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Interference by sucrose in the estimation of ribonucleic acid by the orcinol method
VOL. 2 7 (1958)
SHORT COMMUNICATIONS
2OI
I wish to t h a n k Prof. H. A. KREBS, F.R.S., for his interest and for hospitality in his laboratory, a n d Dr. H. L. KORNB~RG for his e n c o u r a g e m e n t a n d helpful suggestions. This w o r k was s u p p o r t e d b y the Rockefeller F o u n d a t i o n .
Department of Biochemistry, University o/Ox/ord (England)
N . B . MADSEN*
1 E. O. V~rEINMAN, E. H. STRISOWER AND I. L. CHAIKOFF, Physiol. Revs., 37 U957) 252. 2 j , NEEDHAM, Chemical Embryology, Vol. II, P a r t h i , Cambridge Univ. Press, (I93I), p. lO82. a H. L. KORNBERG AND H. A. KREBS, Nature, 179 (1957) 988. 4 H. L. KORNBERO AND N. B. MADSEN, Biochim. Biophys. Acla, 24 (1957) 651. 5 H. L. KORNBERG AND H. BEEVERS, Nature, 18o (1957) 35. 6 H. L. KORNBERG AND H. BEEVERS, Biochim. Biophys. Acta, 26 (1957) 531. 7 H. J. SAZ AND E. P. HILLARY, Biochem. J. 62 (1956) 563. s D. T. O. WONG AND S. J. AJL, ]. Am. Chem. Soc., 78 (1956) 323 o. 9 E. V. McCOLLUM AND N. SIMMONDS, J. Biol. Chem., 33 (1918) 55. 10 A. L. LEHNINGER AND E. P. KENNEDY, J. Biol. Chem., 173 (1948) 753. Received October i o t h , 1957 * Post-doctoral Fellow, National A c a d e m y of Sciences, National Research Council, U.S.A. Present address: Bacteriology Division, Science Service Building, O t t a w a , Canada.
Interference by sucrose in the estimation of ribonucleic acid by the orcinol method A s t a n d a r d procedure for the identification of ribonucleic acid (RNA) in tissue e x t r a c t s is the orcinol m e t h o d 1,2 which has been a d a p t e d b y n u m e r o u s w o r k e r s for the q u a n t i t a t i v e estimation of R N A (see DISCHED). I t has been pointed out on several occasions t h a t sugars o t h e r t h a n ribose interfere in the reaction. The orcinol m e t h o d is, in fact, quite unspecific for ribose, producing a green colour w i t h o t h e r pentoses, and v a r y i n g shades of g r e e n - b r o w n w i t h hexoses 4 and disaccharides 5. The a b s o r p t i o n spectra of several sugars in the orcinol reaction h a v e recently been published s. Of t h e m a n y media tried and being used in biochemical studies on sub-cellular fractions, a q u e o u s sucrose (usually o.25M) is b y far the c o m m o n e s t ; v e r y often R N A e s t i m a t i o n s are p e r f o r m e d on tissue e x t r a c t s p r e p a r e d f r o m sucrose suspensions. As traces of o . 2 5 M sucrose p r o d u c e a h e a v y b r o w n precipitate u n d e r the conditions of the orcinol method, it is obviously of p r i m e i m p o r t a n c e t h a t sucrose should be entirely a b s e n t f r o m the final e x t r a c t containing RNA. Vv'ith the SCHNEIDER7 procedure for e x t r a c t i n g RNA, this means, in effect, t h a t the nucleic acids a n d p r o t e i n s precipitated b y cold trichloracetic acid (TCA) m u s t be washed free of sucrose b e f o r e t h e nucleic acids are extracted b y hot TCA at 9 o°. However, it is possible, particularly if the alcohol-ether extractions are omitted, for sucrose to be left behind with the nucleoprotein precipitate a n d to be s u b s e q u e n t l y t a k e n up into the nucleic acid extract. These traces of sucrose seriously affect the extinction reading at 67o m/~ and the investigator would p r o b a b l y be u n a w a r e of it except in cases of e x t r e m e c o n t a m i n a t i o n . This c o m m u n i c a t i o n describes a routine m e t h o d for checking sucrose c o n t a m i n a t i o n , and also h o w to a p p l y a correction if it occurs. I n all determinations, readings were obtained using a U n i c a m SP-5oo s p e c t r o p h o t o m e t e r , and cells of 1 cm p a t h length. The v o l u m e s of solutions used were as follows: 3 ml i % FeCl 3 in conc. HC1 ; (2 + x) ml w a t e r ; o. 3 ml orcinol (io % in absolute ethanol) ; (t - - x ) ml of t h e test c a r b o h y d r a t e solution. The m i x t u r e s were heated for 45 miD in a boiling w a t e r - b a t h . 1 ml absolute e t h a n o l was added after boiling to t u b e s containing sucrose. U n d e r these conditions, sucrose gives m a x i m u m a b s o r p t i o n at 43 ° and 530 m # ; there is also a small p e a k at 660 m/~. R N A solutions, on the o t h e r hand, follow the a b s o r p t i o n s p e c t r a of p e n t o s e s very closely and give m a x i m a at 42o a n d 67o m # w i t h a m i n i m u m at 52o m/~e. I t was f o u n d t h a t for b o t h sucrose and R N A solutions the extinctions at 52o and 670 m/~ follow Beer's Law. This m e a n s t h a t the ratio of E670/E520 is c o n s t a n t for b o t h s u g a r s over the wide range of c o n c e n t r a t i o n s studied. F o r sucrose this ratio was found to be o.321 whereas for R N A solutions it w a s 4.1o. Thus, in the d e t e r m i n a t i o n of R N A b y the orcinol procedure, routine m e a s u r e m e n t of the ratio of E670/Es~ 0 eliminates the u n c e r t a i n t y as to w h e t h e r sucrose contamin a t i o n is occurring. A similar procedure h a s been applied b y BROWN4 for eliminating hexose interference in p e n t o s e estimations. Using m i x t u r e s of R N A and sucrose of k n o w n concentration, it w a s found t h a t strict a d d i t i v i t y of the extinctions either at 52o m/~ or at 67o m p could n o t be obtained. A slight deviation from additivity in m i x t u r e s a p p e a r s to be the general rule (c[. s). At 52o m/~ the deviation
202
SHORT COMMUNICATIONS
VOL. 27 (I958)
f r om a d d i t i v i t y w a s + 8 % a n d a t 670 m # + 13 %. I n o t h e r words, s u c r o s e - R N A m i x t u r e s gi ve r e a d i n g s a t 670 m/z on t h e a v e r a g e 13 % h i g h e r t h a n w o u l d be e x p e c t e d f r o m t h e i n d i v i d u a l e x t i n c t i o n values. H o w e v e r , to t h e first a p p r o x i m a t i o n , one c a n n e g l e c t t h e s e d e v i a t i o n s w h e n a p p l y i n g a c o r r e c t i o n for t h e i n t e r f e r e n c e of sucrose in t h e orcinol r e a c t i o n . R e g r e s s i o n lines w e r e c a l c u l a t e d for t h e d e p e n d e n c e of t h e e x t i n c t i o n v a l u e s a t 520 m p a n d 670 m # on b o t h su crose a n d R N A c o n c e n t r a t i o n s . Thus, e q u a t i o n s of t h e following form were obtained : E a = K~x • Cx w h e r e E x = e x t i n c t i o n a t w a v e - l e n g t h ~ m/z for a I cm p a t h - l e n g t h ; K x~ = c o n s t a n t for c o m p o n e n t x a t w a v e - l e n g t h ~ m # ; C x = c o n c e n t r a t i o n of c o m p o n e n t x i n / ~ g a d d e d to a final v o l u m e of 6. 3 ml. T h e v a l u e s of K~ f o u n d a t t h e t w o w a v e - l e n g t h s for sucrose a n d for R N A w e r e :
Sucrose RNA
67o m#
52o m p
0.586" lO -3 4.15o. l O - 3
1.824 • lO -3 1.o12" IO-3
Therefore, a s s u m i n g a s t r i c t a d d i t i v i t y of e x t i n c t i o n s a t b o t h 520 m # a n d a t 670 m # t h e following e q u a t i o n c a n be o b t a i n e d c o r r e c t i n g t h e e x t i n c t i o n a t 67o m # for t h e p r e s e n c e of sucrose in RNA estimations : C o n c e n t r a t i o n of R N A = 3'11" EeT° - - ES~° × IOs # g i n final v o l u m e of 6. 3 ml. 11.92 T h e efficiency of t h i s c o r r e c t i o n is i l l u s t r a t e d b y t h e T a b l e I w h e r e t h e e x t i n c t i o n s a t 520 m # a n d 670 m / , of s u c r o s e - R N A m i x t u r e s of k n o w n c o n c e n t r a t i o n s a re shown. The sucrose c o r r e c t i o n r e d u c e s t h e error in t h e d e t e r m i n a t i o n s f r o m b e t w e e n 3o-7o % t o u n d e r 5 %. I n c o m m o n w i t h t h e w o r k of BROWN 4 on hexoses, it is s u g g e s t e d t h a t r o u t i n e m e a s u r e m e n t of R I q A colours a t 52o m/z as well as a t 670 m/z w i l l r e m o v e t h e u n c e r t a i n t y of i n t e r f e r e n c e b y sucrose an d, p r o v i d i n g t h a t t h e i n t e r f e r e n c e is n o t gross, wil l also c o r r e c t for it. TABLE I INFLUENCE
OF ADDED
SUCROSE
RNA added (ttg)
Sucrose added (l~g)
E eTo
65.5 65.5 65.5 131.o 131 .o 131.o
97.6 195 .2 292.8 39o.4 488.0 585.6
o.342 o.4oo 0.453 o.818 0.865 0.928
ON THE
DETERMINATION
E ~~o
o.272 o.493 o.681 0.963 1.14o 1.33 °
RNA ]ound ( uncorr.) (#g) 83.o 96.o lO8 .o 196.o 2o6.o 220.0
OF
RNA
BY
THE
ORCINOL
METHOD
error (%)
RNA round (corrected) lag
error (%)
28 48 65 5° 57 68
66. 5 63.I 62 .o 132.6 I3°.° 131.o
i -5 3.6 5.3 1.2 0.7 o.o
I a m g r a t e f u l to t h e B e l t M e m o r i a l T r u s t e e s for t h e a w a r d of a J u n i o r F e l l o w s h i p , a n d t o Dr. A, L. GREENBAUM for h e l p f u l c o m m e n t s . T. ~'. SLAT~R D e p a r t m e n t o/ B i o c h e m i s t r y , U n i v e r s i t y College, L o n d o n ( E n g l a n d ) 1 M. BIAL, Deut. med. Wochschr., 28 (19o2) 253. 2 W. MEJBAUM, Z . P h y s i o l . Chem., H o p p e - S e y l e r ' s , 258 (1939) 117. a Z. DlSCHE, in E. Chargaff a n d J. N. D a v i d s o n , T h e N u c l e i c A c i d s , A c a d e m i c Press, Inc., N e w Y o r k , 1955. 4 A. H. BROWN, A r c h . B i o c h e m . B i o p h y s . , I i (1946) 269. 5 M. SORENSEN AND G. HAUGAARD, B i o c h e m . Z., 260 (1933) 247. 8 T. F. SLATER, A n a l y s t , (I957), in t h e press. 7 W. C. SCHNEIDER, J . B i o l . Chem., 161 (1945) 293. 8 H. W. KNUDSON, V. W. MELOCHE AND C. JUDAY, I n d . E n g . Chem., A n a l . E d . , 12 (194 o) 715 . R e c e i v e d O c t o b e r 26th, 1957
SHORT COMMUNICATIONS
2OI
I wish to t h a n k Prof. H. A. KREBS, F.R.S., for his interest and for hospitality in his laboratory, a n d Dr. H. L. KORNB~RG for his e n c o u r a g e m e n t a n d helpful suggestions. This w o r k was s u p p o r t e d b y the Rockefeller F o u n d a t i o n .
Department of Biochemistry, University o/Ox/ord (England)
N . B . MADSEN*
1 E. O. V~rEINMAN, E. H. STRISOWER AND I. L. CHAIKOFF, Physiol. Revs., 37 U957) 252. 2 j , NEEDHAM, Chemical Embryology, Vol. II, P a r t h i , Cambridge Univ. Press, (I93I), p. lO82. a H. L. KORNBERG AND H. A. KREBS, Nature, 179 (1957) 988. 4 H. L. KORNBERO AND N. B. MADSEN, Biochim. Biophys. Acla, 24 (1957) 651. 5 H. L. KORNBERG AND H. BEEVERS, Nature, 18o (1957) 35. 6 H. L. KORNBERG AND H. BEEVERS, Biochim. Biophys. Acta, 26 (1957) 531. 7 H. J. SAZ AND E. P. HILLARY, Biochem. J. 62 (1956) 563. s D. T. O. WONG AND S. J. AJL, ]. Am. Chem. Soc., 78 (1956) 323 o. 9 E. V. McCOLLUM AND N. SIMMONDS, J. Biol. Chem., 33 (1918) 55. 10 A. L. LEHNINGER AND E. P. KENNEDY, J. Biol. Chem., 173 (1948) 753. Received October i o t h , 1957 * Post-doctoral Fellow, National A c a d e m y of Sciences, National Research Council, U.S.A. Present address: Bacteriology Division, Science Service Building, O t t a w a , Canada.
Interference by sucrose in the estimation of ribonucleic acid by the orcinol method A s t a n d a r d procedure for the identification of ribonucleic acid (RNA) in tissue e x t r a c t s is the orcinol m e t h o d 1,2 which has been a d a p t e d b y n u m e r o u s w o r k e r s for the q u a n t i t a t i v e estimation of R N A (see DISCHED). I t has been pointed out on several occasions t h a t sugars o t h e r t h a n ribose interfere in the reaction. The orcinol m e t h o d is, in fact, quite unspecific for ribose, producing a green colour w i t h o t h e r pentoses, and v a r y i n g shades of g r e e n - b r o w n w i t h hexoses 4 and disaccharides 5. The a b s o r p t i o n spectra of several sugars in the orcinol reaction h a v e recently been published s. Of t h e m a n y media tried and being used in biochemical studies on sub-cellular fractions, a q u e o u s sucrose (usually o.25M) is b y far the c o m m o n e s t ; v e r y often R N A e s t i m a t i o n s are p e r f o r m e d on tissue e x t r a c t s p r e p a r e d f r o m sucrose suspensions. As traces of o . 2 5 M sucrose p r o d u c e a h e a v y b r o w n precipitate u n d e r the conditions of the orcinol method, it is obviously of p r i m e i m p o r t a n c e t h a t sucrose should be entirely a b s e n t f r o m the final e x t r a c t containing RNA. Vv'ith the SCHNEIDER7 procedure for e x t r a c t i n g RNA, this means, in effect, t h a t the nucleic acids a n d p r o t e i n s precipitated b y cold trichloracetic acid (TCA) m u s t be washed free of sucrose b e f o r e t h e nucleic acids are extracted b y hot TCA at 9 o°. However, it is possible, particularly if the alcohol-ether extractions are omitted, for sucrose to be left behind with the nucleoprotein precipitate a n d to be s u b s e q u e n t l y t a k e n up into the nucleic acid extract. These traces of sucrose seriously affect the extinction reading at 67o m/~ and the investigator would p r o b a b l y be u n a w a r e of it except in cases of e x t r e m e c o n t a m i n a t i o n . This c o m m u n i c a t i o n describes a routine m e t h o d for checking sucrose c o n t a m i n a t i o n , and also h o w to a p p l y a correction if it occurs. I n all determinations, readings were obtained using a U n i c a m SP-5oo s p e c t r o p h o t o m e t e r , and cells of 1 cm p a t h length. The v o l u m e s of solutions used were as follows: 3 ml i % FeCl 3 in conc. HC1 ; (2 + x) ml w a t e r ; o. 3 ml orcinol (io % in absolute ethanol) ; (t - - x ) ml of t h e test c a r b o h y d r a t e solution. The m i x t u r e s were heated for 45 miD in a boiling w a t e r - b a t h . 1 ml absolute e t h a n o l was added after boiling to t u b e s containing sucrose. U n d e r these conditions, sucrose gives m a x i m u m a b s o r p t i o n at 43 ° and 530 m # ; there is also a small p e a k at 660 m/~. R N A solutions, on the o t h e r hand, follow the a b s o r p t i o n s p e c t r a of p e n t o s e s very closely and give m a x i m a at 42o a n d 67o m # w i t h a m i n i m u m at 52o m/~e. I t was f o u n d t h a t for b o t h sucrose and R N A solutions the extinctions at 52o and 670 m/~ follow Beer's Law. This m e a n s t h a t the ratio of E670/E520 is c o n s t a n t for b o t h s u g a r s over the wide range of c o n c e n t r a t i o n s studied. F o r sucrose this ratio was found to be o.321 whereas for R N A solutions it w a s 4.1o. Thus, in the d e t e r m i n a t i o n of R N A b y the orcinol procedure, routine m e a s u r e m e n t of the ratio of E670/Es~ 0 eliminates the u n c e r t a i n t y as to w h e t h e r sucrose contamin a t i o n is occurring. A similar procedure h a s been applied b y BROWN4 for eliminating hexose interference in p e n t o s e estimations. Using m i x t u r e s of R N A and sucrose of k n o w n concentration, it w a s found t h a t strict a d d i t i v i t y of the extinctions either at 52o m/~ or at 67o m p could n o t be obtained. A slight deviation from additivity in m i x t u r e s a p p e a r s to be the general rule (c[. s). At 52o m/~ the deviation
202
SHORT COMMUNICATIONS
VOL. 27 (I958)
f r om a d d i t i v i t y w a s + 8 % a n d a t 670 m # + 13 %. I n o t h e r words, s u c r o s e - R N A m i x t u r e s gi ve r e a d i n g s a t 670 m/z on t h e a v e r a g e 13 % h i g h e r t h a n w o u l d be e x p e c t e d f r o m t h e i n d i v i d u a l e x t i n c t i o n values. H o w e v e r , to t h e first a p p r o x i m a t i o n , one c a n n e g l e c t t h e s e d e v i a t i o n s w h e n a p p l y i n g a c o r r e c t i o n for t h e i n t e r f e r e n c e of sucrose in t h e orcinol r e a c t i o n . R e g r e s s i o n lines w e r e c a l c u l a t e d for t h e d e p e n d e n c e of t h e e x t i n c t i o n v a l u e s a t 520 m p a n d 670 m # on b o t h su crose a n d R N A c o n c e n t r a t i o n s . Thus, e q u a t i o n s of t h e following form were obtained : E a = K~x • Cx w h e r e E x = e x t i n c t i o n a t w a v e - l e n g t h ~ m/z for a I cm p a t h - l e n g t h ; K x~ = c o n s t a n t for c o m p o n e n t x a t w a v e - l e n g t h ~ m # ; C x = c o n c e n t r a t i o n of c o m p o n e n t x i n / ~ g a d d e d to a final v o l u m e of 6. 3 ml. T h e v a l u e s of K~ f o u n d a t t h e t w o w a v e - l e n g t h s for sucrose a n d for R N A w e r e :
Sucrose RNA
67o m#
52o m p
0.586" lO -3 4.15o. l O - 3
1.824 • lO -3 1.o12" IO-3
Therefore, a s s u m i n g a s t r i c t a d d i t i v i t y of e x t i n c t i o n s a t b o t h 520 m # a n d a t 670 m # t h e following e q u a t i o n c a n be o b t a i n e d c o r r e c t i n g t h e e x t i n c t i o n a t 67o m # for t h e p r e s e n c e of sucrose in RNA estimations : C o n c e n t r a t i o n of R N A = 3'11" EeT° - - ES~° × IOs # g i n final v o l u m e of 6. 3 ml. 11.92 T h e efficiency of t h i s c o r r e c t i o n is i l l u s t r a t e d b y t h e T a b l e I w h e r e t h e e x t i n c t i o n s a t 520 m # a n d 670 m / , of s u c r o s e - R N A m i x t u r e s of k n o w n c o n c e n t r a t i o n s a re shown. The sucrose c o r r e c t i o n r e d u c e s t h e error in t h e d e t e r m i n a t i o n s f r o m b e t w e e n 3o-7o % t o u n d e r 5 %. I n c o m m o n w i t h t h e w o r k of BROWN 4 on hexoses, it is s u g g e s t e d t h a t r o u t i n e m e a s u r e m e n t of R I q A colours a t 52o m/z as well as a t 670 m/z w i l l r e m o v e t h e u n c e r t a i n t y of i n t e r f e r e n c e b y sucrose an d, p r o v i d i n g t h a t t h e i n t e r f e r e n c e is n o t gross, wil l also c o r r e c t for it. TABLE I INFLUENCE
OF ADDED
SUCROSE
RNA added (ttg)
Sucrose added (l~g)
E eTo
65.5 65.5 65.5 131.o 131 .o 131.o
97.6 195 .2 292.8 39o.4 488.0 585.6
o.342 o.4oo 0.453 o.818 0.865 0.928
ON THE
DETERMINATION
E ~~o
o.272 o.493 o.681 0.963 1.14o 1.33 °
RNA ]ound ( uncorr.) (#g) 83.o 96.o lO8 .o 196.o 2o6.o 220.0
OF
RNA
BY
THE
ORCINOL
METHOD
error (%)
RNA round (corrected) lag
error (%)
28 48 65 5° 57 68
66. 5 63.I 62 .o 132.6 I3°.° 131.o
i -5 3.6 5.3 1.2 0.7 o.o
I a m g r a t e f u l to t h e B e l t M e m o r i a l T r u s t e e s for t h e a w a r d of a J u n i o r F e l l o w s h i p , a n d t o Dr. A, L. GREENBAUM for h e l p f u l c o m m e n t s . T. ~'. SLAT~R D e p a r t m e n t o/ B i o c h e m i s t r y , U n i v e r s i t y College, L o n d o n ( E n g l a n d ) 1 M. BIAL, Deut. med. Wochschr., 28 (19o2) 253. 2 W. MEJBAUM, Z . P h y s i o l . Chem., H o p p e - S e y l e r ' s , 258 (1939) 117. a Z. DlSCHE, in E. Chargaff a n d J. N. D a v i d s o n , T h e N u c l e i c A c i d s , A c a d e m i c Press, Inc., N e w Y o r k , 1955. 4 A. H. BROWN, A r c h . B i o c h e m . B i o p h y s . , I i (1946) 269. 5 M. SORENSEN AND G. HAUGAARD, B i o c h e m . Z., 260 (1933) 247. 8 T. F. SLATER, A n a l y s t , (I957), in t h e press. 7 W. C. SCHNEIDER, J . B i o l . Chem., 161 (1945) 293. 8 H. W. KNUDSON, V. W. MELOCHE AND C. JUDAY, I n d . E n g . Chem., A n a l . E d . , 12 (194 o) 715 . R e c e i v e d O c t o b e r 26th, 1957