  THE  SHOWED  PROCEDURES  CHROMATOGRAPHY  POOL  FOR  WERE STAGE  FROM S T A G E  B L O L O G I CAL FRACTION  AT  WHICH  WAS  FROM  0.5 M  THIS  TIME  ELUATE  GRADIENT  THE  PROCEDURE  COLLECTED  IN  ML/HR. PROFILE,  CONDUCTIVITY  SURVEY.  THREE  COLUMN,  ONLY  ONE  ACT I V I T Y .  POOLED  AND  OF  AND  EQUIPMENT  IDENTICAL 3.  THE  OF  WELL  DEFINED  WHICH WAS  THIS  PEAK  WAS  CONTAMINATION.  LYOPHILIZED.  THE  IN  ALL  IN  OF  THE  RESPECTS WAS  STAGE TO  THE  LYOPHILIZED  M FORMIC  ASSAYS  ON  AND  AN  ACID,  ALIQUOT  SPECIFIC  A LYOPHILIZED  5  THOSE  OF 0.1  DESCRIBED,  WEIGHT  IN  USED  1 ML  WAS M E A S U R E D  PREVIOUSLY  FROM  IN  USED  SAMPLE  DISSOLVED  4  A C T I V ITY  5 6 AS  CALCULATED  A NEW  TO  INDICATION  1 2 1 WERE  ZERO  CHROMATOGRAPHY.  5  DESCRIBED  NO  CONCEN-R  3 0 0 ML)  300 ML).  APPEAR  ELUTION  VOLUME,  ACTIVITY  BIOLOGICAL  TO  TO  BUFFER  ELUTION  THE  AND  ESTABLISHED  RETURNED  THE  12  BEGAN  ACETATE  BEGAN,  OF  THE  FROM  ANY  1 0 9 TO  STAGE  RATE  BIOLOGICAL  ASSOCIATED  FRACTIONS  A  THE  VOLUME,  REPEATED.  1 5 SHOWS  AND  WERE  WAS  PEAK  PEAK  AMMONIUM  5 . 6 MMHOS;  1 6 . 0 MMHOS:  FIRST  DISCONNECTED  AT  HAD  PH 4 . 5  (CONDUCTIVITY,  THE  WAS  BUFFER  ABSORBANCE  M (CONDUCTIVITY,  USED  WHEN  BEGUN.  FROM  ACTIVITY  ALIQUOT  OF  THIS  FRACTION. THE  ELUTION  PURIFICATION  PROFILE  STAGE  SHOWN  CONTAINED  A  IN  FIGURE  SINGLE  1 6 FOR  MAJOR  PEAK  THE AND  FINAL A SMALL  FIGURE SUMMARY  OF P U R I F I C A T I O N  PROCEDURE  SEPTAL  17 OF SALMON C T  WEIGHT  TISSUE  SPECIFIC ACTIVITY (MRC U / M G )  APPROX. ACTIVITY (MRC U )  5 0 KG  ACID EXTRACTION BY ARMOUR C O .  • FINAL  EXTRACT  4 G  150-230  750,000  650  230,000  MOLECULAR S I E V I N G ON S E P H A D E X G - 5 0  v STAGE  —  1 ACTIVITY I ON E X C H A N G E SE-SEPHADEX,  STAGE  3 5 0 MG ON PH 3 . 4  2 ACTIVITY  -  -  -  MOLECULAR S I E V I N G ON S E P H A D E X G - 5 0 V  STAGE  3 ACTIVITY ION E X C H A N G E SE-SEPHADEX,  STAGE  5 5 MG  1200  65,000  ON PH 4 . 5  4 ACTIVITY  -  -  -  MOLECULAR S I E V I N G ON S E P H A D E X G - 5 0  • STAGE  5 ACTIVITY  1 5 MG  4500  65,000  FIGURE  18.  R E G R E S S I O N S OF LOG DOSE ON R E S P O N S E FOR S T A G E 1, 3 AND 5 P R O D U C T S . CALCULATIONS AS D E S C R I B E D FOR F I G U R E 3 . o STAGE T, VSTAGE 3» a STAGE 5.  i  -39PEAK  AT  V  INDICATING  FREE  OF  PROTEIN  T  CHARACTERISTIC DESCRIBED  IN  PRESENT  IN  WELL  THE  TO  FRACTION ASSAY  SECTION  5 6 ARE  AND  THE  CALCULATED  THE  SPECIFIC  STAGE  PURE  THAT  BE  WITH  THE  ONLY  SPECIFIC  CLOSE OF PURE  1 0 0 $ OF  OF  C T .  OF  PURE  EACH IN  THIS  OF  SALMON C T  NEARLY  25  TIMES  YIELD  AT  THIS  THE  A TOTAL  THE  IN  MATERIAL  M R C L)/MG  PORCINE  WAS  FRACTION,  ACTIVITY  HORMONE,  PEPTIDE  BASIS  THE  HOMOGENEITY  C0RRESPONDED  ON T H E  IN  TO 4 5 0 0  PURE  A SINGLE  ON T H E  WAS A  FOR  CONCENTRATION  18.  CONCENTRATION  TESTS  A C T I V ITY  THE  ESSENTIALLY  ASYMMETRY  THE  BIOASSAY  FIGURE  1 7 SUMMARIZES  65,000  MATERIAL  MRC U  APPLIED  IN  DURING  THE  ACTIVE  MINOR  IN  TWO  IN  FIRST  IN  F|GURE  FIGURE THREE  COMPONENT  FINAL  STAGES  RECOVERY  WAS ABOUT  ACHIEVED  FROM  ACTIVITY  OF  THE  18  17.  STAGES  WHICH WERE  1 0 $ , BUT  SEPTAL  TISSUE  OF  USED  IN  ALTHOUGH  HIGH  PROBABLY  WAS  REMOVED,  A 300,000  PURE  AN  AT AT  STAGES EACH  IN  THE  FOLD  WITH  STAGE  TO  IS  AN  RECOVERIES THE  OVERALL  PURIFICATION  AN  1,3  OCCURED  PART  100$.  ESTIMATED  HORMONE  PREPARATION  LOSSES  DUE  ESSENTIALLY  WITH  THE  BIOASSAYS  THE RECOVERY  0  STAGES,  1 5 M R C L ) / G TO  4 5 0 0 M R C U/MG.  SCHEDULE.  THE RESULTS  CT.  SHOWN  RECORDED  THE  PURIFICATION  SALMON  5 ARE  ALSO  IN  ESSENTIALLY  OF  SUMMARY  AND  AS  WAS  4.  FIGURE OF  OF  ABSOLUTE  WAS 1 4 . 5 MG OF  STAGE  VARIED  ACTIVITY  REPRESENTING  7.  AND  TO  5 MATERIAL  THE BIOLOGICAL  SHOWN  THE  USED,  G SHOWEO  PEPTIDE  WAS  STAGE  THE SLIGHT  COLUMN  PEAK.  PEAK  MG/ML,  0.69  THE  THE RESULTS  FRACTION.  THE  CONTAMINANTS. OF  THE  THAT  WAS  SPECIFIC ACTIVITY  OF  (a)  (b)  0 + H— 50 ' 4 8  -4-  C l  FIGURE  19.  58  56  THIN  54  52  LAYER GEL.  WATER,  PYRIDINE  (A) 48 (B)  PAQ 10  TO  M  L  58. 10  AND  C2  CHROMATOGRAPHY  SILICA © -  0  SOLVENT AND  (15:  20  1 ML  OF  OF  SYSTEM;  10 A 2 0  STAGE  K  PRODUCT ACETIC  O-PAQ  ON ACID,  POSITIVE,  POSITIVE.  ALTERNATE  NMOLE  5  BUTANOL,  10: 12: 3).  NINHYDRIN  SAMPLES C-1,  K  10  ARG.  SAMPLES  FRACTIONS  C-2,  FROM  0.1  FROM  NMOLE  FRACTION  54.  ARG.  -40CRITERIA  8.  SINCE BASED ON  OF  ALL  STAGES  ON E I T H E R  LAYER  CT  TH IN  FOR  110°C  (I5t10t12t3)  FROM  FRACTIONS ALLOWED  WERE  THEN  AFTER  CONTAINING SPECIAL  RETAINED ELUTED  TRACINGS FIGURE  19(A).  SUFFICIENT REAGENT  WERE  A PERIOD  REAGENT  54  ALIQUOTS  VEHICLE  THE  ONLY  MATERIAL  THIN  FOR  IN  AND  OF  ALIQUOTS  TO  THE THE  ORIGIN PLATES  ( P A Q ) REAGENT. TO  FOLLOW  DETECT TIME  WITH A  APPENDIX  SIZES  WERE  MANNER.  THE  ARGININE  THE P A Q .  IN  VARIOUS  AND  ACETIC  C. TAKEN  ONE  PEPTIDE  CHANNEL  CONTAINING  BIOASSAYED.  LAYER  CHROMATOGRAMS ARE  52, 54, BY  FLOURESCENT  SAMPLES  U.L  5 HOURS.  A SIMILAR  DETECTION  DISTINCT  WATER,  APPLIED  DESCRIBED  HOWEVER,  FRACTIONS  TEST  IS  ACTIVATED  N.Y.)  UV L I G H T  TO  SYSTEM  PLATES  S P R A Y E D A SECOND  DESIGNED  AND T R E A T E D  WITH  AND ON  WERE  REAGENTS  UNDEVELOPED,  OF  UNDER  GEL  USED  THIN  SOLVENT  10  P H E N A N T H R E N E Q U I NONE  PLATES  THESE  OF  DETECT  A SYSTEM  PYRIDINE,  WAS U S E D . 58  TO  BASED  PROCEDURE.  ROCHESTER  FOR  PRODUCED  FRACTIONS  SYSTEM  IN  SILICA  6061,  MIGRATE  THE  LIKELY  ASCENDING  56,  NINHYDRIN  FRACTION  REGION  CM,  AND O B S E R V A T I O N  OF  AN  SEPARATIONS  A SEPARATION  BEST  A BUTANOL,  A SECOND E X P E R I M E N T  FROM  IN  54,  SPOTS  PREPARATION  THE  52,  S P R A Y E D WITH  DRYING  AS  INDUSTRIES,  BUFFER  50,  TO  20  MINUTES.  30  SIZE  INVOLVED  CHROMATOGRAPHY  RUN x  PRODUCTS  ACID  WAS  LAYER  P R E C O A T E D 20  HAD  S E E M E D MOST  WAS CHOSEN  (15)  (DISTILLATION  IN  OR M O L E C U L A R  CHROMATOGRAMS WERE  ON E A S T M A N  AND  PURIFICATION  SOLUBILITIES  CONTAMINANT.  ON P O R C I N E  OF  CHARGE  DIFFERENTIAL  ANY  AT  HOMOGENEITY.  AND  BOTH  0.1  IN  IN  CONTAINED  REAGENTS.  SPOTS  CONTAINING  56  SHOWN  ALL  AND  THE P A Q BUT  1.0  TWO  NM OLE  -41OF  ARGININE.  NINHYORIN SPOT OF  BE  0.5  ABOUT  RETARDED.  NMOLE IN  DETECTED WITH  THUS  THE  IT  RESULTS.  19B)  THE ANO  EACH  OF  CONFIRMED  50  CASE  THE  THIS  TWICE  AN  ARGININE  CONCENTRATED  CONFIRM  USING  OF  FRACTIONS IN  MORE  TO  BIOASSAY  CHROMATOGRAM  SAMPLE  A P P E A R E D THAT  COMPONENT.  (FIGURE  DONE  1  POSITIVE.  COULD  SINGLE  THE  AS  THIS  56  TO THE  FAINTLY  ONLY  SPOT  A  HAD  SINGLE AN R  SAMPLES BEING  PEAK  CONTAINED  A SECOND  SAMPLE  COMPONENT  SLIGHTLY  ONLY A  EXPERIMENT  MUCH S A M P L E  UNDEVELOPED  THAT  WAS  WITH  SIMILAR  FROM T H E WAS  WAS  SECOND  BIOLOGICALLY  ACTIVE. ACID  AMINO BETWEEN  NUMBERS  CONFIRMED THESE  THE  VARIOUS  5 0 AND  AS  SUFFICIENT  DESCRIBED  OF  THE  THE  MOLE  RATIOS  ON  THIS  BASIS  49~57  FRACTIONS QUANTITY  TABLE  TO  IN  ACTIVE  IN  BETWEEN  IN  5 6 AS  SHOWN  FRACTIONS.  MATERIAL  P E R F O R M E D ON A L T E R N A T E  HOMOGENEITY  ANALYSES  DIFFERENCE  ANALYSES  PEAK.  IT  THE  COMPLETE  THE NO  AMINO  PURE  E  FURTHER RESULTS  ACIDS  IN  THAT  SALMON C T  CHEMICAL  THE THE  IN  CHARACTERI-  ZATION.  D.  CHARACTERIZATION  1 .  METHODS.  A.  AMINO  AcIO  AMINO 52,  54,  THESE  ACID  AND  56  FRACTIONS  HYDROCHLORIC  OF  THE  WHOLE  MOLECULE.  COMPOSITION. ANALYSES OF  THE  WERE  ACID  FOR  WERE  STAGE  5  CARRIED ELUATE.  LYOPHILIZED 17  HOURS  AND AT  OUT  ON  FRACTIONS  1 0 0 ML  ALIQUOTS  HYDROLYSED  1 1 0 ° C.  THE  OF  SIGNIFICANT  WAS C O N C L U D E D  REPRESENTED  ALLOW  SECTION  I V INDICATE OF  FRACTIONS  IN  50, OF  5.7 M  HYDROLYSED  SAMPLES  WERE  COMPLETE  RECOVERY  DESCRIBED  IN  ORIGINAL ACID  SPECIAL  WERE THE  OF  AT  THESE  MOLAR  AT  280  AND  IS  NM  230  IS  PHOTOMETER  AT  THIS TEXT STAGE  PH  OF  2  ARE AT  A  IN  0.1  OF  THE  WHICH  A  FOR  THESIS  OF  ONLY  WERE ACID TO  PARTICULAR  THE  OF  1  AND  THE  BASED THE  ON  A  CM  LOMB  AND I-*  AND  WHOLE  MOLECULE  WITH  A  AND  A  IN  1  A  WERE  A  CM 600  CELL SPECTRO-  BLANK  LATER  DIFFERENT  OF  LYO-  OURING  PRESENTED  AT  THE  IN  VARIOUS  STAGES  DESCRIPTION  OF  THE  IN  READER WAS  OF  CALCULATED  OCCURED  CONTAINS  EXTINC-  WERE  AGAINST  ANALYSES  EHRLICH  MILLIGRAM  56  FRACTION  ANALYSIS  GLUTAMINE  SPECTRONIC  ACID  THE  PROCEDURES  MEASUREMENTS  TEST  PROTEIN)  ANALYSER,  A  AND  THESE  PATHLENGTH  MACHINE  ALLOW  WITH  QUANTITATION  ON  OF  PERFORMED D  A  HYDROLYSIS.  FRACTION  THE  APPENDIX  AMINO  A  ON  CIRCUIT  LATER)  TRYPTOPHAN. IN  THE  HYOROL.YSE S . ( I DENT I F  ACID WAS  OF  IS  AMINO  MODIFIED  EXCEPT  BY  THIS  AND  ALLOW  ACID  J4.L  40  (119)  AJ.  BEEN  DISCUSSED  FORMIC  OF  E_T  BE  OF  M  SAMPLES  PRESENTED  BY  INSURE  AUTOMATED  ACIDS  RESULTS  BAUSCH  STUDIES  THIS  THE  TO  NMOLES.*  COEFFICIENTS  MG/ML  MODIFICATION THESE  WILL  ON  TO  HAD  2  TRYPTOPHAN  1  IN  MODIFICATION.  EVOLUTION  EQUIVALENT  AMINO  ABSORBANCE  BUFFER.  *EXTENSIVE SECTIONS  METHODOLOGY  DETECTION  TO  (ABSORBANCE  NM  280  ALL  SPECIFIC  MEASURED  OF  DETAILED  C E L L S ' TO  DESTROYED  ANO  FROM  COURSE  THE  SPACKMAN  D E A M I DA T E D  OF  CONTAINING  AND  OF  EXTINCTION  WHICH  SAME  TO  WHICH  DOWN  RESIDUES  SOLUTION  THE  OF  FLOW  LEVELS  COEFFICIENTS  230  OXIDIZED  SUBJECTED  METHOD  ARE  WHICH  THE  THE  THEN  PATHLENGTH  WHICH  OF  REAGENT  AIR  SAMPLES  ANALYSER  DETERMINATION  AT  D.  DETERMINATION  TRYPTOPHAN  TION  -^--CYSTINE.  200  ACIDS  ASPARAGINE CATION  AND  BR I D G E - P H O T O T R A N S I S T O R  LONG  ALLOWED  BY  MODEL  AMINO  VACUO  APPENDIX  ANALYSIS  WHEATSTONE  J_N OF  ALIQUOTS  BIOCHROM  OF  DRIED  TO  DETAILS  IDENTIFY  PERFORMED.  OF  THE THE THE  TABLE AMINO  ACIO  ANALYSES  FRACTIONS  OF  50 AMINO  ACIDS  IV FROM  STAGE  52:  ~  5  CHROMATOGRAPHY  "54  5D"  R  NANO-  MOLE*  NANO-  MOLE  NANO-  MOLE  NANO-  MOLE  MOLES  RATIO  MOLES  RATIO  MOLES  RATIO  MOLES  RATIO  5.1  2. 0  8.7  2.0  15.1  2. 0  18.0  2. 0  THREON1NE  12.8  5. 1  22.4  5.1  37.8  5. 0  45.0  5. 0  SER1NE  10.0  4. 0  17.4  4.0  24.6  3. 3  37.0  4. 1  7.6  3. 0  12.9  3.0  22.7  3. 0  26.6  3. 0  PROL1NE  6.2  2. 5  9.3  2.1  13.4  1. 8  17.3  1. 9  GLYC1NE  8.8  3. 5  14.1  3.2  23.2  3. 0  28.8  3. 2  ALANINE  0  0  0  0  4.6  1 .8  9.3  2.1  12.5  1. 7  17.0  1. 9  VAL1NE  2.7  1. 1  4.8  1.1  6.5  0. 9  8.0  0. 9  M E T H 1 O N 1NE  0  0  0  0  0  0  0  0  1 SOLEUC1NE  0  0  0  0  0  0  0  0  ASPARTIC  GLUTAMIC  ACIO  ACID  CYSTINE  L E U C 1 NE T Y R O S 1NE PHENYLALAN  1NE  0  0  5. 2  20.2  4.7  38.5  5. 1  48.7  5. 4  2.4  1. 0  3.8  0.9  6.5  0. 9  7.4  0. 8  0  0  0  0  0  0  0  0  ANALYSES  ON  SHORT  LYSINE  101NE  ARGININE AMMON i A  *WlTH  0  13.0  SEPARATE  Hi ST  0  REFERENCE  TO  ASPARTIC  Ac I D  OR  COLUMN  7.0  2. 0  16.3  2. 0  3.7  1 .0  9.1  1. 1  4.0  1 .1  8.1  1. 0  19.0  5. 3  44.4  5. 5  LYSINE.  -43PHILIZED MOLAR  ANO  EXTINCTION  VALUES  ANO  THE  DETERMINED USE  WEIGHED  WAS  A  (FlSHER  TO  DETERMINE  COEFFICIENTS  MOLECULAR  FROM  THE (W/V)  0.5$  SCIENTIF|C  IN  Co.)  HYDROCHLORIC  A  THIN  10  OF  yUL  B.  GEL  END  FRACTION  GROUP  THE  ONE  BY  NMOLE  SODIUM  THE  SAMPLE  OF  THE  DANSYL  WAS  THEN  BY  AS  APPENDIX  2.  (52).  FRACTIONS  WHICH  IN  METHOD  CONTAINING THIS  HAD  OF  WAS  BEEN  REAGENT  2$  (v/v)  SPRAYED  SPOTTED  IN  5.7  IDENTIFIED WOODS  DETAILS  OF  WAS  ONTO  WITH  MOLECULE  AND  DISSOLVED  DANSYL  OF  BY  THE  WANG  THESE  IN  0.2  CHLORIDE  (132)  METHODS  ARE  ACETONE.  WAS  USED  AMINO  LAYER AND  M  IN  ACID  N-TERMINAL  POLYAMIDE  WAS  HARTLEY(47).  HYDROCHLORIC  M  AND  NATIVE  GRAY  WITH  DERIVATIVE  BY  THE  HORMONE  TREATEO  SAMPLE  AND  CHROMATO-  MODIFIED INCLUDED  IN  RESULTS ACROSS  DISCUSSION. OF THE  THE  DIFFERENCE  THE  SAMPLES  VARIOUS  QUANTITIES  AMINO  ACTIVE  SIGNIFICANT  ABSOLUTE  EHRLICH  E.  RESULTS THE  THE  DESCRIBED  HARTLEY  THE  AS  I P - D I M E T H Y L AM I N 0 B E N Z A L D E H Y 0 E  (68).  ACID  THE  ANO  TO  GRAPHY  PLATE  DANSYL  OF  WHICH  ACID  AMINO  HYOROLYSIS  ACID  POLYPEPTIDE  ETHANOL  95$  THESE  ANALYSIS.  BICARBONATE  RELEASE  THE  FROM  56.  N-TERMINAL  DETERMINED A  LAYER  OF  0 F  CONCENTRATION.  CALCULATED  AC I D . A N A L Y S E S * ,  SOLUTION  CONCENTRATED SILICA  WERE  WEIGHT  AMINO  PROTEIN  PEAK  COULD  DESPITE  ACID  BE THE  ANALYSED.  ANALYSES  ARE SEEN  SHOWN IN  OBVIOUS THE  THE  OF IN  THE  FOUR  TABLE  IV.  MOLE  RATIOS  DIFFERENCES  AVERAGE  VALUES  IN  FOR  No OF THE  THE  TABLE AMINO  AMINO  ACIDS  ACID  COMPOSITIONS  SALMON CT*  V OF F I V E  HUMAN CT (80)  CALCITONINS  PORC1NE CT (96)  BOV1NE CT(17)  OV 1 NE  CT(91  2  3  4  4  4  THREONINE  5  5  2  2  2  SER1NE  4  1  4  4  4  3  2  1  1  1  PROL1NE  2  2  2  2  2  GLYCINE  3  4  3  3  3  A L A N I NE  0  2  1  1  1  •g- C Y S T i NE  2  2  2  2  2  VALINE  1  1  1  1  1  METH1 ON 1NE  0  1  1  1  1  1SOLEUC1NE  0  1  0  0  0  LEUCINE  5  2  3  3  3  TYROSINE  1  1  1  2  3  P H E N Y L A L A N 1NE  0  3  3  2  1  LYS1NE  2  1  0  1  1  HISTIDINE  1  1  1  1  1  ARGININE  1  0  2  1  1  AMMON1 A  5  5  5  4  4  TRYPTOPHAN  0**  0  1  1  1  ASPARTIC  ACID  GLUTAMIC  ACID  * MOLE I N T E G E R S B A S E D ON A V E R A G E S FROM E I G H T **FROM A ~ AND N E G A T I V E E H R L I C H ' S T E S T . 280 NM O  Q  ANALYSES.  -44MOLE  RATIOS  CLOSE  TO  BASED  THE  OF  ALL  SAMPLE  TO  SAMPLE  MILLIGRAM  230  ACID THE  EXTINCTION  NM.  BASED  MOLAR  280  AT  ON  THIS  ANALYSES  ANO  ARE  VALUES  AS  SHOWN  IN  V.  AS  WERE  GOOD  FOR  AND  TABLE  CONSISTENT  VARYING  TIME  VERY THE  FROM  INTERVALS  PER  COEFFICENTS  OF  AT  A  WEIGHT  AT  280  NM  SIMILAR  TYROSINE  FOUND  BY  CONFIRMED  SALMON  CT  OVINE,  AND  AS  HUMAN  RESIDUE  THAT  FIVE  DISULFIDE  DUES.  THE  DETECTABLE METHIONINE,  10""^ THE  OF  WERE  WAS  HORMONE,  THE  RESIDUES,  OF  A  N— T E R M I N U S EXISTED  MOLECULE  PHENYLALANINE  OR  32 AND  FOR  LACKED  CONTAINING  TO  AND  WERE  THAT  THAT  OF  THE  ONLY  COMPLETE OF  MOLECULE  THE  AMINO  EXTINCTION  WITH  OF  AT  IN  THE  TEST  2.0  M  IDENTIFICATION  CONSIST  AND  FROM  EQUAL  COMPOSITIONS  SALMON  NM  3427  MOLAR  FOR  INDICATED  280  CALCITONINS. THE  (1.39)  SINGLE  AROMATIC  EHRLICH  TRYPTOPHAN.  REPRESENTS THE  ML.  I NO I C A T I N G  THE  NM  POLYPEPTIDE  THIS  NM.  ANALYSIS  ABSENCE  DIFFERENCES  SALMON  OF  APPROXIMATELY  ACID  TO  X  OF  MOLECULE.  HORMONES  RING  DRAMATIC  THE  COMPARED  TERMINAL ALL  AMINO  THE  230  0.45  2.0  CONDITIONS  THEREFORE  V  AT  WAS  UNDER  REAGENT  WAS  COEFFICIENTS  TYROSINE  TABLE  MOLECULAR  7000  IN  OF  230  AND  PEPTIDE  AND  PRESENT  (0.292)  NM  MG  0.69  NM  RESIOUE  280  AT  SOLUTION  EXTINCTION  COEFFICIENT  BUT  EIGHT  HYDROLYSES  CONTAINING  ANALYSIS  1500  A  OF  ACIDS  ABSORBANCES  SOLUTION  AT  AMINO  TOTAL  PERFORMED.  , THE A  A  INTEGRAL  YIELDS  NOT  ON  NO  AS  FIVE  TWO  OF  OF  THE  ACID  OF THE  THE  OF  BOVINE, N-  CONFIRMED CHAIN  PROLINE  ALANINE,  TRYPTOPHAN.  PORCINE,  CYSTINE  AMINO  MOST  COMPOSITION  WITH  RESIDUES,  OTHER  RESI-  EIGHTEEN ISOLEUCINE,  ITS  LOW  CONTENT  OF  - 4 5 A ROMA T I C  AND  ADDITIONAL SIX  FOR  HYDROPHOBIC  RESIDUES  PORCINE)  RESIDUES  CONTAINING  PROBABLY  WEIGHT  OF  MOLECULES  OF  LOW  AROMATIC  AND  ON  SEPHADEX  THAN  AND  THE  COULD  LESS LARGER  AMMONIA  WERE  RESULTED  TO  (TWO  FROM  MOLECULE PROVIDE  RESIN  THAN  FOR  AND  BY  OXIDIZED  OF  TWO  THAT  ENCOUNTERED  METHODS.  A.  PERFORMIC  PROBLEMS  OXIOATION.  FROM  AND  ONE  WITH  THE  TO  OF  WHICH ON  4 . 5  THE  HORMONE  RESIDUES WHICH  PRESENT  WAS  CHARACTERIZED.  ARGININE  FOR  IN  THE  SHOULD  SEQUENCE  MOLECULE  AVOID  ON  FIVE  PE P T I D E S .  CYSTEINE  WORK  THE  ORIGINALLY  TO  3.4  TRYPSIN  SIZE  ACIDIC  CHARGE  BASIC  TRYPTIC  WITH  SEQUENCE  IN  BINDING  CONVENIENT  MOLECULE  WERE  HI S T I D I N E )  ACID  CONTENTS ( 6 0 )  THESE  CALCITONINS  OF  LYSINES  HIGH  ARE  BUT  DEGRADATIONS.  DURING  ACID  OTHER  PERFORMIC  OTHER  ONE  CONTENT  CALCITONINS,  OF  VERSUS  SEPHADEX G - 5 0 .  FIVE  OF  THREE  APPARENT  OF  PH  THE  DIGESTION  OF  EDMAN  WITH  AND  THE  AND  HIGHER  TOTAL  CHANGE  NUMBER  I S O L A T I ON  PEPTIDES  1.  BUFFER  ARGININE  SUGGESTED FOUR  THE  THE  A  MOST  SMALL  TO  ON  OF  (NINE  HYOROPH|LIC  OTHER  COLUMN.  OF  A  THE  ONE  ANY  OF  THAT  DUE  PRESENCE  INTERCHANGE BEEN  INDICATED  THE  WITH  (17).  THE  WAS  IN  DETERMINATION FIRST  IN  SE-SEPHA0EX  P R E P A R A T I ON THE  AS  THOSE  PRESENCE  GROUPS  HYDROPHOBIC  RADIUS  INCREASING  LYSINES,  GREATER  E.  FOUND  FOR  OBSERVED  CT  TENDENCY  ACCOUNTING  SECOND THIS  THIS  RESIDUES  AMIDATED,  THE  HYDRATED  REINFORCE  RESJDUES  SALMON  THE  HYDROXYL  ACCOUNTS  MOLECULAR  RETARDED  AND  WAS  DISULFIDE WHICH  PORCINE  CT  HAD ( 8 , 9 ) .  -46PERFORMIC HYDROGEN THE  ACID  PEROXIDE  MIXTURE  TO  WAS  TO  STAND  PREPARED  9.5  ML  OF  AT  ROOM  BY  90$  ADDING FORMIC  TEMPERATURE  0.5  ML  OF  A C I D AND FOR  FOUR  30$  ALLOWING IT  HOURS.  0 WAS  THEN  STAGE WAS  COOLED  5 MATERIAL  SHAKEN,  B.  C ANO  CARRIED  TO  RESIDUE  OUT  AS  FOR  PREVIOUSLY  STEP.  DETERMINATION SYSTEM  THE  OF  ARE  DIGESTION TRYPSIN  10  AMMONIUM  3 ML  4  TO  0°C.  MG  I N 2 ML  OF  OF  POOLED  THE  MIXTURE  C.AND  LYO-  DISTILLED AND  A  0.1  BUFFER  RESIDUES  THE M  WITHOUT  WERE DONE AS SYSTEM ON  THE  BEFORE,  WAS  AIR  BUT  USING  WHICH AL L 0 WE 0  (38)  A SINGLE SAMPLE.  IN APPENDIX  MG/ML  TR6EA)  DETAILS  OF  D.  WAS  20  yLL OF  OXIDIZED MATERIAL BUFFER  1:50  SAMPLE  FORMIC  AT  (w/w). WAS  DISSOLVED  IN 1 M M HYDROCHLORIC  CHLORIDE.  OF  OXIDIZED MATERIAL  TRYPSIN.  10  OF  RATIO  HOURS OF  MG  THE  D E S C R I B E D , BUT  ANALYSES  DESCRIBED  BICARBONATE  SUBSTRATE FOR  10  3 HOURS AT 0°  REOISSOLVED  (WORTHINGTON,  IN CALCIUM TO  ALL  WITH  CONCENTRATION  ADDED  WAS  FOR  A N A L Y S I S OF  S I N G L E COLUMN, THREE  1 MM  STANO  TO  ACID ANALYSIS.  OXIDATION  c.  ADDED  AND R E L Y O P H I L I Z E D . T H I S L A S T S T E P WAS R E P E A T E D S A M P L E WAS T A K E N FOR A M I N O A C I D A N A L Y S I S .  AMINO  THIS  1 ML  IN A V E S S E L PRECOOLEO  PREPARATION  A  0  ALLOWED THE  PHILIZED. WATER 10 UX  TO  T H I S STOCK I N 1 ML  P H 8.0 AFTER  LYOPHILIZED  A C I D AND  STORED  TO  TO  A C I D WHICH SOLUTION  OF 0.5$  G I V E AN  INCUBATION ANO  A  ENZYME  TO  AT  C  REDISSOLVEO  FROZEN.  WAS  (w/v)  37°  WAS  IN  -47D.  HIGH  VOLTAGE  ELECTROPHORESIS.  THE  INITIAL  SEPARATION  VOLTAGE SYSTEM  DESCRIBED  ^IL  300  ELECTROPHORESIS  S A M P L E S OF  APPROXIMATELY ORIGIN  LINE  WHATMAN OARO  M  APPENDIX  AFTER  C.  PLACED  IN  AND  APPROXIMATELY UNTIL  THE  THE  X 57  SUCH  CM S T R I P  THAT  SAMPLE,  THE  REAGENT,  PAQ  CATHODE  TO  ALSO  THEN  WAS REMOVED  OF  C.  THE  PEPTIDES  WERE  SEGMENTS  CUT  OUT  ANO  OF  TANK PER  CM S H E E T  OF  OF  THE  THE  BUFFER  75$  OR  THE  BOTH  OF  FOR  TO THE  THE  FURTHER  V  PAPER THE  DRY.  A  SHEET  APPLIED WITH  DESCRIBED  UNDEVELOPED  SHEET  POINT.  OEVELOPED AS  IN  4000  OF  ALLOWED OF  ANO 21).  THE  RUN AT  CENTER  STAN-  REAGENTS  CENTIMETER  SHEET  TWO  DESCRIBED  AND  6.5f  THE  (SEE FIGURE  AS  AND  PH  3 CM OF  A PREDETERMINED  FROM  SAVED  TO  A 57  PH 6.5  REMOVED  CADM I U M - N I N H Y O R I N  APPENDIX  OF  AT  THE  CONTAINING  5 }XL S P O T S  APPROXIMATELY  REMAINOER  IN  DEVELOPING  WITH  MARKERS R E A C H E D  P H 1.9  APPLIED  EMPLOYED  5 MILLIAMPERES  HIGH  SOLUTION  MOBILITIES  SATURATION  WAS  AND  WAS BY  EXPERIMENTS  END  WITH  TEST  RELATIVE  I t CONTAINED  AND  WERE  ELECTROPHORESIS  ELECTROPHORETOGRAM 5  DIGEST  FLANKED  MARKERS WERE  WAS  WIDTH  THE  SOLUTIONS OF  FOR  PEPTIDE  F  P A P E R AND  ACID  DYE  0  PEPTIDES  P H 6.5  C.  TRYPTIC  CM FROM  CALCULATION  VISIBLE  M  THE  ( H V E ) AT  APPENOIX  THE  0.3  32  3 MM  AMINO  ALLOW  IN  OF  STRIP  EITHER  IN CONTAINING  PURIFICATION  OR  ELUTION. ELECTROPHORESIS UNDEVELOPED FROM OF  PAPER  WHATMAN  STRIPS  AT  P H 1.9  FROM T H E  CHROMATOGRAMS BY 3  MM  ON AN  ORIGIN  WAS  P E R F O R M E D ON  THE  EXCIZED,  P H 6.5  E L E C T R O P H O R E TOGRAMS AND  SEWING  THE  LINE  10  STRIPS  ONTO  CM FROM THE  NEW  ANODE  SHEETS AND  -48REMOVING  THE  AREA  OF  THE  NEW  SHEET  BENEATH  THE  SEWED  IN  t STRIP.  ELECTROPHORESIS  DESCRIBED  AND  PHORETOGRAM S VELOPED  THE DR I E D  CENTER  AND  ELUTED  E.  PAPER  WHICH  SYSTEM  AND  TO  THE  TOP  OF  SHEETS  PAPER. ABOUT  SOLVENT UNDER  DRYING  THE  DEVELOPED  WITH  UNDEVELOPED  FOR  ELUTION  OR  2.  RESULTS  AND  SIMILAR OF  THE  AMINO TO  TWO  TYROSINE  WAS  RESULTANT  ELECTRO-  OF  PEPTIOES  REACH  TO  THAT  THE  THE  UNDE-  WERE  CUT  OUT  BUTANOL,  (131).  SEWED  THE  ELECTROPHORESIS  ACETIC  AT  THE  WHATMAN x  46  TO  SOLVENT  SHEETS  THE  GRAIN  FRONT  OF  THE  PAPER.  DIVIDED  AND  THE  USED  FOR  CONTAINING  THE  7  CM  PAPER.  MM  CM  57  A  STRIPS  ORIGIN, 3  IN  ACID,  UNDEVELOPED  ON  OF  BOTTOM  WERE  BY  CHROMATOGRAPHY  PERPENDICULAR  THE  SEGMENTS  PAPER  STANDARD  REAGENTS  FURTHER  SEPARATED  OF  CONDITIONS  CHROMAT0GRAMS  THE  THE  PREVIOUSLY  (58).  SHEETS  FROM  FLOW  TO  BE  WERE CM  46  CUT  THESE  HOURS  14  X  15  WERE  NOT  (30S6S20S24)  WATER  FROM  THE  ACID  CONSISTING  ELECTROPHORETOGRAMS  THAT  AS  SEGMENTS  DECENDING  FROM  THESE  THE  CONTAINED  ACETIC  COULD  SUBJECTED  PHASE  PYRIOINE  OF  DEVELOPED.  WHICH M  PERFORMED  CHROMATOGRAPHY.  WERE  SINGLE  0.5  THEN  STRIPS  AND  STRIP  WITH  PEPTIDES ALONE  EDGE  WAS  SUCH OF  THE  REQUIRED AFTER  EDGE  STRIPS  ELECTROPHORETOGRAMS.  PEPTIDES  WERE  CUT  OXIDIZED  SAMPLE  OUT  PURIFICATION.  DISCUSSION. ACID OF  ANALYSIS THE  -G—CYSTINE  NATIVE  RESIDUES  CHLOROTYROSINE  OF  THE  HORMONE ANO  (128).  EXCEPT  PARTIAL CYSTEIC  FOR  THE  CONVERSION ACID  WAS  WAS LOSS OF PRESENT  FIGURE OUTLINE  20  OF P R O C E D U R E S U S E D TO I S O L A T E SALMON CT.  SEPARATION  PROCEDURES  TRYPTIC  PEPTIDES  PEPTIDES  ELUTED  HVE AT PH~5T3" A-2, A-3,  A-1,  -•ST-1 A  A-4  -•ST-1  -•HVE AT P H 1 .9 B-1  -•ST-4A  CHROMATOGRAPHY C-1,  C-2  +  ST-3  HVE AT P H 1.9  D-l,  D-2  —  -•ST-4 >> ST-2  FROM  : = = = = =  = i z  OK  n U  @R  OH  MG  n u fi  l\  YEL/g|  A-l  FR|P -0-O.@ —  '+  o  DNP  1  SCT Y E L Q A-3 (ST-1)  YELO A - 4 (ST-IA)  o  O D  OG  o OCA  FIGURE  21.  P H 6 . 5 E L E C T R O P H O R E T O G R A M OF S A L M O N C T T R Y P T I C PEPTIDES. C O M P O S I T E OF S E V E R A L R U N S . O - N I N HYDRIN P O S I T I V E , POSITIVE, © - P A Q AND NINHYDRIN P O S I T I V E . SINGLE LETTER DESIGNATIONS USED FOR A M I N O A C I D S . OTHER A B B R E V I A T I O N S T A , T A U R I N E ; C A , C Y S T E I C A C I D ; M G , METHYL GREEN; DNP, « - D N P - L Y S i N E ; O G , ORANGE G ; Y E L , Y E L L O W N I N H Y D R I N COLOR; F R , F A S T R E D ; Y R , Y E L L O W C H A N G I N G TO R E D . S I N G L E L E T T E R D E S I G N A T I O N S ( E G . A-3) I N D I C A T E P E P T I D E C O N T A I N I N G REGIONS. TWO L E T T E R D E S I G N A T I O N S ( E G . S T - 1 ) I N D I C A T E ISOLATED P E P T I D E S . DOTTED L I N E I S N E U T R A L .  OK  OH  0°« 0 8?„ OT: OD OE  0  YR  ov O L  §  M  OY  0-  DNP|  o  (ST-4A)  1 h  H  O C A  FIGURE  22.  PH  1.9  OTHER  ELECTROPHORETOGRAM DATA  AS  DESCRIBED  OF IN  REGION  FIGURE  A-2. 21,  FIGURE  23.  PAPER ACETIC 20:  ACID,  24).  DOTTED  I  CHROMATOGRAM  OF  REGION  PYRIDINE,  WATER  OTHER  LINE  IS  DATA  AS  SOLVENT  A-1  OESCRIBED FRONT.  IN  SYSTEM. IN  BUTANOL, (30? FIGURE  6T 21  FIGURE  24.  PH 1.9 E L E C T R O P H O R E T O G R A M OF REGION DATA AS D E S C R I B E D IN F l G U R E 21.  C-1.  OTHER  - 4 9 -  WITH HAD  A MOLE OCCURED  WITH  BY  AND  SHOWN  SINCE  OF  ARE  ALL  SINGLE  NATIONS  OF  DATA  SAMPLE.  THE  A  AND  EXPECTED  WAS S U I T A B L E  TWO  TO  IN  (ST-4A)  IN  REGION  FURTHER HYDRIN  OXIDATION  FOR  DIGESTION  ISOLATEO MORE  WERE  REGION A - 1 WAS  POSITIVE WAS  YIELD  TWO  PURIFIED ISOLATED  BY  ONE  AS  PAPER  (ST-3). H V E AT  TO  IN  IN  CONTAIN  POSITIVE WERE  REGIONS BUT  WERE  TWO  20.  AND SIX  MINOR  PEPTIDE  SHOWN  IN  YIELDED 23,  FIGURE 2 2 ,  ST-4. MAJOR  BUT  IN  FOR  TWO  NIN-  ONLY  REGION  SHOWN  THE  SUBJECTED  MAJOR  SECOND  PH 1 . 9 AS ST-2  AND  CON-  BY H V E  CHROMATOGRAPHY  FIGURE  THE  THE  ST-1A),  FIGURE  CHROMATOGRAPHY  PEPTIDES,  SHOWN  COMPONENT  FROM  REGIONS  MAJOR  AND  (ST-1  PH 1 . 9 AS  SHOWN  WHILE  NINHYORIN  FOUR  THESE  DESIG-  SEPARATION  FROM A S I N G L E  TO  OBTAINED  WITH P A Q WHICH  THE  OUTLINED  H V E AT  THIS  REGIONS  WAS  AS  SUBJECTED  HOMOGENEOUS  DIGEST  THAN  IN  AS  EXPERIMENT  FOR  FOUR  ONLY  PEPTIDES  SEPARATED A - 2 BY  ONLY  INITIAL  OF  BE  REGIONS  USED  SEEN  SHOWN  ONE  NOT  STAGES  SINGLE-NUMBER  2 1 YIELDED  TWO  MAPS  THAN  LETTER,  THE  SPOTS  PURIFICATION  FURTHER  TRYPTIC  BE  PEPTIDE  COULD  ARE  WERE S E P A R A T E D  CHROMATOGRAPHIC  CONTAINING  FURTHER.  PURIFICATION.  THESE  SINGLE  FIGURE  MINOR  DIGESTION  AND  PRESENTED  COMPONENT.  CONTAINED  FURTHER  THIS  THE  PARENTHESIS  TWO  CONTAMINANTS  TO  IN  SINGLE  SHOWN  OTHER  WAS  THE  B A S E D ON MORE  PEPTIDE  INVESTIGATED  WERE  OF  TO  PH 6 . 5 SHOWN  REGIONS  BY  2 1 THRU 2 4 .  THE  REFER  TAINING  AND  MATERIAL  PRODUCED  COMPOSITES  DESIGNATIONS  TO  THE  ELECTROPHORETIC  FIGURES  IN  FIGURES  NOT  1 , 8 INDICATING  THAT  PEPTIDES  A SERIES  AT  OF  TRYPSIN. THE  A  RATIO  ONE  (C-1)  FIGURE  THUS  2 4  THE  PEPTIDES  RATHER  1  15 .  2 25 . 3 MOLECULAR  FIGURE  25.  4 5 6 7 8 9 10  WEIGHT  LOGARITHMIC PLOT ( A T P H 6.5 RELATI MOLECULAR WEIGHT C O R R E S P O N D TO UNI  x  15 .  2 25 . 3  10  OF E L E C T R O P H O R E T I C M O B I L I T Y VE TO A S P A R T I C A C I D ) V E R S U S FOR P E P T I D E S . ALIGNMENTS T C H A R G E S AS I N D I C A T E D . (88)  I  o x >CD  o  < _)  tr  6 7 8 9 10 MOLECULAR  FIGURE 2 6 .  WEIGHT  X  10  L O G A R I T H M I C P L O T OF E L E C T R O P H O R E T I C M O B I L I T Y ( A T P H 1.9 R E L A T I V E TO S E R I N E ) V E R S U S M O L E C U L A R WEIGHT FOR P E P T I D E S . A L I G N M E N T S C O R R E S P O N D TO U N I T C H A R G E S AS I N D I C A T E D . ( 8 8 )  -50T HA N  THE EXPECTED  FROM  THE PAPER  ACTERIZATION  FOUR.  STRIPS  AS  ACID  ANALYSIS  TO  DETERMINE  OF  THE  ANALYSES  THE TO  WERE  THE  PEPTIOES  1.  METHODS.  A.  AMINO  IN  BUT USING  MM D I A M E T E R  SYSTEM  USED  DESCRIBED  IN  PHORETIC  WHOLE  TAKEN  COMPOSITION H V E WERE AND  FOR  ANO  CHAR-  AMINO  THE  COMPARED  TO  DETERMINE  AMlDATED.  OBTAIN  THESE  WHETHER  N — T E R M INAL  DATA  ON  THE  ORDER  OF  MOLECULE.  FOR A N A L Y S I S WITHOUT A  WAS  A  AIR  SINGLE  COLUMN  TO  LOG  WAS C A R R I E D  OXIDATION.  COLUMN-TWO INCREASE  THE  AS  DESCRIBED  ANALYSES  BUFFER  SYSTEM  SENSITIVITY.  WERE  AND  THE  A  DETECTION  CIRCUIT  AS  D.  OF  AMIDE  GROUPS.  ( 8 8 ) HAS  SHOWN  THAT  MOBILITY  OUT  AMPL I F | E R - P H O T 0 T R A N S I S T O R  APPENDIX  DETERMINATION 0FF0RD  TO  ALLOW  SECTION.  WERE  CHARGE  WERE  TO  ELUTION  ANALYSIS.  PREPARATION  PERFORMED  PEPTIDES  NET  OUT  ADEQUATE  BY  PEPT I P E S .  DURING  RESIDUES  THE  REC0VERE0  NEXT  THEIR  PEPTIOES  CARRIED  ACID  PREVIOUSLY,  ELUTEO  CALCULATE  ACIDIC  THE  TRYPTIC  THE  NOT  B.  IN  OF  COMPOSITIONS  6  OF  WERE  QUANTITIES  ALIQUOTS  MOBILITIES  OR  IN  DESCRIBED  CHARACTERiZATION  F.  THESE  RELATIVE  TO  PEPTIDES  ASPARTIC  OF  ACID  KNOWN (U  ELECTRO)  AT  PH  6.5  ASP OR  TO  FALL SHOWN  SERINE INTO IN  (j-lg^ )  CERTAIN FIGURES  R  AT  PH 1 . 9 ANO  POPULATIONS 25  AND 26.  WHEN  KNOWN  MOLECULAR  PLOTTED  PEPTIDES  WHICH  WEIGHT  GRAPHICALLY L I E  NEAR  A  AS GIVEN  T A B L E VI AMINO  ACID  ANALYSES  AMINO  Acios  OF  TRYPTIC  MOLE  PEPTIDES  FROM  SALMON  ST-3  ST-4  CT  RATIOS  ST-1  ST-1A  1.9  1.7  0  0  0  0  1.0  1.0  0  0  1.0  1.0  THREONINE  0.8  0.8  0  0.8  2.9  2.8  SERINE  1.8  1.7  0.8  0  1.1  0.8  0  0  2.1  1.2  0  0  PROLINE  0  0  0  1.1  1.1  1 .0  GLYC1NE  1.1  1.0  0  0  2.0  2.1  ALANiNE  0  0  0  0  0  0  VAL1NE  1.0  1.1  0  0  0  0  ME T H I O N I N E  0  0  0  0  0  0  1SOLEUC1NE  0  0  0  0  0  0  LEUCINE  1.7  1.7  1.8  1.0  0  0  TYROSINE  0  0  0  1*  0  0  P H E N Y L A L A N i NE  0  0  0  0  0  0  LYSINE  0.9  0.8  1.0  0  0  0  0  0  0.8  0  0  0  0  0  0  1.0  0  0  10$  75$  70$  10$  CYSTEIC  ACID  ASPARTIC  GLUTAMIc  H  ACID  Ac 1D  1 ST 1 D1 NE  ARG1N1NE  5.5$  $  YIELD  *  DETERMINED  **  **  APPROXI  AS  CHLOROTYROSINE  MATIONS  ST-2  35$  ST-4A  -51LINE  ON  THESE  INDICATED ITIES ABLE  OF  FOR THE  DATA  CULAR DATA  CHARGE  IN  AND  DETERMINE IN  END  LINE  THE  NET  GROUP  NOT  FORM  OF  OF  NMOLE  0.7  OF  THEIR  OF  4  AND  DISCUSSION.  SHOWS  FIGURES ON  THE  AND  RELATIVE  MOBIL-  FROM  AVAIL-  THE  TO  THE  KNOWING  BY  MOLE-  COMPOSITION  AM I N O  GENERALLY GROUPS  THE  NET  ACID  P0SSfBLE  PRESENT  OF  SIX  OF  AMINO  TO  HAD  ONLY  BUT  THE  SOURCE  OF  THE  EXTRA  THE  YIELDS  OF  PEPTIDES  EDGE  THEY  PEPTIDES  COULD  BE  SPECIFICITY  OF  KNOWN AND  ANALYSES YIELDS  ESTIMATES STRIPS  As  OF  THE  OF  EACH  VARIES IN SHOWN  WERE  IDENTICAL  ARGININE  SINCE  USEFUL  PEPTIDES.  WERE  AND  MAJOR  ST-1A A N D ST-4A  ANALYSES  PEPTIDES  E .  LYSINE  WERE  DETERMINED  APPENOIX  AND  ACID  ROUGH  THE  WERE  IN  APPROXIMATE  MANNER*  ACID  THE  DEVLOPED  TWO  PEPTIDES  DESCRIBED  SIDE  THE  ARE  THE  THE  COMPOSITIONS  CARBOXYL  PEPTIDES  AMINO  ACID  CHARGE  HYDROLYSIS.  SAMPLES  AS  INDETERMINANT  THEIR  WAS  NET  COMPARED  PEPTIDE.  CARB OXYL  RESIDUES  THE  LOST  THE  DETERMINED IT  THE  ANALYSIS.  FROM  CENTAGE  AS  AND  AMINO  THE  TO  DETERMINED  YIELD  OF  PRIOR  RESIDUES  THE  THE  HAVE  CALCULATED  SECTION  THE  C—TE RMINAL  VI  WERE  SAMPLES  IDENTIFICATION  TRYPTIC  PH.  CHARGE  OR  N-TERMINAL  TABLE  GIVEN  COMPOSITION  AMIDE  RESULTS  THE  FROM  SUBSEQUENT  2.  TO  PRECEDING,  WHETHER  FOR  SHOWN  AT  CALCULATED  DA N S Y L A T I O N  TRYPSIN  BEEN  PEPTIDES  HYDROLYSED  THE  THE BY  THE  THE  OF  HAVE  TRYPTIC  GIVE  ANALYSIS  c  THAT  WEIGHTS TO  BEEN  GRAPHS  TO  RESIDUES.  SIX PEPTI.DE.  THE IN  THE  PER-  AN  DETERMINING IN  THE  QUITE  LOW  THOSE  OF  TABLE AND ST-1  TABLE NET  VI I  C H A R G E S OF SALMON C T 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 ( 8 8 )  PEPT1OES  ASP (PH 6 . 5 )  SER (PH 1 . 9 )  MOLECULAR WE 1GHT  NET CHARGE  ST-1  0.30  1220  -1  ST-1 A  0.59  1220  -2  ST-2  -0.41  854  +1.5 +3  777  +1  734  +1 +1  734  0 +1  1.31 ST-3  -0.41  ST-4  -0.44 0.52  ST-4A  0 0.*47  -52AND  ST-4.  THE  DIFFERENT  FROM  SHOWN  IN  EACH ON  ASPARTIC  NET  RATHER  HAD  THAN  N— TERM INAL  IT  RESIDUES FURTHER  THE  AND  SINCE  IT  NET  +1  THE  THAT IN  OF  THE  C-TERMINUS  WAS  INDICATION  THAT  IT  WAS  PROLINE  IN  THIS  OTHER  NET  CONTAINED  CALCITONINS,  RESIDUE  AN  RESIDUE  OF  N— TERM INAL CHARGE  GLUTAMINE  AT  THE  WAS BUT  THIS  6.5  RATHER  PEPTIOE  PARTIAL  ACID  IT  WAS  AFTER  NEITHER BEEN  LYSINE  THE  C - T E R - ,  INDICATED ABSENCE  OF  BASIC  PEPTIDE.  SUGGESTED  THAT  PROLYLAMIDE,  THREONINE.  AS  CONFIRMED.  C-TERMINAL INDICATING  GLUTAMIC  THAT  BLOCKED—A  WAS  THAN  ACIO  ACID  PEPTIDE  +1  THE  CYSTEIC  NOT  WAS  OXIDATION  ASPARAGINE  WAS  A  NEGATIVE CONTAINED  THIS  AND  MORE  THAT  C-TERMINAL  PROBABLY  LEUCINE PH  HAVE  MOBILITY  SPITE  ITS  C-TERMINAL  ONLY  ITS  THAT  OF  CONTAINED  AS  INDICATED  DANSYL-CYSTEIC  ST-4  ANO  THE  A  THAT BOTH  QUITE  PEPTIDE..  THEY  THAT  CONTAINED  COULD  CHARGE  THAT  PERFORMIC  CONTAINED  ONE  ADDITIONAL  INDICATED  OF  HAVE  PEPTIDES THE  WERE  HOWEVER,  MAJOR  LIKELY  HAD  THIS.  FACT  TO  SUGGESTED  ST-1  THEREFORE THE  AND  SUGGESTED  HAD ITS  OF  I D E N T I FI C A T I ON  AND  A  N— TERM INAL ST-3  ACID.  CONFIRMED  PRESENCE  8L0CKED IN  ASPARTIC  PEPTIDE.  CARRIED  THE  MOLECULE  AND  ARGININE  MINAL  MAJOR  DURING  NATIVE  PEPTIDE  DANSYLATION NOR  THE  APPEARED  OCCURED  COMPOSITION  RESIDUES  OF  THUS  PEPTIOES,  CORRESPONDING  PEPTIDES  IT  PEPTIDES  APPEAREO  THE  MINOR  MINOR  MAJOR  EACH  THE  CHARGES  THE  ASPARAGINE  THE  THE  THE  THEY  THAN  ACID.  THAT  OF  OF  CONTAINED  DEAMIDATION AND  VII,  CHARGE  CALCULATED  CHARGE  THOSE  TABLE  NEGATIVE  THEY  MOBILITIES  ACID.  THIS SEEN THE  PEPTIDE ARGININE,  THAT  IT  PEPTIDE  10  FIGURE  27.  SALMON  CT  TRYPTIC  • '/ }• / • / : POSSIBLE AM I N O  AC I O S .  PEPTIDES  CONSERVATIVE  15  ALIGNED  WITH  SUBSTITUTIONS.  20  HUMAN  25  CT.  SINGLE  !  •  LETTER  POSSIBLE NOTATION  30  HOMOLOGIES, USED  FOR  -53HAD  ST-2  AN  LYSINE,  BUT  DATA  PH  DID  AT  LEUCINE  8ECAUSE  THE  HISTIDINE  FIT  ANY  DID  6.5  HOWEVER  LEAST  N— T E R M I N A L  ONE  A  THE  TWO  ANO  THE  THE  ANALYSIS  OF  WAS  AMIDATED  SINCE  THUS  FACT  ALL  THAT  STRUCTURE  THE  NET  WHOLE TWO  IN  OBTAINED  AMMONIA  DATA  FIGURE  FROM  C — T E RM I N A L THE  HAD  THAT  BEEN  AT  AMIDATED,  RESIDUES  WERE  SEEN  IN  SUGGESTED  THAT  ONLY  ONE  TWO  G L U TAM I N E S  ALL  WAS  CONSISTENT  WHICH  TRYPTIC  IT  (88).  INDICATING  FOR  27  MOBILITY  WELL  RESIDUES  MOLECULE  AVAILABLE  A  LINES  CHARGE  ACID  ACCOUNT  AND  PRESENT  THE  ASPARAGIN E S ,  WOULD  INDICATED  INFORMATION  FIVE  WELL  OF  POSITIVE  GLUTAMIC  ONLY  THE  AMIDE  OF  NOT  CARRY  OF  C—TE RMINAL  OF  AS  OBSERVED  WITH  OF  A  AMMONIA.  SUMMARIZES  DIGESTION  AND  THE THE  SALMON  CALCITONIN. THIS CENTRAL OF  INFORMATION  PEPTIDES,  HUMAN  CT  SIGNIFICANT EVEN  IN  ONLY  FOUR  THE  ONLY  POINTS  AND  MENT,  IT  SEEMED  CT  ENDS  OF  THE  OTHER  OF  THE  ORDERING  TO  SHOWN  FIGURE  THE  IN  HUMAN  AND  EVEN  POSSIBLE WITHOUT  SALMON  OF  KNOWN  THE  ALLOWS  IN  THIS  WAS  TWO  SEQUENCES  28  HOMOLOGY  SIGNIFICANT WAS  THE  MOLECULES  HOWEVER,  CALCITONINS,  EXTENSIVELY.  ALLOW  COMPARED  THE  NO  CLEAR,  TWO  NOT  BETWEEN  AND  PORCINE  THE  DIFFERED  ORDER  BETWEEN  REGION  CT  THOSE  WHEN  ARRANGEMENT,  SALMON  THAT  THE  HOMOLOGY  THIS  CENTRAL  BUT  DOES  ANY  REGION.  POSSIBLE  SALMON  MOLECULES  HOMOLOGY  BETWEEN  IN  EITHER  FURTHER  MOLECULE  WERE  BUT  THE  THAT  AT IN  ARRANGE-  SEQUENCE SIMILAR CENTRAL  DATA, TO REGIONS  C-S-hW i-S-T-i-V-t-S-A- :¥- W- R - N - t > N- N-:£r H- R-iP.r S-G-M-G-f:-G-P-E-T-P-NH  PORCINE  BoV  2  S-N-: L s - T - i - V - : t ' i - S - A - : 4-W-K-D- i - N - N - : Y - H - R - ^ S-G-M-G-iF>G-P-E-T-P-NH2  INE  r  ti-s-T-C-V-L:-G4&-I  SALMON  fiUT-N-T-G- S-G-T-P- NH  2  Ls-T-i-M-r'L-G-T-: :-T Q-D-: ;^.N-K-:F'-H-T-.^-P-Q-T-A-. j:-Gr-V-G-A-P-NH :  HUMAN  2  10  FIGURE  28.  ALIGNMENT HUMAN  CT  OF  SALMON  (80).  LOGIES  AND  SALMON  CT.  V:Y  CT  SINGLE  (84)  WITH  CONSISTENT  CONSERVATIVE  20  15  PORCINE  SUBSTITUTIONS,  LETTER  CT  HY0R0PH0B I C  NOTATION  USED  25  ( 9 6 ) , BOVINE RESI DUES,  j>Xv! U N I Q U E FOR  AMINO  CHARGE ACIDS.  30  CT  (17),  , OTHER  ANO  HOMO-  PLACEMENT  IN  - 5 4 G.  SE Q U E N C E  OF  SALMON  SEQUENCES WHILE CARRIEO  OUT  ANNOUNCED EDMAN  THE  WORK  THE  BY  ON  NI A L L  SEQUENCE  MATERIALS  HAD  THE  WORK  THE  SALMON  THE  COMPLETE E_T  DEGRADATIONS.  PENDENT  C A L C I T O N IN  MAMMALIAN  OF  WORK,  BEEN  TRYPTIC  SEQUENCE  AJL IT  WAS  THUS  OF  THE ON  FROM  THE  THEIR IN  MAMMALIAN  HORMONES  OF  KNOWN  STRUCTURE.  MATION  ON  THE  CAN  OBTAINED  BY  ANALYSIS  FLGURE  THESE  BOTH  SIMILAR. FIRST  9  AND  RESIDUES  POSITIONS  2  ALL  FOUR,  AND  FOR  BIOLOGICAL  REPORTED, THE  AND  RETAINS  THAT  BRIDGE  (PROLYLAMIDE ) ,  EVEN OF  WAS  REMOVAL  BIOLOGICAL  AND  AJL  THAT  THE  HUMAN 1-7  THE  AND  SINCE  SEQUENCE WITH  OF  THE  CONSIDERA8LEINFORTO  BIOLOGICAL  ACTIVITY  THE  VARIES  THAT  BELL  REDUCED  WITH  SlEBER  C-TERMINAL  JET  THE AL  AMIDE  RESIDUES  IS  TO  ESSENTIAL  ( 1 0 ) HAVE  THEY  AND  IT  OFFER IS  ARE  (11.5)  THE  ALSO  IDENTICAL  WORK  OF  INDICATES  RESULTS  IN  A  POSITIONS  NO  POSSIBLE  BIOASSAY.  SYNTHETIC  IN  THE  AT  COMMON  IS  AJL  BUT  MOLECULES  FROM  FOR  M E R C AP T OE T H A N OL  OCCUREO, THE  VERY  ONLY  IT  ET  ACTIVITY,  DURING  ARE  IDENTICAL  INDICATES  FOUR  THE  ARE  HORMONE  ACTUALLY  THE  MOLECULES  BRIDGE  WHEN  EVIDENCE  ACTIVITY.  THE  ( 1 6 ,1 2 3 ) .  REFORMED OF  OF  DISULFIDE  BIOLOGICAL  ( 5 0 ) AND  OF  STARTING  SEQUENCES,.,  HORMONES  EVIDENCE  REDUCTION  RESIDUES  E_T  THE  ENDS  FOUR  THE  ACTIVITY  THAT  GUTTMANN  8.  MOST  EVIDENCE  C-TERMINAL  THE  ANO  HOWEVER,  HORMONE  THE  OF  INDE-  t  C-TERMINAL  THREE  WAS  SEQUENTIAL  SOURCE  i N— T E R M I N A L  HORMONE  ALIGNED  2 8  STRUCTURE  OF  BEING  CONTINUE  REPORT.  SHOWN  BE  TO  SAME  IS  OF  WAS  SALMON  SINCE  HORMONE  RELATIONSHIP  KNOWN  AUTOMATED  FUTILE  PARTICULARLY  CONFIRMED  TO  PEPTIDES  ( 8 4 )BASED  OBTAINED  COMPLETEO  COMPARED  CALCITONINS.  THAT  9 7 $ LOSS 2 8 , 3 0  -55ANO  ALSO  31  SHOW  CONSIDERABLE  HOMOLOGY  BETWEEN  THE  FOUR  MOLECULES. IN ARE  THE  MORE  SALMON  SUBTLE.  ANO  POSITIONS SALMON  CENTRAL  10,  ANO  14,  HUMAN  HOMOLOGIES  THE  HYDROPHOBIC  AND  LEUCINE 9,  12,  16,  TYPE  THIS  WOULD  THE  BONDING  TO  AND  SUCH  CONCLUSION  A  FORMATION  SEEN  SINCE  ISREARRANGED  MAY  MEMBRANE REDUCED. RESIDUES TI O N A L  BE  IMPORTANT  WHERE IT  IS  THE  WATER  LIKELY  WOULD  PLAY  CHANGE.  THE  A  OF  SHOWS  THAT  AQUEOUS AN  THE  DILUTE  AQUEOUS  FORCES BY  THE  ANO  TYPE  RECEPTOR  SITE  ON  REGULARLY  EVIDENCE  IN  COIL THAT  THIS  CONFORMATION  THIS  IS  OF  SUPPORTS  RANDOM  A - H E L I C A L  ROLE  WORK  MOLECULE  THAT  THE  HYDROGEN  THE  SOLUTION,  CONCENTRATION  MAJOR  (72).  AND  PORCINE  THE  OF  PROTEINS  IN  ON  MAY  STRUCTURE  DER'WAALS  INTERNALLY  THAN  TYROSINE,  RESIDUES  GLOBULAR  VAN  THE  POSITIONS  TERTIARY  CONFORMATION BY  IN  SUBSTITUTIONS.  IN  EXTERNALLY.  THAT  ONLY  A  THE  16.  INTERESTING  HYDROPHOBIC  SUGGESTS  AT  SAME  PHENYLALANINE,  LARGER  INTO  HE  MORE  BETWEEN  P O S I T ION  THE  SPHERE  HE IN  IN  REGULARITY  GROUPS  (14)  2-CHLOROETHANOL.  MENT  AS  MAINTAINED  PREDOMINATES  RANDOMNESS  IN  MICELLULAR  EOELHOCH  ARE  S I MILARITIES  HERE  LEUCINE 25  THE  CONSERVATIVE  THESE  HYDRATION  AND  THE  SUCH  22.  EXISTS  HOWEVER.  MARKED  HYDROPHOBIC THE  MOLECULES  AND  23,  S T A B L I L I Z A T I ON  BE  BREWER  IN  WITH  THE  THE  HOMOLOGY  ARE  RESIDUES  UNIMOLECULAR  BETWEEN  21,  HOWEVER,  FREQUENTLY  SOLUTIONS  ONE  MOLECULES ,  19  TO  OF  HORMONES—THE  18,  OCCUR  CONTRIBUTE THE  ONLY  UNGULATE  THE  4,  REGION  OF  THE  REARRANGE CELLULAR  SIGNIFICANTLY PLACED  INDUCING  CONFIRMING  SUCH THE  HYDROPHOBIC A  CON FORM A  IMPORTANCE  -56OF  THESE  BY  BREWER  IN  PORCINE  OF  ACTIVITY.  OF  HUMAN  AND  RESIDUES  SINCE  LOSS IN  CANT IN  THAT THE  A  OTHER  POSITIONS THE AN  M A T I ON  ACTIVITY AND  HUMAN  MRC  U/MG  OF C T  WHEN  87),  AND  TO  ANO  ALL  FOUR  CALCITONINS  HAVE  IN  GROUP  OF  IS  NOT  RESIDUES THE  OF  FOR  REASONS  (80)  ALL  ASSAYED ACTIVITY WHEN  IT  THE  THE  HAVE IN  VALINE  POSITION  25  ACTIVITY RESIDUES  OTHER  S I G N I F I -  ACIDIC  POSITION HERE  RESIDUES BUT  15  INDICATING  ACTIVITY.  AS  THROUGHOUT  THE  UNLIKELY  FOUR  BUT  MUCH  THAT  THEIR  BETWEEN IN  THE  OFFER  ARE  LITTLE  INFORM  BIOLOGICAL  BETWEEN  WHILE  2500  DISCUSSED  CHARACERISTICS  ( 1 5 ) , BOVINE  CT  RAT  MOLECULES  HIGHER  ACTIVITIES  THE  ASSAYED  IS  STRUCTURAL  PORCINE  CT.  FOR  SCATTERED  ACTIVITY,  FOR  AT  (80),  ACTIVITY.  BETWEEN WHAT  THREE  ASPARAGINE  MOLECULES  CRITICAL  THE  8  MOLECULES  THREONINE ONLY  LOSS  90$  WITHOUT  THE  ESSENTIAL ARE  IN  OXIDIZED  ARE  AN  A  ACTIVITY  CALCITONINS  CONTAINS  IN  BIOLOGICAL  MOLECULES.  BOVINE  TYROSINE  POSITION  HYOROPHILIC  SUBSTITUTIONS  SALMON  CORRESPONDING (65,  HUMAN  BIOLOGICAL  THE  SALMON  WORK  IN  BE  INDICATION  ON  METHIONINE  MAY  CHARGED  FOR  THE  RESULTS  HYDROPHOBIC  AND  SIMILARITIES  REQUIRED  THE  CALCITONINS  MOLECULE  ARE  OF  METHIONINE  AND  PORTION  OXIDATION  REDUCES  CORRESPONDS  CHARGED  FURTHER  THE  SALMON  PORCINE  IS  CONTRAST  CONSERVATIVE  CENTRAL  GIVE  AND  SALMON  HUMAN,  THE  IN  ACTIVITY  TYROSINASE  OF  ALSO  PORCINE  UNGULATE  THE  IS  THAT  WITH  SIGNIFICANTLY  HERE.  (16)  SHOWS  OXIDATION  BOVINE,  THE  BIOLOGICAL  CALCITONIN  POSITION  RESIDUES IN  WHICH  (13)  CT  THIS  FOR  SALMON  CT  100 CT  AND  4500  MRC  MOUSE  (89)  THE  (17) ANO  HAS  300 A  U/MG SALMON  -57HORMONE  AN  (65). AND  HAS  THE  BASIS  MAMMALIAN  FOR  ITS  HOMOLOGOUS  OF  AND  27,  POTENTIALLY  LYSINE, AND TO  24 ITS  BE  BY  A  HIGHLY  MOLECULES THE  MOLECULE  THREE  SEEM  WELL  SOLUTION.  I T\ T H E  POSITIONS IN  A  REGION  STRUCTURE  HORMONES,  23  IN  BE  FREE  THUS  AND  THE  SUITED BREWER  TO ET  THE  THE  C-TERMINAL  REGION  AL  AN  IN  A  OF  THE  A - H E L I C A L  (17)  HAVE  IN  24,  CHARGED  SPACED  THE  10,17•  MOLECULE IS  IN  MOLECULE  IS  OCCUR  THREE IN  TO END  THE  MOLECULE  MAMMALIAN  DOES BE OF  EXIST  THIS  INVOLVED THE  IF  27.  IN  DIFFERENCE  IN  SUGGESTED  IN  AT  MOLECULE  STRUCTURE  THE  SALMON  PROLINE  SALMON  FACT  NEAR  POSITION  MARKED  BETWEEN  20,  INVOLVE  SALMON  EARLIER  EXPECTED  C-TERMINUS FOR  THE  17,  OF  MOLECULES.  THE  RESIDUES  RESULTING  ARE ARE  10,  SALMON  DIFFERENCE  WHERE  AND  DIFFERENCE  MAMMALIAN  CONTRAST  BE  WHICH  BINDING  THE  BASIS  P 0 S I T I 0NS  IN  CHARGE  OISCUSSEO  WOULD BUT  IN  IN  OF  HYDROPHOBIC  POSITION  CONFORMATION. POSITION  END  CONTAIN  WOULD  RESIDUES  FOURTH  THIS  EQUALLY  CT  STRUCTURAL  SUBSTITUTED  THE  CT  SALMON  ELIMINATED  MOLECULE  AND  GLUTAMINE  HYDROPHILIC.  HYDROPHOBIC MAMMALIAN  ARGININE  THE  BE  SUBSTITUTIONS  RESIDUES  HISTIDINE  MICELLULAR  THESE  ON  POSITIONS:  THE  REPLACED  C — T E R M IN A L  SEVEN  SITE.  WHERE  IS  ONLY OF  AND  SALMON  SIGNIFICANT  20  THE  THE  PORCINE  SOME  ACTIVITY. CAN  OF  BETWEEN  ESTABLISH  HIGHLY  POSITION  LAST  COULD  CONSERVATIVELY  CHARGED  RECEPTOR  THE  NOR  FOUR  HISTIDINE COULD  ON  ACTIVITY  DIFFERENCES  SIMILARITIES  LEAVES  29.  THE  BIOLOGICAL  SHOW  THIS  THE  HORMONES  POSITIONS  INTEREST.  TIMES  250 OF  HIGHER  WHICH  THOSE  NEITHER  26,  THAN  EXAMINATION  POSITIONS ONLY  MORE  WOULD  AQUEOUS THAT  FIGURE  29»  A  POSSIBLE  CONFIGURATION  DETERMINED  FROM  PHOTOGRAPH, POSSIBLE  OPEN  CLOSED  HYDROPHOBIC ARE  FIGURE  3 0 .  A  SHOWN  POSSIBLE  DETERMINED DATA  FIGURE  31  A  AS  POSSIBLE AS  CHAIN.  SALMON  AS  CT  MODEL.  LOWER  CONFORMATION  BONDING. IN  OF  MOLECULAR  UPPER  PHOTOGRAPH, EMPHASIZING  HYDROPHOBIC  RESIDUES  BLACK.  CONFIGURATION FROM  A  OF  MOLECULAR  PORCINE  CT  MODEL.  OTHER  AS  ABOVE.  DETERMINED DATA  A  CONFIGURATION FROM  ABOVE.  A  OF  MOLECULAR  HUMAN MODEL.  CT  AS OTHER  -=58T HE  APPARENTLY  OURING  GEL  TERTIARY ON  HIGH  FILTRATION  STRUCTURE.  CONFORMATIONAL  MOLECULES EDMUND  MOLECULAR  WERE  MAY  MODELS,  N.J.).  THESE  IN  CONFIGURATIONS  MODELS  CONFORMATIONS THE  HYDROPHOBIC  SEEN  THAT  FOLD  THE  SHOWN  EVEN  MANY  ALLOW  IONIC  SHOWN  HIGH  SHOWN  AND  FROM  NEAR THE  POSSIBLE  RESIOUES  ARE  PRESENT  CHARGE  AND  PROBABLY  WOULD  RECEPTORS,  OTHER  FACTORS  WERE  TO  POSSIBLE,  SIDE  CHAIN  HUMAN  INTERACTION  AS  IT  SEEMED  OF  CLARIFY  THE  REGIONS.  ONES BONDING  ABSENCE  OF  IS  CT  CLEARLY  TAIL  IS  NOT EIGHT  SUGGESTING  THAT  SUCH  STABILITY  SUCH  BETWEEN LIKELY  HORMONES  TO  ONLY  DETERMINING  THE  BE  SINCE  LITTLE  DIFFERENCES  THE  IS  THE  CAN  APOLAR  SALMON  STRUCTURE  RELATIVELY  IT  POSSIBLE  THE  TAIL  31  AND COIL  HYDROGEN  C T .  PROLINES  BARRINGTON,  AND  BUT  OF  (L.ANDE-  BONDING.  IS  GROUPS.  HYDROPHILIC  BETWEEN  IT  SALMON  MODELS  RANDOM  COMPACT  C-TERMINUS  INVOLVED  ACT I V I T I E S  INTO  AND  CO.,  CONTRAST  MOLECULES  AND  HUMAN  HYDROPHOBIC FOR  OR—HEL I C A L  THE  BUT  WERE  UNDERTAKEN  SHIPS.'  THE  CONFORMATIONAL  THEIR  POSSIBLE  IN  CONJECTURE  2 9 , 30,  IN  HORMONE  THIS  FILLING  FIGURES  CHARGED  CT  HAVE  INFLUENCE  B I OLOGICAL  OF  OF  IN  ARE  SALMON  DIFFERENCES  SCIENTIFIC  CHAINS  LONG  PORCINE  ITS  STRUCTURE  SIDE  BETWEEN  GROUPS  SPACE  BLACK  SMALL  DEGREE  IN  A  ARE  SUCH  PORCINE,  EMPHASIZE  THESE  BONDING  DIFFERENT  AND  CONFORMATIONS  A  HYDROPHOBIC CLEARLY  GROUPS IN  USING  (A)  WHICH  HYDROPHOBIC  ACTUALLY  AND  (B)  THE  SHOWN  THE  VALIDITY  EDMUND  ARE  OF  FROM  THE  DIFFERENCES  CONSTRUCTED  MOLECULAR  OPEN  RESULT  TEST  TO  WEIGHT  AS  THESE  THE  THAT THE  (19). WOULD  HORMONES A  NUMBER  ANO OF  RELATIVE  FURTHER  STRUCTURE-FUNCTI  INVESTIGATIONS  0N  RELATION-  IV.  STRUCTURE-FUNCTION  IN  THE  MATIONAL IT  WAS  THE  PRECEDING  VARIATIONS  SUGGESTED  ACTIONS  OF  THAT  THE  ULTIMATE  REQUIREMENT A  HORMONES  SUCH  ACTION FOR  TRANSPORT  SECRETION  WILL CT  BE  AND  THE  FROM  ON  BOTH  ROUTES  BY  WHICH  CHANGES LOW  SHOULD  IN  THE IT  HORMONE  TO  THE  BE  PLASMA  HAVE  REMOVED  FACTORS OF THE  CRITICAL (31)  CALCIUM  LEVELS  (46).  FUNCTION LITTLE  BECAUSE  EFFECT  -59-  ON  THE  DISTAL THE  OF  DURING  DYNAMICS  LEVEL  I N  OF  THE  THIS THE  SITE  TARGET  THE  CHAPTER  REMOVAL  OF  OF  ITS  HORMONE  FOR  A  AND  RESPONDS  BE  MANY  DEFINITIONS  INFORAND  CIRCULATION.  THE AN  OF  REQUIRES  THE  WOULD  TARGET  HORMONE  AND  IN  IN  THE  ONE  TO  THE  DYNAMICS  LIFE FROM  IS  A  HORMONE.  THE  LEVEL  AT  ANIMAL  ACTING  HORMONE  OF  INVOLVED  THESE  THE  ALL  AND  ROLE  CIRCULATING  FAST  ENDOCRINE  WOULD  OF  ONLY  DEFINITION  NORMAL  THAN  IN  CIRCULATION  THE  A  ORGANS  IS  CONFOR-  INDICATED,  PLAYEO  RELEASE  EXOGENOUS  ESPECIALLY IS  WERE  PERFORM.  THE  FACTORS  RATHER  IS  CIRCULATING  EFFECTIVE  THE  IT  BEHAVIOR  CIRCULATING  MAMMALS, IN  THE  UNDERSTANDING  MATION  WHICH  TO  THE  POSSIBLE  RECEPTORS  OF  WHEREBY  IN  ANIMAL  CIRCULATION  REMOVAL  OF  REMOVAL  CONFINED  SECRETION.  A  C A L C I T O N I N S .  OF  TARGET  MUST  INVOLVES  CONTROLLED  OF  THE  CONCEPT  WITHIN  STAGE  THE  HORMONE,  THE  INVESTIGATION  RESPONSE  ON  INCLUDE  ORGANS.  IS  AT  MOLECULE  TRANSPORT  NUMBER  DIFFERENCES  HORMONE  BY  HORMONE  A  A  CALCITONINS  THESE  FOLLOWED  THIS  THE  HORMONES  WHILE  FUNCTIONS  CHAPTER  IN  ORGANS.  OF  I N  I N T R O D U C T I ON  A.  OF  R E L A T I O N S H I P S  HORMONE  THE  SUCH LIKE TO  RAPID  MAINTENANCE  ESSENTIAL RELEASE  ALREADY  OF  HIGH  CT  TO NEW  OF  Time (min.)  FIGURE  32.  DISAPPEARANCE INJECTION  OF  OF  0  51  "INTACT"  CALCITONIN  I )-PORCINE  CT (130).  AFTER  -60CIRCULATING IS  LEVEL  CERTAINLY  ANO  THE  PULSE RATE  RESULTS  IN  BE  USEFUL  THIS  BETWEEN ANO  STUDY THE  THEIR  ;  FROM  FOR  DATA  AS  CURVE WITH  VALS  AN  THE  OF  THE  THE  COMPONENT  EJ_ AL^ THE  FIGURE  SHOWING  A  MUCH THESE  FUNCTION MIGHT  POSSIBLE SALMON  THE OF  A  HORMONE  OF  ELUCIDATE  (1 3 0 ) PIG.  CT,  RELATIONSHIPS  THE  CALCITONINS  THE  OF  DECLINE  SHOWN ( T i =  THE  IN 5  COMPARTMENTS  IN  CT  ACTIVITY  ANALYSE  IN  WHILE  32,  HORMONE IS  IN  THE  CONCENTRATION  TAKEN  AT  SIMILAR  INTERDECLINE  USING  USED  THE  LARGE MODEL  DISAPPEARANCE  SUGGESTED  SLOWER  OF  TWO-COMPONENT,  AUTHORS  AND  COMES  CT  RADIOACTIVITY  A  REPRESENTS THE  A  PLASMA  OF  OF  CLEARANCE  EXPERIMENTS  KINETICS  FIGURE  THE  CT.  THE  CT.  MIN)  ON THE  LABELLEO  PORCINE  HALF-LIFE  WHICH  32,  0 ^ l ) - P O R C I N E  BIOLOGICAL  TO  MIGHT  A  ACTIVITY  HAD  (26).  SALMON  SUGGESTED  CIRCULATING  FROM  IN  UNLABELLED  TWO  THE  INJECTION  EXPERIMENT  RAPID  ALSO  SPECIFIC  WE S T  CT  SHOWN  ASSOCIATED  BELOW  THAT  A  ACTIVITIES.  ON  OF  DECREASES  SHOWN  AND  SYSTEM  LOW  LOWER IT  AT  PLASMA  DIFFERENCES  PORCINE  OF  ENDOCRINE  THEY  HIGH  WITH  OF  A  THAT  INACTIVATED  STRUCTURAL  STUDIES  DOSES  WAS  AT  SHOWED  CT  TH I S  USUALLY  RELEASE  RELEASE  KNOWN  THE  IN  AND  ARE  RAPID  STEADY  SALMON  LEVELS  TO  BEST  SEEN  A  CONTROL.  UNDERTAKEN  THE  AFTER  IS  LITTLE  WAS  EXOGENOUS  WAS  THE  SALMON  BIOLOGICAL  EXPONENTIAL  ON  A  CT  INCUBATIONS  THAT  THE  WHERE  BY  MAMMALS  VITRO  THE  EXERT  CHALLENGE  CLINICALLY.  EXPLANATION ANO  IN  IN  A  IN  SUGGESTED  DIFFERENTLY  MAMMALS  STUDIES  ACTION  RATE  THEREFORE  FOLLOWED  EARLY  PROLONGED  IN TO  HORMONE  (4).  LOWER  TRUE  RESPONSE  OF  AND  THE  THAT  THE  EQUILIBRATION  COMPONENT  IN  -61(T^•= 2 THE  MIN)  37  REPRESENTS  THE  ACTUAL  RATE  OF  DEGRADATION  9$  AND  OF  HORMONE.  Input X21  —-p X  THEIR  ANALYSIS  WEIGHT  FOR  SYSTEM  THERE  ARE  ANO  A  A  CAN  NUMBER  THIS  OF  DATA  INACTIVATION  A  IS  MODEL  COMPARTMENT  THE A  IMPLY  MAJOR TO  MODEL  THE  AVAILABLE  CURRENTLY  ALLOW  THIS  IN  WHILE  IN  FIGURE  COULD  CURVE MODEL  THE  FOR  BUT  ALLOW  THEY  DO  ALSO  (109, IS  DECLINE  BODY  THIS 32,  EQUALLY  INACTIVATION  OVER-  THAT  OF  WELL  117),  AN  SUGGEST  SUCH  AS  INSULIN  (63,  FACTORS  INVOLVED  106)  THEIR  DATA  CT  DO  OXYTOCIN  IN  THE  THE  OF  FOR  AT,LEAST  HAVE  INDICATE  ANALYSIS  COMPREHENSI VE. MODEL.  HORMONES  OF  THEMSELVES  FACTOR  MUST  AND  MORE  WHICH  TYPE  THAT  SEEN  OF  16$  TISSUE  ACTIVITY,  OUTSIDE  OF  1.  DISAPPEARANCE  A  MODELS  COMMON  OF  RESPECTIVELY.  2  PATTERN  AUTHORS  FAILS  COMPLETE  ANALYSIS,  THE  OTHER  RELATIVELY  VARIETY  THIS  OF  VOLUMES  AND  1  PRODUCE  SIMPLIFICATION.  BUT  INDICATED  COMPARTMENTS  MODEL  PRODUCE  ALSO  1,2  PROVIDE  STUDIES  (53,  PROVIDED REMOVAL  55), A OF  OF ADH  FAIRLY THIS  RELATIVE  NOT A  ROUTES  ALL6W  BASIS OTHER (39,  IMPORTANCE. SUCH  FOR  AN  SUGGESTING  POLYPEPTIDE 129)  CLEAR TYPE  OF  OF  AND  PICTURE HORMONE  OF  THE  FROM  -62T HE  CIRCULATION,  REMOVING  HAS  CT  QUITE  COMPLEX,  MAJOR  FACTORS  WITH  RENAL  3.  UPTAKE  THE  BINDING  OTHER  CT  TO  FROM  ALBUMIN  (5$  THESE  FACTORS  IN  OVERALL  PICTURE  IS  DOWN  HORMONE PLASMA  INTO  THE  FIVE  IN  THE  PLASMA  PROTEINS.  HORMONE. BY  OF  TARGET  ORGANS  HORMONE  BY  TARGET  OF  HORMONE  IN  PLASMA.  WAS  NOT  AND  FROM  1  FREE  DIFFUSION  COULD  WE S T WAS  THE  IN  EJT_ A_L  NEARLY  VOLUME  BODY  THAT  TO  INACTIVATION  OF  ORGANS  VOLUME  OF  INJECTED  CALCITONIN  THIS  BY  OF  THE  PLASMA  FACT  T H E IR  THE  FOR  PRESENCE  ANOMALIES THE  VOLUME (^ 31 I  ALSO  PROBABLY  THE  THAT  RESULTS  AS  IN  VOLUME AS  OF  DETER-  '  )-PORCINE  SHOWED  INCREASED  THESE  BEHAVE  RENAL  CERTAIN  PLASMA  STUDY  COMPARTMENT  INCREASED.  PLASMA,  INDICATED  SPECIFIC  WOULD  ENZYMES.  EXPLAIN  WHICH  IT  THE  IS,  ARE  WOULD  OF  FOR  THERE  PROTEINS  SINCE  MODEL  EXISTS  PROTEINS  HORMONE  THE  121).  DISTRIBUTION  WEIGHT).  APPARENT  LARGE  DOUBLE  OF  (67, THESE  OUT  IN  EVIDENCE  PROTEINS  BOUND  FROM  CONSIDERED  CONSIDERABLE  HORMONE  PROTEINS  COMPARTMENT  OF  EVIDENCE  AND  RESULTS  TO  CLEAR  PROTECTED  SUCH  THE  BROKEN  FREE  80UN0  PLASMA  DIFFERENTLY  EXCRETION  OF  FACTOR  OF  CARRIERS.  OF  ORGANS.  ALTHOUGH  NO  BE  HORMONE  M.  HOWEVER,  MANY  ORGANS.  FIRST  E_T  MINED  CAN  EXCRETION OF  OF  BELOW.  INACTIVATION  5.  THE  IT  INACTIVATION AND  OF  BUT  ROLE  INVESTIGATE.  LISTED  2.  4.  BE  BEEN  HORMONE  OTHER  QUITE  THE  EQUILIBRATION  1.  WEST  AND  THAT  THE  WOULD  THE  DOSE BE  OF  - 6 3 EXPECTED AND  IF  ONE  COMPARTMENT  FREE,  AND  COMPARTMENTS.  WHILE  FROM  TRACER  THAT  BOTH  THIS  SITUATION  THE  HORMONE  OF  ONTO  COMPARTMENT CERTAINLY AND  THE  DOES  PLAY  A  REMOVAL  SEVERAL E_T  M.  THE  ( 7 9 ) HAVE  URINE  WITH  PAGET'S  OF  1  5  1  I) - P O R C I N E  INJECTION.  THEIR  AN  AL  CT  ROUTE  REMOVAL,  CONSIDERABLY TARGET  ORGANS  MORE MUST  IT  STILL  ROLE NOT  IS  THE  INFUSION  RESULTS  OF  IS  THAT  AMOUNTS  SIZE  IN  CT  IF  INCREASE  OF  PROTEINS THE  OF  THE  WOULD PLASMA,  COULD  INFLU-  FOR  ONLY  TO  UPTAKE  IMPORTANT, OCCUR  IF  BY  THE  THE  LEAST  ORGANS COMPLEX.  HORMONE  IS  TO  IN  TOTAL VALUES  INJECTIONS TOTAL PART  THUS  IMPORTANCE  VARIOUS MORE  AT  CT  PATIENT  THE  AFTER  ACTIVE.  RELATIVE  AND  NEER  A  HIGHER  1 4 $ 0F  THAT  BIOLOGICALLY  INTO  0 . 1 $ OF  URINE  IN  HOWEVER,  MINOR.  CT  SOMEWHAT  FROM 2 . 5  ITS  EXCRETION  AVAILABLE,  PORCINE  IN  INOICATED  BUT  RENAL  R A 0 I 0 - I MM U N 0 A S S A Y A B L E  OF  CT  OF  RELATIVELY  REPORTED  RANGING  IS  QUESTION  BINDING  THIS  BIOASSAY.  WHICH  CT  CALCITONINS  THE  LABELLED  WAS  THE  OF  ( 1 3 0 )  MATERIAL OF  BY  APPARENT  RETAINING  ACCOUNTED  THIS A  OF  BOTH  INDICATES  INCREASING  THE  BETWEEN  THAT  REPORTED  ET  IN  PLASMA  SUGGEST  PR E C I P I T A B L E  TCA (  FROM  DISEASE  WEST  SHIFT  FOR  AL. ( 6 7 )  DOSES  BOUND  MEASURED  EVIOENCE  DETECTED  ONE  BINDING.  FOLLOWING  INFUSED. FOR  OF  BE  THESE  ROLE  ACTUALLY  CLEAR  AND  INCREASE.  CT  REPORTS  CAN  PROTEINS  MAJOR  COMPONENTS,  L_E G G A T E JET  WOULD  INFORMATION  OF  NO  HIGHER  DIFFERENCES  DEGREE  IS  CT  EXIST  TWO  SAMPLING  BY  HORMONE  WOULD  DETAILED THE  FREE  BINDING  STRUCTUAL  ENCE  WORK  ANO  FREE  PLASMA THERE  STUDIES,  BOUND  LEVEL  IF  CONTAINED  1  EXCRETION IS  APPEARS UPTAKE CAUSE  OF  A  UNCLEAR. TO  BE  BY RESPONSE,  TABLE  DISTRIBUTION  125  OF  I-CT  1  CT % G  ^l-PTH  IN  INJECTION.  G  PER TISSUE  RAT  TISSUES  CT io  AFTER  (36)  PTH io  PER TISSUE  25  AND  INTERVENOUS  TiSSUE  VIII  IN  PTH  TOTAL  TISSUE  MASS  $  IN  TISSUE  6.4  3.2  1 3.9  7.0  *  *  1 3.0  14.7  BLOOD  1.0  1.5  6.5  10.1  BONE  0.3  0.6  4.4  7.5  KIONEY  3.2  22.6  2.6  18.0  NOT  REPORTED.  LIVER SKELETAL MUSCLE  *  TOTAL MASS  -64AND  THE  THOSE  CONSEQUENCES  OF  TARGET TAKEN  UPTAKE  ORGANS UP  BY  COMPLETELY THE  THE  CAUSES  REMOVED  FROM LUISE  DE  DISTRIBUTION  UPTAKE  ORGANS.  THE  ORGANS  OF  THIS  OTHER  OTHER  RESULTS  CATE  BY  OF  THE  IS  AT  ANY EJT  OF  BEST  PTH  IV  AFTER  RADIOACTIVITY  INTO  INJECTION THESE  RATS.  CT  FROM  THE  MOVES  INTO  VARIOUS  OF  THOUGHT B U T I N  TO  BE  TERMS  PLASMA  THE OF  RESULTS AND  PRIMARY  QUITE  A  LARGE  TAINS  A  DISPROPORTIONATELY  AMOUNT THIS  PTH  OF  TAKEN  UP  BY  STORED  TISSUES  IS  TISSUE  TO  MAINTAIN  IT  IS  THE  SUGGESTING  NOT  WORK  THE  THAT  ORGAN  LIVING  THE  SOME  LIKELY  RESPONSE. INDI10  TISSUES  AND  RAPID OF  CT,  FOR  THE  THE  AMOUNT OF  IN  BONE,  IS  SMALL;  ACTUALLY  LIVER CT  OF  )-  HORMONE  SEEMS  IT  . I  (  DISAPPEARANCE  PRESENT  TISSUE  DEGREE  CT  MUCH  TOTAL.  LARGE  TO  ESTABLISHES  TISSUES,  BIOLOGICAL PENDING  ALSO  AND  A  SELECTIVE  CON-  SMALL  UPTAKE  THERE  ACTIVITY  LATER  THAT IS  IS  RELEASE.  CT  NO  BY  RAPIDLY  INDICATION  THEN IF  is  WHETHER  ENDED  OR  IF  CT  IN  THESE  THE  RAPIDLY  INACTIVATED  THE  WILL  ESSENTIALLY  UNIDI RECTI 0NAL,  SERV ING  IF,  OTHER  LOW  BE  CIRCULATING  DESTROYED-,  PLASMA  RESPONSE  THIS  VARIOUS  HORMONE'S  MERELY  AND  OF  THAT  VIII  VARIOUS  THE  ORGAN. ALTHOUGH  THE  PROPORTION  OF  BY  ,125  AMOUNT  TARGET  THE  v  CONFIRM  THE  AMOUNT  IN  FROM  MOST  TABLE  IN  UP  WHILE IS  ON  ^l)-PORCINE  INDICATE  TISSUES.  THE  (  OF  TAKEN  AND  EFFECT  SHOWN  (36)  DIFFERENT  RESPONSE  STORED  POSSIBLE AJ_  QUITE  HORMONE  PHYSIOLOGICAL  /12S MINUTES  ARE  WHEN A  THE  SINGLE  THE  CT  LEVELS. IN  CIRCULATING DOSE  OR  MOVEMENT  THE  TISSUES  LEVEL  PULSE  ON  WAS  WOULD  BETWEEN  THE COULD  REDUCED BE  IT  IS  PLASMA ONLY HAND,  RETURN AND  PROLONGED.  TO  THE  -65FURTHER OF  WORK  PORCINE  FROM  IN  CT  TION  OF A  A  SEEN  OF  SECOND  SLICES  THE  SIMILAR (44,  AND  LIVER,  RAPID  IS  THE  INACTIVATION  HOMOGENATES  IS  RAPID.  OTHER  INACTIVATION  WHICH  75)  INACTIVATION  TYPE  AND  PORCINE T I  FROM  INDICATES  (37)  TISSUE  HOMOGENATES  TYPE  AJL  SUPPORT  IMPORTANT  IN  OF  PORCINE  THE  IDEA  THE  DISTRUC-  CT.  TASHJIAN OF  EJ_  VARIOUS  ALSO  TISSUE THIS  LUISE  PARTICULARLY  HAVE  THAT  DE  BY  CT  RATS,  WORKERS  BY  FOR  OF  MUNSON I N  CT  INACTIVATION  (121)  HUMAN  PORCINE  CT  IN  HOUR.  IF  A  FIST  OCCURS  SHOWED  SERUM.  WE S T  PORCINE  PLASMA  IN  THIS  E_T  AJ_ IN  THE  PLASMA,  DURING  INCUBATIONS  (130 ) HAVE VITRO  AT  REPORTED  37°  OF  C  2 1.5 IN  -  2.0  VIVO  IT  COULD  INACTIVATION THIS  IS  TOTAL THE  SEEN  RESPONSE OR AND  ACTIVE. ACTIVE  EVEN  MODEL  TYPE  OF  MODEL  TO  AND  UP HAS  ON  F  IT  3  AND  1  NEXT  OF  OF  FIGURE  FROM  IS  ORGANS BEEN  PAGE  EFFECT  INSTANT THE  ON  OF  TARGET  POTENTIALLY  MAY  NO  PROVIDES AND  SEEN FREE  HORMONE  REPRESENT  OF  LONGER  BE  DESTROYED.  DISCUSSED CURVE  WHILE  32.  0NATE  BY  TOTAL  FRACTION  THE  UPTAKE  OCCURS  THE  SHALL  EFFECTS  REPRESENTS  REPRESENTS \ ^  YET  OF  10$  DISPROPORTI  OTHER  EXPONENTIAL  COMPARTMENT  ABOUT  EXTENDS  NOT  THE  A  TIME  BY  INACTIVATION  RELATIVELY  INACTIVATION  DOUBLE  A3 1  A  HAVE  THE  OF  COMPONENT  HORMONE  IT  SHOWN  ONLY  ACTION  TAKEN  COMPARTMENT  PROTEINS.  MAY  THE  THOUGH  EQUILIBRATION  WHILE  OF  RATE  SLOW  ONLY  ITS  INJECTION ALL  FOR  THE  IT  SINCE  HORMONE  THE  THIS  IN  QUANTITATIVELY  RELEASE  THE  ACCOUNT  INACTIVATION  ORGANS  SIMILAR  THE  IN  FOR  CAN  BOUND  STILL  FIGURE  HORMONE TO  THE  IN  32. THE  FIT IN PLASMA,  PLASMA  EQUILIBRIUM  BETWEEN  -66-  BOUND  AND  ASSUMED  FREE  TO  HORMONE,  BE  COMPARTMENT  IMMUNE  TO  INCLUDES  2  AND  HORMONE  DIRECT ALL  IN  COMPARTMENT  INACTIVATION  EXTRAPLASMA  OR  SPACES  IS  3  EXCRETION. X  AND  \  0  »I AND  ^1 2  A  f  R  E  T  H  E  COMPARTMENTS.  F  \  R  A  C  T  L  0  N  A  TURNOVER  L  INCLUDES  N  RATES  D I S T R U C T I 0N  BETWEEN IN  THESE  PLASMA  AND  U, 1 EXCRETION, RATES DATA  FOR  ALL  IMPORTANCE  OF  THE  SPECIES STUDIES.  NUMBER IN  PERMIT IS  VARIOUS  AND  SINCE  OF THIS  MAY  OF BE  IN  EARLIER SUCH  IS  EARLY  WORK  THE  MAGNITUDE  OF  (26) THE  OF  FOR OF  EXAMPLE  IN A  OF  THESE THE IS  TOTAL  RESPONSES  FROM  DOSE  THIS.  THAT  A  EVIDENCE  COMPARATIVE  STUDIES  HAVE  STRUCTURAL  VARIATIONS  SUGGESTEO  THE  VARIES  INACTIVATION  WITH  OF  THIS  FACTORS  INDICATING  AREA  ALL  CONSIDERABLE'  PERCENT  COMPARATIVE  CLEARLY  INVOLVEO. IS  DISTRUCTION  DETERMINING  ALLOWED  AN  CORREALATED  RATE.  IN  THESE  THE  DEPARTMENT  BE  ANALYSIS  THERE  MUST  COMBINED  TISSUES.  FACTORS  VARIATIONS  THE  USEFUL  IMPORTANCE  MENTIONED  DIFFERENCES  BETWEEN  NOT MODEL  SPECIES  OONE  VATION  THE  EXTRAPLASMA  VALUABLE  THE  EXCRETED A  THE  RELATIVE TO  REPRESENTS  n  THE  DOES  BUT  PARTICULARLY  BEEN  OF  AVAILABLE  COMPONENTS,  THAT  X  WHILE  IN  INACTI-  RELATIONSHIP (31 )  AND  THE  FIGURE  33.  AREA HUMAN  RESPONSE CT  • - 2 0  IN MRC  CURVES  RATS U,  FOR  (85).  X - 100  VARYING O -  MRC  DOSES  VEHICLE U,  A -  OF  ONLY,  400  MRC  U.  36  i6o  2S6  sod  Dose (MRC mU)  GURE  3 4 .  PLOTS  OF  LOG  CALCITONINS O -  HUMAN  PORCINE •  -  DOSE FROM  C T ,  C T ,  CH I CKEN  VERSUS SEVERAL A -  » - C O D CT.  AREA  BOVINE CT,  RESPONSE  SPECIES C T  (31-)»  A —SALMON  F  ( 8 5 ) . D  C T ,  ~  0  4  8  12  16  Time  FIGURE  35.  INACTIVATION DURING (A) • (B)  RAT  IN  SERUMS  SERUM.  PORCINE  SALMON  CALCITONINS  INCUBATION  SALMON  CHICKEN  OF  SERUM, (c)  HUMAN  RAT  SERUM.  CHICKEN  IN:  SERUM, CT  24  IN:  A -  FROM  SEVERAL SEVERAL  SERUM. • -  A -  CT,  SALMON  A -  PORCINE  SPECIES SPECIES  HUMAN  CT,  CT.  PORCINE  • -  SALMON  28  FROM  PORCINE T -  CT, CT  ~20  ( hr)  SERUM,  • -  SERUM. SERUM,  O -  CHICKEN  SERUM. • -  HUMAN  HUMAN  CT,  CT, v-  • -  PORCINE  PORCINE  CT.  CT.  (85).  - 6 7 IN  VITRO  INACTIVATION  PLASMA.  MORE  EXTENDED  THESE  SET  OF  FROM  AREA  THE  RECENT  RESPONSES  DATA  IN  THE  THE  DOSE  THIS  HAD  A  RESPONSES  LINEAR ALSO  THE  MINED  VATED  USED  AS  EXISTED  RESPONSE  THE  THE  AND OF  3 7  AS  FOR  FOR  TO  THE  AND  A  TYPICAL  CALCULATED  FAIRLY AS  A  SHOWN  IN  RELATIVELY  INACTIVATING STRUCTURAL  CALCITONINS  WAS R E S P O N S I B L E  THE  LOG  SHOWN  SIMILAR  IN  OF FIGURE 3 4 .  DATA.FOR  TWO.  ULTIMOBRANCHIAL  LINES  THESE  INDICATED  TWO  TYPES  THAT  OF  A  C T  CALCITONINS,  COURSE.  WERE THE  INCUBATED  RATE  OF  PATTERN  GROUP  BEING  3 5  CONSTANT  MORE  (B)  AS ANO  THE  DIFFERENTIAL  TWO  IN  THIS  OF  THAT  THE  INACTI-  SHOWN  REGARDLESS  BETWEEN  WITH  (C)  FROM  DETER-  RAPIDLY  SUGGESTING  DIFFERENCE  SERUM  EMERGED  CALCITONINS  FIGURE  FOR  WITH  INACTIVATION  CLEAR  MEDIUM,  FUNDAMENTAL  HOUR)  ULTIMOBRANCHIAL  TIME  AND  AS  THREE  THESE  ULTIMOBRANCHIAL  WAS  RAT  AGAINST  SEEN  PLOTS  CALCITONINS AT  IS  BETWEEN  DIFFERENT  3 5 ( A ) .  RELATIONSHIP  THE  SLOPES  CALCITONINS  THAN  FIGURE  SHOWS  BIOASSAY,A  MAMMALIAN  PRESENTS  (MG$ -  PLOTTED  RELATIONSHIP  THE  SPECIES  BY  ARE  CALCITONINS  THESE  CONFIRMED  THE  AREA  13.3  A  IN  FIGURE " 3 3 .  4 0 0  MARKEDLY  VARIOUS  IN  C T  6.8  AREA  WHEN  HUMAN  INCUBATED  HAVE  BELOW  1 0 0  DICHOTOMY  THAT  FOR  TABLE  3 . 5  CALCITONINS.  AND  THE  HORMONES  (85)  ( M R C MU)  THYROID  CLEAR  THE  2 0  FIGURE  OTHER  OF  EXPERIMENTS  FINDING.  DOSE  WHEN  RATES  THE  PLASMA  SOME TYPES  OF  INACTIVATION  20-  TTime (hr)  FIGURE  3 6 .  COMPARISON AND  CHICKEN  ANCE  RATES  OF CT IN  IN  24  V I TRO  IN  RAT  26  INACTIVATION SERUM  TO  THEIR  OF  SALMON  DISAPPEAR-  NORMAL  AND  NEPHRECTOM I ZED  • -  RAT  SERUM,  RATS  (85). (A)  CHICKEN  T OM I ZED (B)  RATS,  SALMON  TOM I Z E D  CT  CT  RATS,  • • O -  NORMAL RAT  NEPHREC-  RATS.  SERUM,  NORMAL  A -  RATS.  A -  NEPHREC-  -68RATES  IN  SERUM  WAS  FOR  THE  THERE THE  PLASMA. A  IN  FAIRLY VIVO  WAS  A  OF  INACTIVATION SLOWER  THAT  THE  SIMILAR  OF  OF  WAS  THAT  THE  SERUM. WERE  AREA  GIVEN  PLASMA  THAN  THE NOT  WERE  NOT  EXPONENTIAL  LEVELS  DROPPED  THE  CURVE  SIMILAR  TO  THE  NOT  WHICH  HAD  1  MEAN  THAT  HOWE V E R . THE  WAS  C T ,  ALSO SUGGESTING  LIVER  MIGHT  ENZYMES  IS  IN  COURSE  THAT  SAME  LIVER  FROM  A  J_N  SHOWN  KNOWN  BE  WERE  TO  BE  THE  IT  ENOUGH  AND  IN IN  COMPONENT,  BE  ALLOW SINCE  UNMEASURABLE  WOULD  BE  REACHED.  RESULTS  IN  EXPERIMENTS  OF  JE_J_  USING  IN  ENZYMATIC  NORMAL  COMPARED  36  VITRO  NO  PROTECTING  ANO TO  THE IN  DISAPPEARANCE CORRELATION  NOTED  THAT  CIRCULATING BEFORE _AL_  ACTIVITY  INCUBATIONS  THE  MEASUREMENT  LEVELS  WEST  TO  BIOLOGICAL  VIVO  SHOWED  SHOULD TO  OF  CONSIDERED  ULTIMOBRANCHIAL  RATE  FLGURE  THE  BE  THE  EXPERIMENTS  FROM  IN  TO  BINDING  RATES  IN  RESULTING  DATA.  OF  DECREASED  VIVO  SIGNIFICANT  SECOND  THE  THE  INCREASEO  ARE  LARGE  V I TRQ  TIME  DID  PORCINE IN  INACTIVATION  VARIATIONS  RESPONSE  THIS  OF  FROM  IN  RATES  EXPECTED  HOMOGENATES  PERHAPS  DISAPPEARANCE  RATS  IN  CT  INDICATED  LIVER  POSSIBILITY  RESULTED  A  RELATI 0NSHIP,  (37)  SINCE  OF  ENZYMES.  DECREASED  ZED  EFFECT  INACTIVATION  CASES  DISAPPEARANCE  RATES  FOR  CALCITONINS  NEPHRECTOMI  C T .  INACTIVATION  BOTH  THE  THAT  BY  IN  RATHER  THE  AJ_  CT  PLASMA.  DEGRADATION. TWO  JIT  IN  THE  PROTEINS  RAT  THE  OF  AND  THAT  MANY  CALCITONINS  FOR  LUISE  ADDITIONAL  AN  TO  CAUSE  SALMON  THAN  RESPONSIBLE SOURCE  DE  STABILITY  INDEX  RESPONSE  MECHANISM TO  THE  GOOD  DIRECT  RESULTS  MUCH  THUS  OF  RELATIVELY  DOSES THE  HORMONE  THIS  (130)  WITH  PART  OF  REPORTED LOW  DOSES  -69WHERE  HORMONE  LEVELS  CURVES  THEREFORE  RATES,  BUT  FROM  FOR  RATHER  THESE  DETERMINED  NO  INFORMATION  GIVE  COMPARTMENT  RATES  WERE  INDICATE TO  1  THAT  SUGGESTS  THAT  THE  BINDING  PROTEINS  IS  NOT  A  OF  THE  DATA  MAJOR  ABOUT  THE  FACTOR  DIFFUSION  RENAL  RATES  EXCRETION  SIGNIFICANTLY.  CALCITONINS  IN  THESE  INACTIVATION  THE  NOR  2  DIFFER  OF  BIOASSAY.  NEITHER  COMPARTMENT  CALCITONINS  BY  TO  DETERMINING  THIS  PLASMA  THE  AREA  RESPONSES. ALL  PROTEOLYTIC MINING  ENZYMES  THE  SIBLE,  DIFFERENT  THAT BUT  THESE  IT  THE  IT  APPEARED  OF  THE  THAT  THESE  PICTURE THE  ROLE  COULD  OF  THIS SINCE  NEER  WERE  THAT  CARRIER  BETTER  THE  OVERALL  WOULD IT  IN  OCCURED  MORE  SALMON  THE  HORMONE  TO  STILL  RESPON-  OF  BY  INACTI-  UNCLEAR,  BUT  CHARACTERISTICS BEFORE  THIS  ESSENTIAL  OBTAIN  A  STRUCTURE  TO  CLEARER  INFLUENCED  HORMONES.  INFORMATION  DIFFERENCES  RATE  THEREFORE  DETAIL  DETER-  INACTIVATION  ALSO  REQUIRED  WAS  GREATER  IN  BE  OF  WAS  THE  RESPONSE:TO  DIRECTLY  RATE  OF  IN  POSSIBILITY  THE  KNOWLEDGE  TOWARDS  FACTOR  NOT  PROTEINS  THE  BY  THE  OF  VARIATIONS  TO  THERAPEUTICALLY  CLEAR.  STUDIED.  OF  STABLE  ENZYMES  ENZYMES  TYPE  WORK  PHYSIOLOGICAL  OF  ENZYMES  RESPONSE  MAJOR  INDICATIVE  A  BE  HOW  OF  THE  A  POINTED  AS  CALCITONINS.  SEEMED WAS  ABOVE  PLASMA  PLASMA  DEGRADING  PROBLEM STUDY  THE  ENZYMES  VATION.  IN  TI ME . C O U R S E  STRUCTUALLY EXISTED  DISCUSSED  J£_T  AJ_  IN CT  THAN  (79)  MAN IS IS  AS  IS  PARTICULARLY  HAS  SHOWN  WELL  AS  POTENTIALLY PORCINE  CT.  IMPORTANT  THAT  THE MUCH  THE  RAT  AND  MORE  SINCE  RESPONSE  ONE  THAT  THE  VALUABLE OF  THE  MAJOR  -70V A L U E S  OE  D E S I G N I N G  C O M P A R A T I V E  OATA  MORE  ANO  A P P L I C A T I O N , WORK  OF  HUMAN  T A S H J I A N  I N A C T I V A T O R S A IN  LARGE  HUMAN  THE  S T A B L E  NUMBER  OF  AND  C A L C I T O N I N S .  THESE  ENZYMES  BEYOND  THE  ENZYMES  IN  SCOPE  WHICH  A P P E A R E D  KNOWN  STRUCTURAL  S P E C I F I C I T I E S WERE  THOSE  ENZYME  BE  MUCH  WERE  T R Y P S I N ,  PROTHROMBIN  ENZYMES.  MORE  CONFUSION  OTHER  MORE  PLASMA  AT  THOSE  OF  WORK  AMONG  AROMATIC  L E A S T  DAMAGING  TO  THE  WITH  F I T  A  SALMON  THE  R E A C T I O N P I C T U R E ,  AGENTS  P R O T E I N S .  THE  AND  IS  COULD  D I S U L F I D E  AND  KNOWN  OF  P E P S I N , WHICH  EXTENT  A  DEGRADING FOR THE  R E S I D U E S  ENZYMES ANO  BY  SECOND THE  B A S I C  THE  OCCUR,  R E D U C T I O N ,  R E D U C T I V E  TO  AROMATIC  THROMBIN)  WHICH  WAS  AS  HORMONE.  IN  A L L  D E S C R I P T I O N  SUCH A L L  ON  WITH  OF  THIS  E X I S T  ACT  OF  L I M I T E D  MORE  P L A S M A - K A L L IK R E I N S ANO  ROLES  ENZYMES  S P E C I F I C I T I E S  C  COULD  DEGRADATION  C H YM 0 T R Y P S IN ;  INVOLVED  TO  C O N F I N E D  R E S I D U E S  BE  KNOWN  C A L C I T O N I N S  TO  THE  THE  C A L C I T O N I N S  THE  TO  C L I N I C A L  E X P E R I M E N T S .  THEM THE  FOR  AT  TO  ARE  OR  PLASMA  E A R L Y  WAS  BETWEEN  FOR  HUMAN  OF  THE  F I N D IN G  S T U D I E S .  ENZYMES  TO  THAN  L I K E L Y  R E D U C T I V E  THE  E X P E C T E D  PLASMA  ANOTHER TO  ON  PATHWAYS  BE  (AUT0PROTHROMBIN  TYPE  OTHER  IN  THE  THESE  NUMBER  AND  P 0 S S IB L Y  C A L C I T O N I N S  C A L C I T O N I N S  AND  MIGHT  AND  WOULO  ENZYMES  STUDY  MAJOR  C L E A V E D  AND  AS  AS  WHICH  OCCUR  SUCH  THIS  D I F F E R E N C E S .  TO  OF  A  D I F F E R E N T I A T E  KNOWN  GROUP  AND  IN  D E S I G N I N G  I N A C T I V A T I O N  OF  ARE  MAMMALIAN  IN  TO  HORMONES  C H A R A C T E R I Z A T I O N OF  THE  TO  WHICH  CONVERTING  THE  (122)  P R O T E O L Y T I C  SERUM,  ACTED  WHICH  USED  V O E L K E L V A L U A B L E  A P P L I C A T I O N  A C T I V E  WAS  PROVED  PLASMA  I T S  MORE  PLASMA  AND  IS  FROM P L A S M I N  ADDING  EVEN  G L U T A T H I O N E  INTERCHANGE  I N A C T I V A T I O N  WITH  R E A C T I O N S  MAY  OCCUR  AT  0 I FFERENT  CONTINUAL  RENEWAL  IN  THE  v i vo.  PARTICULARLY DIMERIZE  THE  WORK  ROLES  OF  POINT  OF FOR  COMPLEX  OF  THE  SUCH  OF  AN  ASSAY IT  DISULFIDE  INTERCHANGE  WITH  IN  HUMAN  WOULD  WHICH  C T  THE  FACTORS  ON  FIRST FOR  PLASMA  IN  SINCE  BE  POSSIBLE  HAS  APPEARS  THE STEP  BEEN TO  ARE  PROVIDES OF  IN  TYPE  THIS  THE THE  IN  A  THIS  UNOOUBTABLY  OF  STUDY  PROBLEMS WERE  THE  IS  TO  PROPERTIES  INVOLVED  TRIED  NEXT  INACTI-  STARTING  INACTIVATING  WHICH  DESCRIBED  CHARACTERIZES  DIFFERENTIAL  DETAILS  ASSAYING  PROCEDURES  PARTIALLY  THE  AND  COMPONENTS.  THE  ADOPTED  CHAPTER  CALCITONINS,  STUOIES  AND  THIS  THESE  PROCEDURES  ASSAY  VITRO  OF  VARIOUS  PROBLEM.  EVENTUALLY  B.  SOME  VARIOUS  _J_N  ( 8 0 ) .  FURTHER  ESTABLISH  AND AGENT  DESCRIBED  THE  V I VP  REOUCTION  PROBLEM  RE A 6 IL Y  _I_N  THE  PREVALENT  THE  VATION  OF  RATE  IN  AND  SECTION.  PROCEDURES.  WAS  UNEQUIVOCAL  DECIDED  THAT  DATA  INACTIVATION  ON  ONLY  THE  BIOASSAY SINCE  COULD  PROVIDE  PROCEDURES  SUCH  AS  131 TCA  PRECIPITATION  CANNOT  BE  EXPECTED  CONDITIONS OF  THE  SHOWN  OF  THE  I  TO  TO  BE THIS  FRAGMENTS THE  EXPERIMENTS.  ONLY  THE  OF  THE  EARLY  FOR  ASSAY  TO FOR  CT  AND  THE  NOT  WHOLE WERE  OF  RADIOIMMUNOASSAYS  BIOASSAY  EXAMPLE  CALCITONIN  PORTION  COULD  ACTIVE  INACTIVATION  THE  ONE  PROCEDURE  AND  VARIABILITY IN  FOR  LABELLED  CORRESPOND  RADIOIMMUNOASSAYS  CLEARLY  OF  OF  THE  THE  HAVE  MOLECULE  THUS  COMPOUNDED  BY  THE  THE  BEEN  ( 1 8 ,3 4 ,  BETWEEN  MOLECULE.  THE  SPECIFICITIES  STUDIED  DISTINGUISH  UNDER  INACTIVE  PROBLEMS INHERENT  BIOASSAY. STAGES  OF  THE  EXPERIMENTS  4 0 ) .  ATTEMPTS  WERE  MADE  TO  ALLOW  DEVELOP  QUANTITATIVE  DETERMINATION  SAMPLE  AND  PERMIT  FICATION  STAGES.  POSSIBLE  TO  VELOCITIES INVOLVEO  FOR WERE  NECESSARY  WITH  THAT  COLUMN  INSTABILITY  THIS  OF  BECAUSE THE  OF  PH  5  COULD  BE  RETARDED  WHILE  IN  BUFFER  THE  CAUSED  COMPLETE  VATION  WAS  SMALL  TYPE  OF  OR  NO  IN  THE  OF  CT  SUITABLE  WAS  IT  SERUM  INACTIVATION  IN  OR 4  AT  QUANTITIES  PROTEINS  OCCURED  COLUMN  APPEARED  MATERIAL A  OF  A  SERUM  IN  THE  ELUATE TO  A  FOR  SURVEYING  BECAUSE  OF  IN  FOR  SODIUM  HOURS  AT  WERE  BRIEFLY, RAPIDLY  OF  OTHER  EXAMPLE, ACETATE  OF  STUDY  BUFFER INACTITO  1 11OO  1 :1000.  THIS  AND  LED  CONTAINING  INACTIVATOR  FOR  THAT  THE  PROTECTIVE.  PRESENT  MORE  IN  CHARACTERIZED  3 7 ° C,  DILUTIONS  THE  INACTIVATION  VARIETY  FOUND,  ASSAY  BECAME  OCCURRED  UNSATISFACTORY  SEMI-QUANTITATIVE  WERE  SITUATIONS.  THE  FRACTIONS  CONTAIN  CLEARLY  OF  THAT OF  VERY  IT  ( 1 ) HAVE  INACTIVATION AND  PROCEDURES  CONCENTRATION  NUMBER  PRESENCE  IN  AND  SCHLUETER A  THAT  NOT  PURI-  WAS  INCUBATIONS  BIOASSAY.  LOW  THE  REQUIRED  FOR  AT  IT  A  MAXIMUM  MATERIALS.  COMPLEXITY  ANO  SOLUTION.  WHERE  EVENTUALLY  ITS  AND  ESTIMATE'  ANO  IN  SUBSEQUENT  SUCCESS  ANO  WOULD  PRESENT  IN  NUMEROUS  INSIGNIFICANT  PROTEIN  STARTING  WAS  SOLUTIONS  BY  PHENOMENON  SITUATIONS  SINCE  CALCITONINS  INDICATED  INCUBATION  THUS  OF  INACTIVATION  ABOVE  SOME  HOWEVER,  SAMPLES  ALDRED  RESULTS  RECOVERY  STARTING  COMPLEX  WHICH  INACT I VATOR  WITH  THIS;PROCEDURE  OF  IN  THE  FREQUENT  THEIR  PROTEINS  OF  OF  PROCEDURES  CONSTANTS  BIOASSAYS  SOLUTIONS.  TYPE  TOTAL  MET  RATE  SOME  ELUATES  BUFFER  THESE  QUITE  USE  THE  ESTIMATION  MEASURE  EXTENSIVE  EVIDENT  OF  ASSAY  TO  LITTLE THAN  RESULT.  INACTIVATOR  WAS  THE  -73DEVELOPED  WHICH  WAS IT  CONCENTRATION. BUFFER  INCUBATION  OUT  TO  THE  VESSEL  SEE  PROTEINS  IF  THIS  SEEMED  SUITABLE  THE  IN  VESSELS  GLASS,  FACT,  ACID  DETERGENT  FACE  MIGHT  VESSELS WATER pH  7.4-  SINCE  LONGER  WAS  BY  MODIFYING  THE  PROTECTIVE  ENZYMATIC APPROACH  A  GLASS  GREATER  RATE  EXCHANGER,  AND  GLASS  A  TO  INCUBATED  UP,TO  TWO IF  SLOW  AND  UNSUITABLE.  GLASS  PRIOR  NOT  INACTIVATION  EQUALLY  AT  WAS  THE  BE  OF  BLOCKED  INACTIVATION.  FROM  WALLS  THAT  COULD  THIS  CARRIED  RAPID  HYDROXIDE  PERIODS  THE  IN  A  DISTILLED 37°  AT  HOURS THE  SUR-  C  IN  IN  THESE  INCUBATIONS  INACTIVATION  DISULFIDE  INTERCHANGE  AT  PH. INCUBATION  RELATIVELY CONTAINED OF  NO  TO  SILICONIZED  PERIODS  RESULTING  PERIOD  RECORD  FOR  FOR  30  TIONS  CATION  FACTOR  WERE  THIS  MORE  PROTEIN  TESTS  WITH  SUGGESTING  CT  OR  A  CT  IN  EXTRANEOUS  INACTIVATED  SODIUM  BUFFER  RESULTED  THIS  M  A  TRIED.  FRACTIONS MINUTE  AS  BE  OF  GLASS  GLASS  LITTLE  POSSIBLY  HIGH  2  IN  WERE  EXTENDED  OCCUREO THIS  ACTING  WASHED  WITH  GLASS  OF  MEASURED  CAUSE  WASHED  WASHED  COULD  MAJOR  THE  SERIES  PURPOSES.  TO  A  OF  INTERFER  BEING  PRESENT  ACID  TRIS-HCL  VESSELS WERE  BE  RINSE  WAS  WASHED  THAN  A  VARIATIONS  THAT  ADDITION  TO  PROVED  ANO  AND  ADSORPTION  LIKELY  WHICH  LIKELY  TO  ADSORPTION  SlNCE  INACTIVATION  PLASTIC  WAS  VESSELS,  WALLS.  FOR  SENSITIVE  SEEMED  INACTIVATION  THE  THAN  NOT  AT  IN  C  THE  MAJOR  REDUCTION  AND  PH  PROCEDURE  SIGNIFICANT  QUANTITATIVE  WITH  MARKED  37°  SIMPLE  CT  OF  7.4,IT TO  WAS  ACTIVITY  WAS  INDICATE  INACTIVATOR  YIELDS  IN  INACTIVATOR  POSSIBLE WHICH  ALTHOUGH  POSSIBLE.  IN  IN  NO  A TO  USE  FRACDETAILED  GENERAL,  -74ALIQUOTS (ARMOUR WERE  MIXED  ONE  HOUR  WITH  WERE  CONTAINING Co.  AT  EQUAL  VOLUME  TO  7.4  PH  37°C  II.  Two  SAMPLES,  USED  WERE  AND  FOR  A  TWO  CALCULATED  THE  TIME  ACTIVITY  WAS  OF  BIOLOGICAL  ACTIVITY  REMOVED  OF  THE  INACTIVATOR  PROCEDURE  WERE  FOR  INCLUDED  C.  P A R T I AL  1.  S E L E C T i O N QF S T A R T i N G THE  OF AND  PORCINE  CT  OF  THESE  AND  NOT  C T AND  THAT  THIS  SALMON HORMONE  PROBABLY  PHYSIOLOGICAL ZATION  OF  IN  OF  FACTORS THE  FIRST.  ONE IN  OF  THE  USED  FOUR  EACH  GROUP..  PERCENTAGE AS  OF  THE  WITH  BIOLOGICAL  AN  INDEX  THIS  DETAILS  PURIFICATION  WILL  STAGES.  " C A L C I TON I N A S E " .  TO  FAILURE  THIS COULD  SALMON  ( 1 2 2 ) SHOWED  I N HUMAN FACTORS.  THIS  PARTICULARLY  CONTRIBUTED  EFFECT  THESE  DIFFERENCES  VOELKEL  THESE  INTRODUCTION  WERE  AND  MATERIAL.  TASHJIAN  FACTORS  INCUBATED  ASSAYED  VARIATIONS  THE  I N A C T I V A T I N G FACTORS  I N THE  AND  AND  TRIS-HCL  DESCRIBED  THE  CT  ELUATE  TIME  WERE  HOUR  ASSAYS  M  REMAINING  100$  O F JA S E L E C T I VE  PARTIALLY CHARACTERIZED  DESCRIBED  SLIGHT  AND  RESPONSES  I N ONE  D E S C R I P T I O N S OF  P U R I F I C A T I ON  WORK  AS  PORCINE  COLUMN  AS  OF  THE  OF 0.1  ZERO  SAMPLE  AND  TAKEN  VARIOUS  BE  AT  BIOASSAYEO  AVERAGE  PRESENT.  USED  IN THE  THE  7.4,  BASE  DILUTION  ACTIVITY ZERO  FROM  TRIS  TAKEN  DILUTION  E A C H  U/ML  VARIOUS  EACH  FOLD  MRC  I N PH  OF  WITH  D I L U T I O N S OF  USUALLY  2.5  AL0831)  SUITABLY DILUTED  SECOND  RATS  AN  ADJUSTED  HOUR  CHAPTER THE  SOLUTION  PHARMACEUTICAL.  FRACTIONS FOR  A  OF  TO  EFFECTIVE  SERUMS  INDICATED  TO  FURTHER  THE  SALMON ENHANCED  CHARACTERI-  IN D E F I N I N G THE RESPONSIBLE  THAT  IN I N A C T I V A T I N G  I N A C T I V A T E THE  HORMONE.  MOLECULE  RAT  PRESENCE  T H E WORK  CHAPTER  SIGNIFICANTLY  AID  AND  THE  FOR  STRUCTURAL THIS  -75R E SI STANCE THAT  THE  FORM  SO  TO  THAT  ONLY  ONE  ALLOWING  DETERMINE  VAST  SUCH  I N A C T I V A T I N G ENZYMES  INCUBATION TO  INACTIVATION.  NUMBER  THE OF  TYPE  BE  OF  CHARACTERIZATION OBTAINED  COMPONENTS  IN R E L A T I V E L Y  I N A C T I V A T I O N OCCURED  IDENTIFICATION  SPECIFICITY  OF  OF  THE  PRESENT  THE  ENZYME.  IN SERUM  OF  STARTING  MATERIAL  YIELD  SIGNIFICANT  PURIFIED  PRODUCT  COMMERCIALLY  USED  STARTING  PLASMA  FRACTIONS  IT  FIRST  WAS  UNCLOTTED  PLASMA  ABILITIES  SINCE  ENZYMES  WERE  SAMPLES  OF  PREPARED AT  37°C  SAMPLES  PREVIOUSLY  75  (±S.E.M.).  SAMPLE LOWER  OF  ±0.1  MG$. ±0.1  THAN  THE  ZERO  THERE  MRC  A  THE  THE  OF  MIN.,  MIN  TIME  TIME  BOTH LEVEL  BETWEEN  DIFFERENCE  IN  THE  THAT  CLOTTING TO  TEST  THIS,  WERE  INCUBATED  FROM  THE  INCUBATION  B IOASSAYED  THE  VALUE  TWO.  PREPARED  AND  INCUBATION  THE  TO  CT  SAMPLES  WERE  (P<.005),  NEEDED  PLASMA  CALCIUM  VALUES  BE  THE  RABBIT  I N J E C T E D WITH  RELATIVELY  INACTIVATING  WERE  PLASMA  ZERO  75  AND  THE  MATERIAL.  THAT  TAKEN  OF  DEMONSTRATE  PORCINE  WERE  MEAN  WOULD  INACTIVATION.  CORRESPONDING  MG$.  LITTLE  SIMILAR EX I STED  U/ML  BOTH  RATS  TO  HEPARINIZED  75  THE  DIFFERENCE WAS  AND  THE  FIVE  9.3  HAD  FOR  AND  AFTER  LEVEL  HOWEVER,  ALIQUOTS  TIME  WAS  PRESENT.  20  I N J E C T E D WITH  SIGNIFICANT THAT  SERUM  DESCRIBED.  MG$  AS  P O S S I B I L I TY  MIN.  ZERO  RATS  9.2  THE  SERUM  RESPONSIBLE  TEN  WAS  AND  CONTAINING  AT  CALCIUM  NECESSARY,  RABBIT  FOR  WERE  WHICH  THE  QUANTITY  GIVEN  PRODUCTS  BECAUSE AND  PURE  IN A  REACTION  LARGE  COHN  RE Q U I RE 0  BUT  LEVEL  THE ±0.1  MEAN  PLASMA  SERUM FOR  OF  8.5  WAS  THE  AS  SAMPLE  THE  PLASMA  SIGNIFICANTLY THERE  VALUES, AMOUNT  WAS  NO  INDICATING OF  INACTIVATOR  TABLE IX  INACTIVATION OF PORCINE C T DURING INCUBATIONS WITH HUMAN PLASMA FRACTIONS  INCUBAT I ON SOLUTI ON  PLASMA CALCIUM LEVELS ZERO TIME  BUFFER ONLY  1 0 . 2 + 0.1  LYOPHILiZEO PLASMA  8.4 + 0.1  +S.E.M.  (MG$) 3 HOURS  (5)* (5)  9.1  + 0.1 ( 2 )  COHN I  8.3  + 0.1 ( 2 )  COHN I I  8.3  + 0.1 ( 2 )  COHN I I 1-0 COHN I I I  10.1  8.5  + 0.1 ( 2 ) + 0.1 ( 2 )  COHN IV-1  1 0 . 4 + 0.1 ( 2 )  COHN I V - 4  1 0 . 7 + Q.1 ( 2 )  COHN V  *  NUMBER OF ANIMALS INJECTED  8.8  + 0.1 ( 2 )  - 7 6 -  COHN ICAL  FRACTIONS  CORP.,  ADDED  TO  PH  FOR  PLASMA  AS  LISTED  IN TABLE  7 . 4 , 0.1  TRiS-HCL  BUFFER  IN  THOSE  WHICH ANY  3 0 MIN.  CT.  AND  THE  M  THE  EXIST  INSOLUBLE  SAMPLES  WERE  MRC  OUT  MADE  PLASMA VARY IN  OF  AND  TIME  CALCIUMS  TO  INCUBATED AND  AFTER  AT 3  3 7 C.  HOURS  OF  I N J E C T E D AT  SIGNIFICANTLY  THE  DATA  WAS  RED I S S O L V E D  LYOPHILIZED  MARKEDLY  SEVEN  WERE  PRESENT  THAN  ONE  COHN  AND  PRESENT  I N MORE  SAMPLES  OF  WAS  THAN  ONE  THREE  WAS  SLIGHTLY  MORE  AND  THIS  FRACTION  WAS  2.  PRELIMINARY THE  IV-1  INSOLUBLE  WITH  THIS  IV-1  WAS  A  LARGE  COULD  BUFFER. EXTRACTED  PLASMA  ZERO THE  OF  SAMPLE CT  BECAUSE  3  CT  HOURS  IN  THREE  THAT  MORE  ENZYME  TESTS  INACTI VATING FURTHER  OF  BECAUSE  SINGLE  FURTHER  ON  WAS  DILUTED  FRACTION  P0RC I NE  CT  WORK.  EXPERIMENTS. IN  THE  PREVIOUS  PERCENTAGE  OF  IN  THE  BUFFER.  BE  ACHIEVED  IN  AN  FOUR  A  POTENT  FOR  NOT  INACTIVATORS  INDICATED  CHOSEN  DID  REMAINED  FRACTIONS IN  THE  AFTER  EITHER  CARRIED  AS  AMOUNT  FRACTION OR  WERE  TIME  THAT  FRACTION.  EXPERIMENTS  THAT  PURIFICATION  WITH  CONCENTRATION  INDICATING  ONE  PURIFICATION  OBSERVED WAS  A  POOLED.  MEASURABLE  INVOLVED  THAN  THESE  NO  FRACTIONS,  I N MORE  ENZYME  DURING  BY  INCUBATIONS  INCUBATION,  RATS  THE  REMOVED  BIOASSAYS  THE  REDUCED  IV-1  FOR  OF  THE  WAS  ZERO  UP  WAS  AND  0  MU/ML,  AT  USED  I X WERE  (SEE 9 2 ) ,  RESIDUE  SUPERNATES  BIOCHEM-  CONCENTRATIONS  IN PLASMA  ' 20  (NUTRITIONAL  OHIO)  CENTRIFUGATION PORCINE  HUMAN  CLEVELAND,  APPROXIMATING SHAKEN  FROM  PH  7.4 BY  INITIAL TIMES  THE  SECTION  MATERIAL THUS  EXTRACTING EXPERIMENT  WITH  1 0 ML  IN  A  G  WAS  FRACTION  CONSIDERABLE  THE 1  IT  FRACTION OF  ALIQUOTS  FRACTION OF  BUFFER.  FIGURE  37.  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 C O H N F R A C T I O N I V - 1 ON S E P H A O E X G-200. C O L U M N , 2.5 x 9 0 C M ; E L U A N T , P H 7 . 4 , 0.1 M T R I S - H C L BUFFER; TEMPERATURE, 4 ° C; F L O W R A T E , 2 0 C M / H R ; F R A C T I O N S I Z E , 6.8 M L . /OCT R E M O V E D 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 . C  -77AFTER  ADOITION  SHAKEN  FOR  SAVED.  AN  PORCINE  C T AS  VATION  30  MIN  TWO  TWO  SAMPLES 3  ML  FOLLOWED THE  AS  THE  COLUMN  ONLY  ONE  THE TO  THOSE  TWO CASE  SIMILAR.  MU/ML MRC  OF  FOR  WAS  B  THAT  TASHJIAN  THE THE  THE  THIS AND  OBSERVEO  MOLECULAR LARGER  FIGURE  VOELKEL  CT  OF  THE  WERE  HAD  D  NM  AS  TO  PACKING.  IN FIGURE  37.  INDICATED AS CONTAINED  MIXTURE  ACTIVITY ELUTION  I N MANY  LESS  280  ANO  FROM  WAYS HUMAN  INACTIVATORS K  M  APPLIEO  INACTIVATORS  THE A  AT  VALVE  WHOLE  0.1  ML  SOLUTION  CT  ABOVE  CONTAIN  FOR  INACTI VAT0R  ( 1 2 2 ) ON  OF  WAS  INCUBATION  PERCENT  FIRST  IN 7.4,  POOLS  FOR  IN  ADJUSTABLE  ABSORBANCE  FIVE  FINAL  COMPONENT  TAKEN  SEPHADEX  THE  THE  I N 6.8  I S SHOWN  THAT  WEIGHTS  WAS  THREE-WAY  EXPERIMENT  TO  ONLY  SAMPLE  THE  INACTI-  MARKEDLY  SEPHADEX  THE  ASSAYED  THE  IN  A  INTO  THE  CM  THE  COLUMN  EXCEPT  MU/ML.  OF  AND  ITS  WITH  PURIFICATION.  COLLECTED  PREVENT  POOL  INCUBATED  EQUILIBRATED  WAS  DIVIDED  EACH  X 90  THROUGH  THE  THAT  WAS  SUPERNATES  DROPPED  MATERIAL  G-200  TO  THEN  FURTHER  PROTEIN.  COLUMN  SO  2.5  ML/HR  20  OF  I S SHOWN  WERE  A  ELUATE  UPWARDS  ANO  RESULTS  REGIONS AND  THE  FOR  THE  DETERMINE  DECIDED  POOLED  ON  OF  INDEX  IN S E C T I O N  20  C  THE  PROFILE  HOUR  I T WAS  SEPHADEX  RATE  WAS  MRC  CONTAINED IN  A  FIGURE  40  AND  4°  WAS  TO  MIXTURE  AND  I N A C T I V A T I O N RATE  THE  ELUATE  DESCRIBED  DESCRIBED  OF  AN  FLOW  ELUTION  THE  SUPERNATE  SAMPLE  OF  THE  IN  EACH  POOLED  AT  BOTTOM  ELUANT  OF  BE  BUFFER.  THE  CENTRIFUGED  SHOULD  AT  ALIQUOT,  THEN  THE  CONTAINING  FRACTIONS  ML  AND  SAMPLES  CHROMATOGRAPHY  TRIS-HCL  10  PREVIOUSLY  ACTIVITY.  LAST  COLUMN  EACH  ALIQUOT  THE  A  OF  THAN  IN  REMOVED PROFILE. SIMILAR SERUM. EACH  WERE 0.3  IN  -78EACH  CASE  0.7.  THESE  100,000 BE  BE  TO  RULED  OTHER  INDICATE  LESS  THAN  INDICATE  OUT. TO  IN  ENTIRE  ONTO  A  2  EQUILIBRATED ELUATE  X  20  GRADIENT  16.0  NM THE  BUFFER, WAS  THIS  BE  USED  EXTRACTION THE FRACTIONS  TO  CANNOT  RESPONSIBLE  POSSIBILITY  FIRST  THAN  THAN  DATA  WERE  SINCE  CANNOT  IT  THAN  THE  ABOUT  FRACTIONS  REMOVE  A  OF  THIS TO  COMPONENT  CONDITIONS  TO  WHICH  ML)  50  OF  THE  WERE  THE  ALLOWED  MOST  0.5  WAS  THEN  THIS  7.4;  RESIN:-  7.4;  APPLIED  COLUMN  MATERIAL  ML/HR.  10  (PH  M  BEEN  THE  (PH  M  TO  HAD  AND OF  0.1  TO  THE  STEP  IN  NONE THE  ABSORBING  AS  A  OF  BROAD  AT PEAK  DEAE-SEPHADEX  THAT  FOLLOWING  THE  CONTAMINANTS.  POOLED  CM-SEPHADEX  WHICH  RATE  REMOVED  FROM  CONDITIONS  PROVED  OF  SUGGESTED  OF  A  ML)  ON  ADSORPTION  COLUMN  DEFINE  50  REMAINED  NUMBER  WAS  BUFFER  FROM  CONDITIONS  RESULT  BULK  AT  BUFFER  VOLUME,  50$  B  TRIS-HCL  SUBSEQUENTLY  THIS A  POOL  DEAE-SEPHADEX  M  INACTIVATING MATERIAL  EXPERIMENTS ACTIVE  IN  0.1  OF  VOLUME,  MMHOS;  A D S O R B E D AND  GRADIENT.  OF  TRIS-HCL  MMHOS;  BUT  VOLUME  ML  INACTIVATING ACTIVITY  COULD  THE  THIS  INACTIVATING ACTIVITY  COLUMN  UNDER  COLUMN.  INITIAL  BY  GREATER  COMPONENTS  BUT  GREATER  Q  WEIGHTS  INVESTIGATED  7.4,  OF  CONDUCTIVITY, THE  CM  I N 6.8  3.5  280  MORE  PH  WITH  CONDUCTIVITY,  THE  SAME  CASES,  WAS  K 'S  HAD  RESPECTIVELY.  THE  REMAINING  COLLECTED  LINEAR  TO  THAT  B  CONTAIN  MOLECULAR  30,000  BOTH  POOL  COMPONENTS  POOLS. THE  FLOW  SMALLER  K *S  INACTIVATION  APPEARED  A  THE  O  AND  TAKEN  FOR  WHILE  FOR FOR  THE AND  INITIAL USED  IN  ADSORPTION FURTHER  SUCCESSFUL  ELUATE A OF  SERIES THE  PURIFICATION. WERE  USED  IN  OF  SUBSEQUENT SCHEDULE  3.  EXPERIMENTS IN THE  PREPARATION A  OF  PH  ROOM  5  G  0.1  M  WHICH  THIS  OF  HAD  TRIS-HCL ROOM  AND  SEPHADEX  WASHED  BUFFER.  THIS  THE  TWICE  WAS  WATER,  FILTRATE  THE  BY  IDENTICAL  EXCEPT  THAT  ML  TO M  WEIGHT  PEAK  WHICH  WELL  ANO  THE  SALT,  AND  WAS  THE  ABOUT 10  SAMPLE  AND  OF  20  ONE  OF  STIRRED  PH  7.4»  FILTRATES 300 ML  ML  WAS  EXTRACT  ML AT  CLEAR  WAS  AGAIN.  WAS  REPEATED  SUPERNATES.  DEAE-SEPHADEX PH  7.4»  FOR  0.1  WITH  M  AT  THE  TRIS-HCL ML  OF  POOLED.  REDUCED USED  M  HOURS  AND  50  WERE  0,1  TWO  FILTERED  WASHES  A  RESIDUE SHAKEN  30  HOUR  YIELD  POOLED  G  THEN  OF  WAS  APPLIED  DESCRIBED ACID  WAS  COMPONENTS  SEPARATED  THE  THE  WITH  TO  FOR  THE  10  ML  FURTHER  CHROMATOGRAPHY.  FORMIC  MOLECULAR  OF  THIS  THAT  HIGH  WAS  WAS  TWO  AND  TO  E Q U I L I B R A T E D WITH  MIXTURE  OF  ML  TO  BY  10  COLUMN  ADDED  FOLLOWED  COLUMN  ENTIRE  80  MIXTURE  ALL  FOR  C E N T R I F U G A T I ON  AND  ML  VOLUME  0.1  BUFFER  50  AND  MIXED  SHAKEN  OF  WITH  LYOPHILIZATION,  PURIFICATION  THE  WAS  CENTRIFUGED  YIELDING WERE  AND  SAVED.  ML  PREVIOUSLY  TEMPERATURE.  TOTAL  30  IV-1  AND  RESUSPENSION  BUFFER  DISTILLED  THEN  REMOVED  SUPERNATES BEEN  BUFFER  IT WAS  TIMES  "CALCITONINASE".  FRACTION  TRIS-HCL  THREE  POOLED  WHICH  OF  I N T H E I PUR.T F I C A T I ON  DESCRIBED  PURIFIED  COHN  IN ANOTHER  PROCESS  TOTAL  BY  OF  WAS  ARE  SECTION.  HIGHLY  TEMPERATURE.  RESUSPENDED  THE  OF  SAMPLE  7.4,  SUPERNATE  A  NEXT  AND  COLUMN  IN THE USED  CAME  FROM  THE  FRACTIONS  AS OFF  TO  A  SEPHADEX  G-200  P R E C E D I N G S E C T I ON AN  ELUANT.  IN A  SMALLER WERE  THE  SINGLE  BROAD  COMPONENTS  DIVIDED  INTO  TWO  —- lOOn  0  FIGURE  38.  80  Fraction  160  Number  240  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 CM-SEPHADEX C - 2 5 . 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 P H 5.0 AMMONIUM ACETATE FROM 0.1 M TO 0 . 5 M A N D F R O M 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 M L .  -80P 0 0 L S .  THE 20  POOL  CONTAINING  THROUGH 4 5 ,  WHEN  THE  FIGURE  SEE  THIS SOLUTION  WAS  FOR  APPLICATION  CLOUDED.  THE  SUSPENDED  SAMPLE  A 2  X 2 0 CM  0.1  M AMMONIUM  AND  THE  ELUTED  0.1  FROM  0.5  TO A  COLUMN  M  COLLOID  0.1  OF  5.0;  LINEAR  NEXT WAS  AT  THE  APPLIED  ON  WITH  COLUMN BUFFER  THE  WAS AT  PH  COLUMN  ACETATE  TO  5.0  WAS  BUFFER  MMHOS; V O L U M E , 2 5 0  ML)  1 6 . 0 MMHOS; V O L U M E , 2 5 0 M L ) .  CONDUCTIVITY, OF  ACETATE  3.5  BUT  WAS  THE  AMMONIUM  CONDUCTIVITY,  GRADIENT  5.0.  ML.  SOLUTION  EQUILIBRATED  ADSORBED  OF  THE  COLLOID  170  OF  AMMONIUM  REMOVED  C-25  M AMMONIUM  REMAINED  WITH  COLUMN  NOT  PH  (FRACTIONS  VOLUME  PH 5 . 0  SUSPENDED  A LINEAR GRADIENT (PH  A TOTAL  TO  THE  BUFFER  WHICH  M (PH5.0;  SECOND  HAD  COMPONENTS  CM-SEPHADEX,  OF  ACETATE  MATERIAL WITH  TO  I N C L U D I N G THE  WITH 2 5 0 ML  WASHED  37)  AOJUSTED  HYDROXIDE  ENTIRE  LARGE  AMMONIUM  ACETATE  BUFFER  FROM  0.5  M  (PH 5 . 0 ;  CONDUCTIVITY,  1 6 . 0 MMHOS; V O L U M E , 2 5 0 M L )  TO  1.0  M  (PH  CONDUCTIVITY,  2 6 . 0 MMHOS; V O L U M E , 2 5 0 M L )  WAS  EMPLOYED WAS  TO  AND  DESCRIBED. SAMPLE  TO  BIOLOGICAL  IN  FRACTIONS THE  CT  2.0  C O M P O N E N T S WERE  FRACTIONS  AT  T H I S COLUMN WERE  POOLED  A RATE  ARE  MRC  REMAINING THE  EACH  AFTER  PERCENTAGE POOL.  1.0  OF  POOL  REMOVED.  IN F I G U R E  ASSESSED ADDED  MRC  3 0 MIN  ACTIVITY  ELUATE  CF 2 0 M L / H R .  S I X REGIONS  C T WERE  PORCINE  CONCENTRATION  ACTIVITY  FOR  U OF  SHOWN  I N THE  INACTIVATING ACTIVITY  A FINAL  FIGURE  ALL  ML  FROM  C DETERMINED. THE  THAT  IN 6 . 8  RESULTS  ELUATE  CATED  37°  INSURE  COLLECTED THE  THE  5.0;  AS TO  L)/ML, OF  INDIPREVIOUSLY  EACH AND  THE  INCUBATION  REMOVED  H CONTAINED  38.  THE  AT  I S SHOWN MAJOR  J  0.4  K  L  i  Fraction  FIGURE  M  39.  ELUTION PROFILE SEPHADEX G-200. OTHER  CONDITIONS  Number  OF C M - S E P H A D E X PRODUCT ELUANT, 0.05 M ACETIC AS  INDICATED  I N FlGURE  ON ACID; 37.  -81-  INACTIVATOR ACITIVITY ANO WAS SELECTED FOR FURTHER PURIFICATION.  THE ADJACENT POOLS G AND I ALSO CONTAINED SOME  INACTIVATOR, BUT IT SEEMED LIKELY THAT THIS WAS DUE PRIMARILY 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-200  AS PREVIOUSLY  DESCRIBED BUT USING 0 . 0 5 M ACETIC ACID AS AN ELUANT. ELUATE WAS COLLECTED IN  6.8  THE  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  40  SUMMARY OF PURIFICATION PROCEDURE FOR "CALCITONINASE"  COHN FRACTION IV-1 (5G) EXTRACTED WITH PH 7 . 4 , 0.1 M TRIS HYDROCHLORIDE RESIDUE (DiSCARDED )  'SUPERNATE CONTAMINANTS ABSORBED ON DEAESEPHADEX IN PH 7 . 4 , 0.1M TRIS  F l LTRATE'  ^RESIDUE (DiSCARDED)  LYOPHILIZED TO 1 0 ML VOLUME FINAL EXTRACT MOLECULAR SIEVING ON SEPHADEX G - 2 0 0 IN 0 . 1 M FORMIC ACID POOLED HIGH MOLECULAR WEIGHT COMPONENTS ION EXCHANGE ON CM-SEPHAOEX, PH 5 . 0 POOL H LYOPHILIZED 50$*  ^  50$  SAVED  MOLECULAR SIEVING ON SEPHADEX G - 2 0 0 IN 0 . 0 5 M ACETIC ACID POOL M FINAL PRODUCT ( 1 6 . 5 MG)  -82FROM  THE  PRECURSOR  C I T R A T E  S O L U T I O N  OCCURED  IN  R E S U L T E D  THE  FROM  BY  I N C U B A T I O N  (111).  INVOLVED  SCOPE  STUDY  C A T I O N  T H I S  WOULD  G E N E R A T E D  DECIDED  EXAMINED  WAS  FRAGMENTS  COULD  BE  P U R I T Y  AND  BUT  NO  FOR  USE  THUS  CONTAINED  G  MG  OF  P R O T E I N  MG  OF  M A T E R I A L  OF  COHN  P U R I F I C A T I O N .  FIGURE  40  O U T L I N E S  T H E ; M A T E R I A L  FURTHER  S T U D I E S .  IN  FRACTION  IV-1  OVER  THE  FRACTION  T H I S  WEIGHT  E Q U I V A L E N T  R E P R E S E N T S  F R A C T I O N  THE  F I N A L  FROM  TO  AS  R E P R E S E N T S  PRODUCT  TOTAL  THE  M  MG  USED  OEGREE  1 50  A  OF  THE  THE  IT  WAS  OF  THE  D E G R E E  WERE  A T T E M P T E D .  S A M P L E S  TH I S  ML.  FROM  OF  POOL  THE  I V - 1 .  OF  P U R I F I C A T I O N  WAS  USED  M A T E R I A L  FOLD  ONLY  6$  A  FOR  R E D U C T I O N  OF  P U R I F I C A I O N ,  TO  ALL  G  IN  OF  WEIGHT  M A T E R I A L ,  BUT  THE  P L A S M A  2500 T H I S  USED  2.5  FROM  S T A R T I N G  P R O T E I N .  THE  S P O N T A N E O U S L Y  ENZYME  P R E P A R E D  WHICH  OF  OF  OF  P U R I F I -  S T U D I E S .  PER  R E P R E S E N T S  P L A S M A  FURTHER  L Y O P H I L I Z E D  S T A G E S  POOL  16.5  T H E  COHN  COHN  THE  WHICH  BEYOND  M A T E R I A L  WAS  FRACTION  OF  PREPARE  OF  AND  WAS  P R O D U C E D .  C O M P A R A T I V E  ML  SUMMARY  THAT  NUMBER  P U R I F I C A T I O N  110  0.15  16.5  2.5  IN  S O L U T I O N  SYSTEM  R E P R E S E N T E D  M  C H A R A C T E R I S T I C S  CONTAINED  OF  POOL  FURTHER  Y I E L D E D  E Q U I V A L E N T  THAT  SOME  POOL  P R O T E I N  L I K E L Y  I N A C T I V E  T H I S  SINCE  OF  A  M  IN  IT  TYPE  R E L E A S E  E L U C I D A T I O N  H.  S I N C E  POOL  4.  T H I S  TYPE  A C E T A T E  POOL  SODIUM  (w/v)  S I M I L A R  D I F F I C U L T  THEREFORE  P U R I T Y  AND  A  PROVE  S U F F I C I E N T  OF  IN  OF  25$  A  AMMONIUM  I Z A T I ON  R E L A T I O N S H I P S  OF  P E R H A P S  C O N C E N T R A T E D  L Y O P H I L  IN  TOTAL  FOLD  IS  BUT  R E D U C T I O N  NOT  IT  IS  N E C E S S A R I L Y  THE  B E S T  -83ESTIMATE  AVAILABLE SINCE  INACTIVATOR  5.  TESTS  WAS  QUANTITATIVE  OF  POOL  ACETATE  DETERMINE  THE NUMBER  CELLULOSE  M  WERE  STRIPS OF  ACETATE  SUBJECTED  A N D ON  STRIPS  INSTRUMENT  CO.,  DARD  HIGH  SAME  SOURCE.  WERE  APPLIED  SOLUTION TCA  AND  PLASMA  ANN ARBOR,  RESOLUTION  WAS  NEAR  360  CONTAINING DESTAINED  SAMPLES  25  AND  THE STRIPS 0.2$  WITH  (w/v)  FROM  >X  G  0  ALLOWED WERE  TO  THEN  I N 5$  S  P O L Y A C R I L A M IDE  GEL ELCTROPHORESIS  WAS  BRATED  IN APPENOIX  WITH  0.01  M  F.  ACETIC  2 NG/^L OF POOL M M A T E R I A L ACID  WAS  APPLIED  ZONE  SHARPENED  PHORESES  WAS  THE  WERE  GELS  DESCRIBED TWO  TO  ACID IN  THE TOP OF  F O R 25  THEN  AFTER  MIN  CARRIED  REMOVED  FROM  THE GELS  OUT  THE TUBES  THE  I N 5 RL  I N 5$  CARRIED  HAO  BEEN  IN  THE G E L AND  A T 320  FROM  STAN-  FOR IN A (w/ V ) HUMAN  CONTROLS.  1 M SUCROSE  VOLT  WAS  ACID.  A 10 j^L S A M P L E  I N A 32  GELMAN  DEVELOPED  ACETIC AS  GELMAN  MIGRATE  S.TAIN  RUN S I M U L T A N E O U S L Y  TO  A  PROTEIN  F  WERE  DESCRIBED  THE  AT P H 8.6  PONCEAU  3 WASHES  USING  THE B U F F E R  BUFFER  CONTAINING  GELS  AS  EQUILI-  CONTAINING  0.002 M A C E T I C  THE  FIELD.  VOLTS  OUT  APPLICATION ELECTRO-  F O R 70  MINUTES  FOR S T A I N I N G AS  '  IN THE A P P E N D I X . DISTINCT  COMPONENTS  WERE  ON  ELECTROPHORESIS  OUT  STRIPS  MICHIGAN.  THE CATHODE  VOLTS.  PRESENT.  CARRIED  ML  BARBITONE  . SAMPLES  TO 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 IDE  COMPONENTS  #51170 C H A M B E R A N D S E R A P H O R E  M I N AT  OF  POSSIBLE.  CELLULOSE  75  MEASUREMENT  OF P U R I T Y .  SAMPLES  ON  NO  SEPARATED  BY  ELECTRO-  AND  -84PHORESIS  ON  MIGRATED  WITH  A  MOBILITY  HIGHER  SEPARATION OF  HIGH  THIS AND  *  ON  THAT  ACETATE  GELS  AND  A  MAJOR  POOL OF  M  THE  A D E Q U A T E , HOWEVER, TO  WAS FOR  CONTAINED  75  1 0 |IG/ML  OF  MATERIALS  OF  THE  J^G/ML CT  OF  I N 0.1 WERE  PORCINE  CT  (AL0831)  AFTER  THE  TWO  WEIGHT OF  OF  250  SUITABLY  THE  BAND BAND  ALBUMIN.  REVEALED  5  A  NUMBER  OF  HAD THE  MINOR  R E L A T I V E L Y LOW OF  BANDS  MOBILITY.  COMPONENTS  WOULD  PURIFICATION  M  REQUIRE  ACHIEVED  SEVERAL  M  MANNER  NATIVE  CHARACTER-  FROM  ARMOUR  CT  AND C  WAS  TO  THAT  INCUBATION  AND  AT  C T AS  CIBA  BASED OF  EACH  ONE  OF  PRIOR  HOUR  MIXTURES  LTD.,  A  PH  7.4.  THE  DESCRIBED  WEIGHED ON  M  APPROXIMATELY  BUFFER  WERE  B IOASSAYED FOR  POOL  PHARMACEUTICAL  MATERIALS  BY  THE  SIMILAR  SALMON  CT  WITH  THE  MATERIAL  ALIQUOTS  37°  AND  THE  TRIS-ACETATE  PORCINE  CALCITONIN  CALCITONINS  HUMAN  PURE  AT  DILUTED PLASMA  POOL  FROM  DILUTED  PORCINE  INACTIVATOR.  U/MG.  INCUBATION  SIMILARLY  TO  ANALYSIS  OF  IN  PURE  I I I ,SYNTHETIC  ACTIVITY  THE  ALLOW  OUT  CHAPTER  THE  MINOR  I N A C T I V A T I N G ENZYME  THREE  ASSAYING  USED  ILLINOIS.  OF  MAJOR  ENZYME.  CARRIED  DESCRIBED  WERE  BAND  CONTAINED  SELECTiVE INACTiVATiON " C A L C I TON I N A S E " .  MATERIAL  THE  P O L Y A C R I L A M IOE  PURIFICATION STAGES.  THE  WHILE EQUAL  ISOLATION  OF  THE  APPROXIMATELY  THAT  INCUBATION  BUT  STRIPS.  01 2"-GL 0 B U L I N S  THE  CONFIRMED  ISTICS  D  THE  MOBILITY  FURTHER WAS  CELLULOSE  IN  BASLE, CO.  AND  KANKAKEE,  DIRECTLY,  BIOLOGICAL THE TO  ALIQUOTS  THREE  MIXTURES  INCUBATION. WERE  B I OASSAYED.  CALCIUM  LEVELS  AFTER  INJECTION  OF  PORCINE  CT  -85WERE 7 . 9 6  ± 0.04  AFTER  HOUR  ONE  (P  DIFFERENCE CT.  THE  LOSS  OF  THAT  THE  OF  AND  ENZYME  i.  METHODS.  A.  DIGESTION TWO  OF  MG  A CONCENTRATION ADJUSTED  TO  SOLUTION  PER  ACETATE  FOR  C AND  AND  USED  THROMBIN. 0.5  1  ML  ENZYME  ML  BUFFER  THE THAT  7.5  PH  OF  FOR  1.0$  STOCK  CONTAINED  WERE  THE  USEP  PROTEIN  NO  INCUBATION  54$  OF  OF  THE  SIGNIFICANT  THUS  CONFIRMING  PORCINE C T .  WITH  "CALCITONINASE."  FOR  DIGESTIONS.  IN 0 . 0 5  ML  FIRST  PROCEDURE,  C T SUBSTRATE  PER  THE  I N THE M  POOL  TO THEN  PH  OF  THE  SOLUTION  BY  0.1  ML  OF  2.0  M TRIS  THE  RESULTANT  OF  ENZYME WAS  FOR  SOLUTION. I NCUBATEO  PEPTIDES.  NATIVE  SOLUTION OF  PROCEDURE  (78)  FOR 1  AMMONIUM ADOED  POOL  BASE TRIS-  (17  OVER-NIGHT  T H I S PROCEDURE S U P P L I E D BY  WAS  HR) WAS  ClBA  USED  LTD.,  SALMON C T .  T H I S PROCEDURE AQUEOUS  CT  M  ACETIC ACID)  THE  ADDING  WAS  AND  5 MG/ML.  DIGESTION  2 MG/ML  SIGNIFICANT  A LOSS  I N A C T I V A T I O N ASSAY  BY MUTT _E_T AJ. IN  AFTER  OF S Y N T H E T I C P O R C I N E  SECOND  A  MG$  ENZYME.  SOLUTION  PURE  WAS  S E L E C T I V E L Y ON  ANALYSED  DIGESTION  BASLE  OF  THIS  ±-0.08  C A L C I T O N I N S SHOWED  D I S S O L V I N G THE  (0.15  SOLUTION  9.11  AND  CALCITONINS DIGESTED  WITH  OF  TIME  REPRESENTED  ACTIVITY  ACTED  FROM  A MODIFICATION  37°  SALMON  PROCEOURES  CONSISTED  ZERO  INCUBATION.  BIOLOGICAL  PEPTIPES  AT  < 0 . 0 0 1 ) AND  HUMAN  E.  AT  M0/a  WAS  A MODIFICATION  DIGESTION  MG  OF  CT  WAS  BICARBONATE  EVERY  2  M MATERIAL  HOURS. I N 1$  OF  OF  SECRETIN  DISSOLVED AND  10  THE  STOCK  AQUEOUS  JW-  WITH IN 0  F  A  N  SOLUTION  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 P H 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  MG  0 °0 s  J  /I  OA  Ov  8?  OL P  M  £  YO  P®3 :  -  ;  0 T  OE P-2  O DNP  QP-I  TA  OCA  FIGURE  41 •  PH 1 . 9 E L E C T R O P H O R E T O G R A M OF P E P T I D E S FROM P O R C I N E CT PRODUCED BY C A L C I T ON I N A S E " . O T H E R DATA AS I N D I C A T E D IN F I G U R E 2 1 . 11  -87TEND  TO  WERE  CENTRIFUGED  NATES  B E MORE  ONLY  SOLUBLE  WERE  REDISSOLVE  TO  WAS  COMPLETELY WERE  CONCENTRATED THE  THE  UNDERGOING  41  SHOWS  ELECTROPHORESIS ISOLATED IN  SER, WAS  OF  AND  OF  OF P E P T I D E 2  OF  P-1  TO  M  THE  IN THESE  WAS  CONTAINED INDICATING  IN FACT  A  AT  PH  1.9.  BUT ONLY  INDICATED  BY  FLVE  FOUR  ANALYSIS.  WELL  WERE AMINO  ITS COMPOSITION  PRESENT ACID  WAS  I T S N — TERM I N A L  CONTAINED  THAT  PEPTIDASE.  P E P T I D E S PRODUCED  P-2  ONLY  PROCESS  THAT  C T DIGESTS  FURTHER  PEPTIDE  SUPER-  WITH  INTERCHANGE  G L Y ^ , MET, PHE^.AND  PHENYLALANINE.  MET  FRAGMENTS,  DETECTED, FOR  ACID  DIGESTS  IN POLYMERIZATION.  THE SUPERNATE  QUANTITY  GLU, PR0 »  I N POOL  THE PATTERN  THE  ATTEMPTS  SUGGESTING  DISULFIDE  THE P O R C I N E  ENZYME  AND  THE  THE P R E C I P I T A T I O N  RESULTING  P E P T I D E S WERE  SUFFICIENT  ANALYSIS  THAT  Q U A N T I T I E S OF P E P T I D E  INACTIVATING  FIGURE  IN DILUTE  REVERSIBLE,  SOLUTIONS  MOLECULE  FOR P E P T I D E S .  INDICATING  SUPERNATES  ANALYSABLE  T H E WHOLE  THE P R E C I P I T A T E  THE P R E C I P I T A T E S  SUCCESS  HORMONES  REMOVE  ANALYSED  LIMITED NOT  THAN  THR,  GROUP  A S P , THR, S E R , ,  3 ALA,  i-CYS^,  CYSTINE. PR0 , o  THE COMPOSITION  WAS  N — TERM I N A L  ASPARTIC ACID, COMPARISON  OF P O R C I N E  IDENTIFICATION THE THREE  OF  PEPTIDE  ANO P-4  BUT CONTAINED OF  THESE  P E P T I O E S PROOUCED  N — TERM I N A L  T H E N — TERM I N A L ALSO  HAD  ONLY  ASP^, L E U , PHE,  COMPOSITIONS  C A L C I T O N I N SHOWN  THE P E P T I D E S  AN  W,AS A S P ^ , T H R , S E R , G L U ,  O F P-3  ASPARTIC ACID.  AND A R G .  SEQUENCE  ARE  WITH  G L Y ^ , M E T , L E U , P H E , , H I S , ARG  RESIDUE  HIS  V A L , L E U ^ , TYR AND ARG  IN FlGURE  INVOLVED. BY  SPLITS  AN  TO T H E 29  PERMITS  P - 1 , P-2 AT  THE  AND  P-3  C-TERMINAL  -88SIOE P-1  OF  THE  AS  THE  PEPTIDE  ARGININE  C — TERM I N A L  AND  PEPTIDE, SECOND  TWO  P-4  P-3,  AS  THE  AND  IN  P E P T I D E , P-2  RESULTED  ARGININE  RESIDUES  THE  AS  MOLECULE  THE  SMALLER  CENTRAL  FROM  INCOMPLETE  AN  CONTAINS  BOTH  THE  WITH  N — TERM IN A L  PEPTIDE.  THE  SPLIT  CENTRAL  AT  AND  FOURTH  THE C — TERM I N A L  PEPTIDES. THESE A  RESULTS  SPECIFICITY  ARGININE THE AT  FOR  C  CLEARLY  PEPTIDE  RESIDUES,  SPECIFICITY 37°  INDICATED  A  SINCE  BONDS  THAT EVEN  ARG-ASN  AND  THE  BONO  THE ON  OTHER AFTER  R E L A T I V E L Y LARGE  THE  ARG-PHE,  BUT  THAT  17  SPLIT  SPECIFICITY  WAS  THE  WERE  HOURS OF  OF  SIDE  HAD OF  INVOLVED  P-3  REAOILY  EQUAL  M  IN  INCUBATION  PEPTIDE  MORE  NOT  IN POOL  CARBOXYL  FACTORS  AMOUNT  WAS  ENZYME  FOR  REMAINED.  THAN ALL  THE  ARGININE  BONDS. THE  RESULTS  FURTHER  EVIDENCE  TABLE,  SINCE  EITHER  OF  THE  PEPTIDE  HAVE  THE  HAD  HAVE  WHEN  FAILURE  IT OF  TO  THE  THE  DETECTED  IN THE  THIS  MATERIAL.  IF  SUSCEPTABLE  THE  BEEN  READILY  IDENTICAL  OF  THE  THE  CAN A TO  WITH  WERE  SUSCEP-  SUPERNATES  THE  IDENTIFIABLE  PRESENCE  OF  A  NORMALLY  THE  BOND  SUSCEPTABLE  THE  SALMON  CT  TO  ACTION  TRYPTIC  PROLINE  IN SALMON  THE  IN  C — TE R M I N A L  THE  OF  OF  ARGININE  OF  BLOCK  SPLIT  PROVIDED  ONE  ARGININE  RESISTANCE  ENZYME  CT  WERE  HAVE  NEAR  SALMON BONDS  RESIDUE  OCCURS  OF  ARGININE  ISOLATED.  SIDE  THIS  ALL  BEEN  BEEN  PREVIOUSLY  SINCE  OF  WOULD  N — TERM I N A L  ENZYME, DASES  PEPTIDES  MOLECULE  CONTRIBUTED  DIGESTIONS NOT  DIGESTS  I T WOULD  THE  THE  THAT  PRODUCED  PEPTIDES ON  NO  THE  SALMON  SINCE  OF  MAY  THE  OF  PEPTI-  BOND  MOLECULE  (68). ALSO  - 8 9 -  SUGGESTEO  THAT  NOT  ALL  TWO  LYSINES.  THE  INACTIVATING  CT  BASIC  THE  CONTAINS  ARGININE  THE  BY  THE  WHERE  AN  30,000 THE  (11,  WAS  THE  RELEASED  ENZYME  IN  WITH  THE  A  NEXT  THE  CT  WITH  SINCE  HUMAN  CONTAINS  POSSIBILITY  WHICH  BONDS  ON  THE  THE  MOLECULAR LARGER  SECTION  THAT  I S KNOWN  P0SSIBILITY  DURING  MUCH  CONTAINS  ATTACK.  THROMBIN  THIS  AND  COMPATABLE  ENZYME  PORCINE  TO  PEPTIDE  FROM  COMPAR I SON THE  EXPERIMENT THE  OF  " C A L C I TON I N A S E  NATURAL WAS  " C A L C I TON I N A S E " 2 . 5 MG  FIBRINOGEN  DISSOLVED  PREPARED PAGE  HOURS  SUBSTRATE  DESIGNED  CONTAINING  IN  OF  ALSO  CLEARLY THE  ARGININE  TO  CARBOXYL WAS  SIDE  REINFORCED  PURIFICATION WEIGHT  AROUND  PRECURSOR  WERE  STAGES  MOLECULE.  DESIGNED  TO  TEST  POSSIBILITY.  SINCE  ON  WAS  ONLY  BE  FOR  MOLECULE  SUGGESTED  MIGHT  MATERIAL  EXPERIMENTS  THIS  ALSO  7 7 , 78). OF  ACTIVE  AND  SUSCEPTABLE  TYPES  BEHAVIOR  THE  ONLY  C H A R A C T E R I S T I C S OF  ARGININE  CERTAIN  ARGININE  ANO  NO  SINCE  WAS  SPECIFICITY  SPECIFICITY  OF  AN  THIS  " C A L C I TON I N A S E "  ATTACK  F.  RESIDUES  RESIDUES  THIS  SPECIFICITY  AND  EITHER  ROOM  FROM  AND  THIS  THE  ONE  M  UPJOHN  FOR  THROMB I N. THROMBIN  SAMPLE. THERE OF  THE  SUBSTRATE. 1  WHICH  AMMONIUM  ENZYME  1 0 UNITS  TO  COMPARE  FRACTION  TEMPERATURE  SAMPLE  PREPARATION  TO  ON  IN 0 . 1  4 0 JUL OF  8 5 ADDED  AT  COHN  TO  1 1  IS FIBRINOGEN  ACTION TWO  OF 1  BICARBONATE SOLUTION  AFTER  INCUBATION  AN  NO  EVIDENCE  IMPURE  C O . KALAMAZOO,  ML  SAMPLES  I S 5 0 TO 6 0 $  STOCK  WAS  THROMBIN  WERE  DESCRIBED FOR  OF  BOVINE  MICHIGAN  WAS  TWO  CLOTTING THROMBIN ADDED  -90T0  THE  IN  LESS  THERE  CONTROL THAN  WAS  SAMPLE FURTHER AND  10  POOL  M  OF  FACT  THROMBIN  ACTIVITY RETARDS  TESTS  OF  ITS  IT THE  AFTER  A  EITHER  THE  SEVERAL OTHER THAT  ENCES  IN  WAS  TO  WITH  BY  ENZYME  NO  PLASMIN  WITH  THROMBIN  ACTIVITY.  CLOTTED  THE  HAD  FIBRINOGEN BY  IT  CLOTTING  THROMBIN  " C A L C I TON I N A S E " OF  THAT  CAUSE  STILL  INCUBATION  CONTROL.  PROPERTIES.  SIGNIFICANT  FORMATION  THESE  INTRODUCTION  SPECIES  DIFFERENT  CONFIRMED  THE"CALCIT0NINASE"IN  THAT  OF  THE  PLASMIN  (11).  POOL  M  IT  IS  CORRES-  ENZYMES.  DISCUSSION.  SPECIES  THE  CLOT  SEEN  IN  A  CLOT  TH I S  THAT  SOLUTION  NO  ADDED.  SOLUTION  NO  AFTER  STILL  "CALCITONINASE"  HOUR  THE  TO  INCUBATION  ADDED.  WAS  WAS  F O R M A T J ON  " C A L C I TON I N A S E "  INACTIVATING  P R E - I N C U B A T I ON  PONDED  IN  5  THAT  PREVENTS  GENERAL  CT  ACTIVE  PRIOR  UNLIKELY  G.  PORCINE  M  FIBRINOGEN  THEREFORE TO  POOL  CONTAINED  FURTHER SINCE  THROMBIN  SIMILAR  THE  OF  THAT  OR  BOVINE  THE  WAS  THERE  CLOT  TOTAL  IN  SOLUTION  INCUBATION  CLOTTING  THAT  SUGGESTED  OF  THE  FAILURE  INDICATED THE  2 5 0 / ( L OF HOURS  RAPID  HOURS  FORMATION  RETAINED  THIS  THREE  A  CLOT  RAPID  SUBSEQUENT  AFTER  IN  OF  TWO  A  RESULTING  MIN.  SIGN  UNITS  CAUSED  STILL  10  NO  ANO  SAMPLE  OF  MAMMALS  WERE  IN  CALCITONINS  PHYSIOLOGICAL  RATE  THIS TO  DISCUSSED  VARIATIONS  THE  TO  AT  THE WERE  WHICH  STANDPOINT  CHAPTER  THE  CALCITONINS AND  EVIOENCE  RESPONSE DUE THEY  THESE  AT  TO  LEAST  WERE  RESPONSES  OF  FROM  A  WAS  PRESENTED  THESE IN  VARIETY  STRUCTURALLY  PART  TO  INACTIVATED.  OBSERVATIONS  OF  DO  NOT  DIFFERFROM  A  APPEAR  ,  -91-  .'  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 THE  POSITION  OF  THE  MOST  LABILE  ARGININE  BOND  IN  THE  PORCINE  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  CT  AND HUMAN  CT  (FlGURE  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  -93THESE  OR  ENZYME BE  PERHAPS  WHICH  ANY OF  GESTS SIZE THE  MANY  WAS  STUDIED  THE ENZYMES  A P L A S M I N OR AND CHARGE  BEST  MAY  THE  THAT  OBSERVATION POTENT  BE MANY  CORRESPOND EVEN  OF  NOT  THE RESPONSE  INVOLVED. THERAPY IT  FACTORS THE  STILL WOULD  THREE  EXPLAIN  HOW  SINCE  THERE  COMPARTMENT  OF  THEIR  ARE MANY  FROM  THE PLASMA  THE RATE  ENZYME,  THERE  NOT  MOOEL  EFFECTS  THE  DESCRIBED OF  I N THE C T E L I M I N A T E D INCREASE T H E C T TO  DESIGNED  FOR  ENZYMES,  AND A V A R I E T Y  EARLIER  INACTIVATION  IN THE A C T I V E  COURSE  FACTORS  OF C L E A R A N C E .  2  OF  THE  PLASMA  BY A L L PLASMA  ATTACK  INCREASE  OF B I N D I N G  MAY  TO  I N AN  DEGREE  OR  C T WERE  RESULT  OF T H I S  FAMILIES,  THAT  A "SUPER"  MUST  MAGNITUDE  HOWEVER,  RELATE  RELATIVE  OTHER  IN THE RATE  THE  OF  FRACTIONS  INFLUENCES  ANY DECREASE  AND  ENZYME  SUGGESTS MAY  SUG-  THOSE  135),  OF A L L THE  THAT  COMPARTMENT  COHN  WHICH  STRUCTURE  RESISTANT  INFLUENCE  (108,  THESE  OTHER  TO  MOLECULAR  WITH  I M P O S S I B L E TO  CHARACTERIZATION  8E C L E A R E D  ITS  CHARACGERI ZED PLASMA  TWO  AND E V A L U A T I O N  WAS  APPEAR  ENZYMES.  I F , FOR E X A M P L E ,  WHICH  WOULD  PLASMA  BUT  COINCIDE  ONE OF  WELL  NOT  THE  THE S P E C I F I C I T Y  ENZYMES  " C A L C I TON I N A S E S "  A COMPLETE  FULLY  NOT  INVOLVED.  DOES  ENZYME,  DO  TO  AT L E A S T  BE  EARLIER.  FAR PROVED  ANY OTHER  KNOWN  " C A L C I TORI I N A S E S " WILL  CHAPTER  C T I N A C T I V A T I N G FACTORS  OTHER TO  COULD  OF THESE  BELONG  I T HAS THUS TO  MAY  SUGGESTED  EXAMPLES  " C A L C I TON I N A S E "  CONTAINED  IN THIS  CHARACTERISTICS  STUDIED  ANY CASE  ENZYMES  K A L L I K R E IN TYPE  THE " C A L C I TON I N A S E " IN  OTHER  LOOKING  OF AT  IT IS EVIDENT IN THE  C T MOVING  PLASMA  INTO  BY R E N A L  EXCRETION.  WOULD  BE D E P E N D E N T  ON T H E  PLASMA  PROTEINS.  I F THE  -94BINDING  I S VERY  STRONG  COMPARTMENT  3  CANNOT  INTO  LONG  MOVE  TIME  OCCURS  AND  ONLY  WILL  THE AS  RESPONSE  OF ANO  THE  (37)  OF  ALSO  TARGET  ORGAN  THE "SUPER THE  1 1  TO AND  ACTION THESE  WHICH  VATORS.  EVEN  INACTIVATION  DEPEND  DE UP  ON  THE  PLASMA  AND  THE  ON  AS  THE  INFLUENCE  THE  A  LIVER  THE  IS  IF, LARGE  DURATION  INACTIVATED  DE  LUISE TO  ET  AL  PLASMA  OR  IF CT  UNDER COULD  REACH  EFFECTIVENESS  WHICH  THE  BY  MIGHT  A  SYSTEM  STUDIES  ON  FACTOR  THIS  AND  SOME  OF  EFFECT  THIS  CHAPTER.  REQUIRES USTNG  OF  ORGAN,  PRECEEDING  I N J_N  IMPORTANT  EFFECT  (36),  REVERSIBLE  TARGET  OR  I S AN  WOULD  E_T _AL  EXCRETEO  THE  INACTIVATED VITRO  LARGE  RESPONSE.  CHARACTERISTICS AN  FAIRLY  A  INACTIVATION.  WERE  BE  THE  2.  IT  IN  I S WEAK »  THIS  RESISTANT  LIVER  INFLUENCING  IN  THE  ORGAN  COMPARTMENT  IS REVERSIBLE.  UPTAKE  ACTION  IN  WHETHER  CALCITONINS  PROLONG  AND  LUISE BY  LIVER  DISCUSSED  NOT  OF  CHARACTERISTICS  STRUCTURAL  ARE  WORK  HAPPENS  THE  ITS  I N A C T I V A T I ON  WHAT  TO  CT  WERE  HOW  URINE.  RESISTANT  FACTOR  STRUCTURAL  THE  UPTAKE  FINAL IS  2  A  RESPONSE  TARGET  BINDING  ANO  LAST  THE  THE  COMPARTMENT  IS TAKEN  THAT  CONDITIONS RETURN  CT  THE  ARE  SLOWLY  THE  SINCE  HAND  IN  EXCRETION  IT MIGHT  REACHES  OTHER  IN  ON  WILL  SHOWED  INACTIVATION THESE  IN  THE  WHETHER  THE INTO  DEPENDS  RESPONSE  ON  ON  APPEAR  IS SUGGESTED  PERCENTAGE  HORMONE  REMAIN  FOR  THUS  USELESS  THE  WILL  IS UNAVAILABLE  ESSENTIALLY  RAPIDLY  WILL  CT  THE  2.  IF  2.  MOVE  PROPORTION  BE  IT  OF  COMPARTMENT  AFTER  COMPARTMENT CT  WHERE  MOST  ELIMINATION  EITHER WHICH BONE  EVALUATING OF  MOLECULES  HAS  NO  EXPLANTS  S I NCE' R E P E A TED  INACTI(105) SMALL  -95DOSES 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 INACTIVATING THE MORE LABILE CALCITONINS. THIS TYPE OF INACTIVATION, 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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J . 89, 3 4 9 ( 1 9 6 3 ) .  135.  C O L E M A N , R.W., M A T T L E R , L . A N D 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 I N ( K A L L I KE IN0GEN ) - K A L L I K R E E N E N Z Y M E 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 . INVEST. 4 8 , 11 ( 1 9 6 9 ) .  TRANS-  DANSYL-AMINO BIOCHIM.  A P P E N D I C E S  A.  STAT I S T I C A L METHODS C U R V E S BY C O M P U T E R *  CALCULATION REQUIRED  THE  FOLLOWING  DOSE  2.  THE  WEIGHT  OF  3.  THE  PLASMA  CALCIUM  PRACTICE  0.3  ML  FOR  RAT  THE  PROBLEM  THE  WEIGHT  TO  DOSE  WAS  USED  MUST  ALSO  AS  A  CONTROL.  AS  SHOWN  RAT  MEASURED  GIVEN  WAS  CALCULATED  FOR  AN  G RAT  80  ADJUSTING  BIOASSAY  $  WEIGHT,  I N THAT  FOR  INJECTED  THE  THIS  DATA  VEHICLE  THE  SUM  OF  BY  THE  NUMBER  THE  RESPONSE  BETWEEN  EACH  AS  PLASMA OF  AND  INJECTED  THE  L O G J Q  (RDOSE/WEIGHT).  -107-  QF  RAT  WAS  VEHICLE THEN  THE  MEASUREO  OF THIS  DAATA  WAS  ONLY  CARRIED END  OUT  OF  THE  FIRST  VEHICLE  INJECTED  (DUM/FNV) I N  PLASMA  LOG  INJECTED  CONSTANT  VEHICLE  CALCULATED  THE  DATA  OF  RATS  WAS  RAT.  SAMPLE  CALCULATED  CALCIUMS  WAS  IF A  THE  A  AVOIDED  PROGRAM  WERE  OF  CORRECTED  THIS  WITH  I N C L U D E D AT  RESPONSE  (RESP)  VRESAV  SAMPLE  THEN'iCALCULATED  THE  SET  CALCULATIONS  PRINTOUT SHEETS MEAN  THE  BASIS  WAS  IN WEIGHT  DATA  FROM  THE  DOSE  BUT  VARIATIONS  RATS  ON  VOLUME.  INCLUDE  18.  I N MG  THE  INJECTION  RAT  FOR  AND  EACH  LINE  GIVEN  INJECTED,  ANIMAL.  AS  DOSE-RESPONSE  MG,  LEVEL  RECORDING  IN THE  WEIGHT  IN  EACH  DIVIDED  RAT  THE  COMPENSATE  RATS  FOR  A  FOR  APPENDIX.  (CA)  C T GIVEN  DOSE  BY  OF  DESIGNED  DIFFERENCE  FOR  LOG  HOUR.  INJECTION  (VRESAV)  LINE  OF  DATA:  THE  1  OF  CALCULATION  REGRESSION  1.  AFTER IN  OF  FOR  DOSE  DOSE THEN  AS  THE  CALCIUM (NDOSE)  WAS  DIVIDED  BY  THE  PLACEO  IN  THE  -108EXECUTION THE BY  MEAN  ARRAY VALUES  T H E NUMBER  WERE TO  PLACED  THE A R R A Y .  THE  RESPONSE  PLACED  IN  IN  (RESP)  T H E XSQ  DISPLAYED  BELOW  WERE  CARRIED  THEN THE  DATA  OF  THE PARAMETERS  Y  FOR  OUT FOR  FOR OF  TERMS  DIVIDED  BY  AS  SHOWN  IN  49.  LINE  CALCULATED IS  IN  WHERE  B  ATION  OF A G I V E N  THCN GRAPH  AND MOST  THE  LINE  IN  THE OF  THE METHODS  DOSE  EQUAL  SUM  OF T H E XSQ  BY  THE  REGRESSION FROM  THE ARRAY FIGURES  REGRESSION IS  IN  BETWEEN WERE  ALSO  SQUARES  SUMMED  SIMILAR  PROCEDURES  ESTIMATES AND OF  DESCRIBED  BY  COEFFICIENT TO  ( B ) OF  T H E SUM  TERMS  (B=  RESPONSES,  OF T H E SXY/SUM WERE  YC,  =  BX  +  COEFFICENT,  X  IS  THE  AND  USED  THE  TEXT.  OF  AND  YC  T H E MEAN  AND  (MND)  ALLOWED C A L C U L A T I O N  FORMULA  RESPONSES  THE R E M A I N I N G  POSSIBLE  DIFFERENCE  POPULATIONS  BX  57  WAS  OF THE P O I N T  Y •  AND  XY.  POINT  THE ESTIMATED  THE ESTIMATED  FLACED  BY  IT  THESE  THE A R R A Y .  TO T H I S  DIVISION  THE MEAN  THEN  T H E PRODUCT  SAMPLE  THE SAMPLE  RESPONSE.  IN  WERE  TND).  (FNE)  THE C A L C U L A T E D  DETERMINATION  THE EQUATION  AND  BY  THE D I F F E R E N C E  THE R E G R E S S I O N  THE  XY  THEN  TERMS  THE  (MRES).  T H E MEAN  YSQ  ESTIMATES  (115).  AS  VALUES AS  (X)  (NDOSE)  (Y)  INCLUDED  THESE  AND  (TRES  DETERMINED  SAMPLES  T O G E T H E R WITH  AND  NECESSARY  INTERVAL  ON X  AND  VALUES OBTAINED  SNEDECOR  WERE  DOSES  T H E COLUMNS  OF  THEIR  IN  RESPONSE  THE ARRAY  Y.  CALCULATED  FROM  T H E LOG OF E A C H DOSE  DEVIATIONS  AND  TOTALS  (MRES AND MND)  DEVI AT IONS  THE  X  THE  OF E X P E R I M E N T A L  IN  CALCULATE  BETWEEN  ANO  DOSE, FOR  THE  TO P L O T  CALCULATIONS  IN  AND MRES GIVEN  XSQ)  THEN  MRES,  IS  DOSES  DEVIMEAN WERE  BOTH THE C O M P U T E R  THE PROGRAM  WERE  -109AIMED 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. (B-g)/SB  FOLLOWS THE  T-0  SlNCE  I STRI BUTI ON WHEN g IS THE POPULATION  REGRESSION COEFFICIENT AND S B IS THE STANDARD DEVIATION OF THE SAMPLE REGRESSION COEFFICIENT, IN THE NULL HYPOTHESIS WHERE 3 l I S ZERO T IS EQUAL TO B / S B .  S B IS THE SQUARE ROOT  OF VARIANCE OF THE SAMPLE REGRESSION COEFFICIENT WHICH"IS EQUAL TO  SYXSQ/SUMXSQ.  SUMXSQ  is  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 (RESP -  YC)  OR  SDYXSQ  (SDYXSQ/(N-2)).  DIVIDED BY THE DEGREES OF FREEDOM  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 C C C C C  s  /  MA IN  01-15-70  C  C NV IS NUMBER OF VEHICLE SAMPLES. NT  0006 0C07  ccoa occs  0010 001 1 0012 0013 0C14 001 5 0016 CC17 0018 0019 0C2C 002 1 0022 0023 0C24 0025 0026 0C27 0C28 0029 003C 0C31 0032 0033 0034 0035 0036 0037 0038 0039 0040 004 1 0042 0043  PAGF 0001  CASS1 MAIN PROGRAMME PROGRAMME TC COMPUTE THE LINEAR REGRESSION OF LOGCCSE/RESPONSE PROGRAMMER PETER GRAYSTDNE PHYSIOLGGY DEPT. UBC FEBRUARY 1968 MCDIFIEU RY RON CDCR JANUARY 1970 REFERENCE CHAPTER 6. SNEDECOR , STA T I ST I C AL METHODS t I CWA STATE COL  r  0CC1 0002 0003 COCA 0005  15:01:51  o /  \  PRESS  IS NUMBER OF TOTAL SAMPLES INC NV  C COLS 79,80 CF RUN IDENTIFIER CARD SETS THE X AXIS CRIGIN CALL PLCTS REAL NOOSE,MNC.MKfS INTEGER DATE, STD 0 I MENS I CN P.DOSE (100 I, HEIGHT 1100 ),CA( 100), TITLE 1 15 I.NDOSEI IOC) DIMENSION XI 100),Y<100),XSO(100),YSQI100),XY(1001,DYX(100I.DYXSQI1 COO),YC(100),RESP(100).DEVI100I.DEVSCI100) 1 READ(5,10> DATE,NV,NT,TITLE, STO 10 F0RMAT(1X,I6,1X,I3,1X,I3.1X,15A4,2X,I2( IF(OATE.LT.0IGC TC 999 WRI TE(6,1010ATE.NV.NT,TITLE READI5.20) (ROOSE(I).HEIGHT!I),CA(11,1 = 1,NT) 20 FORMAT!1X.F8.3,1X.F8.3,1X.F6.2I IF(NV.EO.OIGO TO 25 FNV=NV DUM=0. 00 30 1 = 1,NV • DUM = OUMt-CA(I) 30 CONTINUE VPESAV=OUM/FNV GO TO 35 25 VRESAV=0. 35 NVP = NV+1 TPES=0. TNO=0. no 40 l=NVP,NT NCCSE(II = A L 0 G 1 0 I R 0 O S e m / H E I G H T ( I I * 1 0 0 0 . ) RESP(I)=VRESAV-CA(I) C THE ARRAYS NOOSE AND RESP NOH CONTAIN THE LOG DOSE AND RESPONSE DATA TNO=TND + NOOSE ( I ) TRES = TPES + RESP( I) 40 CONTINUE FNE=NT-NV MNC=T NO/FNE MRES=TRES/FNE C MND CONTAINS MEAN CF LOGOCSE AND MRES CONTAINS MEAN OF RESPONSE SUMXSO=0. SUI-YSC-O. SUMXY=0. DO 50 I=NVP,NT XI I)=NOOSE(Il-MND XSOII)=X(I)*X(I) Y(I)=RESP(Il-MRES SUMXSQ=SUMXSC*XSQU 1 XY(I 1=XI I 1»Y( I 1 Y S C ( I ) = YI I l*Y( I ) SUMYSQ=SUMYSQ+YSQ(I)  .  • • -  -  ••  ...  :  ,  .  J  FORTRAN IV G COMPILER 0044 0C45 0C46 0047  '  oc48  .  00*9 CC5C 0051 0052 0053 0C54 0055 0056 0057 0058 0C59 0C6C 0061 0062 0063 0C64 0065 0066 0067 0068 0069 0070 0071 0072 0073 0074 0C75 0C76 0077 0078 0C79 0080 0081 0082 0083 0081 0085 0C86 0087 0088  MAIN  01-15-70  15:01:51  PAGE 0002  SUM XY=SLMXY + XYt I) SXYSO=SUMXY*SUMXY C SOYXSQ IS SUM OF RESP<Il-YCI I ) RMSQ=SORT(SUMXSQ) 50 CONTINUE C SAMPLE REGRESSION COEFFICIENT OF Y ON X IS B (Y=BX) ,. .. B=SUMXY/SUMXSO DCF =FNE-2. C MEAN SCIIARE OEVIATICN FROM REGRESSION IS SYXSQ SYXSC=50YXS0/C0F . C SAMPLE STANDARD OEVI AT ICN FROM REGRESSION IS SYX SYX=SORTISYXSQ) C SAMPLE STANDARD DEVIATION OF THE REGRESSION COEFFICIENT IS SB SruSYX/RMSQ T=B/SR SLAM=SQRTISUMXY/FNEl/B DO 7C I=NVP,NT YC( I )=B*X( I KMRES 70 CONTINUE C YC CONTAINS ESTIMATED RESPONSES WRITEI6.100) FGRPATI//3X,109H RAWDOSE WEIGHT CA RDG LOGDCSE RESP 100 C LD DVN RES OVN LD SC RES SQ LD*RES EST RESP) WRITE(6,110)1RC0SE!I I . W E I G H T ! I ) , C A ( I ) . N D 0 S 6 ( I l , R E S P U ) , X < I I , Y l l ) , X . C S Q I I ) , Y S Q ( I ) , X Y ( I ) , Y C ( I ) , I = NVP,NTI FORMAT( 1X.11F10.4) 110 WP. ITE (6, 120 ) F0PMAT(1X,3HSUM) 120 WRITE(6.130)TNO.TRES,SUMXSQ,SUMYSO,SUMXY FCPMAT(31X,2F10.4,2CX,3F10.4/I 130 WRITEI6,140) FORMAT!IX,4HMEAN) 140 WRITE16,150)MND,MKES FORMAT! 31X.2F1C.4//) 150 WRITE 16,160)B FORMAT!IX,55HSAMPLE REGRESSION COEFFICIENT OF RESPONSE ON LOGDOSE 160 CIS.F10.4//) . WPITF(6,170ISYXSQ FORMAT!IX,40HMEAN SQUARE DEVIATION FRCM REGRESSION I S , F 1 0 . 4 / / I 170 WR ITEI6.180ISYX FORMAT!IX,44HSAMPLE STANDARD DEVIATION FROM REGRESSION I S . F 1 0 . 4 / / I 180 WRITE(6,190)SB 190 FORMAT 1IX,5BHSAMPLE STANOARO DEVIATION CF THE REGRESS1CN C O E F F I C I E C NT I S . F 1 0 . 4 / / I WP.ITE(6,2C0IT,DCF 200 FORMAT!IX,13H.T,TEST, T IS,F10.4,13H DOF (N-2) I S . F 1 0 . 4 / / I WP.ITEI6.220) SLAM 220 FORMAT!IX,10H LAMBDA I S . F 8 . 4 / / ) CALL REGPL!NDCSE,RESP,NVP.NT,TITLE,DATE,YC,B,STD,T,SLAM 1 WP.ITE(6,55) 55 FORMAT(1H1) GO TO 1 999 CONTINUE CALL PLOTND  .....  EXECUTION  TERMINATED  ©  tRUN -LOAC»+«NFL+»BPLOT 9=-P EXECUTION BEGINS RAT 171167 6 28 PORC RAWCOSE 0.0060 0.C060 0.C06C 0.C0600. CC600.0060  o.cceo  O.COfiO 0.0080-  o.coeo-  0.0120• 0.C12C0.C1200.01200.01200.01200.01500.0150. 0.01500.C150, 0.01500.0150  WE IGHT 80.0000 80.0000 80.CO0O 80.0000 80.0000 80.0000 80.0000 80.00CO 80.0000 80.0000 80.0000 80.0000 80.0000 80.0000 BO.0000 80.0000 BO.0000 80.0000 BO.OOCO 80.0000 80.0000 80.0000  CA ROG B.4000 3500 4000 2000 6000 5000 8.5000 8.5000 8.3000 8.6000 9.0000 9.0000 8.3000 8.3000 7.9000 7.9000 7.3000 8.20C0 8.2000 8.0000 7.8000 7.7000  4fcK  KUMAR  LOGOOSE - 1 . 1249 - 1 , 1249 - 1 , 1249 - 1 , 1249 -1, 1249 - 1 . 1249 - 1 . ooco 0000 0000 0000 8239 8239 8239 -0.8239 -0.B239 -0.B239 -0.7270 -0.7270 -0.7270 -0.7270 -0.7270 -0.7270  RESP 1.8333 1.8833 0.8333 1.0333 0.6333 0.7333 1.7333 1.7353 1.9333 1.633,3 1. 2333 1 .2333 1.9333 1.9333 2.3333 2.3333 2.9333 2.0333 2.0333 2.2333 2.4333 2.5333  LO OVN -0.2133 -0.2133 -0.2133 -0.2133 -0.2133 -0.2133 -0.CR84 -0.0884 -0.C884 -0.C884 0.C877 C.C877 0.0877 0.C877 0.0877 0.0877 0.1846 0. 1846 0.1846 6.1846 0.1846 0.1846  RES DVN 0.0523 0.1023 -C.94 77 -C.7477 -1.1477 -1.0477 -0.0477 -0.0477 0.1523 -0.1477 -0.5477 -0.5477 0.1523 0.152 3 0.5523 0.5523 1.1523 0.2523 0.2523 0.4523 0.6523 0.7523  LO SO 0.0455 0.C455 0 .0455 0.0455 0.0455 0.0455 0.0078-; 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 0.0341 0.0341 0.0341  RES SO U.U027 O.C105 0.8982 0.5591 1.3173 1.0977 O.C023 0.0023 0.0232 0.0218 "0.3000 0.3000 0..0232 0.0232 0.3050 0.3050 1.3277 0.C636 0.063b 0.204b 0.4255 0.5659  EST RESP LD*.RES 1.2116 -0.0112 1.2116 -0.0218 1.2116 0.2022 1.2116 0.1595 1.2116 0.2449 1.2116 0.2235 1.5451 0.C042 . 0.0U42 T4~yr~ -0.0135 5451 0.0131 5451 -0.0480 0151 -0.0480 0151 0.0134 0151 2.0151 U.0134 2.0151 0.0484 2.C151 0.0484 2.2738 0.2127 2.273* 0.0466 2.273B 0.0466 0.083b 2.2733 0.1204 2.2738 0.1389 2.2738  SUM  -20.0550  39.1831  -0.9116  1.7811  MEAN  SAMPLE REGRESSION COEFFICIENT OF RESPONSE CN LOGDOSE IS MEAN SCUARE DEVIATION FROM REGRESSION IS  0.4409  SAMPLE STANOARO DEVIATION OF THE REGRESSION COEFFICIENT IS  LAMBOA IS  4.5095 DOF (N-21 IS  0.0972  2.6692  0.1944  SAMPLE STANOARO OEVIATICN FROM REGRESSION IS  .T.TEST, T IS  0.5549  2C.0000  0.5919  7.8424  1.4813  ; i' 111 - i j j - r  X!  i  i  i j  |  -JJ T u .  !  !  1  ! ' < i ; ' ' * I• ; 1 ' t 1 M l ! 1 1 ; 1 1|• M < 1 11 | * i ' : i ' r i • ' i i i i ! i i i i ! 1 ' 1 t 1 ! i ; i • 1 i i i i I 1 1 i .  }i ill!  ; •  i i '  i i  1  i  I ' l l • i :  i  i -  i 1  T i  <  '  1  1 1 1 1  1  i  !  i  !  i  . 1  . . i i  , i . ; i ! i i '  i. 1  -  • i i I i1 ! ! M i 1 : 1i 1• '; • • ! iI1:1 i' i ' i 1 1  ;  :  1  I  '  .  I  -  i i i  J 1 1 [ 1 • • 1.j i; ; -14-' i 1 ! 1  1 , .  1  I  1  • ! 1  !  !  1 i  1  i i  1  :,:al ;  T!3T  - 1  It.'j.  • : 1  1.' I •  M M  . :  1  i  1 ' : •'  ! i ! i i ! •  1 1  "  , ! i '  J  |  i  ' | l 1  1  -  !  1  1 1  ' 1 1 .  i  •  -rrrr • , i  1 ; . . 1  |  M  • '  J-J . M |  : i :  L-l-l I'll  •-  '  1  ;  M  :  ; i  — ;  M M  i  i! : : : j M  : i• i  i  1  • •  •  \  J!M  ,,|  i  • 1  „  X  1 .  1  i :  i  i  liii M M  1  1  I 1 M M  '  1  1  1  1  I ,  1  i_i ' i !. 1 i !i ;1 | i 1 i 1' 1 i i ! ! ' it  1  l_  1  1 ' ! 1 l 1 i :'! i 1  11  1  i  1  i  I i i i i  I  i j  1 | i ,  11  !  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M M 1 I i M i r i 1 1 1 i ' i i M M I !  1 : ' i i ' i i  •• ; i ••  T'l III!  : ; i . ; ! i , i 1 M < M  n • i i i r M  M  ii  ! m _  • !in  i  • l 1 ! , i •  I i i . i i >  x M I'M +• i IpT M l , 1 M i"i  i  M*  I'M  M M  ! t  . *  ', 1  r •  . i  -  '  M i '  •  f I  • i .  ru  I t ' ' ; i i i : •  i •  i '  1 1  i  i ' • • l_; -l-t-tT-f-h  1  1  1  1 1  1  ' i i M i l , i ; i i i i M • i '. l : i  I 1MM" i M M  |  1  1 . . . J i t '  i  t  i '  i • - , : i i i  '-i-i '  1 < 1  > i i ' i '. 1  I i  •  1  M i ! t ; •  ;  1  | |  i i  I  1  M l ]  I  !  "i ; i i i i_  1 • :  U~LT  , M i  !  |  1  i !  j i • ••  1  1  1• ,! 1• 1i i 1  -  '.~  1  M M  |  m  i I  M ' f 1 1 ii i { •. i .  ' II I | M 1 | 1 1 ' • 1 :1 t i l l | 1I | 1 .. _i ! .!_ | M i l | | 1 1 ii | I i: l i l !M • i l • 1M  1  i  i  ; l,i , i i i |  • i  • • 1  I  j  Ir'  iii  I'M  i  "it!  | 1  i Tll - nM • M  . i  1  I I1 I  .  X.  n  > . i  |  J : -T+rr*  1 _J_L  1  j—  i - i i  • 1  | \  1  jit 1 1  i , M i :  M i l  1 1 !  1  -Xr  1 1 . ;  i  1  i ..1.  1  ii. !.  : . : '!  i 1  1  I'M  ;  M l ;  i  1 j  __  _  j j  M M  i!  r  T  j  i i • I'M  _  -p  ;  1  i i  1 1 1 1 j • ' 1 i  •!  " ••'"; i  ; • : i i . : I T' ,~'~\  • i i M  1  i-  M  !'[M ': M J U-J_ . i. 1  4-  ";  . i. i  _  1  i i ,  1 1  ! L) 1. I | 1 - H~r 1 !  'Ml  ! I  i  1  _jT  i  • 1  r  ~pr  _  [  T  _  1 MM iMl , 1 1 'X ~X^:~4  '  M  j i  - • i  I I I .  i  1  M  i 1 1 i,  1  |  1  1  M M i  4+  : i  1  M M  M M  , t . r_  •i . !  i •  x-  •  -uU-  i :  1  1  ii  'Mi  : . i  1  -  ;  i ii i  '  ; 1  ! 1  i  I  44  j  f  1  ' I I I  i  • 1 1 • 1 ':i .• 1 1 •  41"i" i  1 • 1  t  t _ | )T I |  •  1 I  1  1 MJ l I M ! 1 | ! !• 1 ! i I  1 •  r  _  1i 1 i i 1 |  i  1  • . i  : i l ;  1 i  t  "!~  M j i • , i t : i t 1 : M j  i i  1 III' ' 1 I I 1 I  1 1 1 < i  ' T ' T i "  : i • . ! M  , 1 1  | |  1  :  . i • M I M 1 J j . ;.. I I I ! ' : i ' i " . i : : M i ' ~M.~M'  I ' ' :  . . i .  i i  1  !.:.  I ' l l 1 i  -  • i  1 rrr  I.I  i"i~r  n~  44 rt <  +Ti i :  r 4+ :ir Xi' ! ! 1"1~n?.i~ -4 Xi p\ .j' i " in. XJl  1 | 1  . - i •i,>_Ll. , : 'i  • t •  i"  t : -  i • . . i >'ii i i l l ' ! f 1 ; i | 1 i \ ;. • • • i i | 1 1 !1 . : • •; 1 i 1 ! 1 1 »  ;i  M .:J:i± _:_L.1..L  1  1  I'll • i ! • i. i  ;  ; i  i l l ! ' ;  4T  , > i i  i i . ' 1 | M  W  i 1  i  i i ;  TT-H  3n PTT  rr xfl: 3gr ex X3  iri  fH-  ro-l=M  "IE -tr  tr  :CT>  J_LJJ.  -ICE  ±CD.  -H4  i n  TT-Pr •n-0 L  4 T  :  -MS-'-  7TH'  4-I--0  m 1N33  41  -H-i  •on  'SI.  mi  b c l  m :M±:  Mf  •at  t-r4+  •P4-T  •rrr  ±H-  XDXl  -110SUMXY AS  SQUARED  AS  IN L I N E  INDICATED  PREVIOUSLY.  SYXSQ  CALCULATED  THE  SAMPLE  FOR  SYX,N  ERROR TEXT  FOR  AS  STANDARD SUMXSQ  THE  ESTIMATED  IN CHAPTER  THAT  B.  1.  OF  ABOVE  WERE Y  SIMPLE  P E S I GN  FOR  ON  PREPARATIVE  SEPARATION  OF  CONDITIONS  THE  1  COLUMN  2 $ OF HAS  MAXIMUM  CALCULATE X  CALCULATIONS LIMITS  OF  AND  SYX  (LINE  THE  VALUES  THE  AS AS  CALCULATED  SHOWN  IN  SHOWN  RANGES  5 2 )  STANDARD THE  ALLOWED  AS  INDICATED  BE  SOLUBILITY  OR,  I S TO  TOGRAPHY, REQUIRED  BUT THIS  ERRORS/AND THE  OF  VOLUME  ABOUT  ON  WHICH  SUCH  LEADS  PROCESS IN  TO  LARGE  ADDITION  TO  M U L T I P L I E S THE  METHOD  LOSSES FOR  IF A  ON  HIGH  UNDER  EXAMPLE,  A  2.5 X  AREA  OF  ABOUT  10  ML.  THE  A  COLUMN  IS HIGH, BROAD, SAMPLES  BY  VESSEL  HANDLING  OF  HIGH  FOR  WALLS,  LARGER  BY  VISCOSITY ONE  LABOR  CHROMAANO  ACCIDENTS  TIME AND  ETC.  SAMPLES  A  PROTEIN  LIMITED  PEAKS.  THE  CM  ALLOWING  REPETETIVE  MULTIPLYING  ONLY  100  CM,  BE  THE  SKEWED  BE  AMOUNT  MAY  BY  POTENTIAL  5  DEGREE  SUCH  A P P L I C A T I O N SHOULD  FOR  FOR  L I M I T A T I O N S WHEN  IS REQUIRED.  IF SOLUBILITY  INCREASES BEST  OF  CERTAIN  COLUMN .  TECHNIQUE  PARTICULARLY  SAMPLE  LENGTH.  VALUABLE  HAS  SCALE,  PROCESSED  SAMPLE  SOLUTION  ZONE  IS A  IT  COMPONENTS  INEXPENSIVE- SEPHADEX  A . C R O S S - S E C T I 0NAL  SAMPLE  CAN  THE  BE  REGRESSION.  SELECTED  A  THEN  SQUARE  OF  CHROMATOGRAPHY  A  OF  FROM  A. L A R G E ,  USED  W HICH  I S THE  TO  CONFIDENCE  P U R I F I C A T I O N , BUT  COLUMN  MAY  USED  FURTHER  PROTEIN  TO  T  CHAPTER.  SEPHAOEX  OF  4 5 .  DEVIATION  AND  DETERMINATION IN  SHOWN  IS  TO  glass fiber disc screen -1/8 in.dia.  1/8 in. pipe thread  FlGURE  43  -2 in.-  1/4H-  END-PIECES FOR 10 CM COLUMN--DIMENSIONS. TWO VIEWS SHOWN. DISCS MACHINED FROM 1 IN LUC ITE SHEET.  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). END PIECES DESIGNED AS SHOWN IN  FlGURE  Two  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.  -112SHOWN. CIRCULAR PIECES OF NYLON WINDOW SCREEN AND GLASS FIBER PAPER  (GFP-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 CROSSSECTION 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 1 5 0 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 OF THE COLUMN WAS 4 L AND V 14 L. Q  T  EXCELLENT SEPARATION WAS ACHIEVED AND THE STABILITY WAS SUCH THAT THE COLUMN SELDOM REQUIRED REPOURING.  -113C.  TECHNIQUES  1.  APPARATUS HIGH  MLCHL BY A  VOLTAGE  VERTICAL  RYLE  EJ_ AJ.  DETAILED THE  SHANOON  GLASS  LIDS  THE  ELECTRODES TANKS  WHICH  MOUNTED  EXTERNALLY  INSTITUTE SUPPLY  200  (DON  WAS  TO  TO  PREVENT THE  USED  TO  IN  22.5  PAPER  GLASS  GLASS  THE  TROUGHS  (CATHODE)  AND  DEPTH  TO  A  WERE  OF  THE  APPOXIMATELY TO  FOUR  CONNECTORS  MODEL  EPSK-200  D.C.  CONSTANT  VOLTAGES  UP  ONTARIO)  OF  A CANADIAN  CHROMOTANKS.  WERE  SIMILAR  BOTTOM  ATTACHED  WALLS  COILS. HOLDERS  THE  FROM  BUFFER  ELECTRODE  8UFFER  CONTAINS  WERE  CONTACTING  COOLING  FROM  MAINTAIN  THESE  UPPER  CENTIMETERS  THE  IN. DEEP)  GLASS  ELECTRODES ON  X  THE  THE  DESCRIBED  IN A L L - G L A S S  ONTARIO).  SUPPORT  BUFFER  MILLS,  9IN.  (WESTON,  AS  THE  RESEARCH POWER  TO  5  K V AT  MA. THE  TOXIC  ENTIRE  VAPORS.  CONNECTED TO  CONSTRUCTED x  IN  BY . L E G G E T T - B A I L E Y  WERE  (22. 5'I'N.  OUT  MODIFIED  USED  TWO  BOTH  75)  TECHNIQUE ( 6 8 ) .  CONTAINED  CENTIMETERS.  (74,  CARRIED  THIS  ELECTRODES UPPER  ELECTROPHORESIS.  WAS  MONOGRAPH  SUPPORTING  PLACED  PAPER  OF  RACKS  RODS  PLATINUM IN  THE  LABORATORIES  GLASS  LUCITE  PLACED  APPARATUS  (110),  CHROMATANKS  TROUGHS, AND  STRIP  APPARATUS  WITH  VOLTAGE  ELECTROPHORESIS  DISCUSSION  CONSOLIDATED FITTED  HiGH  FOR  VIEW  TO  THE  ELECTRICAL THE  SYSTEM  THE AN  HOOD  WAS  PLACED  WAS  EQUIPED  INTERLOCK  PAPER  DURING  SAFETY  IN A WITH  SYSTEM  OPERATION  WHILE  FUME  HOOD  TO  REMOVE  LUCITE  OOORS  ALLOWING  THE  MINIMIZING  OPERATOR THE  HAZARD. TWO  VOLATILE  BUFFER  SYSTEMS  USED  IN THE  PRESENT  -114-  STUDY WERE FIRST DESCRIBED BY AMBLER  THE BUFFERS AND  (134).  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: PH  VARSOL  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  -115ARE  WORKING  AS  EXPECTED  AND  ARE  USED  IN C A L C U L A T I N G  RELATIVE  MOBILITIES. IN  A D D I T I O N TO  PHORETOGRAM THE OF  IS SPOTTED  CONDITIONS THE  THE  DURING  AT  FOR  METHYL  GREEN  ( C . I . NO.  STAIN)  ORANGE  STAIN)  AND  CURRENT GREEN  RUN  PH  AND  6.5  TO  20  IS NEUTRAL AND  TO CM  AT  THESE FROM  RUNS  THE  PH  AND  MOVES  THE  PH  1.9  ONLY  ANO. « - D I N I T R O P H E N Y L - L Y S I N E  MARKERS  ARE  USED,  GREEN  30  TAURINE  IN THE  IS  TO  3.  USED  THE  WHEN  REQUIRED  OR  NEEDED.  STOCK  THE  CM  STANDARD  NEUTRAL  FOR  FROM  METHYL  THE T-DINITROONLY  BY  ELEC-  C A L C U L A T I O N S OF  THE  METHYL AND  THE  ORIGIN.  IS NEUTRAL  AT  GREEN: METHYL  THE THIS  PH  ANO  LINE.  REAGENTS.  MAXIMUM  WHEN  REAGENT  SENSITIVITY  INFORMATION  THIS  SOLUTION  CERTIFIED  ACID  C A D M I UM-N I N H Y D R I N  WAS. U S E D  WAS  AMINO  INDICATE  DETECTION  TRAVEL  ANGELES).  ELECTROPHORETOGRAM  RUNS  TO  BIOLOGICAL  THE  ORIGIN.  FOR  I S ALLOWED  LENGTH  BIOLOGICAL  UNTIL  LINE  AT  INDICATE;  USED:  CERTIFIES  NEUTRAL  MOBILITY.  TO  ARE  CERTIFIED  THE  RELATIVE  ELECTRO-  (CALBIOCHEM, LOS  I T S L O C A T I O N ON  INDICATE  DYES  MARKERS  FISHER  THIS  EACH  DETERM I NE: T H E  TO  THREE  16230,  APPLIED  MARKER  FlSHER  42590,  A MARK  MIXTURES  VISIBLE  RUN  ( C . I . NO.  TO  TRO E N D 0 S M 0 S I S , USED  ACID  - D I N I T R O P H E N Y L - L Y S I NE  PHENYL-LYSINE  IS  RUNS  I S NORMALLY  HAS  WITH THE  RUN.  G  AMINO  REAGENT  WAS  CONTAINING 5  REAGENT)  IN 250  ON  ML  G  OF (1  HEILMAN TO  CADMIUM  BY  MIXING  ACETATE  GLACIAL  (54)  NMOLES/CM  THE , N — TERM IN A L  PREPARED  OF  5  E_T A L  ACETIC  )  WAS  AMINO  ACID  15  OF  ML  (FLSHER ACID  ANO  A  -116500  ML  DISTILLED  (PIERCE PAPERS UNTIL AN  WATER  CHEMICAL WERE  CO.)  DIPPED  THE ACETONE  OVEN  A T 60°  GENERAL BELOW  C  WITH  100  ML  IN ACETONE  IN THIS WAS  MINUTES  AND  BEEN  SHOWN  TO  GIVE  ALLOWED  GRADE). TO  WAS  FOR COLOR  DEVELOPMENT.  AMINO  THE COLORS  THEN  A I R DRY  THE P A P E R  P E P T I D E S 'HAVING THE N-TERMINAL  HAVE  (W/V) N I N H Y D R I N  (FISHER REAGENT  SOLUTION  REMOVED.  F O R 20  O F 0.5$  ACIDS  INDICATED  PLACEO IN IN  LISTED WITH  REAGENTo  THIS  YELLOW RED  (STABLE  1 WEEK)  G L Y , S E R , THR, C Y S , PRO,  ORANGE  SER, HIS  SLOW  RED  "FAST  RED  THESE  COLOR  PROCEDURES  CYSTEIC  (OVERNITE)  ( L E , VAL L E U , L Y S , ARG, A S P , GLU, T Y R , P H E , M E T , M E T SO2, TRP, ALA  DIFFERENCES NEEDED  TO  ARE U S E F U L  IDENTIFY  IN P R E D I C T I N G  THE  THE D A N S Y L - D E R I V A T I V E S OF THE  PEPTIDES. BECAUSE (0.1 THE  PAQ REAGENT  SENSITIVITY  WAS  USED  WERE  PAQ REAGENT  SOLUTION  DIPPED  WAS  CONTAINING  SCIENTIFIC, w/v  ROUTINELY  NINHYDRIN REAGENT  REAGENTS  10$  ITS HIGH  NMOLES/C M ^ ) AND THE E X T R A  SPECIAL  THE  OF  CO.,  SOOIUM IN THIS  FOR A R G I N I N E  INFORMATION USED  IT PROVIDED,  IN COMBINATION  FOR P E P T I D E  D E S C R I B E D BY YAMADA PREPARED  PEPTIDES  BY M I X I N G  WITH  DETECTION. AND  I..TANO  EQUAL  THE  (133).  VOLUMES  0.02$ ( W / V ) P H E N A N T H R E N Q U I N O N E  HIGHEST  HYOROXIDE MIXTURE  P U R I T Y ) IN ANHYDROUS I N 60$  AND  ETHANOL.  ALLOWED  TO  OF A  (FLSHER  ETHANOL  THE P A P E R S  A I R D R Y 20  A  AND WERE  MINUTES  -117PRIOR  TO E X A M I N A T I O N AFTER  AN  BE  DEVELOPED  20  ML  WITH  WITH  8 0 ML ALL  60° C  AM 1 NO Ac 1o  1.  HYDROLYSIS.  2  MOORE  AND  PYREX  WERE  STEIN  TO 2 0 NMOLES CULTURE  WITH  HOT  NARROW  (1MM),  FROZEN  IN  THE  TUBES  NECK THE AT  WITH SEALED  110° C  PARK, AND  AND  WERE  WAS  HOT  THEN  A TEMP-BLOK AFTER  A VACUUM ACID  LYOPHILIZED  FLAME NECK.  ICE-ACETONE  TUBES  FROM  IN WATER  IN T H I S  COLOR ON  DRYING  BAKER  AND  0.5  OF 5.7 M  H E A T E D NEAR  CONTENTS  OUT  TO P R E V E N T BEFORE  THE OVER  SODIUM  CONSTANT B O I L I N G REAGENT  TOP  TO Y I E L D WERE  AND  THE  HYDROLYSIS OUT FOR  WERE  A  THEN  BUBBLING  INSTRUMENTS, TUBES  THE  SEALING  FLAME.  CARRIED  FROM L A B - L L N E  ADAMSON  ML  THE  GAS-OXYGEN  CONTAINING  1 2 X 7 5 MM  DRAWN  ROUTINELY  WAS  DESCRIBED  IN  AND  1 0 " ^ TORR  DESSICATOR  USED  IN THEN  MIXTURE  HYDROLYSIS  AS  OF S O L U T I O N S  REDISSOLVED  WALLED  A MODERATELY  IN  ACETATE  ACETONE.  OUT E S S E N T I A L L Y  ALIQUOTS  E V A C U A T E D TO ABOUT  HYDROCHLORIC OISTILLED  CARRIED  GAS-OXYGEN  ILLINOIS.  DRIED  MIXING  OF D I S T I L L E D  A VIOLET-RED  T H E . T U B E S WERE  HEAVY  A DRY  IN  IN  BY  COULD  ,  (77).  TUBES ACID.  FINE,  PAPER  R E A G E N T MADE  5 0 ML  NINHYDRIN  OF P R O T E I N  HYDROCHLORIC A  THE  G OF CADMIUM  AND  PRODUCED  DRYING  ANALYS i s  HYDROLYSES BY  1  UV L A M P .  MINUTES.  20  D.  ACID  (w/v)  i  PEPTIDES  FOR  NINHYDRIN  CONTAINING  ACETIC  OF 0 . 5  A LONGWAVE  2 0 MINUTES  AN A C I D  OF G L A C I A L  REAGENT AT  ADDITIONAL  OF A S O L U T I O N  ML  150  UNDER  17  ON  HOURS  MELROSE  OPENED  HYOROXIOE.  THE  AZEOTROPE  GRADE  HYDROCHLORIC  -118-  AciD  FROM  ALLIED  DEIONI2ED  DISTILLED, WERE HOT  WASHED TAP  THEY ACID, FROM  2.  RINSED THE  AIR  ACID  DISSOLVED AND  THEN  RINSE  TO  FOR  APPLICATION  3.  AUTOMATED  AMINO AND  TO  2 THE  ANALYSIS  AMINO  A  OF  COMMERCIAL  THEIR  50  FROM X 8,  TIMES  DISTILLED  IN 1 M AND  WITH WATER.  HYDROCHLORIC  DRIED  DIRECTLY  CONTAINING CYSTEINE  PHOSPHATE THE  5 JU-  AND  FROM  AIR  BUFFER  FOR  0  F  ^  AT  FOUR THE  WERE  PH  6.5  HOURS  TO  SAMPLES  HYDROCHLORIC  M  WERE ACID  COLUMN.  AN  AUTOMATEO  SPACKMAN SYSTEMS  AND  THESE OF  INCLUDE  ON  HAVE  THE  THE  OF  EQUIPMENT  SEPARATION  CITRATE  VARY  OF  BUFFERS  AMINO  FOR (118)  BEEN  BASIS  MARKET  POLYSTYRENE RESINS,  SERIES  SINCE  THERE  INSTRUMENTS  ANCILLARY  SYSTEM  JIT_ AJ_ I N 1 9 5 8  IS STILL  INSTRUMENTS  SULFONATED A  OF  ANALYSES PERFORMED.  VARIETY  FEATURES  BY  REPORT  PROCEDURE  ACID  PROCEDURE,  COMMON  ELUTION DOWEX  ORIGINAL  DESIGN  HYDROLYSIS  OVEN.  ALTERNATE  OF  THE  ACID  AS -G*—CYSTINE.  CAME  MAJORITY  IN  C  ANALYSER  DETAILED  THE  BASIC  M TO  WITH  REPORTED  THIS  0.1  OPEN  ALTHOUGH'SEVERAL  NUMBER  SAME  FOR FOUR  WITH  OVERNITE  WITH  ANALYSIS.  FIRST  ACID  PH  USED  RINSED  RINSES  SAMPLES  RECOVERY  TO  TUBES  DILUTED  CYSTEINE.  STAND  COMPLETE  THE  OF  60°  LTD.  AND  STAND  IN A  I N 2 0 0 /J.L O F  ADJUSTED  FOUR  IN THE  HYDROLYSED  ALLOWED  INSURE  MORE  OXIDATION  DRIED,  BY  ALLOWED.TO  ONCE  THE  DETERGENT  FOLLOWED  THEN  CANADA,  WATER.  IN STRONG  WATER  WERE  CHEMICALS  OF  THE  ARE  NOW  WHICH  USE  PRIMARILY AVAILABLE. ACIDS  SIMILAR  TO  FOLLOWED  BY  BY  -119DETECTION ELUTED  AND  AMINO  COLORED  ACIDS  REACTION  RECORDED.  MIX  THE A  THIS  ELUANT  MIXTURE  REACTION  THROUGH  AND  THE  ABSORBANCE  AMINO  ACIDS  AND  AT  ORDER  IN  THE  ELUATE  OF  THE  COLUMNS  USED.  MOST  AND CAN AND  FLOW,  RECORDER  FLEXIBILITY  MAJOR  VARIABLES  MAJOR  VARIABLE  ARE  ROUTINELY  AMINO  DETECTION  LIMITS  POSSIBLE  ONLY  THEM.  THIRD  A  FOR  FOR  THE  IMINO  IONIC  DETECT  VARIABLE  AND  1  TO  SINCE AMINO  WHICH  HAS  AT  THE PHOTO-  FOR  THE  ACIDS. THE  IN THE  AMINO  ACIDS  TEMPERATURE OF  THE  BUFFERS  SYSTEMS  FOR  CONTROLLING NINHYDRIN CYCLE  IN THE  10  TO  BORE  I N THE  FOR  THESE  ACIDS PROVED  OF  THESE  TO  QUITE  SECOND  50  TO  REPORTED  LEVELS  NOT  THE  INSTRUMENTS  HAVE  NMOLES  COLUMN.  OF  THE  RANGE  THEY  THE  I S ONE  COMMERCIAL  ALTHOUGH  TO  ELUATE.  ACCELERATE  SYSTEMS  MOST  PUMP  NARROW  INSTRUMENTS.  QUANTITATION  THE  OF  PROGRAMMED  PROGRAMMING  FROM  BUFFER  STRENGTH  HAVE  IN A  1  THE  OF  AND  LINE '  IS RECORDED  CHANGES  COMMERCIAL  VARYING  ON  CELLS  REGENERATION  AND  M  WITH  S E P A R A T I O N OF  AND  SIGNIFICANCE TO  FLOW NM  THE  ACID,  TO  570  BY  AMOUNT  PHOTOMETER  COIL  BATH  TEMPERATURE  THESE  A  THE  A N I N H Y D R I N PUMP  AT  MACHINES  AND  A  REAGENT  OF  IS SENSITIVITY. USEO  LITTLE  WATER  OF  PH  IN THE  OF  OF  AND  OF  NANOMOLES  ARE  THE  AND  THROUGH  VARIED  COMMERCIAL  BUFFERS  THE  NM  DEGREE BE  SWITCHING THE  440  THIS  SERIES  WHERE  BY  OF  THE  REQUIRES A  COLUMN  OF  I N A' B O I L I N G A  REACTION  C A R R I E D OUT  WHICH  THE  METER  THE  ARE  PASSED  TO  THE  I S MEASURED  PROPORTION  THENCE  ON  NINHYDRIN REAGENT.  SYSTEM  I S THEN  TUBING  A  PROCEDURES  FLOWING  CONSTANT  TEFLON  WITH  PRODUCT  THESE  CONTINUOUSLY MOVE  QUANTITATION BASED  LIMITS  IT IS  QUANT I T A T E CRITICAL  IS  250  -120RELIABILITY THE  SYSTEM THE  SINCE  REQUIRES  WORK  WAY  IN  FOR  AMINO  THIS  LABORATORY  ACID  AVAILABLE  ACID  ANALYSIS ANALYSES  PROTEIN  AN  SINGLE  USING  ANALYSIS  QUANTITATION BRING  AT  THIS  OF  THE  OF  THIS  (52)  ALLOWS  THAN  HALF  THE  50  OR  BELOW  PROCEDURE  THE IN  1  STUDY AMINO  STUDIES  AND  IF  IS  LIMITING  FACTOR  THE  IN  WHICH  AREAS  THOSE  IN  PRESENT  OF  OF  ACID THE  NECESSARY  CLEARLY  LEVEL  WITH  BE  AMINO  USE  THE  DETERMINATION  MATERIAL  NMOLE  MUST  OTHER  LEVEL.  LINE  PROCEDURE  BY  SEQUENCE  NMOLE  UNDER-  REQUIRED  THE  THE  ANALYSIS.  WORK  UNDER  IF  LEVELS  OUTSTRIPED  OF  USE  GOOD  QUANTITIES.  PROTEIN  ADVANCES  FAR  AT  IT  ONE  SENSITIVE  PROTEIN  THE  LITTLE  OTHER  PROTEINS  ALL AT  AND  HIGHLY  LIMITED  OUT  EXAMPLE  LESS  A  I S OF  ACHIEVE  THESIS  EQUIPMENT  HAVE  METHOD  TO  THE  QUANTITIES  STUDY.  CHEMISTRY  PEPTIDES  THIS  HEART  CARRIEO  THE  As  IN  QUITE  AVAILABLE  DANSYL-EDMAN  SAMPLES  REQUIRED  ONLY  I S THE  FOR  ANALYSIS.  TO  IN  DETERMINING  AVAILABLE  SENSITIVITY  ANALYSIS. SINCE  ARE  COMMERCIALLY IN  FIVE  DESCRIBED  WERE  THE  INCREASED  THE  WAS  ON  FOR  A  ROUTINE ESSENTIAL  OTHER  AVAILABLE  TECHNIQUES. To NEWLY HAD  ACHIEVE  THIS,MODIFICATIONS  DEVELOPED THE  COMMERCIAL  HIGHEST  FLEXIBILITY  IN  SENSITIVITY  WERE  THE  MORE  COMMON  THE  RECORDER  EXPERIMENTS  PROGRAMMING.  7  LOW  A  THE  HIGH  (5.5MV  LEVELS  CARRIEO  FULL  BASES FLOW DEGREE SCALE  INDICATED  OUT  BlOCHROM  THE  SENSITIVITY  PATHLENGTH  MM) A N D  CIRCUIT AT  MACHINE,  AVAILABLE  LONG  WERE  AND OF  THIS  CELLS OF  THE  200,  A WHICH  DESIRED HIGH  (14  MM  VERSUS  AMPLIFICATION  DEFLECTION).  THAT  ON  THIS  SYSTEM  IN  EARLY WAS  -121I N A D E QU A T E ANO  THE  SINCE  SIGNAL  RELATIVELY  TO  SMALL  FLUCTUATIONS  IN  WAS  THE  THE  PHOTOMETER  TO  THE  THE  CHILD  SEMICONDUCTOR,  OF  THE  ONE  OF  OF  THIS  RESISTANCE D.C.  EXTREMELY WAS  STABLE  PROVIDED THE  A L L OWE 0  NOISIER  WERE  USED  FLOW  CELL,  WITH  A  U  THE SYSTEM 2  T03  TWO  TO  MM  NMOLE  COLUMN  ACIDS  WERE  SET  570 FLOV  NM  SIDE  THE  OF  THE  NOISE  MM  LEVEL THE  GAIN  ABSORBANCE 7  IN  CELLS  AND  AMPLIFIER.  USED  DESIGNED FA I R -  AT  FLOW  DRAINING VARIABLE  CIRCUIT,  WHICH  HIGH  HAVE  GAIN FULL  NM  AN  CIRCUITS THEY SCALE)  SEPARATE  570  ALLOWED  WHILE  IN THESE  THREE  WAS  VARIATION  SECOND  (55MV  IN  RECORDER,  BRIOGE  8 ASEL INE,  MERCURY  THE  THE  CIRCUIT.  THE  A  TUBES  PROBLEM  ( F P T 1OO,  GAIN  OF  THIS  WAS  OF  THE  RANDOM  C A L I F O R N I A ) WHICH  BRIDGE  RECORDER  WITH  CIRCUIT  A  THE  REDUCTION  OF  THROUGH  OUTPUT.  THE  PART  ENOUGH  POOR.  THE  GROUND  BY  RECORDER  CIRCUITS  WITH  A 440  CELL  ANO  AT  CARRIED  OUT  USING  14  MM  NM  CELL. IN TABLE  ACCEPTABLE LEVEL. SYSTEM  TO TO  THE  IN  A M P L I F I E D BY  VIEW,  GREAT  WAS  PH0TOMULTIPLIER  OPPOSITE  CONTROL  ON  SYSTEM  HIDDEN  NOT  PH0T0TRANSIST0R  SUPPLIED  ANALYSES  AND  OFF  THE  A MAJOR  A WHEATSTONE THE  STILL  DETECTION  MOUNTAIN  MEASURE  AT  THE  A  WAS  NEARLY  GREATLY  OUTPUT  ALLOWED WAS  WAS  ON  THE  UNDETECTABLE  IN  OF  OUTPUT  CURRENT  IN  DEVELOPED  RESISTANCE  ADJUSTMENT PART  SIDE  OF  LINE.  PROBLEM  ON  AS  NEWLY  WERE  NOISE  WHICH  THIS  RATIO  BASE  BASED  USED  A  NOISE PEAKS  INHERENT  OVERCOME  SENSITIVITY  QUANTITATION  As WAS  SEPARATED  I V WERE  INDICATED USED.  ON  A  52  THE  WAS IN  POSSIBLE  THE  ACIDIC  X 0.9  CM  TABLE AND  COLUMN  AT  THE  NEUTRAL  THIS  THE STANDARD AMINO  OF.B|0-RAD  -122AMINEX  RESIN  A-6  AT  PH  ANO  AS  DESCRIBED  3.25  AT  PH  52°  USING  C  0.2  PREPARED  4.40  IN  DISTILLED,  PH CITRATE  (AMINO  ACID  (A.A.A.  A  GRADE,  78.4  G  49.4  ML  30.0  ML  ACID  CANADA,  BUFFER  3.25  PH  TOTAL  RUN  TIME  M  SODIUM  0.2  AN  ADDITIONAL  ON  A  x  14  0.9  USING  C  PREPARED  PH  G  LTD.)  BIO-RAD.)  AND  52°  4.40  78.4  VOLUME  PH  WITH  3.25  ACID  MIN.  60  GRADE  REAGENT,  CHEMICALS  OCTANOIC  THE  WATER  CALIFORNIA)  HYDROCHLORIC  FINAL  DEIONIZED  DIHYDRATE  ANALYSIS  (BAKER-ADAMSON ALLIED  BUFFERS  LABORATORIES,  RICHMOND, CONC.  CITRATE  BELOW.  SODIUM BIO-RAD  SODIUM  N  A  AS  WAS  PUMPED  BUFFER  4.40 OF  AT  FOR  A  AN  0.5  ML  0.5  ML  4.0  L  4.0  L  RATE  OF  ADDITIONAL  HOURS  AND  40  HYDROXIDE  AND  RE-EQUIL  2  70  MINUTES.  BASIC  CM  COLUMN  BIO-RAD  0.35  OF  SODIUM  N  DESCRIBED  CITRATE  ACIDS  REQUIRED  WERE  SEPARATED  RESIN  A-5  BUFFER  FOR  REGENERATION  I B R A T I 0N  AMINEX  FOR  MIN.,  100  MINUTES.  AMINO  ML/HR  70  AT  PH  AT  5.28  BELOW.  5.28  SODIUM CONC.  CITRATE  HYOROCHLORIC  OCTANOIC FINAL  VOLUME  ELUTION TOTAL  OF  AT  A  &  GRADE,  A  137.2G  BIO-RAD)  REAGENT)  26  B|0-RAD)  ML  0.5  ML  4 . 0 L  TIME  NINHYDRIN PUMPED  (B  GRADE,  1  THESE  CYCLE  ACI0  (A.A.A.  ACID  (A.A.A.  DIHYDRATE  ACIDS OF  110  REAGENT  RATE  OF  35  AT  70  ML/HR  REQUIRED  70  MIN  AND  A  MIN. PREPARED ML/HR  AND  AS  INDICATED  MIXED  WITH  BELOW THE  WAS  ELUATES  FOR  180a  AAA—, 15K'  6.8K< IN4004  Recorder  10K  ne  5F  10K  M  AAA-t 20K 20K  20K  AAA—±  He 5 F M  FFTIOO  —  ,  FPT100  Recorder  r  Recorder  IN4004  AAA—I 180a  FIGURE  45  IMPROVED CIRCUIT  PHOTOTRANSISTOR OIAGRAM.  DETECTOR  FOR  AMINO  ACID  ANALYSER—  -123-  D E T E C T I ON.  N I N H Y D R I N REAGENTt •  METHYL CELLOSOLVE (SEQUANAL GRADE, PIERCE  CHEMICAL  3  L  1  L  80  G  CO.,  • --Ro:G^|i|0»-O!ifeysiiL4,N:O I s) • • • 4  M SODIUM  ACETATE. ( F I S H E R  NINHYDRIN STANNOUS (A.A.A.  AND  THE  WAS  REAGENT  OF  CAREFUL  A  ANALYSES  COULD  ALTHOUGH ORIGINAL AND  THE  OF  THE  TO  BE  GAIN  AMINO MUCH  THEREFORE REDESIGN WHICH  WAS  SYSTEM. THE  SHOWN  NOW  SYSTEM  WAS  A  OF  MARKED  THE  IMINO  ACID  ON  TO  REST  INCREASE  IN FIGURE TO  HAMILT0N(51)  COME HAD  THE  THE  45  THE  GAIN  AND  TO  PRIMARILY  PRESENTED  ENCOUNTERED  IN HIGH  A  THESE OF  5  THUS  WITH  PROCEDURES WORKING  PRODUCT  OF  MIN  TWO  DAY.  TOO  OVER  THE  HIGH,  U N S A T I S F A C T O R Y WHEN  T H E PHOTOTRANSISTOR THE  UNDER  STILL  WAS  PARTICULARLY  30  IMPROVEMENT  WERE  BASELINE  FOR  HOUR.  NORMAL  REQUIREMENTS  THAN  KNOWN  DIFFICULTIES  AUTOMATED  IN A  USED,  6  G  WITH  A MINIMUM  ALMOST  OUT  IN THE  LTD.)  OF  CARRIED  ACIDS.  DECIDED  TOTAL  ALR  MIXTURE  REQUIRED  OF  QUIETER  AS  A  THE  NITROGEN.  USE  LEVEL  ABSORBANCE  UNDER  FULL  THIS  WAS  LIQUID  ANALYSIS  MATERIAL  NOISE  MAXIMUM SINCE  THE  AND  1.60  BUBBLING  STORED  AND  BE  BY  CANADIAN  COMPLETE  PLANNING  CHEM.)  BIO-RAD)  WAS  PROTEIN  PIERCE  GRADE,  DIHYDRATE  REMOVED  ( L GRADE,  CONDITIONS NMOLES  CHLORIDE GRADE,  OXYGEN NITROGEN  (REAGENT  CERTIFIED)  440  CHANNEL  IS LESS  CIRCUIT SYSTEM  IN THIS CUT FROM  DOWN THE  DETAILED  THAN  HAD  AND  PROVED  IT  CIRCUIT THE  THAT  WAS BY  NOISE  HYDRAULIC DISCUSSION  SENSITIVITY  ANALYSIS  OF  -124-  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 ( 1 1 7 ) 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 CONFIGURATION 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 REQUIREMENTS FOR THE VOLTAGE STABILIZER SUPPLYING THE LAMPS TO ABOUT  -125ONE  FOURTH  WHICH  HAD  OF  ITS  OCCURED  IT  ALSO  TO  INSTABILITIES  SYSTEM  RANDOM  IN  THE  ADDITIONAL  WAS  THE  DETERMINATION PROCEDURE  TO OF  THAT ALL  REQUIRED  IONIC  STRENGTH  BELOW  AND  (0.8  ELEVATION  WHICH AN  OF  DEVENYI  VETWEEN  THE  ACCELERATED  NOISE  OPERATIONS. CHANNELS  OF  IN  A  SODIUM  MATERIAL  SINGLE  WHICH  (38)  ACIDS  USE  OF  LAMP  SWITCHING  HALVED  OF  IN  N  DURING  ELIMINATED  DUE  LAMPS.  AMINO  THE  AND  VARIATIONS  CHANCE  ADOPTION  SIMILAR  CAPACITY  PREVIOUSLY  ELIMINATED  AN MENTS  RATED  COLUMN  ALLOWED  A  SINGLE  RUN.  THIRD  BUFFER  WITH  IONS)  PREPARED  OPERATING  REQUIRE'  TEMPERATURE  AS  TO  THIS INCREASED SHOWN  58°  C.  THE  160  G  PH 6 . 5 5 SODIUM  HYDROXIDE  CITRIC  ACID  OCTANOIC .  FINAL  (A.A.A.  PUMPING  RATES FOR  TO  AT BE  THIS  INCREASED  NINHYDRIN. THE  ELEVATED.  QUANTITATIVE  AMINO  OF  THE  ANALYSIS  WAS  PH  60  THESE 5  NMOLE  WAS  REDUCED MIN;  PH  TO  6.45,  NOT  A  50  FOLD  POSSIBLE  TO  95  ML/HR  80  OF  ACID  TIME  LESS  TEMPERATURE  SYSTEM  SAMPLE  MODIFICATIONS  LEVEL,  STILL  OXIDIZED  PROTEIN.  TO  THIS  COMPLETE  4.40,  0.5  BIO-RAD)  WAS  FOR USED  SALMON  ANALYSIS  REQUIRED 5  MIN;  RECYCLE,  ALLOWED INCREASE WORK  AT  HOURS  ROUTINE IN THE  BUFFER FOR  CT  ANO  ON  AS  FOR  THAN  ALLOWED  A  (PH 70  AND  THE  LITTLE  AS  COMPLETE 3.25,  50  MIN;  MIN).  OPERATION  NMOLE  THE  ALLOWED  SENSITIVITY, 1  M  L  4  OF  NMOLES  259 c  BIO-RAD)  GRADE,  ANALYSES  2  REAGENT)  VOLUME  VISCOSITY  ML/HR  CERTIFIED  GRADE,  (A.A.A.  ACID  REDUCED  40  (FISHER  AT BUT  LEVEL.  THE IT  - 1 2 6 -  BASELINE PROBLEM NOISE  NOISE SINCE  FREE  FROM THE  AND  THE  HYDRAULIC  S O L I D STATE  COULD  PROVIDE  SYSTEM  WAS  ELECTRONICS ALMOST  THE  MAJOR  WERE E S S E N T I A L L Y  UNLIMITED  GAIN.  LINEAR  AMPLIFICATION  OF  THE  OUTPUT  SIGNAL  HOWEVER, M A G N I F I E D  NOISE  AS  THE  SIGNAL  DUE  THE  AMINO  ACID  AS  WELL  CALCULATIONS OF  T H I S SYSTEM  (L/L OF A  OF  = E~  BASED  THE  TRANSMITTED  LIGHT  (L)  THAT  OF  (c)  AND THE  THE  FUNCTION  OF  COMPOUND,  PATHLENGTH  QUANTITY  MEASURED,  NMOLE  FROM  PARTICLES LARGE  WHICH CAUSE  CHANGES  TIVELY  SMALL  SIGNAL  TO  WITH  AN  BECOMES  IS 1 0 $ 0F  EFFECT. RATIO WHICH  SUCH  DIRECTLY  46  DESIGNED USE  SHOWS BY  WITH  OF  THE  THE  BY  ARE  DIAGRAM  ELECTRONIC  X ARE OF  INTENSITY ( L ) IS  OF  Q  THUS  CONSTANT, CONCEN-  FROM  BUBBLES  HAVE  ONLY  AN  FUNCTION THAT  T01  AND BUT  A  RELA-.  IMPROVE  OF  PEAK  ITS  INPUT.  HEIGHT AND  LONGER  THAT EXAGGERATED.  A LOG-AMPLIFIER  DETECTION  THE  AMPLIFIER  S E R V I C E S , VANCOUVER,  PHOT0-TRANSIST0R  0  RECORDER  BASELINE,  USING  NO  (JJT) OF  COMPOUND  THE  IN A  CONCENTRATION  BASELINE  CIRCUIT  THAT  P O S S I B L E TO  A L O G - A M P L I F I E R MEANS  TECHCAL THE  IS THEREFORE SYSTEM  THE  CHANGE  IN THE  IT  THE  AND  Q  SOMETIMES  TO  OF  RESULT  IS A LOGARITHMIC  V A R I A T I O N S NEAR  FIGURE  l  SCALE.  FLUCTUATIONS  PROPORTIONAL  OF  FUNCTION  BOTH  FULL  LAW  C O E F F I CENT  A CONCENTRATION  2 0 NMOLES  LARGE  NOISE  OF  WHEN JJt,  I S A LOG  ELUATE  INCIDENT LIGHT  ABSORPTION  IN CONCENTRATION  OUTPUT  USE  SMALL  1 5 TO  RATIO  CONCENTRATION  (x).  IN T H I S S Y S T E M  DEFLECTION  THE  THE  TRATION, AND  TO  PRODUCTS.  I N THE  BEER-LAMBERT  THAT  X  DISSOLVED  FOR  THE  STATES  C  THE  THE  ON  WHICH  U  NINHYDRIN  CONCENTRATIONS  )  0  LOGARITHMIC  I,  ARE  TO  THE  SYSTEM.  B.C.  -127FURTHER  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  (FlSHER  REAGENT  GRADE).  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 INCONVENIENCE.  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 NINHYDRIN ALLOW COMPLETE 4  AMINO  ACID ANALYSES IN SLIGHTLY OVER  HR ON SAMPLES OF LESS THAN  THE LOG-AMPLIFIER IS USED. MEANS  THAT THE AUTOMATED  1  NMOLE  WHEN THE FULL GAIN OF  THE DECREASED-ANALYSIS TIME ALSO  SYSTEM  ANALYSES DURING A NORMAL DAY.  ALLOWS  COMPLETION OF THREE  A FURTHER ADVANTAGE OF THE  - 1 2 8 SYSTEM AND  IS  THAT  THE  CONCENTRATION  TRATION PEAK THE  FROM  HEIGHT USE  AREA ARE  ALLOWS  BETWEEN  OF  E.  BETWEEN  IN  THE  AND  N— T E R M I N A L  TWO  AMINO  PROCEDURE  OF  THE  SAMPLE  TO  OF  PEPTIDE  CLEANED  CHEMICAL, 1 0 JJJ-  OF  SULFONYL REAGENT SEALED FOR  2  ACID. ACID AFTER  AFTER  CAL  DRYING AND  HOURS  TEDIOUS OF  OF  IN  FOR  PROCESS  OF  SHOWN  AREA  NO  BY OF UNITS  AND  SIGNIFICANT  OF  DANSYL  DETERMINED AND  5 0  WAS  BY  HARTLEY  MM  PER A  MODIFICATION AN  ( 4 7 ) .  APPROXIMATELY  PYREX  CULTURE  OESCRIBED  DRIED  SODIUM  IVATTVES.  IN  1  TUBE  FOR  NMOLE WHICH  THE  VACUUO  AND  BICARBONATE  OEIONIZED;  ALIQUOT  (BAKER  DISTILLED  WATER.  ( 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 ~ ANGELES)  MG/ML)  WAS  THEN  THE  THE  IN  INTEGRATING  LOS  5 0 JUL  CONCEN-  COMPENSATED  HAVE  HEIGHT  VARIATION  BLOCHEM,  IN  OF  ACCURACY  METHODS  M  0.2  (1  DRIED  X  4  SAMPLE  GRADE)  ACETONE  ADDED  THE  WERE GREY  A  CHLORIDE  AND  OF  SAME  OF  PARA F I L M .  HOURS  FOUR  IN  THE  CHLORIDE,  WITH  THE  PEAK  METHODS.  OF  THE  1 0 JU.L  REAGENT  GRADE  WAS  BY  TUBES.  DANSYL  BE  BE A N A L Y S E D C O N T A I N I N G  PLACED  IN  AND  SYSTEM  ACIDS  DANSYL  REDISSOLVED  CAN  IDENTIFICATION  THE  WAS  HEIGHT.  INACCURACIES  THIS  OF  HYDROLYSIS  ACIDS  THE  BETWEEN  DETERMINATION  PEAK  FACTORS  AND  MEASUREMENTS  BEEN  OF  COMPARISONS  P R E P A R A T I ON  HAD  DIRECT  AMINO  CORRECTION  ELIMINATED.  VARIATION  RELATIONSHIP  MEASUREMENTS  CALCULATIONS  HEIGHT  LINEAR  A  TUBE  ADDED  SAMPLE  WAS  VACUUM  DESICCATOR  OF  CONSTANT DRAWN  HYDROLYSIS  AT  OUT  IN  THEN  AND  1 0 5 ° C  THE  INCUBATED OVER  BOILING AND  FLSHER  3 7 ° C  SULFURIC  HYDROCHLORIC  FLAME THE  AT  TUBE  SEALED.  TUBE  WAS  OPENED  -129AND  DRIED  RESIDUE  IN  WAS  A  VACUUM  THEN  DISSOLVED  ACETIC  ACID  MIXTURE  PLATES  USED  FOR  THE  WAS  MODIFIED  BY  TRADING  SYSTEMS  NATURAL  AMINO  AMINO FROM  SQUARE USED  TIME  ANO  COST,  COST  THE  IS  IN  CONTAINING  SIDE  THE.  LISTED  LTD.  SAME  WOODS  HYDROXIDE.  TO  THE  P O L Y A M I DE  BASIC  FROM  A  TO  THE  SAMPLES.  SINCE  IS  REALLY  ONE  TO  50  POINT.  IS  FOUR  FOUR  THE  TH I S CAN  IS  OF  THE SQUARE  M  0.5 EACH  0.5  TO  BOTH  WASHED  SAMPLE  APPLIED  SOLVENT  ALL  REDUCES BE  PLATE  SIMILAR  OF  CM  5  SIGNIFICANT.  OF  PMOLES  PLATES  SAMPLE.  NINE  PLATES  A  TWO-  USING  PICOMOLE/^LL  60  SIDE  THE  INTO  AS  (132)  POLYAMIDE  SINGLE  CUT  THOUGH  DERIVATIVES  IDENTIFICATION  ARE  C O R N E R , A N D  10  ON  TAIWAN)  POSI T I V E  NOT  WANG  PROCEDURE  TAIPEI,  RUN  DANSYL  AND L  0  F  DANSYL  CM  IN  OF  THE  THE  SYSTEMS  OPPOSITE  USED  ARE  BELOW.  1.  1.5^  2.  AQUEOUS  FORMIC  ACID  3.  BENZENE, ACETIC ACID, 9*1 N-HEXANE, N-BUTANOL, ACETIC  4.  0.1  THE  BASIC  M  AMMONIUM  HYDROXIDE,  TWO-DIMENSIONAL  ACID,  3*3*1  ETHANOL,  RUN  CONSISTS  9*1 OF  IN  DIMENSION  (1)  FOR  3  TO  5  MINUTES,  FOLLOWED  IN  DIMENSION  (2)  FOR  5  TO  7  MINUTES.  EXTREME  THE  PLATE  TAKEN  THE  ACETONE-GLACIAL  AND  PLATES  TO  ONE  UNKNOWN AT  THE  CONTAINING  APPLIED  SIDES  OF  THE  CHROMATOGRAPHY  FACTOR  SOLUTION  ACID  AN  APPLICATION  DERIVATIVES  ARE  STANDARD  SODIUM  OF  IDENTIFYING THAT  CO.  WHICH  THE  JAL  10  FOR  WHICH- ALLOW  ACID  CM  15  RUNNING  A  TO  THIN-LAYER  SOLVENT  REUSED  FOR  HARTLEY(52).  (CHENG-CHIN  PLATES  USED  SIMILAR  DIMENSIONAL,  ORIGINAL  (3*2)  IN  OVER  SEPARATION.  SYSTEM  PRODUCED  DESSICATOR  TO  DRY  THROUGHLY  AFTER  SOLVENT  SOLVENT BY  SOLVENT  CARE ( 1 ) .  MUST  (1) (2) BE  •P •V  BY BH BK# M  BH M « BK«  NH  W  A0  #  *K  # T #  NY OY«E »S * •D  #  H  _Q±L  -M, t 2 .  (A)  G  aH R  OH  '  (B)  t2, t3.  NH  NH >aH »R  OH OH (C)-»-1, t 2 , t1  (0)  t2, t3, t4.  NH UK  OH  (E) FIGURE ON  MIAGRATION  47.  POLYAM IOE  D E S C R I B E D S I N G L E  IN  L E T T E R  FOR  MOST  OH,  DNASYL  L A Y E R S T H E  IN  TEXT  NOTATION  D E R I V A T I V E S . H Y D R O X I D E ;  O F  t 2 , t4. DANSYL  V A R I O U S ANO WERE FOR  T H E  O F  AMINO  S O L V E N T S  RUN  D I M E N S I O N S  IN  P A R E N T  A D D I T I O N A L NH,  D E R I V A T I V E S  S O L V E N T S .  DANSYL  T H E  AMINO  A C I D S  A B B R E V I A T I O N S A M I N E ;  BY,  A C I O S  A R E  A R E  INDICATED, USED  I N C L U D E ;  B I S - D N S - T Y R ;  BH, B i s - D N S - H i s ; BK, B i S - D N S - L Y S ; C A , D N S - C Y S T E I C NY, N - D N S - T Y R ; OY, O - D N S - T Y R ; a H , a - D N S - H l s ; e K ,  A C I D ; e-DNS-LYS.  -1 3 0 FlGURE SPOTS OF  VISIBLE  PROL INE,  GLYCINE, TIVELY  AS  CAN  OF  SERINE?  AT  THIS  AT  AND  MAY 3  TO  SPOTS  STAGE  THE  SOLVENT ONLY  SINCE THE  ACID  IT  THE  (FIGURE  MAY AND  A  DERIVATIVES BETTER  BE  AFTER  WASHED  WASHING  OF  TWICE  BE  RUN  THE  SHOWN  IF  DERIVA-  THE  (2)  AND  (4)  ARE  AND  IS  CC— H I S T I D I N E  DIMENSION  BE  BY  THIS  REUSE IN  A  PROCEDURE  IF  IS  AS  SHOWN  FOR  ANO  BY  STANDING  THESE  M  (E).  47  OVERNITE 1  ARE  PREFERABLE FlGURE  IN  OF  (2)  RUNNING  ( 2 ) , AND  MIXTURE  LARGE  MINUTES  5  E-LYSINE  SOLVENT  HIDDEN  HYDROXIDE  CC— H I S T I D I N E , SEPARATED  (B).  47  UNRESOLVED.  TO  5  POSI-  BE  STILL  DANSYL  IN  CYSTINE,  7*HE  STREAK  FOR  FROM  FlGURE  IN MAY  FROM  IN  THREONINE  E-LYSINE  SEPARATION FOR  IDENTIFICATION  DERIVATIVES  SOLVENT  ALSO  IF  HYDROXIDE.  DERIVATIVE  DIMENSION  47D).  MAY  ARE  SEPARATED  RUNNING  IMMEDIATELY  GIVES  (1)  IN  OERIATIVES IDENTIFIED,  SEPARATE ACID:  POSI-  BE  MAY  (3)  BE  01— H I S T I D I N E ;  STREAK  DERIVATIVES  BY  EXPECTEO  PLATES  SOLVENT  ( 1)  DERIVATIVES (4)  ACID  BE  ALSO  DANSYL  OC— H I S T I D I N E MAY  CAN  AND  STAGE  HYDROXIDE  SOLVENT  MINUTES  5  CYSTEIC  AND  SEPARATED  THIS  CAN  DI-DANSYL  TO  DERIVATIVES  PHENYLALANINE,  THE  ASPARTIC  FROM  FLOURESCENT  DANSYL  ALANINE  SOLVENT  E-LYSINE  AT  DANSYL  47C).  ARGININE  THE  FROM  FROM  THE  HYDROXIDE.  MINUTES  8  0-TYROSINE  CYSTEIC  BE  ACID  THE  RUNNING  STAGE  ABOUT  GLUTAMIC  E-LYSINE  (FIGURE  THIS  FOR  AND  BY  POINT.  DANSYL  THE  DANSYL  AND  AND  BENEATH THE  THIS  THE  VALINE,  AMINE  ARGININE  OF  LEUCINE,  TRYPTOPHAN  AT  OF  STAGES.  HISTIDINE  (2)  OC-TYROSINE  TWO  AND  MADE  DIMENSION  POSITIONS  LYSINE  DANSYL BE  THE  THESE  METHIONINE,  TYROSINE,  TIONS  AFTER  IDENTIFIED  CANNOT  SHOWS  I S O L E U C I NE ,  OF  TIVES  (A)  47  IN  AMMONIUM  -131HYDROXIDE THE THE  AND ACETONE, STANDARD  AMINO  DANSYL  6.5 /J.MOLES  1  ML  O F 0.1  M  1  ML  OF DANSYL ALLOWED  OF  SODIUM  STAND  WITH  CONTAINED  HISTIDINE  MADE  U P TO  SOLUTIONS SOLUTION  F.  WERE USED  A  5  X 7 5 MM  10  ML  OF MONOMER  4.6 NIUM  (0.5$  N ACETIC  TUBES  FROM  FROM  ALIQUOT  THE BIS-.  HYDROCHLORIC  CALBlOCHEM,  VOLUME  LOS ANGELES  IN ACETONE.  TO  PRODUCE  THE  THE  ABOVE  STANDARD  CHROMATOGRAPHY.  (61). USING  (409$  v/v  w/v  THE GELS  1 ML  OF  A  CARRIED  WERE  PREPARED  SOLUTION  ACR I L A M I OE » 0 . 6 $  IN DISTILLED  OUT  WATER),  CONTAINING w/v 1 0 ML OF  N , N , N' N ' - TE TRAM E THYL E NE D IAM IN E I N  AND 6  P E R S U L F A T E ) FOR EACH  PURCHASED  M  G E L E L E C T R O P H O R E S I S WAS  STOCK  ACIO)  AN  GEL E L E C T R O P H O R E S I S .  1  1  SOLUTIONS  PRIMARILY 5.7  MIXTURE  BICARBONATE  PERJ4.L.  CONCENTRATION  N ,N - M E T H Y L E N E B I S A C R I LAM I D E CATALYST  THE  WITH  T H E CC-H I, S T I 6 I N E D E R I V A T I V E ( 4 7 ) .  IN EQUAL  OF JOHNS  GLASS  IN  FOLLOWING  ANO MIXED  T H E STOCK  CONTAINED  FROM  DISSOLVED IN  (6 MG/ML).  HYDROLYSED  IN THIN-LAYER  PREPARED  BY THE WERE  DERIVATIVE  PURCHASED  P O L Y A C R I L A M I DE  IN  WAS  MIXED  THE METHOD  WERE  AND THE SODIUM  WHICH  SIMILAR  POL Y A C R I LAM I DE  USING  OF  TO P R O O U C E  WAS  ACID  OF ACETONE.  SAMPLE  FOR 17 HOURS  ANGELES)  IN ACETONE  OVERNITE ML  DERIVATIVE  S-DANSYL-LYSINE AND  8  USED  BICARBONATE. SOLUTION  650 PMOLES  THE HISTIDINE  ACID  ACIDS  OF AN A M I N O  CHLORIDE  TO  PRECIPITATED THUS  AMINO  A C I D S ( C A . L B I O C H E M , Los  PROCEDURE.  WAS  1:1.  EASTMAN  ML  OF C A T A L Y S T  TUBE.  ORGANIC  2  ORGANIC CHEMICALS,  (0.6$  w/V  REAGENTS  AMMO-  WERE  ROCHESTER,  N.Y.,  -1 AND  AMMONIUM  SOLUTION BEFORE  PERSULFATE DEGASSEO  WAS  BEING  PLACED  LAYERED  CAREFULLY  ON  PUMP  ELIMINATE  THE  TO  POLYMERIZE. MODEL GELS WAS  PLACED  TOWARD  0.01  M  THE  ACETIC  EQUILIBRATION OF  PROTEIN  ACETIC  ACID  VISE  ACID  THE  PROTEIN  CHICAGO THE  lN  1 2 . 5 $  FOR  WAS IN  FOR  WAS-'-, T H E N  THE  GELS  ALLOWED  IN  1  OF  OUT  THE  AT  WERE  1 2 . 5 $  TCA  IN  STAIN (COLAB 1 2 . 5 $  GELS  SHANOON ONTARIO). MIGRATION  AFTER  THE  BUFFER IN  2 0 JA.G  0 . 0 0 2  M  AND  THE  FIELD  FOR  2 5  MIN.  3 2 0 v  FOR  7 0  MIN  FOR USED  CRACKED  3 0  MIN  WAS  TO  0 . 0 5 $  LABORATORIES,  WERE  WITH  GEL  THEN  TCA  TO  EQUILIBRATED  FRESH  VOLT  A  SAMPLE  SUCROSE  M  3 2  IN  VOLTS.  TUBES  R250  THE  WERE  WITH  INFUSION  WESTON,  THAT  3 2  TOP A  THE  ILLINOIS) SOLUTION  OF  IN  THE  STAINING. BLUE  SO  AT  OUT  L T D . ,  GELS  THE  WAS  AND  CARRIED  PLACED  BRILLIANT  STAINING  TO  WATER AN  CARRIED  GEL  OESSICATOR  WITH  REPLACED 1 0 JJ.L  VACUUM  GEL  CANADA  THE  A  THE  THE  WAS  HOURS  3  SHARPENED  GELS  TCA.  AND  REMOVED.  HEIGHTS,  IN  OF  IN  GRADE.  DISTILLED  APPARATUS  APPLIED  ZONE  THE  COOMASSIE  THE  BUFFER  WAS  TUBES  AND  TOP  (COLAB  CATHODE  ELECTROPHORESIS THE  IN  DISSOLVED  APPLICATION  AND  TUBES.  MINISCUS,  APPARATUS  REAGENT  MINUTES  3 0  THE  THE  FlSHER  ELECTROPHORESIS  1 2 7 3 4 WERE  WAS  FOR  IN  32-  ( 2 2 ) .  A  FIX (w/v) INC.,  AFTER  OESTAINED  IN  1  HOUR  OVERNITE  -1 33SiNGLE  G.  LETTER  A  -  ALANiNE  B  -  ASPARTIC  C  -  CYSTEINE  D  -  ASPARTIC  ACID  E' -  GLUTAMIC  ACID  F  -  PHENYLALAN1NE  G  -  GLYC1NE  H  —  HISTIDINE  I  -  ISOLEUCINE  K  -  LYS1NE  L  -  LEUCINE  M  -  METHIONINE  N  —  ASPARAGINE  P  -  ACID  NOTATION  OR  FOR  ASPARAGINE  PROLINE  Q - G L U T A M I N E R  -  ARGI  S  -  SERINE  T  -  THREONINE  V  -  VALINE TRYPTOPHAN  WX  NINE  -  UNKNOWN  OR  "OTHER"  Y - T Y R O S I N E Z  -  GLUTAMIC  ACID  OR  GLUTAMINE  AMINO  ACIDS.  (59)  

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