DANIEL W. MILESAND DANW. URRY
4448
4.15 A distant from the nucleus in question make a minor contribution (say about 15%) to the field a s p metry. So far we have ignored the effect of dipole field due to water molecules. If two water molecules complete a distorted coordination octahedron of a thallium atom with four iodine atoms of a square con-
figuration, the inclusion of the effect of the dipole field merely amounts t o the partial compensation of the field asymmetry due to the two iodine atoms in the complex anion. Therefore, the effect is not important at least for the estimation of the order of magnitude of the asymmetry parameter.
Reciprocal Relations in the Circular Dichroism of Adenosine Mononicotinate
by Daniel W. Miles and Dan W. Urry Institute for Biomedical Research, Education and Research Foundation, American Medical Association, Chicago, Illinois 60610 (Received M a y 29, 1967)
Adenosine 5-mononicotinate has been used as a model system with which to demonstrate reciprocal relations in optical rotation. These relations arise from the manner in which the total rotational strength of a molecule sums to zero for the electric-electric- and magnetic-electric-coupled oscillator terms and may be used on an empirical basis for determining the juxtaposition of spectrally isolable chromophoric groups in complex biological systems. I n the adenosine 5’-mononicotinate model system, hypochromism is used to demonstrate that the adenyl and nicotinyl moieties are interacting in an approximate stacked or card-pack conformation in accord with the limitations imposed by the molecular structure. Solvent-eff ect studies indicate that the interaction is primarily hydrophobic in nature. The experimental variables of pH, solvent dielectric, and temperature are used to demonstrate that the interacted state gives rise to complex circular-dichroism bands of large ellipticities in accord with the reciprocal relations. The almost exactly equal but opposite in sign rotational strengths of the resolved bands at 257 and 271 mp are to some degree fortuitous for coupling with higher energy transitions, and static terms are expected to contribute.
Introduction In an idealized case in which a pair of electronic transitions derive their rotational strength, each from coupling with the transition dipole moment of the other, the rotational strength of the first due to interaction with the second, R12,is of the same magnitude but opposite in sign to the rotational strength of the second due to interaction with first, R21, ie., R12 = -Rzl. One never has the ideal case of an isolated pair of interacting transitions; however, should two electronic transitions, each occurring in a different group, have frequencies which are only slightly displaced, then The Journal of Physiccll Chemistry
their interaction may dominate and one approaches the ideal case. This is because the rotational strengths are inversely proportional to the differences in frequency, ( v z z - v l Z ) - - l , of the interacting transitions. As an experimental parameter is varied which brings two different chromophoric groups into juxtaposition, one might expect that close lying transitions, in particular, exhibit a coupling in which the circular dichroism peak due to a transition in the first chromophore becomes more positive while the circular dichroism peak of a close-lying electronic transition in the second chromophore becomes more negative in a reciprocal manner.
RECIPROCITY IN THE CIRCULAR DICHROISM OF ADENOSINE MONONICOTINATE
This coupling has been called “reciprocal relations” in optical rotation.‘ To the extent that the absorption hands overlap, there is an excitational degeneracy with an exciton splitting resulting in the interaction of electronic transitions of slightly different energies, which may give rise to rotatory power. When the two chromophores are identical, the splitting becomes marked and there is, in certain cases, a near conservation of rotational strength over the absorption band.’-‘ Adenosine 5’-mononicotinate (AMN) is an excellent molecule with which to study the interaction of the two chromophoric groups. Attachment of adenine to the 1’carbon and attachment of the nicotinate by ester linkage to the 5’ carhon on the same D-ribose moiety places both bases on the same side of the ribose ring. This structure allows the bases to interact in a stacked manner (Figure 1). As the relative orientations of the adenosyl and nicotinyl moieties can be changed only by rotations about three bonds, the number of allowed conformations is greatly limited when compared to dinucleoside phosphates and dinucleotides. Structurally, therefore, this molecule provides a relatively simple model system for studying the interaction of two bases. As such, adenosine 5‘-mononicotinate presents an opportunity to demonstrate the reciprocal relations, for it contains two nonidentical chromophores with closely spaced electronic transitions. In this communication we wish to demonstrate the reciprocal relations and emphasize the utility of this relationship when attempting to assess the proximity of two spectrally isolable moieties in a complex system such as an enzyme in which the active site or the catalytic mechanism involves the interaction of two groups with closely placed electronic transitions. One such example would be diaphorase, where the reduced nicotinamide moiety interacts with and reduces the flavin group. Should binding of nucleotides and coenzymes to proteins involve aromatic amino acids, this may be determined by the observation of a complex CD band exhibiting reciprocal behavior. Also, of course, the proximity of the two bases in solutions of dinucleotides may be assessed. In the latter case, we have demonstrated the interactions of the bases in flavin adenine dinucle otides and nicotinamide adenine dinucleotides.’.’ Experimental Section Adenosine 5’-mononicotinate was obtained from Sigma Chemical Company, Lot No. 36B-0850; the reported purity was better than 99%. The water used was distilled, deionized, and finally glass distilled. Dioxane of spectral grade was from Matheson Coleman and Bell. Adenosine 5‘-monophosphate and nicotin-
4449
Figure 1.. Molecular model of adenosine 5’-mononicotinate, demonstrating how readily the aromatic groups achieve a stacked conformation.
amide mononucleotide were also obtained from Sigma Chemical Company. Absorption- and circular-dichroism curves were determined on a Cary Model 14 spectrophotometer and on a prototype circular-dichroism attachment built by Cary Instruments for the Cary Model 60 spectropolarimeter, respectively. The CD unit was calibrated using the Cary Model 1401 circular-dichroism attachment for the Model 14. The standard used was an aqueous solution of d-10-camphor sulfonic acid (J. T. Baker, Lot No. 9-361) with an eL - sR of 2.2 a t 290 mp. Determinations of pH were made on a Radiometer pH meter, Model 25SE. Sample temperatures were maintained with a Haake KT-62 kryothermat and were monitored, while spectra were being run, with a YSI Model 42SC telethermometer. The monitoring was achieved by means of a thermocouple inserted through a rubber cap into the cell solution. Curves were resolved into Gaussian functions using the DuPont 310 curve resolver. Cell-path lengths were calibrated using solutions of chromate in 0.05 N KOH. (1) D. W.Urry. Pioe. Nd. A d . Sci. U. S.. M. 640 (1965). (2) M. M. Warshaw, C. A. Bush. and I. Tinoeo, Jr.. B k h e m . B i p (1965). .ohm. _Rea. Cmm... 18.633 . . .
(3) K. E. Van Holde. J. Brahrns, and A. M. Michelaon. J . Mol. Bel.. 12, 726 (1965). (4) C. A. Bush and J. Brahms. J . C h m . Phya., 46,79 (1967). (5) C. A. Buah and I. Tinooo. Jr., J . Mol. Bbl., 23,601 (1967). (6) D. W. Miles and D. W. Urry. in preparation. (7) D. W. Miles. D. W. Urry, and H.Eyring, in preparation.
Volum 71. Number 13 Deeernber 1867
DANIELW. MILESAND DANW. URRY
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8
- AMN ( n o
-_
ADENOSINE M O N O N I C O T I N A T E 2 M NoCl pH effect
salt)
AMN+2N NaCl
6
6
4 4 2 2
0
7
0
3 0
0
-w
2-,
X
S
X I
-2
-4
-4 -6 -6
-8
t
-8
I
1
L
I
220
I
I
240
I
I
260
280
1
I
300.
Figure 2. Circular dichroism curves for adenosine mononicotinate (AMN) without salt () and in the presence of 2 N NaCl (- - - -). The circular dichroism curves of adenosine 5’-monophosphate (AMP) (- . - . ) and nicotinamide mononucleotide (NMN) ( . . . . ) are included to demonstrate the low rotations of nucleotides containing the adenine and nicot,inamide chromophores. All measurements were performed in unbuffered aqueous solutions at neutral pH.
Results and Discussion In aqueous solution at neutral pH, adenosine .5’mononicotinate exhibits circular-dichroism curves which are more complex and of larger magnitude than exhibited by either adenosine 5’-monophosphate (AMP) or nicotinamide mononucleotide (NMN). Nor can the AMN curve be explained as a sum of AMP and NMN8 (see Figure 2). Several experimental parameters may be systematically varied in order to determine the extent to which the complex CD curve of AMN is derived from interactions of the adenine and nicotinate moieties. At neutral pH, both groups are uncharged. On lowering the pH both groups become protonated. Should the two bases be juxtaposed, one would expect a charge repulsion to disperse the bases. As is shown in Figure 3, decreasing the pH results in a marked decrease in the magnitude of the circular-dichroism The Journal of Physical Chemistry
I
I
I
220
240
260
I
I
I
280
I
300
Figure 3. The circular-dichroism curves of adenosine mononicotinate in 2 M NaCl at various pH values. The buffer was 0.1 M phosphate.
bands. The pH effect is reversible. As protonation of adenosine produces relatively small eff ectsjgone expects the two bases to be sufficiently close that a charge repulsion brings about a change in their relative orientation. The presence of such a large pH effect, the hydrophobic nature of the bases, the conformations allowed by the structure of AMN, and the well-known base stacking in nucleic acids suggests that the bases may be held together in a stacked manner by hydrophobic forces.lO If this is the case, decreasing the dielectric of the medium should also lead to a decrease in the complexity of the CD curve. As dioxane has a static (8) The base of NMN contains an amide substituent at position C3 on the pyridine moiety, and the attachment of the nicotinamide moiety is to the 1’ carbon of ribose in NMN rather than by ester linkage to the 5‘ carbon. The curve is included to demonstrate the low rotation, which may be expected to be greater for 1’ attachment than for 5’ attachment. (9) C. Y . Lin, D. W. Urry, and H. Eyring, Bhchem. B w p h y s . Res. Comm., 17, 647 (1964). (10) The use of the term hydrophobic is intended to encompass those forces which cause relatively nonpolar groups to adhere in polar media. I n particular, it is taken to include van der Waals forces (exclusive of hydrogen bonding) as well as those forces arising from solvent-solvent orientation effects.
RECIPROCITY IN THE CIRCULAR DICHROISM OF ADENOSINEMONONICOTINATE
4451
ADENOSINE YONON I C O T I N ATE
7:3 5 3 0:I
4
2
?
E@
x
0
u
-2
-4
-6
I
260
1
1
280
t
1
4
300
X(rnp)
Figure 4. The circular dichroism curves of adenosine mononicotinate in different water-dioxane mixtures.
dielectric const,ant of 2.2 and a molar refractivity of 22 as compared to 85 and 3.7, respectively, for water and as dioxane is miscible in all proportions with water, it provides a suitable system for determining the presence of hydrophobic interactions such as dispersion forces. Figure 4 shows the extent of the solvent effect. Addition of dioxane results in a decrease in magnitude and complexity of the CD bands until, in pure dioxane only, a simple positive curve is observed. The process is reversible, that is, the same results are obtained whether one starts with pure water or pure dioxane. Just as decreasing the dielectric constant of the medium decreases the hydrophobic forces holding the bases together, increasing the dielectric constant should enhance the interaction. As may be seen in Figure 2 2 N NaCl increases the amplitude of the CD bands. Information on the relative orientation of bases may be obtained from the absorption spectrum. As one goes from the disordered to the ordered structure, a hypochromism is observed if the bases are stacked in a card-pack fashion, whereas a hyperchromism is observed when the bases are ordered in a head to tail fashion."-13 As may be seen in Figure 5, decreasing dioxane concentration leads to a hypochromism; also, increasing the pH and increasing salt lead to a hypochromism. Thus the bases are oriented more nearly in a card-pack fashion. We may also assess the extent to which intermolecular base stacking may occur. The variation in magnitude of the CD extrema accompanying a hundredfold change in concentration is less than 3%. This is little more than the expected experimental
2
I
a - Z N NoCl(pH=7) b - water (pH=?) C - water : d i o x a n e 5 . 5 by Y/V d - pH:2.0(H3P04)
240
260
280
300
X(rnp1
Figure 5. The absorption curves of adenosine mononicotinate in various solvent systems. A marked hypochromism results upon decreasing dioxane concentration, or increasing the pH and increasing the salt concentration.
error. Over the concentration range dictated by solubility and path length (10-5-10-3 M ) , there is no concentration effect. Thus, both in circular dichroism and absorption, the pH effect, the dioxane solvent effect, as well as the salt effect demonstrate that the bases within a single AMN molecule are oriented in a stacked manner by hydrophobic forces and that the complexity of the CD curve is dependent on this interaction. Temperature is another experimental parameter which may be varied to demonstrate that the complex CD band is dependent on the relative orientation of adenine and nicotinate, and, therefore, that the complexity is a result of the coupling of electronic transitions in one base with those of the other. In addition, the temperature effect will allow an estimate of the enthalpy and entropy of the interaction in aqueous media. The effect of temperature on the circular dichroism of Ah3N is given in Figure 6 , where the magnitudes of the nega(11) W. Rhodes, J . Am. Chem. SOC.,83, 3609 (1961). (12) I. Tinoco, aid., 82, 4785 (1960); J . Chem. Phys., 34, 1067 (1961). (13) M.Kashrt, Radiation Res., 20, 55 (1963).
Volume 71, Nu aber 13 December 1957
DANIELW. MILESAND DANW. URRY
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ADENDSIN E
8
MONON I C O T I N P T E
in 2 M NoCl T E M P E R A T U REFFECT E
6 4
2
? O 0 X
E -2 -4
-6 -8
220
-10 I
!20
~
l
240
260 X(rnpL)
~
l
280
~
I
300
Figure 6. Circular dichroic spectra of adenosine !j’-mononicotinate a t various temperatures in 2 M NaCl.
tive and positive extrema are found to decrease in a proportionate way. Thermal disruption of the base stacking results in lower rotational strengths. Assuming a two-state model the enthalpy, A H , and entropy, AS, changes tire approximately 12 kcal/mole and 38 eu when going from an ordered to disordered state in aqueous medium. As may be seen by comparing Figures 3-5, the disordered state is dependent on the experimental parameter used to demonstrate the transition. The differences in the disordered states are likely due to differences in the average base-sugar orientations. Attempts to resolve the absorption curves of each chromophore into the expected number of component bands and then to use these bands to describe the interacting systems involve the assumption that the frequencies of the transitions are not altered for the base pair. Because the initial resolution into component bands is itself very doubtful and because of likely shifts on interaction, we have chosen to simultaneously fit hoth the absorption and circular-dichroism curves with a> minimum of Gaussian functions. Such resolution does allow a gross assignment of bands to a given moiety and presents a spectral delineation of dipole strengths, rotational strengths, and anisotropies, which are indicative of types of transitions. It is The Journal of Physical Chemistry
,
260 X(rnpL)
300
,
Figure 7. Resolution of the CD curve into a set of Gaussian curves, which may also be used to describe the corresponding absorption curve of Figure 8.
expected that the anisotropies of magnetic transitions will be low, as the procedure attempts to associate as much dipole strength as possible with a given rotational strength. The procedure is to fit the CD curve with a minimum set of Gaussian functions. These functions are then put in the positive mode and their heights are varied in an attempt to fit the absorption curve. One then shifts back and forth between absorption- and circular-dichroism curves until what appears to be a minimum set of Gaussian functions will simultaneously fit both sets of data by changing only sign and height of the function. Figures 7 and 8 give the resulting set of Gaussian functions. Bands are resolved a t 271, 257, 240,228, and 222 mp. Comparison of the AMN absorption in the ordered and disordered states shows that while there is a significant hypochromism there is no marked change in the position of the band near 260 mp (Figure 5). No dramatic shift in transition frequencies is expected when going from the noninteracted bases to the stacked bases, The nicotinate group has an absorption band a t 264 mp in decyl nicotinate,14 whereas the absorption for adenosine is at 259 mp. It is to be expected that the 257-mp band is the result of a transition in the (14) When the nitrogen substituent is a ribose as in nicotinamide mononucleotide (NMN), the band is a t 266 mp.
RECIPROCITY IN THE CIRCULAR DICHROISM OF ADENOSINE MONONICOTINATE
-~
4453
~~~
~~
Table I : Critical Values for Resolved Gaussian Curves
--Wavelength of extrema, ----pm
7
271
Molar extinction coeff X IO3 Dipole strength (Di) x 10-a6 Molar ellipticity x 103 Rotational strength (Ri) X 10-40 Anisotropy lIli/Dil X lo-'
5.2 3.3 -11.1
-5.4 1.6
7
257
240
228
222
13.1 11.1 8.0 5.2 0.5
3.4 2.7 -2.7
3.0
... ...
1.5 5.6 2.1
-1.6 0.6
1.4
12.2 4.1 Large
which the two nitrogen-containing aromatic groups are held in a stacked orientation by hydrophobic forces. As adenine and nicotinate moieties have electronic transitions a t 259 mp and 264-266 mk, respectively, when dispersed in aqueous solutions, it is expected that these transitions will derive a substantial part of their rotational strength from coupling one with the other when in a stacked conformation. Should this be the case, it is necessary that the CD extremum resulting from one of the transitions becomes more positive while that of the other becomes more negative.' This is qualitatively the case for each experimental variable (Figures 2-4). Considering the coupled oscillator contribution to optical rotation, the rotational strength of the 271-mp band, R271, of the nicotinic acid moiety due to coupling with the a electronic transitions in the adenine moiety may be written as the following sum R271 =
R27lm
(1)
a
220
260
3 Ob
X(mp1 Figure 8. Resolution of the absorption curve into a set of Gaussian curves, which by varying height and sign will simultaneously describe the CD curves of Figure 7 .
adenine moiety and the 271-mp band is the result of a transition in the nicotinate moiety. This assignment is further supported by the relative intensities of the transitions. The adenine absorption band in AMP is severalfold more intense than the band in decyl nicotinate and in NM?;. This is the relative intensity observed in the resolved bands (Figure 8 and Table I). Thus, we take the 271-mp band to be a transition in the nicotinyl moiety and the 257-mp band to be a transition in the adenyl moiety. Reciprocal Relations In the foregoing discussion it has been determined that the AXIN complex circular-dichroism curve with large ellipticities is characteristic of the conformation in
Similarly, the rotational strength of the 257-mp transition in the adenine moiety due to coupling with the /3 transitions in the nicotinate moiety is written R257 =
c R267.B B
(2)
The rotational strength of the a' transition in adenine due to coupling with the p' transition in nicotinate may be written
As the numerator does not change sign on interchanging the indices, and the denominator does, it is apparent that the rotational strength of the /3' transition in nicotinate due to coupling with the a' transition in adenine is the same value but opposite in sign, i.e. Ru*pl =
-R@J~I
(4)
They vary in a reciprocal manner. When two transitions are of closely placed frequencies, the denominator in eq 3 becomes small, causing the term Ru~Sjto become larger. The rotational strength never goes to Volume 71, Number 13 December 1967
4454
infinity for t,ransitions occurring a t the same frequency as an excitat
