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A method of estimation of 2-deoxyribose
VOL. 24 (1957)
PRELIMINARY NOTES
439
A m e t h o d of e s t i m a t i o n of 2-deoxyribose I t h a s b e e n p r o p o s e d t h a t m a l o n a l d e h y d e (propanedial) is f o r m e d d u r i n g t h e o x i d a t i o n of 2-deoxys u g a r s b y p e r i o d a t e 1-4. R e c e n t l y , 2 - t h i o b a r b i t u r i c acid (2-thio-4,6-dioxypyrimidine) h a s been e m p l o y e d as a r e a g e n t for identification of malonaldehydeS, e. I n t h i s l a b o r a t o r y , h i g h l y sensitive a n a l y t i c a l p r o c e d u r e s for 2 - d e o x y s u g a r s h a v e been developed 7 w h i c h are b a s e d u p o n p e r i o d a t e o x i d a t i o n a n d s u b s e q u e n t e s t i m a t i o n of t h e r e s u l t i n g m a l o n a l d e h y d e w i t h 2 - t h i o b a r b i t u r i c acid. T h i s r e p o r t describes t h e e s t i m a t i o n of 2-deoxyribose. To 3.5 ml of a n a q u e o u s solution c o n t a i n i n g o.2-12. 5 / , g of 2-deoxy-D-ribose*, in a t e s t tube, a d d o. 5 m l of a solution of o . o 2 5 M periodic acid in o . I 2 5 N sulfuric acid a n d m i x well. A f t e r 2o rain a t r o o m t e m p e r a t u r e , t h e m i x t u r e is s h a k e n m o m e n t a r i l y w i t h i.o m l of a solution of i.o g of s o d i u m arsenite dissolved in 5 ° m l o . 5 N HC1. A f t e r 2 min, a n a l i q u o t of I.O ml, or less, of t h e r e s u l t i n g solution is t r a n s f e r r e d to a 15 × 125 m m t e s t t u b e c o n t a i n i n g 2.0 ml of a q u e o u s 0.6 % 2 - t h i o b a r b i t u r i c acid** ( a d j u s t e d to p H 2.o u s i n g i . o N N a O H a n d i . o N HC1). T h e v o l u m e is b r o u g h t to 3.o ml a n d t h e c o n t e n t s of t h e t u b e are m i x e d . A b l a n k is p r e p a r e d b y m i x i n g 2.o m l of t h e t h i o b a r b i t u r i c acid r e a g e n t w i t h i.o m l distilled 1.0 w a t e r . T h e t u b e s are fitted w i t h t e a r - d r o p c o n d e n s e r s a n d i m m e r s e d in a boiling w a t e r b a t h for 2o min. A f t e r cooling in t a p w a t e r a t r o o m t e m p e r a t u r e for 0.8 z rain, t h e i n t e n s i t y of t h e p i n k solution is m e a s u r e d A a t 532 m/~ w i t h a B e c k m a n D U S p e c t r o p h o t o m e t e r . 0.6 T h e color is stable for a t least one h o u r . T h e (3 a b s o r p t i o n s p e c t r u m (Fig. I), o b t a i n e d w i t h a W a r r e n recording s p e c t r o p h o t o m e t e r , is in a g r e e m e n t w i t h (j, 0 . 4 s p e c t r a r e p o r t e d b y o t h e r w o r k e r s for t h e c o n d e n s a tion p r o d u c t of m a l o n a l d e h y d e a n d 2 - t h i o b a r b i t u r i c acidS, °. T h e optical d e n s i t y w a s p r o p o r t i o n a l to con0 0.2 c e n t r a t i o n u p to a t least 2. 5 # g 2-deoxy-D-ribose/ml. M a l o n a l d e h y d e bis-bisulfite, s o d i u m salt s w a s 0 utilized in s t u d i e s d e m o n s t r a t i n g t h e c o m p l e t e con450 4"~5 5~o ~,5 5~o 5~,5eoo v e r s i o n of 2 - d e o x y - n - r i b o s e to m a l o n a l d e h y d e ; t h e WAVE LENGTH (mp) l a t t e r w a s f o u n d to be stable in t h e excess of peri o d a t e in t h e a b o v e p r o c e d u r e 7. Fig. I. Visual s p e c t r a of p r o d u c t s obT h e c h a r a c t e r i s t i c s p e c t r u m (Fig. i) h a s also t a i n e d b y h e a t i n g p e r i o d a t e reaction been d e r i v e d f r o m a l i q u o t s of 5 % trichloracetic acid m i x t u r e s w i t h 2 - t h i o b a r b i t u r i c acid. (TCA) h y d r o l y s a t e s of c o m m e r c i a l p r e p a r a t i o n s of A = 2.o # g 2-deoxyribose ; B = 2.o p g deoxyadenosine*** and highly polymerized salmon deoxyadenosine; C = 4° pg DNA. d e o x y r i b o n u c l e i c acid (DNA)* b y h e a t i n g a t 9 o ° C for 15 rain prior to a n a l y s i s 9. T h e a b s o r b e n c y a t 532 m/~ for t h e d e o x y a d e n o s i n e s a m p l e a c c o u n t s for a 96 % recovery of deoxyribose. T h e s m a l l a b s o r b e n c y for t h e D N A s a m p l e is a reflection of i n c o m p l e t e h y d r o l y s i s b y TCA. F u r t h e r s t u d i e s on t h e a p p l i c a t i o n of t h i s p r o c e d u r e for t h e e s t i m a t i o n of 2 - d e o x y s u g a r s in v a r i o u s biological m a t e r i a l s are in progress. V . S . WARAVDEKAR Laboratory o/Chemical Pharmacology, National Cancer Institute, L . D . SASLAW
National Institutes of Health, Bethesda, Md. (U.S.A.) 1 C. F. HUEBNER, S. R. AMES AND E. C. BUBL,J. Am. Chem. Soc., 68 (1946) 1621. 2 L. A. MANSON AND J. O. LAMPEN, J. Biol. Chem., 191 (1951) 95s j . T. EDWARD AND D. M. WALDRON, J. Chem. Soc., (1952) 3631. 4 V. E. CRAMER ANn G. A. NEVILLE, J. Franklin Inst., 256 (1953) 379. 5 S. PATTON AND G. W. KURTZ, J. Dairy Sci., 34 (1951) 669. s W . G. JENNINGS, W. L. DUNKLEY AND H. G. REIBER, Food Research, 2o (1955) 13. 7 V. S. WARAVDEKAR AND L. D. SASLAW (in p r e p a r a t i o n ) . 8 L. D. SASLAW AND V. S. WARAVDEKAR ( s u b m i t t e d for publication). 9 W. C. SCHNEIDER, J. Biol. Chem., 161 (1945) 293. R e c e i v e d F e b r u a r y 5th, 1957 * O b t a i n e d f r o m M a n n R e s e a r c h L a b o r a t o r i e s , Inc., N.Y.C. ** A 3 % solution of 2 - t h i o b a r b i t u r i c acid ( E a s t m a n K o d a k Co., Rochester, N.Y.) in h o t water, is percolated t h r o u g h a c o l u m n of W o e l m acid a l u m i n u m oxide (activity g r a d e I) a n d allowed to crystallize a t r o o m t e m p e r a t u r e . T h e w h i t e r e c t a n g u l a r c r y s t a l s (C4H4N~SO 2. I ~ H z O ) are w a s h e d w i t h cold w a t e r a n d air-dried. * * * O b t a i n e d f r o m California F o u n d a t i o n for Biochemical R e s e a r c h , L o s Angeles, Calif.
PRELIMINARY NOTES
439
A m e t h o d of e s t i m a t i o n of 2-deoxyribose I t h a s b e e n p r o p o s e d t h a t m a l o n a l d e h y d e (propanedial) is f o r m e d d u r i n g t h e o x i d a t i o n of 2-deoxys u g a r s b y p e r i o d a t e 1-4. R e c e n t l y , 2 - t h i o b a r b i t u r i c acid (2-thio-4,6-dioxypyrimidine) h a s been e m p l o y e d as a r e a g e n t for identification of malonaldehydeS, e. I n t h i s l a b o r a t o r y , h i g h l y sensitive a n a l y t i c a l p r o c e d u r e s for 2 - d e o x y s u g a r s h a v e been developed 7 w h i c h are b a s e d u p o n p e r i o d a t e o x i d a t i o n a n d s u b s e q u e n t e s t i m a t i o n of t h e r e s u l t i n g m a l o n a l d e h y d e w i t h 2 - t h i o b a r b i t u r i c acid. T h i s r e p o r t describes t h e e s t i m a t i o n of 2-deoxyribose. To 3.5 ml of a n a q u e o u s solution c o n t a i n i n g o.2-12. 5 / , g of 2-deoxy-D-ribose*, in a t e s t tube, a d d o. 5 m l of a solution of o . o 2 5 M periodic acid in o . I 2 5 N sulfuric acid a n d m i x well. A f t e r 2o rain a t r o o m t e m p e r a t u r e , t h e m i x t u r e is s h a k e n m o m e n t a r i l y w i t h i.o m l of a solution of i.o g of s o d i u m arsenite dissolved in 5 ° m l o . 5 N HC1. A f t e r 2 min, a n a l i q u o t of I.O ml, or less, of t h e r e s u l t i n g solution is t r a n s f e r r e d to a 15 × 125 m m t e s t t u b e c o n t a i n i n g 2.0 ml of a q u e o u s 0.6 % 2 - t h i o b a r b i t u r i c acid** ( a d j u s t e d to p H 2.o u s i n g i . o N N a O H a n d i . o N HC1). T h e v o l u m e is b r o u g h t to 3.o ml a n d t h e c o n t e n t s of t h e t u b e are m i x e d . A b l a n k is p r e p a r e d b y m i x i n g 2.o m l of t h e t h i o b a r b i t u r i c acid r e a g e n t w i t h i.o m l distilled 1.0 w a t e r . T h e t u b e s are fitted w i t h t e a r - d r o p c o n d e n s e r s a n d i m m e r s e d in a boiling w a t e r b a t h for 2o min. A f t e r cooling in t a p w a t e r a t r o o m t e m p e r a t u r e for 0.8 z rain, t h e i n t e n s i t y of t h e p i n k solution is m e a s u r e d A a t 532 m/~ w i t h a B e c k m a n D U S p e c t r o p h o t o m e t e r . 0.6 T h e color is stable for a t least one h o u r . T h e (3 a b s o r p t i o n s p e c t r u m (Fig. I), o b t a i n e d w i t h a W a r r e n recording s p e c t r o p h o t o m e t e r , is in a g r e e m e n t w i t h (j, 0 . 4 s p e c t r a r e p o r t e d b y o t h e r w o r k e r s for t h e c o n d e n s a tion p r o d u c t of m a l o n a l d e h y d e a n d 2 - t h i o b a r b i t u r i c acidS, °. T h e optical d e n s i t y w a s p r o p o r t i o n a l to con0 0.2 c e n t r a t i o n u p to a t least 2. 5 # g 2-deoxy-D-ribose/ml. M a l o n a l d e h y d e bis-bisulfite, s o d i u m salt s w a s 0 utilized in s t u d i e s d e m o n s t r a t i n g t h e c o m p l e t e con450 4"~5 5~o ~,5 5~o 5~,5eoo v e r s i o n of 2 - d e o x y - n - r i b o s e to m a l o n a l d e h y d e ; t h e WAVE LENGTH (mp) l a t t e r w a s f o u n d to be stable in t h e excess of peri o d a t e in t h e a b o v e p r o c e d u r e 7. Fig. I. Visual s p e c t r a of p r o d u c t s obT h e c h a r a c t e r i s t i c s p e c t r u m (Fig. i) h a s also t a i n e d b y h e a t i n g p e r i o d a t e reaction been d e r i v e d f r o m a l i q u o t s of 5 % trichloracetic acid m i x t u r e s w i t h 2 - t h i o b a r b i t u r i c acid. (TCA) h y d r o l y s a t e s of c o m m e r c i a l p r e p a r a t i o n s of A = 2.o # g 2-deoxyribose ; B = 2.o p g deoxyadenosine*** and highly polymerized salmon deoxyadenosine; C = 4° pg DNA. d e o x y r i b o n u c l e i c acid (DNA)* b y h e a t i n g a t 9 o ° C for 15 rain prior to a n a l y s i s 9. T h e a b s o r b e n c y a t 532 m/~ for t h e d e o x y a d e n o s i n e s a m p l e a c c o u n t s for a 96 % recovery of deoxyribose. T h e s m a l l a b s o r b e n c y for t h e D N A s a m p l e is a reflection of i n c o m p l e t e h y d r o l y s i s b y TCA. F u r t h e r s t u d i e s on t h e a p p l i c a t i o n of t h i s p r o c e d u r e for t h e e s t i m a t i o n of 2 - d e o x y s u g a r s in v a r i o u s biological m a t e r i a l s are in progress. V . S . WARAVDEKAR Laboratory o/Chemical Pharmacology, National Cancer Institute, L . D . SASLAW
National Institutes of Health, Bethesda, Md. (U.S.A.) 1 C. F. HUEBNER, S. R. AMES AND E. C. BUBL,J. Am. Chem. Soc., 68 (1946) 1621. 2 L. A. MANSON AND J. O. LAMPEN, J. Biol. Chem., 191 (1951) 95s j . T. EDWARD AND D. M. WALDRON, J. Chem. Soc., (1952) 3631. 4 V. E. CRAMER ANn G. A. NEVILLE, J. Franklin Inst., 256 (1953) 379. 5 S. PATTON AND G. W. KURTZ, J. Dairy Sci., 34 (1951) 669. s W . G. JENNINGS, W. L. DUNKLEY AND H. G. REIBER, Food Research, 2o (1955) 13. 7 V. S. WARAVDEKAR AND L. D. SASLAW (in p r e p a r a t i o n ) . 8 L. D. SASLAW AND V. S. WARAVDEKAR ( s u b m i t t e d for publication). 9 W. C. SCHNEIDER, J. Biol. Chem., 161 (1945) 293. R e c e i v e d F e b r u a r y 5th, 1957 * O b t a i n e d f r o m M a n n R e s e a r c h L a b o r a t o r i e s , Inc., N.Y.C. ** A 3 % solution of 2 - t h i o b a r b i t u r i c acid ( E a s t m a n K o d a k Co., Rochester, N.Y.) in h o t water, is percolated t h r o u g h a c o l u m n of W o e l m acid a l u m i n u m oxide (activity g r a d e I) a n d allowed to crystallize a t r o o m t e m p e r a t u r e . T h e w h i t e r e c t a n g u l a r c r y s t a l s (C4H4N~SO 2. I ~ H z O ) are w a s h e d w i t h cold w a t e r a n d air-dried. * * * O b t a i n e d f r o m California F o u n d a t i o n for Biochemical R e s e a r c h , L o s Angeles, Calif.