7782
J. Phys. Chem. 1992,96,1182-7181
TABLE I V Free Energies of Hydration for Sekted Transition Metal Iona at 298 K free energy of hydration experimentalb computed ion radius‘ (kcal/mol) (kcal/mol) error (%) cu2+
Zn2+ Ni2+
co2+ Fez+ Cr2+ V2+
Mn2+
0.72 0.74c 0.78 0.82 0.83 0.84‘ 0.88 0.91
-480.6 -483.3 -470.0 -478.9 -447.9 -427.4 -409.5 -436.4
-508.6 -497.9 -478.1 -460.2 -456.1 -452.6 -436.8 -425.9
-5.8 -3.0 -1.7 +3.9 -1.8 -5.9 -6.7 +2.4
“Goldschmidt radii. Experimental free energies corrected for ligand field stabilization using data provided in ref 24. ‘Pading radius. (Table 1 in ref 23). Our model predictions for all ions are within 6.7%of the experimental values. The results in Tables I11 and IV clearly demonstrate the versatility of the new approach to determining solvation free energies which, in principle, can be extended to any ion of known radius and any solvent whose bulk dielectric constant, dipole moment, and refractive index are known. Acknowledgment. This work was funded by the Defense Nuclear Agency of the Department of Defense, through Tulane University’s Center for Bioenvironmental Research. Any opinions, findings, and conclusions expressed in this paper are those of the authors and do not necessarily reflect the views of the sponsors.
References and Notes (1) Born, M. Z . Phys. Chem. 1920, 1, 45. (2) Latimer, W. M.; Pitzer, K. S.;Slansky, C. M. J. Phys. Chem. 1939, 7. 108. Stokes, R. H. J . Am. Chem. Soc. 1964,86,979. Rashin, A. A.; Hoing, B. J. Phys. Chem. 1985, 89, 5588. Rashin, A. A: J . Phys. Chem. 1990, 94, 1725. Bokris, J. OM.; Reddy, A. K. N. Modern Electrochemistry; Plenum New York, 1977; Vol. 1. (7) Noyes, R. M. J. Am. Chem. Soc. 1%2,84, 513. (8) Abraham, M. H.; Lizzi, J. J. Chem. Soc., Faraday Trans. I 1978,74, 1608. (9) Abraham, M. H.; Lizzi, J. J. Chem. Soc., Faraday Trans. 1 19’18.74, 2858. (10) Lizzi, J.; Meszaros, L.; Ruff, J. J . Chem. Phys. 1979, 70, 2491. ( 1 1) Abraham, M.H.;Lizzi, J.; Kriitof, E. Awr. J. Chem. 1982,35, 1273. (12) Liui, J.; Ruff, I.; Dogonadze, R. R.; Kalman, E.; Kronyshev, A. A.; Ulstmp, J. The Chemical Physics of Solvation, Part A; Elsevier: Amsterdam, 1985; p 119. (13) Markin, V. S.;Volkov, A. G. J. ElecrroaMI. Chem. 1987, 235, 23. (14) Laidler, K. J.; Pegis, C. Proc. R. Soc. London 1957, A241, 80. (15) Booth, F. J. Chem. Phys. 1951, 19, 391. (16) Padova, J. Electrochim. Acta 1967, 12, 1227. (17) Guggenheim, E. A. Proc. R. Soc. London 1957, A155, 80. (18) Gur, Y.; Ravina, I.; Babchin, A. J. J . Colloid Interface Sci. 1978,64, 333. (19) Newman, J. Ind. Eng. Chem. Fundam. 1968, 1, 514. (20) Pauling, L. The Nature of the Chemical Bond, Cornell University Press: Ithaca, NY, 1939. (21) Goldschmidt, N. M. Chem. Ber. 1927.60, 1263, (22) Halliwell, H. F.; Newberg, S.C. Trans. Faraday Soc. 1%3,59, 1126. (23) Rosseinsky, D. R. Chem. Rev. 1965, 65, 467. (24) Dunn, T. M.; McClure, D. S.;Pearson, R. G. Some Aspects of Crystal Field Theory; Harper and Row: New York, 1965.
Picosecond Laser- Induced Fluorescence Polarization Studies of Mitoxantrone and Tetraklsporphine/DNA Complexes Yizhong Shen, P.Myslinski, Teresa TreszczanoWicz,+Yixian Liu, and J. A. Koningstein* The Ottawa- Carleton Chemistry Institute, Department of Chemistry, Carleton University, Ottawa, Ontario, Canada KlS 5B6 (Received: March 2, 1992; In Final Form: May 15, 1992)
The picoaecond time-resolved fluorescence spectra of complexes of the drug molecules tetrakis(4-N-methylpyridy1)porphine (H2TMPyP)and mitoxantrone (MX) complexed with DNA in phosphate buffer solution are reported. Timeresolved emission spectra of the drug molecules permit the assignment of fluorescence bands from intercalated MX and HzTMPyP as well as that of externally bound H2TMPyPto DNA. The assignment of fluorescence spectra is facilitated by the results of both isotropic and anisotropic emission spectroscopies. The decay of emission of the intercalated drug molecules is 240 f 50 ps for MX and 10.0 f 0.5 ns for HzTMPyP,while for externally bound H2TMPyPthe decay time is 1.5 f 0.5 ns.
I. Introduction Complexes of mitoxantrone (MX) and tetrakis(4-N-methylpyridy1)porphine (HzTMPyP, Figure 1) to deoxyribonucleicacid (DNA) have been studied in detail using different techniques (UV-visible, fluorescence, circular dichroism, nuclear magnetic resonance, and resonance Raman).l-s In this work we present the results of a picosecond timaresolved spectroscopic study of the emission spectra of MX/DNA and H2TMPyP/DNA complexes in buffer solutions. The absorption spectra of these compounds in the visible region are characterized by bands between 500 and 730 nm. Red-shifted fluorescence can be induced from MX and H2TMPyP molecules in solution with laser radiation tuned to the wavelengths of these absorption bands. We have chosen the method of polarized timaresolved fluorescence spectroscopy in order to obtain information on solutions of MX and Visiting scientist funded by the Network of Centres of Excellence Programme in association with the Natural Sciences and Engineering Research Council of Canada, Institute of Physical Chemistry, Polish Academy of Sciences, Warsaw, Poland. *Towhom correspondence should be addressed.
0022-3654/92/2096-7182S03.00/0
H2TMPyP, compared to solutions of MX/DNA and H2TMPyP/DNA complexes. This information is obtained from picosecond laser-induced fluorescence decay measurements. Information about H,TMPyP is also obtained from the concentration20 and temperaturedejmdent intensities of the flubands. Theae data permit one to (i) obtain the emission spectra of intercalated and externally groove bound H,TMPyP to DNA, (ii) separate the light signals from MX and HzTMPyP free in solution from those of molecules complexed to DNA or different binding components of H2TMPyP, and (iii) obtain information about proton tautomerization in H2TMPyP.
II. ExperimentdSectim Instruments. Fluorescence spectra were induced with tunable radiation from a 6 - p laser source6 or from the fixed frequency radiation of a continuous wave Ar+ ion laser. The main elements of the picosemnd setup were a NdYAG laser (Quantronix Model 116 Head with InfraAction Corp. Model ML-SOD modelocker), a second harmonic generation (SHG) crystal,and a synchronously pumped dye laser which generates 6-ps pulses tunable between 560 and 680 nm (Rh6Gor DCM dye). At 630 nm, the laser used 0 1992 American Chemical Society
Mitoxantrone and Tetrakisporphine/DNA Complexes
The Journal of Physical Chemistry, Vol. 96, No. 19, 1992 7783 Circular
U C H $ W C H , C H , O H
I
OH
II
0
Dichroism
Spectrum
I
NHCH~CHZM~CH&M~OH
Mitoxantrone
-0.50
I
I
300
400
X
L ' +d CH3'
500
1
600 7 0 0
(nm)
Figure 2. Circular dichroism spectrum of a H2TMPyP/DNA complex (r(c) = 10) in buffer solution. 3"
HZTM4rP Figure 1. Molecular structures of MX and H2TMPyP.
for excitation generates stable picosecond pulses with 250 mW of average power. The repetition rate of the laser pulses was reduced by passing the l i t beam through a low-voltage travelling wave electrooptic modulator (Lasemetrics Model 3 130F). The repetition rate of the laser was 270 kHz. The emitted light from the sample was dispersedby a Jarrell-Ash double monochromator which operated in the subtractive mode. The light was detected by a Hamamatsu R1564U multichannel plate photomultiplier cooled to -20 OC. Once amplified, the signals generated by the single-photon detector were fed into a constant fraction discriminator (Tenelec Model 3130F). The time to amplitude converter output was digitized (Canberra Model ADC8075) and accumulated in a 4K multichannel analyzer PC board working under multitasking graphic/software for PC/AT microcomputer. The entire time response of the system was 42 ps. Time-resolved fluorescence spectra were obtained by recording the signal contained in a fixed number of selected channels as a function of wavelength. Sample Haodlhg. DNA and drug samples were gifts from Dr. Y.Yevdokimov. The DNA was isolated from chicken erythrocytes. Molecular mass of the DNA is (5-7) X lo", the ratio of o.d.(260 nm)/o.d.(280 nm) is 2.0. Emission spectra were measured from solutions which were prepared from a stock buffer solution containing MX or H2TMPyP and a buffer solution of the DNA with concentration of 150 @/mL. Phosphate buffer (0.01 M phosphate, 0.3 M NaCl, pH 7.0) was used to dilute all drug and DNA solutions. The composition of the MX/DNA and H,TMPyP/DNA complexes were expressed in terms of an r(c) number, where r(c) represented the molar ratio of DNA base pairs to MX or H2TMPyPmolecules. The r(c) number ranged from 1.8 to 1600 for the H2TMPyP/ DNA complexes and was approximately 30 for the MX/DNA samples. Fresh samples were prepared before each measurement. A b sorption spectra and occasionally circular dichroism (CD) spectra were recorded for quality control. The spectra were taken from solution in air-tight quartz cells.
III. Results and Discussion Abeorption and Emission Spectn. There exist extensive studie~'*&'"'~ on the nature of bonding between H2TMPyP, MX,and DNA. For H2TMPyP/DNA complexes, the porphine molecule may be either intercalated or externally bound to the DNA. This conclusion was reached from spectroscopic studies including UV-visible absorption circular dichroism, flow dichroism, fluorescence,electron spin resonance, nuclear magnetic resonance, and resonance Raman.'J3-'' The overall results from these ex-
periments suggest that intercalated complexes are formed between planar four-coordinated porphine [M = H2, Cu(II), and Ni(II)] and G-C base pairs of DNA. Conversely, for a porphine having axial ligands, a groove-bound complex is formed with A-T base pairs of DNA. Intercalation results from a r-r overlap of electrons of the porphine and the purine/pyrimidine ring of DNA. The porphine is externally bound when the nonplanar porphine fits into the major/minor groove and interacts with the DNA electrostatically via the negative groove surface potential of the DNA duplex.'J8J9 A CD spectrum of a H2TMPyP/DNA complex with r(c) = 10 is shown in Figure 2. The positive signal at 420 nm has been attributed to externally bound H2TMPyP,and the negative signal at 440 nm is associated with the intercalated H2TMPyPS2 If blue radiation of a continuous-wave Ar+ ion laser enters a phosphate buffer solution of H2TMPyP, in additioin to the red fluorescence of this compound, the Raman band of water with a shift of 3500 cm-I is also observed. By using the intensity of the Raman shift as an internal standard, values of the relative cross section of fluorescence as a function of concentration of pure H2TMPyP or H2TMPyP/DNA complexes have been obtaindam The spectroscopic data show that aggregation of H,TMPyP takes place in the buffer solution for H2TMPyP concentration greater than 2.5 X l(rs M. From the value of the cross section of emission from H2TMPyP/DNA complexes with r(c) > 6400, it was detenninedmthat H2TMPyPmolecules occupy only one specific site of the DNA molecule due to intercalation. Upon a decrease in the value of r(c), another site of the DNA molecule becomes gradually occupied where the molecules are externally groove bound to DNA. An equilibrium distribution for the H2TMPyP molecules over these two sites is achieved for r(c) < 3200 complexes. Contribution to the overall fluorescence signal from nonbound H2TMPyP molecules becomes detectable for smaller values of r(c). Figure 3A is an absorption spectrum of H2TMPyP/DNA complexes at r(c) 10. Figure 3B is an absorption spectrum of MX/DNA complexes at r(c) = 30. Absorption spectra of H2TMPyP/DNA and MX/DNA complexes are red-shifted in comparison to spectra of H2TMPyP and MX molecules in the buffer solution. However, larger differences exist in the fluorescence spectra of the complex of H2TMPyP/DNA and H2TMPyP in the buffer solution in comparison with complexed and nonbonded MX. IsotropicFlwuesceace Decry Meewrements and Tcme-Rdved Spectra. Figure 4A shows the fluorescence spectra of H,TMPyP/DNA complexes (r(c) 45) in a phosphate buffer solution. The sample was excited with picosecond radiation at 57 1.7 nm, and fluorescence was measured in the spectral region from 620 (16 130 cm-I) to 760 nm (13 160 cm-I) and recorded from 0 to 6 ns after the excitation. Whereas the spectrum of the complex shows peaks at 662 and 718 nm, the emission of H2TMPyPin the buffer solution (not shown here) is characterized
-
-
1184 The Journal of Physical Chemistry, Vol. 96, No. 19, 1992 2.0
-A
TABLE I: Radiative Decay Times and Relative Contribution to the Fluorescence Intensity of Intercalated and Externally Bound Porphine Of H,TMPVP/DNA” complex r(c) decay time (ns) contribution (56) 112 9.1 84 intercalated 1.5 16 external 45 10.2 81 intercalated 1.7 18 external 11 10.0 79 intercalated 1.9 21 external 5.4 10.6 56 intercalated 1.1 11 external 4.1 33 non-bonded
HzTMPyP/DNA
I 5 -
z
E
B
1.0-
am a
0,5t 3 00
\ 400
5 00
600
Shen et al.
700
Xhm)
“Estimated error: in decay times h0.5 ns; in contribution 10%. TABLE II: Temperature-Dependent Contribution to Fluorescence by Intercalated and Externally &und HzTMPyP of HzTMPyP/DNA Complexes’ contribution (%)
MWDNA
?
complex r(c)
intercalated
external
112
84 92 81 90 19 93
16 8 18 9 21 7
45 11
temp (“C) 20 50 20 50 20 50
Estimated error: in contribution 10%. 50 0
600
700
000
X(nm)
Figure 3. Absorption spectra of H2TMPyP/DNA (r(c) = 10) and MX/DNA complexes (r(c) = 30) in buffer solution.
by a featureless broad band between 625 and 790 nm.I4 The fluorescence s p e c ” of H,TMPyP/DNA buffer solution contains contributions of several species: intercalated bound H,TMPyP, externally bound H,TMPyP, and nonbonded H,TMPyP. From the isotropic decay of solutions with different r(c) numbers, the following lifetimes of fluorescence (q)of these species were obtained: 10.0 f 0.5, 1.5 f 0.5, and 4.0 f 0.5 ns. The isotropic decay of HzTMPyP in buffer solution ( M, 20 “C) is monoexponential and the lifetime of fluorescence (711) is 4.0 f 0.5 ns, so 4.0 ns is corresponding to nonbonded H2TMPyP. For solutions with r(c) > 10 complexes, the emission from nonbonded H,TMPyP can no longer be detected with the picosecond setup. The decay of the spectrum shown in Figure 4A is biexponential (T,, = 10.2 ns and tfl = 1.7 ns), so the spectrum is made up of contributions of intercalated and externally bound H2TMPyP. The emission of the 10.24s component was shown in the time-resolved spectrum given in Figure 4B. Here the emission is recorded in the time period of 9-1 5 ns after the picosecond laser pulse. During that period the emission of 1.7-11s component is too weak to contribute to the overall spectrum. From an amplitude ratio 1(662 nm)/Z(718 nm) of the 10.2-ns component, we conclude that the 10.2-ns component is intercalated bound H2TMPyP.20 The spectroscopic signature of externally bound H,TMPyP was obtained as follows: an analysis was made of the amplitudes at t = 0 of the fluorescence with decay times of 10.0 and 1.5 ns at 11 different wavelengths of the red emission. The intensity amplitudes as well as the isotropic decay times of all compounds used in the investigation were obtained by analyzing the measured fluorescence decay curves using the standard nonlinear leastsquares iterative convolution method based on the Marquardt algorithm. Adequacy of the exponential decay fitting was judged by inspection of plots of weighted residuals and by the statistical parameter x 2 (the reduced chi square) and SVR (the serial variance ratio).,’ A plot of the t = 0 intensity amplitudes for the emission processes (with radiative decay times of 1.5 and 10.0 ns) versus wavelength is shown in Figure 4C. Within the experimental error
of the analysis, the spectrum for intercalated H,TMPyP is the same as that shown in Figure 4B. The emission of externally bound H,TMPyP is stronger than that of intercalated H2TMPyP. It is relevant that an earlier studyI4 found the intensity of H,TMPyP fluorescence in the presence of poly(d(A-T)) to be stronger in comparison to that of H,TMPyP and poly(d(G-C)) DNA. This strengthens the present assumption that intercalated H,TMPyP shows a preference for bonding with G-C base pairs. An assignment of three components in the H2TMPyP/DNA buffer solution is rfl(intercalated H,TMPyP) = 10.0 f 0.5 ns q(externa1ly bound H2TMPyP) = 1.5 f 0.5 ns rfl(free H2TMPyP) = 4.0 f 0.5 ns Shown in Table I is the result of an analysis of contribution to the fluorescence by intercalated and externally bound H,TMPyP and nonbound components with different r(c) values. As shown in Table 11, the intensity of fluorescence of the intercalated HzTMPyP increases with an increase of temperature. For an increase in the r(c) number from 11 to 110, the fluorescence of the intercalated H,TMPyP increases over that of externally bound porphine at both 20 and 50 OC. Figure 5A shows the picosecond laser (at 590.2 nm) induced emission spectrum of MX/DNA complexes with r(c) =S 30 together with the emission spectrum of MX in the buffer solution. The fluorescence spectrum of the complex is red-shifted but the contours are similar. The intensity of the shoulder toward the longer wavelengths is weaker for the MX/DNA complex. The isotropic fluorescence decay of the red emission of the complex solutions yields so(MX/DNA) = 240 f 50 ps, while for a lW5 M MX in the buffer solution the radiative decay of MX is characterized by T ~ ( M X = ) 160 f 50 ps. The time-resolved spectra of MX/DNA complexes (r(c)= 30) was shown in Figure 5B. The spectrum was recorded for points in time between 480 ps and 2.5 ns after the onset of the picosecond laser pulse which induced the emission. For comparison, another MX/DNA spectrum recorded between 0 and 2 ns was shown Figure 4B. MX is intercalated and may also be externally bound to DNA.3Z The contribution to the overall spectrum of emission from externally bound MX and free MX cannot be detected. tropic Fluoresceace Ikcay Me~surementaIn this section, the results of polarized fluorescence spectrum are discussed, and
The Journal of Physical Chemistry, Vol. 96, No. 19, 1992 7785
Mitoxantrone and Tetrakisporphine/DNA Complexes I
I
I
'
I
I
I
'
A 318.00-
m
5212.000
106.00-
0.00
620
643
667
713 wavelength( nm) l
F
736
690
'
1
7
1
r
1
i
I8000-
760
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'
B
I-
z
3
s
318.00
-
6oooL 6000-
A A
m
42
6212.00 1 0
u
i
12000 12000-
600 600
700
t COUN
106.00
0-2ns
MXJDNA
400 0.00
620
643
667
690
713
736
760
wavelength( nm)
300
42 200
318
I
t
100
Xhm)
Figure 5. (A) Fluorescence spectra of MX and MX/DNA complexes in buffer solution. (B) Time-resolved fluorescence spectrum of MX/ DNA complexes in buffer solution.
620
643
667
690
713
736
760
(nm) Figure 4. (A) Fluorescence spectrum of H2TMPyP/DNA complexes in buffer solution. (e) Timeresolved spectrum (recorded 9-15 ns after the excitation) of H,TMPyP/DNA complexes in buffer solution. (C) Fluorescence spectra of intercalated (broken line) and externally bound (solid line) H,TMPyP to DNA.
all experimental data are shown in Figures 6 and 7. The time-dependent anisotropy for molecular emission is given by22 r(t) = W2(u'(O).u'(t))
(1)
where P2 is a Legendre polynomial of order 2 and the angular brackets stand for a spatial ensemble average. In addition, Z(0) is the transition moment at a time t 3: 0 at which the short laser
pulse induces the emission, and Z ( t ) is the transition moment at a time 1. If an analyzer is placed between the sample and the monochromator, then
where Ill(t) and I, ( t ) are time-dependent fluorescence intensities polarized parallel and perpendicular to the electric vector of the laser respectively (see also section 11). For a rigid spherical rotor r ( t ) = r(0) exp(-t/.r,)
(3)
where .rr is the rotational decay time and r(0) is the amplitude of rotational correlation. For the molecules with parallel transition moments for emission and absorption, r(0) = 0.4; in the case of
Shen et al. so0 -
300 -
H 2TMPyP
MX
1,Itl
400
'+.,
; 0
-
t 2
4
6
e
ns
2
4
6
8
ns
r(t) 0,s 0,41 0.3
I
2
I
B
H2T MP y P I DN A
I
4
3
6 n s
5
counts,
MX/DNA
400
t
0
5
IO
15
20
25
30 nr
Figure 7. (A) Decay curve of the fluorescence polarized parallel and
0 I
I
3
2
4
I
6
ns
Figure 6. (A) Decay curve of the fluorescence polarized parallel and
perpendicular to the polarization of the exciting light and rotational relaxation curve of MX in buffer solution. (B) Decay curve of the fluorescence polarized parallel and perpendicular to the polarization of the exciting light and rotational relaxation curve of MX/DNA complexes in buffer solution. the directions of the transition moments for emission and absorption are not parallel but are at an angle r(0) = 0.2(3 cosz e - 1) (4) For rigid nonspherical rotors, r(t) decays are more complex and eq 3 becomeszz 5
r(t) = 40)Cui exp(-t / 7,) I=
I
(5)
Eai = 1 The data for MX in the buffer solution (Figure 6A) indicated
perpendicular to the polarization of the exciting light and rotational relaxation curve of H2TMPyP in buffer solution. (B) Decay curve of the fluorescence polarized parallel and perpendicular to the polarization of the exciting light and rotational relaxation curve of H2TMPyP/DNA complexes in buffer solution. that only one rotational relaxation time can be detected: 300 f 100 ps. Therefore, MX must be a rigid molecule because r(0) = 0.4. The experimental results obtained in this laboratory by the method descibed in ref 20 indicate that bonded and nonbonded MX were present in solution of the r(c) = 30 MX/DNA complexes. However, deconvolution of the isotropic decay of the red emission of the r(c) = 30 MX/DNA complexes indicated that within the approximation of the method used, the emission was from only one species: intercalated MX. Conversely, the anisotropic fluorescence measurements of MX/DNA show a rapid decay from 0 to 5 ns, followed by a leveling off to r(r = 10 ns) = 0.13 (Figure 6B). The initial rapid decay time is 200 i 50 ps from r(0) = 0.4 which was thought to be due to the presence of nonbonded MX as well as wobbling of MX in DNA.23J4f7A p parently, the experimental data for the temporal behavior of r(t),
Mitoxantrone and Tetrakisporphine/DNA Complexes which was not deconvoluted, are more sensitive to the presence of small amounts of nonbonded MX than the deconvolution procedure applied to the isotropic decay. This is probably due to the fact that the rotational relaxation time, as well as the duration of fluorescence decays, are smaller than 400 ps and comparable with the response time of the instrument. Polarized emision for a buffer solution of H2TMPyPare shown in Figure 7A. Contrary to the polarized fluorescence measurements for MX in the buffer, for H2TMPyPr(0) = 0.1. However, whereas MX does not exist as tautomer, H2TMPyPdoes, and the rate of proton tautomerization and the excitation wavelength for fluorescence can influence the temporal behavior of the rotational decay function as explained below. An assignment3' has been proposed for the absorption and fluorescenoespectra of the compound tetraphenylporphyrin (TPP). The position of Q(x)(O,O) and Q(x)(l,O) are at 647 and 591 nm, and bands of the absorption spectra at 549 and 515 nm are assigned to Q(y)(O,O) and Q(y)(l,O), respectively. Maxima in the fluorescence spectrum at 652 and 719 nm are assigned to Q(x)(O,O) and Q(x)(O,l). A comparison of these data with the position of bands of the fluorescence and absorption spectra of H2TMPyPshow similarities, and it is assumed that the assignment made for TPP is also valid for H2TMPyP. The polarized fluorescence data of Figure 7 were obtained for fluorescence at 660 nm, and the wavelength of the exciting radiation was at 566 nm. This suggests that in the experiments fluorescence was excited with radiation in resonance with overlapping absorption bands composed of transitions between Q(x)(l,O) S(0) and Qcv)(O,O) S(0) electronic surfaces. The direction of polarization of the red emission is parallel to the x-axis and from eq 4 we get 0.4 > r(0) > 4 . 2 . In case the rate of proton tautomerization is faster than the fluorescence decay, the emission is ( x y ) polarized. For the combination where absorption occurs via a transition moment which is parallel to they (or x) axis, followed by (xy) polarized emission
-
-
r(0)
0.2
X
2/wJ(3
cos2 0 - 1) d0 = 0.1
The observed value r(0) = 0.1 is presently interpreted in terms of the fast tautomerization process. The rotational decay time for a M H2TMPyP in the buffer solution is 300 f 75 ps. The temporal behavior of r(t) of a chromophore imbedded in DNA is complicated. For instance, for ethidium bound to DNA there are at least three different processes which contribute to the depolarization of the emission of the chromophore. Rapid initial decay has been attributed to wobbling of the drug in the intercalation site.23-25-27Further decay of the polarization anisotropy in the 1-50-ns region results primarily from torsional motion of DNA and the long-time decay arises from bending of the DNA helii?3.26*2"30Similar to the results of MX/DNA, the initial decay of r(t) for the r(c) a 37 H2TMPyP/DNA (Figure 7B) is also in part due to the presence of nonbonded porphine. However,the isotropic decay of intercalated porphine is the longest of the decays of all species present in the solution and the temporal behaviour for r ( t ) where 2 ns < t < 15 ns (r(t) = 0.1) is within the experimental error equal to that for intercalated MX for 4
The Journal of Physical Chemistry, Vol. 96, No. 19, 1992 7787 ns < t < 10 ns (r(t) = 0.12). The absolute value of r(t) = 0.1 to 0.12 for the drug/DNA complexes reported in this work is considerably smaller than the proposed limiting value of 0.23 associated with internal motions of the DNA and bound chrom~phore.~~ Acknowledgment. This research is supported by grants of the Natural Sciences and Engineering Research Council and the Network of Centres of Excellence Programme in association with NSERC. We thank Dr. A. G. Szabo and Dr. D. T. Krajcarski for their assistance with the iterative convolution procedure. We thank Dr. Y. Yevdokimov for supplying the DNA and the drug samples. We also thank Nichole Michelin for helpful discussions.
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