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 T h e s i s submitted i n p a r t i a l f u l f i l m e n t of the requirements f o r the degree of Master of A r t s i n the department of chemistry U n i v e r s i t y of B r i t i s h Columbia May 1949 ABSTRACT A review i s made of the b i o c h e m i s t r y of s u l f u r and of i m i d a z o l e s . The r e s u l t s obtained by v a r i o u s workers on the f r a c t i o n a l a n a l y s i s of s u l f u r i n p r o t e i n s haye been com-p i l e d and the p o s s i b i l i t y t h a t a p o r t i o n of the p r o t e i n s u l f u r at present unaccounted f o r i n c e r t a i n p r o t e i n s may be a s c r i b e d to the presence of t h i o l i m i d a z o l e s has been c r i t i -c a l l y examined. The study suggests that many of the c y s t i n e d e t e r m i n a t i o n s r e c o r d e d i n the l i t e r a t u r e may be h i g h due to the i n t e r f e r e n c e of t h i o l i m i d a z o l e breakdown p r o d u c t s , A review of the chemistry of the t h i o l i m i d a z o l e s i s presented and the o r i g i n of the r e c e n t r e v i v a l of i n t e r e s t i n these compounds d i s c u s s e d , A method has been developed whereby i t i s p o s s i b l e to separate added e r g o t h i o n e i n e from those f r a c t i o n s of a p r o t e i n h y d r o l y s a t e that i n t e r f e r e w i t h the Hunter d i a z o t e s t . I t has a l s o been found that t h i s b e t a i n e i s des t r o y e d by b o i l -i n g w i t h h y d r o c h l o r i c a c i d and w i t h many other h y d r o l y z i n g agents i n the presence of p r o t e i n breakdown p r o d u c t s . In an attempt to f i n d a s u i t a b l e h y d r o l y z i n g agent which would ensure the s t a b i l i t y of the t h i o l i m i d a z o l e r i n g many methods were t r i e d but thus f a r without complete success. U n t i l such a procedure i s evolved no f i n a l c o n c l u s i o n can be reached as to the presence or absence of the t h i o l i m i d a z o l e r i n g i n the p r o t e i n molecule. ACKNOWLEDGEMENT I wish to express my g r a t i t u d e to Dr., B. A.' Ea g l e s under whose guidance t h i s work has been c a r r i e d out f o r the a s s i s t a n c e and ad v i c e that made i t p o s s i b l e . TABLE OP CONTENTS Page A b s t r a c t I. H i s t o r i c a l 1. A review of the b i o c h e m i s t r y of s u l f u r • 1 P o l k Lore • • • • • • • • • • 1 A l c h e m i s t r y • • • • • • • • • • • • • • 2 E a r l y Q u a n t i t a t i v e • • • • • . 2 S u l f a t e s . • 3 C y s t i n e • • • • • • • • • • • • • • • • 4 Methionine • • • • • • • • • • 6 Thiamin . . . . . . . . • 8 B i o t i n • 9 G l u t a t h i o n e . . • • 11 Taurine • • • • • • • • • • • • . . . . 12 Mucin • • • • • • • • • • • • • • . . • 13 Heparin • • • • • • • • • . . 13 A s t e r u b l n • • • • • • • • • • • • • • • I S Toad Poisons . . . . . . . . . . . • . 14 Sulfones 14 La n t h i o n i n e • • • • • • • • • • . . . . 14 A Thio E t h e r . . . • • • 15 D j e n k o l i c a c i d • • • • • * . . • • • • 15 Urochrome • • • • • • • • • • • • • • • 16 Page P e n i c i l l i n • • • • • • • • • • • • • • • 1 6 Thio-sugar • • • • • • • • • • • • • • • 16 Odours and F l a v o u r s • • • • • • • • • • 1 6 Thiocyanates • • • • • • • • • • • • • • l " 7 S y n t h e t i c Drugs • • • • • • • • • • • • 18 S u l f u r B a c t e r i a • • • • • • • • • • • • 19 2. A review of the b i o c h e m i s t r y of the im i d a z o l e s • • • • • • • • • • • • • • • 19 Purin e s • • • • • • • • • • • • • • • • 19 H i s t i d i n e • • 20 Histamine • • • • • • • • • • • • • • • 21 Carnosine and Anserine • • • • • • • • • 2 2 C r e a t i n e • • • • • • • • • . • • • • • • 25 A l l a n t o i n • • • • • • • • • • • • • • • 23 P i l o c a r p i n e • • • • • • • • • • • • • • 2 4 B i o t i n . . . . . . . • • • 24 T r i m e t h y l H i s t i d i n e . . . . . . . . . . 25 3« A review of the chemistry of the t h i o l i m i d a z o l e s • • • • • • • • • • • • 2 5 E r g o t h i o n e i n e from Ergot • • • • • • • • 2 5 E r g o t h i o n e i n e from Blood • • • • • • • • 27 I s o l a t i o n 3 « . . . . . . . . . . . . . 29 N u t r i t i o n Experiments • • • • • • • • # 3 0 Phy s i o l o g y • . • , ... , t # • • ... . 33 Pathology . • . • • • • • . • • • • • • 35 A n t i - t h y r o i d E f f e c t . . 3 6 Page A n a l y s i s . . . . . . . • . • • . • • • 40 Snth e s i s . . . • • • • • • • 4 2 I I , C o n s i d e r a t i o n of Problem • • • • • • • • • 4 5 I I I * E x p e rimental . • • • • • • • • • • • • • • 6 5 P r e p a r a t i o n of e r g o t h i o n e i n e • • • • • 63 Synth e s i s of t h i o l h i s t i d i n e • • • • • • 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 of the method • • • • • • • • 6 9 I n t e r f e r e n c e by Amino A c i d s • • • • • • 74 S e p a r a t i o n of T h i o l i m i d a z o l e s from Hy d r o l y s a t e s « ^ * 75 Se p a r a t i o n o f E r g o t h i o n e i n e from Amino A c i d s • • • • • • • • • • • • • • 7 7 F u r t h e r S t u d i e s o f H y d r o l y s a t e s • • • • 78 Methods of H y d r o l y s i s • • • • • • • • • 79 Grote's Reagent 0 . • • • • • • • • • • 80 IV. Summary and Co n c l u s i o n s • • • » • < » * & * ( ; 81 B i b l i o g r a p h y • • • • • • • • • • • • • • • 8 3 A STUDY OP UNKNOWN SULFUR CONTAINING COMPONENTS OP PROTEINS I H i s t o r i c a l 1* A Review of the Bi o c h e m i s t r y of S u l f u r F o l k Lore The importance of the r o l e of s u l f u r i n the f u n c t i o n -i n g of l i v i n g t h i n g s has been r e c o g n i z e d s i n c e the time of the e a r l y Greeks when Herc u l e s was s a i d to have used s u l f u r i n c l e a n s i n g the s t a b l e s of Augea* S u l f u r s p r i n g s i n v a r i o u s p a r t s of the world have maintained r e p u t a t i o n s of great h e a l i n g powers s i n c e the time of our e a r l i e s t r e c o r d s and s u l f u r has been used s i n c e time immemorial i n a v a r i e t y of f o l k r e c i p e s as an i n t e r n a l medicament. (1) P o s s i b l y the most famous of these r e c i p e s i s the s u l f u r and molassas " s p r i n g t o n i c " . I n a l l these s u l f u r c o n t a i n i n g pharmaceuticals the a c t i v e p r i n c i p l e i s a very s m a l l f r a c t i o n reduced t o the -2 or -4 v a l e n c e . The v a l u e of the s u l f u r c o n t a i n i n g medicaments i s probably very much over estimated by the populace but p h a r m a c e u t i c a l t e x t s r e c o g n i z e 1 2 them as v a s c u l a r s t i m u l a n t s and nervous s e d a t i v e s when used e x t e r n a l l y and as l a x a t i v e s i n t e r n a l l y , (2) A l c h e m i s t r y The f i r s t e x t e n s i v e e xperimental work on the b i o -chemistry of s u l f u r was c a r r i e d out by the i a t r o c h e m i s t s of the r e n a i s s a n c e . Each a l c h e m i s t had h i s own i n t e r p r e t a t i o n of the c o n f u s i n g l i t e r a t u r e on the s i g n i f i c a n c e of h i s "elements" but f i n a l l y i n the e a r l y s i x t e e n t h century P a r a c e l s u s put h i s con-cept of the importance of s u l f u r i n an understandable form. He co n s i d e r e d the " h y p o s t a t i c a l p r i n c i p l e s " , mercury, s u l f u r , and s a l t t o be p a r a l l e l to the Greek elements, f i r e , a i r , and e a r t h . These three elements made up a l l t h i n g s i n c l u d i n g the seven metals. S u l f u r imparted to the substances i n which i t oc c u r r e d the p r o p e r t y of i n f l a m a b l l i t y and bound together the mercury and s a l t i n the same manner as the s o u l of man u n i t e d the body and s p i r i t . Other a l c h e m i s t s c o n s i d e r e d s u l f u r i d e n -t i c a l w i t h the qu i n t e s s e n c e , (3) Although none of these a l -chemical i d e a s of s u l f u r b i o c h e m i s t r y has been accepted by modern s c i e n c e , the f a c t that these m y s t i c i s t s c o n s i d e r e d s u l f u r of importance r a i s e d i t to a p l a c e o f prominence i n the minds of s c i e n t i f i c men. E a r l y Q u a n t i t a t i v e When, i n the n i n e t e e n t h century men f i n a l l y began to study the chemistry of l i v i n g t h i n g s e x t e n s i v e l y on a c a r e f u l l y reasoned b a s i s , they found that they c o u l d make no progress un-t i l they had broken the l i v i n g t i s s u e down i n t o simpler u n i t s . The f i r s t methods used i n doing t h i s , such as ash i n g and 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 agents, were v e r y d r a s t i c and r e s u l t e d u s u a l l y i n o x i d i z i n g a l l the s u l f u r to the s u l f a t e . In t h i s form the t o t a l s u l f u r was determined but not h i n g was le a r n e d of the numerous d i f f e r e n t forms i n which i t might o c c u r . Some thought t h a t they had enough knowledge from t h i s work to w r i t e a g e n e r a l formula f o r p r o t e i n s thus, ( C 4 o H 6 2 ° 1 2 N 1 0 ^ x s y p z * G r a d u a l l y however l e s s d r a s t i c methods of a n a l y s i s were developed which r e v e a l e d that s e v e r a l d i f f e r e n t forms of s u l f u r r e s u l t e d from the same treatment. S u l f u r was thus shown t o occur i n l i v i n g t i s s u e s l i n k e d In a v a r i e t y o f ways. Much has now been done i n t h i s a n a l y t i c a l phase of 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 not complete f o r s u l f u r c o n t a i n i n g products are s t i l l o f t e n o btained from n a t u r a l products by treatments which would not y i e l d these s p e c i f i c f r a c t i o n s from any of the s u l f u r c o n t a i n i n g u n i t s now known to occur. S u l f a t e s The commonest form of s u l f u r i n the s o i l I s the i n o r g a n i c s u l f a t e i o n and i n t h i s form i t a l s o occurs i n l i v i n g c e l l s t o a l i m i t e d e x t e n t . The r o l e p l a y e d by p l a n t s i n r e d u c i n g n i t r a t e s o f the s o i l i n order t o y i e l d n i t r o g e n i n a form a v a i l a b l e t o animals i s g e n e r a l l y a p p r e c i a t e d but mention i s seldom made of the f a c t t h a t , as f a r as can be determined, animals are a l s o dependent on p l a n t s f o r t h e i r t o t a l supply of reduced s u l f u r , (4) Inasmuch as s u l f a t e s cannot be reduced by animals any s u l f u r t h a t becomes completely o x i d i z e d by the body processes cannot be r e c l a i m e d but must be ex c r e t e d i n t h i s 4 form and t h e r e f o r e 75-95$ of the s u l f u r e x c r e t e d I s i n the form of s u l f a t e . S u l f a t e s , l i k e n i t r a t e s , are present i n animal t i s s u e s i n l i m i t e d amounts and have but a l i m i t e d e f f e c t on the osmotic c o n c e n t r a t i o n . The g r e a t e s t use of s u l f a t e s by animals i s f o r the purpose 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 h y d r o x y l groups. They are combined w i t h these groups as e t h e r a l s u l f a t e s and exc r e t e d thus i n the u r i n e . There are a few other compounds of b i o l o g i c a l importance which c o n t a i n o x i d i z e d s u l f u r but they w i l l be d i s c u s s e d i n d i v i d u a l l y . C y s t i n e In 1810 W o l l a s t o n (5) i s o l a t e d a substance from b l a d d e r c y s t s which proved t o be the f i r s t n a t u r a l s u l f u r c o n t a i n i n g organic compound s t u d i e d and a l s o the f i r s t amino a c i d , (6) He gave i t the name c y s t i c oxide and t h i s name has si n c e been m o d i f i e d to c y s t i n e . In s p i t e o f the l a b i l i t y of the s u l f u r i n t h i s compound, n e i t h e r W o l l a s t o n nor any one e l s e n o t i c e d i t u n t i l 1837 and thus i t was the f i r s t of a long s e r i e s of compounds c o n t i n u i n g t o extend even t o the present time, i n which one s u l f u r atom was at f i r s t mistaken f o r two atoms of oxygen. When the c o r r e c t e m p i r i c a l formula was f i r s t worked out by Thaulow (7) ve r y l i t t l e was known of org a n i c s t r u c t u r e s or even of atomic weights. Thaulow fs formula C 6 H 1 2 H 2 ° 4 S 2 w a s c o r r e c t only because h i s e r r o r s I n t a k i n g the atomic weight of oxygen as e i g h t and i n not r e c o g n i z i n g the double nature of t h i s molecule compensated each o t h e r . P o s s i b l e s t r u c t u r a l formulae f o r c y s t i n e were bandied about a l l through the course of the v a r i o u s r a d i c a l and type t h e o r i e s t h a t were s u c c e s s i v e l y proposed d u r i n g the n i n e t e e n t h century but i t was not u n t i l 1903 t h a t Erlenmeyer (8) f i n a l l y e s t a b -l i s h e d by s y n t h e s i s the s t r u c t u r e now accepted, GRg-CH-C-OH $ NH2 0 S NH2 >0 C y s t i n e CH2-CH-CK)H Durin g t h i s p e r i o d of n e a r l y a century, over which work was being done to e s t a b l i s h the s t r u c t u r a l formula o f c y s t i n e , others were s t u d y i n g i t s o c c u r r e n c e . A l l the r e a c t i o n s i n d i c a t e d that much of t h e , s u l f u r i n p r o t e i n s qccured as c y s t i n e but no one had been a b l e t o i s o l a t e i t from t h i s source. One attempt r a f t e r another f a i l e d because of the f a i l u r e t o observe v a r i o u s simple t e c h n i c a l procedures which were l a t e r found t o be e s s e n t i a l f o r i t s i s o l a t i o n and i t was onl y a f t e r twelve other amino a c i d s had been i s o l a t e d t h a t , i n 1899, Morner f i n a l l y succeeded In o b t a i n i n g c y s t i n e from p r o t e i n s . For a long p e r i o d c y s t i n e and the c l o s e l y r e l a t e d c y s t e i n e were the on l y forms of s u l f u r known to occur i n p r o t e i n s and t h e r e f o r e many of the analyses done i n t h i s p e r i o d were m i s l e a d i n g . I t was found however t h a t c y s t i n e i s present at l e a s t i n s m a l l amounts In n e a r l y a l l p r o t e i n s except the protamines. The k e r a t i n s have a very l a r g e percentage of c y s t i n e and i t i s b e l i e v e d t h a t i t has an important f u n c t i o n i n t h i s p r o t e i n 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 l i g h t . Tanning i s more a t h i c k e n i n g of the s k i n than an i n c r e a s e i n the pigment con-t e n t and the added p r o t e c t i o n would come from the i n c r e a s e d amount of c y s t i n e - c o n t a i n i n g k e r a t i n p r e s e n t . C y s t i n e a l s o 6 seems t o have another more dynamic f u n c t i o n I n the body. Experiments done on the e f f e c t of i o d o a c e t a t e on enzymes i n d i c a t e t h a t -SH groups are e s s e n t i a l t o enzyme a c t i o n and c y s t e i n e i s the onl y amino a c i d c o n t a i n i n g t h i s group t h a t i s known t o occur i n p r o t e i n s . C y s t i n e has been shown t o f i l l much of but not the e n t i r e need of an organism f o r s u l f u r and the a b i l i t y of most animals t o s y n t h e s i z e c y s t i n e from other forms.1 of reduced s u l f u r emphasises i t s importance. Methionine Even b e f o r e MoWer succeeded i n i s o l a t i n g c y s t i n e from p r o t e i n s i t was r e c o g n i z e d that they c o n t a i n e d more than one form o f s u l f u r . As e a r l y as 1846 Pleitmann (10) observed t h a t o n l y a f r a c t i o n of the t o t a l p r o t e i n s u l f u r i s a l k a l i l a b i l e . 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 i n c o n t r a s t to the remainder which he mistakenly termed o x i d i z e d . In 1898 S h u l t z (11) was able t o gather t o g e t h e r the r e s u l t s o f s e v e r a l workers who had made comparative d e t e r m i n a t i o n s of d i f f e r e n t s u l f u r f r a c t i o n s . From these r e s u l t s S h u l t z t r i e d u n s u c c e s s f u l l y to f i n d a constant r e l a t i o n s h i p between the t o t a l s u l f u r and the a l k a l i l a b i l e f r a c t i o n . Another form of s u l f u r , c i f - t h i o - l a c t i c a c i d , which seemed to have been d e r i v e d from something other than c y s t i n e was I s o l a t e d by Suter, Friedmann and F r a n k e l (12) from s e v e r a l p r o t e i n s , A review has been g i v e n by Johnson (13) of the papers, p u b l i s h e d up t i l l 1911, con c e r n i n g these v a r i o u s s u l f u r l i n k a g e s . Most, but not a l l , of these s u l f u r r e a c t i o n s , u n e x p l a i n e d at the time, have s i n c e been accounted f o r by the presence of methionine. I t was the f a c t t h a t c y s t i n e does not supply a l l the s u l f u r requirements of c e r t a i n microorganisms t h a t l e d t o the d i s c o v e r y of methionine by Mue l l e r (14), M e u l l e r , who was engaged In s t u d i e s concerned w i t h the n i t r o g e n and a c t i v a t o r requirements o f s t r e p t o c o c c i , found that t h e r e was a substance i n c a s e i n h y d r o l y s a t e s necessary f o r t h e i r growth t h a t was not i n c l u d e d i n some other h y d r o l y s a t e s . In 1922 he succeeded i n i s o l a t i n g from c a s e i n a f a c t o r which was named methionine. Although I t turned out t h a t t h i s amino a c i d was not an e s s e n t i a l f a c t o r f o r the growth of the s t r e p t o c o c c i , i t proved t o be necessary f o r most s p e c i e s of animals. Methionine can be conver t e d i n the body to c y s t e i n e by the f o l l o w i n g r e a c t i o n s ; the process however i s not r e v e r s i b l e , 9H3 H C ^ T ^ C H 2 s s 6H 2 CHNH2 i ~ S H2 •Ho t}R2 CH 20H 6H N H 2 LO CH 2 -CS 3 C H 2 + CHNH 2 - H 2 6 » 0 C f C H-lffl 2 CH-NH2 1,0 Of \ ) H ' P \J) Q OH C( . ^OH X0H X)H Methionine Homo- Serine Gystethione c y s t e i n e — " s \ <?H2 CH 2 CH 20H CH 2 CHNH 2 CH 2 CH 2SH • CHNH2 JO 2*up CHNH2 + CHNH 2 OH X)H X 0 H Homo- C y s t e i n e s e r i n e The v a r i o u s i n t e r m e d i a t e products o f these r e a c t i o n s have been 8 I s o l a t e d from n a t u r a l s ources. The a n a l y t i c a l d ata a v a i l a b l e shows that i n g e n e r a l p r o t e i n s c o n t a i n a l a r g e r f r a c t i o n of methionine than c y s t i n e and the former accounts f o r the m a j o r i t y of s u l f u r i n f o o d s t u f f s . Mention should a l s o be made of the r o l e p l a y e d by methionine i n t r a n s m e t h y l a t i o n . The body would appear to be inc a p a b l e of s y n t h e s i z i n g methyl groups and i s , t h e r e f o r e , dependent on the r e l a t i v e l y few compounds i n the d i e t which c o n t a i n these groups i n a l a b i l e form, Du Vigneaud and co-workers (15) have shown by t e s t s w i t h deuturium that the methyl group o f methionine i s used i n s y n t h e s i z i n g c h o l i n e and c r e a t i n e and i t i s probably a l s o necessary f o r the s y n t h e s i s of a d r e n a l i n , a n s e r i n e and other methylated p r o d u c t s . I t has been found (16, 17) that b e t a i n e may act as a source o f methyl groups i n the r a t and i t i s p o s s i b l e t h a t e r g o t h i o n e i n e and 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 , hypaphor-i n e , and t r i g o n e l l i n e i n the v a r i o u s l o c a t i o n s i n which they are found may perform s i m i l a r f u n c t i o n s . Thiamin Thiamin, another e s s e n t i a l m e t a b o l i t e c o n t a i n i n g s u l f u r , i s a substance of paramount importance. Lack o f t h i a -min i n the d i e t i n t e r f e r e s w i t h many metabolic f u n c t i o n s u l t i m a t e l y r e s u l t i n g i n b e r i - b e r i , Eijkmann was one of the f i r s t 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 pigeons c o u l d contract i t and t h e r e f o r e be used as an experimental animal f o r i t s study. Other workers e s t a b l i s h e d the f a c t t h at the di s e a s e was due to a d e f i c i e n c y of some substance and i n 1898 9 Jansen and Donath (18) succeeded i n i s o l a t i n g the v i t a m i n i n c r y s t a l l n e form. I t was the f i r s t v i t a m i n to be obtained i n pure form but i t was not s y n t h e s i z e d u n t i l n e a r l y f o r t y y e a r s l a t e r (19). N - C - N H 2 H C I . C H 3 I  I) /C=C-CH2 —GHgOH CBV-C C-CH2-NN I I I Cfi CH-S Thiamin h y d r o c h l o r i d e N=CH 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 w i t h the s y n t h e s i s of c o c a r b o x y l a s e , t h i a m i n pyrophosphate, and the p r o s t h e t i c groups of many c a r b o x y l a s e s . These compounds of the v i t a m i n are necessary f o r the breakdown of p y r u v i c a c i d and hence f o r the process of g l y c o l y s i s . However, a l a c k of t h i a m i n i s evidenced p a r t i c u l a r l y i n the nerves as p o l y n e u r i t i s . T h i s seems to be a d i r e c t e f f e c t on the neurones, not a secondary r e s u l t of a g e n e r a l l y d i s r u p t e d metabolism. A theory has been proposed (20) that the d e f i c i e n c y d i s r u p t s the f o l l o w i n g s e r i e s 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 a c e t y l c h o l i n e . ATP + Thiamin—•.—=> A d e n y l i c a c i d •»• c o c a r b o x y l a s e p p c a r b o x y l a s e 0 2CH3-C-CT-0H + 0 2 cocarboxylase * 2CH3-C^ + 2 C O 2 P C H 3 C H 3 CH 3-C N + CH3-CH 2-N(CH3)30H *CH3-CH2 — N — C - C H 3 OH C H 3 6 A c e t y l c h o l i n e B i o t i n The t h i r d and only other form of s u l f u r known t o be e s s e n t i a l i n the d i e t i s the v i t a m i n b i o t i n . Work l e a d i n g to 10 the d i s c o v e r y of t h i s v i t a m i n progressed a l o n g t h r e e separate channels and hence i t has been v a r i o u s l y d e s i g n a t e d as b i o t i n , coenzyme R, and v i t a m i n H, Working w i t h the c h a r c o a l adsorbable f r a c t i o n of b i o s , the b i o s I I B of M i l l e r (21), Kogl and Tonnis (22) succeeded i n 1935 In g e t t i n g a minute amount of a c r y s -t a l i n e product which c o n t a i n e d most of the a c t i v i t y of the o r i g i n a l adsorbate. T h i s they c a l l e d b i o t i n but there was too l i t t l e of i t to make a f u l l study p o s s i b l e . Meanwhile A l l i s o n , Hoover and Burk (23), o b t a i n e d an e x t r a c t , coenzyme R, which was a c t i v e i n promoting the growth of Rhizobium' t r i f o l i i , a legume nodule organism. A l s o the work of Boas (24), on the e f f e c t of a d i e t c o n t a i n i n g much raw egg-white l e d to the d i s c o v e r y of an anti-egg-white i n j u r y f a c t o r , v i t a m i n B i o -t i n and coenzyme R were shown to be i d e n t i c a l i n 1939 and i n 1940 Szent-Gyorgyi (25) demonstrated that v i t a m i n H had s i m i l a r p r o p e r t i e s . The v i t a m i n was s y n t h e s i z e d i n 1943 (26) by a l o n g and d i f f i c u l t process and the s t r u c t u r e was shown to be: Szent-GySrgyi found t h a t the v i t a m i n must be s u p p l i e d to p r a c t i c a l l y a l l s p e c i e s of animals and micro-organisms that he s t u d i e d . In c e r t a i n i n s t a n c e s the amounts r e q u i r e d were so i n f i n i t e s m a l that a d e f i c i e n c y c o u l d o n l y be demonstrated by f e e d i n g raw egg-white. Native egg white has a s m a l l amount o f H H H ^OHp-CHjj-OHjj-CHo-Cf 'OH N—C—C H H H 2 As y e t l i t t l e i s known about the metabolism of b i o t i n . 11 a p r o t e i n , a v i d l n , which combines w i t h b i o t i n t o prevent i t s a s s i m i l a t i o n . Owing to the wide range of substances i n which b i o t i n o c c u r s , the minute amount u s u a l l y r e q u i r e d , i t s occurrence In the bound s t a t e i n which i t i s a v a i l a b l e to o n l y a few organ-Isms, and due to the f a c t that the i n t e s t i n a l f l o r a seem to be a b l e to s y n t h e s i z e i t , i t i s d i f f i c u l t to determine the exact b i o t i n requirements of l i v i n g organisms. R a p i d l y growing t i s s u e s such as embryos and tumors c o n t a i n a l a r g e amount of b i o t i n and s e v e r a l attempts have been made though u n s u c c e s s f u l l y to prevent or cure cancers by r e d u c i n g the b i o t i n Intake. G l u t a t h i o n e The t r i - p e p t i d e , g l u t a t h i o n e or g l u t a m y 1 - c y s t e n y l -g l y c i n e , i s another s u l f u r c o n t a i n i n g compound t h a t has been found i n a very wide range of t i s s u e s and c e l l s . C-C—CH2-CH2-G CH V OH Ed H X) ? H 2 H S 1 H G l u t a t h i o n e (Reduced Form) Gl u t a t h i o n e may be r e v e r s i b l y o x i d i z e d by removing the hydrogen atoms from the sulphur to form d i s u l f i d e : 2GSH 2H + GS-SG T h i s occurs i n n e u t r a l s o l u t i o n at the r a t h e r low p o t e n t i a l of -0.233 I n d i c a t i n g that the compound might p l a y a p a r t In the t r a n s f e r of hydrogen near the carbohydrate end of g l y c o l y s i s . However, though r e a c t i o n s between a s c o r b i c a c i d and g l u t a t h i o n e have been noted i n v i t r o no one has y e t found evidence to e s t a b l i s h the r o l e of g l u t a t h i o n e i n any s p e c i f i c hydrogen 12 t r a n s f e r r e a c t i o n . The o n l y proven f u n c t i o n of g l u t a t h i o n e i n l i v i n g c e l l s i s as a coenzyme f o r the r e a c t i o n forming l a c t i c a c i d from methyl g l y o x a l , 0 p g l u t a t h i o n e OH 0 CH,—<f— C — H + HpO s p e c i f i c enzyme * CH,-C— C-OH, * ° H Because t h i s one f u n c t i o n does not seem to account f o r the presence of such r e l a t i v e l y l a r g e and u n i v e r s a l q u a n t i t i e s as occur i n l i v i n g t h i n g s i t i s g e n e r a l l y assumed that g l u t a t h i -one does a c t as a hydrogen c a r r i e r . There i s f o r example 34-37mg/l00ml i n blood, 60mg/l00ml i n yeast and 70mg/l00ml i n l i v e r . I t seems probable t h a t organisms use the p e p t i d e g l u t a t h i o n e r a t h e r than c y s t e i n e as a hydrogen c a r r i e r because of i t s much g r e a t e r , s o l u b i l i t y . G l u t a t h i o n e was f i r s t d i s c o v e r e d by Hopkins i n 1921 (27), At f i r s t he c o n s i d e r e d i t to be g l u t a m y l - c y s t e i n e but because of evidence presented by Hunter and E a g l e s (28), Hopkins (29) made another study and r e v i s e d ' h i s o r i g i n a l f o r m u l a . There are , as w e l l as the s i x w i d e l y d i s t r i b u t e d s u l f u r compounds to which r e f e r e n c e has a l r e a d y been made, a number of others which are l e s s common and presumably l e s s important to l i v i n g organisms. Taurine Taurine l i k e g l y c i n e may occur conjugated w i t h c h o l i c a c i d In the b i l e s a l t s . I t a l s o occurs i n very h i g h c o n c e n t r a t i o n s i n the 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 where i t may a c t by an e f f e c t on the osmotic c o n c e n t r a t i o n . Presumably, t a u r i n e i s formed from c y s t e i n e by d e c a r b o x y l a t i o n and o x i d a t i o n of the s u l f u r . 13 0 NHg-CHg-CHg-S-OH Tau r i n e Mucin The g l y c o p r o t e i n mucin may have two d i f f e r e n t types of s u l f u r c o n t a i n i n g p r o s t h e t i c groups, c h o n d r o i t i n s u l f u r i c a c i d or m u c o i t i n s u l f u r i c a c i d . The former i s composed of a molecule of s u l f u r i c a c i d , w i t h one of amino-galactose and one of g l u c o n i c a c i d . M u c o i t i n s u l f u r i c a c i d i s the corresponding compound, w i t h glucose s u b s t i t u t e d f o r g a l a c t o s e . Mucin i s important i n m a i n t a i n i n g a moist smooth s u r f a c e on the mucous membranes; f r e e c h o n d r o i t i n s u l f u r i c a c i d i s used pharma-c o l o g i c a l l y i n the treatment of mi g r a i n e . H e p a r i n H e p a r i n i s another s u l f u r c o n t a i n i n g substance i n which s u l f u r i c a c i d i s combined w i t h a car b o h y d r a t e . I t i s produced by the l i v e r i n smal l amounts and i n h i b i t s the con-v e r s i o n o f prothrombin i n t o thrombin. I t s a c t i o n i s d i f f e r e n t from that o f dicoumerol which prevents the f o r m a t i o n of prothrombin. Both of these drugs are used m e d i c i n a l l y as a n t i c o a g u l a n t s , h e p a r i n having the prompter a c t i o n but r e q u i r -i n g hyperdermic i n j e c t i o n . In mucin the s u l f u r i c a c i d apparent-l y a c t s o n l y as a l i n k between the p r o t e i n and the carbohydrate but i n h e p a r i n i t seems to p l a y a more a c t i v e p a r t f o r the a c t i v i t y of the h e p a r i n i s d i r e c t l y p r o p o r t i o n a l to the percentage s u l f u r (30). A s t e r u b i n A s t e r u b i n i s another o x i d i z e d s u l f u r compound, 14 i s o l a t e d from s t a r f i s h and i d e n t i f i e d by Akermann i n 1935 (31)* Nothing i s known of i t s f u n c t i o n . H OBs W 0 • C H 3 — N — C — N — C H g — C H g — S — O H A s t e r u b i n CH 3 H 6 Toad Poisons Some of the toad p o i s o n s c o n t a i n s u l f u r but i t i s b e l i e v e d t h a t these s u l f a t e groups are not e s s e n t i a l t o t h e i r a c t i o n , The for m u l a of b u f o t h i o n i n e , the commonest of these p o i s o n s , i s as f o l l o w s : 0,S-Oi w ii—C-0-Nr_ 3 Buf o t h i o n i n e , o_0<A ,-JU? T ^ C H 3 } 3 S °| |( I ° G " ^ C H 3 \ / ^ N 7 H Sulfones L e f e v r e and Rangier (32) found that 1% o f the s u l f u r of d e p r o t e i n a t e d b l o o d serum i s i n the s u l f o n i c form but no study seems to have been made as to the exact nature of t h i s f r a c t i o n , S u l f a t i d e s S u l f a t l d e s c o n s t i t u t e another n a t u r a l l y o c c u r r i n g group of 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 . S u l f a t e occurs i n c e r t a i n of the 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 of the b r a i n (33), P o s s i b l y s u l f a t e r e p l a c e s the phosphate i n these compounds. Lan t h l o n i n e As w e l l as the two accepted amino a c i d s , c y s t i n e and methionine t o which r e f e r e n c e has been made, there are th r e e 15 other s u l f u r c o n t a i n i n g compounds that have been I s o l a t e d from p r o t e i n a t e o u s m a t e r i a l s and t h e r e f o r e c o n s i d e r e d t o be p o s s i b l e c o n s t i t u e n t s of the p r o t e i n molecule. Of these the be s t known Is l a n t h i o n i n e which may be i s o l a t e d from h a i r a f t e r h y d r o l y s i s w i t h sodium carbonate, Whether or not l a n t h i o n i n e i s formed d u r i n g the h y d r o l y s i s from c y s t e i n e i s a matter of debate; i t i s probably of b i o l o g i c a l s i g n i f i c a n c e , however, f o r I t would l i k e w i s e be formed In the i n t e s t i n a l tract", C — C — CHg- S-CH 2— G— Gf L a n t h i o n i n e HO' N H 2 NH 2 OH A Thio E t h e r A compound very s i m i l a r to l a n t h i o n i n e but c o n s i s t i n g of a mixed et h e r of c y s t e i n e and homocysteine has been i s o l a t e d by Horn and Jones (34) from the weed A s t r a g a l u s v u l g a r i s . T h i s compound i s of p a r t i c u l a r i n t e r e s t because i t occurs w i t h the isomorphous compound c o n t a i n i n g selenium when the weed i s grown- on s e l e n i f e r o u s s o i l * 0 V 0 SG—OH- C H 2 - S- CHg—CH2—GH—Q~f H0 X NH 2 (Se) NH 2 OH . D j e n k o l i c A c i d D j e n k o l i c a c i d , i s o l a t e d from the d j e n k o l nut by Van Veen and Hyman (35) i s another reduced s u l f u r compound th a t i s thought t o occur i n p r o t e i n s . As f a r as has been determined I t has not y e t been i s o l a t e d from any other source. The formula g i v e n f o r i t i s as below, <\ NH2 NH 2 0 HO-C—CH—CH 2-S-GH 2-S-CH 2-CH—C-OH D j e n k o l i c a c i d 16 tJrochrome TJrochrome i s an impure f r a c t i o n o f the u r i n e which c o n t a i n s the pigment u r o b i l i n and a p o l y p e p t i d e w i t h a r a t h e r h i g h content o f s u l f u r . I t must, t h e r e f o r e , be c o n s i d e r e d i n stud y i n g the forms of s u l f u r o c c u r i n g i n the u r i n e . P e n i c i l l i n 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 but they a l l c o n t a i n s u l f u r i n a t h i a s i n e r i n g . The a c t i o n o f p e n i -c i l l i n i n i n h i b i t i n g the growth of b a c t e r i a i s not y e t under-stood nor i s i t known whether the s u l f u r i s e s s e n t i a l f o r the a n t i b i o t i c e f f e c t . S y n t h e s i s of G - p e n i c i l l i n was r e c e n t l y e f f e c t e d through the combined e f f o r t s of s e v e r a l l a b o r a t o r i e s both B r i t i s h and American and i t s s t r u c t u r e i s now d e f i n i t e l y e s t a b l i s h e d (36), Thio-sugar A t h i o sugar b e l i e v e d to be a t h i o - m e t h y l - r i b o s e has been i s o l a t e d from y e a s t by M a n d e l l and Dunham (37) and f u r t h e r s t u d i e d by Suzuki (38) and by Levene and Sobotka (39), I t i s known t h a t the sugar occurs I n ye a s t 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 completely e l u c i d a t e d y e t and n o t h i n g i s known of i t s s p e c i f i c f u n c t i o n . Odours and F l a v o u r s Some of the sharp f l a v o r s and odors of n a t u r a l 0 H G - p e n i c i l l i n The a n t i b i o t i c , p e n i c i l l i n , c o n t a i n s a mixture of 17 substances are due to s u l f u r c o n t a i n i n g substances. The com-pound c h i e f l y r e s p o n s i b l e f o r the f l a v o r of mustard i s a l l y l -i s o t h i o c y a n a t e : CH2=CH-CH2-N=C=S A somewhat s i m i l a r compound g i v e s the d i s t i n c t i v e odour and f l a v o u r to g a r l i c . I t i s a l l y l - d i s u l f i d e : CH2=CH-CH2-S-S-GH2-CH=CH2 One o f the d i s t i n c t i v e components of asparagus i s be methyl mercaptan. T h i s compound may Aabsorbed from the g a s t r o -i n t e s t i n a l t r a c t and ex c r e t e d by the kidneys w i t h i n f i f t e e n minutes of i t s i n g e s t i o n . Although the odour o f methyl mercaptan i s not un-p l e a s a n t , that of i t s homologue, b u t y l mercaptan, Is extremely so and t h i s forms the p r i n c i p l e component of the skunk's odour, Thiocyanates A simple compound c o n t a i n i n g reduced s u l f u r t h a t occurs w i d e l y d i s t r i b u t e d i n v a r i a b l e amounts i n a l l body f l u i d s i s t h i o c y a n a t e . I t may enter the body i n the d i e t but most of i t would appear t o be formed endogenously, p o s s i b l y as a d e t o x i f i c a t i o n product o f cyanides (40), I t i s sometimes used m e d i c a l l y i n the treatment o f h y p e r t e n s i o n but has no known f u n c t i o n i n the q u a n t i t i e s i n which i t i s normally present i n the body. However, i t i s p o s s i b l e that t h i o c y a n a t e s are used i n the b i o l o g i c a l s y n t h e s i s o f c e r t a i n n a t u r a l s u l f u r compounds such 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 between i t and a number of other o r g a n i c compounds occur very e a s i l y . 18 The t w e n t y - f i v e s u l f u r compounds d e s c r i b e d above together w i t h e r g o t h i o n e i n e (see s e c t i o n I, 3) comprise most of the forms i n which s u l f u r i s known to occur commonly i n p l a n t s and animals but do not by any means account f o r a l l the r e a c t i o n s t h a t have been observed t o occur between n a t u r a l s u l f u r c o n t a i n i n g substances and v a r i o u s l a b o r a t o r y r e a g e n t s . I t i s t r u e that s e v e r a l of the compounds t h a t have been l i s t e d are a p p a r e n t l y of very l i m i t e d importance but c e r t a i n of them p l a y a very a c t i v e r o l e i n metabolism and i t i s probable that some of the as y e t unknown compounds of s u l f u r w i l l a l s o be found to be of great importance. S y n t h e t i c Drugs In a d d i t i o n to the n a t u r a l l y o c c u r r i n g s u l f u r com-pounds a number of s y n t h e t i c substances have been found to possess 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 proved v a l u a b l e i n medicine. By f a r the most important c l a s s o f these compounds i s t h a t o f the sulfonamides, A f u l l d i s c u s s i o n of these compounds i s beyond the scope of t h i s r e p o r t but i t i s i n t e r -e s t i n g to note t h a t , a l t h o u g h t h e r e are s e v e r a l d i f f e r e n t t h e o r i e s on t h e i r exact mode of a c t i o n , most of the t h e o r i e s agree i n assuming that the sulfonamides i n t e r f e r e w i t h the f u n c t i o n of some normal m e t a b o l i t e , t h a t i s the sulfonamide group i s s i m i l a r i n s t r u c t u r e t o but d i f f e r e n t i n f u n c t i o n from the substance t a k i n g p a r t i n normal metabolism. Thio u r e a , t h i o u r a c i l and c e r t a i n d e r i v a t i v e s of these compounds are used i n the treatment of t h y r o t o x i c o s i s . T h i s a c t i o n w i l l be more f u l l y d i s c u s s e d i n a l a t e r s e c t i o n of 19 t h i s r e p o r t . Sulfonmethane and sulfonnethylmethane are b o t h used as h y p n o t i c s and i n some cases as antispasmodics. They are u s u a l l y known by t h e i r t r a d e names, s u l f o n a l and t r i o n a l . S u l f u r B a c t e r i a Although only a b r i e f mention can be made here of the T h i o b a c i l l i and T h i o b a c t e r i a l e s , the review of the b i o -chemistry of s u l f u r would not be complete i f n o t h i n g were s a i d of them. V a r i o u s s p e c i e s of these remarkable b a c t e r i a are able to o x i d i z e hydrogen s u l f i d e , s u l f u r or t h i o s u l f a t e s and w i t h the energy so obtained they can reduce c a r b o n - d i o x i d e . In a good many s p e c i e s l i g h t i s r e q u i r e d f o r t h i s p r o c e s s . These b a c t e r i a s y n t h e s i z e the reduced carbon i n t o a l l the o r g a n i c compounds needed to b u i l d t h e i r protoplasm i n c l u d i n g the v i t a m i n s , t h i a m i n , b i o t i n , n i c t o t i n i c a c i d , pantothenic a c i d , p y r i d o x i n e and r i b o f l a v i n (41). 2, A Review of the B i o c h e m i s t r y of the Imidazoles The i m i d a z o l e s form one of the most Important c l a s s e s of n a t u r a l h e t e r o c y c l i c compounds f o r t h i s r i n g occurs as an i n t e g r a l p a r t of the p u r i n e r i n g as w e l l as independantly i n a number of commonly o c c u r r i n g compounds. Purines Only a very b r i e f discussion of the purines i s p o s s i b l e h e r e . With the p y r i m i d i n e s they occur a t t a c h e d to the pentoses i n n u c l e i c a c i d s and i n t h i s form probably p l a y a 20 key r o l e i n the c o n t r o l l i n g mechanism o f c e l l s . I t has been shown that n u c l e i c a c i d s which presumably can v a r y o n l y i n the p o s i t i o n s of the l i n k a g e s and the bases c o n t a i n e d t h e r e i n are the c o n t r o l l i n g f a c t o r i n the p r o d u c t i o n of a s p e c i f i c type of c a p s u l e by pneumococci (42). P u r i n e s a l s o occur i n the p r o s t h e t i c groups and co-enzymes of a l a r g e number of enzyme systems and here a g a i n p l a y a very a c t i v e p a r t i n c e l l metabo-l i s m . There i s some evidence f o r b e l i e v i n g t h a t the p u r i n e s are s y n t h e s i z e d by the body from the simple Imidazole r i n g of h i s t i d i n e f o r e x c r e t i o n of the p u r i n e breakdown product, a l l a n t o i n , by the r a t ceases i f the d i e t i s l a c k i n g i n h i s t i d i n e , H i s t i d i n e The most important o f the simple i m i d a z o l e compounds i s the amino a c i d h i s t i d i n e , H i s t i d i n e was simultaneously i s o l a t e d i n 1896 by K o s s e l (43) from s t u r i n e and by Hedin (44) from a mixture of p r o t e i n s . I t i s (3imidazole « amino-proprionic a c i d and s t r u c t u r a l l y i t i s c l o s e l y r e l a t e d to a r g i n i n e though i t has now been e s t a b l i s h e d t h a t these are not i n t e r - c h a n g e a b l e In mammalian metabolism, H i s t i d i n e i s e s s e n t i a l i n the d i e t of the young growing animal but i t seems that i t can be s y n t h e s i z e d to some extent and the need f o r i t i n the a d u l t probably v a r i e s with the s p e c i e s . I t occurs i n almost a l l p r o t e i n s but i n p a r t i c u l a r l y 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 , hemoglobin and i n most p r o -tamines. The metabolic path of h i s t i d i n e i s but p o o r l y 21 understood. I t may l e a d through the removal of u r e a to glutamic a c i d and hence to g l y c o g e n e s i s or through the removal of ammonia to u r o c a n i c a c i d but i n normal metabolism n e i t h e r of these products seem to be formed i n s u f f i c i e n t q u a n t i t y to account f o r an a p p r e c i a b l e p o r t i o n of the t o t a l h i s t i d i n e m e t a b o l i z e d . Histamine Histamine, the d e c a r b o z y l a t i o n product o f h i s t i d i n e , i s a compound of very great medical importance. I t occurs n a t u r a l l y i n ergot and as a p u t r i f a c t i o n product i n meat and f e c e s . That formed by b a c t e r i a l a c t i o n In the i n t e s t i n e may be absorbed i n t o the blood stream but i t i s u s u a l l y d e t o x i -f i e d at a s u f f i c i e n t r a t e to prevent n o t i c e a b l e e f f e c t s . I f , however, histamine 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 c o n t r a c t i o n of smooth muscle, s t i m u l a t i o n of the s a l i v a r y , g a s t r i c , p a n c r e a t i c and l a c r i m a l glands and a f a l l . i n b l o o d p r e s s u r e due to i n c r e a s e d p e r m e a b i l i t y of the c a p i l l a r i e s ; a l l these are symptoms c h a r a c t e r i s t i c of a n a p h y l a c t i c shock, asthma and a l l e r g y . The s i m i l a r i t y of the symptoms of these d i f f e r e n t c o n d i t i o n s which may be brought about by any of a wide v a r i e t y of d i f f e r e n t substances has been 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 . The theory most g e n e r a l l y accepted at present s t a t e s t h a t , i n cases where only a s l i g h t immunity i s b u i l t up, most of the antibody formed w i l l be h e l d to the c e l l s u r f a c e s . I f antigen, i s now 22 i n t r o d u c e d i n t o the b l o o d stream i t w i l l not be n e u t r a l i z e d t i l l i t reaches the c e l l s u r f a c e where the r e a c t i o n causes the c e l l t o r e l e a s e the h i s t a m i n e i n t o the b l o o d . Thus i t i s always h i s t a m i n e t h a t causes the observed e f f e c t s . Evidence f o r t h i s theory comes from the f a c t t h a t compounds which w i l l r e a c t w i t h histamine or which w i l l cause the development of a n t i b o d i e s f o r histamine are e f f e c t i v e i n the treatment of a l l e r g y and a n a p h y l a c t i c shock. In s m a l l e r c o n c e n t r a t i o n s histamine may be a normal hormone s t i m u l a t i n g c e r t a i n glands to s e c r e t e and the removal of i t from the b l o o d 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 — CHg— CH- Cf-OH N N N-C-OHg-CHg-NHg Carnosine H Carnosine and a n s e r i n e are two compounds c o n t a i n i n g h i s t i d i n e t h a t occur very w i d e l y i n v e r t e b r a t e muscle t i s s u e . The 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 been e x t e n s i v e -l y s t u d i e d by C i f f o r d (45) and Hunter (46), Hunter has a l s o developed a c o l o r i m e t r i c method f o r i t s d e t e r m i n a t i o n based on Knoop's d i a z o r e a c t i o n (47), P r a c t i c a l l y n o t h i n g i s known of the f u n c t i o n of e i t h e r c a r n o s i n e or a n s e r i n e a l t h o u g h they are present i n s i g n i f i c a n t and constant q u a n t i t i e s . The o n l y c l u e t h a t we have at present i s t h a t they are both strong b u f f e r s , Carnosine i s / B a l a n y l - h i s t i d i n e , a d i p e p t i d e of h i s t i d i n e l i n k e d t o the 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 but has a methyl group i n the number t h r e e p o s i t i o n , that i s on one of the n i t r o g e n s of the r i n g . T h i s i s i n c o n t r a s t to e r g o t h i o n e i n e which i s methylated on the amino n i t r o g e n . C r e a t i n i n e % 0 — C H g N. N n N H C r e a t i n i n e i s not g e n e r a l l y c o n s i d e r e d to be r e l a t e d to h i s t i d i n e because m e t a b o l i c a l l y i t i s formed by the de h y d r a t i o n o f c r e a t i n e which i n t u r n i s . s y n t h e s i z e d from g l y c i n e , a r g i n l n e and methionine. C r e a t i n i n e does, however, c o n t a i n the im i d a z o l e r i n g w i t h keto, imino and methyl groups a t t a c h e d . Thus i t forms an i n t e r m e d i a t e i n what i s a p o s s i b l e path o f f o r m a t i o n of i m i d a z o l e s from guanidlne compounds or v i s a v e r s a . A l l a n t o i n Another i m i d a z o l e o c c u r r i n g i n the u r i n e of c e r t a i n animals i s a l l a n t o i n , a d i - k e t o s u b s t i t u t e d i m i d a z o l e r i n g H 0 0=*Q — C — N - C — N H 2 N NH A / C i i 0 w i t h a u r e a s i d e chain*. I t i s thus a s u b s t i t u t e d hydantoin* A l l a n t o i n has a pronounced s t i m u l a t i n g e f f e c t on the growth of t i s s u e s and t h i s i s d o u b t l e s s the f u n c t i o n o f the l a r g e amounts found i n p l a n t and animal embryos. I t has a l s o been 24 found that a l l a n t o i n w i l l g r e a t l y h asten the h e a l i n g of many wounds and i t has been used e x t e n s i v e l y , p a r t i c u l a r l y i n the treatment of war wounds. Reference has a l r e a d y been made to the p o s s i b i l i t y of a metabolic r e l a t i o n s h i p between h i s t i d i n e , a l l a n t o i n and the 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 a pharma-c o l o g i c a l use i n the treatment of e p i l e p s y and chorea. T h e i r 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 t h e i r s i m i l a r i t y t o the b a r b i t u r a t e s . P i l o c a r p i n e H H H X<?— 9 A C — C H 2 - C H 3 VN 3^ H2sA S t i l l another i m i d a z o l e of importance i n pharma-co l o g y Is p i l o c a r p i n e , a n a t u r a l a l k a l o i d found i n the leaves of a Sou£h American shrub. In pharmacy I t i s one of the most important of the drugs used to s t i m u l a t e the parasympathetic nervous system b e i n g used In the treatment of glaucoma, b r o n c h i t i s , and s k i n d i s o r d e r s . The demethylated compound p i l o c a r p i d i n e occurs w i t h p i l o c a r p i n e i n the same shrub and has a s i m i l a r but weaker p h y s i o l o g i c a l a c t i o n , B i o t i n The b i o c h e m i s t r y of b i o t i n has been d i s c u s s e d w i t h the other s u l f u r c o n t a i n i n g compounds but i t may be p o i n t e d out t h a t I t a l s o c o n t a i n s a s u b s t i t u t e d i m i d a z o l e r i n g . In b i o t i n t here i s an oxygen atom i n the 2 p o s i t i o n , the p o s i t i o n c o r r e s p o n d i n g to that occupied by the s u l f u r of e r g o t h i o n e i n e . T r i m e t h y l H i s t i d i n e 0 HC • C- CHg- CH- Ct' \ 1 Ja* 0 T r i m e t h y l h i s t i d i n e T r i m e t h y l - h i s t i d i n e i s a b e t a i n e that has been I s o l a t e d from c e r t a i n e d i b l e mushrooms. In s p i t e o f 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 of note because i t i s a p o s s i b l e i n t e r m e d i a t e i n the h y p o t h e t i c a l s y n t h e s i s of ergo-t h i o n e i n e from h i s t i d i n e , 3. A Review of the Chemistry of T h i o l l m l d a z o l e s E r g o t h i o n e i n e from E r g o t The h i s t o r y of the t h i o l l m l d a z o l e s i s i n some ways s i m i l a r t o that of c y s t i n e , E r g o t h i o n e i n e was e a r l y i s o l a t e d from a n o n - p r o t e i n source and In s p i t e of many e f f o r t s to i s o l a t e i t or a r e l a t e d compound from p r o t e i n s and i n s p i t e of many i n d i c a t i o n s o f the presence o f a t h l o l l m i n a z o l e i n p r o t e i n s no one has y e t succeeded i n p r o v i n g any of these compounds to be a n a t u r a l c o n s t i t u e n t of p r o t e i n s , C. Tanret (48) was the f i r s t to i s o l a t e a n a t u r a l t h i o l i m l d a z o l e when he e x t r a c t e d e r g o t h i o n e i n e from ergot of rye i n 1909, He s t u d i e d the p h y s i c a l and chemical p r o p e r t i e s of the compound and worked out the e m p i r i c a l formula, C gH^^OgS, In f u r t h e r s t u d i e s Barger and Ewins (49) gave good evidence f o r the f o l l o w i n g s t r u c t u r a l f o r m u l a . 26 0 HC — C-CHg- CH - OCo*" f ( C H 3 ) 3 SH Although there was never much doubt of the v a l i d i t y of t h i s formula i t was only proved by s y n t h e s i s d u r i n g the past year by Winegard (50). H a r r i n g t o n and h i s co-workers (51, 52) had p r e v i o u s l y succeeded 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 two d i f f e r e n t methods but were unable to methylate t h i s compound to form the b e t a i n e , e r g o t h i o n e i n e . Barger and Ewlns based t h e i r s t r u c t u r e on the f o l l o w i n g evidence, l e a t i n g e r g o t h i n -e i n e w i t h c o n c e n t r a t e d a l k a l i s p l i t s o f f trimethylamine l e a v -i n g an u n s a t u r a t e d a d d ; t h i s shows t h a t i t i s a b e t a i n e . 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 a c i d to remove the s u l f h y d r y l group and reduced w i t h sodium to s a t u r a t e the double bond, y i e l d s () (4imidazole) p r o p r i o n i c a c i d which has been s y n t h e s i z e d , Barger and Ewin assumed t h a t the assumed that the s u l f u r was a t t a c h e d to carbon 2 of the r i n g because i t s l a b i l i t y i n the presence of f e r r i c c h l o r i d e and s t a b i l i t y i n a l k a l i i s l i k e t h a t of known compounds so c o n s t i t u t e d . T h i s was l a t e r confirmed by the s y n t h e s i s of t h i o l h i s t i d i n e . In these two papers, the one by Tanret and the other by Barger and Ewins there i s a f a i r l y complete study of the most important r e a c t i o n s of e r g o t h i o n e i n e . I t i s p r e c i p i t a t e d by s i l v e r and mercury s a l t s , by phospho-tungstic a c i d , and b a s i c l e a d a c e t a t e but not by n e u t r a l l e a d a c e t a t e , p i c r i c a c i d or t a n n i c ' a c i d . Iodine a l s o causes a p r e c i p i t a t i o n but by o x i d i z i n g the e r g o t h i o n e i n e to a d i s u l f i d e . I t i s s o l u b l e 27 i n water and d i l u t e a l c o h o l and s l i g h t l y so i n p y r i d i n e hut i n s o l u b l e i n a b s o l u t e a l c o h o l and other o r g a n i c s o l v e n t s . Barger 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 has no marked p h y s i o l o g i c a l a c t i o n though they d i d not g i v e t h e i r evidence. Though t h i s maybe c o r r e c t , at the present time the statement i s sub j e c t to some doubt and f o r some time i t d i d disco u r a g e f u r t h e r work on the compound. E r g o t h i o n e i n e from Blood In 1925, Bulmer, E a g l e s and Hunter (53) found t h a t they o b t a i n e d c o n s i s t e n t l y lower r e s u l t s i n determining the u r i c a c i d i n the bloo d by p r e c i p i t a t i o n o f the u r i c a c i d w i t h s i l v e r l a c t a t e a c c o r d i n g to the method of F o l i n and Wu (54) than they d i d u s i n g the d i r e c t method of Benedict (55). They a l s o found that the r e d u c i n g substance which was I n t e r f e r i n g w i t h the d i r e c t method o c c u r r e d i n . h i g h c o n c e n t r a t i o n i n the c o r p u s c l e s . The unknown substance was found to be present i n the blood of the r a b b i t , dog, c a t , ox, and guinea p i g . Immediately a f t e r the p u b l i c a t i o n of these r e s u l t s Benedict (56) made a defense o f the d i r e c t method on the ground t h a t i t was much simpler than the i n d i r e c t method and t h a t the r e s u l t s agreed c l o s e l y i n most c a s e s . He s t a t e d that he had long r e c o g n i z e d the presence of another r e d u c i n g substance i n blood and that he w i t h h i s a s s o c i a t e s had been working on the i s o l a t i o n of i t f o r some time. A paper p u b l i s h e d i n 1926 by Bened i c t , Newton and Behre (57) announced t h a t a substance which they c a l l e d t h i a s i n e had been i s o l a t e d from b l o o d by Mrs. Dugdale i n 1921 and t h a t they had now confirmed t h i s as 28 the substance which i n t e r f e r e d w i t h the d i r e c t u r i c a c i d d e t e r m i n a t i o n . They gave p h y s i c a l c o n s t a n t s f o r the compound and p o s t u l a t e d the e m p i r i c a l formula as, G i 2 H 2 0 N 4 ° 3 s « Meanwhile, i n 1925, Hunter and E a g l e s (58) had independently i s o l a t e d the i n t e r f e r i n g substance and they gave the e m p i r i c a l formula, C6HX1N2O3 and a r a t h e r d i f f e r e n t set of p h y s i c a l c o n s t a n t s . For example the m e l t i n g p o i n t was gi v e n as 224-225 C f o r t h i s compound w h i l e the m e l t i n g p o i n t of t h i a s i n e was g i v e n as 162-163. Both groups found that the r e s p e c t i v e compounds o c c u r r e d almost e n t i r e l y i n the c o r p u s c l e s and hence would l i t t l e e f f e c t the ur.ic a c i d d e t e r m i n a t i o n . In a l a t e r paper Hunter and E a g l e s (59) named the substance t h a t they had i s o l a t e d , sympectothion ( f i r m l y bound s u l f u r ) and submitted the r e v i s e d formula C I Q H 3 O % s 2 ° 4 w h i c h i s two molecules of e r g o t h i o n e i n e p l u s one of water. However befo r e t h i s had been p u b l i s h e d Newton, Benedict and Dakin submitted evidence on December 6, 1926 (60) to show t h a t t h i a s i n e was 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 and on December 23 E a g l e s and Johnson (61) showed that sympectothion was fundamentally the same as bot h the above compounds. The d i f f e r e n c e s i n the p h y s i c a l c o n s t a n t s of t h i a s i n e and sympecto-t h i o n had a r i s e n because Benedict and h i s co-workers had i s o l a -t e d the h y d r o c h l o r i d e w h i l e Hunter and Ea g l e s had i s o l a t e d the f r e e base. In the above paper Newton et a l . suggested t h a t the name e r g o t h i o n e i n e be shortened to t h i o n e i n e but p o s s i b l y due to c o n f u s i o n w i t h t h i o n i n e , 7aminophenothiazine, t h i s name has not been g e n e r a l l y accepted. 29 I s o l a t i o n " Since these three o r i g i n a l methods of i s o l a t i n g e r g o t h i o n e i n e were developed, s e v e r a l m o d i f i c a t i o n s have appeared i n the l i t e r a t u r e . The f i r s t was developed by E a g l e s i n 1928 (62) and was based p r i m a r i l y on a combination of some of the r e a c t i o n s used by Tanret (48) w i t h those o f Hunter and E a g l e s ( 5 8 ) . E r g o t was e x t r a c t e d w i t h a l c o h o l , the f a t s and a l k a l o i d s removed and the e x t r a c t was c l a r i f i e d w i t h barium hydroxide. The e r g o t h i o n e i n e was s u c c e s s i v e l y p r e c i p i t a t e d w i t h b a s i c l e a d a c e t a t e , mercuric c h l o r i d e , l e a d a c e t a t e and sodium hydroxide and phosphotungstic a c i d . I t was f i n a l l y i s o l a t e d as the f r e e base by c r y s t a l i z a t i o n from a b s o l u t e a l c o h o l . A y i e l d of 0.65$ of pure e r g o t h i o n e i n e was o b t a i n e d from the e r g o t . In 1929 Hopkins (63) developed a method f o r the 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 and thus brought i n t o prominence the r e l a t i v e l y s p e c i f i c r e a c t i o n between t h i o l groups and cuprous i o n s . By a p p l y i n g the use of t h i s reagent to the p r e c i p i t a t i o n of e r g o t h i o n e i n e from b l o o d W i l l i a m s o n and Meldrum (64) were ab l e t o shorten the procedure c o n s i d e r a b l y . The p r o t e i n s and some o f the other i n t e r f e r i n g substances are removed w i t h d I l u t e a c i d and " d i a l l z e d i r o n " . The g l u t a t h i o n e i s then o x i d i z e d by a e r a t i o n and the e r g o t h i o n e i n e p r e c i p i t a t e d as the cuprous s a l t , P i r i e (65) m o d i f i e d t h i s method somewhat so as to make i t a p p l i c a b l e to ergot which i s r i c h e r i n e r g o t h i o n e i n e than i s blood but which has more i n t e r f e r i n g substances. 30 With t h i s method P i r i e got a y i e l d o f 0.18$ e r g o t h i o n e i n e . I t must be p o i n t e d out, however that w h i l e these methods hfive been g e n e r a l l y compared on the b a s i s of the percentage y i e l d , i t has r e c e n t l y been shown by Lawson and Rimington (66) and by Hunter et a l . (67) that there i s c o n s i d e r a b l e v a r i a t i o n i n the normal e r g o t h i o n e i n e content of ergot and hence t h i s b a s i s of comparison i s not r e l i a b l e . R e c e n t l y Hunter and co-workers (68) has combined the copper 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 used by Hunter and E a g l e s i n the o r i g i n a l I s o l a t i o n of e r g o t h i o n e i n e from b l o o d and has o b t a i n e d a 0.26$ y i e l d of e r g o t h i o n e i n e from e r g o t . In t h i s method the ergot I s e x t r a c t e d w i t h water, much of the i m p u r i t y removed wi t h uranium a c e t a t e , and the e r g o t h i o n e i n e p r e c i p i t a t e d w i t h cuprous o x i d e . I t i s then f r e e d of the copper w i t h hydrogen s u l f i d e , t r e a t e d w i t h c h a r -c o a l and c r y s t a l i z e d from a b s o l u t e a l c o h o l . Experiments N u t r i t i o n A f t e r the i d e n t i t y of the e r g o t h i o n e i n e i n the b l o o d had been e s t a b l i s h e d , E a g l e s and h i s co-workers proceeded to study the p o s s i b l e f u n c t i o n s of i t i n the body. F i r s t E a g l e s and Cox (69) proved t h a t e r g o t h i o n e i n e c o u l d not r e p l a c e h i s t i d i n e i n the d i e t and t h i s work has s i n c e been supplemented by Neuberger and Webster (70) who showed that even t h i o l h i s t i -d i n e cannot take the p l a c e of h i s t i d i n e . F u r t h e r evidence of 31 the d i s t i n c t metabolsim of 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 and Broom (71) who found that t h i o l h i s t a m i n e has onl y one two-thousandth the p r e s s o r e f f e c t of histamine on the r a t and l e s s than one f i f t y - t h o u s a n d t h the e f f e c t on the c a t . The e f f e c t of t h i o l h i s t a m i n e on the r a b b i t blood sugar was not d e t e c t a b l e . I f the body has not the mechanism to remove the s u l f u r from 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 that i t c o u l d add s u l f u r to the i m i d a z o l e r i n g and t h e r e f o r e the metabolism of t h i o l i m i d a z o l e s i s most l i k e l y q u i t e d i s t i n c t from t h a t o f h i s t i d i n e . I f i t i s of a d i s t i n c t o r i g i n the t h i o l i m i d a z o l e might come from the d i e t or might be s y n t h e s i z e d In the animal body, p o s s i b l y from t h i o c y a n a t e and o r n i t h i n e . 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 evidence has been gathered which f a v o r s the theory of exogenous o r i g i n . Some of the most important evidence f o r the exogenous o r i g i n o f e r g o t h i o n e i n e i s i n the paper by E a g l e s and Vars who c a r r i e d out a study of the c o n d i t i o n s governing the e r g o t h i o n e i n e content o f p i g s b l o o d . The content of the blood of g r a i n f a t t e n e d p i g s from a Toronto a b a t t o i r had been found to be very h i g h i n comparison w i t h t h a t of garbage f e d p i g s from the S p r i n g s i d e Farm, New Haven. T h i s l e d them to a s e r i e s o f d i e t a r y experiments which showed t h a t w h i l e there 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 i n the bloo d of p i g s on a garbage or c a s e i n d i e t there was a .very c o n s i d e r a b l e amount when they were on a d i e t o f grasses, c o r n or other g r a i n s . 32 The Hunter d i a z o t e s t (72) which w i l l be d e s c r i b e d i n a l a t e r p a r t of t h i s r e p o r t was known at t h i s time and E a g l e s and Vars (73) a p p l i e d i t d i r e c t l y to a sample of z e i n h y d r o l y z e d w i t h s u l f u r i c a c i d . The r e s u l t s i n d i c a t e d the 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 the i n t e r f e r e n c e of h i s t i d i n e prevented the drawing of d e f i n i t e c o n c l u s i o n s . T h i s r e p o r t w i l l l a t e r demonstrate that h i s t i -d ine i s not the o n l y i n t e r f e r i n g substance. On the b a s i s of t h i s work Ea g l e s and Vars p o s t u l a t e d the presence i n c e r t a i n p r o t e i n s of a t h i o l i m i d a z o l e which would most probably occur as the amino a c i d t h i o l h i s t i d i n e . The amino a c i d might be methylated i n the animal body. T h i s i s the o r i g i n of the t h e s i s that has been the b a s i s o f t h i s r e p o r t . In 1935 P o t t e r and Pranke (74) c a r r i e d out an expanded s e r i e s of d i e t a r y experiments u s i n g r a t s as the experimental animal. The v e r y s t r i k i n g d i f f e r e n c e s t h a t they ob t a i n e d i n the e r g o t h i o n e i n e content of the b l o o d w i t h d i f f e r e n t d i e t s are summarized i n Table I , I t i s i n t e r e s t i n g to note that they found c o n s i d e r a b l e d i f f e r e n c e between two samples of the same g r a i n . P o t t e r and Pranke conclude from these r e s u l t s that the e r g o t h i o n e i n e of the b l o o d i s e n t i r e l y exogenous but do not venture t o suggest i n what form i t may o c c u r . 33 Table I, E r g o t h i o n e i n e content of b l o o d of r a t s D i e t Minimum mg/lOOml Maximum mg/lOOml Mean mg/lOOml Number of t e s t s C o r n 1 0.69 1.23 0.93 27 Wheat 0.12 0.66 0.43 57 Oats 1 1.18 1.30 1.22 7 2 0.46 0.87 0.59 7 B a r l e y 0.15 0.15- 0.15 9 Potato 0.08 0.08 0.08 7 S t o c k 2 0.98 1.28 1.17 6 Low T h i o n e i n e 3 0.08 0.08 0.08 12 1. The g r a i n d i e t s c o n t a i n e d as w e l l as 82$ of the g r a i n , 10$ c a s e i n , 3$ l a r d , 2$ cod l i v e r o i l , 2$ d r i e d yeast and 1$ McCollum's s a l t mixture. 2. Stock d i e t c o n t a i n e d 25$ wheat, 25$ c o r n , 20$ o a t s , 10$ f i s h meal, 5$ wheat germ, 5$ a l f a l f a , 5$ peas, 3$ cod l i v e r o i l , 1$ NaCl, 1$ CaC0 3. 3. Low t h i o n e i n e d i e t c o n t a i n e d 45$ sucrose, 20$ c a s e i n , 10$ y e a s t , 10$ corn s t a r c h , 10$ b u t t e r , 3$ s a l t s , 2$ cod l i v e r o i l . P h y s i o l o g y Because e r g o t h i o n e i n e had now been shown to be a normal c o n s t i t u e n t of mammalian b l o o d , i t was f e l t t h a t the statement of Barger and Ewins t h a t e r g o t h i o n e i n e had no p h y s i o l o g i c a l a c t i o n should be v e r i f i e d and more f u l l y s t u d i e d . 34 T a i n t e r (75) d i d t h i s by t e s t i n g the a c t i o n of e r g o t h i o n e i n e i n j e c t e d i n t r a v e n o u s l y on the blood p r e s s u r e , p u l s e r a t e , b i l a r y s e c r e t i o n , smooth muscle c o n t r a c t i o n and b l o o d sugar l e v e l u s i n g b o t h the r a b b i t and c a t . He found no e f f e c t i n any case, A few y e a r s l a t e r T r a b u c c h i (76) c a r r i e d out another s e r i e s of t e s t s u s i n g v a r i o u s experimental animals. Apparent-l y T r a b u c c h i was not acquainted w i t h the work of T a i n t e r , He a l s o found e r g o t h i o n e i n e without a c t i o n on the u t e r i n e muscle of the r a b b i t but noted an a c t i o n on the f r o g ' s h e a r t . Bathed i n a 0,1$ s o l u t i o n of e r g o t h i o n e i n e i n Ringer's s o l u t i o n the h e a r t beat f a s t e r w h i l e a 1% s o l u t i o n caused a • r a p i d decrease i n the amplitude of the beat and caused i t to stop i n d i a s t o l e , T rabucchi a l s o s t u d i e d the e f f e c t of e r g o t h i o n e i n e on pigeons w i t h b e r i - b e r i because an e a r l y i n c o r r e c t formula f o r t h i a m i n gave i t a s t r u c t u r e r a t h e r c l o s e l y r e l a t e d to and p o s s i b l y d e r i v e d from e r g o t h i o n e i n e (77), He found t h a t 2,5mg of e r g o t h i o n e i n e i n t r a m u s c u l a r l y d a i l y r a i s e d the temperature of the b i r d one degree, prevented f u r t h e r decrease i n weight and s t e a d i e d the walking but d i d not d e l a y death; F i n a l l y , T r a b u c c h i found that e r g o t h i o n e i n e , l i k e c y s t e i n e and g l u t a t h i o n e , a c t s as an a n t i d o t e i n cyanide p o i s o n i n g . There was a theory that cyanide acted by p o i s o n i n g the g l u t a t h i o n e and c y s t e i n e - c y s t i n e r e d u c t i o n - o x i d a t i o n systems'but T r a b u c c h i s a i d that i f so e r g o t h i o n e i n e must a l s o a c t i n a s i m i l a r manner. The evidence presented by P I r i e (78) to show t h a t both t h i o l h i s t i d i n e and e r g o t h i o n e i n e are 35 c a t a l y s t s of the o x i d a t i o n of g l u t a t h i o n e and c y s t e i n e , might h e l p e x p l a i n t h i s e f f e c t . Thus, w h i l e T a i n t e r , on the b a s i s of h i s work, agreed w i t h Barger and Ewins, T r a b u c c h i concluded that e r g o t h i o n e i n e 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 , Braun, Mason and Brown (79) i n v e s t i g a t e d the p o s s i b i l i t y t h a t e r g o t h i o n e i n e might act i n p l a c e of i n s u l i n but got negative r e s u l t s . The f a c t t h a t n e i t h e r t h i o l -h l s t i d i n e nor e r g o t h i o n e i n e occurs i n i n s u l i n had a l r e a d y been 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 (80) u s i n g the bromine o x i d a t i o n method of Zahnd and C l a r k (81), Pathology S e v e r a l workers have found the e r g o t h i o n e i n e content of the bloo d and u r i n e to var y w i t h d i s e a s e . S a l t (82) found 3-12mg/l00ml of r e d b l o o d c o r p u s c l e s i n normal and n e p h r i t i c persons but found i t t o be from 7-15mg/l00ml i n f i f t e e n d i a b e t i c c a s e s , S u l i v a n and Hess (83) found 90mg per l i t e r of e r g o t h i o n e i n e i n normal u r i n e and n o t i c e d t h a t w i t h d i f f e r e n t d i s e a s e s i t might be e i t h e r h i g h e r o r lower, being: h i g h i n both cancer and a r t h r i t i s . 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 and cancer has bee n f u r t h e r s t u d i e d by the la t e : Herbert Winegard and h i s a s s o c i a t e s but, as f a r as i s known t h e i r r e s u l t s have not y e t been p u b l i s h e d (52), A study of the d i a z o r e a c t i o n o f the blood was made by Nakayama (84) and he a r r i v e d at the con-c l u s i o n t h at e r g o t h i o n e i n e was probably the c h i e f cause of the r e a c t i o n . In d i s e a s e s where he found a s t r o n g d i a z o 3 6 r e a c t i o n i n the b l o o d he found a weak one i n the u r i n e . In a l l t h i s work on pathology each f i g u r e i s based on about f i f t e e n i n d i v i d u a l t e s t s but i t i s d o u b t f u l i f the d i e t s were p r o p e r l y c o n t r o l l e d . I f these r e s u l t s are s i g n i f i c a n t and the c o n c e n t r a t i o n of e r g o t h i o n e i n e i n the b l o o d and u r i n e does vary on a constant d i e t then i t must e i t h e r be s y n t h e s i z e d or r e t a i n e d to a v a r y i n g degree. Wakayama^s work makes the l a t t e r theory seem more pr o b a b l e . A n t i - t h y r o i d E f f e c t What may prove to be a v e r y Important p h y s i o l o g i c a l f u n c t i o n of e r g o t h i o n e i n e was f i r s t demonstrated by Lawson and Rimmington (66) i n 1947. The d i s c o v e r y i n 1943 by Mackenzie and Mackenzie (85) t h a t t h i o u r e a has a g o i t r o g e n i c a c t i o n l e d to the i n v e s t i g a t i o n of many r e l a t e d s u l f u r com-pounds i n an e f f o r t to f i n d something more a c t i v e and l e s s t o x i c . Astwood and h i s co-workers (86, 87, 88) t e s t e d a long s e r i e s of compounds f i n d i n g s e v e r a l which were a c t i v e , the l e a s t t o x i c of which, p r o p y l - t h i o u r a c i l , i s now w i d e l y used. Even t h i s substance may, however, cause a g r a n u l o c y t o s i s and other t o x i c e f f e c t s . Lawson and Rimington n o t i c e d that a l l , the a c t i v e compounds c o n t a i n e d a carbon atom l i n k e d to two n i t r o g e n s and a s u l f u r atom and they suggested that the compound e r g o t h i o n e i n e which c o n t a i n s t h i s group and occurs In the blood n a t u r a l l y without t o x i c e f f e c t s might be u s e f u l In c l i n i c a l treatments. Lawson and Rimington t h e r e f o r e proceeded to t e s t the e f f i c i e n c y of e r g o t h i o n e i n e as a t h y r o i d i n h i b i t o r . They 37 found t h a t t h e i r r e s u l t s v a r i e d when they used the r a t growth method of determining i t s a c t i v i t y but were more c o n s i s t e n t when they measured the i o d i n e content of the t h y r o i d gland of r a t s a dministered e r g o t h i o n e i n e by subcutaneous i n j e c t i o n . The l a t t e r method was based on the theory that these drugs act by p r e v e n t i n g the accumulation of i o d i n e i n the t h y r o i d g l a n d . T h e i r r e s u l t s , g i v e n In Table I I , i n d i c a t e t h at the a c t i v i t y of e r g o t h i o n e i n e i s comparable to t h a t of t h i o u r a c i l . In as much as v a r i a b l e q u a n t i t i e s of e r g o t h i o n e i n e of the same order as those a d m i n i s t e r e d are normally present i n the blood these workers found i t d i f f i c u l t to p r o p e r l y c o n t r o l t h e i r t e s t s and suggest t h a t some of the v a r i a t i o n s encountered by them may have been due to t h i s d i f f i c u l t y . T a ble H i E f f e c t of E r g o t h i o n e i n e on the Iodine of the T h y r o i d Gland Dosage mg/Kg body wt. Iodine content i n g/lOOmg of wet t h y r o i d E r g o t h i o n e i n e Mean D e v i a t i o n T h i o u r a c i l Mean D e v i a t i o n 0.2 0.5 0.75 2.0 20.0 13.5 21.0 5.0 4.03 4.11 4.00 1.76 19.6 8.0 4.65 6.4 1.49 2.40 2.25 1.20 C o n t r o l 58.2 ± 3.48 g/lOOmg of wet t h y r o i d Astwood and S t a n l e y (89) c a r r i e d t h i s work f u r t h e r 38 by t e s t i n g the c l i n i c a l e f f e c t i v e n e s s of e r g o t h i o n e i n e , b o t h as a t h e r a p e u t i c agent i n t h y r o t o x i c o s i s and as a p o s s i b l e cause of simple g o i t e r . Measuring the a c t i v i t y of the e r g o t h i o n e i n e by i t s e f f e c t on the r a t e of uptake of r a d i o -a c t i v e Iodine by the t h y r o i d g l a n d , they found no change of r a t e i n f i v e normal persons on the a d m i n i s t r a t i o n of 50 to 400 mg of e r g o t h i o n e i n e o r a l l y or i n t r a v e n o u s l y . Persons i n j e c t e d w i t h m e r c a p t o t h i a z o l e or methyl t h i o u r a c i l , on the other hand showed a sharp break i n the r a t e of uptake and l e v e l i n g of the r a d i o - a c t i v i t y of the t h y r o i d gland on a d m i n i s t r a t i o n of the drugs. Owing to the f a i l u r e of these experiments on humans, Astwood and S t a n l e y t r i e d t o repeat the work of Lawson and Rimington on r a t s but c o u l d f i n d no change i n the weight or i o d i n e content of the t h y r o i d s of r a t s t r e a t e d w i t h e r g o t h i -oneine whether i t was a d m i n i s t e r e d In the food or by i n j e c t i o n . They a l s o t r i e d f e e d i n g an impure c o n c e n t r a t e of e r g o t h i o -neine to see i f the e f f e c t observed by Lawson and Rimington c o u l d have been due to some i m p u r i t y i n t h e i r e r g o t h i o n e i n e but a g a i n w i t h negative r e s u l t s , Lawson and Rimington (90) had proceeded at the same time to use e r g o t h i o n e i n e c l i n i c a l l y In the treatment of two cases of human t h y r o t o x i c o s i s but got no response i n e i t h e r c a s e . They suggest the p o s s i b i l i t y that t h i s f a i l u r e and those of Astwood and S t a n l e y may have been due to o x i d a t i o n of the e r g o t h i o n e i n e to the d i s u l f i d e . 39 Latner and Mowbray (91) have made a study of the e r g o t h i o n e i n e content of the b l o o d of s i x t h y r o t o x i c o s i s p a t i e n t s when f a s t i n g and found i t below normal i n f o u r cases and a low normal i n the other two. They suggest that the d i f f i c u l t i e s of Astwood and S t a n l e y may be due to the f a c t t h a t e r g o t h i o n e i n e does not seem to be bound by serum p r o t e i n as i s t h i o u r a c i l and hence i t c o u l d be more e a s i l y e x c r e t e d by the kidney. More i n d i r e c t support f o r the t h e s i s t h a t e r g o t h i o -neine has g o i t r o g e n i c p r o p e r t i e s comes from the work of Monti and V e n t u r i (92) who s y n t h e s i z e d and t e s t e d three t h i o l -b enzimidazoles f i n d i n g them o f comparable a c t i v i t y to methyl t h i o - u r a c i l . I f these t h r e e compounds are a l l a c t i v e i t seems probable that s u b s t i t u t i o n of b e t a i n e f o r the benzene r i n g would not a f f e c t the a c t i v e grouping to a great e x t e n t . H-N QV:: HOCH2Vv/N-CHg-OH C>\X<=I> s 5 a H Thio-Benzimidazole 1,3 d i (hydroxymethyl)- 1,3 - d i - p h e n y l -t h i o b e n z i m i d a z o l e t h i o b e n z i m i d a z o l e Warner-Jauregg and Koch (93) have c a r r i e d out s t u d i e s on the g o i t r o g e n i c e f f e c t of s e v e r a l n a t u r a l p r o d u c t s . Among the a c t i v e compounds found was a heat l a b i l e compound from white cabbage. They p o s t u l a t e that t h i s i s a b e n z y l -t h i o u r e a but have not i s o l a t e d i t and t h e r e f o r e i t i s p o s s i b l e t h a t i t may be r e l a t e d to the t h i o l - i m i d a z o l e s . 40 A n a l y s i s S e v e r a l methods and m o d i f i c a t i o n s t h e r e o f have been developed f o r the d e t e c t i o n and d e t e r m i n a t i o n of t h i o l -i m i d a z o l e s . The f i r s t and s t i l l the most g e n e r a l l y u s e f u l method i s an a d a p t a t i o n of the d i a z o ^reaction which was p u b l i s h e d by Hunter i n 1928 (72). T h i s t e s t g i v e s a y e l l o w 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 pu r p l e r e d on the a d d i t i o n of c o n c e n t r a t e d a l k a l i . T y r o s i n e when t r e a t e d i n t h i s way g i v e s a p i n k c o l o r v e r y s i m i l a r t o t h a t of the t h i o l i m i d a z o l e s and h i s t i d i n e g i v e s a y e l l o w c o l o r which may obscure the d e s i r e d t e s t and make q u a n t i t a t i v e e s t i m a t i o n of t h i o l i m i d a z o l e d i f f i c u l t . The method has been s l i g h t l y m o d i f i e d r e c e n t l y t o i n c r e a s e i t s accuracy w i t h the modern p h o t o e l e c t r i c c o l o r i m e t e r (94), A l s o another m o d i f i c a t i o n has been proposed by Latner (95) f o r use i n the d e t e r m i n a t i o n of er g o t h i o n e i n e i n b l o o d . In 1929 Behre and Benedict (96) brought out another method based on the o r i g i n a l method of i s o l a t i o n o f e r g o t h i o -neine used by Benedict, Newton and Behre (56). T h i s method i s based on the p r e c i p i t a t i o n of u r i c a c i d and e r g o t h i o n e i n e w i t h s i l v e r l a c t a t e and the s e p a r a t i o n o f these two substances by f i r s t d i s s o l v i n g the u r i c a c i d w i t h a c i d sodium c h l o r i d e and subsequently the e r g o t h i o n e i n e w i t h sodium c y a n i d e . The er g o t h i o n e i n e i s then determined c o l o r i m e t r i c a l l y by the c o l o r developed w i t h P o l i n and Wu's t u n g s t i c a c i d reagent (54), T h i s t e s t i s , however, c o n s i d e r e d r a t h e r l e s s s p e c i f i c than t h a t developed by Hunter. 41 A q u a n t i t a t i v e study of the r e a c t i o n s of the v a r i o u s forms of s u l f u r present i n p r o t e i n s has been c a r r i e d out by Zahnd and C l a r k e (97) and Blumenthal and C l a r k e ( 8 1 ) , 1 T h i s study i n c l u d e d an i n v e s t i g a t i o n o f the e f f e c t of o x i d a t i o n by bromine and i t was found that o n l y that s u l f u r i n ^C=S, ^C-SH, and ^C-SH l i n k a g e s was l a b i l e i n t h i s r e a g e n t . T h i s s u l f u r i n c l u d e s t h a t o f the t h i o l l m l d a z o l e s but not of any other commonly o c c u r r i n g s u l f u r compound. Bromine o x i d a t i o n has been used by Du Vignead, S i f f e r d and M i l l e r (80) to prove that t h e r e i s no t h i o l h i s t i d l n e i n i n s u l l i n but the presence of a bromine l a b i l e s u l f u r f r a c t i o n has not y e t been e s t a b l i s h e d as proof of the presence of t h i o l l m l d a z o l e s . Other methods of a n a l y s i s which as y e t do not seem to have been used fo r ' the d e t e r m i n a t i o n of t h i o l l m l d a z o l e s but which might prove u s e f u l i n t h i s f i e l d of work are the o x i d a t i v e procedure of Kitamura and Masuka (98) and the c o l o r i m e t r i c method of Grote, The technique of Kitamura and Masuka u t i l i z e s the o x i d a t i o n o f s u l f u r by means of hydrogen p e r o x i d e . In t h e i r study on the i n f l u e n c e of HgOg on v a r i o u s s u l f u r compounds they found t h a t , whereas RCHgSH and t h i o -phene groups were s t a b l e i n the presence o f t h i s reagent, substances c o n t a i n i n g the f o l l o w i n g are q u a n t i t a t i v e l y o x i d i z e d : -NM3-SR, 0=C-SH, S=C~SH, =N-C-S, -0-C=S, -N=6-S-C=N-, and °jS=S, Grote (99) developed an i n t e r e s t i n g method of d i f f e r e n t i a t i n g s u l f u r compounds, u s i n g the product r e s u l t i n g 1 See a l s o s e c t i o n I I , 42 from t h e a c t i o n of l i g h t on sodium n i t r o p r u s s i d e . T h i s reagent which may a l s o he made by t r e a t i n g n i t r o p r u s s i d e w i t h bromine, g i v e s a green c o l o r p a s s i n g through t u r q u o i s e , b l u e and p u r p l e to crimson w i t h )C=S, -N=S, or )S=S groups. The r e a c t i o n i s s e n s i t i v e to Ippm of t h i o u r e a . The reagent a l s o g i v e s a t r a n s i e n t blue c o l o r w i t h >C-SH groups and w i t h S-S-Crgroups on r e d u c t i o n . The r e s u l t s o b tained i n u s i n g t h i s reagent w i t h e r g o t h i o n e i n e are d i s c u s s e d In the e x p e r i -mental s e c t i o n of t h i s r e p o r t . S y n t h e s i s I t has been p o i n t e d out t h a t Barger and Ewins (49) demonstrated the s t r u c t u r e - o f e r g o t h i o n e i n e by means of i t s r e a c t i o n s but d i d not c o n f i r m t h i s s t r u c t u r e by s y n t h e s i s . When i t was d i s c o v e r e d t h a t e r g o t h i o n e i n e was a normal c o n s t i t u e n t of the b l o o d i t became d e s i r a b l e to s y n t h e s i z e both to provide m a t e r i a l f o r experimentation and to prove the s t r u c t u r e . S e v e r a l attempts were made but no one succeeded u n t i l November 1948 when Winegard (50) f i n a l l y developed a method. The d e t a i l s of h i s proceedure do not seem to have been p u b l i s h e d as yet and i t i s o n l y known that the s y n t h e s i s i n v o l v e s the use of diazomethane. Other t h i o l i m i d a z o l e s had, however been s y n t h e s i z e d many years b e f o r e t h i s . The f i r s t was t h i o l h i s t a m i n e s y n t h e s i z e d by Pyman (71) from dibenzamidoketo butane. The steps of the s y n t h e s i s are p a r a l l e l to those used s h o r t l y a f t e r w a r d by Ashley and H a r r i n g t o n (51) i n the s y n t h e s i s of t h i o l h i s t i d i n e . I t was Pyman w h o " f i r s t developed the method 4 3 method of s y n t h e s i z i n g the t h i o l i m i d a z o l e r i n g by the use of t h i o c y a n a t e , the method which has been used by a l l subsequent 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 shley 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 a c c o r d i n g to the method o f WIndaus, D o r r i e s and Jenson (100) b r e a k i n g the i m i d a z o l e r i n g . One benzamido group I s e l i m i n -ated t o g i v e a ketone and the r i n g reassembled w i t h t h i o -cyanate t o g i v e t h i o l h i s t i d i n e . H 0 0X G=-C-CH 2-CH~C / t 3 £ N Jm NH 2 X0CH 3 CI C H C&V— C - C H p - C H C ' « a I OH NHg 0 NHg V i i C=G~CE2-CE~([*r N N-JJ NH2 " H S H KCNS H 0 Co l d > q—C,—<JHg — CH— C NaCO HN HN HN IP C O C H 3 \CH30H [HCI p HC1 =C CH 2-CH-C 0 HN v0CH^ Harrinton:- and Overoff (52) t r i e d u n s u c c e s s f u l l y to form e r g o t h i o n e i n e 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 i n c i d e n t a l l y developed another method to s y n t h e s i z e the amino a c i d s t a r t i n g w i t h a s p a r t i c a c i d . 44 .0 l \ ) H CHNHo + 0. G- CH* i -CH, Cv |\)H CH 2 CH-N^ PCI C — 0 C-CH, o' H CH—N \ C N^ \ CHNHo X3-SH CHg-NHg r ,KCNS CHo « , 2 CHNHo I OH HC1 X)H cr CHg CH-N C —0 cf 0 It c-CHp-N' I G-C=0 6 9=2 CHHH-C-CB I CJ-0-02H5 *o ,Phthallmide CHr> * | ^  0 CH-NH-C-CH 3 % ° - C 2 H 5 S t i l l another s y n t h e s i s o f t h i o l h i s t i d i n e was worked out by Dey (101) u s i n g ph£/ia/o - a > -)odo-acetonylimide and malonic ester# ,0 OH / C -0 -c x 0 CHoI + Na- 6—N -q ^ Jo N c r .0 9 f o H ^ -C-CHo-C N 0 kc-6 OH HBr CH^C-CHg-CH-C^ ( KCNS H 2N-CH 2-C-CH 2-CH- C^  9 HN N NH 2 \)H S i H NH 2 OH 45 I I , C o n s i d e r a t i o n of Problem I t has been p o i n t e d out t h a t the d i s c o v e r y of methionine made p o s s i b l e the e x p l a n a t i o n of p r e v i o u s l y r e c o g -n i z e d r e a c t i o n s of p r o t e i n s and accounted f o r a l a r g e f r a c t i o n of the p r o t e i n s u l f u r that was known to be d i f f e r e n t from that of c y s t i n e or c y s t e i n e . I t i s l i k e w i s e of importance t h a t i n the l i g h t of the l i t e r a t u r e a r e i n v e s t i g a t i o n of the r e l a t i o n s h i p between the t o t a l s u l f u r of p r o t e i n s and the s u l f u r accounted f o r by the known amino a c i d s be undertaken to see i f t h e r e e x i s t s a f r a c t i o n of the t o t a l s u l f u r t h a t might occur as a t h i o l i m i d a z o l e . The u n e x p l a i n e d r e a c t i o n s of p r o t e i n s u l f u r must a l s o be s t u d i e d to o b t a i n an estimate of the amount and nature of the unknown f r a c t i o n s . The evidence i n f a v o r o f the h y p o t h e s i s t h a t t h i o -l i m i d a z o l e s occur i n p r o t e i n s come from the f e e d i n g e x p e r i -ments and the study made by C l a r k e et a l . (81, 97), S u l l i v a n and Hess (103) have found t h a t normal human u r i n e c o n t a i n s about 90 m g / l i t e r as measured by c o l o r i m e t r i c a n a l y s i s . I f i t i s assumed that t h i s e r g o t h i o n e i n e i s e n t i r e l y exogenous then an average d a i l y i n t a k e o f e r g o t h i o n e i n e would be at l e a s t 135 mg, and the food must c o n t a i n on the average i n the neighborhood of 0,01$ e r g o t h i o n e i n e . I f t h i s were c o n c e n t r a t e d i n the p r o t e i n i t would be i n a c o n c e n t r a t i o n of about 0,15$, The work of Zahnd and C l a r k e and of Blumenthal 46 and C l a r k e x i n d i c a t e s an even h i g h e r c o n c e n t r a 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 . P r o t e i n s , p r o t e i n h y d r o l y s a t e s and f r a c t i o n s t h e r e o f 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 , n i t r i c a c i d and w i t h bromine to y i e l d d i f f e r e n t f r a c t i o n s of the t o t a l s u l f u r p r e s e n t . Of p a r t i c u l a r i n t e r e s t - i s the f r a c t i o n found to be o x i d i z a b l e by bromine, a reagent which w i l l o x i d i z e the s u l f u r of e r g o t h i o n e i n e or t h a t o c c u r r i n g I n the ;C=S ° * C — S H or ~ N*C- SH l i n k a g e s but not t h a t o f c y s t i n e or methionine. I f the bromine o x i d i z a b l e s u l f u r i n p r o t e i n s occurs as a t h i o l i m i d a z o l e i t i s d i f f i c u l t to r e c o n c i l e the f a c t that Blumenthal and C l a r k e f i n d as much of t h i s f r a c t i o n i n the h y d r o c h l o r i c a c i d h y d r o l y s a t e s as i n the whole p r o t e i n w i t h the work of L e f e v r e and Rangier (32) who ,found t h a t t h i o l l m l d a z o l e s are d e s t r o y e d by t h i s treatment. I t has been found i n t h i s l a b o r a t o r y that e r g o t h i o n e i n e does not g i v e 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 w i t h 2 h y d r o c h l o r i c a c i d but I t i s p o s s i b l e that the breakdown products are s t i l l o x i d i z a b l e by bromine. Prom the r e s u l t s of t h e i r work Blumenthal and C l a r k e conclude t h a t t h e r e i s a l s o another unknown form of s u l f u r i n p r o t e i n s which i s l a b i l e i n a l k a l i n e plumbite but not o x i d i z a b l e by n i t r i c a c i d . Some of the r e s u l t s o b t a i n e d by Blumenthal and C l a r k e are g i v e n i n Table I I I , I t w i l l be n o t i c e d t h a t the bromine o x i d i z a b l e s u l f u r content of the albuminoids i s p a r t i c u l a r l y h i g h but that i t a l s o occurs to some extent i n a l l the other p r o t e i n s s t u d i e d , 1 See a l s o s e c t i o n I, 3, " I s o l a t i o n " . 2 See s e c t i o n I I I , " S e p a r a t i o n of e r g o t h i o n e i n e from amino a c i d s " . 47 Table I I I S u l f u r i n P r o t e i n s from Blumenthal and C l a r k e P r o t e i n T o t a l S P r e e x i s t i n g S u l f a t e S C o r r . S0 4 S by B r 2 C o r r . S 0 4 S by HN0 3 A l k a l i l a b i l e S G e l a t i n 1 0.20 0.06 0.01 0.03 0.03 G e l a t i n 2 1.10 0.99 0.00 0.04 0.02 Blood a l b . 1.10 0.07 0.05 0.83 0.62 Lactalbumin 1.22 0.00 0.02 0.81 0.65 Egg white 1.60 0.03 0.04 0.68 0.43 Z e i n 0.52 0.16 0.02 0.25 0.22 C a s e i n 0.63 0.00 0.01 0.09 0.09 Wool • 3.06 0.00 0.21 3.06 3.07 Horn 3.60 0.09 0.26 3.51 3.59 Some of the r e s u l t s o b t a i n e d from s u l f u r and amino a c i d analyses of p r o t e i n s by v a r i o u s workers u s i n g s e v e r a l d i f f e r e n t methods are compiled i n Table iVA. For convenience the r e s u l t s found f o r each p r o t e i n have been averaged although i t i s r e a l i z e d t h a t f i g u r e s thus o b t a i n e d may be m i s l e a d i n g . The p r o t e i n s were do u b t l e s s o f v a r i a b l e p u r i t y , some of the r e s u l t s have been c o r r e c t e d f o r the a d d i t i o n of water d u r i n g h y d r o l y s i s and those of Block and B o i l i n g have been c a l c u -l a t e d on a common b a s i s of s i x t e e n percent n i t r o g e n i n the p r o t e i n s . One of the most s t r i k i n g f a c t s shown by t a b l e i s the v a r i a b i l i t y of the va l u e s found p a r t i c u l a r l y f o r c y s t i n e and c y s t e i n e and f o r s u l f u r i t s e l f . The average f i g u r e s f o r 48 s u l f u r i n z e i n account f o r twenty-two percent more s u l f u r r e p o r t e d as known amino a c i d s than i s found i n the t o t a l s u l f u r but on the other hand i f the f i g u r e g i v e n by B a e r n s t e i n f o r the t o t a l s u l f u r I s c o r r e c t there i s more than 20% of the s u l f u r not accounted f o r . The g e n e r a l p i c t u r e shows,only a sm a l l f r a c t i o n o f the t o t a l s u l f u r unaccounted f o r i n most of the p r o t e i n s c o n s i d e r e d and because most of-these f i g u r e s were c a l c u l a t e d from percentages of the amino a c i d s on the assumption that no c o r r e c t i o n had been made f o r the water of h y d r o l y s i s , t h i s s m a l l f r a c t i o n may be I n s i g n i f i -c a n t . P r e - e x i s t i n g s u l f a t e i n the p r o t e i n s may a l s o g i v e r i s e to a p a r t of the s u l f u r unaccounted f o r . T h i s s u l f a t e i s u s u a l l y not d e t e c t a b l e but the few f i g u r e s a v a i l a b l e f o r i t , show that i n some p r o t e i n samples i t may comprise up to twenty percent of the t o t a l s u l f u r . Where one sample of a p r o t e i n has been analyzed f o r a l l known f r a c t i o n s and t o t a l s u l f u r , the r e s u l t s are u s u a l l y i n goodvagreement and t h i s has l e d B a e r n s t e i n (104) and Brand (105) to d i s c o u n t the p o s s i b i l i t y of unknown s u l f u r c o n t a i n i n g u n i t s b e i n g p r e s e n t . However, the d i e t a r y and bromine o x i d a t i o n experiments i n d i c a t e the presence i n c e r t a i n p r o t e i n s of as y e t unaccoun-ted f o r forms of s u l f u r . I t i s p o s s i b l e t h a t a p o r t i o n of t h i s s u l f u r e x i s t s as t h i o l i m i d a z o l e . P o s s i b l e e x p l a n a t i o n s of the d i s c r e p a n c i e s which e x i s t i n the f i g u r e s o b tained by the r e s p e c t i v e groups of workers employing d i f f e r e n t t e c h -niques to assay the nature and amounts of the v a r i o u s forms of unknown s u l f u r from p r o t e i n s may be o f f e r e d . 49 Table IVA D i s t r i b u t i o n of S u l f u r i n P r o t e i n s P r o t e i n Sample C y s t i n e Methio- T o t a l S S unaccounted f o i & Cys- nine S $ of % of t e i n e S p r o t e i n s u l f u r Egg I 1 0.51$ 1.09$ 1.75$ 0.15$ 8.6$ Albumin 2 2 0,60 0.98 1.60 0.02 1.2 33 0,46 1.13 1.60 0.01 0.6 43 0.47 1.12 1.85 0.26 14.1 5 3 0.40 1.40 6* 0.35 0.99 75 0.53 1.11 8 a 6 0.24 b 0.27 c 0.27 9 7 0.26 107 0.26 11 a 8 0.63 b 0.64 129 1.36 ' 139; 1,20 1 4 i o 2,0 15 1 0 1.66 16 1 0 1,71 1 7 i o 1.18 18 1 1 1.60 19 a 1.12 b 0.87 50 Table IVA Continued P r o t e i n Sample C y s t i n e & Cys-t e i n e S Methio-nine S T o t a l S S unaccoi % of p r o t e i n anted f o r % of s u l f u r Egg Albumen Cont'd 2 0 2 4 o,25 21 a b c Average 0,82 0,96 0.97 0,95 oTJ? 6.42 l.Oo 1.66 C a s e i n 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 4 3 0.08 0.87 5* 0.07 0.73 6 5 0.08 0.79 7? 0.08 8 8 0.08 9 8 0.08 10 9a 0.68 b 0.64 l l 1 0 1.2 1 2 1 1 0.63 1 3 1 2 0,09 0.69 0.78 0,0 0.0 1 4 1 4 0.06 1 5 1 5 0,08 1 6 1 6 0.08 1^18 0,07 • 51 Table IVA Continued P r o t e i n Sample C y s t i n e Methio- T o t a l S S unaccounted for & Cys- nine S % of % of • t e i n e S p r o t e i n s u l f u r C a s e i n 19 18 0.07 Cont'd 1 9 2 0 0.18 2 0 2 1 0.09 2 1 2 2 0.08 2 2 2 4 0,63 22 2& 0.58 b 0.58 c 0.58 Cv0~6 Average 6.09 0.68, 0.53 L a c t - l 2 1.01 0.56 1.56 -0.01 -0.6 albumin 2 3 0.83 0.60 1.42 -0.01 -0.7 3 4 1.15 0.55 4 5 0.91 0.57 5 a 0.56 b 0.65 0 1.02 6 8 a 0.86 b 0.86 7 9 a 0,52 b 0.50 8 1 1 1.22 9 1 2 0.82 0.60 1.42 0.0 0.0 1 0 1 4 0.68 52 Table IVA Continued P r o t e i n Sample C y s t i n e Methio- T o t a l S S unaccounted f o r & Cys- nine S % of % of t e i n e S P r o t e i n S u l f u r L a c t - 0.61 albumin Cont'd 1 2 1 7 0.65 1 3 1 Q 1.06 , . 2 0 14 1.00 1 5 2 1 0.70 , 23 16 a 0.81 b 0,81 -c 0.81 d 0.82 . , ,«25 17 a 0.38 b 0.38 c 6.36 Average 0.82 5 7 5 0 1.40 0.08 . "575 Wool I 1 3.8 0.13 3 . 9 0 . 0 0 . 0 2 4 3.5 3 6 a 3.4 b 3.4 c 3.8 2.7 b 2 . 7 10 5 3.06 6 1 4 2 . 6 Average 3 7 2 " 0.13 3.5 0 , 2 578 53 Table IVA Continued P r o t e i n Sample C y s t i n e Methio- T o t a l S S unaccounted f o r & Cys- nine S % of % of t e i n e S p r o t e i n s u l f u r G e l a t i n I 1 0.05 0.24 0.5 0.2 40.0 2 2 0.21 3 4 0.05 0.21 4 5 0.043 0.21 5 1 ! 0.19 b 0.13 0.17 c 0.18 Average 0.048 0.19 oTB" 0.25 l o i j o E d e s t i n i 1 0.40 • 0.53 1.2 0.3 25.0 0.48 0.44 0.99 0.07 7.0 3 * 0.27 0.45 0.31 b 0.34 c 0.34 • 5 7 0.36 6 7 0.33 7 7 0.37 8 9 a 0.37 b 0.47 9 1 3 0.85 - 1 0 1 4 0.33 11 0.36 54 Table IVA Continued P r o t e i n Sample C y s t i n e Methio- T o t a l S S unaccounted f o r & Cys- nine S % of % of t e i n e S p r o t e i n s u l f u r E d e s t i n 1 2 1 6 0,32 Cont'd I 3 1 7 0.35 1 4 1 8 0.20 I S 1 9 0.26 1 6 2 ° 0.48 1 7 2 1 0.32 1 8 2 2 0.32 19 2 I 0.48 . b 0.48 c oTBT 0.48 IToT Average 0.4S 0719" 19.'' Z e i n i 1 2 2 0.24 0.51 0.47 0.36 -0.39 -108. 3 2 0.50 0.42 0.93 0.01 1.1 4 4 0.21 0.49 5 5 0.27 0.62 6 7 0.28 7 9 a 0.54 b 0.50 8 9 a 0.48 b 0.47 9 0.24 10 0.27 55 Table IVA Continued P r o t e i n Sample C y s t i n e Methio- T o t a l S S unaccounted, f oi & Cys- nine S % of % of t e i n e S p r o t e i n s u l f u r Z e i n 11 0.22 Cont'd 12 0,13 13 0.23 14 0.42 15 . - 0,45 16 a 0.36 b 0.36 c 0.32 WW 1 '• Average 0.27 0,46 0.60 -0713 Serum I 1 1.7 0.28 1.7 -0.3 -18. albumin A 1.63 3 6 a 1.52 b 1.52 c 1.62 4 7 1.62 5 1.89 10 6 1.75 7 1 1 1.10 8 1 2 1.74 0,18 1.94 0.02 1.0 9 1 2 1.68 0.28 1.96 0.00 0.0 Average 1.63 0"7£5 1772 -0.16 -9.3 56 Table IVA Continued P r o t e i n Sample C y s t i n e Methio- T o t a l S S unaccounted f o r & Cys- nine S % of % of t e i n e S p r o t e i n s u l f u r G l i a d i n I 1 0.69 0.5 1.2 0.0 0,0 2 2 0.26 0 . 4 3 0.99 0.30 30.0 3 4 0 . 4 5 4 7 0.58 5 8 a 0.60 b 0.61 6 1 4 0.55 7 1 5 0.58 8 1 6 0,58 9 " 0.65 i o l s 0.62 l l 1 9 0.38 1 2 2 0 0.73 IS 2* 0.57 Average 0.56 074"6" 1.1 bTT 9TT" 57 Footnotes to Table IVA. 1. From Block, R, J , and B o i l i n g , D., The De t e r m i n a t i o n o f  the Amino A c i d s , M i n n e a p o l i s , Burgess P u b l i s h i n g Co.,1940. C y s t i n e - c y s t e i n e by phospho 18 t u n g s t i c a c i d , c o l o r i m e t -r i c a l l y . Methionine by homocysteine method. S u l f u r by BurgessrParr s u l f u r bomb. 2. From B a e r n s t e i n , H. D., J . B i o l . Chem., 97, 663-8 and 669-74 (1931). C y s t i n e - c y s t e i n e by B a e r n s t e i n ' s gasometric method. Methionine by B a e r n s t e i n ' s v o l a t i l e i o d i d e method. S u l f u r by peroxide f u s i o n i n Pa r r bomb. 3. B a e r n s t e i n , H. D., J . B i o l . Chem., 115, 25 (1936). Methods not a v a i l a b l e . H y d r o l y s i s by h y d r o - l o d i c a c i d . 4. Cohn, E. J . , Ergeb. P h y s i o l . , 33, 781 (1931). Methods not a v a i l a b l e . 5. F i g u r e s s e l e c t e d from the l i t e r a t u r e by Dr. B. A. E a g l e s . 6. S u l l i v a n , M. X. and Hess, W. C., P. S. Pub. H e a l t h  Reports, s u p p l , 86* (1930). a, S u l l i v a n t e s t . b, Okuda t e s t ( I o d o m e t r i c ) • c, F o l i n - Looney t e s t , 7. F o l i n , 0. and Marenzi, J . B i o l . Chem., 83, 103-108 (1929). Method a m o d i f i c a t i o n of th a t of F o l i n and Looney. 8. G r a f f , M a c u l l a and G r a f f , J . B i o l . Chem., 121, 81-86 (1937), Method a m o d i f i c a t i o n o f t h a t o f V i c k e r y and White, 58 9, B a e r n s t e i n , H. D., J . B i o l , Chem., 115, 31 (1936). a. V o l a t i l e i o d i d e method. b. Homocysteine method. 10. S h u l t z , P. M., Z . p h y s i o l . Chem., 25, 16-25 (1898). 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 not a v a i l a b l e . 11. Blumenthal, D. and C l a r k e , H. T., J . B i o l . Chem., 110, 343 (1935). Method not g i v e n . 12. Brand, E., Ann. N.Y. Acad. Sc., 47. 187-228 (1946). C y s t i n e - c y s t e i n e by photometric i o d i n e method. Methionine by homocysteine method. S u l f u r by method of P r e g l (Saschek)• 13. Osborne, T. B. and Campbell, J . Am. Chem. Soc., 18, 609 (1896). Method not a v a i l a b l e . 14. V i c k e r y , H. B . and White, A., J . B i o l . Chem., 99, 701 (1933). O r i g i n a l method i n v o l v i n g I s o l a t i o n o f c y s t e i n e w i t h cuprous o x i d e . 15. Loc. c i t . , Method of P o l i n and Marenzi. 16. Loc. c i t . , Method of S u l l i v a n . 17. Loc. c i t . , Method of Okuda. 18. Loc. c i t . , Method of P o l i n and Looney. 19. Loc. c i t . , Method of Jones, G e r s d o r f f and M o e l l e r . 20. Loc. c i t . , B a e r n s t e i n ' s gasometric method. 59 21. V a s s e l l , B . , J . B i o l . Chem., 140, 323 (1941). O r i g i n a l method i n v o l v i n g a c o l o r developed w i t h p-amino-d ime thy 1-anl l i ne . 22. Loc. c i t . , P o l a r o g r a p h i c method. 23. K a s s e l l and Brand, J . B i o l . . Chem., 125, 115 (1938). a. Photometric method. b. Microphotometric method, c . S u l l i v a n method, d. B a e r n s t e i n method. 24. Csonka, F. A., and Denton, C. A., J . B i o l . Chem., 165, 329 (1946). Method a m o d i f i c a t i o n o f th a t o f McCarthy and S u l l i v a n . 25. Horn, M, J . , Jones, D, B,, and Blum, A. E., J . B i o l . Chem., 166, 315 and 521 (1946). a. M o d i f i e d method of McCarthy and S u l l i v a n w i t h a c i d h y d r o l y s i s . b. M o d i f i e d method of McCarthy and S u l l i v a n w i t h papain h y d r o l y s i s . c . M i c r o b i o l o g i c a l assay. 60 Table IVB Deviations from the Mean of Cystine-Cysteine Results by Various Methods Method Egg albumin Casein rH O Wool Edestin Zein Serum albumin Gliadin Average Deviation from mean i n % of sulfur of protein Vickery and White -.03 -.15 -.7 -.02 -.03 -.02 -.05 Folin and Marenzi -.16 -.16 -.01 -.01 +.02 -.01 +.03 +.01 +.00 +.01 -.01 +.02 +.01 -.02 Sullivan -.18 -.01 -.26 -.02 -.22 +.2 -.03 -.03 -.05 -.11 +.00 -.08 Okuda -.15 -.17 -.18 +.2 +.00 +.00 -.11 +.07 -.06 Folin and Looney -.15 -.02 +.20 +.22 +.6 +.00 -.14 -.14 -.01 +.04 +.01 Jones, Gersdorf and Moeller -.02 -.09 -.05 -.18 -.08 Baernstein's gasometric +.18 +.06 +.09 +.08 -.01 +.00 +.17 +.14 +.12 +.23 +.14 -.30 .15 +.08 Vassell +.00 -.13 -.01 -.05 Polarograph -.01 -.01 -.01 Block and Boiling +.09 +.00 +.6 +.06 -.03 +.07 +.13 +.05 Graff -Kjeldahi +.21 -.01 +.04 -.5 +.04 +.05 Graff BaSC>4 +.22 -.01 +.04 -.5 +.05 +.05 Baernstein -.02 +.04 +.05 -.01 + .01 +.11 +.01 Photometric +.00 -.02 +.11 +.05 +.03 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 i s des-t r o y e d by the u s u a l methods o f h y d r o l y s i s . The product of t h i s breakdown has not as y e t been i d e n t i f i e d but i t i s p o s s i b l e that i t c o u l d r e a c t both w i t h bromine and behave as one of the other 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. The f a c t that the accuracy of some o f these methods was judged by the degree t o which they accounted f o r a l l the s u l f u r of p r o t e i n s lends p l a u s i b i l i t y to t h i s theory (106, 107). I f the breakdown products of t h i o l l m l d a -z o l e s are measured w i t h the known amino a c i d s i t seems most l i k e l y t h a t I t Is the d e t e r m i n a t i o n s of c y s t e i n e that are at f a u l t f o r more bromine o x i d i z a b l e s u l f u r i s found i n the albuminoids than c o u l d be present I n the methionine f r a c t i o n of these p r o t e i n s and the r e s u l t s o b t a i n e d In the determina-t i o n of c y s t e i n e by d i f f e r e n t methods are much more more v a r i a b l e than In methionine d e t e r m i n a t i o n s . In one method G r a f f , M a c u l l a and G r a f f (108) have determined c y s t e i n e by i s o l a t i n g i t from the h y d r o l y s a t e s w i t h CugO and measuring bot h the n i t r o g e n and the s u l f u r c o n t e n t s . T h i s method would measure any u n s p l i t t h i o l h i s t i d i n e or e r g o t h i o n e i n e present i n the h y d r o l y s a t e w i t h the c y s t e i n e but i f they were i n c l u d e d there would be a d i s c r e p a n c y between the v a l u e s as determined by n i t r o g e n and by s u l f u r . The r e s u l t s of G r a f f e t a l . show no s i g n i f i c a n t d i s c r e p a n c y but S h u l t z and Vars (109) have p o i n t e d out that a l l r e s u l t s o b t a i n e d by t h i s method do not agree so c l o s e l y . By the barium s u l f a t e method they found h e p a t i c p r o t e i n to 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 but G r a f f 62 and B a r t h found i t t o have 4,65$ by K j e l d a h l d e t e r m i n a t i o n s . T h i s d i f f e r e n c e i n the f i n d i n g s c o u l d be e x p l a i n e d i f a l a r g e f r a c t i o n of t h i o l h i s t i d i n e were p r e s e n t . In Table IVA are l i s t e d 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 s i x t e e n d i f f e r e n t methods. Most of these methods are based on the d e t e r m i n a t i o n of the r e d u c i n g power of the -SH group and would probably not d i f f e r e n t i a t e c y s t e i n e s u l f h y d r y l groups from those of t h i o l i m i d a z o l e breakdown p r o d u c t s . The d e v i a t i o n s from the mean of the r e s u l t s o b t a i n e d by these d i f f e r e n t methods are compared In Table IVB, I f o n l y c e r t a i n methods do not measure s u l f u r from the t h i o l i m i d a z o l e s these would be expected to show a v a r i a b l e but c o n s i s t e n t l y n e g a t i v e d e v i a t i o n . In the case of those methods f o r which enough f i g u r e s are a v a i l a b l e t o make s t a t i s t i c a l treatment a p p l i c a b l e t h i s i s t r u e of the methods developed by V i c k e r y and White, by Jones, G e r s d o r f f and M o e l l e r and, except f o r the r e s u l t from wool, of S u l l i v a n ' s procedure, A m o d i f i c a t i o n of the V i c k e r y and White method by G r a f f et a l , shows r e s u l t s h i g h e r than the average and i t , t h e r e f o r e , seems probable that the d e v i a t i o n shown by the. o r i g i n a l method a r i s e s from f a c t o r s other than the d i f f e r -ences i n the s u l f h y d r y l groups. S i m i l a r l y the Jones, Gers-d o r f f and M o e l l e r method i s a m o d i f i c a t i o n of the one developed by F o l i n and Looney and taken t o g e t h e r the two methods do not show a c o n s i s t e n t d e v i a t i o n . The method of S u l l i v a n , on the other hand, i s based on a v e r y s p e c i f i c 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 low r e s u l t s o b t a i n e d by 63 i t may be of c o n s i d e r a b l e s i g n i f i c a n c e . U n f o r t u n a t e l y t h e r e has been no m i c r o b i o l o g i c a l method f o r the assay o f c y s t i n e or c y s t e i n e developed as y e t (110) but i t would be i n t e r -e s t i n g to see I f r e s u l t s o b t a i n e d by t h i s method agree w i t h those g i v e n by S u l l i v a n ' s method. I t I s p o s s i b l e t h a t i t has 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 has thus f a r prevented the f o r m u l a t i o n of a m i c r o b i o l o g i c a l method. A t h i r d p o s s i b l e e x p l a n a t i o n o f the d i f f e r e n c e s between the s u l f u r content of p r o t e i n s and t h e i r i n d i c a t e d content of amino a c i d s and t h i o l i m i d a z o l e s Is t h a t c y s t e i n e and h i s t i d i n e might occur l i n k e d as a t h i o e t h e r . On h y d r o l y s i s by the u s u a l l a b o r a t o r y methods the t h i o ether might y i e l d c y s t e i n e and a d e r i v a t i v e of h i s t i d i n e but i t i s p o s s i b l e that i t would g i v e s e r i n e and t h i o l h i s t i d i n e by enzymatic h y d r o l y s i s . I f t h i s be t r u e i t i s d i f f i c u l t , t o e x p l a i n the nature of the bromine o x i d i z a b l e f r a c t i o n observed by Zahnd and C l a r k e , HC =C-CHo- CH-C-OH s m2 /> CHg-CH-Cr0H H y p o t h e t i c a l t h i o ether H I , E x p e r i m e n t a l P r e p a r a t i o n of e r g o t h i o n e i n e In order to c a r r y out the proposed search f o r t h i o l i m i d a z o l e s i t was f e l t t h a t the f i r s t r e q u i s i t e was a 64 supply of e r g o t h i o n e i n e and t h i o l h i s t i d i n e f o r use i n con-t r o l s . Seventy m i l l i g r a m s of e r g o t h i o n e i n e prepared by Dr. B. A. E a g l e s i n 1928 were a v a i l a b l e . R e c r y s t a l i z a t i o n of t h i s m a t e r i a l from a l c o h o l showed t h a t i t had s u f f e r e d r e l a t i v e l y l i t t l e decomposition s i n c e o r i g i n a l l y prepared but i t was, n e v e r t h e l e s s , f e l t t h a t a l a r g e r supply was d e s i r a b l e . Twelve hundred grams of ergot were ob t a i n e d through the g e n e r o s i t y of the Upjohn Company of Kalamazoo, Michigan, A p r e l i m i n a r y t e s t of the e r g o t h i o n e i n e content of t h i s ergot was made by e x t r a c t i n g a f i n e l y ground sample w i t h b o i l i n g water, c l a r i f y i n g the f i l t r a t e w i t h n e u t r a l l e a d a c e t a t e and a p p l y i n g the Hunter t e s t . In comparison w i t h some of the e r g o t h i o n e i n e prepared by E a g l e s t h i s t e s t i n d i c a t e d o n l y 1 mg of e r g o t h i o n e i n e per gram of e r g o t . In t h i s t e s t the e x t r a c t i o n may have been incomplete, the c l a r i f i c a t i o n w i t h n e u t r a l l e a d a c e t a t e l e s s s a t i s f a c t o r y than the uranium a c e t a t e used by Hunter (67) i n h i s method of d e t e r m i n i n g e r g o t h i o n e i n e i n ergot and the v i s u a l comparison i n a c c u r a t e . However, i t does seem that t h i s sample had a r a t h e r lower e r g o t h i o n e i n e content than those from which P i r i e (65) was a b l e to e x t r a c t 1.8 mg/g and Hunter et a l . (68) 2.6 mg/g. D u r i n g the course of t h i s work a paper by Lawson and Riming-ton (66) has been p u b l i s h e d which d e s c r i b e s even g r e a t e r d i f f e r e n c e s i n the e r g o t h i o n e i n e content of ergot from d i f f e r e n t sources and more r e c e n t l y Hunter has found v a l u e s f o r the e r g o t h i o n e i n e content of ergot o n ' A l b e r t a r y e v a r y i n g 65 from 0.184 to 0.474$ (67). Nine hundred grams of the ergot were t r e a t e d a c c o r d i n g to P i r i e ' s method (66). Only a s m a l l amount of white p r e c i p i t a t e formed on the a d d i t i o n of cuprous oxide and a l l subsequent steps gave c o r r e s p o n d i n g l y low but t y p i c a l y i e l d s . However, the f i n a l product of about 40 mg. was i n s o l u b l e i n 3:4 acetonetwater and gave an orange c o l o r by the Hunter t e s t (94) such as might be g i v e n by a mixture of h i s t i d i n e and e r g o t h i o n e i n e . E v i d e n t l y o n l y a s m a l l amount of e r g o t h i o n e i n e was e x t r a c t e d from the ergot and much of the s u l f u r was removed from i t d u r i n g p u r i f i c a t i o n . These d i f f i c u l t i e s may have been caused by the f a c t t h a t the procedure was s e v e r a l times i n t e r r u p t e d and the complete i s o l a t i o n c a r r i e d out over a p e r i o d of s e v e r a l weeks. The remaining three hundred grams were e x t r a c t e d a c c o r d i n g to the method of E a g l e s (62), Again s m a l l but t y p i c a l p r e c i p i t a t e s of the e r g o t h i o n e i n e f r a c t i o n were obtained a t each s t e p . T h i s time, a g a i n , a s m a l l y i e l d , 53 mg, was obtained but t h i s substance gave on l y a c o l o r t y p i c a l . o f h i s t i d i n e by the Hunter t e s t . I t i s thought t h a t i m p u r i t i e s formed i n the a l c o h o l d u r i n g d r y i n g w i t h sodium may have o x i d i z e d the e r g o t h i o n e i n e . S y n t h e s i s of t h i o l h i s t i d i n e Attempts 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 have thus f a r been as u n s u c c e s s f u l as those to i s o l a t e e r g o t h i o n e i n e . The method of Ashley and H a r r i n g t o n (51) was chosen as the most s u i t a b l e . In the f i r s t attempt the r e a c t a n t s exploded 66 w i t h c o n s i d e r a b l e v i o l e n c e d u r i n g b e n z o y l a t i o n . T h i s may have been due to carbon d i o x i d e r e l e a s e d by the f o r m a t i o n of a c i d or to a s m a l l r i s e i n temperature a f t e r most of the i c e had melted. In subsequent runs the r e a c t i o n v e s s e l was p e r i o d i c a l l y opened although e x c e s s i v e p r e s s u r e was never n o t i c e d . In the second attempt 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 a good y i e l d was obtained from the b e n z o l y a t i o n but t h a t of methyl c 8 -dibenzamido-Y-ketoval-erate was very s m a l l . F a i l -u re here appears to have been due to the f a c t t h a t the r e a c t i o n , was not c a r r i e d out i n anhydrous c o n d i t i o n s . One more e f f o r t has been made to date to c a r r y out t h i s s y n t h e s i s . T h i s time y i e l d s were ob t a i n e d as i n d i c a t e d by Ashley and H a r r i n g t o n i n a l l except the f i n a l step when no t h i o l h i s t i d i n e c r y s t a l i z e d out. The Hunter t e s t when a p p l i e d to the impure s o l u t i o n of products showed o n l y the y e l l o w c o l o r t y p i c a l of h i s t i d i n e and i t i s c o n s i d e r e d p o s s i b l e that the r e a c t a n t s may have been contaminated w i t h i r o n which would c a t a l y z e the removal of the s u l f h y d r y l 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 There are no r e c o r d s a v a i l a b l e of any attempt to i s o l a t e t h i o l l m l d a z o l e s from p r o t e i n s by methods p a r a l l e l to those used f o r the i s o l a t i o n of these compounds from other sources. T h i s , the s i m p l e s t method of approach, was, t h e r e -f o r e , the f i r s t s t u d i e d i n t h i s work. U n f o r t u n a t e l y a t e s t f o r e r g o t h i o n e i n e c o u l d not be made at the t e r m i n a t i o n of each step due to i n t e r f e r e n c e from amino a c i d s and humus. The 67 f i r s t t e s t s , made on z e i n , human h a i r and f i n g e r n a i l s , h y d r o l y z e d w i t h s u l f u r i c a c i d and c l a r i f i e d w i t h c h a r c o a l , gave a p p a r e n t l y p o s i t i v e c o l o r s w i t h the Hunter t e s t but no pu r p l e p r e c i p i t a t e , A c o n t r o l u s i n g pure e r g o t h i o n e i n e , however, showed that when s m a l l q u a n t i t i e s were used the er g o t h i o n e i n e was almost q u a n t i t a t i v e l y removed by the c h a r c o a l (Darco), I t may be t h a t another brand o f c h a r c o a l c o u l d be found which would be capable of ads o r b i n g the co l o u r e d i m p u r i t i e s without d e s t r u c t i o n of e r g o t h i o n e i n e but t h i s has not as y e t been i n v e s t i g a t e d . A t e s t on egg white, h y d r o l y z e d w i t h s u l f u r i c a c i d i n the presence of t i t a n o u s c h l o r i d e and t r e a t e d a c c o r d i n g to P i r i e ' s (65) method f o r the i s o l a t i o n o f e r g o t h i o n e i n e from e r g o t , gave a s t r o n g r e d - p u r p l e c o l o r by the Hunter t e s t but a g a i n no purple p r e c i p i t a t e . However, when t e s t e d a g a i n a week l a t e r t h i s product gave a d e f i n i t e l y n e g a t i v e t e s t and no f r a c t i o n of a sample of egg white d i v i d e d i n t o albumin, g l o b u l i n , mucin and p r o t e i n f r e e f i l t r a t e gave a p o s i t i v e t e s t . These r e s u l t s , b a f f l i n g a t the time, may have been due to the I n t e r f e r e n c e of t y r o s i n e as d i s c u s s e d i n a f o l l o w i n g s e c t i o n . F o l l o w i n g these p r e l i m i n a r y f a m i l i a r i z a t i o n t e s t s a s e r i e s of t e s t s was made i n an e f f o r t to determine which p r o t e i n s would be the most s a t i s f a c t o r y f o r a d e t a i l e d study. The method used was ag a i n c h i e f l y based on the e a r l i e r methods f o r i s o l a t i n g e r g o t h i o n e i n e . The p r o t e i n was hydro-l y z e d w i t h h y d r o c h l o r i c a c i d and 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 by S u l l i v a n and Hess (103) i n the i s o l a t i o n of c y s t i n e . The h y d r o l y s a t e was n e u t r a l i z e d , c l a r i f i e d w i t h b a s i c l e a d a c e t a t e and 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 w i t h cuprous oxide as i n P i r i e ' s procedure. The p r e c i p i t a t e 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 the copper f r e e f i l t r a t e . The p r o t e i n c o n t a i n i n g sub-stances t e s t e d and the r e s u l t s of the c o l o r r e a c t i o n o b tained are g i v e n i n Table V, Table V Hunter Diazo R e a c t i o n on a P r o t e i n H y d r o l y s a t e F r a c t i o n Substance Tested R e s u l t Substance Tested R e s u l t Z e i n 1 P o s i t i v e Cabbage Negative Corn G l u t i n i n D o u b t f u l Peccan D o u b t f u l A l e . & H2O2 Probable F i l b e r t s Negative s o l u b l e c o r n Bad Corn (whole)' 3 Negative B r a z i l Nut Negative Wheat G l u t i n i n Negative Ox Hoof Probable Wheat G l i a d i n P o s s i b l e Ox Horn Negative Oat G l i a d i n Negative Ox Lung P o s s i b l e Hordein Negative Ox B r a i n P o s i t i v e Pea Albumin Negative Ox F i b r i n Negative Pea (whole) P o s s i b l e 1 Prepared a c c o r d i n g - t o method o f Mason and Palmer (111), 2 The f i l t r a t e from the p r e c i p i t a t i o n of the 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 might 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 Moldy and d i s c o l o r e d k e r n e l s which were present i n the c o r n used. 69 No odor of trlmethylamine was n o t i c e d i n any case and no pu 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 although o b s c u r i n g p r e c i p i t a t e s of humus were o f t e n p r e s e n t . I t was not d e f i n i t e l y known i f these 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 p o s i t i v e t e s t under these c o n d i t i o n s . By the time the t e s t s l i s t e d i n the t a b l e had been completed work c a r r i e d out c o n c u r r e n t l y on Improving the method of i s o l a t i o n r e v e a l e d t h a t these r e s u l t s were probably o n l y a measure of the t y r o s i n e l e f t i n s o l u t i o n a f t e r n e u t r a l i z a t i o n and the p r o j e c t was abandoned. R e v i s i o n of the method The o b j e c t i v e o f t h i s second l i n e of endeavour was to t e s t and modify each step of the above procedure so as to make accurate and r e l i a b l e r e s u l t s p o s s i b l e . D i f f i c u l t y was experienced because i t was i m p o s s i b l e to d e t e c t anything but i n o r d i n a t e l y h i g h c o n c e n t r a t i o n s of e r g o t h i o n e i n e u n t i l a f t e r the f r a c t i o n a t i o n was complete. T h e r e f o r e , i f two steps of the procedure were at f a u l t i t might not be p o s s i b l e to d e t e c t improvement i n one u n l e s s the other were simultane-o u s l y c o r r e c t e d . In each t e s t a s e r i e s of d i l u t i o n s were made, u s u a l l y l / l , l / l O , l/lOO, and l/lOOO i n order t o d i l u t e out i n t e r f e r i n g substances. Because there was most evidence from d i e t a r y experiments f o r the presence of t h i o l i m i d a z o l e s i n z e i n t h i s p r o t e i n was used i n the f o l l o w i n g t e s t s , S u l l i v a n and Hess (103) have found t h a t 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 to 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 decreases the d e s t r u c t i o n 70 of c y s t i n e . In the i n i t i a l work i n t h i s study t h i s r e d u c i n g compound was used and i t was now c o n s i d e r e d d e s i r e a b l e to i n v e s t i g a t e i t s s p e c i f i c e f f e c t on t h i o l i m i d a z o l e s . The f i r s t t e s t s gave n e g a t i v e c o l o r s w i t h the h y d r o l y s a t e s of z e i n both w i t h and without t i t a n i u m w h i l e r e p e t i t i o n of the t e s t s gave weakly p o s i t i v e r e s u l t s i n both c a s e s . None of these r e s u l t s was as s t r o n g as those g i v e n by the i n i t i a l t e s t s w i t h z e i n but there was no evidence of any improvement due to the t i t a n i u m and i t was, t h e r e f o r e , omitted from subsequent h y d r o l y s e s , Barger and Ewins (49) have shown t h a t the s u l f u r of 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 i n the presence of f e r r i c i o n s . Because the i r o n reduced i n t h i s r e a c t i o n would be r e o x i d i z e d by a i r i t was f e a r e d that small amounts of i r o n i f present 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 -l y z e the o x i d a t i o n of the s u l f u r . In work performed thus f a r care had been taken to a v o i d any possible, contamination w i t h i r o n but i t was deemed d e s i r a b l e to i n v e s t i g a t e the n e c e s s i t y of t h i s p r e c a u t i o n . The f i r s t t e s t a g a i n gave negative r e s u l t s both when the p r o t e i n was h y d r o l y z e d i n d i s t i l l e d reagents and when i t was h y d r o l y z e d i n 0,03$ f e r r i c c h l o r i d e . However, when 6,5$ f e r r i c c h l o r i d e was b o i l e d w i t h a h y d r o l y -sate t h a t was known to g i v e a strong r e d d i a z o t e s t , the r e s u l t a n t product gave a d i s t i n c t l y n e g ative c o l o u r . T h i s was co n s i d e r e d to support the h y p o t h e s i s that the substance b e i n g measured was a t h i o l i m i d a z o l e . P r e c a u t i o n s were con-t i n u e d f o r the avoidance of a l l p o s s i b l e contamination w i t h i r o n . 71 A s e r i e s of t e d t s were made i n an e f f o r t to d e t e r -mine the best c o n d i t i o n s fbr h y d r o l y s i s . The r e s u l t s o f these t e s t s are summarized i n Table VI a c c o r d i n g to the i n t e n s i t y of the c o l o r formed but because of 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 of the e f f e c t of d i l u t i o n the r e a l s i g -n i f i c a n c e of these r e s u l t s i s d i f f i c u l t to a s s e s s . The r e s u l t s do not f a l l i n t o any d e f i n i t e p a t t e r n although some of the stro n g e s t c o l o r t e s t s were o b t a i n e d by h y d r o l y s i n g w i t h h y d r o c h l o r i c a c i d . H y d r o l y s i s w i t h p e p s i n ( D i f c o . ) , papain ( D i f c o . ) , and p a n c r e a t i n ( N u t r i t i o n a l Biochemicals) were t r i e d but none of the p r e p a r a t i o n s d i g e s t e d the z e i n completely and the r e s u l t s were I n c o n c l u s i v e but d e f i n i t e l y not as str o n g as those w i t h h y d r o c h l o r i c a c i d . In many of these t e s t s a t r a n s i e n t p i n k c o l o r was obtained at the i n t e r f a c e on adding a l k a l i . I t i s not known whether t h i s was caused by decomposition of the c o l o u r e d compound In the s t r o n g a l k a l i or by d i l u t i o n of i t beyond the l i m i t o f s e n s i t i v i t y . I t w i l l be n o t i c e d that r e l a t i v e l y low r e s u l t s were obtained i n s e r i e s VI of Table VI where the a l c o h o l and water s o l u b l e f r a c t i o n of the c o r n was used. T h i s f r a c t i o n , the f i l t r a t e o b t a i n e d on the removal of z e i n a f t e r t r e a t i n g the a l c o h o l e x t r a c t of c o r n w i t h water, i s t h a t i n which any f r e e e r g o t h i o n e i n e from the c o r n would probably appear. The c o l o r o b tained i n t h i s s e r i e s was d i s t i n c t and r e l a t i v e l y f r e e of i n t e r f e r e n c e . In f r a c t i o n a t i n g the c o r n a l a r g e volume of t h i s s o l u t i o n was o b t a i n e d so t h a t , although i t Table VI E f f e c t of C o n d i t i o n s of H y d r o l y s i s on the I n t e n s i t y of the C o l o r Given by the Hunter Test Temperature °C 100 125 140 150 165 210 20 Time h r s . 1.5 5.0 2.5 6.0 16.. 28. 18. 2.5 2.0 0.17 0.0 S e r i e s Expressed as e q u i v a l e n t amount of e r g o t h i o n e i n e In per cent of z e i n . I I I 0.13 0.08 0.13 0.13 0.08 0.17 0.13 0.08 0.17 0.08 0.08 0.4 0.2 0.13 0.04 I I I ? IV 0.04 0.04 0.32 V 0.08 0.2 VI 0.08 0.13 0.08 VI I 0.0 0.0 9 Key to S e r i e s I. 3g of z e i n i n 15ml of 20$ HC1. I I . 3g of z e i n i n 15ml of 20$ H2SO4. I I I . 3g of z e i n i n 15ml of 40$ H2SO4. IV. 3g of z e i n i n 15ml of 50$ formic a c i d , 7$ HC1. V. 3g of z e i n i n 15ml of 20$ NaOH. VI. 8ml of a l c o h o l and water s o l u b l e f r a c t i o n of corn w i t h 7ml of 37$ HC1. V I I . 3g of r e s i d u e from r e s o l u t i o n of 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 to thatt from an equal weight of z e i n , i t must have c o n t a i n e d a g r e a t e r amount of the c o l o u r g i v i n g compound than the z e i n prepared from the same amount of c o r n . I f t h i s compound i n the a l c o h o l and water 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 i t would amount to 0,07$ of the t o t a l weight o f the c o r n . However, c o n c e n t r a t i o n of t h i s s o l u t i o n d i d not 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 i n t e n s i t y of the r e a c t i o n nor d i d h y d r o l y s i s w i t h h y d r o c h l o r i c a c i d d e s t r o y i t as i t was l a t e r found to d e s t r o y e r g o t h i o n e i n e . When the z e i n was r e - p u r i f i e d by f o u r s u c c e s s i v e p r e c i p i t a t i o n s from a l c o h o l , the c o l o u r developed by a h y d r o l y s a t e prepared from the p u r i f i e d product was not a p p r e c i a b l y weakened. Work was, t h e r e f o r e , continued w i t h the p r o t e i n but i t i s hoped a l s o to i n v e s t i g a t e f u r t h e r the nature of the c o l o u r g i v i n g substance i n the s o l u b l e f r a c t i o n . T e s t s were made u s i n g d i f f e r e n t combinations of the p r e c i p i t a t i n g agents t h a t had been found of value i n the i s o l a t i o n of e r g o t h i o n e i n e from ergot and b l o o d . S e v e r a l combinations of b a r y t a , phosphotungstic a c i d , b a s i c l e a d a c e t a t e , mercuric c h l o r i d e and cuprous oxide were t r i e d but i n no case was there l e s s i n t e r f e r e n c e or a stronger c o l o u r than t h a t i n the t e s t s performed w i t h the method o r i g i n a l l y o u t l i n e d . During t h i s study i t became c l e a r t h a t the method employed f o r removing m e t a l l i c s a l t s w i t h hydrogen s u l f i d e was a p o s s i b l e source o f 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 e r g o t h i o n e i n e w i t h copper, the 74 s e n s i t i v i t y of the Hunter t e s t was g r e a t l y reduced i f the copper was not completely removed* However f i l t r a t i o n of the copper s u l f i d e was o f t e n d i f f i c u l t due to i t s c o l l o i d a l nature and much e r g o t h i o n e i n e was d e s t r o y e d when the process was prolonged. Ten d i f f e r e n t s u b s t i t u t e s were used f o r the gas i n s e p a r a t i n g the e r g o t h i o n e i n e and copper but only c u p f e r r o n was found to g i v e complete removal of the copper without d e s t r u c t i o n o f the e r g o t h i o n e i n e or i n t e r f e r e n c e w i t h the Hunter d i a z o t e s t . I n t e r f e r e n c e by Amino A c i d s E a r l y i n the course of t h i s study an I n v e s t i g a t i o n had been made of p o s s i b l e sources of i n t e r f e r e n c e w i t h the Hunter t e s t . I t was found that h i s t i d i n e gave a y e l l o w c o l o r and t y r o s i n e a p i n k c o l o r c l o s e l y resembling that g i v e n by t h i o l i m i d a z o l e s i n shade and s e n s i t i v i t y . An attempt was made to d i s t i n g u i s h these c o l o r s s p e c t r o s c o p i c a l l y but there were no d i s t i n c t a b s o r p t i o n bands, A spectrophotometer might serve to d i f f e r e n c i a t e the c o l o u r s when presen t i n combinations but i t would not be s u i t a b l e f o r r o u t i n e work. I n t e r f e r e n c e due to h i s t i d i n e was e a s i l y r e c o n i z a b l e and was o n l y s e r i o u s i n a q u a l i t a t i v e t e s t when i t was s u f f i c i e n t l y i n t e n s e to obscure 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 c o l o u r 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 to about 0,003 mg/ml and would be d e f i n i t e l y p o s i t i v e i f a l l the t y r o s i n e of z e i n were presen t i n the h y d r o l y s a t e . The h y d r o l y -sate was, t h e r e f o r e , 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 f t e r s tanding a few hours and i t was thought to c o n t a i n o n l y 75 the 0.41 mg/ml s o l u b l e i n water. T h i s amount c o u l d not account f o r the r e a c t i o n s observed when the h y d r o l y s a t e was d i l u t e d w i t h one thousand p a r t s of water. A f t e r the i n c o n c l u s i v e r e s u l t s a l r e a d y o u t l i n e d , a c l o s e r check of the e f f e c t of t y r o s i n e was made at the suggestion of Dr. G, Hunter, T e s t s w i t h M i l i o n r s reagent showed t h a t t y r o s i n e i s not completely p r e c i p i t a t e d by n e u t r a l i z i n g a p r o t e i n h y d r o l y s a t e but th a t a l a r g e p a r t may remain i n s o l u t i o n , t h e r e f o r e , a l l the r e s u l t s o b t a i n e d h e r e t o f o r e , except those from the work on the d e s t r u c t i v e e f f e c t of hydrogen s u l f i d e where no h y d r o l y s a t e was i n v o l v e d , may have been o n l y a measure of the s o l u b l e t y r o s i n e . S e p a r a t i o n of T h i o l i m i d a z o l e s from Hydrolysates I t v/as now necessary t o d e v i s e a method f o r com-p l e t e l y s e p a r a t i n g t y r o s i n e and h i s t i d i n e from any t h i o -l i m i d a z o l e l i k e l y to be p r e s e n t , B a s i c l e a d a c e t a t e was used 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 or t h i o l h i s t i d i n e w i t h out any h i s t i d i n e a c c o r d i n g to the method of Hunter and Raragosky (112), The t h i o l i m i d a z o l e s were then f r e e d w i t h hydrogen 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 s i l v e r (51, 53) or b a r y t a and s i l v e r (113) l e a v i n g the t y r o s i n e i n s o l u t i o n . S e v e r a l f r a c t i o n a t i o n s were made v a r y i n g the order and pH of the p r e c i p i t a t i o n s . T e s t s were made at s e v e r a l stages i n the f r a c t i o n a t i o n and a c o n t r o l sample w i t h e r g o t h i o n e i n e added to the z e i n was run w i t h the t e s t . The procedure used and the r e s u l t s o b tained i n one of the most ext e n s i v e of these t e s t s i s g i v e n i n the accompanying c h a r t . In no case 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 fat soluble water soluble vaouum d i s t i l neutralize with 2.5 N NaOH allow to settle 4 hrs f i l t e r discard humus and tyrosine Test: - T B 0.3 K c 0.3 disoard supernatant* (histidine) P disoard PbS add 5ml 20# Pb(QAo)2 and 2.5 ml 2.5 N NaOH v wash with water treat with IN H2SO4 supernatant neutralize with 2.5 N NaOH treat with excess 2C$ AgN03 Testt T 0 0.3 K . 0.3 Testt T . 0.000 K B 0.000 Test: T • 0.01 K m 0.01 Testt T 8 0.001 K e 0.000 treat with Ba(0H)2 & AgN03 Test: T m 0.02 K B 0.01 disoard Ag2S<^ fi l t r a t e Test: T B 0.000 0.000 77 t e s t f o r a t h i o l i m i d a z o l e observed on completion of t h e f r a c t i o n a t i o n . T e s t s performed on the c o n t r o l sample wi t h added e r g o t h i o n e i n e , at v a r i o u s p o i n t s i n the f r a c t i o n a t i o n when they were p o s i t i v e , were not always as s t r o n g as those on the h y d r o l y s a t e prepared without added e r g o t h i o n e i n e . S e p a r a t i o n of E r g o t h i o n e i n e from Amino A c i d s Because of the f a i l u r e of these attempts to i s o l a t e any t h i o l i m i d a z o l e from z e i n , even when i t was added at the begi n n i n g , the r e a c t i o n s w i t h pure e r g o t h i o n e i n e and amino a c i d s were s t u d i e d . 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 now made that the c o n v e n t i o n a l method of h y d r o l y s i s w i t h hydro-c h l o r i c a c i d completely d e s t r o y s the t h i o l i m i d a z o l e r i n g , A paper by Le f e v r e and Rangier (32) t h a t r e c e n t l y became a v a i l -a ble g i v e s evidence s u p p o r t i n g t h i s r e s u l t . In c o n t r a s t , s u l f u r i c a c i d was found to g i v e very l i t t l e d e s t r u c t i o n . N e u t r a l i z a t i o n o f the s u l f u r i c a c i d was u s u a l l y accomplished w i t h barium hydroxide i n order t h a t no s a l t s should be l e f t i n s o l u t i o n . However, i t was found t h a t barium s u l f a t e c o - p r e c i p i t a t e d e i g h t y percent of the e r g o t h i o n e i n e from the s o l u t i o n s used. N e u t r a l i z a t i o n w i t h 2,5 N sodium hydroxide was found to be b e t t e r although about ten percent was s t i l l l o s t . No d i f f i c u l t y was experienced i n s e p a r a t i n g the er g o t h i o n e i n e from h i s t i d i n e w i t h b a s i c l e a d a c e t a t e but s e v e r a l methods were t r i e d f o r the removal of t y r o s i n e b e f o r e a s u c c e s s f u l one was found. Lead w i l l p r e c i p i t a t e t y r o s i n e from a more a c i d s o l u t i o n than t h a t r e q u i r e d f o r the 78 p r e c i p i t a t i o n o f e r g o t h i o n e i n e but the s e p a r a t i o n c o u l d not be made q u a n t i t a t i v e . Both s i l v e r and b a r y t a were t r i e d as s e l e c t i v e p r e c i p i t a n t s but no e r g o t h i o n e i n e c o u l d be re c o v e r e d from the p r e c i p i t a t e s . F i n a l l y i t was found t h a t mercuric s u l f a t e , Hopkins-Cole reagent (114), s a t i s f a c t o r i l y removed er g o t h i o n e i n e from t y r o s i n e . The e r g o t h i o n e i n e was f r e e d w i t h hydrogen s u l f i d e and the mercuric s u l f i d e c e n t r i f u g e d o f f . F u r t h e r S t u d i e s of H y d r o l y s a t e s The methods developed i n t h i s work w i t h mixtures of known amino a c i d s were now a p p l i e d to p r o t e i n h y d r o l y s a t e s . When e r g o t h i o n e i n e was added to the p r o t e i n none c o u l d be i s o l a t e d , but when i t was added a f t e r h y d r o l y s i s most c o u l d be r e c l a i m e d . Thus i t seems that t h i s t h i o l i m i d a z o l e i s de s t r o y e d by s u l f u r i c a c i d when p r o t e i n breakdown products are present although i n pure s o l u t i o n the d e s t r u c t i o n had been shown to be n e g l i g i b l e , A number of a l t e r n a t e methods of h y d r o l y s i s have been t e s t e d by f r a c t i o n a t i o n a c c o r d i n g to the method o u t l i n e d i n Chart I I and the r e s u l t s are l i s t e d i n Table V I I . At the present time no s a t i s f a c t o r y method has been found although i t seems that i t Is p o s s i b l e to i s o l a t e a s m a l l amount of t h i o -l i m i d a z o l e a f t e r h y d r o l y s i s w i t h f o r m i c a c i d or combined enzymes. Work i s being c o n t i n u e d i n t h i s f i e l d . 79 Table V I I Methods of H y d r o l y s i s Temperature 175 C 165 C 125 C 40 C Time 12 h r s 1.75 h r s 8 h r s 24 h r s Agent Expressed as the f r a c t i o n of added e r g o t h i o n e i n e r e c l a i m e d . NaOH 20$ 0.0 H 2 S 0 4 20$ 0.0 H 3 P O 4 20$ 0.0 KOH 20$ 0.0 T r i c h l o r o a c e t i c 70$ w i t h SnS0 4 10$ T r i c h l o r o a c e t i c 70$ w i t h T i 2 ( S 0 4 ) 3 0.0 0.0 Papain O.O 1 T r y p s i n 0.0 Pep s i n 0.0 Pep s i n & T r y p s i n 0.'022 Formic 50$ w i t h HC1 7$ Pormamide 90$ 0,0 0.01 0.01 1 A s m a l l but d i s t i n c t p u r p l e p r e c i p i t a t e formed i n 24 h r s . 2 Hydrolyzed 24 h r s w i t h p e p s i n at pH 2,0 and a f u r t h e r 24 h r s w i t h t r y p s i n at pH 8.0, 80 Chart H Ifethod of Fractionation Currently Used 0.2 g zein or egg albumin 1 mg ergothioneine 1 ml hydrolyzing agent discard fat soluble hydrolyze extract with ether water soluble disoard humus & tyrosine^ discard filtrate«-(tyrosine) disoard HgS <-disoard f i l t r a t e , (histidine) discard PbS04 a-neutrallze with 2.5 N NaOH f i l t e r Hopkins-Cole HgS04 reagent wash ppt. with 3# HgS04 " treat with H2S, centifuge aerate r add 0.5 ml of 2C$ Pb (0Ao)2 and 0.25 ml 2.5 N NaOH per ml of solution wash ppt. with water treat with H2SO4 " oheok neutrality perform Hunter test G r o t e 1 s Reagent In an e f f o r to o b t a i n a t e s t which might serve t o c o n f i r m the f i n d i n g s o b t a i n e d when employing the d i a z o r e a c t i o n developed by Hunter f o r t h i o l i m i d a z o l e s , a study of the Grote r e a c t i o n was undertaken. T h i s t e s t i s s a i d to g i v e d i s t i n c t i v e c o l o u r s w i t h both C-SH and C S groups. 81 In so f a r as time p e r m i t t e d , experiments c a r r i e d out employ-i n g t h i s t e s t on t h i o l i m i d a z o l e s were without r e s u l t . No c o l o u r at a l l was observed. I t i s p o s s i b l e that f u r t h e r i n v e s t i g a t i o n may pro v i d e the proper c o n d i t i o n s e n a b l i n g t h i s t e s t to 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 . I t is> of course, p o s s i b l e , however, that under no circumstances i s the r e a c t i o n g i v e n by t h i o l i m i d a z o l e s . Imidazoles have been shown by Langley and Sexton (115) to have at l e a s t two tautameric forms and presumably t h i o l i m i d a z o l e s , l i k e t h i o - u r e a , would have t h r e e . These forms i n c l u d e both C-SH and C=*S groups but the C-SH does not r e a c t w i t h sodium n i t o p r u s s i d e and i t may be th a t n e i t h e r groups r e a c t s w i t h Grote's r e a g e n t . H C = C-R ' H C = C - R I I I I SH \ / SH HC=^C-R I \ HN NH V s IV Summary and Con c l u s i o n s A study of the r e s u l t s o b t a i n e d by v a r i o u s workers on the f r a c t i o n a l a n a l y s i s of p r o t e i n s suggests that many of the c y s t i n e d e t e r m i n a t i o n s r e c o r d e d i n the l i t e r a t u r e may be h i g h due to the i n t e r f e r e n c e of t h i o l i m i d a z o l e breakdown 82 products* Examination of the c o l o r g i v e n by the Hunter d i a z o t e s t w i t h e r g o t h i o n e i n e by means of a simple g r a t i n g specto-scope d i d not r e v e a l any d i s t i n c t a b s o r p t i o n bands which c o u l d be used to c h a r a c t e r i z e the c o l o r . Some I n d i c a t i o n of the presence of f r e e e r g o t h i o n -eine i n c o r n has been found a l t h o u g h other c o n s i d e r a t i o n s make i t s occurrence seem u n l i k e l y . Time has not p e r m i t t e d a complete I n v e s t i g a t i o n of t h i s s u b j e c t as y e t . I t has been shown t h a t e r g o t h i o n e i n e may d i s a p p e a r from a s o l u t i o n d u r i n g p u r i f i c a t i o n employing c h a r c o a l as a d e c o l o r i z i n g agent. The method u s i n g hydrogen s u l f i d e g e n e r a l l y employed f o r s e p a r a t i n g m e t a l l i c s a l t s from combination w i t h e r g o t h i o -neine i s another p o s s i b l e source of d e s t r u c t i o n of the b e t a i n e . S e p a r a t i o n may be more s a t i s f a c t o r i l y accomplished when copper i s removed by c u p f e r r o n , A survey has been made of the r e s u l t s o b t a i n e d by the 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 h y d r o l y s a t e s of a number of p r o t e i n s i n an attempt to o b t a i n f i g u r e s on 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 . The r e s u l t s of t h i s survey a r e , however, c o n s i d e r e d u n r e l i a b l e , A method has been developed whereby e r g o t h i o n e i n e may be separated from the f a c t o r s i n a p r o t e i n h y d r o l y s a t e which i n t e r f e r e w i t h the Hunter d i a z o t e s t ( t y r o s i n e and h i s t i d i n e ) but i t has not y e t been p o s s i b l e t o o b t a i n a t e s t f o r 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 to which e r g o t h i o n e i n e has been added b e f o r e h y d r o l y s i s . E r g o t h i o n e i n e i s d e s t r o y e d by prolonged b o i l i n g i n h y d r o c h l o r i c a c i d even when the r e d u c i n g agent t i t a n o u s c h l o r i d e i s p r e s e n t . The t h i o l i m i d a z o l e r i n g i s a l s o broken by b o i l i n g i n other h y d r o l y z i n g agents when p r o t e i n breakdown products are p r e s e n t . T h i s may e x p l a i n ' the f a i l u r e o f e a r l i e r attempts to i s o l a t e t h i o l i m i d a z o l e s from p r o t e i n s . V a r i o u s techniques and procedures have been employed i n an attempt to determine the presence of the t h i o l i m i d a z o l e r i n g i n p r o t e i n s . I t has not been p o s s i b l e as y e t to d e v i s e a s a t i s f a c t o r y technique f o r the d e t e r m i n a t i o n of t h i o l i m i d a -z o l e s . During t h i s study the i n f l u e n c e of v a r i o u s i n t e r -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 reagent has been found to gi v e no c o l o u r w i t h 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 f o r the d i f f e r e n c i a t i o n of s u l f u r groups. 83 Bibliography 1, Davison, F. R,, Synopsis of Materia Medlca, Toxicology and Pharmacology, 3rd ed., St. Louis, C. V. 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