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Anomalous rotary dispersion of acridine orange-native deoxyribonucleic acid complexes
PRELIMINARY
397
NOTES
Anomalous rotatory dispersion of acridine orange-native deoxyribonucleic acid complexes BLOUT AND STRYER1,2 have shown that a symmetric dye, acriflavine, exhibits optical activity when bound to an asymmetric helical polymer, poly (=,L-glutamic acid). Bound to polyglutamate in its random-coil form the dye shows no optical activity. Optical activity has now been observed (Fig. I) with acridine orange bound to native, helical DNA. Bound to heat-denatured, coiled DNA the dye shows no apparent optical activity (Fig. I). The magnitude of the rotation depends upon the ratio of bound acridine orange (D) to native DNA nucleotides (P) in the complex. When D/P 1/56 there is no measurable rotation ([=]i (acridine orange) < IOO°). At higher ratios a Cotton effect (l~ig. I) appears with an inflection at 504 m/~ and maximum and minimum at 515 and 49 ° m/~, respectively. The magnitude of this Cotton effect increases until D/P is abou ~0.25. At still higher ratios this first Cotton effect is gradually replaced by a second Cotto i effect with an inflection at 465 m/, a n d m a x i m u m at 450 m/~ and a minimum at 480 m/~ The magnitude of this second Cotton effect increases until D/P ---- I, corresponding to m a x i m u m dye binding. The variation of rotation with D/P provides information about the origin of tile a s y m m e t r y induced in the dye. Direct coupling of the asymmetric field of the polymer with the chromophore is not: in itself sufficient to account for this variation. There is considerable evidence based on spectral shifts that electronic coupling between nearby bound dyes can occur in the acridine orange-DNA system 3 5. If light interacts with J I I I I
'
L '
I I
'
~.
J I r I rl~-r-'
I I i If]
'
'
+500
19 " ' ~ o
-500
/
=
: P/D:/
-IOOO
P/D:§6
P/D:1.5
~--~
i i i i i i
ii,
,
,
i
i
~
:
¸
T--r--
P k
+5ooi
DENATURED
-500
-tooo
350
,
400
450
50o
550
I
600 X m/z
.... ,,,,,
400 450 500 Wavelength (m~)
550
600
F i g . i. A n o m a l o u s r o t a t o r y d i s p e r s i o n of a c r i d i n e o r a n g e - D N A complexes. Optical rotation d e t e r m i n e d w i t h a R u d o l p h M o d e l 80 S p e c t r o p o l a r i m e t e r u s i n g 1 - c m c o r e x a n d 2 . 5 - c m a n n e a l e d , s t r a i n - f r e e q u a r t z c e l l s a n d a x e n o n - a r c l i g h t s o u r c e . S l i t r e a d i n g s ~ 0. 3 r a m . P r o b a b l e e r r o r : i n r e a d i n g s = .'- o . o o 2 °, i n m o l a r r o t a t i o n s = ± 5 o ° . M o l a r r o t a t i o n is r o t a t i o n in d e g r e e s f o r I-cm path of I M DNA-bound acridine orange. Molar rotations are corrected for the rotation of D N A a t h i g h D N A / d y e r a t i o s . D N A / d y e r a t i o s ( P / D ) , e s t i m a t e d r a n g e of e r r o r -L o . I , w e r e d e t e r m i n e d b y s p e c t r o p h o t o m e t r i c t i t r a t i o n u s i n g W o r t h i n g t o n B i o c h e m i c a l s Co. h i g h l y p o l y m e r i z e d c a l f - t h y m u s D N A a s d e s c r i b e d p r e v i o u s l y 2, 3 . 2 o 4 ° H M a c r i d i n e o r a n g e i n i m M s o d i u m c a c o d y l a t e b u f f e r ( p H 6.7). D e n a t u r e d D N A w a s h e a t e d a t IOO ° C f o r i o r a i n a n d c o o l e d r a p i d l y . All r e a d i n g s w e r e t a k e n a t 25 ° + I ° C.
Biochim. Biophys. Acta, 5 ° (1961) 3 9 7 - 3 9 0
398
PRELIMINARY NOTES
such coupled dyes as a u n i t a n d the u n i t does not possess a center or plane of s y m m e t r y , the absorption would be accompanied b y a n o m a l o u s r o t a t o r y dispersion. To briefly summarize the spectral evidence: the spectrum of the b o u n d acridine orange changes from the monomeric form (~ma:¢= 504 m/x, 55 ooo) at low D / P to the aggregated or strongly coupled form (~-may= 464 mix, e = 13 5oo) at high D/P. The disappearance of the m o n o m e r e x t i n c t i o n follows an e q u a t i o n of the form e~0¢ = 415oo ( I - - D / P ) ~ -~ 13 500 (Fig. 2) which would be expected if acridine orange distributes r a n d o m l y on the D N A b i n d i n g sites a n d a " m o n o m e r " dye is a dye b o u n d to a site with e m p t y sites as neighbors, i.e., - - 0 - - - where - indicates an e m p t y a n d O a dye-filled site. The second Cotton effect which has an inflection at the aggregated dye absorption m a x i m u m a n d which is m a x i m a l at D / P = I is clearly due to strongly coupled dyes. The origin of the first Cotton effect with an inflection at the " m o n o m e r " dye m a x i m u m a n d which is m a x i m a l at D / P a b o u t o.25 is less obvious. Recent theoretical work °, 7 has established t h a t weak coupling m a y occur between chromophores, such as the nucleotide bases of DNA, resulting in hypochromism with no change in absorption m a x i n m m . There is some justification, therefore, for assuming t h a t a configuration of the type (A) - - O - O - - could have sufficiently weak coupling to exhibit a "monomer" spectrum a n d yet sufficiently strong coupling to cause optical activity. The p r o b a b i l i t y of the r a n d o m occurrence of a dye in such a configuration would be ( I - - D / P ) 3 ( D / P ) a n d a fit of this equation to the rotation d a t a (~1 varies as ( I - - - D / P ) 3 ( D / P ) ) for the first Cotton effect is shown in Fig. 2. The agreement between experiment a n d theory must be, to a certain extent, fortuitous since it is probable t h a t configurations such as (B) O 0--O a n d m a n y others will also c o n t r i b u t e to the observed rotation. In addition it has been shown a 5 t h a t the d i s t r i b u t i o n of acridine orange on DNA is not completely r a n d o m . The fraction of dyes involved in configuration (B) is (I D/P)a(D/P) z (in general (I D/p)m(D/P) r~-I where n n u m b e r of filled and m -~ n u m b e r of e m p t y sites) and e
- -
....
"~
-~,4oo . ~
2
. . .
DNA pO43-/AO RATIO 3 4 5 6
i .......
/
,2oo
__
2oso
~
- ~- 6 o o o o g
"',¢/.
,oo !
a~o
1-1-~-,~7°°°°
f'L" ',
-J ,oooo
0 ~_
0
0.[
0.2
0.3
0.4
0.5
0,6
(,-%)2
0.7
O.B
09
0 I.O
~"
Fig. 2. Variation of optical rotation and absorption with acridine orange (AO)/DNA ratio. Molar rotations at 49o and 51.5 m/t multiplied by - - i , and 1.5 i5 respectively to put them on the same scale. Theoretical equations: monomeric dye (n = i, m -- 2) e -- 41 500 (l-- D/P) ~ + 13 50o; case (A) (~ = 2, m = 3), [O¢]M- I. [O'104 (I--D/P)a(D/P); case (B)(n = 3, m--5), V~¢M= 7.65 't°4 (l D/P)S(D/P) 2.
Biochi~Hl l?iophvs. :[ (t(~, 5o ( ~)0 [) 397 39~)
399
PRELIMINARY NOTES
fit of the equation for case (B) to the experimental data is as good as for case (A). Other choices for n and m lead to considerably poorer fit. To treat this problem exactly it would be necessary to determine the orientation of each dye relative to the DNA, compute by existing methods s the optical rotatory contribution of each configuration, weight according to the probability of occurrence of the particular configuration, and sum over all possible configurations which contribute to rotation. In the absence of such detailed information, by considering only the dependence of the rotation upon D/P we can say that it is quite likely that configurations of the type (A) or (B) are the cause of the first Cotton effect. In this statistical treatment the binding sites are not specified except insofar that they lie in a regular, helical array. They m a y be the phosphate groups a-5 or spaces between nucleotide bases 9, etc. Although the statistical model appears to account satisfactorily for the variation of rotation with D/P, the possibility that changes in polymer conformation, charge density, orientation and/or location of dye on the DNA could also account for this effect cannot at present be excluded.
Section on Physical Chemistry, Laboratory of Neurochemistry, National Institute of Mental Health, Bethesda, Md. (U.S.A.)
D. M. NEVILLE, Jr. D. F. BRADLEY
1 E. R. BLOUT AND g. STRYER, Proc. Natl. Acad. Sci. U.S., 45 (1959) 1591. 2 L. STRYER AND E. R. BLOUT, J . , 4 m . Chem. Soe., 83 (1961) 1411. 3 D. F. BRADLEY AND G. FELSENFELD, Nature, 185 (I959 } 192o. 4 D. F. BRADLEY AND M. K. \VOLF, Proe. Natl. ~4cad. Sei. U.S., 45 (1959) 944. 5 A. L. STONE AND D. F. BRADLEY, .]..4m. Chem. Soc., in t h e press. e I. TtNOCO, J. ,4m. Chem. Soc., 82 (196o) 4785 . 7 W. T. SIMPSON AND D. L. PETERSON, J. Chem. Phys., 26 (1957) 588. s I. TINOCO AND R. \V. WOODY, .[. Chem. Phys., 32 (196o) 461. 9 L. S. LERMAN, J. ~1ol. Biol., 3 (1961) I8.
Received April 27th , 1961 Biochim. Biophys. ,4cla, 50 (1961) 397 399
Dissociation of ribonucleic acid and protein synthesis in bacteria deprived of potassium Mutants of Escherichia coli defective in potassium transport (Trx-) have been described 1, 2. In media with low potassium content strain B-2o7 (ref. I) fails to grow but the corresponding wild type (TrK +) responds with a normal growth rate. A study of the need ol potassium for growth and multiplication requires a simple and effective way of depleting cells of internal potassium. TrK mutants are particularly suitable for this type of study since after several washings they lose much of their internal potassium whereas wild-type cells retain considerable internal potasA b b r e v i a t i o n s for m e d i a : m e d i u m A is t h e m i n i m a l m e d i u m d e s c r i b e d b y DAVIS AND MINGIOLI3 s u p p l e m e n t e d w i t h glucose (o.25 %). AHL M c o n t a i n s in a d d i t i o n L-histidine, L-leucine, a n d L - m e t h i o n i n e r e q u i r e d b y s t r a i n B-2o 7 for g r o w t h . S o d i u m - A c o r r e s p o n d s t o m e d i u m A, e x c e p t for t h e s u b s t i t u t i o n of s o d i u m for p o t a s s i u m p h o s p h a t e s , a n d c o n t a i n s a p p r o x . 2/~g/ml of c o n t a m i n a t i n g t r a c e s of p o t a s s i u m . Sodium-AHLM is s u p p l e m e n t e d w i t h h i s t i d i n e , leucine, a n d m e t h i o nine (each 50 /~g/ml).
Biochim. Biophys. Acta, 50 (1961) 399-4o2
397
NOTES
Anomalous rotatory dispersion of acridine orange-native deoxyribonucleic acid complexes BLOUT AND STRYER1,2 have shown that a symmetric dye, acriflavine, exhibits optical activity when bound to an asymmetric helical polymer, poly (=,L-glutamic acid). Bound to polyglutamate in its random-coil form the dye shows no optical activity. Optical activity has now been observed (Fig. I) with acridine orange bound to native, helical DNA. Bound to heat-denatured, coiled DNA the dye shows no apparent optical activity (Fig. I). The magnitude of the rotation depends upon the ratio of bound acridine orange (D) to native DNA nucleotides (P) in the complex. When D/P 1/56 there is no measurable rotation ([=]i (acridine orange) < IOO°). At higher ratios a Cotton effect (l~ig. I) appears with an inflection at 504 m/~ and maximum and minimum at 515 and 49 ° m/~, respectively. The magnitude of this Cotton effect increases until D/P is abou ~0.25. At still higher ratios this first Cotton effect is gradually replaced by a second Cotto i effect with an inflection at 465 m/, a n d m a x i m u m at 450 m/~ and a minimum at 480 m/~ The magnitude of this second Cotton effect increases until D/P ---- I, corresponding to m a x i m u m dye binding. The variation of rotation with D/P provides information about the origin of tile a s y m m e t r y induced in the dye. Direct coupling of the asymmetric field of the polymer with the chromophore is not: in itself sufficient to account for this variation. There is considerable evidence based on spectral shifts that electronic coupling between nearby bound dyes can occur in the acridine orange-DNA system 3 5. If light interacts with J I I I I
'
L '
I I
'
~.
J I r I rl~-r-'
I I i If]
'
'
+500
19 " ' ~ o
-500
/
=
: P/D:/
-IOOO
P/D:§6
P/D:1.5
~--~
i i i i i i
ii,
,
,
i
i
~
:
¸
T--r--
P k
+5ooi
DENATURED
-500
-tooo
350
,
400
450
50o
550
I
600 X m/z
.... ,,,,,
400 450 500 Wavelength (m~)
550
600
F i g . i. A n o m a l o u s r o t a t o r y d i s p e r s i o n of a c r i d i n e o r a n g e - D N A complexes. Optical rotation d e t e r m i n e d w i t h a R u d o l p h M o d e l 80 S p e c t r o p o l a r i m e t e r u s i n g 1 - c m c o r e x a n d 2 . 5 - c m a n n e a l e d , s t r a i n - f r e e q u a r t z c e l l s a n d a x e n o n - a r c l i g h t s o u r c e . S l i t r e a d i n g s ~ 0. 3 r a m . P r o b a b l e e r r o r : i n r e a d i n g s = .'- o . o o 2 °, i n m o l a r r o t a t i o n s = ± 5 o ° . M o l a r r o t a t i o n is r o t a t i o n in d e g r e e s f o r I-cm path of I M DNA-bound acridine orange. Molar rotations are corrected for the rotation of D N A a t h i g h D N A / d y e r a t i o s . D N A / d y e r a t i o s ( P / D ) , e s t i m a t e d r a n g e of e r r o r -L o . I , w e r e d e t e r m i n e d b y s p e c t r o p h o t o m e t r i c t i t r a t i o n u s i n g W o r t h i n g t o n B i o c h e m i c a l s Co. h i g h l y p o l y m e r i z e d c a l f - t h y m u s D N A a s d e s c r i b e d p r e v i o u s l y 2, 3 . 2 o 4 ° H M a c r i d i n e o r a n g e i n i m M s o d i u m c a c o d y l a t e b u f f e r ( p H 6.7). D e n a t u r e d D N A w a s h e a t e d a t IOO ° C f o r i o r a i n a n d c o o l e d r a p i d l y . All r e a d i n g s w e r e t a k e n a t 25 ° + I ° C.
Biochim. Biophys. Acta, 5 ° (1961) 3 9 7 - 3 9 0
398
PRELIMINARY NOTES
such coupled dyes as a u n i t a n d the u n i t does not possess a center or plane of s y m m e t r y , the absorption would be accompanied b y a n o m a l o u s r o t a t o r y dispersion. To briefly summarize the spectral evidence: the spectrum of the b o u n d acridine orange changes from the monomeric form (~ma:¢= 504 m/x, 55 ooo) at low D / P to the aggregated or strongly coupled form (~-may= 464 mix, e = 13 5oo) at high D/P. The disappearance of the m o n o m e r e x t i n c t i o n follows an e q u a t i o n of the form e~0¢ = 415oo ( I - - D / P ) ~ -~ 13 500 (Fig. 2) which would be expected if acridine orange distributes r a n d o m l y on the D N A b i n d i n g sites a n d a " m o n o m e r " dye is a dye b o u n d to a site with e m p t y sites as neighbors, i.e., - - 0 - - - where - indicates an e m p t y a n d O a dye-filled site. The second Cotton effect which has an inflection at the aggregated dye absorption m a x i m u m a n d which is m a x i m a l at D / P = I is clearly due to strongly coupled dyes. The origin of the first Cotton effect with an inflection at the " m o n o m e r " dye m a x i m u m a n d which is m a x i m a l at D / P a b o u t o.25 is less obvious. Recent theoretical work °, 7 has established t h a t weak coupling m a y occur between chromophores, such as the nucleotide bases of DNA, resulting in hypochromism with no change in absorption m a x i n m m . There is some justification, therefore, for assuming t h a t a configuration of the type (A) - - O - O - - could have sufficiently weak coupling to exhibit a "monomer" spectrum a n d yet sufficiently strong coupling to cause optical activity. The p r o b a b i l i t y of the r a n d o m occurrence of a dye in such a configuration would be ( I - - D / P ) 3 ( D / P ) a n d a fit of this equation to the rotation d a t a (~1 varies as ( I - - - D / P ) 3 ( D / P ) ) for the first Cotton effect is shown in Fig. 2. The agreement between experiment a n d theory must be, to a certain extent, fortuitous since it is probable t h a t configurations such as (B) O 0--O a n d m a n y others will also c o n t r i b u t e to the observed rotation. In addition it has been shown a 5 t h a t the d i s t r i b u t i o n of acridine orange on DNA is not completely r a n d o m . The fraction of dyes involved in configuration (B) is (I D/P)a(D/P) z (in general (I D/p)m(D/P) r~-I where n n u m b e r of filled and m -~ n u m b e r of e m p t y sites) and e
- -
....
"~
-~,4oo . ~
2
. . .
DNA pO43-/AO RATIO 3 4 5 6
i .......
/
,2oo
__
2oso
~
- ~- 6 o o o o g
"',¢/.
,oo !
a~o
1-1-~-,~7°°°°
f'L" ',
-J ,oooo
0 ~_
0
0.[
0.2
0.3
0.4
0.5
0,6
(,-%)2
0.7
O.B
09
0 I.O
~"
Fig. 2. Variation of optical rotation and absorption with acridine orange (AO)/DNA ratio. Molar rotations at 49o and 51.5 m/t multiplied by - - i , and 1.5 i5 respectively to put them on the same scale. Theoretical equations: monomeric dye (n = i, m -- 2) e -- 41 500 (l-- D/P) ~ + 13 50o; case (A) (~ = 2, m = 3), [O¢]M- I. [O'104 (I--D/P)a(D/P); case (B)(n = 3, m--5), V~¢M= 7.65 't°4 (l D/P)S(D/P) 2.
Biochi~Hl l?iophvs. :[ (t(~, 5o ( ~)0 [) 397 39~)
399
PRELIMINARY NOTES
fit of the equation for case (B) to the experimental data is as good as for case (A). Other choices for n and m lead to considerably poorer fit. To treat this problem exactly it would be necessary to determine the orientation of each dye relative to the DNA, compute by existing methods s the optical rotatory contribution of each configuration, weight according to the probability of occurrence of the particular configuration, and sum over all possible configurations which contribute to rotation. In the absence of such detailed information, by considering only the dependence of the rotation upon D/P we can say that it is quite likely that configurations of the type (A) or (B) are the cause of the first Cotton effect. In this statistical treatment the binding sites are not specified except insofar that they lie in a regular, helical array. They m a y be the phosphate groups a-5 or spaces between nucleotide bases 9, etc. Although the statistical model appears to account satisfactorily for the variation of rotation with D/P, the possibility that changes in polymer conformation, charge density, orientation and/or location of dye on the DNA could also account for this effect cannot at present be excluded.
Section on Physical Chemistry, Laboratory of Neurochemistry, National Institute of Mental Health, Bethesda, Md. (U.S.A.)
D. M. NEVILLE, Jr. D. F. BRADLEY
1 E. R. BLOUT AND g. STRYER, Proc. Natl. Acad. Sci. U.S., 45 (1959) 1591. 2 L. STRYER AND E. R. BLOUT, J . , 4 m . Chem. Soe., 83 (1961) 1411. 3 D. F. BRADLEY AND G. FELSENFELD, Nature, 185 (I959 } 192o. 4 D. F. BRADLEY AND M. K. \VOLF, Proe. Natl. ~4cad. Sei. U.S., 45 (1959) 944. 5 A. L. STONE AND D. F. BRADLEY, .]..4m. Chem. Soc., in t h e press. e I. TtNOCO, J. ,4m. Chem. Soc., 82 (196o) 4785 . 7 W. T. SIMPSON AND D. L. PETERSON, J. Chem. Phys., 26 (1957) 588. s I. TINOCO AND R. \V. WOODY, .[. Chem. Phys., 32 (196o) 461. 9 L. S. LERMAN, J. ~1ol. Biol., 3 (1961) I8.
Received April 27th , 1961 Biochim. Biophys. ,4cla, 50 (1961) 397 399
Dissociation of ribonucleic acid and protein synthesis in bacteria deprived of potassium Mutants of Escherichia coli defective in potassium transport (Trx-) have been described 1, 2. In media with low potassium content strain B-2o7 (ref. I) fails to grow but the corresponding wild type (TrK +) responds with a normal growth rate. A study of the need ol potassium for growth and multiplication requires a simple and effective way of depleting cells of internal potassium. TrK mutants are particularly suitable for this type of study since after several washings they lose much of their internal potassium whereas wild-type cells retain considerable internal potasA b b r e v i a t i o n s for m e d i a : m e d i u m A is t h e m i n i m a l m e d i u m d e s c r i b e d b y DAVIS AND MINGIOLI3 s u p p l e m e n t e d w i t h glucose (o.25 %). AHL M c o n t a i n s in a d d i t i o n L-histidine, L-leucine, a n d L - m e t h i o n i n e r e q u i r e d b y s t r a i n B-2o 7 for g r o w t h . S o d i u m - A c o r r e s p o n d s t o m e d i u m A, e x c e p t for t h e s u b s t i t u t i o n of s o d i u m for p o t a s s i u m p h o s p h a t e s , a n d c o n t a i n s a p p r o x . 2/~g/ml of c o n t a m i n a t i n g t r a c e s of p o t a s s i u m . Sodium-AHLM is s u p p l e m e n t e d w i t h h i s t i d i n e , leucine, a n d m e t h i o nine (each 50 /~g/ml).
Biochim. Biophys. Acta, 50 (1961) 399-4o2