Resonance Raman spectroscopy of proflavin (II)

Spectrochtmlea Acta, Vol. 43A, No, 1l. pp. 1385-1392, 1987. Printed in Great Britain.

i84-8539/87 $3,00+0,00 PergamonJournals Ltd.

Resonance Raman spectroscopy of proflavin (II) RICHARD H. CLARKEand SOOKHEEHA Department of Chemistry, Boston University, Boston, MA 02215, U.S.A.

(Received 19 December 1987; in final forrn 22 March 1987; accepted 23 April 1987) Abstract--To understand molecular structure for a complex of a dye molecule of DNA receptor, the depolarization ratios and band widths of the Raman spectra of proflavin in different DNA media were analyzed. According to these experimental analyses, it is possible that the proflavin molecule lies near the phosphate group, having a strong interaction with the phosphate group to restrict tumbling motion and inplane motion. Refinement of the bandshape, the improvement of the vibrational bandshape theory and modifications of the reorientational theory should allowa detailed picture of the intercalation of proflavinto DNA.

INTRODUCTION

Resonance Raman spectroscopy, examining the vibrational modes of the chromophore portion of the molecule, has been employed for the study of complex molecules of biological interest I-1-4]. Yet to our knowledge, few Raman studies have been made on the effect of the binding of dye molecules to DNA, or the spectroscopy of the dye molecule itself, partly due to the strong fluorescing nature of the dye molecule. Evidence for the formation of an intercalation complex was found from many studies ['5-7] with the aromatic dye molecule placed between base-pairs with some distortion of the overall stacking pattern; the phosphate backbone is distorted and DNA lengthens when the DNA allows dye molecules to occupy the space between base-pair stacks. Intercalation causes DNA unwinding from a combination of pulling along the DNA double helix axis and unwinding in order not to break the sugar-phosphate backbone I"8-10]. Hence, the interactions between dye and DNA are believed to alter cell metabolism, diminishing, and in some cases, terminating cell growth [11, 12]. In Raman spectroscopy, water is an excellent solvent since it scatters very weakly in the region 300-3100 cm-1. Hence many biological systems can be observed close to their natural states. As a first step to probe structural features of dye and DNA receptors in our laboratory, Raman spectra of proflavin in solid and solution were obtained. The observed band region lies in 300-1700 cm- 1. The bands of the 350-700 cmare considered to arise from out-of-plane ring vibrations composed of the C-C-C, C - C - N torsion and C-H bendingand 1000-1700 cm- 1 from in-plane C-C stretching and C - C - C bending. The Raman bands higher than 1700 cm- t in frequency are not measured due to the strong fluorescence interference. In order to understand which vibrational motion is sensitive to stacking in an intercalation model, the calculation of the normal modes of proflavin were undertaken to compare with the experimental results and the observed Raman bands were assigned in the previous paper [13]. From the results of calculation, the bands

not observed due to fluorescence interference are the C-H stretching modes in the 2800-3200 cm -~ region. Further, the depolarization ratios of the observed Raman bands have been measured; the dependence of depolarization ratio on medium (or environment) has been used to obtain qualitative structuralinformations on the binding of proflavin with DNA. Good quality (high signal to noise ratio) resonance Raman spectra of proflavin at a concentration of 1.5 • 10- 5 M in the solutions of different nucleic acid at 1.02 x 10 -4 M were obtained in our laboratory. The excitation frequency of the laser (457.9 nm) is within the visible absorption region. The absorption spectrum of proflavin is well known [7] and its first excited state maximum is at 440 nm; its second excited state maximum is at 280 rim. The Raman bands obtained lie between 300 and 1700 cm- ~. The values Of depolarization ratios are useful not only in assigning bands but also for obtaining information on intermolecular interactions 1"14, 15-1, since the intensity of Raman scattered radiation is influenced by the internal field caused by molecular interactions. The depolarization ratio (p) is the ratio of the intensity parallel to incident light polarization with the intensity of Raman ban~ perpendicular to incident light polarization, p = lh/Io. The Raman intensities can be discussed in terms of the polarizability derivative tensor elements. The polariz. ability derivative tensor, (co'- c9~/8Q), is the rate of change of polarizability tensor 9 with respect to normal coordinate Q at the equilibrium configuration of the molecule. Since the polarizability tensor :, has been regarded as a function of the nuclear coordinates, it is a function of the normal coordinate Q for a particular vibrational mode. The depolarization ratio also can be expressed as 2 2 2 p = (3~,2 + 5y~,)/(45cx +4y,)

(1)

where a, 7~ and 7,~ are called isotropic (trace), quadropole (anisotropic symmetry) and magnetic d!pOl.e components (anisotropi c antisymmetry) of the scattering tensor, respectively. The observed vertical intensity (Iv) contains the mean value term and anisotropy term

1385

1386

RICHARDH. CLARKEand SOOKHEEHA

and the observed horizontal intensity (lh) contains that of the vibrational motion. Changes in depolarization ratios have been observed in the vibrational band when proflavin molecules experience the difference in environmental from the simple nucleic acid to D N A which has complicated structure due to distortion of electronic distributions. Figure 1 shows spectra (lh, I,) obtained for proflavin and calf thymus DNA wkh proflavin in the range 1400-1700 cm -1, The Raman band at the 1645 cm -~ position can be correlated with the combination band of 568.2 era- 1 and 1084,2 c m - t, and this band belongs to At symmetry. The parallel intensity of the spectra increases or perpendicular intensity decreases relatively as the proflavin molecule experiences the larger nucleic acids derivatives. The tabulation of depolarization ratios of Raman bands in Tables i - 4 was obtained from a mixed solution of proflavin with various nucleic acid solutions described above for inplane (Tables 1, 2) vibrations and out-of-plane (Tables 3, 4) vibrations, The values of the depolarization ratios in each table for pure proflavin sotution are slightly different from one another, and the values of Table 1

tend to be smaller. The change in the depolarization ratios is considered to be due to the change of the environment of the proflavin molecule. The out-ofplane vibrations are less susceptible to the change of

I DNA+ oroftavin

t_. 1400

_~

1475

_

~,

15~0

1625

1700

Frequency (cm"1)

Fig. 1. The horizontal and vertical Raman intensities.

Table I. Depolarization ratios of proflavin for different media At cm- 1

Pr

T

C

G

A

dT

dC

dG

dA

pT

pC

pA

DNA

1645.7 1580.9 1479.2 1429.4 1369.9 595.9

0.46 0.45 0.46 0.42 0.41 0.36

0.47 0.51 0.54 0.50 0.47 0.43

0.53 0.50 0.52 0,50 0.49 0.40

0.51 0.54 0.56 0.51 0.49 0.48

0.51 0.53 0.55 0,53 0.44 0.44

0.56 0,53 0.54 0.52 0.51 0.49

0.57 0.52 0.53 0.51 0.53 0.51

0.61 0.59 0.57 0.54 0.52 0.48

0.60 0.56 0,56 0,55 0.54 0.52

0.63 0.59 0.63 0.59 0.59 0.59

0.62 0.60 0.64 0.62 0.58 0.58

0.63 0.61 0.65 0.61 0.57 0,57

0.68 0.61 0.69 0.64 0.63 0.61

DNA: 1.02 x 10-* M. Proflavin: 1.50 x 10 -~ M. Pr: proflavin, T: thymine, 0: guanine, A: adenine, dC: deoxyoytidylic mono-phosphate, pA: polydeoxyadenylic acid, dG: deoxyguanidine monophosphate, pC: polydeoxycytidylic acid

Table 2. Depolarization ratio of protlavin for different media B2 em- ~

Pr

T

C

G

A

dT

dC

dG

dA

pT

pC

pA

DNA

1498.1 1393.3 1325,2 1291.1 1217.0 1160.0 1110.1 949.0

0.45 0.47 0,45 0.47 0.48 0.48 0.46 0.43,

0.51 0.53 0.47 0.53 0.52 0.53 0.54 0.57

0.47 0.55 0.50 0.51 0.50 0.54 0.52 0.54

0.46 0.54 0.53 0.56 0.53 0.57 0.50 0.55

0.55 0.56 0.53 0.54 0.54 0.51 0.56 0.50

0.54 0.57 0.55 0.55 0.55 0.54 0.57 0.58

0.49 0.58 0.57 0.59 0.54 0.56 0.59 0.57

0,59 0.60 0.57 0,60 0.60 0.57 0,60 0.60

0.59 0.59 0.65 0,57 0.58 0.56 0.62 0.55

0.60 0.68 0.68 0,64 0.65 0.60 0.67 0.6S

0.63 0.65 0.67 0.67 0.64 0,64 0.67 0.64

0.65 0.69 0.70 0.66 0.69 0,63 0.71 0.69

0.63 0.71 0.72 0.69 0.71 0.64 0.73 0.70

Table 3. Depolarization ratio of proflavin for different media B1 em- t

Pr

T

C

G

A

dT

dC

dG

dA

pT

pC

pA

DNA

759.9 650.6 494.1 353.5

0.38 0.33 0.47 0.42

0.40 0.44 0.55 0.47

0.42 0.46 0.51 0.48

0.40 0.42 0.50 0.46

0.41 0.41 0.50 0.45

0.44 0.49 0.53 0.51

0.43 0.5l 0.56 0.53

0.45 0.47 0.55 0.52

0.47 0.51 0.54 0.55

0.53 0.53 0.59 0.57

0.57 0.57 0.57 0.56

0.54 0.54 0.58 0.57

0.58 0.57 0.60 0.58

1387

Resonance Raman spectroscopy of proflavin (II) Table 4. Depolarization ratio of proflavin for different media A2 crn - t

Pr

T

C

G

A

dT

dC

dG

dA

pT

pC

oA

DNA

856.8 424.9

0.41 0.37

0.45 0.42

0.43 0.43

0.46 0.45

0.45 0.46

0.49 0.53

0.51 0.50

0.49 0.50

0.50 0.52

0.55 0.55

0.54 0.53

0,57 0.56

0.59 0.58

environment than those of in-plane vibrations according to Tables 1 and 2. The differences of the changes in depolarization ratios of inner ring modes and outer ring modes are appreciable, and the change in depolarization ratio is small between the single-stranded synthetic DNA and natural calfthymus DNA. Figure 2 is a graph of depolarization ratio vs structural complication of nucleotide to see the effect of medium easily. It also shows the changes in the depolarization ratios (mostly increases) in the vibrational bands when proflavin molecules experience the difference in environments from the simple nucleic acid to more structurally complicated DNA. We believe that scattering tensors (polarizability derivative) are subjected to the influence of DNA intercalation and tensor elements also become asymmetric due to the distortion of the tensor. Some Raman bands are more susceptible to change in the environments than the others according to the way of intercalation and specific binding sites; this information can be a valuable tool to study how the proflavin molecule behaves in DNA. The solutions of proflavin and nucleotide, proflavin and nucleic acid monophosphate, proflavin and singlestranded DNA of mono bases, and DNA and proflavin solution were prepared as discussed in a previous paper. The perpendicular and parallel intensities to the incident radiation of vibrational bands for each solution have been obtained. The depolarization ratios of all solutions for every band were measured and compared with one another. The value of the depolarization ratio increases as the proflavin was introduced to the more complicated structure of DNA. Guanine and adenine-proflavin solution has about the same change and thymine and cytosine has about the same change in depolarization ratio. The outer ring motion shows slightly larger change than inner ring vibrational motion. In-plane modes are generally more restricted than out-of-plane modes as the molecule

r .o

<)

0.6

0.~ N

r

r

0 a

0.4

I

I

I

pr

T

dT

, I

pT

1

intercalates and interacts with DNA. The solutions of proflavin and nucleotide, proflavin and nucleic acid monophosphate, proflavin and single-stranded DNA of mono bases, and DNA and proflavin solution were prepared as discussed in a previous paper 1-13]. The perpendicular and parallel intensities to the incident radiation of vibrational bands for each solution have been obtained. The depolarization ratios of all solutions for every band were measured and compared with one another. The value of the depolarization ratio increases as the proflavin was introduced to the more complicated structure of DNA. Guanine and adenine--proflavin solution has about the same change and thymine and cytosine has about the same change in depolarization ratio. The outer ring motion shows slightly larger change than inner ring vibrational motion. In-plane modes are generally more restricted than out-of-plane modes as the molecule intercalates and interacts with DNA. Finally, we have chosen to study the rotational and vibrational bandwidth, since the width of a Raman spectral line arising from internal vibrations of molecules is determined by both the vibrational and rotational relaxation processes and intermolecular interaction will significantly affect the bandwidth. The nature and extent of intermolecular interactions vary for different environments and are dependent upon such factors as molecular size, shape, polarity and electronic structure. In order to probe the effects of strong interaction on the vibrational bands of a molecule, we have chosen to study the rotational and vibrational (isotropic) band shape to determine the qualitative nature of binding. Because of intercalation, one would expect that the vibrational line shape and widths will be significantly affected by intermolecular interactions. Intercalation of the proflavin to DNA will restrict interactions such as reorientations and collisions with other molecules due to the structural (steric) hindrance. Polarized and depolarized Raman spectra of proflavin were obtained in various nucleotide media. Three bands from the totally symmetric modes were selected to study vibrational broadening as well as five bands from each of the symmetric modes for reorientational broadening. Totally symmetric vibrational bands and reorientational bands were separated. The isotropic (C~o(t)) and the anisotropic (C,,~,, ((3)) correlation functions can be separated using the Fourier transformation of the polarized (1,) and depolarized (I~~ components of a Raman band. The expressions 1-16-20] are given by

DNA

Fig. 2. Depolarization ratios of proflavin vs nucleotide.

Iv = l v v - (4/3)Iv n, Iee~, = Iv H

(2)

1388

RICHARDH. CLARKEand SOOKHEEHA

and

V -7

(, c~,o =

Jl~,o(co) exp ( - kot)dco

Monochromator

(3) Scattering center E

c~

=- f lo.,,o (co)exp ( - iwt) dco

. . . . . . . .

L

(4)

P

SL Y

where 1•o and Io,i,o are the normalized functions of the polarized (lp) and depolarized (la,p) components of the observed Raman intensity, respectively. From these experimental analyses, we have come to understand certain features of proflavin intercalation with DNA, such as the position of proflavin, intercalation preference of proflavin over base pair and motions in the D N A base pair stack.

z

TM

L . . . . .

. . . . . . . .

Laser beorn

Fig, 3. Geometry of optical system.

EXPERIMENTAL

Spectra were obtained from a sample of proflavin with various nucleotide and nucleic acids in 2-mm diameter quartz tube. Proflavin (3,6-diaminoacridine hemisulfate) from Aldrich Chemical Company was purified from an aqueous solution after treatment with charcoal, concentrated, chilled overnight at 4~ and filtered. The precipitate was rinsed with ethyl ether and dried in air. Stock solution of proflavin of concentration 5.0 • 10 -3 M was diluted when needed. Calf thymus DNA from Sigma Chemical Co. was used without further purification to prepare a stock solution of 4.02 • 10- a M in a cold room environment (approximately 4"C) by constant stirring for several days which was diluted when needed. The synthetic homopolymeric acid and polydeoxycytidilic acid from Sigma Chemical Co. were used without further purification. The synthetic homopolymeric DNA solutions were prepared without sonication in order to have a system as close as possible to the natural state. The Raman spectrometer was based on a Spex 1451X double monochromator with two gratings of 110 x 110 mm size and 1800 lines mm -t equipped with a cooled spectral EMI 9558 photomultiplier. Excitation was provided by the 457.9 mm line from a Coherent lnnova 20 argon laser. The spectra were measured on a Camac Crate controlled by a RSX/PDPI l Digital Computer system. Data acquisition and manipulation were undertaken by the program called Ram. Samples were prepared in room temperature, and spectra were taken under liquid nitrogen temperature with a laser power level of 500 mW to avoid photodecomposition of the sample. A snapshot of the DNA-proflavin intercalation in room temperature, hence, was obtained. The experiment was rerun every time to cheek the time dependence of spectra; no change in spectra were seen. The path o f the incident light and the arrangement of the light-collecting system is specified by X, Y and Z and X is taken to be in the direction of the polarization of the laser beam. The distance between the scattering center and the entrance slit, SL, is 350 ram. With the present system, the widths of the image of the scattering region on the entrance slit is about 1.5 times that of the scattering region itself. TM in Fig. 3, a tunable excitation monochromator filter 1460 from Spex Industries, was used to reject spurious lines and background lines. Here M, P and L are mirror, polarizer and lens, respectively. The output signal from photomultiplier was converted into analog voltage by means of a picoammeter, and multiple scans (3 cm -~ per s) were collected and averaged on a Camao Unit controlled by RSX/PDPll on Digital computer system. The spectral slit width of the monochromator was about 5--10cm- t B A N D W I D T H ANALYSIS

species A t. There are significant changes in line widths for different modes and they have certain trends as the medium varies. These changes in band shape will be discussed in terms of rotation for depolarized bands and of collision and dipole--dipole moments for vibrational broadening. Approximate vibrational and reorientational relaxation times were calculated to compare with collisional and structural relaxation time. As mentioned in previous paper ['13], there arc 63 vibrational modes: 21 totally symmetric (i.e. preserving the symmetry of the molecule when it vibrates) of symmetry species At of in-plane modes, 21 asymmetric species Bz of in-plane modes, 11 asymmetric species B1 of out-of-plane modes, and 10 asymmetric species A2 of out-of-plane modes. All modes are both Raman and i.r. active. Figure 4 illustrates the polarized and depolarized intensities of the Raman band of proflavin at 1580 cm - L The half widths (hwhh) of the Raman band at half height were measured from polarized and depolarized Raman bands in each medium a n d slit corrections were made. The three isotropic band

I.sO

>~ 0)

1550

1565,

1580

1~95

1610

Frequency (cm -I)

Vibrational relaxation bands were separated from reorientational band width for totally symmetric

Fig. 4. Iso and anisotropic Raman band at 1580 cm-~.

Resonance Raman spectroscopy of proflavin (II) widths (1580,0, 1396,4 and 586.5 cm-1) are obtained for totally symmetric bands belonging to AI symmetry. These isotropic bands widths were separated from the polarized spectra by subtracting the contribution of reorientation motion of the molecules seen in the depolarized spectra. The six anisotropic bands widths are measured from the depolarized Raman bands and each band belongs to a different symmetry. These values are tabulated in Tables 5 and 6. Lorentzian curve fitting was not undertaken since the correction is within the experimental error [21] and the trends of changes in bandwidth are of interest in our analysis. The observed Raman band widths were corrected according to the equation of Dijkman. The isotropic part of the Raman spectra ofproflavin solution provides information about the molecular vibrational relaxation which is closely related to intermolecular interactions. There are several factors considered to be important to vibrational Raman band broadening: energy exchange (or transfer) caused by dipole-dipole moment interactions, phase relaxation caused by elastic collision, and dissipation of vibrational energy to the environment (or lattice). In order that the environment picks up the oscillator energy, the energy of the fluctuating motion of the lattice should be comparable to the oscillator energy, and the spectral power density in this lattice fluctuation is very small compared to the oscillator energy range of the molecule. We will consider two major contributions to vibrational relaxation; these are collision of molecules and resonant energy transfer due to dipole--dipole interaction whose mechanism for the exchange of resonance energy between the interacting molecules. It is safe to assume that (Aco/2)/so= l/~lso = 1/~L,~b to well within experimental error. The Raman band widths (hwhh) of proflavin in various nucleic acid media are tabulated in Tables 5 and 6. Table 5 illustrates the isotropic bandwidths of totally symmetric modes (A1). One can notice that the Raman bands broaden as proflavin experiences interaction with more complicated and heavier molecules in

1389

the medium, In order to discuss this result, we assume a binary collision process and an induced dipole-dipole interaction in the solution. When molecules are in low density liquid and gas, the range of interaction is several atomic radii and the dipole-dipole interaction would be the major interaction that causes the Raman line broadening, However, in the high density liquid, the majority of collisions have a large impact on line broadening due to electronic overlapping that causes the polarizability distortion. Here, collision is considered to be a very short range interaction so that two molecules have significant electronic overlap. Assuming the collisions and induced dipole--dipole to be the major relaxation mechanisms, then our experimental observed medium dependence of bandwidth is quite reasonable: as the structure of the medium molecules become complicated, the collision frequency increases, causing band width to increase, However, the narrowing effects have been observed when the proflavin molecule interacts with single stranded polynucleotide and DNA. It is assumed that intercalation of the proflavin molecule with DNA prevents free motion and collision of proflavin with neighboring atoms. Hence, the induced dipole-dipole interaction dominates the vibrational relaxation when proflavin is intercalated with DNA and it is possible that the induced dipole-dipole interaction has been increased throughout the change of medium from the values of line widths of proflavin solution to proflavin-polynucleotide solution, Schematic vibrational modes are illustrated to facilitate the comparison with bandwidths. The band at 1580.9 cm- t is a stretching mode and shows more line narrowing (17 ~) than inner ring stretching at 1369.9 cm- 1 (12,2~) and in-plane bending at 595.9cm -t (12,4~) when proflavin interacts with homopolymeric single-stranded DNA. This result agrees with the observation of CHEN and MORRISfrom the study of hypochromism that the interaction is localized on the outer rings. There is not much change in values of band widths between the polynucleotide and DNA, and the bandwidths with

Table 5. Bandwidths of totally symmetricmodes cm - 1

Pr

T

C

G

A

dT

dC

dG

dA

pT

pC

pA

DNA

1580.9 1369.9 595.9

8.2 7.0 9.8

8.7 7.5 10.I

8.7 7.8 10.3

8.8 7.8 1.05

8,2 8.0 10.8

9.9 8.9 10.6

10.8 8,4 11.0

9.4 9.2 10.9

10.5 8.2 9.4

7.2 8.2 9.6

8.7 7.0 9.4

9.7 7.5 9.7

8.7 7.8

Table 6. Anisotropic bandwldths era- 1

Pr

T"

C

G

A

dT

dC

dG

dA

pT

pC

pA

DNA

1580.9 1369.9 595.9 1498.1 759.9 424,9

9.5 8.1 12.9 12.9 10.6 11.2

8.9 7.0 10.6 11.5 9.9 10.5

8.8 7.0 11.5 10.9 9.3 10.2

8.5 7.5 11.0 9.8 9.8 10.7

8,2 7.2 11.2 9.8 9.5 10.0

7.7 7.0 10.9 8.8 8.9 9.4

7.8 7.1 10.5 8.2 8.7 10.0

8.0 7.1 10.2 9.2 8.1 9.8

8.1 7.0 8.5 9.5 8.7 9.5

6.5 5.9 7,9 7.9 7,8 8.9

6.8 6.2 8,0 8.0 7.5 8.7

5.8 6.3 6.4 7.8 7.7 8.0

6.4 6.4 8.2 7.9 7.4 7.9

1390

RICHARD H. CLARKE and SOOKHEE HA

DNA are close to the average value of three different homopolymeric synthetic DNA. These results suggest that proflavin interacts with a single strand of DNA in a stable form of intercalation so that collision motions of proflavin are restricted. Further, intercalation with double-stranded DNA adds only induced dipoledipole disturbance into the proftavin molecules. Table 6 shows the anisotropic bandwidths of symmetric and asymmetric modes of vi.bration. These anisotropic bandwidths are related to rotational motion of the molecules as shown in the theory section. Usually, the rotations of the molecule about its major symmetry axis (z axis in proflavin) are called spinning, and the rotations about its minor axis (y axis in proflavin) are called tumbling. They exhibit different dynamic behavior in terms of symmetry. The point group of profiavin is Ca,.. There are four vibrational modes, one totally symmetric (i.e. preserving the symmetry of the molecule) of symmetry species At, and three nontotally symmetric of symmetry species Bt, B2 and A2. For A~ modes, where ~ 4 : 0 , the rotation of the proflavin molecule about the major symmetry axis does not change the polarizability tensor. Hence Al modes do not provide information on the spinning motions of the molecule. Rotation about the perpendicular axis (y axis) does however modulate the tensor and therefore tumbling motions can be studied using the At modes. On the other hand, the modes o f the rest of the symmetric species change for both kinds o f rotations, and so these contain information on both spinning and tumbling motions. In-plane modes in Table 6, especially the modes of A l species, shows more line narrowing effect as the proflavin experiences the change of medium from small molecules to large and complicated DNA. One can see that for symmetry about the major symmetry axis (z axis), the totally symmetric Raman modes are insensitive to spinning reorientation and tumbling reorientations are diminished visibly as intercalation takes place. From the depolarization ratio experiments, the out-of-plane modes show more polarized nature and less line narrowing effect when proflavin molecules interact with nueleotide and DNA. Hence, it is safe to state that it is still possible for the proflavin molecule to have spinning rotation to a restricted extent within the plane of the stack. Some efforts [22, 23] have been made to calculate the diffusion constants D l_ and DI, indicating the spinning motion and tumbling motion, respectively, from the line widths using the anisotropic polarizability tensor in term of the probability density. However, for the groups lower than Ca the diffusion constants are not separable. Several authors 1-24-27] reported that the bandwidth reflects the density dependence of the system observed. Our experimental results agree with this finding, since the values of bandwidths of all proflavin vibrations decrease by increasing the molecular weight of the medium (larger density). There is a slight difference between the out-of-plane modes and inplane modes in degree of increases of band broadening.

In-plane mode bandwidths are more susceptible than out-of-plane modes bandwidths. As we expected, there is measurable line narrowing when the proflavin interacts with a polynucleotide or DNA because a medium such as polynucleotide and DNA has more complicated structure, causing intermolecular binding or steric hindrance. Such intermolecular binding affects and hinders the orientational motion. It is possible to postulate that reorientation occurs only with the process of the structural break-up and reformation of groups, and this phenomenon is unlikely to happen to the intercalation of proflavin with DNA or polynucleotide. It is also possible that proflavin solution concentration is already high enough and reorientation motion is restricted. Some authors [28-30] introduced the structural relaxation time z~ to understand the relation between the molecular orientation and density of the solution. When the structural relaxation time is larger than the reorientational time, the molecule reorients until the direction of its angular velocity is abruptly changed by a collision. Such a system is called collision limited. On the contrary, when T~ < vo,, reorientation is structurally limited. In this case, the molecule is held fixed until structural break-up occurs or lower angular orientation occurs. During the time that its environment is randomized, the molecule is free to execute a rotational jump until it is again trapped by the local structure. This comparison will be a good test for the proflavin intercalation to DNA and the way a proflavin molecule interacts with a polynucleotide if the method of measurement of v, is developed. It is known that r, is related to viscosity or density, namely related to the structure of the system. From our experimental results, we assume that z~ < zpDNA~< "eDNA'The approximate relaxation times for reorientational motion proflavin with nucleotides (dA, dG, dT, dC), with homopolymeric DNA and with DNA are tabulated in Table 7. The bandwidths are averaged in each category before calculation and reorientational times were obtained by means of the relation %, = 1/2nCcoo, (cm-l) where C is the speed of light and coo,is the averaged half width. From the Table 7 the r, is assumed to be about 2.0 ps. The temperature factor should also be taken into consideration, since the experiment was performed under liquid nitrogen temperature. The bandwidths in DNA and polynucleic acid are not much different than in isotropic bandwidths. F r o m this fact, we may conclude that the intercalation model suggested by GEORGHIOU [31] explains our experimental results. Since the spinning reorientation is more feasible in an intercalated system, the proflavin molecule would be situated to have less distortion when it is in motion. If the tumbling reorientation is a more feasible mechanism, the proflavin molecule is likely to lie in the middle of the stack. The possible hydrogen bonding between the hydrogens of proflavin and the phosphate groups of DNA will allow the reorientation about the major axis (z axis) without severe distortion of the proflavin molecule. From the findings that the depolarization

Resonance Raman spectroscopy of proflavin (1I)

1391

Table 7. Relaxation times for original bands

"~pDNA TDNA

cm- t

dN

pDNA

DNA

ran

1580.9 1369.9 595.9

7.90 7.05 10.0

6,4 6.1 7.4

6,4 6.4 8.2

2.2 2.5 1.8

2.7 2.9 2.4

2,8 2.8 2.2

1498.1 759.9 424.9

8.9 8.6 9.7

7.9 7.7 8.5

7.9 7.4 7.9

2.0 2.1 1.8

2.2 2.3 2.1

2.2 2.4 2.2

dN: bandwidth of nucleotide, unit era-t. pDNA: homopolymeric single stranded DNA, unit cm-t.

T: relaxation time in unit of pc. ratios of the polynucleotide system and the D N A system do not show much difference and reorientation about the major axis is suppressed, we may support a model that the proflavin intercalates into DNA to the one side of the stack close to the phosphate group of the helix backbone. CONCLUSION

Our Raman studies, together with the calculation of the vibrational modes, have allowed specific information on proflavin-DNA intercalation. The calculation of vibrational modes allows assigning of the observed Raman bands of proflavin molecules and facilitate the analysis. The appropriate concentration of about 10- ~ M ofproflavin solution to work with was decided from the fluorescence study. These overall influences of molecule-medium interactions are seen as the increase in the depolarization ratios macroscopically. In At in-plane modes, the range of the depolarization ranges lies between 0.45 and 0.68. B2 in-plane modes, antisymmetric, has tensor elements ~ and ~y and ~2 = 0. These two elements become asymmetric ~yz ~ ~zr, hence the ~,~ ~ 0, and depolarization ratios increase. The range of depolarization ratios is 0.45--0.73 and these values are slightly larger than those of A1. Out-of-plane modes, BI and A2 symmetry, have ~xy and c~yx.The depolarization ratios are up to 0.58 and have smaller changes than in in-plane vibrations. In every symmetry species, the outer ring vibration motions show larger values of depolarization ratios than inner ring vibrations; this means the outer ring is more susceptible to the influence of the medium, for example, depolarization ratio 0.69 for outer ring stretching and 0.64 for inner ring stretching in AI modes. Interaction with pyrimidine nucleotides, single-ring aromatic base (CMP, TMP) gives a smaller value of the depolarization ratio than purine nucleotides (two-ring aromatic bases, G M P and AMP) and this may indicate that there is no specific binding preference of proflavin with respect to the nucleic acid pair. This phenomenon explains that the depolarization ratios in homopolymeric singlestranded DNA (pT, pC and pA) in Tables 3 and 4 are not much different. The depolarization ratios in single-stranded homopolymeric DNA-proflavin do not vary much from tha'c

of double helix calf thymus DNA-proflavin, and the polarizability scattering tensor elements of in-plane modes become asymmetric, which gives higher values of depolarization ratios as the proflavin molecule intercalates with DNA. Also, the depolarization ratio increases drastically when the medium changes from nucleic acid phosphate to single stranded DNA. These phenomena can be explained by the model proposed by GEORGH1OU. The proflavin molecule is situated between the base-pair stack by intercalation. One end of the pro flavin molecule is near the negatively charged phosphate group available upon strong interactions with the proflavin molecule. The other end of profla~in is far from the phosphate group of the other side of the backbone. The polarizability ellipsoid will be distorted asymmetrically when the molecules undergo vibrational motion. An example is the C - C outer ring stretching band at 1492.0 cm- 1. The stretching of the C-C bond of one outer ring of the molecule is hindered because of its proximity to a phosphate group, while the C-C bond stretching in the other outer ring is relatively unrestricted. This is believed to cause the increase of the asymmetricity in the polarizability tensor. The Raman studies, depolarization ratios and bandwidths analysis suggest a few interesting points. 1. There are no specific binding preferences of proflavin toward the base pairs of DNA. Intercalation is rather due to the structure of DNA and its availability to give binding sites to proflavin. 2. The vibrational motion of in-plane modes is more influenced than those of out-of-plane modes by intercalation. The vibrational motions of out-of-plane modes are more polarized and relatively easier. 3. The outer ring is more susceptible to intercalation than the inner ring. In other words, the interaction is localized on the outer rings. 4. Tumbling motion (rotation about the minor axis) is largely suppressed relative to spinning motion (rotation about major axis). From these results, we may build an intercalation model in which proflavin molecules lie near phosphate groups, probably having strong interactions with the phosphate group to restrict tumbling motion and in-plane vibrational motion. Refinement of the bandshape, the improvement of vibrational bandshape theory and modification of reorientational theory to

1392

RICHARDH, CLARKEand SOOKHEEHA

calculate the diffusion constant in low symmetry molecules should allow an extremely detailed picture of the binding of proflavin to DNA.

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