COMPLEX FORMATION AND ENERGY TRANSFER FROM PHOTOEXCITED THEOPYRONENE “‘0 DEOXYRIBGAUCLEIC ACID Thesis for the Degree of Ph D MICHIGAN STATE UNIVERSITY ' s LAum'A‘ _- 1971 This is to certify that the thesis entitled Complex Formation and Energy Transfer from Photoexcited Thiopyronine to Deoxyribonucleic Acid. presented by S. Lalitha has been accepted towards fulfillment of the requirements for PA“ D= degreeinml- " ("is 4 Hum Major 8 5AA. 197/ I)ate 0-7639 “A w... __4...._.-H‘ ABSTRACT COMPLEX FORMATION AND ENERGY TRANSFER FROM PHOTOEXCITED THIOPYRONINE TO DEOXYRIBONUCLEIC ACID BY S. Lalitha Thiopyronine is an effective photosensitizer which inactivates bacteria upon illumination with visible light [Photochem. Photobiol., g, 364 (1964)]. Therefore, the present study was undertaken to obtain information about the mechanism of energy transfer from the photoexcited dye to the deoxyribonucleic acid (DNA). At first, some aspects of the excited states of thiopyronine molecules were investigated. Electron para- magnetic resonance (EPR) and optical studies of randomly distributed molecules of thiopyronine photoexcited into the triplet state have been carried out at 77°K. The zero field Splitting (ZFS) parameter 0* and the phosphorescence lifetime of the lowest triplet state decrease as the con- 6 to lO-ZM. The decrease centration is increased from 10- of this D* values is discussed in terms of intramolecular charge transfer and the delocalization of the triplet electrons over adjacent molecules. At 77°K, thiopyronine S. Lalitha also exhibits delayed fluorescence and an emission probably from electron hole recombination. Furthermore, upon illum- ination in the main absorption band a radical is generated both at 296°K and 77°K. Secondly, thiopyronine-DNA complexes were studied with optical and EPR techniques. From the dependence of the ZFS parameter of the thiopyronine triplet state on the molecular environment, it is concluded that there exist two types of complexes. For low DNA phOSphate to dye ratios (P/D) the thiopyronine molecules are probably stacked at the surface of the macromolecule. For high P/D ratios thiOpyronine behaves like an isolated (inter- calated) molecule. The initial energy transfer from the dye to the DNA may involve the photoinduced radical or energy available through electron-hole recombination (470 nm) and delayed fluorescence (590 nm) of the thio- pyronine excited with visible light. The phosphorescent triplet state of thiopyronine cannot be directly involved in the energy transfer. COMPLEX FORMATION AND ENERGY TRANSFER FROM PHOTOEXCITED THIOPYRONINE TO DEOXYRIBONUCLEIC ACID By S.ALalitha A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biophysics 1971 DEDICATION to my parents and sister ii AC KNOWLEDGME NTS The author wishes to express her appreciation to Dr. A. Haug for his unfailing enthusiasm and guidance throughout these investigations. The helpful advice of the committee members, Dr. E. Eisenstein, Dr. M. Jost, and Dr. B. Rosenberg is also appreciated. The author wishes to thank David Graber for synthesizing the mole- cule. The encouragement of other members in the labora- tory is also gratefully acknowledged. The author would like to thank Dr. H. T. Tien for enabling her to start the graduate studies in the department. I would also like to express my thanks to Dr. A. Lang for enabling me to carry out these investigations in the MSU/AEC Plant Research Laboratory. This work was SUpported under Contract No. AT(ll-l)- 1338. iii TABLE OF CONTENTS Page LIST OF TABLES . . . LIST OF FIGURES O C O O O O O C O O O O 0 Vi ORGANIZATION OF THESIS . . . . . . . . . . viii < GENERAL INTRODUCTION . . . . . . . . . . . 1 PART A: SPECTROSCOPY OF THE LOWEST PHOSPHORESCENT STATE OF THIOPYRONINE. . . . . . . . . . . 3 METHODS AND MATERIAL . . . . . . . . . . . 7 RESULTS 0 O O O O O O O O O O O O O 0 1]- Absorption Measurements . . . . . . . . . 11 Heat of Transition. . . . . . . . . . . 20 Emission Measurements. . . . . . . . . . 20 EPR Measurements . . . . . . . . . . . 31 DISCUSSION 0 O O C C O O I O O O O O O 39 PART B: COMPLEX FORMATION AND ENERGY TRANSFER FROM PHOTOEXCITED THIOPYRONINE TO DEOXYRIBONUCLEIC ACID . 48 Introduction. . . . . . . . . . . . . 48 MATERIALS AND METHODS. . . . . . . . . . . 51 RESULTS 0 O I O O O O O O O O O O O O 53 Absorption Spectra of the DNA/dye Complex . . . 53 Binding Constant Determination. . . . . . . 53 EPR Measurements . . . . . . . . . . . 66 Emission Characteristics of the NA-Dye Complex . 66 DISCUSSION 0 O O O O O O O O O O O O O 77 LIST OF REFERENCES. . . . . . . . . . . . 82 iv Table 1. LIST OF TABLES Page Thermodynamic constant for the dimerisation of thiopyronine . . . . . . . . . . . l9 Phosphorescence lifetimes of thiopyronine in ethanol and phosphate buffer/glycol (pH 7.6) O O O I O O O I O O O 0 3O Corrected phosphorescence peaks and relative intensities of thiopyronine in ethanol, at 77°K, as a function of concentration . . . 30 The Am==l canonical peaks for thiopyronine (5 x 10-3 M) in phosphate buffer/glycol at 9239 MHz 0 I O O O I O O I O O O 32 ZFS energies and g-tensor components derived from the Am = l EPR spectra of thiopyronine (3 x 10-3 M) in phosphate buffer/glycol . . 38 ZFS parameter of thiopyronine in phosphate buffer/glycol (P.B.G.) and in ethanol at 9239 MHz as a function of concentration . . 45 Binding constant of thiopyronine to DNA deter- mined by different methods . . . . . . 67 The ZFS parameter D* for various DNA/dye ratios determined at 77°K in phosphate buffer/ ethylene glycol (pH = 7.6) for a thiopyro- nine concentration of 3 x 10-5 M . . . . 70 Phosphorescence maximum and lifetime of various DNA-dye ratios, for a concentration of thiopyronine of 4 x 10‘5 M in phosphate buffer/glycol (pH = 7.6) . . . . . . 76 ‘7 LIST OF F IGURES Figure Page 1. Molecules structurally related to thiopyro- nine 0 I O O I O O O I O O O O O 5 2. Absorption spectra in ethanol. -------- 10'5 M thioxanthene, 10'5 M thiopyronine . 13 4 3. Absorption spectra of 10- M thi0pyronine as a function of temperature. . . . . . . . 15 4. Absorption spectra of 2.5 x 10-4 M thiopyronine as a function of temperature . . . . . . l7 5. Absorption spectra of 10-5 M thiopyronine -.-.-.-.-. pH 7.6, ---------- pH 1, and pH 13 O O O O O O O O O O 22 6. Fluorescence spectra at room temperature thiopyronine (excitation 564 nm), ---------- thioxanthene (excitation 265 nm) in ethanol at a concentration of 10‘5 M . . 24 7. Phosphorescence Spectra at 77°K thioxanthene,-.-.-.-.-.-. thiopyronine, --------- thiopyronine in ethanol at a con- centration of 10-5 M. ._ . . . . . . . 26 8. Phosphorescence spectra at 77°K in phosphate buffer/glycol thiopyronine 10“2 M, -------- thiopyronine 10-5 M . . . . . . 28 9. Variation of the position of the Am = 2 tran- sition (Hmin) with concentration of thiopyro- nine in ethanol at 77°K. . . . . . . . 34 vi Figure Page 1. Absorption spectra at room temperature in phosphate buffer thiopyronine 2 x 10-5 M, -------- P/D = 0.5 ........ P/D = 50 I O C O O O O O I O O O 55 2. Difference spectra at room temperature in phosphate buffer with a thiopyronine con- centration of 2 x 10'5 M. -.-.-.-.-. P/D = 1, -------- P/D = 5, P/D = 50 . 57 3. Difference spectra as a function of salt con- centration for a P/D of 16 with a thiopy- ronine concentration of 10'5 M. c. 0.1 M NaCl, b. 0.01 M Nacl. a. 0.001 M Nacl . 59 4. Meltin curves for DNA concentration of 5 x 10' M in phosphate buffer as a function of P/D. DNA, -o-o-o-o- P/D = 24, and -+-+-+-+- P/D = 124. . . . . . 61 5. Spectrometeric titration of DNA with thiopyro- nine at 564 nm. DNA concentration is 2 x 10'5 M. For each 10 ul of thiopyronine added, the concentration of thiopyronine increased by 3.7 x 10'7 M . . . . . . 63 6. Changes of the fluorescence intensity for different amounts of DNA. Thiopyronine concentration is 7 x 10'6 M. . . . . . 65 7. Variation of Hmin with P/D for a thiopyronine concentration of 3 x 10'5 M in phosphate buffer/glycol and a microwave frequency of 9239 MHz . . . . . . . . . . . . 69 8. Fluorescence spectra at room temperature 2 x 10‘5 M thiopyronine, ---------- P/D = 150 o o o o o o o o 72 9. Phosphorescence spectra in phosphate buffer/ glycol at 77°K a) ........ thiopyronine 10'2 M, b) thiopyronine 10-5 M, c) -'-'-'-‘-' - P/D = 24, d) -------- P/D = 124. In the blue region the intensity (b,c) is multiplied by a factor of 10 for illustration purposes. . . . . . . . 74 vii ORGANI ZATION OF THES I S The two parts of this thesis are presented indi- vidually in the format of a scientific paper. The references are, however, combined at the end of the thesis. Part of Section A has already been published under the title "Spectrosc0py of the Lowest Phosphorescent State of Thiopyronine" by S. Lalitha and A. Haug, J. Amer. Chem. Soc. 23, 3340 (1971). Section B has been submitted for publication. viii GENERAL INTRODUCTION Thiopyronine, a three-ring hetero-aromatic molecule belonging to the class of xanthene dyes, has been known to be a potent mutagenic and photosensitizing agent of bacterial-5 and viruses.6 Other structurally similar 7,8 dyes, e.g., acridines are also mutagenic and carcino- genic.9 Numerous investigations indicate that the dye molecules complex with the deoxyribonucleic acid (DNA) 10-13 and inhibit the translation mechanism. The dye molecules may intercalate and/or attach to the surface 14-18 of the DNA macromolecule. The principal forces stabilizing this interaction are Coulombic and van der Waals.19 Relaxation kinetics based on temperature jump methods and circular dichroism (CD) experiments for various polynucleotides and DNA have been utilized to study the interaction forces as a function of temper- ature.20-22 The presence of dyes such as acridine, methylene blue, or thiopyronine make living cells such as bacteria, 1,10 viruses sensitive to visible light. These dyes act as photosensitizers. Molecular oxygen is attributed to enhance the photosensitizing effect of these dyes espe- cially in the case of proteins.23 The mechanism of energy transfer from the dye to the DNA and vice versa has eluded many investigators. Several pathways have been suggested for the latter type of transfer. Singlet as well as triplet energy transfer have been proposed and a few of these results have been 24'26 Little explained on the basis of the exciton model. is known about the mode of energy transfer from the dye to the DNA, i.e., the most likely mechanism to explain the visible light sensitization of the ultraviolet absorb- ing macromolecules (DNA and protein). Some aspects of this sensitization have been investigated in the case of proteins23 and DNA.27 Since thiopyronine was found to be an efficient photosensitizer, the present work was undertaken to probe, if possible, into the mechanism of energy transfer from the dye to the DNA. Part A is an attempt to char- acterize thiopyronine with respect to its spectroscopic properties, since it was felt that an investigation of the energy levels of the dye might suggest possible energy states available for the transfer. In Part B experiments on the interaction between the dye and DNA are reported. PART A SPECTROSCOPY OF THE LOWEST PHOSPHORESCENT STATE OF THIOPYRONINE One- and two-ring hetero-aromatic molecules, due to their involvement as building blocks for biological I molecules, have drawn the attention of many investi- gators. Use of spectroscopic techniques such as electron paramagnetic resonance (EPR), absorption, and emission to study these molecules have been wide.28-30 However, the study of three-ring hetero-aromatic molecules has been limited to a few of biological interest such as quinox- 32 33 34:35 ' 31 o o o n o aline, phenoxazine, riboflaVin, and acridines. SpectroscoPic methods have provided some insight into the singlet and triplet levels of the molecules.36-38 In the following, I report the study of the hetero- aromatic molecule thiopyronine, which contains a sulfur bridge connecting two benzene rings (figure 1) in a random matrix at 77°K by optical and EPR methods. Our findings demonstrate that the phosphorescence properties as well as the zero field splitting (ZFS) parameters are con- centration dependent. These properties are discussed in .mcwcouwmownu ou pmumamu maamusposupm mmasomaoz .H musmflm «1:82 wozn.0_1% _ +l wzthz 13 the intensity of the emission decreased. Thiopyronine incorporated in PVA films revealed this emission both at 296°K and 77°K. The emission could be obtained by directly exciting the molecule in the 564 nm absorption band. For this purpose Corning cut-off filters (3484, 3486, A < 520 nm) were employed. By utilizing neutral density filters, the intensity of the emission was found to be prOportional to the incident light intensity. The blue emission of thiopyronine was not as concentration dependent as the red peak (Table 3). The excitation spectrum of this long-lived emission revealed a peak at 280 nm and a small one at 350 nm. The intensity of this emission was also proportional to that of the impinging light intensity. The decay of the emission could be analyzed in terms of two exponential components, one with a lifetime of 0.23 second and another one with a half-life of 1.0 second. At a concentration of 10-4 M, the intensity ratio of these two components was 15:1 respectively. The emission is quenched at higher 30 Table 2 Phosphorescence lifetimes of thiopyronine in ethanol and phosphate buffer/glycol (pH 7.6) Concentration (M) Lifetime in msec (i 10%) Ethanol Phosphate buffer/glycol 9 x 1010 350 - 9 x 10'9 321 - 9 x 10'8 233 228 9 x 10"7 170 134 9 x 10'6 157 107 9 x 10’5 144 89 9 x 10'4 127 81 Table 3 Corrected phosphorescence peaks and relative intensities of thiopyronine in ethanol, at 77°K, as a function of concentration. . (nm) Intensity (nm) Intensity Concengration Amaxz Arbitary Amax.2 Arbitary - Units ' Units 5 x 10'6 703 10 460 1 5 x 10'5 720 16 480 3 5 x 10’4 760 41 480 4 5 x 10‘3 800 82 - - 31 concentrations (; 10-2M) and is apparently insensitive to the oxygen content and pH (1-12) of the sample. Thioxanthene has a phosphorescence maximum at 450 nm. The decay of the triplet state of thioxanthene could be characterized as two exponential decays with lifetimes of 60 msec and 1.0 second. The intensity ratio of these two components were 100 : 1 respectively. EPR Measurements 3 For a concentration of 3 x 10- M the Am = 1 transitions of thiopyronine are given in Table 4. Since the Am = 1 transitions were weak such high con- centrations of thiopyronine had to be used. Varying the concentration of thiopyronine to lower values, resulted in a shifting of the Am = 2 position to lower field strengths (Figure 9). This change in the position of the Am = 2 transition was found to be greater in ethanol than in phosphate buffer. This triplet state could be excited through the 564 nm absorption band. With neutral density filters it was found that the pOpu- lation of the triplet state is proportional to the inci- dent light intensity. Positioning the field on the Am = 2 transition, the lifetime of the triplet state was measured 2 with a mechanical chopper. For a concentration of 10- M, the lifetime of 23 msec obtained with EPR techniques agreed with those measured with the phosphoroscope. Table 4 32 The Am = l canonical peaks for thiopyronine (5 x lO-3M) in phosphate buffer/glycol at 9239 MHz. Field Position Component Gauss Hl(x) 3178 H2(x) 3392 Hl(y) 3651 H2(y) 2959 Hl(z) 3739 H2(Z) 2863 33 .xobn um Hocmsum ca mafia Ioumm0flnu mo cowumuucmocoo suw3 AcHEmV coauwm Icmuu N Sq may no COAUHmom 0:» mo coaumwum> .m musmflm 34 5:22.850 1 332 o. .10. To — - u 1 can. «my 13an 0.0. one. 35 The intrinsic splitting in the molecular triplet state caused by the dipolar spin-spin interaction of the two unpaired electrons is given by the spin Hamiltonian, H = D (522 - (1/3) 52) + E (5x2 - Syz) 7 = - (xsx2 + ys 2 + zs 2) 8 where X + Y + X = 0. D = (1/2) (x + Y) - z = - (3/2) z 9 E = (1/2) (y - X) 10 0*2 = (D2 + 3E2) 11 A resonance in the low field Am = 2 region is observed at H i.e., the minimum field value at which the micro- min’ wave absorption occurs (l/ZgB)2 [(hv)2 - (4/3) 0*2 CG ll . ] 12 min . or *2 (3/4) [(hv>2 - (zge>2 Hm' 2] 13 in D II The zero field splitting (ZFS) parameters are evaluated in terms of the Am = l resonance fields. x = (98,2 / (6 hv) [H2(x)2 - H1(x)2] 14 with similar expressions for Y and Z. 36 Line shape analysis has shown that ._. + Hmin HO + 0.3A- 0.1 15 where H0 is the low field maximum and A is given by A = AHo (1.5 I 0.2) 16 AHo is the difference from the first maximum and the first zero crossing of the signal.42’43 One could also obtain the value of g , and XX’ gyy 922’ the value of the g-tensor in the three principal directions by a method of iteration. 82(x)2 - Hl2 = 2 (ge/gxx)2 (0' - 38') Ho 17 where HO = hv/ge , D' = D/geB , E' = E/geB H ( )2 - H ( )2 = 2 < / )2 (0' + 3E') H 18 2 Y 1 Y 9e gyy 0 H2(z)2 - 81(2)2 = 4 (ge/gzz)2 D'HO 19 (gxx/ge)2 = 20102 - 2D'E' + 2E'2)/[Hl(X)2 + 82(x)21 20 2 2 I n .2 2 2 (9yy/ge) = 2(Ho + 2D E + 2E )/[H1(y) + H2(y) ] 21 37 2 (9122/99)2 = 20302 + 0' - E'z)/Hl(z)2 + 82(z)2 22 The value of D and E obtained by this method could be used to compute D* and compared with that resulting 44 The values calculated by this from the Am = 2 signal. technique are tabulated in Table 5. For thioxanthene a 0* value of 0.1097 cm“1 was obtained from the Am = 2 transition. The radical of thiopyronine appeared at 3285 gauss with a microwave frequency of 9239 MHz. When thiopyronine incorporated into PVA films was examined in the phosphoroscope a third emission was observed at 590 nm. The position of the peak coincided with the fluorescence maximum. The intensity of this emission was proportional to the impinging light intensity. Table 5 38 ZFS energies and g-tensor components derived from the Am = l EPR spectra of thiopyronine (3 x 10- in phosphate buffer/glycol Parameter Value x 0.0066 1 0.0001 cm-1 y 0.0216 1 0.0004 cm"1 z 0.0273 2 0.0004 cm"1 D 0.0410 : 0.0004 cm‘1 E 0.0075 i 0.0002 cm‘1 9 2.003 gxx 1.994 yy 9 1.999 22 DISCUSSION The strong absorption band of thiopyronine at 564 nm results from the interaction of the chromophore, i.e., the dimethyl amino groups with the ring system. These groups can act as electron donors or acceptors due to the non-bonding electrons in the nitrogen atoms.45 The positively charged sulfur atom can easily accept an electron. The molecule is symmetric and this further facilitates formation of resonance structures with the positive charge oscillating between the nitrogen and sulfur atoms. Compared with other structurally related molecules such as acridines the direction of the long wavelength absorption band is probably determined by the 46 Thioxanthene with a sulfur line joining the nitrogens. atom in the unsubstituted ring system has an absorption in the ultraviolet region. As the concentration of the dye is increased a shoulder appears at 530 nm. The optical density at this wavelength increases as the concentration is increased un- til it: becomes a predominant peak. Dyes tend to aggre- gate forming dimers and higher aggregates. Since increas- ing the concentration does not result in a single 39 40 isosbestic point, it seems to imply that a single kind of complex is not formed. Higher aggregate formation might result. Keeping the concentration the same (Figure 3) and raising the temperature is equivalent to diluting the solution, which is consistent with the increase in the 564 nm absorption peak. The increase in the 564 nm intensity indicates a larger number of monomer units. Utilizing the information from the absorption spectra one could calculate the heat of dimerization and the related thermodynamic properties. Since the values for thiopyronine are comparable to other dyes such as thionine and methylene blue, it is quite likely that the molecules are separated by a distance of 3 A°.47"49 In a medium with pH 50.5 the long wavelength absorption peak disappears, hydration of thiopyronine in the carbon bridge is possible, a reaction similar to that of thioxanthene.50 At pH 313 a nucleophilic attack by OH- at the carbon bridge could lead to a disruption of the resonance structure. Starting with a solution at a high or low pH, the optical density at 564 nm can be restored by neutralizing the acid or base as the case may be. However, this reaction is not completely reversible since it depends on the time elapsed before neutralizing, e.g., if the solution is allowed to stand 41 for 15 minutes and then neutralized, only 50% of the original intensity, measured at 564 nm, is restored. At 77°K as the concentration is increased, the phosphoresence peak shifts to longer wavelength with a concomitant increase in intensity. There have been reports that on dimerization of dyes such as eosin Y and rhodamine B, the phosphorescence peak shifts by 10-20 nm, its shape remains unaltered. The shift in the case of thiopyronine is much larger, for a three-fold increase in concentration,the shift is of the order of 110 nm. It has been reported that in cases where only a shift occurs with no change in spectral shape, the intermolec- ular separation is the same in both the ground and excited states.51 Spectral shape changes have been reported for acridine orange. It is quite likely that in the case of acridine orange, thiopyronine,and possibly methylene blue forming higher aggregates the intermolec- ular separations are different in the ground and excited states. In the case of eosin and rhodamine the large side groups may hinder the formation of higher aggregates. Phosphorescence spectra for a few of the acridine, xanthene dyes show that most of the dyes have their lowest triplet state close to the red spectral region..35'52 , The intensity of the phosphorescence emission increased considerably as the concentration is increased (Table 3). Due to N-fold aggregation, the monomer 42 excited state is expanded into N-fold levels. The spread- ing of the singlet level enhances the rate of intersystem crossing resulting in increased phosphorescence emission.51 The dependence of the triplet lifetime on concentration may imply that aggregates favor quenching. For a com- parable concentration, the lifetimes in ethanol are longer than those measured in phosphate buffer (Table 2). It has been reported that aqueous solution favors dimer- ization.47 Flash photolysis experiments of aqueous solutions of 6 thiopyronine (5.5 x 10- M) at room temperature resulted in triplet states species with absorption peaks at 340, 370, 410, 470, 480, and 690 nm.41' 53 These authors cal- culated the theoretical triplet levels and also predicted that the extreme reactivity of the dye is due to the large yield of triplet states. They also found a con- centration dependence for the decay of the triplet levels. In contrast to the lifetime, which drastically depends on concentration, the ZFS parameters are not measurably influenced by adjacent randomly distributed molecules once they are a critical distance apart. Figure 9 shows that below a certain concentration in ethanol (310-4 M) the Hm. 1n does not change appreciably with concentration. The lowest D* value of 0.0656 cm—1 is probably that of a thiopyronine molecule isolated from adjacent thiopyronine molecules. From presently available 43 theoretical and experimental data the D* values of hetero- cyclics and substituted aromatic compounds seem to be comparable to those of the aromatic analogs, e.g., quinox- aline 0* = 0.1055 cm'l,5S naphthalene 0* = 0.1031 cm‘1.56 Substituents such as amino and methyl groups do not seem to alter appreciably the ZFS parameter.57 Thiopyronine 4 in ethanol (10- M) has been found to have a 0* value of 0.0656 cm-l, that is lower than that of its aromatic 1 5 >.8 related nitrogen substituted acridine orange D* = 0.0732 -1 59 analog anthracene (D* = 0.0737 cm- and structurally cm Apparently the intramolecular interaction between the dimethyl amino groups and the positively charged sulfur contributes to a further delocalization of the triplet electrons as compared to the aromatic analog or the nitro- gen substituted acridine molecule. Experimental data indicates that the stronger the charge transfer character- istics the greater is the delocalization of the triplet 44,60 1 electrons. D* values of 0.0502 cm"1 and 0.0772 cm- have been reported in the case of intermolecular charge transfer complexes of l, 2, 4, 5 tetracyanobenzene with durene and mesitylene in random matrix.44 Upon increasing the concentration, the D* values decrease indicating further delocalization of the triplet electrons contributing to the triplet state, probably over two or more adjacent molecules. This seems to be consistent with the fact that on increasing the 44 concentration a transition from a random to a more ordered distribution of molecules takes place (Figure 9). Simi- larly an increase in D* with lowering of dye concentration has been reported for other dyes.59 It is also possible that the triplet electrons are more delocalized intra- molecularly as a result of increased intermolecular interactions at higher concentrations. A D* value of 0.0064 cm"1 has been observed for crystalline ion radical salts, this value has been attributed to the two electrons being distributed over four molecules.61 At all concentrations measured, the D* value of thiopyronine in phosphate buffer/glycol is lower than that in ethanol (Table 6). This, probably, is due to dimerization as mentioned earlier. The emission of thiopyronine in the blue region with a peak at 470 nm has been found to be independent of oxygen content and pH of the sample. This emission is quenched at higher concentrations. Preliminary exper- iments at 77°K with structurally related molecules such as proflavine and methylene blue also reveal two emissions, in the blue and red regions of the spectra. Besides emission from the lowest triplet state a long-lived emission at wavelength shorter than the main absorption 62-67 This band have been reported by many workers. emission cannot be attributed as delayed fluorescence since delayed fluorescence occurs at 590 nm. Lewis 45 Table 6 ZFS parameter of thiopyronine in phosphate buffer/glycol (P.B.G.) and in ethanol at 9239 MHz as a function of concentration Concentration (M) Solvent D* (cm-1) 4 x 10‘3 ethanol 0.0423 1 0.001 1 x 10‘3 ethanol 0.0446 i 0.001 1 x 10'4 ethanol 0.0639 : 0.002 1 x 10'5 ethanol 0.0656 : 0.002 3 x 10‘3 P.B.G. 0.0394 : 0.001 6 x 10‘4 P.B.G. 0.0401 1 0.002 6 x 10'5 P.B.G. 0.0430 1 0.002 6 x 10'6 P.B.G. 0.0434 1 0.002 46 assigned the emission in the blue region to emission from a higher triplet state and suggested that it is quite possible that such emission may occur in dyes.62 How- ever, it is rather doubtful that this emission results from a higher triplet state as rapid decay would occur to the lowest one. This emission is probably a re- combination luminescence. EPR experiments have shown that aggregation favors the delocalization of the triplet electrons. It is quite possible that the solute aggregates may favor the transfer of the photoinduced electron to an adjacent molecule, followed by recombi- nation luminescence. At higher concentrations (a 10-2 M) other quenching processes cause this luminescence to decrease to negligible values. The minimum energy necessary to generate a free electron in a dye aggre- gate has been estimated to be of the order of 3.1 eV.65 This is consistent with our findings that the blue emission can be observed only with excitation of at least 360 nm (3.4 eV). Since this kind of emission has been found in other dyes it is likely that aggregation favors the photo- induced electron-hole generation and emission of recombi- nation luminescence in the blue spectral region. The two components observed in the decay pattern is similar to those observed in the case of p-dimethoxyben- 68 69 zene and benzene solution. The two components have been attributed to two conformers of the emitting state. 47 Due to environmental effects, it is possible that the emitting state occurs in two conformations. It is quite possible that the thiOpyronine molecule exists in two different conformations giving rise to two components in the decay pattern. The peak at 590 nm observed with PVA films incor- porating thiopyronine at 77°K is probably delayed fluor- escence. The peak occurs at the same wavelength as the fluorescence peak. Delayed fluorescence has been found 70'71 As the intensity of the in acridine and proflavine. delayed fluorescence and the lowest triplet state are proportional to the incident light intensity, it is probably delayed emission of the eosin type. PART B COMPLEX FORMATION AND ENERGY TRANSFER FROM PHOTOEXCITED THIOPYRONINE TO DEOXYRIBONUCLEIC ACID Introduction Dyes such as thiopyronine, methylene blue, and acridine have been known to be photosensitizers.l The dye molecules bind with the DNA phosphates and/or inter- calate between the base pairs. X-ray studies have indi- cated that intercalation of the dye molecules does occur with changes in the length of the DNA macromolecule.72 Relaxation, sedimentation, and CD studies have been used to probe further into the mechanism of dye/DNA or poly- 16'22'73'74 The relaxation and CD nucleotide binding. studies indicate that external binding as well as insertion of dye molecules between base pairs and poly- nucleotides takes place. The sedimentation experiments show that on binding of the dye, the length of DNA increases. Comparing the binding characteristics at 10°C and 30°C it is observed that a higher fraction of the dye is bound externally at the higher temperature 48 49 with a concomitant increase in length. This increase is smaller than that at a lower temperature. In the case of the proflavine/DNA complex, the heat of binding for inter- calation is -7.8 kcal whereas that for outside binding is -9.8 kcal. The external binding depends on the salt concentration.20'22 A large number of investigations have been carried out to understand the nature of binding.17 However, few reports exist regarding the mechanism of energy transfer from the photoexcited DNA or dye to the other moiety of the complex. ThiOpyronine was chosen for our investi- gations since it is a potent biological photosensitizer. Moreover, the photosensitization experiments with thio~ pyronine have been carried out only in in_zixg_sys- tems.2-4'75'76 In this part, we report studies of the mechanism of energy transfer from the photoexcited thio- pyronine to the DNA molecule. Firstly, it is established that the dye complexes with the DNA molecule. The exis- tence of more than one type of complex is shown by spec- troscopic techniques such as absorption and EPR spectro— scopy. The bound dye molecules may transfer energy absorbed in the visible region to the DNA by at least three energetically possible pathways. The DNA is characterized by a broad phosphorescence band between 26 380 nm to 600 nm. Because thiopyronine has electronic states responsible for delayed fluorescence (590 nm) and 50 for electron-hole luminescence (470 nm) the dye may trans- fer the absorbed light energy to the triplet state of the DNA. A radical can be induced by exciting into the main absorption band of thiopyronine. It is feasible that this radical may be involved in producing the photo- chemical lesion of the DNA bases. The type and proba- bility of a particular transfer process depends on the dye concentration and the environmental conditions (ionic strength, temperature). Direct energy transfer from the triplet state of the dye to that of the DNA is not energetically possible. MATERIALS AND METHODS ThiOpyronine was prepared as mentioned in Part A. Calf thymus DNA was purchased from Sigma Chemical Company. Both DNA and thiopyronine were dissolved in 0.02M phos- phate buffer (pH 7.6). The concentration of thiopyronine was measured at 564 nm (e = 7.4 x 104)41 and that of DNA at 260 nm (e = 6.6 x 103).77 Solutions of different DNA phosphate/dye (P/D) ratios were prepared by adding a known concentration of DNA to a known concentration of dye (v/v) followed by thorough mixing. At high con- centrations of thiopyronine (a 10'-3 M) a precipitate is formed. Unless otherwise mentioned, sodium chloride was also added to all solutions to make up a final concen- tration of 0.1M. For emission measurements at 77°K, redistilled ethylene glycol/phosphate buffer (2:3 v/v), was employed. The absorption spectra were measured with a Cary-l4 spectrometer. To determine the binding constant of thio- pyronine to DNA at room temperature, a solution of DNA (2 x 10.5 M) was optically titrated (A = 564 nm) by add- ing small aliquots of thiopyronine. 51 52 Moreover, since the dye fluoresces strongly, the fluorescence intensity could also be utilized to obtain information about binding. An Aminco-Bowman spectro- fluorimeter was employed to measure the fluorescence spectra at room temperature. For a constant dye con- centration of (7 x 10-6 M) its fluorescence quenching was determined as a function of DNA concentration. Equilibrium dialysis was carried out in Lucite cells at 4°C. The concentration of DNA used in these experiments was 10-3 M. Using a Gilford 2400 spectrometer the melting curves were obtained in the DNA absorption region for various DNA-dye ratios with and without 0.1 M NaCl. Evaporation of the samples were minimized by covering the cuvette with parafilm. The temperature was con- tinuously monitored at a second sample placed in the cell compartment containing a calibrated thermometer. The phosphorescence spectra and lifetimes as well as the EPR data were obtained as outlined in Part A. RESULTS Absorption Spectra of the DNA/dye Complex The absorption spectra of thiopyronine and the DNA-dye complex are shown in Figure 1. With increasing DNA concentration the long wavelength absorption shifts towards the red. With thiopyronine as reference the difference spectra of the DNA/dye complex do not show an isosbestic point (Figure 2). The negative peak in the difference spectra is sensitive to salt concentration (Figure 3). The melting temperatures of DNA increased concomi- tantly with the dye concentration. In the absence of NaCl the melting temperature of the DNA-dye complex is as shown in Figure 4. In 0.1M NaCl, the melting temper- ature increased only by two degrees upon adding thiopy- ronine to obtain a final P/D ratio of 30. Binding Constant Determination The results of the optical titration carried out with a constant concentration of DNA are shown in Figure 5. The fluorescence quenching observed as a function of DNA concentration is portrayed in Figure 6. 53 54 .om u o\m ooooooooo moonn\m '''''' 5: 'OHxN m wcficoumcoflnu ummmsn muocmmonm ca musumummfimu Eoon um muuommm coaumu0mnm .H musmflm 55 660 ..."Oooooou ~---‘ \ 1 BIO o” a"° O — m A '0 E C d I '— 0 ° 6 fi — '° .1 o... m 0.... z \\\‘o... 3 L 460 4|O Figure 2. 56 Difference spectra at room temperature in phosphate buffer with a thiopyronine con- 5 centration of 2 x 10- M. oP/D 1, -------- p/0 = 5, —— P/D = 50. Optical Density 57 0.4 r- -o.4_ # 450 500 550 Wavelength . n m 600 650 Figure 3. 58 Difference spectra as a function of salt con- centration for a P/D of 16 with a thiopyronine 5 concentration of 10- M. c. 0.1 M NaCl, b. 0.01 M NaCl. a. 0.001 M NaCl. A00 0.3 0.2 0.l 0.0 “0.2 59 550 nm 600 Figure 4. 60 Melting curves for DNA concentration of 5 x 10.5 M in phosphate buffer as a function of P/D. DNA, -o-o-o-o- P/D = 24, and -+-+-+-+- P/D = 124. 61 06 mmDChmmc—zmp mm mm 0h mm mm — q _ , . . . IOI I no \1 00V ¢N_uo\n_ 62 .2 hnoa x n.m >3 pmmmmuocw one: Ioummofinu mo cowumuucmucoo map .pmppm mafia nonsaofisu no as OH some now .2 mice x m ma cofiumuucmocoo dza .Ec vmm no new: Ioumcoflnu nuHB 420 no coaumuufiu oaumumEouuommm .m musmflm 63 CON ounce “2.22.595 _a on. 00. . a Q'. 00 64 .2 mloa x n ma coflumuu tamocoo mchouamofins .dzo mo macsoem ucmumm imam now muflmcmucfi mocoommuosam ecu mo mmmcmno .m musmfim 65 ON 5:3}302 Yo. m. 42o o. ill I! fl H m0 0.. 11.1.9 66 The binding constant as determined by different techniques is given in Table 1. Details of the calculations can be found in the respective articles by Gellert, et al.,78 79 80 L6ber, and Cavalieri and Nemchin. EPR Measurements Keeping the dye concentration constant, the Am = 2 position of the triplet state of the DNA-dye complex shifted towards lower magnetic fields (Figure 7). The ZFS constant D* was calculated from the following relation, where Hm. In is the field strength at which the transition occurs, v the frequency of the microwaves used, and g = 2.0023 (Table 2). 8 is the Bohr's magneton and h Planck's constant. 0*2 = (3/4) [(h 0)2 - 4 (g B)2 Hminzl Exciting thiopyronine into its main absorption band a radical is generated at 296°K and 77°K, as determined by EPR spectrosc0py. Emission Characteristics of the DNA-Dye Complex The fluorescence spectra of thiOpyronine and DNA- dye complex are given in Figure 8. The long-lived emission spectra and lifetimes of the free and bound dye are shown in Figure 9. These spectra were determined as a function of the P/D ratio. For a constant dye concentration the 67 Table 1 Binding constant of thiopyronine to DNA deter- mined by different methods Method K (liter mole-l) Absorption 3 x 104 Fluorescence 2 x 104 Equilibrium-dialysis 2 x 103 68 with P/D for a thiopyronine 5 Figure 7. Variation of Hmin concentration of 3 x 10- M in phosphate buffer/glycol and a microwave frequency of 9239 MHz. Hmin (Am=2) |630 IS 20'— lanol- 69 J l l J l |00 P/D |20 I40 |60 |80 70 Table 2 The ZFS parameter D* for various DNA/dye ratios determined at 77°K in phosphate buffer/ethylene glycol (pH = 7.6) for a thiopyronine concentration of 3 x 10-5 M * D P/D (cm-1) 3 0.0467 10 0.0486 20 0.0528 60 0.0636 88 0.0643 71 Figure 8. Fluorescence spectra at room temperature 2 x 10.5 M thiopyronine, ......... p/0 = 150. 72 800 700 600 1 - 500 _ _ _ _ _ _ 400 3:5 36.1.95 H WAVELENGTH (nm) 73 .mmmomusm coaumuu ImsHHfi now OH 00 uouocm m an poflamfluass ma Ao.nv huwmcmucfi on» cowmwu moan ecu CH .vma uoE ........ 8.e~uo\a.l.ll.ulxo .2 mica mcficoummownu Illllllll an .2 NIOH mcflcouamoflsu ........ Am .Monh um HoomHm \ummmsn mumsmmonm ca muuoomm mocmommuozmmozm .m musmflm 74 700 800 900 WAVELENGTH (nm) 600 400 AllSNBlNI 75 phosphorescence maximum undergoes a blue shift and the lifetime of the complex increases as the DNA concentration is enhanced (Table 3). However, varying the DNA concen- tration, the position of the peak and the lifetime of the blue peak do not alter measurably (Figure 9). The excit- ation spectrum of the blue emission of the DNA-dye complex is similar to that of thiopyronine. At 77°K, DNA has a triplet state with a peak at 470 nm which decays with a half life of 0.3 second. At 77°K, the DNA-dye complex (P/D = 100) exhibits delayed fluorescence with a peak at 590 nm. For the phosphorosc0pe speeds available (1 rev/25 msec) the emission can be characterized by a lifetime of 100 msec, and the intensity depends linearly on that of the inci- dent exciting light. 76 Table 3 Phosphorescence maximum and lifetime of various DNA-dye ratios, for a concentration of thiopyro- nine of 4 x 10'5 M in phosphate buffer/glycol (pH = 7.6) P/D A max L?§:::?e 0.5 7400 91 5. 7300 93 100 7000 142 DISCUSSION The shift in the absorption maxima to longer wave- length at higher values of P/D indicates that thiopyro- nine is bound to the DNA. The changes observed are probably due to alteration of the absorption band of the dye due to binding with the DNA. The difference spectra as well as the absorption spectra do not reveal a single isosbestic point, thereby indicating that a single type of binding does not occur. The negative peak (550 nm) in the difference spectrum increases at higher salt concentration. With increasing dye con- centration, the change in melting temperature and the difference spectra of the DNA-dye complex are sensitive to ionic strength. This supports the idea that dye molecules are externally bound to the polymer under certain conditions.15 The binding constants (K) determined by different techniques agree within error of measurements. The equil- ibrium dialysis method consistently gives lower values for K since some of the dye molecules (about 15%) are adsorbed on the dialysis bag. It is noted that the 77 78 binding constant of thiOpyronine to the DNA is comparable to that of acridine derivatives.79 Regarding the nature of the DNA-dye complex our EPR experiments indicate that there are at least two types of complexes (Figure 7). Depending on the dye concentration and ionic strength the dye molecules can intercalate and/or bind to the phosphate groups of the DNA.81'82 For low P/D ratios, the ZFS parameter is lower than that for high P/D ratios (Table 2). For lower DNA concentration, the ZFS parameter decreases thus demon- strating a delocalization of the electrons contributing to the triplet state. Such a delocalization becomes possible when the triplet electrons can be distributed over neigh- boring molecules. The adjacent molecules are probably thiOpyronine molecules, because thiopyronine can form aggregates as evidenced by the behavior of its lowest triplet state (Part A).90 It has to be pointed out that this triplet state is that of the DNA-dye complex as demon- strated by other spectroscopic measurements. For high P/D ratios one may tentatively assume that the dye molecules intercalates between the base pairs, resulting in isolated thiopyronine molecules as characterized by larger D* value. Figure 7 shows that the Hm. changes in sharply at a critical P/D ratio. Going from a high P/D ratio to a low P/D one, the thiOpyronine molecules probably stack up externally. The apparent transition 79 may indicate that the stacking of additional thiopyronine molecules is possibly a co-Operative process accompanied by conformational change of the macromolecule.16’83’84 The information obtained from EPR experiments is further confirmed by the phosphorescence data. For a low P/D ratio, the phosphorescence maximum approaches that of the dye at that concentration, for a high P/D ratio, the lifetime of the photoexcited triplet state is longer than that at a low P/D ratio, indicating that for low DNA con— centrations, dye stacking on the phosphates takes place, favoring quenching processes. These measurements demon- strate that quenching due to the interaction between dye— DNA base pairs is appreciably smaller as compared to that of dye-dye complexes, as evidenced by the concentration dependence. Delayed fluorescence could be seen only at high con- centrations of DNA. As the delayed fluorescence was only measurable for times longer than several milliseconds it cannot be the result of singlet-singlet annihilation. It may be generated from triplet-triplet annihilation or re— combination processes.70'71 Delayed fluorescence has been reported for other dye-DNA complexes.85 Based on our experiments, we would like to suggest that at least three possibilities exist for the energy transfer from the photoexcited dye to the DNA. First, DNA has been shown to have a broad phosphorescence peak at 470 nm and its triplet state can be characterized by a 80 lifetime of 0.3 second. Since the DNA-thiopyronine com- plex exhibits a long-lived emission at 470 nm, probably resulting from an electron—hole recombination luminescence, it is energetically feasible that energy can be trans- ferred to the DNA base pairs. The second possibility is energy transfer from the excited state emitting delayed light (590 nm), since the phosphorescence band of the DNA macromolecule reaches up to 600 nm.86 Thirdly, since illumination of thiopyronine with visible light generates a radical in the presence and absence of DNA, it is probable that this radical is in- volved in the photoprocesses occurring in the DNA-dye com- plex. This assumption is supported by the following facts. Free radicals can be induced by exciting thio- pyronine in its main absorption band only, which is consistent with the action spectrum for photosensitization of E. coli in the presence of thiOpyronine.lo Moreover, illuminating acridine derivatives in a rigid matrix up to the longest wavelength absorption band, the dye molecules become initially photoionized. The radical ions and electrons thus generated may recombine87’88 or may become involved in photochemical alterations of the DNA-dye system. 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