UBC Theses and Dissertations

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UBC Theses and Dissertations

A study of unknown sulfur containing components of proteins Black, F. L. 1949

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L £"!> £7 mi  A STUDY OP UNKNOWN SULFUR CONTAINING COMPONENTS OF PROTEINS  by  P. L . B l a c k  A Thesis  submitted i n p a r t i a l  fulfilment for  of the requirements  the degree o f Master o f A r t s  i n t h e department  University  of chemistry  of B r i t i s h  May 1949  Columbia  ABSTRACT  A review of i m i d a z o l e s .  i s made o f t h e b i o c h e m i s t r y o f s u l f u r a n d  The r e s u l t s  the f r a c t i o n a l a n a l y s i s piled  t o the presence  examined.  of t h i o l i m i d a z o l e s has been suggests  presented  t h a t many o f t h e c y s t i n e  breakdown p r o d u c t s ,  of the chemistry  of the t h i o l i m i d a z o l e s i s  and t h e o r i g i n o f t h e r e c e n t r e v i v a l  of interest  t h e s e compounds d i s c u s s e d , A method h a s b e e n d e v e l o p e d  to  criti-  i n t h e l i t e r a t u r e may b e h i g h d u e t o  interference of thiolimidazole A review  in  that a p o r t i o n of the p r o t e i n  The s t u d y  determinations recorded the  i n p r o t e i n s haye b e e n com-  a t p r e s e n t u n a c c o u n t e d f o r i n c e r t a i n p r o t e i n s may be  ascribed cally  of sulfur  and t h e p o s s i b i l i t y  sulfur  o b t a i n e d by v a r i o u s workers o n  separate  added e r g o t h i o n e i n e f r o m  whereby i t i s p o s s i b l e those  fractions  of a  p r o t e i n h y d r o l y s a t e t h a t i n t e r f e r e w i t h the Hunter d i a z o It  has a l s o been found  that t h i s betaine i s destroyed by b o i l -  ing with hydrochloric acid agents attempt ensure  i n the presence to f i n d  were t r i e d a procedure  o f p r o t e i n breakdown p r o d u c t s .  i s e v o l v e d no f i n a l  w h i c h would  complete  success.  Until  such  c o n c l u s i o n c a n be r e a c h e d a s  o r absence o f t h e t h i o l i m i d a z o l e r i n g  molecule.  I n an  o f t h e t h i o l i m i d a z o l e r i n g many methods  but thus f a r without  to the presence protein  and w i t h many o t h e r h y d r o l y z i n g  a s u i t a b l e h y d r o l y z i n g agent  the s t a b i l i t y  test.  i n the  ACKNOWLEDGEMENT  I w i s h t o e x p r e s s my g r a t i t u d e E a g l e s under carried  whose g u i d a n c e  t h i s work h a s b e e n  out f o r the assistance  made i t p o s s i b l e .  t o Dr., B. A.'  and a d v i c e  that  TABLE OP  CONTENTS  Page Abstract I.  Historical 1.  A review  of the biochemistry  P o l k Lore  • • • • • • • • • •  Quantitative  • • • •  ••  Methionine  • • • . 2  . . . . . . . .  Biotin  •  4  6 •  8  . . • • 11  • • • • • • • • • • • •  Mucin • • • • • • • • • • • • •  . . . . •  • • • • • • • • •  Asterubln  3  9  Glutathione  Heparin  • • • •  • • • • • • • • • •  Thiamin  Taurine  2  . • • • • • • • • • • • • •  1 1  Sulfates Cystine  •  • • • • • • • • • •  Alchemistry Early  of s u l f u r  • • • • • • • • • • •  Toad P o i s o n s  . . . . . . . . .  12  . . • 13 . . 13  •  • • • I S  . . • . 14  Sulfones  14  Lanthionine  • • • • • • • • • •  A Thio Ether  . . . •  Djenkolic  acid  • • • • • * . .  Urochrome  • • • • • • • • • • • •  . . . .  14  • • 15 • • • • 15 • • • 16  Page Penicillin  • • • • • • • • • • • • • • •  1  Thio-sugar  • • • • • • • • • • • • • • •  16  Odours and F l a v o u r s Thiocyanates  2.  • • • • • • • • • • 1 6  • • • • • • • • • • • • • • l"  S y n t h e t i c Drugs  • • • • • • • • • • • •  18  Sulfur  • • • • • • • • • • • •  19  Bacteria  A review  of the biochemistry  imidazoles Purines  of the  • • • • • • • • • • • • • • •  19  • • • • • • • • • • • • • • • •  19  Histidine  • •  Histamine  • • • • • • • • • • • • • • • 21  Carnosine Creatine  20  and A n s e r i n e • • • • • •  Allantoin  • • • • • • • • • 2 2  • • • . • • • • • • 25  • • • • • • • • • • • • • • •  Pilocarpine  3«  6  23  • • • • • • • • • • • • • • 2 4  Biotin  . . . . . . . • • •  24  Trimethyl Histidine  . . . . . . . . . .  25  A review  of the chemistry  thiolimidazoles  of the  • • • • • • • • • • • • 2 5  Ergothioneine  from Ergot  • • • • • • • • 2 5  Ergothioneine  from Blood  • • • • • • • • 27  Isolation  « . . . . . . . . . . . . .  3  N u t r i t i o n Experiments Physiology  •  • • • • • • • •  . • , ... ,  t  #  29 #  30  • • . . . . 33  Pathology . • . • • • • • . • • • • • • 35 Anti-thyroid Effect. .36  7  Page  II, III*  Analysis  . . . . . . . • . • • . • • •  Snthesis  . . .  • • • • • • • 4 2  C o n s i d e r a t i o n of Problem Experimental  • • • • • • • • • 4 5  . • • • • • • • • • • • • • • 6 5  Preparation of ergothioneine Synthesis  of t h i o l h i s t i d i n e  • • • • • 63 • • • • • •  65  I n i t i a l work w i t h p r o t e i n h y d r o l y s a t e s  66  R e v i s i o n o f t h e method  • • • • • • • • 6 9  I n t e r f e r e n c e b y Amino A c i d s  • • • • • •  Separation of T h i o l i m i d a z o l e s from Hydrolysates « ^ Separation of Ergothioneine Amino A c i d s Further  • • • •  0  Bibliography  • • • • 78  • • • • • • • • •  . • • • • • • • • • •  Summary a n d C o n c l u s i o n s  * 75  from  Studies of Hydrolysates  Grote's Reagent  74  • • • • • • • • • • 7 7  Methods o f H y d r o l y s i s  IV.  40  • • • » • < » * & * ( ;  79 80 81  • • • • • • • • • • • • • • • 8 3  A STUDY OP UNKNOWN SULFUR CONTAINING COMPONENTS OP PROTEINS  I  Historical  1*  A Review o f t h e B i o c h e m i s t r y Folk  of Sulfur  Lore The i m p o r t a n c e o f t h e r o l e o f s u l f u r i n t h e f u n c t i o n -  ing of l i v i n g early  things  has been r e c o g n i z e d  G r e e k s when H e r c u l e s was  cleansing  t h e s t a b l e s o f Augea*  s a i d t o have u s e d Sulfur  springs  o f t h e w o r l d have m a i n t a i n e d r e p u t a t i o n s s i n c e t h e time o f our e a r l i e s t used  records  sulfur i n i n various  of great  healing  recipes sulfur  (1)  P o s s i b l y t h e most  i s t h e s u l f u r and m o l a s s a s containing  small  parts powers  and s u l f u r has been  s i n c e time immemorial i n a v a r i e t y o f f o l k r e c i p e s  i n t e r n a l medicament.  very  s i n c e the time of the  as a n  famous o f t h e s e  "spring tonic".  I n a l l these  pharmaceuticals the a c t i v e p r i n c i p l e i s a  f r a c t i o n r e d u c e d t o t h e -2 o r -4 v a l e n c e .  of the s u l f u r c o n t a i n i n g  medicaments  i s probably very  e s t i m a t e d by t h e populace but p h a r m a c e u t i c a l t e x t s 1  The  value  much  over  recognize  2 them a s v a s c u l a r  stimulants  and n e r v o u s s e d a t i v e s when u s e d  e x t e r n a l l y and a s l a x a t i v e s i n t e r n a l l y ,  (2)  Alchemistry The chemistry  confusing  cept  Each alchemist  literature  salt  h a d h i s own i n t e r p r e t a t i o n o f t h e  on t h e s i g n i f i c a n c e  These t h r e e  seven metals. occurred  i n an understandable  of inflamabllity  Other a l c h e m i s t s  the quintessence,  modern s c i e n c e , t h e f a c t  (3)  that  o f importance r a i s e d  minds o f s c i e n t i f i c  sulfur, a i r , and  i n which i t  a n d bound t o g e t h e r t h e  considered  Although  these  sulfur  iden-  none o f t h e s e a l -  has been accepted  by  m y s t i c i s t s considered  i t t o a p l a c e o f prominence i n t h e  men.  Quantitative When, i n t h e n i n e t e e n t h  study  He  including the  t o the substances  ideas of sulfur biochemistry  Early  mercury,  form.  i n t h e same manner a s t h e s o u l o f man u n i t e d  body a n d s p i r i t .  sulfur  put h i s con-  t o t h e Greek e l e m e n t s , f i r e ,  S u l f u r imparted  mercury and s a l t  chemical  Paracelsus  e l e m e n t s made up a l l t h i n g s  the property  t i c a l with  of h i s "elements" but  the "hypostatical p r i n c i p l e s " ,  t o be p a r a l l e l  work o n t h e b i o -  o u t by t h e i a t r o c h e m i s t s o f t h e  o f the importance of s u l f u r  earth.  the  experimental  i n the e a r l y sixteenth century  considered and  extensive  o f s u l f u r was c a r r i e d  renaissance.  finally  first  the chemistry  reasoned b a s i s , they  of l i v i n g found  century  t h i n g s e x t e n s i v e l y on a  that they  til  they had broken t h e l i v i n g  The  first  men f i n a l l y b e g a n t o  methods u s e d i n d o i n g  tissue this,  carefully  c o u l d make no p r o g r e s s u n down i n t o  simpler  such as a s h i n g and  units.  3 treatment w i t h s t r o n g a c i d s and o x i d i z i n g  a g e n t s , were v e r y  drastic  a l l the sulfur  and r e s u l t e d  sulfate.  usually  i n oxidizing  I n t h i s f o r m t h e t o t a l s u l f u r was d e t e r m i n e d b u t  n o t h i n g was l e a r n e d o f t h e numerous d i f f e r e n t might o c c u r .  Some t h o u g h t t h a t  (C oH62°12 10^x y z* N  s  p  4  analysis  forms i n which i t  t h e y h a d enough knowledge  t h i s work t o w r i t e a g e n e r a l f o r m u l a  f o r proteins  were d e v e l o p e d w h i c h r e v e a l e d t h a t  t h u s shown t o o c c u r i n l i v i n g  tissues  linked  methods o f  several  f r o m t h e same t r e a t m e n t .  from  thus,  G r a d u a l l y however l e s s d r a s t i c  forms o f s u l f u r r e s u l t e d  ways.  to the  different  Sulfur  was  In a variety of  Much h a s now b e e n done i n t h i s a n a l y t i c a l p h a s e o f  s u l f u r b i o c h e m i s t r y , but i t i s s t i l l containing products are s t i l l  not complete f o r s u l f u r  often obtained from  p r o d u c t s by t r e a t m e n t s which would n o t y i e l d t h e s e fractions  f r o m any o f t h e s u l f u r  containing units  natural specific now known t o  occur. Sulfates The inorganic cells  commonest f o r m o f s u l f u r  sulfate  form a v a i l a b l e is  extent.  of the s o i l  s e l d o m made o f t h e f a c t  reduced  sulfur,  The r o l e  a n i m a l s any s u l f u r  occurs i n l i v i n g  i n order t o y i e l d n i t r o g e n i n a  that,  a p p r e c i a t e d but mention  as f a r as c a n be d e t e r m i n e d ,  d e p e n d e n t on p l a n t s f o r t h e i r (4)  I sthe  played by plants i n  t o animals i s g e n e r a l l y  animals are also  by  i o n and i n t h i s form i t a l s o  to a limited  reducing nitrates  i n the s o i l  Inasmuch a s s u l f a t e s  t o t a l supply o f  cannot be r e d u c e d  t h a t becomes c o m p l e t e l y o x i d i z e d  by t h e  body p r o c e s s e s c a n n o t be r e c l a i m e d b u t must be e x c r e t e d i n t h i s  4 f o r m a n d t h e r e f o r e 75-95$ o f t h e s u l f u r of  sulfate.  Sulfates,  like  tissues  i n limited  osmotic  concentration.  is  f o r the purpose  hydroxyl groups.  nitrates,  e x c r e t e d I s i n t h e form  are present i n animal  amounts a n d h a v e b u t a l i m i t e d  effect  The g r e a t e s t u s e o f s u l f a t e s b y a n i m a l s  of d e t o x i f y i n g aromatic  and h e t e r o c y c l i c  They a r e c o m b i n e d w i t h t h e s e g r o u p s a s  e t h e r a l s u l f a t e s and e x c r e t e d thus i n t h e u r i n e . few  o t h e r compounds o f b i o l o g i c a l  oxidized  on t h e  s u l f u r but they  will  importance  There  are a  which c o n t a i n  be d i s c u s s e d i n d i v i d u a l l y .  Cystine In  1810 W o l l a s t o n  bladder c y s t s which proved  (5) i s o l a t e d  t o be t h e f i r s t  c o n t a i n i n g o r g a n i c compound s t u d i e d acid,  (6)  sulfur  noticed series  i n this  i t until  natural  and a l s o  He g a v e i t t h e name c y s t i c  s i n c e been m o d i f i e d t o c y s t i n e . the  a substance  In spite  1837 a n d t h u s  i t was t h e f i r s t  When t h e c o r r e c t  H  S  w  atomic weight  a  s  correct  only  weights. because  o f oxygen as e i g h t  double nature o f t h i s molecule Possible through  s t r u c t u r a l formulae  of a long  even t o t h e present mistaken  f o r two  e m p i r i c a l f o r m u l a was  (7) v e r y l i t t l e  s t r u c t u r e s o r even o f atomic H  amino  compound, n e i t h e r W o l l a s t o n n o r any one e l s e  worked o u t by Thaulow  6 12 2°4 2  the f i r s t  of the l a b i l i t y of  t i m e , i n w h i c h one s u l f u r a t o m was a t f i r s t  C  sulfur  o x i d e a n d t h i s name h a s  o f compounds c o n t i n u i n g t o e x t e n d  atoms o f o x y g e n .  from  first  was known o f o r g a n i c Thaulow s f  his errors  formula In taking the  and i n not r e c o g n i z i n g t h e  compensated each  other.  f o r c y s t i n e were b a n d i e d  t h e c o u r s e o f t h e v a r i o u s r a d i c a l and type  about a l l  theories  t h a t were s u c c e s s i v e l y p r o p o s e d d u r i n g but  i t was n o t u n t i l  l i s h e d by s y n t h e s i s  1903 t h a t E r l e n m e y e r t h e s t r u c t u r e now  GRg-CH-C-OH $ NH2 0 S NH2 >0 CH2-CH-CK)H During t h i s period work was b e i n g cystine,  but  that  various be  over which  i t s occurrence.  much o f t h e , s u l f u r  i n proteins  to isolate  another f a i l e d  A l l the reactions q c c u r e d as c y s t i n e  i t from t h i s  source.  because o f the f a i l u r e  and i t was o n l y  succeeded I n o b t a i n i n g  found t o  a f t e r twelve  i n 1899, Morner  c y s t i n e from p r o t e i n s .  For a  c y s t i n e a n d t h e c l o s e l y r e l a t e d c y s t e i n e were t h e  f o r m s o f s u l f u r known t o o c c u r  i n proteins  and t h e r e f o r e  many o f t h e a n a l y s e s done i n t h i s p e r i o d were m i s l e a d i n g . was f o u n d however t h a t c y s t i n e i s p r e s e n t amounts I n n e a r l y  a l l proteins  k e r a t i n s have a v e r y believed  that  except  a thickening  It  at least i n small  the protamines.  The  l a r g e p e r c e n t a g e o f c y s t i n e and i t i s  i t has an important  function i n this protein i n  p r o t e c t i n g the animal from u l t r a - v i o l e t  tent  One  t o observe  s i m p l e t e c h n i c a l p r o c e d u r e s w h i c h were l a t e r  long period only  a century,  amino a c i d s h a d b e e n i s o l a t e d t h a t ,  finally  estab-  Cystine  essential f o r i t s isolation  other  (8) f i n a l l y  accepted,  of nearly  were s t u d y i n g  no one h a d b e e n a b l e  attempt r a f t e r  century  done t o e s t a b l i s h t h e s t r u c t u r a l f o r m u l a o f  others  indicated  the nineteenth  light.  o f t h e s k i n t h a n an i n c r e a s e  T a n n i n g i s more  i n t h e pigment  con-  and t h e a d d e d p r o t e c t i o n w o u l d come f r o m t h e i n c r e a s e d  amount o f  cystine-containing k e r a t i n present.  Cystine  also  6 seems t o have a n o t h e r more d y n a m i c f u n c t i o n I n t h e b o d y . Experiments  done o n t h e e f f e c t -SH g r o u p s  o f i o d o a c e t a t e o n enzymes  indicate  that  a r e e s s e n t i a l t o enzyme a c t i o n a n d  cysteine  i s t h e o n l y amino a c i d  known t o o c c u r i n p r o t e i n s . much o f b u t n o t t h e e n t i r e the a b i l i t y  c o n t a i n i n g t h i s group  that i s  C y s t i n e h a s b e e n shown t o f i l l need o f an organism  f o r s u l f u r and  o f most a n i m a l s t o s y n t h e s i z e c y s t i n e f r o m  forms. o f r e d u c e d 1  sulfur  other  emphasises i t s importance.  Methionine  MoWer s u c c e e d e d i n i s o l a t i n g c y s t i n e  Even b e f o r e from p r o t e i n s one  form o f s u l f u r .  that  a s 1846 P l e i t m a n n  different  sulfur  unsuccessfully sulfur  fractions.  to find  oxidized.  In  something  determinations of  From t h e s e r e s u l t s  labile  a c i d , which  has b e e n g i v e n b y J o h n s o n  fraction.  tried  Another  form of  seemed t o h a v e b e e n d e r i v e d  several proteins,  of these  A review  (13) o f t h e p a p e r s , p u b l i s h e d up  1911, c o n c e r n i n g t h e s e v a r i o u s s u l f u r  but not a l l ,  Shultz  o t h e r t h a n c y s t i n e was I s o l a t e d b y S u t e r ,  F r i e d m a n n a n d F r a n k e l (12) f r o m  till  i n contrast  a c o n s t a n t r e l a t i o n s h i p between t h e  and t h e a l k a l i  s u l f u r , cif-thio-lactic from  i s alkali  S h u l t z (11) was a b l e t o g a t h e r t o g e t h e r t h e r e s u l t s o f  s e v e r a l w o r k e r s who h a d made c o m p a r a t i v e  total  sulfur  w h i c h he m i s t a k e n l y t e r m e d  than  (10) o b s e r v e d  T h i s f r a c t i o n he c a l l e d u n o x i d i z e d s u l f u r  the remainder  1898  As e a r l y  t h e y c o n t a i n e d more  only a f r a c t i o n of the t o t a l p r o t e i n  labile. to  i t was r e c o g n i z e d t h a t  linkages.  Most,  sulfur reactions, unexplained at the  t i m e , have s i n c e been a c c o u n t e d  f o r by t h e presence o f  methionine. I t was t h e f a c t s u l f u r requirements discovery  t h a t c y s t i n e does not supply a l l t h e  o f c e r t a i n microorganisms  of methionine  by M u e l l e r  engaged I n s t u d i e s c o n c e r n e d requirements  (14),  of streptococci,  f o u n d t h a t t h e r e was a for their  i n c l u d e d i n some o t h e r h y d r o l y s a t e s .  Although  from  M e u l l e r , who was  w i t h t h e n i t r o g e n and a c t i v a t o r  i n c a s e i n hydrolysates necessary  isolating  substance  g r o w t h t h a t was n o t  I n 1922 he s u c c e e d e d i n  c a s e i n a f a c t o r w h i c h was named  I t turned out t h a t t h i s  methionine.  amino a c i d was n o t a n e s s e n t i a l  f a c t o r f o r t h e growth o f the s t r e p t o c o c c i , necessary  that led to the  f o r most s p e c i e s o f a n i m a l s .  i t proved  Methionine  c o n v e r t e d i n t h e body t o c y s t e i n e b y t h e f o l l o w i n g  t o be  c a n be reactions;  t h e p r o c e s s however i s n o t r e v e r s i b l e ,  9H3 s S 2 •Ho CH -CS CH-lffl ' P  H s t}R CH + CH-NH  H  2  2  2  3  2  .  C( X  2  \J)  0H  C^T ^CH 6H 6HNH 0 Of OH 2  CH 0H CHNH 1,0 Q ^OH 2  - H26  2  »  2  2  CHNH 2 LO Cf \)H  i  ~  X)H  Methionine  Homocysteine  — " \ <?H CH CH CHNH CHNH JO 2*  Serine  Gystethione  s  2  2  2  2  2  2  OH  CH 0H CH CHNH 2  u p  2  X)H Homoserine  The  CH SH • CHNH 2  +  2  X  0H  Cysteine  various intermediate products  o f t h e s e r e a c t i o n s have b e e n  8 I s o l a t e d from n a t u r a l  sources.  The a n a l y t i c a l d a t a  shows t h a t  i n general proteins contain a larger  methionine  than cystine  majority  of sulfur Mention  methionine  and t h e former  i n transmethylation.  c o n t a i n t h e s e groups  i n a labile  o f methionine  of a d r e n a l i n ,  groups  groups  form,  i s used  which  Du V i g n e a u d a n d that the  i n s y n t h e s i z i n g c h o l i n e and  necessary f o rthe synthesis  a n s e r i n e and o t h e r m e t h y l a t e d p r o d u c t s .  ( 1 6 , 17) t h a t  i n t h e r a t and i t i s p o s s i b l e  and t r i g o n e l l i n e  that  e r g o t h i o n e i n e and  i n the various locations  a r e f o u n d may p e r f o r m  I t has  b e t a i n e may a c t a s a s o u r c e o f m e t h y l  the other b e t a i n e s , s t a c h y d r i n e , t r i m e t h y l h i s t i d i n e , ine,  t o be  and i s , t h e r e f o r e ,  few compounds i n t h e d i e t  and i t i s p r o b a b l y a l s o  been found  The body w o u l d a p p e a r  (15) h a v e shown b y t e s t s w i t h d e u t u r i u m  m e t h y l group creatine  accounts f o r t h e  s h o u l d a l s o be made o f t h e r o l e p l a y e d b y  on the r e l a t i v e l y  co-workers  f r a c t i o n of  i n foodstuffs.  incapable of synthesizing methyl dependent  available  similar  hypaphor-  i n which  they  functions.  Thiamin Thiamin, sulfur, min  another e s s e n t i a l metabolite c o n t a i n i n g  i s a substance  i n the d i e t  i n t e r f e r e s w i t h many m e t a b o l i c  ultimately resulting first  i n beri-beri,  Eijkmann  t o work w i t h t h i s d i s e a s e f i n d i n g t h a t  contract for  o f paramount i m p o r t a n c e .  i t and t h e r e f o r e be u s e d  i t s study.  Other workers  functions  was one o f t h e pigeons  could  as an e x p e r i m e n t a l a n i m a l  established  d i s e a s e was due t o a d e f i c i e n c y  Lack o f t h i a -  the f a c t  that the  o f some s u b s t a n c e a n d i n 1898  9 J a n s e n and  Donath  c r y s t a l l n e form. pure form but later  (18)  succeeded i n i s o l a t i n g the v i t a m i n i n  I t was  i t was  the  not  first  v i t a m i n t o be  synthesized u n t i l  obtained  nearly forty  in  years  (19). N - C - N H 2 H C I  . C H 3  II I) C=C-CH2 GHgOH CBV-C C-CH2-N I I I Cfi CH-S Thiamin N=CH —  /  N  One  of the c h i e f  f u n c t i o n s of t h i a m i n i s concerned  synthesis of cocarboxylase, prosthetic  hydrochloride  with  t h i a m i n p y r o p h o s p h a t e , and  g r o u p s o f many c a r b o x y l a s e s .  the  T h e s e compounds o f  v i t a m i n are necessary  f o r t h e breakdown o f p y r u v i c a c i d  hence f o r the p r o c e s s  of g l y c o l y s i s .  thiamin i s evidenced This  seems t o be  secondary  following  a direct  result  t h e o r y has  particularly effect  as  series  (20)  and  polyneuritis.  the neurones, not  of a g e n e r a l l y d i s r u p t e d metabolism.  been proposed  the  However, a l a c k o f  i n the nerves on  the  a A  that the d e f i c i e n c y d i s r u p t s the  of r e a c t i o n s * p r e v e n t i n g the  s y n t h e s i s of  acetylcholine. ATP  +  Thiamin—•.—=> A d e n y l i c a c i d  pp 2CH3-C-CT-0H + 0  •»•  carboxylase cocarboxylase  2  0 * 2CH3-C^  P CH -C 3  N  cocarboxylase  C  + CH3-CH -N(CH3)30H 2  OH  H  *CH -CH2 —  3  C  +2 C O 2  H  3  N — C - C H 3  3  C  H  3  6  Acetylcholine Biotin The  third  and  e s s e n t i a l i n the d i e t  o n l y o t h e r f o r m o f s u l f u r known t o  i s the v i t a m i n b i o t i n .  be  Work l e a d i n g t o  10 the d i s c o v e r y c h a n n e l s and coenzyme R,  of t h i s v i t a m i n progressed hence i t has  and  been v a r i o u s l y designated  v i t a m i n H,  Working w i t h  f r a c t i o n of b i o s , the b i o s (22)  s u c c e e d e d i n 1935  t a l i n e product original little  of  was  Burk  This  of a d i e t  discovery and  of  biotin,  charcoal  (21), Kogl  most o f t h e  activity  c a l l e d b i o t i n but  study  Also  an  possible.  adsorbable and  of  Tonnis  the  t h e r e was  Meanwhile  too  Allison,  e x t r a c t , coenzyme R,  t h e work o f B o a s  c o n t a i n i n g much raw  an a n t i - e g g - w h i t e  which  Szent-Gyorgyi The  difficult  (25)  and  H  egg-white l e d to  H  identical  synthesized  the H  the  the  i n 1939  Bioand  d e m o n s t r a t e d t h a t v i t a m i n H had  v i t a m i n was  process  ( 2 4 ) , on  a  injury factor, vitamin  coenzyme R were shown t o be  properties. and  as  a c t i v e i n p r o m o t i n g the growth o f Rhizobium' t r i f o l i i ,  effect  1940  they  (23), obtained  legume n o d u l e o r g a n i s m .  tin  separate  I n g e t t i n g a m i n u t e amount o f a c r y s -  i t t o make a f u l l  H o o v e r and  the  I I B of M i l l e r  which c o n t a i n e d  adsorbate.  along three  i n 1943  s t r u c t u r e was  (26)  shown t o  in  similar  by  a  long  be:  ^OHp-CHjj-OHjj-CHo-Cf 'OH  N—C—C H As Szent-GySrgyi practically studied.  yet  H  little  H  i s known a b o u t t h e m e t a b o l i s m o f  found t h a t the a l l species  2  v i t a m i n must be  o f a n i m a l s and  In c e r t a i n instances  i n f i n i t e s m a l that f e e d i n g raw  the  Native  egg  to  micro-organisms that  amounts r e q u i r e d were  a d e f i c i e n c y c o u l d o n l y be  egg-white.  supplied  biotin.  w h i t e has  demonstrated  he  so by  a s m a l l amount  of  11 a p r o t e i n , a v i d l n , w h i c h combines w i t h  b i o t i n to prevent i t s  a s s i m i l a t i o n . Owing t o t h e w i d e r a n g e o f b i o t i n occurs, In the  t h e m i n u t e amount u s u a l l y r e q u i r e d ,  i t s occurrence  bound s t a t e i n w h i c h i t i s a v a i l a b l e t o o n l y  Isms, and able  substances i n which  to  due  to the  synthesize  fact  s u c h as  the  intestinal  i t , i t is difficult  b i o t i n requirements of tissues  that  living  embryos and  flora  a few  seem t o  to determine the  organisms.  Rapidly  tumors c o n t a i n  organbe  exact  growing  a l a r g e amount  of  b i o t i n and  s e v e r a l a t t e m p t s h a v e b e e n made t h o u g h u n s u c c e s s f u l l y  to prevent  or cure  c a n c e r s by  reducing  the  biotin  Intake.  or  glutamy1-cystenyl-  Glutathione The  tri-peptide, glutathione  glycine, i s another  sulfur containing  compound t h a t h a s  f o u n d i n a v e r y w i d e r a n g e o f t i s s u e s and  Ed  C-C—CH2-CH2-G H  CH  X) ? H S  cells.  V 2  been  OH H  1  H Glutathione Glutathione  may  atoms f r o m t h e  occurs  r e v e r s i b l y o x i d i z e d by  sulphur  2GSH This  be  2H  to form  removing the  disulfide:  i n n e u t r a l s o l u t i o n at the  the  rather  compound m i g h t p l a y  of hydrogen near the  carbohydrate  However, t h o u g h r e a c t i o n s b e t w e e n a s c o r b i c have been noted i n v i t r o e s t a b l i s h the  hydrogen  + GS-SG  -0.233 I n d i c a t i n g t h a t transfer  (Reduced Form)  no  one  has  r o l e of glutathione  yet  i n any  end  low  p o t e n t i a l of  a part of  the  glycolysis.  a c i d and  glutathione  found evidence specific  In  to  hydrogen  12 transfer reaction. in  living  lactic  cells  i s as  Because t h i s presence of  one  glutathione OH s p e c i f i c enzyme * C H , - C — ° H  + HpO  one  f u n c t i o n does not  such r e l a t i v e l y  in living  does a c t  liver.  things  as  60mg/l00ml i n y e a s t  i t s much g r e a t e r , s o l u b i l i t y .  discovered t o be  by  H o p k i n s i n 1921  glutamyl-cysteine  H u n t e r and  Eagles  There i s f o r  but  Glutathione At  as w e l l as  which are  to l i v i n g  peptide  he  because  first considered  because of evidence presented (29)  the  made a n o t h e r  study  it by  and  six widely d i s t r i b u t e d  s u l f u r compounds t o w h i c h r e f e r e n c e  important  the  formula.  There are,  number o f o t h e r s  example  70mg/l00ml  was  first  as  glutathi-  a hydrogen c a r r i e r  (27),  (28), Hopkins  revised' his o r i g i n a l  and  organisms use  t h a n c y s t e i n e as  the  universal quantities  i t i s g e n e r a l l y assumed t h a t  a hydrogen c a r r i e r .  rather  0 C-OH,  seem t o a c c o u n t f o r  l a r g e and  I t seems p r o b a b l e t h a t  glutathione of  glutathione  a coenzyme f o r t h e r e a c t i o n f o r m i n g  34-37mg/l00ml i n b l o o d , in  only proven f u n c t i o n of  a c i d from methyl g l y o x a l ,  0 p CH,—<f— C — H *  occur  The  has  already  l e s s common and  b e e n made, a  presumably  less  organisms.  Taurine Taurine cholic  like  a c i d I n the  concentrations where i t may  g l y c i n e may  bile  i n the  a c t by  salts.  occur  conjugated  I t also occurs  o x i d a t i o n of the  i n very  high  muscle t i s s u e of c e r t a i n i n v e r t e b r a t e s  an  effect  on  the  osmotic  P r e s u m a b l y , t a u r i n e i s f o r m e d f r o m c y s t e i n e by and  with  sulfur.  concentration. decarboxylation  13 0 NHg-CHg-CHg-S-OH  Taurine  Mucin The of  g l y c o p r o t e i n m u c i n may h a v e two d i f f e r e n t  sulfur containing prosthetic  acid  or mucoitin s u l f u r i c  molecule  of sulfuric  of g l u c o n i c a c i d .  acid.  The f o r m e r  Mucoitin sulfuric substituted  i n m a i n t a i n i n g a moist  membranes; f r e e cologically  chondroitin sulfuric i s composed o f a  a c i d , w i t h one o f a m i n o - g a l a c t o s e  compound, w i t h g l u c o s e important  groups,  acid  a n d one  i s the corresponding  f o rgalactose.  Mucin i s  smooth s u r f a c e o n t h e mucous  chondroitin sulfuric  i n the treatment  types  acid  i s used  pharma-  of migraine.  Heparin Heparin which s u l f u r i c produced  i s another  acid  by t h e l i v e r  that  c o n t a i n i n g substance i n  i s combined w i t h a c a r b o h y d r a t e .  v e r s i o n o f prothrombin from  sulfur  i n s m a l l amounts a n d i n h i b i t s into  thrombin.  o f dicoumerol which prevents  prothrombin.  Both  o f these drugs  Itsaction  injection.  the con-  i s different  the formation of  a r e used  a n t i c o a g u l a n t s , h e p a r i n h a v i n g t h e prompter i n g hyperdermic  It i s  m e d i c i n a l l y as action but r e q u i r -  I n mucin t h e s u l f u r i c  acid  apparent-  l y a c t s o n l y as a l i n k between t h e p r o t e i n and t h e c a r b o h y d r a t e but  i n h e p a r i n i t seems t o p l a y a more a c t i v e p a r t f o r t h e  activity  of the heparin i s d i r e c t l y  percentage  proportional to the  sulfur (30).  Asterubin A s t e r u b i n i s another  oxidized  sulfur  compound,  14 isolated Nothing  f r o m s t a r f i s h a n d i d e n t i f i e d b y Akermann i n 1935 ( 3 1 ) * i s known o f i t s f u n c t i o n . H  OBs W  0 CH —N—C—N—CHg—CHg —S—OH CH H 6 3  •  Asterubin  3  Toad  Poisons Some o f t h e t o a d p o i s o n s  believed  that these  sulfate  action,  The f o r m u l a  poisons,  i s as f o l l o w s :  groups are n o t e s s e n t i a l t o t h e i r  o f b u f o t h i o n i n e , t h e commonest o f t h e s e  ,-JU?  , o_ <A  T ^ 3C H  0 , S - O i0 w ii—C-0-Nr_ 3 °| |( I ° "^CH \/^N H }  contain sulfur but i t i s  S  Buf o t h i o n i n e  3  G  3  7  Sulfones L e f e v r e and R a n g i e r of deproteinated blood study  (32) f o u n d  t h a t 1% o f t h e s u l f u r  serum i s i n t h e s u l f o n i c  seems t o h a v e b e e n made a s t o t h e e x a c t  f o r m b u t no  nature  of this  fraction, Sulfatides S u l f a t l d e s c o n s t i t u t e another  naturally  g r o u p o f compounds c o n t a i n i n g o x i d i z e d s u l f u r . occurs  Sulfate  i n c e r t a i n o f t h e c e r e b r o s i d e s and p h o s p h o - l i p i d s o f  the b r a i n (33), these  occurring  P o s s i b l y s u l f a t e r e p l a c e s t h e phosphate i n  compounds. Lanthlonine As w e l l a s t h e two a c c e p t e d  methionine  amino a c i d s , c y s t i n e a n d  t o w h i c h r e f e r e n c e h a s b e e n made, t h e r e a r e t h r e e  15 other  s u l f u r c o n t a i n i n g compounds t h a t h a v e b e e n I s o l a t e d f r o m  proteinateous constituents Is  materials  and t h e r e f o r e  considered  of the p r o t e i n molecule.  t o be p o s s i b l e  Of t h e s e t h e b e s t  l a n t h i o n i n e w h i c h may be i s o l a t e d f r o m h a i r a f t e r h y d r o l y s i s  with  sodium c a r b o n a t e ,  during  Whether o r n o t l a n t h i o n i n e  the h y d r o l y s i s from c y s t e i n e  i s probably  of biological  l i k e w i s e be f o r m e d I n t h e  s i g n i f i c a n c e , however, f o r I t w o u l d intestinal  tract", Lanthionine  2  2  A Thio  2  Ether  A compound v e r y of a mixed e t h e r  vulgaris.  This  with  s i m i l a r to lanthionine but c o n s i s t i n g  o f c y s t e i n e and homocysteine has been  i s o l a t e d by Horn and Jones  occurs  (34) f r o m t h e weed  t h e i s o m o r p h o u s compound c o n t a i n i n g  0 X  G—OHNH  Djenkolic  0 C H - S - CHg—CH —GH—Q~f (Se) NH OH . 2  2  that  Acid a c i d , i s o l a t e d from the  V e e n a n d Hyman (35) i s thought  2  2  Djenkolic Van  s e l e n i u m when  soil*  V S  Astragalus  compound i s o f p a r t i c u l a r i n t e r e s t b e c a u s e i t  t h e weed i s grown- on s e l e n i f e r o u s  H0  i s formed  i s a matter o f debate; i t  C — C — CHg- S - C H — G — G f HO' N H NH OH  i s another reduced  t o occur i n p r o t e i n s .  djenkol  nut by  s u l f u r compound  As f a r a s h a s b e e n  determined I t has n o t y e t been i s o l a t e d from any other The  known  formula given  source.  f o r i t i s as below,  <\ NH2 NH 0 HO-C—CH—CH -S-GH -S-CH -CH—C-OH 2  2  2  2  Djenkolic  acid  16 tJrochrome TJrochrome i s a n impure f r a c t i o n o f t h e u r i n e contains high  t h e p i g m e n t u r o b i l i n and a p o l y p e p t i d e  content  studying  of sulfur.  I t must, t h e r e f o r e ,  t h e forms o f s u l f u r o c c u r i n g  with a  which rather  be c o n s i d e r e d  in  i n the urine.  Penicillin  G-penicillin H  0 The  antibiotic,  penicillin,  contains  a mixture of  s e v e r a l a c t i v e compounds w i t h d i f f e r e n t s i d e c h a i n s b u t t h e y all  contain  cillin  sulfur i n a thiasine ring.  i n inhibiting  The a c t i o n o f p e n i -  the growth o f b a c t e r i a i s not y e t under-  s t o o d n o r i s i t known w h e t h e r t h e s u l f u r i s e s s e n t i a l f o r t h e antibiotic effected  effect.  Synthesis  o f G - p e n i c i l l i n was  t h r o u g h t h e combined  both B r i t i s h  and A m e r i c a n  e f f o r t s of several  and i t s s t r u c t u r e  recently laboratories  i s now  definitely  established (36), Thio-sugar A thio  sugar b e l i e v e d  been i s o l a t e d from y e a s t studied  by Suzuki  known t h a t  t o be a t h i o - m e t h y l - r i b o s e  by M n d e l l a  and Dunham  (39), I ti s  l i n k e d t o adenine but the  p o s i t i o n o f the d i f f e r e n t groups has not been elucidated  (37) a n d f u r t h e r  (38) a n d b y L e v e n e a n d S o b o t k a  the sugar occurs I n yeast  completely  y e t and n o t h i n g i s known o f i t s s p e c i f i c  O d o u r s and  has  function.  Flavours  Some o f t h e s h a r p f l a v o r s a n d o d o r s o f n a t u r a l  17 substances  a r e due t o s u l f u r  pound c h i e f l y  c o n t a i n i n g substances.  responsible f o r the flavor  T h e com-  o f mustard i s a l l y l -  isothiocyanate: CH =CH-CH -N=C=S 2  A  2  somewhat  similar  odour and f l a v o u r t o g a r l i c .  compound g i v e s t h e d i s t i n c t i v e It i s allyl-disulfide:  CH =CH-CH -S-S-GH -CH=CH 2  One  2  2  of the d i s t i n c t i v e  2  components o f a s p a r a g u s i s be  methyl mercaptan. intestinal  tract  T h i s compound m a y a b s o r b e d f r o m t h e g a s t r o A  and e x c r e t e d by t h e k i d n e y s  within  fifteen  minutes o f i t s i n g e s t i o n . Although pleasant,  t h e odour o f m e t h y l mercaptan i s n o t u n -  t h a t o f i t s homologue, b u t y l m e r c a p t a n , I s e x t r e m e l y  so and t h i s f o r m s t h e p r i n c i p l e  component o f t h e s k u n k ' s  odour,  Thiocyanates A simple occurs widely fluids  compound c o n t a i n i n g r e d u c e d  distributed  i s thiocyanate.  sulfur  that  i n v a r i a b l e amounts i n a l l body  I t may e n t e r  t h e body i n t h e d i e t b u t  most o f i t w o u l d a p p e a r t o be f o r m e d e n d o g e n o u s l y , p o s s i b l y a s a d e t o x i f i c a t i o n product  o f cyanides  used m e d i c a l l y i n t h e treatment  (40),  I t i s sometimes  o f h y p e r t e n s i o n b u t h a s no  known f u n c t i o n i n t h e q u a n t i t i e s i n w h i c h i t i s n o r m a l l y present are used  i n t h e body.  However, i t i s p o s s i b l e t h a t  i n the biological  compounds s u c h  thiocyanates  synthesis of certain natural  as e r g o t h i o n e i n e f o r i n the l a b o r a t o r y r e a c t i o n s  b e t w e e n i t a n d a number o f o t h e r o r g a n i c compounds o c c u r easily.  sulfur  very  18 The together of  twenty-five  with ergothioneine  the forms i n which  plants  and  reactions  that  i s true  are play  a very  s e c t i o n I , 3)  do  not  by  any  s u b s t a n c e s and  s e v e r a l of of very  some o f t h e  as y e t  f o u n d t o be  of  the  various  great  i s that  the  synthetic  f a r the  on  their  A full  scope o f  that  sulfur w i l l  also  although there  exact  these  the  d i s c u s s i o n of but  thiouracil  i t is  inter-  several different theories  i s the  the  sulfonamide  different i n function  i n normal metabolism. and  c e r t a i n d e r i v a t i v e s of  compounds a r e u s e d i n t h e  treatment  of t h y r o t o x i c o s i s .  action w i l l  discussed  i n a later  more f u l l y  in  these  sulfonamides i n t e r f e r e with that  to  compounds  mode o f a c t i o n , most o f t h e  substance t a k i n g part Thiourea,  class of  are  some n o r m a l m e t a b o l i t e ,  be  be  s u l f u r com-  proved valuable  this report  group i s s i m i l a r i n s t r u c t u r e t o but from the  i t i s probable  s u b s t a n c e s have been f o u n d  most i m p o r t a n t  agree i n assuming t h a t f u n c t i o n of  them  naturally occurring  sulfonamides,  to note t h a t ,  theories  c e r t a i n of  Drugs  compounds i s b e y o n d t h e esting  listed  importance.  p o s s e s s a p h y s i o l o g i c a l a c t i o n t h a t has By  in  reagents.  compounds t h a t h a v e b e e n  unknown compounds o f  pounds a number o f  of  laboratory  l i m i t e d importance but  In a d d i t i o n to the  medicine.  most  means a c c o u n t f o r a l l t h e  a c t i v e r o l e i n m e t a b o l i s m and  Synthetic  comprise  have b e e n o b s e r v e d t o o c c u r b e t w e e n n a t u r a l  that  apparently  (see  above  s u l f u r i s known t o o c c u r commonly  a n i m a l s but  sulfur containing It  s u l f u r compounds d e s c r i b e d  section  these  This of  19  this report. S u l f o n m e t h a n e and  sulfonnethylmethane  a s h y p n o t i c s and  i n some c a s e s  u s u a l l y known by  their  are both  as a n t i s p a s m o d i c s .  t r a d e names, s u l f o n a l and  used  They  are  trional.  Sulfur Bacteria Although the T h i o b a c i l l i  o n l y a b r i e f m e n t i o n c a n be made h e r e  and  Thiobacteriales,  chemistry  o f s u l f u r w o u l d n o t be  o f them.  Various  energy  species of these remarkable  so o b t a i n e d  These b a c t e r i a organic  s y n t h e s i z e the reduced  compounds n e e d e d t o b u i l d  a c i d , p y r i d o x i n e and  riboflavin  imidazoles form  of n a t u r a l h e t e r o c y c l i c  biosaid  are  or t h i o s u l f a t e s  and  carbon-dioxide.  carbon  into  process. a l l the  t h e i r protoplasm  nictotinic  acid,  including  pantothenic  (41).  A Review of the B i o c h e m i s t r y  The  bacteria  i s required for this  the v i t a m i n s , t h i a m i n , b i o t i n ,  2,  sulfur  they can reduce  I n a good many s p e c i e s l i g h t  of the  c o m p l e t e i f n o t h i n g were  able to o x i d i z e hydrogen s u l f i d e , with the  the review  of  one  of the  Imidazoles  o f t h e most I m p o r t a n t  compounds f o r t h i s  i n t e g r a l p a r t of the p u r i n e r i n g  classes  r i n g o c c u r s as  as w e l l a s  independantly  an in a  number o f commonly o c c u r r i n g compounds. Purines  O n l y a very b r i e f d i s c u s s i o n of t h e p u r i n e s i s possible here. the pentoses  With the p y r i m i d i n e s they  i n nucleic  a c i d s and  i n this  occur  attached  to  form probably p l a y  a  20 key  role  i n t h e c o n t r o l l i n g mechanism o f c e l l s .  shown t h a t n u c l e i c p o s i t i o n s of the the c o n t r o l l i n g  l i n k a g e s and factor  g r o u p s and  s y s t e m s and lism. are  here  i n the p r o d u c t i o n of a s p e c i f i c (42).  Purines also  occur  for believing  by  type  i n the  t h e body f r o m  the  t h a t the  simple  the r a t ceases  i f the d i e t  most i m p o r t a n t  o f the  metabo-  purines  Imidazole  f o r e x c r e t i o n o f t h e p u r i n e breakdown  allantoin,  are  co-enzymes o f a l a r g e number o f enzyme  T h e r e i s some e v i d e n c e  histidine  the bases c o n t a i n e d t h e r e i n  again play a very active part i n c e l l  s y n t h e s i z e d by  been  a c i d s which presumably can v a r y o n l y i n the  o f c a p s u l e by p n e u m o c o c c i prosthetic  I t has  ring  of  product,  i s lacking  in  histidine, Histidine The is  t h e amino a c i d h i s t i d i n e ,  isolated  i n 1896  from  a mixture  acid  and  it  has  by  Kossel  structurally  now  i m i d a z o l e compounds  H i s t i d i n e was  (43)  of p r o t e i n s .  simple  from  sturine  I t i s (3imidazole  i t i s closely  related  been e s t a b l i s h e d t h a t these  simultaneously and  by H e d i n  (44)  « amino-proprionic to a r g i n i n e though  are not  inter-changeable  I n mammalian m e t a b o l i s m ,  Histidine animal but and  i s e s s e n t i a l i n the d i e t i t seems t h a t i t c a n be  o f the young  growing  s y n t h e s i z e d to  some e x t e n t  the need f o r i t i n the a d u l t p r o b a b l y v a r i e s w i t h  species.  I t o c c u r s i n almost  a l l p r o t e i n s but  h i g h c o n c e n t r a t i o n i n h i s t o n e s , h e m o g l o b i n and tamines.  The  metabolic  path  of h i s t i d i n e  the  i n particularly i n most  i s but  poorly  pro-  21 understood. I t may acid  and  lead  through the removal  hence t o g l y c o g e n e s i s or t h r o u g h  ammonia t o u r o c a n i c a c i d b u t these products account  of urea to  glutamic  the removal  i n normal metabolism  seem t o be f o r m e d i n s u f f i c i e n t  of  n e i t h e r of  quantity to  f o r an a p p r e c i a b l e p o r t i o n o f t h e t o t a l  histidine  metabolized. Histamine  Histamine, is  a compound o f v e r y g r e a t m e d i c a l i m p o r t a n c e .  naturally feces. be  the d e c a r b o z y l a t i o n product  i n e r g o t and  That  absorbed  fied  into  the blood  at a s u f f i c i e n t  however, h i s t a m i n e  rate  stream  but  to prevent  i n meat  and  In the i n t e s t i n e  may  i t is usually  all  these are  asthma and different  l a c r i m a l glands  symptoms c h a r a c t e r i s t i c The  similarity  c o n d i t i o n s w h i c h may  wide v a r i e t y  and  of d i f f e r e n t  capillaries;  be b r o u g h t  symptoms o f about  by any  only a slight  immunity  at present is built  f o r m e d w i l l be h e l d t o t h e c e l l  up,  shock, these of  a  has b e e n e x p l a i n e d by  c o n s i d e r i n g them a l l as a n t i g e n - a n t i b o d y r e a c t i o n s . most g e n e r a l l y a c c e p t e d  blood  of anaphylactic  of the  substances  salivary,  a fall.in  to i n c r e a s e d p e r m e a b i l i t y of the  allergy.  If,  i s I n j e c t e d h y p e r d e r m i c a l l y i t causes  p a n c r e a t i c and  p r e s s u r e due  detoxi-  noticeable effects.  c o n t r a c t i o n o f smooth m u s c l e , s t i m u l a t i o n o f t h e gastric,  histidine,  I t occurs  as a p u t r i f a c t i o n p r o d u c t  f o r m e d by b a c t e r i a l a c t i o n  of  states  that,  most o f t h e  surfaces.  The  theory  i n c a s e s where antibody  I f antigen,  is  now  22 introduced till  into  the b l o o d  i t reaches  the c e l l  stream  i t will  for  this  t h a t causes  t h e o r y comes f r o m  react with histamine  and  histamine to  be  s e c r e t e and  the blood.  the observed  the f a c t  effects.  causes Thus i t i s Evidence  t h a t compounds w h i c h  will  cause the development  are e f f e c t i v e  a n a p h y l a c t i c shock.  may  into  or which w i l l  antibodies f o r histamine allergy  neutralized  s u r f a c e where t h e r e a c t i o n  the c e l l to r e l e a s e the histamine always h i s t a m i n e  not be  i n the treatment  of of  In smaller concentrations  a n o r m a l hormone s t i m u l a t i n g c e r t a i n  the removal o f i t from  the blood  glands  stream,  may  t h e r e f o r e , cause s e r i o u s c o m p l i c a t i o n s , Carnosine  and  Anserine  0 G =  C — C H g — CH-  N  N  Cf-OH  N-C-OHg-CHg-NHg  Carnosine  H Carnosine histidine The ly  and  that occur very widely  compounds c o n t a i n i n g  i n v e r t e b r a t e muscle  d i s t r i b u t i o n of c a r n o s i n e i n p a r t i c u l a r has s t u d i e d by  developed  Cifford  (45)  a colorimetric  Knoop's d i a z o r e a c t i o n of  a n s e r i n e a r e two  c l u e t h a t we  (47),  i n significant  Carnosine  histidine  linked  (46),  Hunter has  also  Practically  and  constant  quantities.  i s that they  i s /Balanyl-histidine,  on  n o t h i n g i s known  carnosine or anserine although  have at p r e s e n t  buffers,  Hunter  been e x t e n s i v e -  method f o r i t s d e t e r m i n a t i o n b a s e d  the f u n c t i o n of e i t h e r  are present  and  tissue.  are both  The  they only  strong  a d i p e p t i d e of  t o t h e r a t h e r r a r e (? amino a c i d .  Anserine i a  23 s i m i l a r b u t h a s a m e t h y l g r o u p i n t h e number t h r e e  position,  that  i s in  i s o n one o f t h e n i t r o g e n s  contrast  to ergothioneine  of the r i n g .  This  w h i c h i s m e t h y l a t e d o n t h e amino  nitrogen. Creatinine  % 0 —CHg N. N  n N H Creatinine  i s not g e n e r a l l y  to h i s t i d i n e because m e t a b o l i c a l l y dehydration glycine, contain  the imidazole  attached.  is.synthesized  Creatinine  r i n g with keto,  of imidazoles  from  does, however,  imino and m e t h y l groups  Thus i t f o r m s a n i n t e r m e d i a t e  path o f formation visa  and m e t h i o n i n e .  t o be r e l a t e d  i t i s formed by t h e  o f c r e a t i n e which i n turn  arginlne  considered  i n what i s a p o s s i b l e  f r o m g u a n i d l n e compounds o r  versa. Allantoin Another imidazole  occurring  animals i s a l l a n t o i n , a di-keto H 0=*Q — N  A /C  i n the urine  of c e r t a i n  substituted imidazole  ring  0  C — N - C—NH NH  2  ii  0 with a urea  s i d e chain*.  I t i s thus a s u b s t i t u t e d  A l l a n t o i n has a pronounced of  t i s s u e s and t h i s  s t i m u l a t i n g e f f e c t on t h e growth  i s doubtless  amounts f o u n d i n p l a n t  hydantoin*  the f u n c t i o n o f the large  a n d a n i m a l embryos.  I t has also  been  24 found that  allantoin will  wounds and  i t has  treatment  o f war  the p o s s i b i l i t y  g r e a t l y h a s t e n t h e h e a l i n g o f many  been used wounds.  extensively,  R e f e r e n c e has  particularly  i n the  a l r e a d y b e e n made t o  o f a m e t a b o l i c r e l a t i o n s h i p between  histidine,  a l l a n t o i n and t h e p u r i n e s . Other h y d a n t o i n d e r r l v a t i v e s have found c o l o g i c a l use  i n the treatment  o f e p i l e p s y and  b e n e f i c i a l a c t i o n i n these d i s e a s e s r e f l e c t s to the  a pharma-  chorea.  their  Their  similarity  barbiturates.  Pilocarpine H X  <?— 9 N H2  A H  V ^ sA  H C—CH2-CH  3  3  Still cology  another  Is p i l o c a r p i n e ,  o f a Sou£h American important nervous  i m i d a z o l e of importance a natural alkaloid  shrub.  bronchitis,  and  to s t i m u l a t e the  I n the treatment  skin disorders.  The  occurs with p i l o c a r p i n e  of  has  b u t weaker p h y s i o l o g i c a l  i n the  leaves  o f t h e most  parasympathetic glaucoma,  demethylated  pilocarpidine a similar  found i n the  I n pharmacy I t i s one  of the drugs used  system b e i n g used  i n pharma-  compound  same s h r u b  and  action,  Biotin The  b i o c h e m i s t r y o f b i o t i n has  been d i s c u s s e d w i t h  t h e o t h e r s u l f u r c o n t a i n i n g compounds b u t out t h a t  It also contains a substituted  i t may  imidazole r i n g .  b i o t i n t h e r e i s an o x y g e n atom i n t h e 2 p o s i t i o n , corresponding to that  o c c u p i e d by  the  be p o i n t e d  sulfur  the  In  position  of e r g o t h i o n e i n e .  Trimethyl  Histidine 0  HC  • C- CHg-  \  1  CH- Ct'  Ja*  Trimethyl  0  Trimethyl-histidine Isolated  from c e r t a i n e d i b l e  histidine  i s a betaine that mushrooms.  has been  In spite of i t s  l i m i t e d d i s t r i b u t i o n i t i s worthy o f note because i t i s a possible  intermediate i n the hypothetical  thioneine  3.  of ergo-  from h i s t i d i n e ,  A Review o f t h e C h e m i s t r y o f  E r g o t h i o n e i n e from The similar  synthesis  Thiollmldazoles  Ergot  h i s t o r y of the thiollmldazoles  t o that  of cystine,  i s i n some ways  E r g o t h i o n e i n e was e a r l y  isolated  f r o m a n o n - p r o t e i n s o u r c e and I n s p i t e o f many e f f o r t s t o i s o l a t e i t o r a r e l a t e d compound f r o m p r o t e i n s  and i n s p i t e o f  many i n d i c a t i o n s o f t h e p r e s e n c e o f a t h l o l l m i n a z o l e i n proteins  no one h a s y e t s u c c e e d e d i n p r o v i n g any o f t h e s e  compounds t o be a n a t u r a l (48) he  was t h e f i r s t  extracted  studied and  constituent  to isolate a natural  ergothioneine from ergot  the p h y s i c a l  Barger and Ewins  following  formula,  C. T a n r e t  thiolimldazole  o f r y e i n 1909,  and c h e m i c a l p r o p e r t i e s  worked out t h e e m p i r i c a l  studies  of proteins,  when He  o f t h e compound  CgH^^OgS,  In further  (49) g a v e g o o d e v i d e n c e f o r t h e  structural formula.  26 0  HC —  C-CHg- CH - OCo*"  f SH Although formula  (CH ) 3  t h e r e was i t was  by Winegard previously different  3  n e v e r much d o u b t o f t h e v a l i d i t y  only proved  (50).  by  Harrington  and  h i s co-workers  methods but  were u n a b l e  to methylate Barger  s t r u c t u r e on  the  eine with concentrated i n g an u n s a t u r a t e d  f o l l o w i n g evidence, alkali  add;  splits  this  shows t h a t i t i s a  s u l f h y d r y l g r o u p and  reduced  double  () ( 4 i m i d a z o l e )  been s y n t h e s i z e d ,  its  the  s u l f u r was  lability  in alkali T h i s was  silver  basic acid by  saturate  E w i n assumed t h a t t h e  of f e r r i c  c h l o r i d e and  and  leav-  the the  p r o p r i o n i c a c i d which  i n the presence  later  has  assumed because stability  t h a t o f known compounds so c o n s t i t u t e d .  confirmed two  by  the  papers,  Ewins there  synthesis of the  one  is a fairly  by  mercury  or t a n n i c ' a c i d .  salts, not  by  thiolhistidine.  Tanret  complete  reactions of ergothioneine.  l e a d a c e t a t e but  oxidizing  sodium t o  2 of the r i n g  is like  and  t o remove  to carbon  most i m p o r t a n t by  and  ergothin-  betaine.  attached  In these by B a r g e r  Barger  with  compound  Ewlns based  leating  acid  had  two  o f f trimethylamine  T h i s compound, i f o x i d i z e d w i t h n i t r i c  bond, y i e l d s  this  and  year  (51, 52)  s u c c e e d e d i n s y n t h e s i z i n g t h i o l h i s t i d i n e by  a  that  this  s y n t h e s i s d u r i n g the past  to form the b e t i n e , e r g o t h i o n e i n e . their  of  and  study  of  the  phospho-tungstic  acid,  and  picric  a l s o causes a p r e c i p i t a t i o n  the e r g o t h i o n e i n e  other  It i s precipitated  by n e u t r a l l e a d a c e t a t e ,  Iodine  the  to a d i s u l f i d e .  but  It i s soluble  27 i n w a t e r and d i l u t e insoluble  i n absolute Barger  no  a l c o h o l and s l i g h t l y a l c o h o l and o t h e r  organic  solvents.  and Ewins a l s o s t a t e d t h a t e r g o t h i o n e i n e h a s  marked p h y s i o l o g i c a l a c t i o n t h o u g h t h e y d i d n o t g i v e  evidence.  Though t h i s m a y b e  statement  correct,  from  they  obtained consistently  uric  acid  lower  and Hunter results  that  with the direct  the reducing  method o c c u r r e d  The unknown s u b s t a n c e  i n the blood  of the rabbit, after  results  agreed  closely  was f o u n d  t o be p r e s e n t  the p u b l i c a t i o n o f these  results  of the direct  than  the i n d i r e c t  i n most c a s e s . of another  method o n t h e g r o u n d method a n d t h a t t h e  He s t a t e d t h a t he h a d reducing  substance i n  and t h a t h e w i t h h i s a s s o c i a t e s h a d b e e n w o r k i n g  i s o l a t i o n o f i t f o r some t i m e . Benedict,  which they Mrs.  Interfering  pig.  long recognized t h e presence blood  ( 5 5 ) . They  d o g , c a t , o x , and g u i n e a  (56) made a d e f e n s e  i t was much s i m p l e r  Newton and B e h r e  called  with  in. high concentration i nthe  corpuscles.  Immediately  w h i c h was  acid  a n d Wu (54)  method o f B e n e d i c t  substance  that  i n determining the  a c c o r d i n g t o t h e method o f F o l i n  they d i d u s i n g the d i r e c t  a l s o found  that  i t did  (53) f o u n d  i n t h e b l o o d by p r e c i p i t a t i o n o f t h e u r i c  lactate  Benedict  time t h e  Blood  I n 1925, B u l m e r , E a g l e s  than  their  f u r t h e r work o n t h e compound.  Ergothioneine  silver  at the present  i s s u b j e c t t o some d o u b t a n d f o r some t i m e  discourage  by  so i n p y r i d i n e h u t  on t h e  A p a p e r p u b l i s h e d i n 1926 (57) a n n o u n c e d t h a t a  t h i a s i n e h a d been i s o l a t e d  substance  from b l o o d by  D u g d a l e i n 1921 a n d t h a t t h e y h a d now c o n f i r m e d  t h i s as  28 the  substance  which i n t e r f e r e d w i t h t h e d i r e c t  determination. and  uric  They gave p h y s i c a l c o n s t a n t s f o r t h e compound  postulated the e m p i r i c a l  formula as, G i 2 2 0 4 ° 3 « H  N  M e a n w h i l e , i n 1925, H u n t e r and E a g l e s independently  acid  isolated  the i n t e r f e r i n g  s  (58) h a d  substance  and t h e y  gave  t h e e m p i r i c a l f o r m u l a , C6HX1N2O3 and a r a t h e r d i f f e r e n t s e t of p h y s i c a l constants.  F o r example t h e m e l t i n g p o i n t was  g i v e n a s 224-225 C f o r t h i s  compound w h i l e  o f t h i a s i n e was g i v e n a s 162-163.  Both  r e s p e c t i v e compounds o c c u r r e d a l m o s t and h e n c e w o u l d  little  In a l a t e r substance sulfur) is  that  paper  and s u b m i t t e d  groups found  entirely  t h e ur.ic a c i d  H u n t e r and E a g l e s  they had i s o l a t e d ,  two m o l e c u l e s  before  effect  the m e l t i n g point  the r e v i s e d  i n the corpuscles  determination. (59) named t h e  sympectothion formula  However  and D a k i n  (60) t o show  that  i d e n t i c a l w i t h e r g o t h i o n e i n e a n d o n December 23  E a g l e s and J o h n s o n fundamentally differences  o n December 6, 1926  bound  s  3  o f e r g o t h i o n e i n e p l u s one o f w a t e r .  evidence  t h i a s i n e was  (firmly  CIQH O% 2°4which  t h i s h a d b e e n p u b l i s h e d Newton, B e n e d i c t  submitted  that the  (61) showed t h a t  sympectothion  was  t h e same as b o t h t h e above compounds.  i n the p h y s i c a l constants of thiasine  t h i o n had a r i s e n because Benedict  The  and  sympecto-  and h i s c o - w o r k e r s h a d  isola-  t e d t h e h y d r o c h l o r i d e w h i l e Hunter and E a g l e s had i s o l a t e d the f r e e base.  I n t h e above p a p e r  Newton e t a l . s u g g e s t e d  t h e name e r g o t h i o n e i n e b e s h o r t e n e d due  to c o n f u s i o n w i t h  thionine,  has  not been g e n e r a l l y a c c e p t e d .  that  to t h i o n e i n e but p o s s i b l y  7aminophenothiazine,  t h i s name  29 Isolation " Since  these  three  o r i g i n a l methods o f  e r g o t h i o n e i n e were d e v e l o p e d , appeared i n the in  1928  (62)  was  (58).  E r g o t was  a l k a l o i d s removed and hydroxide.  The  with basic  and  was  e x t r a c t was was  those  clarified  with  and and  barium  successively precipitated c h l o r i d e , lead acetate  phosphotungstic  Hopkins  prominence the  acid.  I t was  (63)  developed  and  finally absolute  was  obtained  a method f o r  relatively  specific  cuprous i o n s .  By  and  and  Meldrum  considerably. substances  The  (64)  ergothioneine  o x i d i z e d by  precipitated  Pirie  (65)  as  modified  the  and  but  w h i c h has  brought  other  a e r a t i o n and  cuprous  of  this  blood  the  procedure  interfering  "diallzed  iron".  the  salt,  t h i s method somewhat so a s  make i t a p p l i c a b l e t o e r g o t w h i c h i s r i c h e r i s blood  from  shorten  some o f t h e  a r e removed w i t h d I l u t e a c i d  g l u t a t h i o n e i s then  thus  a p p l y i n g the use  were a b l e t o  p r o t e i n s and  the  r e a c t i o n between  to the p r e c i p i t a t i o n of ergothioneine  Williamson  than  some  ergot.  t h i o l groups and  The  of  a l c o h o l , the f a t s  i s o l a t i o n of g l u t a t h i o n e u s i n g cuprous oxide  reagent  Eagles  o f Hunter  o f 0.65$ o f p u r e e r g o t h i o n e i n e  I n 1929  into  by  a combination  with  extracted with  the  developed  f r e e b a s e by c r y s t a l i z a t i o n f r o m  A yield  from the  (48)  lead acetate, mercuric  as t h e  alcohol.  first  Tanret  ergothioneine  sodium h y d r o x i d e isolated  The  b a s e d p r i m a r i l y on  o f t h e r e a c t i o n s u s e d by Eagles  s e v e r a l m o d i f i c a t i o n s have  literature.  and  isolating  more i n t e r f e r i n g  in  to  ergothioneine  substances.  30 W i t h t h i s method P i r i e g o t I t must be  pointed  out,  a yield  o f 0.18$  ergothioneine.  however t h a t w h i l e  these  b e e n g e n e r a l l y compared on t h e b a s i s o f t h e it  has  r e c e n t l y b e e n shown by  H u n t e r e t a l . (67) the  that  there  normal e r g o t h i o n e i n e  of comparison i s not Recently  Lawson and  methods hfive  percentage  Rimington  yield,  (66)  and  i s considerable variation  content  of  ergot  and  H u n t e r and  co-workers  (68)  has  combined  and  Eagles and  ergot.  has  obtained  I s o l a t i o n of ergothioneine  a 0.26$ y i e l d ergot  precipitated with  of ergothioneine  Is extracted with  i m p u r i t y removed w i t h  ergothioneine freed  original  I n t h i s method t h e  much o f t h e  cuprous oxide.  crystalized  Hunter  from from  water,  u r a n i u m a c e t a t e , and  of the copper w i t h hydrogen s u l f i d e ,  c o a l and  basis  reliable.  c o p p e r p r e c i p i t a t i o n w i t h c e r t a i n r e a c t i o n s u s e d by  blood  in  hence t h i s  the  i n the  by  It is  the then  treated with  char-  from absolute a l c o h o l .  Experiments  Nutrition After had  the  Cox  of the and  ergothioneine  proved that ergothioneine  i n the d i e t  N e u b e r g e r and  dine cannot take  and  Webster  c o u l d not  t h i s work has (70)  who  i n the  blood  h i s co-workers proceeded  p o s s i b l e f u n c t i o n s o f i t i n the body.  (69)  histidine by  identity  been e s t a b l i s h e d , Eagles  study and  the  First  s i n c e been  Further  Eagles  replace  showed t h a t e v e n  the p l a c e of h i s t i d i n e .  to  supplemented thiolhistievidence  of  31 the d i s t i n c t  metabolsim o f the t h i o l i m i d a z o l e s has been  f u r n i s h e d by Dale,  Gaddum a n d Broom  (71) who f o u n d  that  t h i o l h i s t a m i n e h a s o n l y one t w o - t h o u s a n d t h t h e p r e s s o r of histamine the e f f e c t rabbit  o n t h e r a t and l e s s  on the c a t .  blood  sugar  than  The e f f e c t  effect  one f i f t y - t h o u s a n d t h  o f t h i o l h i s t a m i n e on t h e  was n o t d e t e c t a b l e .  I f t h e body h a s n o t  t h e mechanism t o remove t h e s u l f u r f r o m t h i o l i m i d a z o l e s i t seems u n l i k e l y and  t h a t i t c o u l d add s u l f u r  to the imidazole  ring  t h e r e f o r e t h e m e t a b o l i s m o f t h i o l i m i d a z o l e s i s most  likely  quite d i s t i n c t  a distinct  origin  from t h a t o f h i s t i d i n e .  t h e t h i o l i m i d a z o l e m i g h t come f r o m t h e d i e t  or might be s y n t h e s i z e d I n t h e a n i m a l t h i o c y a n a t e and o r n i t h i n e . evidence  I f i t i sof  has been gathered  body, p o s s i b l y from  A c e r t a i n amount o f c i r c u m s t a n t i a l which f a v o r s the theory  o f exogenous  origin. Some o f t h e most i m p o r t a n t exogenous  o r i g i n of ergothioneine  and  V a r s who c a r r i e d  the  ergothioneine  blood  out a study  content  evidence  i s i n t h e paper by E a g l e s  of the conditions  of pigs blood.  governing  The c o n t e n t  o f g r a i n f a t t e n e d p i g s from a Toronto  been found  f o r the  a b a t t o i r had  t o be v e r y h i g h i n c o m p a r i s o n w i t h t h a t o f g a r b a g e  f e d p i g s f r o m t h e S p r i n g s i d e Farm, New H a v e n .  T h i s l e d them  t o a s e r i e s o f d i e t a r y e x p e r i m e n t s w h i c h showed t h a t t h e r e was p r a c t i c a l l y  no e r g o t h i o n e i n e  on a g a r b a g e o r c a s e i n d i e t  i n the blood  while of pigs  t h e r e was a .very c o n s i d e r a b l e  amount when t h e y were on a d i e t grains.  of the  of grasses,  corn or other  32 The in  Hunter d i a z o  a later part  test  of t h i s r e p o r t  E a g l e s and  Vars  (73)  applied  hydrolyzed  with  sulfuric  (72) was  which w i l l  known a t  i t directly  acid.  The  be  this  to a  described  time  sample o f  results indicated  presence of a t h i o l i m i d a z o l e though they f o u n d t h a t interference  of h i s t i d i n e prevented the  conclusions. dine  i s not  This the  report w i l l  only  t h i s work E a g l e s and proteins as  the  i n the  that  has  animal body.  been the  I n 1935  Potter  different  i n the  and  The  This  basis  On  the  histi-  basis  i s the  very  these r e s u l t s that do  the  not  o r i g i n of  content  c a r r i e d out r a t s as  an the  I,  blood  It i s interesting  Pranke conclude  of the  suggest  they  with  d i f f e r e n c e between  and  ergothioneine  venture to  of the  i n Table  Potter  be  the  s t r i k i n g differences that  summarized  same g r a i n .  occur  report.  (74)  they found c o n s i d e r a b l e  samples of t h e  exogenous b u t  The  of  presence i n c e r t a i n  amino a c i d m i g h t  of t h i s  Pranke  ergothioneine  d i e t s are  to note that  occur.  the  s e r i e s of d i e t a r y experiments using  experimental animal. obtained  drawing of d e f i n i t e  substance.  amino a c i d t h i o l h i s t i d i n e .  expanded  the  demonstrate that  Vars p o s t u l a t e d  zein  the  o f a t h i o l i m i d a z o l e w h i c h w o u l d most p r o b a b l y  methylated thesis  later  interfering  and  blood  is  two from  entirely  i n what f o r m i t  may  33  Table  I,  Ergothioneine  content  Minimum mg/lOOml  Maximum mg/lOOml  Mean mg/lOOml  Number of t e s t s  1  0.69  1.23  0.93  27  Wheat  0.12  0.66  0.43  57  1  1.18  1.30  1.22  7  2  0.46  0.87  0.59  7  Barley  0.15  0.15-  0.15  9  Potato  0.08  0.08  0.08  7  Stock  0.98  1.28  1.17  6  0.08  0.08  0.08  12  Diet  Corn  Oats  Low  2  Thioneine  1. 10$  The  3  grain diets contained  c a s e i n , 3$  lard,  1$ M c C o l l u m ' s s a l t 2. 10$  cod  oil, 3.  1$  Low  yeast,  NaCl,  corn  of the g r a i n ,  o i l , 2$ d r i e d y e a s t  and  mixture. contained  1$  25$  wheat, 25$ alfalfa,  c o r n , 20$  5$ p e a s , 3$  oats, cod  CaC0 . 3  thioneine diet  10$  rats  as w e l l as 82$  liver  f i s h m e a l , 5$ wheat germ, 5$  liver  10$  Stock d i e t  2$  of blood of  c o n t a i n e d 45$  s t a r c h , 10$  s u c r o s e , 20$  b u t t e r , 3$  salts,  2$  casein,  cod  liver  oil. Physiology Because e r g o t h i o n e i n e had  now  n o r m a l c o n s t i t u e n t o f mammalian b l o o d , statement o f B a r g e r and  b e e n shown t o be i t was  felt  that  Ewins t h a t e r g o t h i o n e i n e had  p h y s i o l o g i c a l a c t i o n s h o u l d be  verified  and  more f u l l y  a the  no studied.  34 Tainter  (75)  injected bilary  d i d t h i s by  i n t r a v e n o u s l y on  the blood  t h e r a b b i t and  years  later  l y T r a b u c c h i was also found  not  cat.  He  ergothioneine pulse rate,  found  B a t h e d i n a 0,1$ solution  blood no  sugar  effect  incorrect closely  i n the  found  raised  on  in  c y s t e i n e and  poisoning.  another  Apparent-  the work of T a i n t e r ,  the f r o g ' s  a 1%  He  the u t e r i n e muscle  in  heart. Ringer's  s o l u t i o n caused a •  o f the beat  and  a l s o s t u d i e d the  f o r thiamin  temperature  delay death;  like  amplitude  Trabucchi  t o and  f u r t h e r decrease not  faster while  gave i t a  caused i t to  effect  of  Finally,  one  found  that  daily  prevented  s t e a d i e d the walking  Trabucchi  a theory  degree,  but  did  ergothioneine,  an a n t i d o t e i n  that cyanide  (77),  a c t e d by  cyanide poisoning  cysteine-cystine reduction-oxidation  systems'but T r a b u c c h i  show t h a t b o t h  and  intramuscularly  g l u t a t h i o n e , a c t s as  t h e g l u t a t h i o n e and  i n a similar  of the b i r d  i n weight  T h e r e was  structure rather  p o s s i b l y d e r i v e d from ergothioneine  t h a t 2,5mg o f e r g o t h i o n e i n e  the  animals.  a c t i o n on  a c t i o n on  out  p i g e o n s w i t h b e r i - b e r i because an e a r l y  formula  related  carried  s o l u t i o n of ergothioneine  in diastole,  ergothioneine  with  without an  the h e a r t beat  r a p i d decrease stop  noted  (76)  experimental  acquainted  ergothioneine  o f the r a b b i t but  to  pressure,  Trabucchi  s e r i e s of t e s t s u s i n g various  act  a c t i o n of  case, A few  He  the  s e c r e t i o n , smooth m u s c l e c o n t r a c t i o n and  l e v e l using both any  testing  said  manner.  t h a t i f so e r g o t h i o n e i n e must The  evidence  thiolhistidine  and  presented  by  ergothioneine  PIrie are  also (78)  35 catalysts help  o f t h e o x i d a t i o n o f g l u t a t h i o n e and c y s t e i n e , might  explain this  effect.  o f h i s work, a g r e e d that  ergothioneine  Thus, w h i l e  w i t h B a r g e r and Ewins, T r a b u c c h i  but  that ergothioneine  got negative  hlstidine  concluded  has s e v e r a l p h y s i o l o g i c a l a c t i o n s ,  B r a u n , Mason a n d Brown possibility  T a i n t e r , on t h e b a s i s  results.  (79) i n v e s t i g a t e d t h e  might  The f a c t  nor ergothioneine  occurs  act i n place that neither  of insulin thiol-  i n i n s u l i n had already  e s t a b l i s h e d by du Vigneaud, S i f f e r d  and M i l l a r  been  (80) u s i n g t h e  b r o m i n e o x i d a t i o n method o f Zahnd a n d C l a r k ( 8 1 ) , Pathology S e v e r a l workers have found of the blood found  and u r i n e t o v a r y  persons b u t found  f i f t e e n d i a b e t i c cases, per  with disease.  3-12mg/l00ml o f r e d b l o o d  nephritic  liter  being: h i g h  diseases  i n both  i t t o be f r o m 7-15mg/l00ml i n (83) f o u n d  been p u b l i s h e d  Herbert  and cancer has  W i n e g a r d and h i s  a s f a r a s i s known t h e i r r e s u l t s h a v e n o t y e t (52),  A study  b l o o d was made by Nakayama  of the diazo r e a c t i o n ofthe  (84) a n d he a r r i v e d  c l u s i o n t h a t e r g o t h i o n e i n e was p r o b a b l y the r e a c t i o n .  o r lower,  and a r t h r i t i s .  s t u d i e d by the late:  a s s o c i a t e s but,  90mg  i n normal u r i n e and n o t i c e d t h a t  T h i s r e l a t i o n between e r g o t h i o n e i n e beenfurther  S a l t (82)  i t m i g h t be e i t h e r h i g h e r  cancer  content  c o r p u s c l e s i n normal and  S u l i v a n a n d Hess  of ergothioneine  with different  the ergothioneine  at the con-  the c h i e f cause o f  I n d i s e a s e s where h e f o u n d  a strong  diazo  36  reaction all  i n the  b l o o d he  t h i s work on  fifteen  pathology each f i g u r e  individual tests  properly controlled. the  concentration of  v a r y on  a constant  retained theory  to  of  M a c k e n z i e and  are  significant b l o o d and  t h e n i t must e i t h e r  be  of  (85)  e f f o r t to  of  The  urine  does  synthesized  find  his  Important  or  latter  many r e l a t e d  (86,  several  87,  88)  by  goitrogenic sulfur and  i s now  Rimington noticed  a s u l f u r atom and  they  without  toxic  long  the  widely  used. and  that a l l , to  suggested that  compound e r g o t h i o n e i n e w h i c h c o n t a i n s t h i s g r o u p and  In c l i n i c a l  a  however, c a u s e a g r a n u l o c y t o s i s  Lawson and  blood n a t u r a l l y  less  tested  compounds c o n t a i n e d a c a r b o n atom l i n k e d and  com-  w h i c h were a c t i v e ,  which, p r o p y l - t h i o u r a c i l ,  effects.  a  s o m e t h i n g more a c t i v e  co-workers  Lawson  i n 1943  t h i o u r e a has  of  physiological  demonstrated by  discovery  that  investigation  s u b s t a n c e may,  active  a very first  i n 1947.  compounds f i n d i n g  toxic  nitrogens  e f f e c t s m i g h t be  two  the occurs useful  treatments.  Lawson and the  and  Wakayama^s work makes t h e  p r o v e t o be  Astwood and  other toxic  In the  were  Effect  (66)  l e d to the  Even t h i s  the  diets  e r g o t h i o n e i n e i n the  Mackenzie  pounds i n an  least  i t i s d o u b t f u l i f the  e r g o t h i o n e i n e was  Rimmington  series  In  seem more p r o b a b l e .  function  toxic.  urine. about  I f these r e s u l t s  diet  What may  action  but  i n the  i s b a s e d on  a varying degree.  Anti-thyroid  and  f o u n d a weak one  e f f i c i e n c y of  Rimington t h e r e f o r e proceeded to  e r g o t h i o n e i n e as  a thyroid  test  inhibitor.  They  37 found  that their  r e s u l t s v a r i e d when t h e y u s e d t h e r a t g r o w t h  method o f d e t e r m i n i n g  i t s activity  when t h e y m e a s u r e d t h e of r a t s The  administered  iodine content  a c t by p r e v e n t i n g t h e  b a s e d on  of  given In Table  of ergothioneine  thyroid  subcutaneous  the theory  accumulation  Their results,  activity  of the  e r g o t h i o n e i n e by  l a t t e r method was  gland.  b u t were more c o n s i s t e n t  injection.  that these  i o d i n e i n the II, indicate  i s comparable  gland  to t h a t of  drugs thyroid  that  the  thiouracil.  I n as much as v a r i a b l e q u a n t i t i e s o f e r g o t h i o n e i n e o f  the  same o r d e r  i n the  as  those  administered  blood  these workers found  their  t e s t s and  by  them may  Table  H i  suggest  Effect  i t difficult  that  h a v e b e e n due  to t h i s  mg/Kg body  wt.  to p r o p e r l y  control encountered  difficulty.  of Ergothioneine  Iodine  present  some o f t h e v a r i a t i o n s  Thyroid  Dosage  are normally  on t h e  Iodine of  the  Gland  content  in  g/lOOmg o f wet  thyroid  Ergothioneine Mean Deviation  Thiouracil Mean Deviation  0.2  20.0  4.03  19.6  1.49  0.5  13.5  4.11  8.0  2.40  0.75  21.0  4.00  4.65  2.25  5.0  1.76  6.4  1.20  2.0  Control  58.2  ± 3.48  Astwood and  g/lOOmg o f wet  Stanley  (89)  carried  thyroid  t h i s work f u r t h e r  38 by  testing  the c l i n i c a l  e f f e c t i v e n e s s of ergothioneine,  as a t h e r a p e u t i c a g e n t cause of  simple  i n t h y r o t o x i c o s i s and  goiter.  Measuring the  e r g o t h i o n e i n e by  i t s effect  active  the  rate 400  I o d i n e by  in five mg  on  thyroid  of e r g o t h i o n e i n e  orally  injected with mercaptothiazole o t h e r h a n d showed a s h a r p  the  they  Stanley t r i e d  or  o f the  of the  w h e t h e r i t was  see  of these  c o u l d h a v e b e e n due but  to repeat  time  to u s e  cases case. those of  effect  the  o f Astwood and  the  and  on  to  and or  treated with ergothi-  I n the  by  f o o d o r by of  Lawson and  some i m p u r i t y i n t h e i r  injection.  ergothioRimington  ergothioneine  results,  Rimington  (90)  had  ergothioneine c l i n i c a l l y  suggest  on  change i n the weight  observed  o f human t h y r o t o x i c o s i s b u t They  to  Persons  t h e work o f Lawson  no  administered  again with negative Lawson and  of  e x p e r i m e n t s on humans,  f e e d i n g an impure c o n c e n t r a t e  i f the  change  thyroid gland  t h y r o i d s of r a t s  to  no  or methyl t h i o u r a c i l ,  i o d i n e content  neine  found  intravenously.  could f i n d  They a l s o t r i e d  the  a d m i n i s t r a t i o n o f 50  R i m i n g t o n on r a t s b u t  oneine  of  drugs.  Owing t o t h e f a i l u r e Astwood and  activity  break i n the r a t e of uptake  l e v e l i n g o f the r a d i o - a c t i v i t y a d m i n i s t r a t i o n of the  as a p o s s i b l e  the r a t e of uptake of r a d i o -  gland,  n o r m a l p e r s o n s on  both  S t a n l e y may  e r g o t h i o n e i n e to the  In the treatment  got  the p o s s i b i l i t y  proceeded at the  no  response  that t h i s  h a v e b e e n due  disulfide.  of  same two  i n either  failure  and  to o x i d a t i o n  39 Latner ergothioneine  and  Mowbray  content  of  the  p a t i e n t s when f a s t i n g and c a s e s and the  a low  does not  as  i s t h i o u r a c i l and  by  the  of  i t below normal i n  other  two.  Stanley  seem t o be  four  They s u g g e s t may  be  due  bound by  that  to the  fact  serum p r o t e i n  more e a s i l y  excreted  kidney.  n e i n e has  goitrogenic  Venturi  (92)  who  support  f o r the  properties synthesized  thesis that  comes f r o m t h e and  tested  thio-uracil.  I f these three  seems p r o b a b l e t h a t r i n g would not  s u b s t i t u t i o n of betaine  a f f e c t the  QV::  compounds a r e  ergothio-  work o f  three  to  f o r the  benzene extent.  C>\X<=I>  HOCH V /N-CHg-OH v  5  s  methyl  a l l active i t  a c t i v e grouping to a great  2  Monti  thiol-  b e n z i m i d a z o l e s f i n d i n g them o f c o m p a r a b l e a c t i v i t y  H-N  the  six thyrotoxicosis  h e n c e i t c o u l d be  More i n d i r e c t  and  found  o f Astwood and  ergothioneine  have made a s t u d y o f  blood  normal i n the  difficulties  that  (91)  a  H Thio-Benzimidazole  1,3 d i ( h y d r o x y m e t h y l ) thiobenzimidazole  W a r n e r - J a u r e g g and studies  on  Among t h e  the  goitrogenic  that  (93)  e f f e c t of  but  i t may  have not be  They p o s t u l a t e  a heat that  i s o l a t e d i t and  r e l a t e d to  the  have c a r r i e d  out  several natural  a c t i v e compounds f o u n d was  from white cabbage. thiourea  Koch  1,3 - d i - p h e n y l thiobenzimidazole  labile  this  is a  therefore  thiol-imidazoles.  products.  compound benzyl-  i t i s possible  40  Analysis S e v e r a l methods a n d m o d i f i c a t i o n s t h e r e o f have b e e n developed  f o r t h e d e t e c t i o n and d e t e r m i n a t i o n o f t h i o l -  imidazoles.  The f i r s t  and s t i l l  t h e most g e n e r a l l y u s e f u l  method i s a n a d a p t a t i o n o f t h e d i a z o ^reaction w h i c h was p u b l i s h e d b y H u n t e r i n 1928 ( 7 2 ) .  This test  gives a yellow  c o l o r w i t h t h i o l l m l d a z o l e s which changes t o a p u r p l e r e d on t h e a d d i t i o n o f c o n c e n t r a t e d a l k a l i . in  t h i s way g i v e s a p i n k c o l o r v e r y  thiolimidazoles obscure  and h i s t i d i n e  the desired test  similar  t o that of the  g i v e s a y e l l o w c o l o r w h i c h may  a n d make q u a n t i t a t i v e  thiolimidazole d i f f i c u l t . modified recently  T y r o s i n e when t r e a t e d  estimation of  The method h a s b e e n  slightly  t o i n c r e a s e i t s a c c u r a c y w i t h t h e modern  photoelectric  colorimeter (94),  been proposed  by L a t n e r  Also another  m o d i f i c a t i o n has  (95) f o r u s e i n t h e d e t e r m i n a t i o n o f  ergothioneine i n blood. In  1929 B e h r e a n d B e n e d i c t  method b a s e d neine used is  based  with by and  Newton a n d B e h r e  on t h e p r e c i p i t a t i o n o f u r i c lactate  dissolving  subsequently  ergothioneine  the u r i c  acid  with Polin  two  sodium  substances chloride  sodium c y a n i d e .  The  c o l o r i m e t r i c a l l y by t h e  a n d Wu's  tungstic  a c i d reagent (54),  considered rather less  by Hunter.  T h i s method  and e r g o t h i o n e i n e  acid with acid  the ergothioneine with  i s , however,  that developed  (56).  and t h e s e p a r a t i o n o f t h e s e  i s then determined  c o l o r developed This test  out another  o n t h e o r i g i n a l method o f i s o l a t i o n o f e r g o t h i o -  by B e n e d i c t ,  silver  first  (96) b r o u g h t  specific  than  41  A quantitative various out  by  This  forms of Zahnd and  in  ^C=S,  reagent. not  s u l f u r present Clarke  study i n c l u d e d  o x i d a t i o n by  an  (97)  b r o m i n e and  This  there  i s no  a bromine l a b i l e  established  as  proof of  the  O t h e r methods o f t o have been u s e d f o r ' the  of  method o f  Masuka u t i l i z e s peroxide.  the  been c a r r i e d Clarke  effect only  and  presence of  I n t h e i r s t u d y on  in this  in this field  not  Masuka  do  substances  containing  oxidized:  -NM3-SR,  -N=6-S-C=N-,  and  Grote differentiating also  the  0=C-SH,  yet  not  seem  o f work a r e (98)  and  s u l f u r by  means o f  the  influence  of  presence of are  the been  the  the  technique of Kitamura  following  (80)  thiollmldazoles  of  i n the  Bromine  Miller  and  hydrogen  HgOg on  compounds t h e y f o u n d t h a t , w h e r e a s RCHgSH and  phene g r o u p s were s t a b l e  but  thiollmldazoles.  determination of  oxidation  sulfur  i n i n s u l l i n but  a n a l y s i s w h i c h as y e t  The  1  of  thiollmldazoles  Vignead, S i f f e r d  Grote,  (81),  that  labile  the  the  s u l f u r compound.  p r o c e d u r e o f K i t a m u r a and  colorimetric  has  s u l f u r f r a c t i o n has  which might prove u s e f u l  See  that  was  thiolhistidlne  presence of  sulfur  found that  o t h e r commonly o c c u r r i n g Du  of  B l u m e n t h a l and  linkages  sulfur includes  to prove that  1  and  ^C-SH  b e e n u s e d by  oxidative  i n proteins  i t was  o x i d a t i o n has  but  reactions  i n v e s t i g a t i o n of the  ^C-SH, and  o f any  study of the  various thio-  t h i s reagent,  quantitatively  S=C~SH, =N-C-S,  -0-C=S,  °jS=S, (99)  d e v e l o p e d an  i n t e r e s t i n g method  s u l f u r compounds, u s i n g  section  II,  the  of  product r e s u l t i n g  42  from the reagent  a c t i o n of l i g h t w h i c h may  on  a l s o he  sodium n i t r o p r u s s i d e .  made b y  treating nitroprusside with  bromine, g i v e s a green c o l o r p a s s i n g and  purple  reaction  to crimson  with  )C=S,  through turquoise,  -N=S,  )S=S  or  t o Ippm o f  thiourea.  gives a t r a n s i e n t blue  color with  >C-SH g r o u p s and  t h i s reagent  with  The  ergothioneine  mental s e c t i o n of t h i s  results  The  The  reagent  also  with  obtained  are d i s c u s s e d  blue  groups.  i s sensitive  S - S - C r g r o u p s on r e d u c t i o n .  This  i n using  In the  experi-  report.  Synthesis I t has demonstrated the r e a c t i o n s but  been p o i n t e d out  t h a t B a r g e r and  structure-of ergothioneine  d i d not  confirm  this  discovered that ergothioneine  constituent  of the blood  both  until  was  S e v e r a l a t t e m p t s were  November 1948  method.  The  when W i n e g a r d  details  i n v o l v e s the use  of  made b u t (50)  and  before  s y n t h e s i z e d by  synthesize  and no  not  one  a  seem t o h a v e synthesis  diazomethane.  this.  The  Pyman (71)  first  however b e e n  was  from dibenzamidoketo butane.  afterward  Ashley  and  I t was  synthesized  thiolhistamine  synthesis are p a r a l l e l to those used  thiolhistidine.  the  succeeded  developed  steps of the by  to prove  i t i s o n l y known t h a t t h e  Other t h i o l i m i d a z o l e s had, many y e a r s  a normal  finally  o f h i s p r o c e e d u r e do  b e e n p u b l i s h e d as y e t  synthesis.  i t became d e s i r a b l e t o  to provide m a t e r i a l f o r experimentation  structure.  (49)  b y means o f i t s  s t r u c t u r e by  When i t was  Ewins  Harrington  (51)  i n the  The  shortly  synthesis  Pyman w h o " f i r s t d e v e l o p e d  the  of  method  4 3  method o f s y n t h e s i z i n g t h e t h i o l i m i d a z o l e r i n g b y t h e u s e o f t h i o c y a n a t e , t h e method w h i c h h a s b e e n u s e d by a l l s u b s e q u e n t proceedures. To s y n t h e s i z e t h i o l h i s t i d i n e A s h l e y  and H a r r i n t o n  s t a r t e d w i t h h i s t i d i n e methyl e s t e r and b e n z o y l a t e d i t according breaking ated  t o t h e method o f WIndaus, D o r r i e s a n d J e n s o n the imidazole r i n g .  One b e n z a m i d o g r o u p I s e l i m i n -  t o g i v e a ketone and t h e r i n g r e a s s e m b l e d w i t h  cyanate t o give  (100)  thio-  thiolhistidine.  H 0 G=-C-CH -CH~C t 3 N Jm NH 0CH C H /  2  X  2  3  0  X  £ CI  Cold NaCO  >  H 0 q—C,—<JHg — C H — C HN O C HN HN  IP  H 3  C  \CH30H [HCI  C&V— C - C H p - C H C  ' «  I  a  NHg  0  NHg  OH  HC1  =C 0  p  CH -CH-C HN 0CH^ 2  v  V i i  KCNS  C=G~CE2-CE~([*  r  N-JJ  N  NH2  "  H  S H  H a r r i n t o n : - and O v e r o f f to form ergothioneine i n c i d e n t a l l y developed amino a c i d  (52) t r i e d u n s u c c e s s f u l l y  by m e t h y l a t i n g  t h i o l h i s t i d i n e but  a n o t h e r method t o s y n t h e s i z e t h e  starting with  aspartic acid.  44 .0 C |\)H CH  cr  v  l\)H G- CH*  CHg  2  + 0. CHNHo  PCI  i  -CH,  CH-N^  CH-N C-CH, C —0  C—0  o'  cf  0 H CH—N C  \  It  c-  CHg-NHg  CHp-N' I GC=0 6  r  \  X3-SH N^  ,KCNS CHo « , 2  CHNHo  HC1  , P h t h a l l m i d e CHr> * |^ 0 CH-NH-C-CH CHHH-C-CB  9=2  CHNHo I OH  I  CJ-0-0 H 2  X)H  %°-  5  *o  Still o u t b y Dey (101) malonic  another  synthesis of thiolhistidine  using  ph£/ia/o-a>-)odo-acetonylimide  C  2 5 H  was w o r k e d and  ester# ,0  0  -c -q  0  x  ^  9  OH / C CHoI + Na- 6 — N  Jo  N  foH -C-CHo-C N  cr  ^  c-  k  6 OH HBr .0 CH^C-CHg-CH-C^ HN N NH \)H 2  S i  H  (  KCNS  9  0 H N - C H - C - C H - C H - C^ NH OH 2  2  3  2  2  45  II,  Consideration  I t has  of  Problem  been p o i n t e d  m e t h i o n i n e made p o s s i b l e nized  reactions  of the of  protein  of p r o t e i n s  light  of the  a c c o u n t e d f o r by  to  i f there  m i g h t o c c u r as  total the  the  The limidazoles ments and and  Hess  (103)  a b o u t 90 If  t h e n an least  average d a i l y  135  mg,  and  the  n e i g h b o r h o o d o f 0,01$ i n the  food  The  of  the  undertaken  s u l f u r that  to o b t a i n  reactions an  feeding  e t a l . (81,  estimate  thio-  experi-  97),  n o r m a l human u r i n e by  Sullivan contains  colorimetric analysis. is entirely  of ergothioneine  must c o n t a i n  on  the  Clarke  and  of  exogenous  w o u l d be  at  average i n  I f t h i s were  i n a concentration  work o f Zahnd and  the  the h y p o t h e s i s t h a t  ergothioneine.  p r o t e i n i t w o u l d be  that  and  unexplained  ergothioneine  intake  that  unknown f r a c t i o n s .  Clarke  as m e a s u r e d this  total  come f r o m t h e  have found t h a t  i t i s assumed t h a t  The  studied  i n favor  s t u d y made by  mg/liter  importance  known amino a c i d s be  occur i n proteins  the  of  a r e i n v e s t i g a t i o n of  a thiolimidazole.  evidence  recog-  d i f f e r e n t from  s u l f u r of p r o t e i n s  nature of the  of  accounted f o r a large f r a c t i o n  e x i s t s a f r a c t i o n of the  amount and  discovery  of p r e v i o u s l y  known t o be  o f p r o t e i n s u l f u r must a l s o be of  the  It i s likewise  r e l a t i o n s h i p between the  see  and  literature  sulfur  that  explanation  s u l f u r t h a t was  c y s t i n e or c y s t e i n e .  i n the  the  out  of  the  concentrated  a b o u t 0,15$,  Blumenthal  46  and  Clarke  x  limidazoles fractions nitric the  i n d i c a t e s an  even h i g h e r  i n proteins.  Proteins,  thereof  a c i d and  total  the  ;C=S  s u l f u r of or  o c c u r s as  reconcile  the  Of  ~ *CN  fact  ergothioneine SH  I f the  that  but  give  not  Clarke  Lefevre  and  that  find  laboratory  that  this  In  of  sulfur i n to as much o f  Rangier  by  which  occurring  acid hydrolysates  t h i o l l m l d a z o l e s are destroyed  been found i n t h i s  does not  or that  bromine o x i d i z a b l e  B l u m e n t h a l and  whole p r o t e i n w i t h t h e work o f  I t has  linkages  hydrochloric  the  bromine, a reagent  a thiolimidazole i t i s d i f f i c u l t  f r a c t i o n i n the  ,found t h a t  and  d i f f e r e n t f r a c t i o n s of  particular interest-is  o x i d i z a b l e by  or methionine.  proteins  this  the  °*C—SH  cystine  thio-  protein hydrolysates  w i t h bromine to y i e l d  sulfur present.  oxidize  of  have been t r e a t e d w i t h a l k a l i n e p l u b i t e ,  f r a c t i o n f o u n d t o be will  concentration  as  in  (32)  the  who  treatment.  ergothioneine  a p o s i t i v e Hunter t e s t a f t e r b o i l i n g  with  2 hydrochloric  acid  but  I t i s p o s s i b l e that  p r o d u c t s are  still  o x i d i z a b l e by  bromine.  the  breakdown  Prom t h e  o f t h e i r work B l u m e n t h a l and  Clarke  a l s o a n o t h e r unknown f o r m o f  s u l f u r i n p r o t e i n s which i s  labile  i n a l k a l i n e p l u m b i t e but  Some o f t h e given  i n Table I I I ,  oxidizable high  but  proteins 1 2  r e s u l t s obtained  See See  It w i l l  s u l f u r content  that  of  not  conclude that  results  o x i d i z a b l e by  by  B l u m e n t h a l and  be  noticed  the  i t also occurs to  that  the  is  nitric  Clarke  albuminoids i s some e x t e n t  there  acid.  are  bromine particularly  i n a l l the  other  studied,  a l s o s e c t i o n I , 3, " I s o l a t i o n " . s e c t i o n I I I , "Separation of ergothioneine  f r o m amino  acids".  47  Table  III  Protein  Sulfur  Total  S  i n Proteins  f r o m B l u m e n t h a l and  Preexisting Sulfate S  Corr. S0 S by B r  4  2  Clarke  Corr. S0 S by HN0  4 3  Alkali labile  1  0.20  0.06  0.01  0.03  0.03  Gelatin 2  1.10  0.99  0.00  0.04  0.02  Blood  1.10  0.07  0.05  0.83  0.62  Lactalbumin  1.22  0.00  0.02  0.81  0.65  Egg  1.60  0.03  0.04  0.68  0.43  Zein  0.52  0.16  0.02  0.25  0.22  Casein  0.63  0.00  0.01  0.09  0.09  3.06  0.00  0.21  3.06  3.07  3.60  0.09  0.26  3.51  3.59  Gelatin  alb.  white  Wool  •  Horn  Some o f t h e r e s u l t s o b t a i n e d f r o m s u l f u r a n d amino acid  analyses of proteins  different the it The  by v a r i o u s  methods a r e c o m p i l e d  workers u s i n g  i n Table iVA.  several  F o r convenience  r e s u l t s f o u n d f o r e a c h p r o t e i n have b e e n a v e r a g e d i s r e a l i z e d that proteins  figures  were d o u b t l e s s  r e s u l t s have been c o r r e c t e d hydrolysis lated  t h u s o b t a i n e d may be m i s l e a d i n g . of variable  purity,  f o rthe addition  some o f t h e  o f water  and t h o s e o f B l o c k a n d B o i l i n g h a v e b e e n  o n a common b a s i s  although  of sixteen  percent nitrogen  during  calcui n the  proteins. One  o f t h e most  the  variability  and  cysteine  striking facts  shown by t a b l e i s  o f the values found p a r t i c u l a r l y f o r  and f o r s u l f u r i t s e l f .  cystine  The a v e r a g e f i g u r e s f o r  S  48 sulfur  i n z e i n account  reported  as  s u l f u r but  known amino a c i d s on  the  of  the  s u l f u r not  shows,only a for  total  small  i n most o f  accounted f o r .  proteins  the  on  the  assumption that  water of h y d r o l y s i s ,  cant. rise  Pre-existing to  a part  is usually it,  not  show t h a t  of  no  t h i s small  but  total  i s more t h a n  general  the  and  b e c a u s e most  proteins  may  sulfur.  be  amino  Insignifi-  also This  figures  s a m p l e s i t may  the  of-  b e e n made f o r  f r a c t i o n may  few  picture  s u l f u r unaccounted  s u l f u r unaccounted f o r .  detectable  the  there  c o r r e c t i o n had  s u l f a t e i n the  the  of  total  total  by  from percentages of  i n some p r o t e i n  twenty p e r c e n t  the  The  considered  t h e s e f i g u r e s were c a l c u l a t e d acids  figure given  s u l f u r Is correct  f r a c t i o n of  the  sulfur  than i s found i n the  o t h e r hand i f the  B a e r n s t e i n f o r the 20%  f o r t w e n t y - t w o p e r c e n t more  give sulfate  available c o m p r i s e up  Where one  sample o f  for to a  p r o t e i n has  b e e n a n a l y z e d f o r a l l known f r a c t i o n s and  total  sulfur,  r e s u l t s are  this  has  the  led Baernstein  possibility  (104)  usually and  i n g o o d v a g r e e m e n t and  Brand  (105)  o f unknown s u l f u r c o n t a i n i n g  However, t h e  dietary  i n d i c a t e the  presence i n c e r t a i n proteins  ted  f o r forms of  this of the  and  sulfur.  s u l f u r e x i s t s as  the  discrepancies  respective  niques to  to discount  the  units being  present.  bromine o x i d a t i o n  It i s possible  thiolimidazole. which e x i s t i n the  experiments of  that  as y e t  unaccoun-  a portion  Possible figures  explanations obtained  groups of workers employing d i f f e r e n t  assay the  n a t u r e and  o f unknown s u l f u r f r o m p r o t e i n s  amounts o f may  be  the  various  offered.  of  by  techforms  49 T a b l e IVA  Protein  Egg Albumin  D i s t r i b u t i o n of Sulfur  i n Proteins  S unaccounted f o i % of $ of protein sulfur  Cystine & Cysteine S  Methionine S  0.51$  1.09$  1.75$  0.15$  8.6$  0,60  0.98  1.60  0.02  1.2  33  0,46  1.13  1.60  0.01  0.6  3  0.47  1.12  1.85  0.26  14.1  5  0.40  1.40  6*  0.35  0.99  5  0.53  1.11  Sample  I  1  2  2  4  7  3  8  9  a  0.27  c  0.27 0.26 0.26  7  11  a b  12  0.24  b  7  10  8  0.63 0.64 1.36 '  9  13 ; 9  1 4  6  1,20  io  15  Total  2,0  10  1.66  16 io  1,71  18  1.60  10  1.18  1 7  19  11  a  1.12  b  0.87  S  50  Table  IVA  Protein  Egg Albumen Cont'd  Continued Sample  20  2 4  Methionine S  Total  S  S u n a c c o ia n t e d f o r % of % of protein sulfur  0,82  o,25 21 a  0,96  b  0.97  c  0,95  Average Casein  Cystine & Cysteine S  6.42  l.Oo  1.66  oTJ?  I  1  0.09  0.73  0.75  -0.07  -9.3  2  2  0.18  0.72  0.80  -0,10  -12.5  3  2  0.15  1.70  0.83  -0.02  -2.4  3  0.08  0.87  5*  0.07  0.73  6  0.08  0.79  4  5  7?  0.08  8  8  0.08  9  8  0.08  10 a  0.68  b  0.64  9  l l  1  0  1.2  12  1 1  13  1 2  0,09  14  1 4  0.06  15  1 5  0,08  16  1 6  0.08  1^18  0.63  0,07  0.69  •  0.78  0,0  0.0  51  T a b l e IVA C o n t i n u e d Protein  Sample •  Casein Cont'd  19 18 19  2 0  20 21  2 1  2 2  22  Cystine & Cysteine S  S  S unaccounted f o r % of % of protein sulfur  0.18 0.09 0.08  2 4  0,63  22 &  0.58  b  0.58  c  0.58  Average  Total  0.07  2  Lactalbumin  Methionine S  6.09  0.68,  0.53  Cv0~6  l  2  1.01  0.56  1.56  -0.01  -0.6  2  3  0.83  0.60  1.42  -0.01  -0.7  3  4  1.15  0.55  4  5  0.91  0.57  0.0  0.0  5 a  0.56  b  0.65  0  1.02  6 a  0.86  b  0.86  8  7 a  0,52  b  0.50  9  8  1 1  9  1 2  10  1 4  1.22 0.82 0.68  0.60  1.42  52  T a b l e IVA C o n t i n u e d Protein  Lactalbumin Cont'd  Sample  Methionine S  Total  S  S unaccounted f o r % of % of Protein Sulfur  0.61 1 2  1  13  1 Q  ,  14  7  . 2 0  15  2 1  , 23 16 a  0.65 1.06 1.00 0.70 0.81  b  0,81  c  0.81  d  0.82  -  .  ,«25 17 a  0.38  b  0.38  c  6.36  Average Wool  Cystine & Cysteine S  I 2  1  ,  0.82  5 7 5 0  1.40  0.08  3.8  0.13  3 . 9  0 . 0  0 . 0  0 , 2  578  .  "575  3.5  4  3 a  3.4  b  3.4  c  3.8  6  2.7 b 5 6  2.7  10 1 4  Average  3.06 2.6 372"  0.13  3.5  53  T a b l e IVA C o n t i n u e d Protein  Sample  Gelatin  I  Cystine & Cysteine S  Methionine S  0.05  1  0.24  Total  S  S unaccounted f o r % of % of protein sulfur  0.5  0.2  40.0  0.19  oTB"  0.25  loijo  0.53  1.2  0.3  25.0  0.99  0.07  2  2  3  4  0.05  0.21  4  5  0.043  0.21  0.21  5 !  0.19  b  0.13  1  0.17 c Average  i  Edestin  0.18 0.048 0.40  1  3*  •  0.48  0.44  0.27  0.45  0.31 b  0.34  c  0.34  • 57  0.36  6  0.33  7  77  0.37  8 a  0.37  b  0.47  9  9  -  13  10 11  1 4  0.85 0.33 0.36  7.0  54 T a b l e IVA C o n t i n u e d Protein  Edestin Cont'd  Sample  Cystine & Cysteine S  12  1 6  0,32  I3  1 7  0.35  14  1 8  0.20  1 9  0.26  16 °  0.48  17  2 1  0.32  18  2 2  0.32  IS  2  19 I  0.48  . b  0.48  2  c Average Zein  Methionine S  Total  S  S unaccounted f o r % of % of protein sulfur  0.48  oTBT  0.4S  IToT  0.24  0.51  0.36  -0.39  0.93  0.01  i  1  2  2  3  2  0.50  0.42  4  4  0.21  0.49  5  5  0.27  0.62  6  7  0.28  0719"  19.'' -108.  0.47  7 a  0.54  b  0.50  8 a  0.48  b  0.47  9  9  9  0.24  10  0.27  1.1  55  T a b l e IVA C o n t i n u e d Protein  Zein Cont'd  Sample  11  0.22  12  0,13  13  0.23  14  0.42  15  .  -  Methionine S  0.36  b  0.36  c  0.32  I  1  Total  S  S unaccounted, f oi % of % of protein sulfur  0,45  16 a  Average Serum albumin  Cystine & Cysteine S  0.27  0,46  0.60  -0713  1.7  0.28  1.7  -0.3  WW  1  -18.  A  1.63 3 a  1.52  b  1.52  c  1.62  6  4  7  1.62  5 6 7  1.89 10  1.75  11  8  1.10 1.74  0,18  1.94  0.02  1.0  12  1.68  0.28  1.96  0.00  0.0  Average  1.63  0"7£5  1772  -0.16  -9.3  9  1 2  '•  56 Table  IVA C o n t i n u e d  Protein  Gliadin  Sample  I  1  2  2  3  4  4  7  Cystine & Cysteine S  Methionine S  Total  0.69  0.5  1.2  0.0  0,0  0.26  0.43  0.99  0.30  30.0  1.1  bTT  9TT"  S unaccounted f o r % of % of protein sulfur  0.45  0.58  5 a  0.60  b  0.61  8  0.55  6  1  7  1 5  0.58  8  1 6  0,58  4  9 "  io  S  l s  0.65 0.62  ll  1 9  0.38  12  2 0  0.73  IS * 2  Average  0.57 0.56  074"6"  57  F o o t n o t e s t o T a b l e IVA. 1.  From B l o c k , the  R, J , a n d B o i l i n g , D., The D e t e r m i n a t i o n o f  Amino A c i d s ,  Cystine-cysteine  Minneapolis,  Burgess P u b l i s h i n g  b y phospho 18 t u n g s t i c a c i d ,  Co.,1940.  colorimet-  rically. M e t h i o n i n e b y h o m o c y s t e i n e method. S u l f u r by B u r g e s s r P a r r 2.  From B a e r n s t e i n , 669-74  s u l f u r bomb.  H. D., J . B i o l .  Chem., 9 7 , 663-8 a n d  by B a e r n s t e i n ' s  gasometric  (1931).  Cystine-cysteine  M e t h i o n i n e by B a e r n s t e i n ' s S u l f u r by p e r o x i d e 3.  Baernstein,  f u s i o n i n Parr  H. D., J . B i o l .  Methods n o t a v a i l a b l e . 4.  volatile  iodide  method. method.  bomb.  Chem., 115, 25  Hydrolysis  (1936).  by h y d r o - l o d i c  acid.  Cohn, E . J . , E r g e b . P h y s i o l . , 3 3 , 781 ( 1 9 3 1 ) . Methods n o t a v a i l a b l e .  5.  Figures  s e l e c t e d f r o m t h e l i t e r a t u r e b y D r . B. A. E a g l e s .  6.  S u l l i v a n , M. X. a n d H e s s , W. C., P. S. Pub. H e a l t h Reports,  7.  suppl,  86* ( 1 9 3 0 ) .  a,  Sullivan test.  b,  Okuda t e s t  c,  Folin-  Folin,  (Iodometric)•  Looney  test,  0. a n d M a r e n z i ,  J. Biol.  Method a m o d i f i c a t i o n o f t h a t 8.  Graff, Maculla  and G r a f f ,  Chem., 83, 103-108  o f F o l i n and Looney.  J. Biol.  Method a m o d i f i c a t i o n o f t h a t  (1929).  Chem., 121, 81-86 ( 1 9 3 7 ) ,  of Vickery  and  White,  58  9,  10.  B a e r n s t e i n , H. D., J . B i o l , a.  Volatile  iodide  b.  Homocysteine  Chem., 115, 31  (1936).  method.  method.  S h u l t z , P. M., Z . p h y s i o l . Chem., 25, 16-25 ( 1 8 9 8 ) . A c o m p i l a t i o n from  s e v e r a l e a r l y workers.  Methods n o t  available. 11.  B l u m e n t h a l , D. a n d C l a r k e , H. T., J . B i o l . 343  Chem., 110,  (1935).  Method n o t g i v e n . 12.  Brand,  E.,  A n n . N.Y. A c a d .  S c . , 4 7 . 187-228  C y s t i n e - c y s t e i n e by photometric i o d i n e Methionine by homocysteine S u l f u r b y method o f P r e g l 13.  method.  method. (Saschek)•  O s b o r n e , T. B. a n d C a m p b e l l , 609  (1946).  J . Am. Chem. S o c . , 18,  (1896).  Method n o t a v a i l a b l e . 14.  V i c k e r y , H. B . a n d W h i t e , 701  A., J . B i o l .  Chem., 9 9 ,  (1933).  O r i g i n a l method i n v o l v i n g cuprous  Isolation of cysteine  with  oxide.  15.  L o c . c i t . , Method o f P o l i n a n d M a r e n z i .  16.  L o c . c i t . , Method o f S u l l i v a n .  17.  L o c . c i t . , Method o f Okuda.  18.  L o c . c i t . , Method o f P o l i n a n d L o o n e y .  19.  L o c . c i t . , Method o f J o n e s ,  20.  L o c . c i t . , B a e r n s t e i n ' s g a s o m e t r i c method.  G e r s d o r f f and M o e l l e r .  59  21.  Vassell, B . ,  J. Biol.  Chem., 140, 323  O r i g i n a l method i n v o l v i n g  a color  (1941).  d e v e l o p e d w i t h p-amino-  d ime t h y 1 - a n l l i ne . 22.  L o c . c i t . , P o l a r o g r a p h i c method.  23.  Kassell  24.  and Brand,  J . B i o l . . Chem., 125, 115  a.  Photometric  b.  M i c r o p h o t o m e t r i c method,  c.  Sullivan  d.  B a e r n s t e i n method.  method.  method,  C s o n k a , F . A., a n d D e n t o n , C. A., J . B i o l . 329  Chem., 165,  (1946).  Method a m o d i f i c a t i o n o f t h a t 25.  (1938).  o f McCarthy and S u l l i v a n .  H o r n , M, J . , J o n e s , D, B,, a n d Blum, A. E . , J . B i o l . Chem., 166, 315 a n d 521 ( 1 9 4 6 ) . a.  M o d i f i e d method o f M c C a r t h y a n d S u l l i v a n w i t h hydrolysis.  b.  M o d i f i e d method o f M c C a r t h y and S u l l i v a n papain  c.  hydrolysis.  Microbiological  assay.  with  acid  60 Table IVB  Average  Gliadin  Serum albumin  Zein  O  Edestin  rH  Wool  Casein  Egg albumin  Deviations from the Mean of Cystine-Cysteine Results by Various Methods  Method  Deviation from mean i n % of s u l f u r of p r o t e i n  Vickery and White F o l i n and Marenzi  -.03 -.16 -.16  -.01 -.01  Sullivan  -.18  -.01  Okuda  -.15  F o l i n and Looney  -.15  -.7  +.18  -.02  -.03  +.02 -.01 +.03 +.01  +.00 +.01  -.05  -.02  -.05  -.01  +.02 +.01  -.02  -.11  +.00  -.08  -.11  +.07  -.06  -.01  +.04  +.01  -.26 -.02 -.22  +.2  -.03 -.03  -.17 -.18  +.2  +.00 +.00  +.20 +.22  +.6  +.00 -.14  -.14  -.09  -.05  -.18  -.08  +.23 +.14  -.30 .15  +.08  -.02  Jones, Gersdorf and Moeller Baernstein's gasometric  -.02  -.15  +.06 +.09 +.08  -.01 +.00 +.17  +.14 +.12  Vassell  +.00  -.13  -.01  -.05  Polarograph  -.01  -.01  -.01  Block and B o i l i n g  +.09  +.00  Graff -Kjeldahi  +.21  -.01  +.04  Graff BaSC>4  +.22  -.01  +.04  Baernstein  -.02 +.04 +.05  -.01  + .01  +.11  +.01  +.00  -.02  +.11 +.05  +.03  Photometric  +.6  +.06  -.03  +.07  +.13  +.05  -.5  +.04  +.05  -.5  +.05  +.05  61  I t has been found  that the t h i o l i m i d a z o l e r i n g  t r o y e d by t h e u s u a l methods o f h y d r o l y s i s . t h i s breakdown has n o t as y e t b e e n possible  of  i d e n t i f i e d but i t i s  s u l f u r c o n t a i n i n g amino a c i d s i n c e r t a i n  a n a l y t i c a l methods. t h e s e methods was  The f a c t  judged  a l l the s u l f u r  theory  The p r o d u c t  t h a t i t c o u l d r e a c t b o t h w i t h b r o m i n e and b e h a v e a s  one o f t h e o t h e r  for  i s des-  (106, 1 0 7 ) .  that the accuracy  o f some o f  by t h e d e g r e e t o w h i c h t h e y  of p r o t e i n s lends p l a u s i b i l i t y I f t h e breakdown p r o d u c t s  accounted  to this  of t h i o l l m l d a -  z o l e s a r e m e a s u r e d w i t h t h e known amino a c i d s i t seems most likely fault  that I t Is the determinations  f o r more b r o m i n e o x i d i z a b l e s u l f u r  albuminoids of these  than  c o u l d be p r e s e n t  variable  than  In methionine  G r a f f , M a c u l l a and G r a f f isolating  i s found  obtained  In the  determinations.  I n one method  (108) h a v e d e t e r m i n e d  contents.  c y s t e i n e by  thiolhistidine  T h i s method w o u l d  or ergothioneine  n i t r o g e n and by s u l f u r .  no s i g n i f i c a n t  The r e s u l t s  p o i n t e d out t h a t a l l r e s u l t s  obtained  as  determined  o f G r a f f e t a l . show  d i s c r e p a n c y but S h u l t z and Vars  so c l o s e l y .  present  t h e c y s t e i n e b u t i f t h e y were i n c l u d e d  t h e r e w o u l d be a d i s c r e p a n c y b e t w e e n t h e v a l u e s  hepatic  determina-  methods a r e much more more  t h e n i t r o g e n and t h e s u l f u r  i n the hydrolysate with  agree  fraction  i t f r o m t h e h y d r o l y s a t e s w i t h CugO and m e a s u r i n g  measure any u n s p l i t  by  i n the  I n the methionine  p r o t e i n s and t h e r e s u l t s  t i o n o f c y s t e i n e by d i f f e r e n t  both  of cysteine that are at  (109) h a v e  b y t h i s method do n o t  By t h e b a r i u m s u l f a t e method t h e y  found  p r o t e i n t o c o n t a i n 1-2$ c y s t e i n e n i t r o g e n b u t G r a f f  62  and  B a r t h found  i t t o have 4,65$ b y K j e l d a h l d e t e r m i n a t i o n s .  This difference  i n the f i n d i n g s  c o u l d be  l a r g e f r a c t i o n o f t h i o l h i s t i d i n e were In  Table  IVA  are  listed  explained i f a  present.  the r e s u l t s  of c y s t i n e - c y s t e i n e  d e t e r m i n a t i o n s by  sixteen different  methods a r e b a s e d  on t h e d e t e r m i n a t i o n o f t h e r e d u c i n g power  of  t h e -SH  cysteine  g r o u p and  would p r o b a b l y  s u l f h y d r y l groups from  breakdown p r o d u c t s . results Table from  o b t a i n e d by  IVB,  The  not  those  of  these  thiolimidazole t h e mean o f  the  methods a r e compared  I f o n l y c e r t a i n methods do  but  Most o f  differentiate  d e v i a t i o n s from  these d i f f e r e n t  the t h i o l i m i d a z o l e s  variable  methods.  n o t measure  t h e s e w o u l d be  expected  consistently negative d e v i a t i o n .  In  sulfur  to  show a  In the case  of  t h o s e methods f o r w h i c h enough f i g u r e s a r e a v a i l a b l e t o make statistical developed Moeller  treatment  by V i c k e r y and  and,  procedure, by  applicable this  except  White, by  f o r the r e s u l t  i s true of the  Jones,  Gersdorff  from wool, of  seems p r o b a b l e  ences i n the and  developed methods do Sullivan,  Sullivan's  f a c t o r s other than  s u l f h y d r y l groups.  M o e l l e r method by F o l i n and not  the average  t h a t t h e d e v i a t i o n shown b y  o r i g i n a l method a r i s e s f r o m  dorff  and  A m o d i f i c a t i o n o f t h e V i c k e r y and W h i t e method  G r a f f e t a l , shows r e s u l t s h i g h e r t h a n  therefore,  methods  Similarly  i s a m o d i f i c a t i o n of the  L o o n e y and  taken together  o t h e r hand, i s based  c o l o r r e a c t i o n w i t h c y s t e i n e , and  the  The  on a v e r y low  the.  differ-  the Jones,  show a c o n s i s t e n t d e v i a t i o n .  on t h e  the  and i t ,  Gers-  one the  two  method  of  specific  r e s u l t s obtained  by  63 it  may b e o f c o n s i d e r a b l e  has or  significance.  cysteine  those given has  developed  a s y e t (110) b u t i t w o u l d b e i n t e r -  b y S u l l i v a n ' s method.  b y t h i s method a g r e e  the formulation  A third the  o f amino a c i d s  of proteins  the usual  cysteine that  o f t h e d i f f e r e n c e s between  and t h e i r  indicated  and t h i o l i m i d a z o l e s I s t h a t  h i s t i d i n e might occur  content  c y s t e i n e and  l i n k e d as a t h i o ether.  laboratory  has thus f a r  o f a m i c r o b i o l o g i c a l method.  possible explanation  s u l f u r content  with  I t I s possible that i t  been i n t e r f e r e n c e from t h i o l i m i d a z o l e s t h a t  prevented  On h y d r o l y s i s  methods t h e t h i o e t h e r  might  yield  and a d e r i v a t i v e o f h i s t i d i n e b u t i t i s p o s s i b l e  i t would g i v e  hydrolysis. the  there  b e e n no m i c r o b i o l o g i c a l method f o r t h e a s s a y o f c y s t i n e  e s t i n g t o see I f r e s u l t s obtained  by  Unfortunately  serine  and t h i o l h i s t i d i n e b y enzymatic  I f t h i s be t r u e  i ti s difficult,  to explain  n a t u r e o f t h e b r o m i n e o x i d i z a b l e f r a c t i o n o b s e r v e d by  Zahnd a n d C l a r k e , HC = C - C H o - CH-C-OH  s  Hypothetical  m /> 2  thio  CHg-CH-C 0H r  HI,  ether  Experimental  Preparation  of  In order thiolimidazoles  ergothioneine  t o c a r r y out the proposed  i t was f e l t  that  the f i r s t  search f o r  r e q u i s i t e was a  64  supply  of ergothioneine  trols. Dr.  B.  and  t h i o l h i s t i d i n e f o r use  Seventy m i l l i g r a m s of A. E a g l e s  i n 1928  ergothioneine  were a v a i l a b l e .  prepared  but  little  i t was,  decomposition  nevertheless, f e l t  by  Recrystalization  o f t h i s m a t e r i a l f r o m a l c o h o l showed t h a t i t had relatively  i n con-  suffered  since o r i g i n a l l y  prepared  that a l a r g e r supply  was  desirable. T w e l v e h u n d r e d grams o f e r g o t were o b t a i n e d t h e g e n e r o s i t y o f t h e U p j o h n Company o f K a l a m a z o o , A preliminary test was  made b y  the f i l t r a t e  the Hunter t e s t .  ergothioneine mg  prepared  of ergothioneine  e x t r a c t i o n may neutral  ergothioneine  extracting a finely  water, c l a r i f y i n g applying  of the  with  Hunter  (67)  i n ergot  and  those  a b l e t o e x t r a c t 1.8  ton  the  (66)  course  has  differences different for  the  than  mg/g  and  boiling  some o f  In t h i s  than  test  the  the with  determining inaccurate.  a rather  from which P i r i e  H u n t e r e t a l . (68)  o f t h i s work a paper by  1  uranium  comparison  sample had  and  the  the c l a r i f i c a t i o n  the v i s u a l  ergothioneine  ergot  i n d i c a t e d only  i n h i s method o f  this  content  test  satisfactory  However, i t d o e s seem t h a t  During  this  have been i n c o m p l e t e ,  a c e t a t e u s e d by  of t h i s  neutral lead acetate  gram o f e r g o t .  less  Michigan,  sample w i t h  In comparison w i t h  by E a g l e s  per  lead acetate  ergothioneine  ground  content  through  lower  (65)  2.6  Lawson and  was  mg/g. Riming-  been p u b l i s h e d w h i c h d e s c r i b e s even g r e a t e r i n the  sources  ergothioneine and  ergothioneine  content  of ergot  more r e c e n t l y H u n t e r has content  of ergot  from  found  on'Alberta  rye  values varying  65  f r o m 0.184  t o 0.474$  (67).  N i n e h u n d r e d grams o f t h e according  t o P i r i e ' s method  white p r e c i p i t a t e and  i n 3:4  the Hunter t e s t histidine  and  (94)  ergothioneine.  s u l f u r was  difficulties  53  mg,  out  over  remaining  obtained  g i v e n by  ergot  a mixture  and  fact  that  the  but  Again  ergothioneine  this  again,  substance  These the  complete  small  but  f r a c t i o n were a small  yield,  gave o n l y a c o l o r  the Hunter t e s t .  I t i s thought  alcohol during drying  with  ergothioneine.  thiolhistidine  far  b e e n a s u n s u c c e s s f u l as t h o s e  The  method o f A s h l e y suitable.  of  much o f  Attempts t o s y n t h e s i z e t h i o l h i s t i d i n e have  most  by  s e v e r a l weeks.  (62),  This time,  have o x i d i z e d the of  the  a p e r i o d of  i m p u r i t i e s formed i n the  Synthesis  was  g a v e an o r a n g e c o l o r  i n t e r r u p t e d and  of the  at each s t e p .  s o d i u m may  mg.  typical  E v i d e n t l y o n l y a s m a l l amount  t o t h e method o f E a g l e s  was  but  t h r e e h u n d r e d grams were e x t r a c t e d  t y p i c a l . o f h i s t i d i n e by that  and  extracted from the  s e v e r a l times  typical precipitates obtained  o f a b o u t 40  h a v e b e e n c a u s e d by  isolation carried The  low  of  oxide  removed f r o m i t d u r i n g p u r i f i c a t i o n .  may  p r o c e d u r e was  according  product  s u c h as m i g h t be  was  s m a l l amount  gave c o r r e s p o n d i n g l y  acetonetwater  ergothioneine  the  steps  Only a  the a d d i t i o n of cuprous  However, t h e f i n a l  insoluble  of  f o r m e d on  a l l subsequent  yields.  (66).  e r g o t were t r e a t e d  and  In the  to i s o l a t e  Harrington  first  attempt  (51) was  thus  ergothioneine. chosen as  the r e a c t a n t s  the  exploded  66  with considerable violence during benzoylation. h a v e b e e n due of  acid  ice  t o c a r b o n d i o x i d e r e l e a s e d by  or to a small r i s e  had  melted.  periodically  This  the  may  formation  i n t e m p e r a t u r e a f t e r most o f  the  In subsequent runs the r e a c t i o n v e s s e l  was  opened a l t h o u g h  excessive pressure  was  never  noticed. In the  second attempt  a good y i e l d was  obtained  to  synthesize  from the b e n z o l y a t i o n but  methyl c8 -dibenzamido-Y-ketoval-erate u r e h e r e a p p e a r s t o h a v e b e e n due r e a c t i o n , was  not  One this by  synthesis.  Ashley  and  the  color  impure typical  out  was  t o the  very fact  small. that  b e e n made t o d a t e  T h i s t i m e y i e l d s were o b t a i n e d  Harrington  i n a l l except  crystalized  out.  The  s o l u t i o n of products of h i s t i d i n e  t h e r e a c t a n t s may  and  the f i n a l  of  Fail-  the  to c a r r y  out  as i n d i c a t e d s t e p when  no  H u n t e r t e s t when a p p l i e d  showed o n l y t h e  i t i s considered  have b e e n c o n t a m i n a t e d w i t h  c a t a l y z e the removal o f the  that  i n anhydrous c o n d i t i o n s .  more e f f o r t h a s  thiolhistidine to  carried  thiolhistidine  sulfhydryl  yellow  possible that  i r o n w h i c h would  groups.  I n i t i a l work w i t h p r o t e i n h y d r o l y s a t e s T h e r e a r e no isolate those  fore, for  i s o l a t i o n of these  T h i s , the  the f i r s t  attempt  compounds f r o m  to  c o u l d not  be  Unfortunately  made a t t h e  therea  test  termination of  t o i n t e r f e r e n c e f r o m amino a c i d s and  humus.  to  other  s i m p l e s t method o f a p p r o a c h , was,  s t u d i e d i n t h i s work.  ergothioneine  s t e p due  a v a i l a b l e o f any  t h i o l l m l d a z o l e s f r o m p r o t e i n s by methods p a r a l l e l  used f o r the  sources.  records  The  each  67  first  t e s t s , made o n z e i n , human h a i r  hydrolyzed  with  sulfuric  acid  gave a p p a r e n t l y  positive  colors with  purple p r e c i p i t a t e ,  and f i n g e r  and c l a r i f i e d  with  nails, charcoal,  t h e H u n t e r t e s t b u t no  A c o n t r o l u s i n g pure  ergothioneine,  however, showed t h a t when s m a l l q u a n t i t i e s were u s e d t h e ergothioneine charcoal  (Darco),  c o u l d be f o u n d coloured but  was a l m o s t q u a n t i t a t i v e l y r e m o v e d b y t h e I t may be t h a t a n o t h e r  w h i c h w o u l d be c a p a b l e  i m p u r i t i e s without  t h i s has  brand o f c h a r c o a l  of adsorbing the  destruction of ergothioneine  not as y e t been i n v e s t i g a t e d .  A test  o n egg w h i t e ,  hydrolyzed  with  sulfuric  in  t h e p r e s e n c e o f t i t a n o u s c h l o r i d e and t r e a t e d  to  Pirie's  b u t a g a i n no p u r p l e  a g a i n a week l a t e r test  according  (65) method f o r t h e i s o l a t i o n o f e r g o t h i o n e i n e  f r o m e r g o t , gave a s t r o n g r e d - p u r p l e test  c o l o r by t h e H u n t e r  precipitate.  t h i s product  However, when t e s t e d  gave a d e f i n i t e l y  negative  and no f r a c t i o n o f a sample o f egg w h i t e d i v i d e d  albumin, g l o b u l i n , positive  test.  m u c i n and p r o t e i n f r e e f i l t r a t e  These r e s u l t s ,  baffling  into  gave a  a t the time,  may h a v e  b e e n due t o t h e I n t e r f e r e n c e o f t y r o s i n e a s d i s c u s s e d following  these  preliminary familiarization  s e r i e s o f t e s t s was made i n a n e f f o r t  p r o t e i n s w o u l d be t h e most The  ina  section. Following  a  acid  to determine  tests which  satisfactory f o ra detailed  study.  method u s e d was a g a i n c h i e f l y b a s e d on t h e e a r l i e r  methods f o r i s o l a t i n g  ergothioneine.  lyzed with hydrochloric acid  The p r o t e i n was h y d r o -  a n d t i t a n o u s c h l o r i d e , a method  68  s u c c e s s f u l l y used of  cystine.  basic  lead  by  The  S u l l i v a n and  h y d r o l y s a t e was  a c e t a t e and  cuprous oxide  Hess  (103)  i n the  neutralized,  isolation  clarified  the t h i o l i m i d a z o l e p r e c i p i t a t e d  was  t r e a t e d w i t h hydrogen s u l f i d e and  a Hunter t e s t c a r r i e d  out  on  p r o t e i n c o n t a i n i n g sub-  stances  free f i l t r a t e .  t e s t e d and  are g i v e n i n Table  Table V  the r e s u l t s  The  The  with  precipitate  the copper  as i n P i r i e ' s p r o c e d u r e .  with  of the c o l o r r e a c t i o n  obtained  V,  Hunter Diazo  R e a c t i o n on  a Protein Hydrolysate  Fraction Substance Zein  Tested  1  Result  Substance  Positive  Cabbage  Negative  Tested  Result  Corn  Glutinin  Doubtful  Peccan  Doubtful  Ale.  & H2O  Probable  Filberts  Negative  Negative  Brazil  Negative  soluble Bad C o r n  2  corn (whole)'  3  Nut  Wheat  Glutinin  Negative  Ox  Hoof  Probable  Wheat  Gliadin  Possible  Ox  Horn  Negative  Negative  Ox  Lung  Possible  Hordein  Negative  Ox  Brain  Positive  Pea  Albumin  Negative  Ox  Fibrin  Negative  Pea  (whole)  Possible  Oat  Gliadin  1  Prepared  a c c o r d i n g - t o method o f Mason and  Palmer  2  The f i l t r a t e f r o m t h e p r e c i p i t a t i o n o f t h e a l c o h o l s o l u t i o n of z e i n i n water. T h i s m i g h t c o n t a i n any f r e e e r g o t h i o n e i n e of the c o r n .  3  M o l d y and d i s c o l o r e d corn used.  k e r n e l s w h i c h were p r e s e n t  (111),  i n the  69  No o d o r o f t r l m e t h y l a m i n e  was n o t i c e d i n a n y c a s e  p u r p l e p r e c i p i t a t e s were o b t a i n e d precipitates  although  a n d no  obscuring  o f humus were o f t e n p r e s e n t .  I t was n o t  definitely  known i f t h e s e were e s s e n t i a l c h a r a c t e r i s t i c s o f  a positive  t e s t under these  tests out  listed  conditions.  By t h e t i m e t h e  i n t h e t a b l e had b e e n c o m p l e t e d work  carried  c o n c u r r e n t l y o n I m p r o v i n g t h e method o f i s o l a t i o n  that  these  tyrosine  r e s u l t s were p r o b a b l y  left  i n solution after  revealed  o n l y a measure o f t h e n e u t r a l i z a t i o n and the p r o j e c t  was a b a n d o n e d . R e v i s i o n o f t h e method The to t e s t  objective of this  and r e l i a b l e r e s u l t s  was e x p e r i e n c e d  possible.  Difficulty anything  inordinately high concentrations of ergothioneine  steps of the procedure to detect  improvement  ously corrected.  interfering  were a t f a u l t  Therefore,  i t m i g h t n o t be p o s s i b l e  substances.  and l / l O O O i n o r d e r  B e c a u s e t h e r e was most  from d i e t a r y experiments f o r the presence zein this  p r o t e i n was u s e d  S u l l i v a n and Hess  simultane-  a series of dilutions  l / l O , l/lOO,  until  i f two  i n one u n l e s s t h e o t h e r were  I n each t e s t  made, u s u a l l y l / l ,  in  so a s t o  b e c a u s e i t was i m p o s s i b l e t o d e t e c t  a f t e r t h e f r a c t i o n a t i o n was c o m p l e t e .  out  o f e n d e a v o u r was  and m o d i f y e a c h s t e p o f t h e above p r o c e d u r e  make a c c u r a t e  but  second l i n e  were to dilute evidence  ofthiolimidazoles  i n the following t e s t s ,  (103) h a v e f o u n d  that the a d d i t i o n  of t i t a n o u s c h l o r i d e t o a p r o t e i n d u r i n g h y d r o l y s i s shortens the time  r e q u i r e d f o r h y d r o l y s i s and d e c r e a s e s  the destruction  70  of  cystine.  In the i n i t i a l  work i n t h i s  compound was u s e d and i t was investigate  i t s specific  The f i r s t  effect  r e p e t i t i o n of the t e s t s None o f t h e s e  by t h e i n i t i a l  omitted  on  c o l o r s w i t h the  w i t h and w i t h o u t  gave w e a k l y p o s i t i v e r e s u l t s  r e s u l t s was  a s s t r o n g as t h o s e  and E w i n s  of  ferric  ions.  Because the i r o n reduced  w o u l d be r e o x i d i z e d b y a i r i t was iron  i f present  therefore,  i r o n b u t i t was  The f i r s t  reagents  and when i t was  However,  when 6,5$  i n this reaction small  amounts  I n work performed thus f a r  test  ferric  a g a i n gave  with  hydrolyzed  hydrolyzed  negative  in distilled  i n 0,03$ f e r r i c  c h l o r i d e was  chloride.  boiled with a hydroly-  known t o g i v e a s t r o n g r e d d i a z o t e s t ,  product  considered  presence  t o a v o i d any p o s s i b l e , c o n t a m i n a t i o n  r e s u l t s b o t h when t h e p r o t e i n was  resultant  sulfur  deemed d e s i r a b l e t o i n v e s t i g a t e t h e n e c e s s i t y  this precaution.  s a t e t h a t was  i n the  feared that  the o x i d a t i o n of the s u l f u r .  gave a d i s t i n c t l y  to support  b e i n g m e a s u r e d was tinued  of  i n a t h i o l i m i d a z o l e s o l u t i o n would c a t a -  c a r e had been t a k e n  was  given  no e v i d e n c e  (49) h a v e shown t h a t t h e  t h i o l i m i d a z o l e s i s o x i d i z e d and removed  of  i n both  from subsequent h y d r o l y s e s ,  of  lyze  titanium while  due t o t h e t i t a n i u m and i t was,  Barger  of  reducing  thiolimidazoles.  t e s t s w i t h z e i n b u t t h e r e was  any improvement  this  considered d e s i r e a b l e to  t e s t s gave n e g a t i v e  hydrolysates of z e i n both  cases.  now  study  the h y p o t h e s i s  a thiolimidazole.  f o r the avoidance  negative  the  colour.  that the  This  substance  P r e c a u t i o n s were  con-  of a l l p o s s i b l e contamination  with  iron.  71  A series  o f t e d t s were made i n a n e f f o r t  mine t h e b e s t c o n d i t i o n s f b r h y d r o l y s i s . tests  to deter-  The r e s u l t s  o f these  a r e summarized i n T a b l e V I a c c o r d i n g t o t h e i n t e n s i t y  of t h e c o l o r formed but because o f i n t e r f e r e n c e by i m p u r i t i e s and  the v a r i a b i l i t y  n i f i c a n c e of these results  do n o t f a l l  of the effect results into  of t h e s t r o n g e s t c o l o r  (Difco.),  i s difficult  any d e f i n i t e  the r e a l  to assess.  some  by h y d r o l y s i n g  Hydrolysis with pepsin  and p a n c r e a t i n  sig-  The  pattern although  t e s t s were o b t a i n e d  with hydrochloric acid. papain  of d i l u t i o n  (Nutritional  (Difco.),  Biochemicals)  were t r i e d b u t n o n e o f t h e p r e p a r a t i o n s d i g e s t e d t h e z e i n completely not  and t h e r e s u l t s were I n c o n c l u s i v e b u t d e f i n i t e l y  as s t r o n g as t h o s e w i t h h y d r o c h l o r i c a c i d . I n many o f t h e s e  obtained  at the i n t e r f a c e  tests  a transient  on adding  alkali.  w h e t h e r t h i s was c a u s e d by d e c o m p o s i t i o n compound the  limit  In the strong a l k a l i  I t i s n o t known  o r by d i l u t i o n  o f i t beyond  of sensitivity.  were o b t a i n e d  low r e s u l t s  i n s e r i e s V I o f T a b l e V I where t h e a l c o h o l a n d  soluble fraction  the f i l t r a t e  obtained  o f t h e c o r n was u s e d .  any  free  The  color obtained  This  on t h e r e m o v a l o f z e i n a f t e r  the a l c o h o l e x t r a c t o f c o r n w i t h water,  free  was  of the coloured  I t w i l l be n o t i c e d t h a t r e l a t i v e l y  water  pink color  of interference.  volume o f t h i s  s e r i e s was d i s t i n c t  In fractionating  s o l u t i o n was o b t a i n e d  treating  i s that i n which  e r g o t h i o n e i n e f r o m the c o r n would p r o b a b l y i n this  fraction,  appear.  and r e l a t i v e l y  the corn a l a r g e  so t h a t , a l t h o u g h i t  Table Effect  of Conditions of Hydrolysis  Temperature Time  °C  hrs.  100 1.5  0.13  I  on t h e I n t e n s i t y  125 5.0  E x p r e s s e d as  Series  0.08  II  2.5  6.0  16..  28.  of the Color  Given by the Hunter  140  150  165  210  20  18.  2.5  2.0  0.17  0.0  Test  e q u i v a l e n t amount o f e r g o t h i o n e i n e I n p e r c e n t o f z e i n . 0.13 0.13 0.08  0.17 0.13 0.08  0.08 0.17 0.08  0.4  0.2  0.13  0.04  ?  III IV  0.04  0.04  V  0.08  0.2  VI  0.08  VII  0.0  Key  VI  to  I. II. III. IV.  0.32  0.13 0.0  0.08  9  Series 3g o f 3g o f 3g o f 3g o f acid,  zein i n zein i n zein i n zein i n 7$ HC1.  15ml o f 20$ 15ml o f 20$ 15ml o f 40$ 15ml o f 50$  HC1.  H2SO4. H2SO4. formic  V. VI. VII.  3g o f z e i n i n 15ml o f 20$ NaOH. 8ml o f a l c o h o l and w a t e r s o l u b l e f r a c t i o n o f c o r n w i t h 7ml o f 37$ HC1. 3g o f r e s i d u e f r o m r e s o l u t i o n o f z e i n i n a l c o h o l h y d r o l y z e d w i t h 20$ HC1.  73  gave a r e l a t i v e l y weak c o l o u r compared t o thatt f r o m an weight of z e i n , the  i t must h a v e c o n t a i n e d  c o l o u r g i v i n g compound t h a n  same amount o f c o r n . water  the  a g r e a t e r amount  z e i n prepared  I f t h i s compound i n t h e  s o l u b l e f r a c t i o n were e r g o t h i o n e i n e  0,07$ o f t h e of t h i s  t o t a l weight  s o l u t i o n d i d not  intensity  i t as  z e i n was  corn.  i t was  l a t e r found  r e - p u r i f i e d by  four  product  was  However,  to i n v e s t i g a t e f u r t h e r the nature  to destroy  ergothioneine.  successive  precipitations  the p r o t e i n but of the  a g e n t s t h a t had  i s o l a t i o n of ergothioneine  been found  from ergot  i n no than  case  was  c h l o r i d e and  there  t h a t i n the  less  Work  i t i s hoped colour giving  combinations of of value  and  combinations of baryta, phosphotungstic acetate, mercuric  prepared  fraction.  T e s t s were made u s i n g d i f f e r e n t precipitating  to  concentration  a p p r e c i a b l y weakened.  also  soluble  and  i t w o u l d amount  a hydrolysate  therefore, continued  i n the  with  not  by  was,  substance  alcohol  the  did hydrolysis with hydrochloric  from a l c o h o l , the c o l o u r developed from the p u r i f i e d  from  of  cause a p r o p o r t i o n a l i n c r e a s e i n the  o f the r e a c t i o n nor  acid destroy When t h e  o f the  equal  Several  acid, basic  i n t e r f e r e n c e or a  t e s t s performed with  i n the  blood.  cuprous oxide  lead  were t r i e d stronger  t h e method  the  but  colour  originally  outlined. During  this  study  i t became c l e a r t h a t t h e  employed f o r removing m e t a l l i c was  a p o s s i b l e source  s a l t s with hydrogen  method  sulfide  of d e s t r u c t i o n of t h i o l i m i d a z o l e s .  A f t e r p r e c i p i t a t i o n of ergothioneine  w i t h copper,  the  74  sensitivity  o f t h e H u n t e r t e s t was g r e a t l y r e d u c e d i f t h e  c o p p e r was n o t c o m p l e t e l y removed* c o p p e r s u l f i d e was o f t e n  difficult  n a t u r e and much e r g o t h i o n e i n e was p r o l o n g e d . gas  due t o i t s c o l l o i d a l  was d e s t r o y e d  the ergothioneine  was f o u n d t o g i v e  Hunter d i a z o  complete removal o f the copper  Early  and  b y Amino  i n shade and s e n s i t i v i t y .  were no d i s t i n c t serve  absorption  color  given by  An a t t e m p t was  s p e c t r o s c o p i c a l l y but there  bands,  to d i f f e r e n c i a t e the colours  A spectrophotometer when p r e s e n t  i t would n o t be s u i t a b l e f o r r o u t i n e Interference  and  h i s t i d i n e gave a y e l l o w  tyrosine a pink c o l o r c l o s e l y resembling that  made t o d i s t i n g u i s h t h e s e c o l o r s  but  study an I n v e s t i g a t i o n  sources of i n t e r f e r e n c e with the  I t was f o u n d t h a t  thiolimidazoles  with  Acids  i n the course of t h i s  b e e n made o f p o s s i b l e  Hunter t e s t .  or interference  test.  Interference  had  when t h e p r o c e s s  and copper b u t only  without d e s t r u c t i o n o f the ergothioneine the  of the  T e n d i f f e r e n t s u b s t i t u t e s were u s e d f o r t h e  i n separating  cupferron  However f i l t r a t i o n  might  i n combinations  work.  due t o h i s t i d i n e was e a s i l y  reconizable  was o n l y  serious  i n a q u a l i t a t i v e t e s t when i t was  sufficiently  intense  t o o b s c u r e a weak t h i o l i m i d a z o l e r e a c t i o n .  The  colour  r e a c t i o n f o r t y r o s i n e was s e n s i t i v e t o a b o u t  0,003 mg/ml and w o u l d be d e f i n i t e l y t y r o s i n e o f z e i n were p r e s e n t s a t e was, t h e r e f o r e , after  standing  p o s i t i v e i fa l lthe  i n the hydrolysate.  The h y d r o l y -  always c a r e f u l l y n e u t r a l i z e d and f i l t e r e d  a few h o u r s and i t was t h o u g h t  to contain  only  75  the  0.41 mg/ml s o l u b l e  i n water.  account f o r the r e a c t i o n s  amount c o u l d n o t  o b s e r v e d when t h e h y d r o l y s a t e  d i l u t e d w i t h one t h o u s a n d p a r t s After  This  was  of water.  the inconclusive  r e s u l t s already  outlined,  a c l o s e r c h e c k o f t h e e f f e c t o f t y r o s i n e was made a t t h e suggestion  o f D r . G, H u n t e r ,  showed t h a t  tyrosine  neutralizing  effect  with Milion s r  i s not completely  a protein hydrolysate  remain i n s o l u t i o n , heretofore,  Tests  therefore,  precipitated by  but that  a l l the r e s u l t s  Separation  a measure o f t h e s o l u b l e  of Thiolimidazoles  from  I t v/as now n e c e s s a r y t o d e v i s e separating  limidazole  likely  tyrosine  t o be p r e s e n t ,  any h i s t i d i n e a c c o r d i n g  Basic  (112),  and s i l v e r  Several the  tyrosine.  Hydrolysates a method f o r com-  lead acetate  was u s e d  silver  the tyrosine  f r a c t i o n a t i o n s were made v a r y i n g Tests  with  t o t h e method o f H u n t e r a n d  (113) l e a v i n g  precipitations.  with  ( 5 1 , 53) o r  i n solution.  t h e o r d e r a n d pH o f  were made a t s e v e r a l  stages i n the  sample w i t h e r g o t h i o n e i n e  z e i n was r u n w i t h t h e t e s t .  added t o  The p r o c e d u r e u s e d a n d t h e  r e s u l t s o b t a i n e d i n one o f t h e most e x t e n s i v e i s given  involved,  The t h i o l i m i d a z o l e s were t h e n f r e e d  f r a c t i o n a t i o n and a c o n t r o l the  was  or t h i o l h i s t i d i n e  h y d r o g e n s u l f i d e and- r e p r e c i p i t a t e d w i t h baryta  obtained  a n d h i s t i d i n e f r o m any t h i o -  to p r e c i p i t a t e e i t h e r e r g o t h i o n e i n e  Raragosky  may  e x c e p t t h o s e f r o m t h e w o r k on t h e d e s t r u c t i v e  may h a v e b e e n o n l y  out  a large part  o f h y d r o g e n s u l f i d e where no h y d r o l y s a t e  pletely  reagent  i n t h e accompanying c h a r t .  of these  tests  I n no c a s e was a p o s i t i v e  76 Chart I  Fractionation of Protein Hydrolysate to Separate Thiolimidazoles from Tyrosine and Histidine  Results of Hunter test expressed as amount of ergothioneine to give an equivalent oolour.  Symbols i  T m test hydrolysate, E m oontrol hydrolysate with 5 mg of  ergothioneine added before hydrolysis. 1 g zein 5 ml 2C# HC1  1 hydrolyze 8 hrs at 125 C extract vdth ether discard f a t soluble water soluble vaouum d i s t i l neutralize with 2.5 N NaOH allow to settle 4 hrs filter discard humus and tyrosine disoard supernatant* (histidine)  K  B  0.3  c 0.3  add 5ml 20# Pb(QAo) and 2.5 ml 2.5 N NaOH 2  v  wash with water treat with IN  P  Test: - T  H2SO4  supernatant  Testt T 0 0.3 K . 0.3  disoard PbS  Testt T . 0.000 K 0.000 B  neutralize with 2.5 N NaOH treat with excess 2C$ AgN03  Test: T • 0.01 K 0.01 m  Testt T K  8 e  treat with Ba(0H) & AgN0 2  3  Test: T K  m B  disoard Ag S<^ 2  filtrate  0.001 0.000  0.02 0.01  Test: T B0.000 0.000  77  test  f o r a t h i o l i m i d a z o l e observed  fractionation.  on completion  T e s t s p e r f o r m e d on t h e c o n t r o l  of the  sample  with  added e r g o t h i o n e i n e , a t v a r i o u s p o i n t s i n t h e f r a c t i o n a t i o n when t h e y were p o s i t i v e , were n o t a l w a y s a s s t r o n g a s on t h e h y d r o l y s a t e p r e p a r e d  without  Separation of Ergothioneine Because o f the f a i l u r e any  t h i o l i m i d a z o l e from z e i n ,  beginning,  chloric  f r o m Amino of these  able gives evidence  of hydrolysis with  to give very  with barium hydroxide  s h o u l d be l e f t  i n solution.  from the s o l u t i o n s used.  was  still  A  avail-  In contrast,  l i t t l e destruction. a c i d was u s u a l l y  i n order  t h a t no  However, i t was f o u n d  c o - p r e c i p i t a t e d eighty percent  was f o u n d  hydro-  (32) t h a t r e c e n t l y became  N e u t r a l i z a t i o n of the s u l f u r i c accomplished  now  destroys the thiolimidazole r i n g ,  supporting t h i s r e s u l t .  a c i d was f o u n d  hydroxide  attempts to i s o l a t e  e v e n when i t was added a t t h e  t h e c o n v e n t i o n a l method  p a p e r b y L e f e v r e and R a n g i e r  sulfate  Acids  The r a t h e r s t a r t l i n g d i s c o v e r y was  acid completely  sulfuric  ergothioneine.  t h e r e a c t i o n s w i t h p u r e e r g o t h i o n e i n e a n d amino  a c i d s were s t u d i e d . made t h a t  added  those  salts that  barium  o f the e r g o t h i o n e i n e  N e u t r a l i z a t i o n w i t h 2,5 N s o d i u m  t o be b e t t e r a l t h o u g h  about t e n p e r c e n t  lost. No d i f f i c u l t y was  experienced  i n separating the  ergothioneine from h i s t i d i n e w i t h b a s i c l e a d acetate but s e v e r a l methods were t r i e d a  s u c c e s s f u l one was f o u n d .  f r o m a more a c i d  f o r the removal of t y r o s i n e before Lead w i l l  precipitate tyrosine  s o l u t i o n than t h a t r e q u i r e d f o r t h e  78  p r e c i p i t a t i o n of ergothioneine b e made q u a n t i t a t i v e .  but the separation could not  Both s i l v e r  and b a r y t a were t r i e d  s e l e c t i v e p r e c i p i t a n t s b u t no e r g o t h i o n e i n e from the p r e c i p i t a t e s . sulfate,  Finally  Hopkins-Cole reagent  ergothioneine  from t y r o s i n e .  w i t h hydrogen s u l f i d e Further  was  present  although  shown t o be  was  freed  centrifugedo f f .  i n t h i s work w i t h  mixtures  applied to p r o t e i n hydrolysates.  a d d e d a f t e r h y d r o l y s i s most  Thus i t seems t h a t  by s u l f u r i c  sulfide  removed  a d d e d t o t h e p r o t e i n none c o u l d b e  i s o l a t e d , b u t when i t was  destroyed  mercuric  satisfactorily  The e r g o t h i o n e i n e  methods d e v e l o p e d  When e r g o t h i o n e i n e  that  recovered  Hydrolysates  o f known amino a c i d s were now  be r e c l a i m e d .  (114),  and t h e m e r c u r i c  Studies of  The  i t was f o u n d  c o u l d be  as  this  could  thiolimidazolei s  a c i d when p r o t e i n b r e a k d o w n p r o d u c t s  i n pure  are  s o l u t i o n t h e d e s t r u c t i o n had been  negligible,  A number o f a l t e r n a t e methods o f h y d r o l y s i s h a v e been t e s t e d by f r a c t i o n a t i o n i n Chart I I and t h e r e s u l t s present  according  are l i s t e d  i n Table  VII.  t i m e no s a t i s f a c t o r y method h a s b e e n f o u n d  seems t h a t i t I s p o s s i b l e t o i s o l a t e limidazole enzymes.  t o t h e method o u t l i n e d  a s m a l l amount  after h y d r o l y s i s with formic Work i s b e i n g  continued  acid  i n this  At the  although i t of t h i o -  o r combined  field.  79 Table VII  Methods o f H y d r o l y s i s  Temperature  175  Time  12 h r s  Agent  E x p r e s s e d as t h e f r a c t i o n o f a d d e d ergothioneine reclaimed.  C  165 1.75  C  125  hrs  8 hrs  NaOH 20$ H S0  20$  0.0  H3PO4  20$  0.0  KOH  40  C  24 h r s  0.0  4  2  C  20$  0.0  Trichloro acetic 70$ w i t h S n S 0 10$  0.0  4  Trichloro acetic 70$ w i t h T i ( S 0 ) 2  4  0.0  3  Papain  O.O  Trypsin  0.0  Pepsin  0.0  Pepsin &  Trypsin  F o r m i c 50$ HC1 7$ Pormamide  0.'02  with 90$  1  0.01  2  0.01  0,0  1  A s m a l l but d i s t i n c t  2  Hydrolyzed  purple precipitate  24 h r s w i t h p e p s i n a t pH 2,0  24 h r s w i t h t r y p s i n  a t pH  8.0,  f o r m e d i n 24 h r s . and a f u r t h e r  80  Chart H  Ifethod of Fractionation Currently Used  0.2 g zein or egg albumin 1 mg ergothioneine 1 ml hydrolyzing agent hydrolyze  extract with ether discard f a t soluble water soluble neutrallze with 2.5 N NaOH filter disoard humus & tyrosine^ Hopkins-Cole HgS0 reagent 4  discard filtrate«(tyrosine)  wash ppt. with 3# HgS0 "  4  treat with H2S, centifuge  disoard HgS <aerate r disoard f i l t r a t e , (histidine)  add 0.5 ml of 2C$ Pb (0Ao) and 0.25 ml 2.5 N NaOH per ml of solution 2  wash ppt. with water treat with H2SO4  discard PbS0 a4  "  oheok neutrality  perform Hunter test  Grote s 1  In confirm  give  an e f f o r  the f i n d i n g s  reaction the  Reagent  Grote  developed reaction  to obtain obtained  by Hunter  a t e s t which might  when e m p l o y i n g  d i s t i n c t i v e colours  the diazo  f o r thiolimidazoles,  was u n d e r t a k e n .  This  w i t h b o t h C-SH  test  serve t o  a study of  i s said  to  and C S g r o u p s .  81  In  so f a r a s t i m e p e r m i t t e d ,  ing t h i s colour  t e s t o n t h i o l i m i d a z o l e s were w i t h o u t r e s u l t .  a t a l l was o b s e r v e d .  i n v e s t i g a t i o n may p r o v i d e test  e x p e r i m e n t s c a r r i e d o u t employ-  I t i spossible  that  the proper conditions  t o be s a t i s f a c t o r y i n t h i s c o n n e c t i o n .  No  further enabling  this  I t is> of course,  p o s s i b l e , however, t h a t u n d e r no c i r c u m s t a n c e s i s t h e reaction given  by t h i o l i m i d a z o l e s .  I m i d a z o l e s have b e e n shown by L a n g l e y a n d S e x t o n (115)  t o h a v e a t l e a s t two t a u t a m e r i c  thiolimidazoles,  like  thio-urea,  forms and presumably  w o u l d have t h r e e .  These  f o r m s i n c l u d e b o t h C-SH a n d C=*S g r o u p s b u t t h e C-SH d o e s not  react with  sodium n i t o p r u s s i d e  groups r e a c t s w i t h Grote's  HC=C-R  I  I  SH  a n d i t may be t h a t  neither  reagent.  '  HC=C-R  I I  \  /  SH  HC=^C-R I \ HN NH  V s  IV  Summary a n d  Conclusions  A study o f the r e s u l t s obtained on the  the f r a c t i o n a l analysis of proteins cystine determinations recorded  by v a r i o u s  workers  suggests that  many o f  i n t h e l i t e r a t u r e may b e  h i g h due t o t h e i n t e r f e r e n c e o f t h i o l i m i d a z o l e b r e a k d o w n  82  products* Examination of the test with  ergothioneine  scope d i d n o t  by  r e v e a l any  c o l o r g i v e n by  means o f a  distinct  the Hunter  simple  grating  color.  Some I n d i c a t i o n o f t h e p r e s e n c e o f f r e e i n c o r n has  been found  make i t s o c c u r r e n c e complete  although  seem u n l i k e l y .  I n v e s t i g a t i o n of t h i s I t has  other  s u b j e c t as  p o s s i b l e source be  when c o p p e r i s removed by  by  the  survey  The  has  on  of t h i s  with  of d e s t r u c t i o n of  more s a t i s f a c t o r i l y  ergothio-  the  accomplished  cupferron,  b e e n made o f t h e r e s u l t s  survey  separated  obtained  to  obtain  h i s t i d i n e ) but  i t has  a r e , however, c o n s i d e r e d u n r e l i a b l e ,  been developed  from the  which i n t e r f e r e with  for  a  the d i s t r i b u t i o n of t h i o l i m i d a z o l e s i n p r o t e i n s .  results  be  employing c h a r c o a l as  o f a number o f p r o t e i n s i n an a t t e m p t  A method has may  disappear  a p p l i c a t i o n of the Hunter r e a c t i o n to f r a c t i o n a t e d  hydrolysates figures  may  s a l t s from combination  S e p a r a t i o n may  A  a  method u s i n g h y d r o g e n s u l f i d e g e n e r a l l y e m p l o y e d  i s another  betaine.  permitted  yet.  been shown t h a t e r g o t h i o n e i n e  separating metallic  neine  not  agent.  The for  ergothion-  considerations  Time has  from a s o l u t i o n during p u r i f i c a t i o n decolorizing  specto-  a b s o r p t i o n bands w h i c h  c o u l d be u s e d t o c h a r a c t e r i z e t h e  eine  diazo  whereby  ergothioneine  factors i n a protein hydrolysate  the Hunter d i a z o t e s t  ( t y r o s i n e and  not y e t been p o s s i b l e t o o b t a i n a  test  t h i o l i m i d a z o l e s from a pure p r o t e i n h y d r o l y s a t e nor  from  82  a h y d r o l y s a t e t o w h i c h e r g o t h i o n e i n e h a s b e e n added  before  hydrolysis. Ergothioneine hydrochloric chloride  a  an a t t e m p t  titanous i s a l s o broken  satisfactory During  T h i s may e x p l a i n ' t h e f a i l u r e  of earlier  t h i o l i m i d a z o l e s from p r o t e i n s .  techniques  and p r o c e d u r e s  t o determine the presence  i n proteins.  zoles.  The t h i o l i m i d a z o l e r i n g  are present.  Various  ring  agent  boiling i n  i n o t h e r h y d r o l y z i n g a g e n t s when p r o t e i n b r e a k d o w n  attempts to i s o l a t e  in  by prolonged  e v e n when t h e r e d u c i n g  i s present.  by b o i l i n g products  acid  i s destroyed  have b e e n  employed  of the thiolimidazole  I t has not been p o s s i b l e as y e t t o d e v i s e  technique this  study  f o r the determination  of thiolimida-  the influence of various  inter-  f e r i n g f a c t o r s has been i n v e s t i g a t e d . Grote*s with  reagent  has been found  t o g i v e no c o l o u r  e r g o t h i o n e i n e under the c o n d i t i o n s suggested  differenciation of sulfur  groups.  f o r the  83  Bibliography  1,  Davison, F. R,, Synopsis o f M a t e r i a Medlca, T o x i c o l o g y and Pharmacology, 3rd ed., S t . L o u i s , C. V. 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