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A natural guanine-containing analogue of vitamin B12
452
PRELIMINARY NOTES
VOL. 25 (Z957)
if tmcurring in vivo, could produce modified b o d y p r o t e i n s w h i c h m i g h t interfere w i t h e n z y m i c a c t i v i t i e s or cell p e r m e a b i l i t y , or m i g h t possess a n t i g e n i c properties.
Department o/ Biochemistry, College o/Physicians and Surgeons, Columbia University, New York, and the New York State Psychiatric Institute, New York, N . Y . (U.S.A.)
NIRMAL K. SARKAR DONALD D. CLARKE HEINRICIt WAELSCH
1 H. WAELSCH, P. OWADES, E. BOREK, .~. GROSSOWICZ AND M. SCHOU, Arch. Biochem., 27 (i95 O) 237. 2 p. SIEKEVITZ, J. Biol. Chem., 195 (1952) 549. s H. BORSOOK, E. L. DEASY, A. J. HAAGEN-SMIT, G. KEIGHLEY AND P. H. LowY, J. Biol. Chem., 184 (195 ° ) 529. 4 R. SCHWEET, Federation Proc., 15 (1956) 35 o. s W. BLOCK, Z. physiol. Chem., 296 (lO8) 1954. Received May 3 i s t , 1957
A natural guanine-containing analogue of vitamin B12 A p r e p a r a t i o n c o n t a i n i n g two v i t a m i n BI: a n a l o g u e s h a s been o b t a i n e d from t h e f e r m e n t a t i o n b r o t h s of a Nocardia s t r a i n isolated in o u r l a b o r a t o r y 1. T h e e x t r a c t i o n from t h e m i c r o o r g a n i s m s was p e r f o r m e d b y a u t o c l a v i n g a cell s u s p e n s i o n s u p p l e m e n t e d w i t h cyanide. T h e s u b s t a n c e s u n d e r i n v e s t i g a t i o n h a v e been s e p a r a t e d from v i t a m i n B i t a n d factor B, likewise p r e s e n t in f e r m e n t a t i o n broths, a n d purified b y a p r o c e d u r e i n v o l v i n g : a d s o r p t i o n on charcoal, elution in isopropanol-water, r e m o v a l of v i t a m i n B t t as d i c y a n i d o c o m p l e x in benzyl alcohol, a d s o r p t i o n on i o n - e x c h a n g e r s (Amberlite IRC-5o, A m b e r l i t e I R A - 4 o i , D o w e x I) a n d electrophoresis in a c o l u m n of W h a t m a n cellulose powder:. T h e eluate from t h e cellulose c o l u m n w a s c o n c e n t r a t e d in vacuo a n d t h e red s u b s t a n c e s were precipitated b y a c e t o n e addition. T h e cobalt c o n t e n t of t h e product, dried over CaCI 2 in vacuo a t room t e m p e r a t u r e , w a s f o u n d to be 3.2 % ; two a t o m s of p h o s p h o r u s a n d one molecule of ribose are p r e s e n t per a t o m of cobalt. No benziminazole d e r i v a t i v e s were f o u n d after h y d r o l y s i s by 6 N HCI. H y d r o l y s i s b y 2 N HCI a t zoo ° for 2 - 4 h sets free a p u r i n e whose ultra-violet s p e c t r u m a n d c h r o m a t o g r a p h i c b e h a v i o r are similar to t h o s e of g u a n i n e . Differential s p e c t r o p h o t o m e t r y of t h e h y d r o l y s a t e in acid a n d alkaline solutions a n d FOLZN-CIOCALTKU'S reaction s h o w t h a t t h e p r e p a r a t i o n c o n t a i n s one molecule of g u a n i n e per molecule of ribose. T h e a b s o r p t i o n s p e c t r u m of a q u e o u s solutions of t h e p r o d u c t h a s m a x i m a a t 273, 320, 356, 500, 53 ° m/t over a wide r a n g e of p H v a l u e s (from 1 to 1o). B y a d d i t i o n of c y a n i d e to n e u t r a l a n d alkaline solutions t h e m a x i m a are shifted to 276, 307.5, 367.5, 54 ° a n d 580 m p . P a p e r c h r o m a t o g r a p h y in b u t a n o l - a c e t i c a c i d - w a t e r a n d p a p e r electrophoresis in o . 5 N acetic acid followed b y b i o a u t o g r a p h y s revealed t h e presence of two g r o w t h factors for E. coli i z3/3. Most of t h e a c t i v i t y was d u e to a n electronegative c o m p o n e n t s h o w i n g a n R F value relative to v i t a m i n B t : of a b o u t o.z2. T h e o t h e r factor, w h i c h is p r e s e n t in s m a l l e r a m o u n t , h a s a n R F of a b o u t 0.27 a n d b e h a v e s like v i t a m i n Bz: in electrophoresis. D e a m i n a t i o n b y HNO~ a p p a r e n t l y does n o t affect t h e latter c o m p o n e n t while t h e former is c h a n g e d into a more electronegative g r o w t h factor. A purine, n o t d i s t i n g u i s h a b l e from x a n t h i n e in its c h r o m a t o g r a p h i c b e h a v i o r a n d ultra-violet a b s o r p t i o n s p e c t r u m , h a s been f o u n d after h y d r o l y s i s of t h e d e a m i n a t e d m i x t u r e by 2 N HCI. Therefore we m a y conclude t h a t t h e p r e p a r a t i o n consists m a i n l y of a g u a n i n e - c o n t a i n i n g a n a l o g u e of v i t a m i n B12. T h e c h r o m a t o g r a p h i c a n d electrophoretic properties of this s u b s t a n c e are closely similar to t h o s e described for factor C s. R. BARCHIELLI Laboratori Ricerche Farmitalia, Milan (Italy) G. BORETTI P. J ULITA A. MIGLIACCI A. MINGHETTI 1 A. DI MARCO, C. G. ALBERTI, G. BORETTI, M. GmONE, A. MIGLIACCl AND C." SPALLA, in H. C. HEINRICH, Vitamin B i t und Intrinsic Faktor, E n k e Verlag, z957, p. 55. z H. DELLWEG, E. BECHER AND K. BERNnAtmR, Biochem. Z., 328 (1956) 88. s j . E. FORD, E. S. FIOLDSWORTI~ AND S. K. Kor~, Biochem. J., 59 (1955) 86. Received M a y 27th, z957
PRELIMINARY NOTES
VOL. 25 (Z957)
if tmcurring in vivo, could produce modified b o d y p r o t e i n s w h i c h m i g h t interfere w i t h e n z y m i c a c t i v i t i e s or cell p e r m e a b i l i t y , or m i g h t possess a n t i g e n i c properties.
Department o/ Biochemistry, College o/Physicians and Surgeons, Columbia University, New York, and the New York State Psychiatric Institute, New York, N . Y . (U.S.A.)
NIRMAL K. SARKAR DONALD D. CLARKE HEINRICIt WAELSCH
1 H. WAELSCH, P. OWADES, E. BOREK, .~. GROSSOWICZ AND M. SCHOU, Arch. Biochem., 27 (i95 O) 237. 2 p. SIEKEVITZ, J. Biol. Chem., 195 (1952) 549. s H. BORSOOK, E. L. DEASY, A. J. HAAGEN-SMIT, G. KEIGHLEY AND P. H. LowY, J. Biol. Chem., 184 (195 ° ) 529. 4 R. SCHWEET, Federation Proc., 15 (1956) 35 o. s W. BLOCK, Z. physiol. Chem., 296 (lO8) 1954. Received May 3 i s t , 1957
A natural guanine-containing analogue of vitamin B12 A p r e p a r a t i o n c o n t a i n i n g two v i t a m i n BI: a n a l o g u e s h a s been o b t a i n e d from t h e f e r m e n t a t i o n b r o t h s of a Nocardia s t r a i n isolated in o u r l a b o r a t o r y 1. T h e e x t r a c t i o n from t h e m i c r o o r g a n i s m s was p e r f o r m e d b y a u t o c l a v i n g a cell s u s p e n s i o n s u p p l e m e n t e d w i t h cyanide. T h e s u b s t a n c e s u n d e r i n v e s t i g a t i o n h a v e been s e p a r a t e d from v i t a m i n B i t a n d factor B, likewise p r e s e n t in f e r m e n t a t i o n broths, a n d purified b y a p r o c e d u r e i n v o l v i n g : a d s o r p t i o n on charcoal, elution in isopropanol-water, r e m o v a l of v i t a m i n B t t as d i c y a n i d o c o m p l e x in benzyl alcohol, a d s o r p t i o n on i o n - e x c h a n g e r s (Amberlite IRC-5o, A m b e r l i t e I R A - 4 o i , D o w e x I) a n d electrophoresis in a c o l u m n of W h a t m a n cellulose powder:. T h e eluate from t h e cellulose c o l u m n w a s c o n c e n t r a t e d in vacuo a n d t h e red s u b s t a n c e s were precipitated b y a c e t o n e addition. T h e cobalt c o n t e n t of t h e product, dried over CaCI 2 in vacuo a t room t e m p e r a t u r e , w a s f o u n d to be 3.2 % ; two a t o m s of p h o s p h o r u s a n d one molecule of ribose are p r e s e n t per a t o m of cobalt. No benziminazole d e r i v a t i v e s were f o u n d after h y d r o l y s i s by 6 N HCI. H y d r o l y s i s b y 2 N HCI a t zoo ° for 2 - 4 h sets free a p u r i n e whose ultra-violet s p e c t r u m a n d c h r o m a t o g r a p h i c b e h a v i o r are similar to t h o s e of g u a n i n e . Differential s p e c t r o p h o t o m e t r y of t h e h y d r o l y s a t e in acid a n d alkaline solutions a n d FOLZN-CIOCALTKU'S reaction s h o w t h a t t h e p r e p a r a t i o n c o n t a i n s one molecule of g u a n i n e per molecule of ribose. T h e a b s o r p t i o n s p e c t r u m of a q u e o u s solutions of t h e p r o d u c t h a s m a x i m a a t 273, 320, 356, 500, 53 ° m/t over a wide r a n g e of p H v a l u e s (from 1 to 1o). B y a d d i t i o n of c y a n i d e to n e u t r a l a n d alkaline solutions t h e m a x i m a are shifted to 276, 307.5, 367.5, 54 ° a n d 580 m p . P a p e r c h r o m a t o g r a p h y in b u t a n o l - a c e t i c a c i d - w a t e r a n d p a p e r electrophoresis in o . 5 N acetic acid followed b y b i o a u t o g r a p h y s revealed t h e presence of two g r o w t h factors for E. coli i z3/3. Most of t h e a c t i v i t y was d u e to a n electronegative c o m p o n e n t s h o w i n g a n R F value relative to v i t a m i n B t : of a b o u t o.z2. T h e o t h e r factor, w h i c h is p r e s e n t in s m a l l e r a m o u n t , h a s a n R F of a b o u t 0.27 a n d b e h a v e s like v i t a m i n Bz: in electrophoresis. D e a m i n a t i o n b y HNO~ a p p a r e n t l y does n o t affect t h e latter c o m p o n e n t while t h e former is c h a n g e d into a more electronegative g r o w t h factor. A purine, n o t d i s t i n g u i s h a b l e from x a n t h i n e in its c h r o m a t o g r a p h i c b e h a v i o r a n d ultra-violet a b s o r p t i o n s p e c t r u m , h a s been f o u n d after h y d r o l y s i s of t h e d e a m i n a t e d m i x t u r e by 2 N HCI. Therefore we m a y conclude t h a t t h e p r e p a r a t i o n consists m a i n l y of a g u a n i n e - c o n t a i n i n g a n a l o g u e of v i t a m i n B12. T h e c h r o m a t o g r a p h i c a n d electrophoretic properties of this s u b s t a n c e are closely similar to t h o s e described for factor C s. R. BARCHIELLI Laboratori Ricerche Farmitalia, Milan (Italy) G. BORETTI P. J ULITA A. MIGLIACCI A. MINGHETTI 1 A. DI MARCO, C. G. ALBERTI, G. BORETTI, M. GmONE, A. MIGLIACCl AND C." SPALLA, in H. C. HEINRICH, Vitamin B i t und Intrinsic Faktor, E n k e Verlag, z957, p. 55. z H. DELLWEG, E. BECHER AND K. BERNnAtmR, Biochem. Z., 328 (1956) 88. s j . E. FORD, E. S. FIOLDSWORTI~ AND S. K. Kor~, Biochem. J., 59 (1955) 86. Received M a y 27th, z957