J. Phys. Chem. 1993,97, 6155-6160
6155
Structure Studies of HzTMpyP4 and ZnTMpyP4 Bound to DNA Using Laser-Induced Dichroism in Solution Yixian Liu and J. A. Koningstein' The Ottawa- Carleton Chemistry Institute, Department of Chemistry, Carleton University, Ottawa, Ontario, Canada KIS 5B6
Received: December 15, 1992; In Final Form: February 3, I993
A laser-induced dichroism experimental method (LIDE) was used to study the structure of tetrakis(4-Nmethylpyridy1)porphine (H2TMpyP4) and its zinc derivative (ZnTMpyP4) bound to DNA. On the basis of our LIDE results, the structure of H2TMpyP4 bound to CT DNA is given. Our LIDE results reveal that there are two kinds of externally groove-bound structures for ZnTMpyP4 bound to CT DNA. One is bound in a minor groove with an angle of 30" f 5" between the transition moments at 550 (in the ZnTMpyP4 molecular plane) and 290 nm (in the base pair plane), while the other is bound in a major groove having an angle of 50° f 7" between the transition moments at 564 (in the ZnTMpyP4 molecular plane) and 290 nm.
I. Introduction There is a considerableamount of information available in the literature on the binding modes of H2TMpyP4 and ZnTMpyP4 to nucleic acids in solution.l-1° Using an electric field induced linear dichroism technique, Geacintov et al.9 have reported that benzo[a]pyrene-7,8-dihydrodiol 9,10-oxide (BPDE) does not intercalate to DNA. It was found that the plane of the pyrenelike BPDE chromophore lies on the surface of a cone whose axis is that of the DNA helix and whoseangleis 35" or less. Geacintov et al."J have also reported some experimental results on the orientation of H2TMpyP4 and ZnTMpyP4 bound to CT DNA using a linear dichroism spectral method. There is still no experimentalinformation available in the literature on the spatial structure of H2TMpyP4 and ZnTMpyP4 bound to nucleic acids. Time-resolved polarization spectroscopyusing pump and probe laser beam techniquescan obtain detailed information on relative directionsof transition moments for transitions between electronic surfaces and on the value of the rotational correlation time of molecules in ground and electronic excited statesO1l-l6In this work, we report the experimental results and calculations based on the theory published earlier by this laboratoryl4J6 using laserinduced dichroism for HzTMpyP4 and ZnTMpyP4 bound to CT DNA in solution. Different kinds of experiments were carried out in the visible and UV regions to determine the orientation of the plane of H2TMpyP4 and ZnTMpyP4 bound to CT DNA relative to that of the DNA base pair plane. Our results also reveal that ZnTMpyP4 bound to CT DNA occupies minor and major grooves.
II. Experimental Section A. Instrumentation. Absorption spectra of H2TMpyP4 and ZnTMpyP4 in different solvents were obtained on a Lambda 4B UV-vis spectrophotometer, and laser-induced dichroism experiments (LIDE) were carried out on a Jobin-Yvon double monochromator. An UV filter and UV photomultiplier detector system were also used if an UV probe beam was used. The tunable laser source for UV-vis came from an excimer laser (Lumonics Model TE-320-2 with repetition rate of 25 Hz) simultaneously pumping two tunable dye lasers (Lumonics Model hyperDYE300). One dye laser, as a pump source, was tuned to the visible absorption band (500-600nm) of the sample and another dye laser, as a probe source, was tuned to the Soret band or was frequency doubled to the UV band of DNA using a BBO crystal. These two beams were separated using a quartz prism. The UV light, with pulse duration of 5 ns, can be tuned from 230 to 300
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0022-365419312097-6155!§04.00/0
nm, which is located in the nucleic acid bases' absorption band. The polarization direction of the probe beam was rotated relative to that of the pump beam using a combination of a GlanThompson polarizer with either a broad band polarization rotator (in the visible region) or an UV half-wave plate at 290 nm (in the UV region). After passing through a dichroic mirror, the pump beam and probe beam were coaxial and were focused with a f = 20 cm quartz lens on a quartz sample cell with 1- and 2-cm optical paths. The transmitted probe beam coming from the quartz cell was collected onto the slit of the double monochromator (for the probe in the visible region). The signal was detected by a cooled RCA 31034 photomultiplier with nanosecond response and then amplified by a fast preamplifier (Stanford Research Systems Model SR 445). The UV detector system was used when the probe beam was in the spectral region 260-290 nm. The amplified signal was sent to a boxcar operating with a 3 4 s gate (Stanford Research Associates Models Sr 250, 235, 275, and 245). The scanning of the monochromator, as well as the gate of the boxcar, was either controlled by a Raven PC-XT computer or recorded by a recorder. B. Sample Handling. The buffer solution used for all experiments contained0.01 M sodiumphosphateand 0.3 M NaCl with pH 7.0. The porphyrin samples were gifts from Dr. Y. Yevdokimov (refs 3 and 5). A stock buffer solution containing H2TMpyP4at 1.47 X 10-3 M and another stock buffer solution containing ZnTMpyP4 at 1.1 X M were prepared. As well, a stock solution of CT DNA (Sigma, type I, the absorption ratio of 2601280 is 1.9) with 1 ng of DNA dissolved in 1 mL of buffer solution was prepared. The concentration of the diluted DNA base pairs was found using the molar extinction coefficient t = 6600 M-1 cm-1 (at 260 nm)17 (it was about 4 X 1 V M in our experiments). The chemical structures of H2TMpyP4 and CT DNA are shown in Figure 1. All stock solutions were stored at 5 OC, and fresh samples were made 24 h in advance of each experiment. All of the experiments were conducted at room temperature (20 "C). 111. Results
A. Theoryof Laser-InducedDichroIsmofMoleculesinSolution. In laser-induced dichroism experiments (LIDE), one uses two pulsed laser beams whose wavelengthsare tuned to two absorption bands of a molecule in solution. One of the beams is used as an optical pump and creates excited states; the other beam is employed as a probe beam. In the situation where the rotational correlation time of the molecule is longer than the pulse duration, the pump creates a spatial anisotropyin the distribution of ground 0 1993 American Chemical Society
Liu and Koningstein
6156 The Journal of Physical Chemistry, Vol. 97,No. 23, 1993
1
,003
H2TM P y P4
V -.0061
380
I
I
I
450
400
480
WAVELENGTH ( n m l
Figure 2. Second-derivative absorption spectrum of the H2TMpyP4DNA complexes in the Soret band for r = 200.
DNA
Figure 1. HzTMpyP4 and DNA chemical molecular structures.
states, and this anisotropycan be measured with the probe beam. This is done by measurement of the intensity of the transmitted probe beam with the pump on (I(on)) and off (Z(off)). In earlier publications from this laboratory it was shown that16
In [Z(on)/I(ofnl = -A[g(aG,y,4- 11
(1)
where
A=
(2)
afLN
and g(aG,y,a) = sin' y
sin'
+ v(a)cos'
a']
+
where (4) and
where cr: is the cross section for the ground-state absorption of photons from the probe beam, L is the length of the optical path of the sample, and N represents the total concentration of molecules absorbing the probe beam in the ground state. The a coefficient denotes the strength of the pump beam, aG is an angle of the probing dipole with respect to the pumping dipole, and 7
is the angle between the electric vectors of the pump and the probe beams. An example of a theoretically calculated result based on eq 1 for a = 1 is shown in Figure 3a of ref 16. LIDE can be used to determine the angle between transition dipole moments for absorptions at the wavelength of the pump and probe which, in turn, can give structural information, as will be shown below where we discuss aspects of molecules which are attached to DNA. As is well-known, molecules such as HzTMpyP4 and ZnTMpyP4 can form complexes with the large molecule.6J0.25 In the case of HzTMpyP4, evidence is available that this molecule intercalates and can become externally bound to DNA; for ZnTMpyP4, only an externally bound complex has been fo~nd.~JOJ~ B. LIDE of H2TMpyPcDNA Complexes. The second derivative of the absorption spectrum of the HzTMpyPrDNA complex with r = 200 (Soret band) is shown in Figure 2; the r value is a ratio of the concentration of base pairs of DNA to the total concentration of porphyrins. The absorptionspectrum from 500 to 620 nm is due to the Qv and Qx, bands and the Soret band is at 430 nm. From the second-derivative spectrum, it appears that the Soret band has components which are at 427 and 445 nm, in agreement with CD spectra of H2TMpyP4-DNA?*6.ZS which are assigned to absorptionsassociated with externally bound and intercalated H2TMpyP4, respectively. A recent resonance Raman excitation profile showed that there are four maxima: at 4 19and 427 nm, for the externally bound H2TMpyP4molecule, and at 434 and 441 nm, for intercalated HzTMpyP4.3 These maxima have been assigned to the position of the 0-0 electronic origins in the Soret band of two tautomers for intercalated and externally bound HzTMpyP4 (see Table I). If the sample is exposed to laser radiation in the region 400640 nm, we observe fluorescence which extends from 620 to 750 nm. UV selectiveexcitation and time-resolved spectroscopy show that the fluorescence spectrum of intercalated HzTMpyP4 peaks at 654 and 715 nm, and that of externally bound H2TMpyP4 shows peaks at 667 and 725 nm. The radiative lifetime of F(654) is -10 ns and that of F(667) is -2 ns.5918 If solutions containing the HzTMpyPd-DNA complex are exposed tolaser pulses (Cl-mW power) with 5 4 s duration tuned to thespectral interval 5 10-530nm, effectivepopulationsaturation of the S1(Q,,) state of intercalated HzTMpyP4 occurs. For higher power levels, the Sl(Q,,) state of externally bound HzTMpyP4 can also become populated and LIDE can be performed because the rotational relaxation time of the H2TMpyP4-DNA complex is much longer than the 5 4 s duration of the laser pulses. The results of these LIDE measurements are shown in Figures 3 and 4, where the solid lines are theoretical results based on eq 1. From the absorption spectrum for HzTMpyP4, it follows that A 0.3 at 520 nm, and pumping at 519 nm followed by probing at 427 and 419 nm gives results (see Figure 3) that are consistent with a situation in which the angle between the transition moments for pump and probe beam are parallel (519 and 419 nm) and
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Structure Studies of HzTMpyPeDNA and ZnTMpyP4-DNA
TABLE I: Assignment of Part of the Fluorescence, RRP, LID& and Absorption Spectrum of HzTMpyPd-DNA' abs LIDE RRP flu assignment (nm) (nm) (nm) (nm) 245 UV band UV band 280 290 1 4 A-T base pair 419 OCO tautomer 1 ext 423 Soret band OCO tautomer 2 ext 427 434 OCO tautomer 1 int 437 Soret band OCO tautomer 2 int 44 1 519 Q: 2 4 both tautomers ext 520 Q: 2 4 both tautomers int and ext 525 Q: 2 4 both tautomers int 565 Q: 1 - 0 both tautomers int and ext Qx: 2 4 both tautomers int and ext 600 Qx: 1 - 0 both tautomers int and ext 635 654 Qx: 0 - 1 both tautomers int 667 Qx: 0 - 1 both tautomers ext 715 Qx: 0 - 2 both tautomers int 725 Qx: 0 - 2 both tautomers ext a abs, absorption; LIDE, laser-induced dichroism experiment; RRP, resonance Raman excitation profiles; flu, fluorescence; int, intercalated component; ext, externally bound component.
The Journal of Physical Chemistry, Vol. 97, No. 23, 1993 6157
r = 65
Probe=427nm A=i.5 a=0.64
c.17
t
Pump=525nm
A=1.5 a=0.53 Probe=419nm aG=600
10
0
40
30
20
50
70
60
90
80
y (degrees)
Figure 4. Same as in Figure 3, but the pump is at 525 nm (intercalated HzTMpyP4 molecular plane) and probing is at 419 and 427 nm, which are transitions of groove-bound H2TMpyP4, L = 20 mm.
r = 85 Pump=519nm 0.15
2bo
240
220
3bo
2;10
WAVELENGTH (nm)
Figure 5. Second-derivative absorption spectrum of the HzTMpyP4DNA complexes with r = 200, in the UV region.
0.05
t
O . " ,
0.24
A==0.42 a=1.5
aG-00
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1
.
A=0.9 a=i.5 Probe=419nm
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.
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perpendicular ( 5 19 and 427 nm). Apparently, the pump is tuned to coincide with an electronic transition in the Q 2 4 band of one of the tautomers of the externally bound molecule. If the wavelength of the pump is tuned to 525 nm and that of the probe is at 433.6 and 441 nm, we obtain the result that transition moments are parallel (525 and 441 nm) and perpendicular (525 and 433.6 nm) (results not shown in the text). We conclude that, at 525 nm, we pump into the Q 2 4 band of one of the tautomers of intercalated HzTMpyP4. Shown in Figure 4 is LIDE for pumping at 525 nm, which is in the Q, 2 4 band of one of the tautomers of intercalated H2TMpyP4, while probing the external molecules at 419 and 427 nm. The direction of the transition moment at 419 nm has an angle of 60° f 8 O with respect to the pump transition moment at 525 nm. The angle between transition moments at 419 and 427 nm is 50° f 7O. If the wavelength of the pump is tuned to 519 nm and that of the probe is at 433 and 441 nm, we obtain angles of 40° f 7O (between transition moments at 519 and 433 nm) and 60° f loo (for the transition moments at 519 and 441 nm).
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Pump=518.7nm Probe=ZgOnm
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The second derivative of the absorption spectrum of the HzTMpyP4-DNA complex between 220 and 300 nm is shown in Figure 5 . If the fluorescence in the red spectral region is induced with a laser which is tuned to that interval, we find that for 260 nm the spectrum is primarily that of the intercalated molecule, while that of the externally bound molecule appears if the laser is tuned to -290 nm.I9l26 This observation is in agreement with the interpretation of the CD spectrum of the HzTMpyPd-DNA complex.~8.zsLIDEfor 5 19-nmpumpand 290-nmprobeisshown in Figure 6, where the solid line is a theoretical curve based on
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6158 The Journal of Physical Chemistry, Vol. 97, No. 23, I993
Liu and Koningstein 0.03
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ZnTldpyP4-DNA
r = 60 Pump=550nm Probe=ZBOnm A=0.2: a=0.7 Z
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ZnTbfpy P4-DNA r = 60 Pump=564nm Probe=ZBOnm A=0.2: a=0.7
0.017-
where h I 0
1+
[ I - u(a)] cos2 aE
cos' y ([I - u(a)] sin' aE
+
0
.
-
4
r
r
'= -0.009 v
9
+
uE/uG=1.2
[ l - h(a)] cos' aE) ( 7 )
u: is the cross section for the excited-state absorption of photons from the probe beam, a@ is the angle of the probing dipole with respect to the pumping dipole absorbed at the excited state, and u(a) and h(a) are functions defined as before. The theoretical calculation curves based on eq 6 are shown in Figure 4a,c of ref 16, for A = 1, (I = 1, aE = ' 0 and aE = 90°, respectively. In the absorption spectrum of the ZnTMpyP4-DNA complex, from 530 to 640 nm is due to the Qx and Qy bands and the Soret band is at 438 nm. From the second-derivative spectrum, it appears that the Soret band has only one component which is at 438 nm, but from the CD spectrum? it appears that the Soret band has components which are at 433 and 455 nm, according to the binding modes of ref 22. From our LIDE results, we assign the absorptions to a minor and major groove-bound ZnTMpyP4. If the sample is exposed to laser radiation in the region 400610 nm, we observe fluorescence which extends from 580 to 720 nm of only one externally bound ZnTMpyP4-DNA complex. However, UV wavelength selective excitation and time-resolved spectros~opy~~ show that there are two kinds of fluorescence spectra: at 61 8 and 662 nm and at 620 and 660 nm. The radiative lifetime of F(618) is shorter than that of F(62O).l9 The sccond derivative of the absorption spectrum of the ZnTMpyPrDNA complex between 220 and 300 nm is shown in Figure 7. If the fluorescence in the red spectral region is induced with a laser which is tuned to that interval, we find that at 260 nm the spectrum is primary that of one groove-bound. That of the other (groove-bound) molecule appears if the laser is tuned to -290 nm.19J6 These results are in agreement with that of Pasternack et al. on the CD spectrum in which there were two positive peaks at 433 and 455 nm, respectively.6 If solutions containing the ZnTMpyP4-DNA complex are exposed to laser pulses (
