photobiological properties of methylene violet

Photochemistry and Photobiology, 1997, 66(2): 245-252

Photobiological Properties of Methylene Violet*

Harry Morrisont, Taj Mohammad and Ravi Kurukulasuriya Department of Chemistry, Purdue University, West Lafayette, IN. USA

Received 18 November 1996; accepted 5 May 1997

ABSTRACT

The interaction of methylene violet (MV) and 4-bromomethylene violet (BrMV) with calf thymus and supercoiled a x 1 7 4 phage RF I DNA is reported. Measure- ments employing UV-visible absorption spectroscopy and equilibrium dialysis give evidence for the formation of complexes by each dye with DNA in the dark. They covalently bind to DNA, and MV nicks DNA, when the nucleic acid/dye mixtures are irradiated with visible light in a deoxygenated environment. Quantum efficiencies for singlet oxygen formation are 0.27 and 0.25 for MV and BrMV, respectively. A higher value (0.49) is observed for 44odomethylene violet (IMV).

INTRODUCTION

Methylene violet (MV)$, a phenothiazine dye, has recently been the focus of interest in our laboratory as part of an ongoing project designed to develop novel compounds capable of inactivating viral nucleic acid upon illumination with visible light. One of the broad goals of the project is to provide new methods for the decontamination of whole blood or cellular components without substantially or irre- versibly harming the blood constituents. The phenothiazine family absorbs light in the appropriate region for activation in the presence of hemoglobin but, prior to the commence- ment of this study, very little had been reported on, e.g. , the photochemical, photobiological and photophysical properties of MV (cf: Otsuki and Taguchi (1) for a recent study of the role of intramolecular charge-transfer in the MV long-wavelength absorption band). Methylene violet is of particular interest because it is a neutral species as opposed to the

*Presented, in part, at the 24th Annual Meeting of the American Society for Photobiology, 15-20 June 1996, Atlanta, GA, USA.

?To whom correspondence should be addressed at: Department of Chemistry, Purdue University, West Lafayette, IN 47907- 1393, USA. Fax: 3 17-494-0239.

$ Ahbreviarions: BrMV, bromomethylene violet; BSA, bovine serum albumin: ID, one dimensional; 2D, two dimensional; DMSO, dimethyl sulfoxide; DPBF, 1,3-diphenylisobenzofuran; DQF- COSY, double quantum-filtered correlated spectroscopy; ds, double-stranded; EDTA, ethylenediamine tetraacetic acid, disodium salt; EIMS, electron ionization mass spectrum; HRMS, high-resolution mass spectrum; IMV, iodomethylene violet; MB, methylene blue; MV, methylene violet; NBS, N-bromosuccinam- ide; NOESY, nuclear Overhauser and exchange spectroscopy; loz, singlet oxygen; P/D, phosphatddye ratio.

0 1997 American Society for Photobiology 003 I -8655/97 $S.OO+O.OO

permanently positively charged phenothiazines such as methylene blue (MB). The positive charge of MB is undoubtedly responsible, at least in part, for the inability of this dye to photosensitize intracellular virus (2). In fact, MV has been found to be an RNA and DNA viral photosensitizing agent capable of inactivating intracellular virus (2). We discuss herein observations to date that are relevant to its photobiology and also present pertinent data for the halo- genated derivative, 4-bromomethylene violet (BrMV).

MATERIALS AND METHODS Materials. Methylene violet (Bernthsen), purified as described be-

low, and 1,3-diphenylisobenzofuran were from Aldrich Chemical Co. (Milwaukee, WI). Calf thymus DNA (type I : sodium salt, highly polymerized), polyribonucleotides as 5' potassium salts, dialysis tub- ing (1 2 000 Da cutoff), cleaned according to the literature procedure (3) and Trizma buffer salts (Trizma base and Trizma hydrochloride) were from Sigma Chemical Co. (St. Louis, MO). Electrophoresis- grade agarose was purchased from Bethesda Research Laboratories, Gaithersburg, MD. Supercoiled @XI74 phage RF I DNA was from Sigma and was ca 85% covalently closed circular form (supercoiled RF I) and the remaining open circular (form 11). The DNA was supplied in 10 mM Tris-HCl/l mM ethylenediamine tetraacetic acid (EDTA), pH 7.5 at a concentration of 500 pg/mL according to the supplier's information. Nicked circular CtX 174 DNA (relaxed form), >90% form TI, was obtained from New England Biolabs (Beverly, MA) and was supplied in the above buffer of pH 8.0. Absolute ethanol was from McCormick Distilling Co. The HPLC-grade methanol was from Mallinckrodt Specialty Chemical Co. (Paris, KY); HPLC-grade acetonitrile and EDTA were from Fisher Scientific (Fairlawn, NJ). Phosphate buffer salts and sodium acetate were from J. T. Baker (Phillipsburg, NJ). Water was distilled from glass using a Coming MP-I water still. Sephadex (bead size 20-80 pm) that excludes molecular weights > I00000 was from Pharinacia Fine Chemicals (Piscataway, NJ).

Instrumentation. An SLM SPF-500 spectrofluorometer was used for fluorescence measurements. A Perkin-Elmer Lambda 3B UV/visible spectrophotometer was used for absorbance measurements. The HPLC analysis utilized a Varian 9010 series liquid chromatograph fitted with a Rheodyne 7125 injection port and a 200 pL injection loop. A Varian 9050 variable wavelength detector (set to 280 and/or 610 nm) was used to monitor injections that were recorded and processed by a Hewlett Packard 3395 computing integrator. A Rain- in Microsorb MV C-18 ( 5 pm) analytical (4.6 mm X 25 cm) re- versed-phase stainless steel column was used with a solvent program (unless otherwise specified) consisting of 50% 100 mM sodium acetate buffer (pH 4.5) and 50% acetonitrile at a flow rate of 1.5 mL/min.

Methods. Photolytic DNA-binding studies were done by irradiating 3 mL solutions in corex tubes using a turntable with the samples ca 8 cm from a Canrad-Hanovia (Newark, NJ) 450 W medium pressure mercury lamp (model 679A-36) surrounded by a uranium yellow filter (X > 330 nm). Photochemical reactions at wavelengths

245

246 Harry Morrison et a/.

Mv M!3

B BrMV

f IMV

Figure 1. Chemical structures of MV, BrMV, IMV and MB.

>520 nm utilized a combination of the uranium yellow filter in conjunction with 2% potassium dichromate (pathlength 1.25 cm). Under these conditions MV was excited with the 546 (green) and 578 nm (yellow) mercury lines. Irradiation at 254 nm utilized a Canrad-Hanovia (model 688A-45) low-pressure mercury lamp.

Quantum efficiency experiments for '02 were performed with a Continuum Nd:YAG laser set to provide photons at 532 nm at a frequency of 10 Hz with pulse energies of 0.04-0.08 mJ. The power of the laser and the irradiation time were maintained at approxi- mately 0.8 mW and 20 s, respectively. The laser showed good sta- bility during the course of these experiments. The quantum efficiency measurements for binding of MV to DNA were carried out at 620 nm in a 1 cm2 quartz cuvette on a Nd:YAG tunable dye laser at ambient temperature. The photon flux was measured with an OPHIR power meter model AN/2. Lyophilization was performed on a Neslab Cryocool-equipped Virtis benchtop freeze dryer in Lab- conco lyophilization flasks. Some preparative chromatography utilized a Chromatotron model 7924T made by Harrison Research (Palo Alto, CA).

Gel electrophoresis. The a x 1 7 4 RF I DNA was diluted in ster- ilized 50 mM phosphate buffer, pH 7.0 to a working concentration of 0.12 p,g/pL. Equal volumes of MV in buffer and DNA solution were mixed and 10 pL was placed in small tapered end glass tubes. After degassing with argon employing the syringe-needle technique, the small insert tube was placed into a large Fisherbrandm borosili- cate disposable culture tube and irradiated (A > 520 nm) at room temperature. Each sample was treated with 1.6 pL of bromophenol blue tracking dye (0.25% bromophenol blue and 40% sucrose in water) and the samples were loaded onto a 1% agarose slab gel (6.7 X 10.0 X 0.5 cm) containing 2 pL ethidium bromide (10 mg/mL). The horizontal gel was prepared and run in TBE buffer (Tris, boric acid and EDTA, pH 8.0). The electrophoretic separation was carried out in buffer containing 2 pL of ethidium bromide (10 mg/mL) at a constant voltage of 6 V/cm using an EC-105 power supply for ca 5 h. The gels were photographed using a Polarcid MP-3 Land Cam- era and Polaroid high-speed film type 57.

Preparation of MV, BrMV and iodornethylene violet (ZMV). Com- mercial MV (84%; impurities are 13% N-monodemethylated MV and 3% N,N-didemethylated MV) was further purified by flash chromatography on silica gel (230-400 mesh) with a gradient elution using 0-10% methanol in chloroform or by using the Chromatotron (2 mm rotor) by eluting with chloroform and then a chloroform- methanol mixture (90:lO). Purity was determined by HPLC to be >99%. 4-Bromomethylene violet and IMV were prepared as described below and purified on silica gel or by preparative TLC with chloroform. Their structures are shown in Fig. 1.

BrMV. A mixture of MV (64 mg, 0.25 mmol), N-bromosuccinim- ude (NBS) (49 mg, 0.28 mmol) in acetic acid (1.5 mL) was irradiated, in the dark, with ultrasound radiation (4). After 6 h the reaction mixture was diluted with water (15 mL) and refrigerated. The solid was collected by filtration and washed with water to give 80 nig of crude product, the HPLC analysis of which indicated the presence of MV, BrMV and dibromoMV in 27, 55 and 18%, respectively. Comparable results were obtained when NBS was replaced with Br2/ IVaOAckIOAc (5). Purification involved initial crystallization from i ,2-dichloroethane followed by flash chromatography on silica gel

60 (mesh 230400) using gradient elution with methanol (0-10%) in chloroform. This provided BrMV in >99% purity; electron ionization mass spectrum (EIMS): d z (rel. int.) 336/334 (100/77, M*.); high-resolution mass spectrum (HRMS): 333.9781 (required for C14HIIBrN20S 333.9775). For NMR data, see below.

IMV. To a 2 mL solution of 0.2 M sodium iodide was added: 6 mL of 0.06 M purified MV in absolute EtOH, a freshly prepared 5 mL aqueous solution of 0.1 M potassium iodate, 0.03 M potassium iodide and 3 mL of 1 M HCI, all at room temperature. The IMV begins to precipitate after 10 min of stirring. The reaction was run for 3 h, after which the precipitate was collected, washed with 2 mL water and dried in vacuo overnight, to provide 157 mg of 65% pure IMV. The IMV was purified in a manner analogous to that used for BrMV to give 97.7% pure material (by HPLC); 500 MHz 'H-NMR

6.90 (dd, J = 2.5 and 9.0 Hz, IH, H-8), 7.02 (d, J = 9.5 Hz, lH,

EIMS: d z (re]. int.) 382 (100, M+.); HRMS: 382.9722 (required for CI4H1 ,IN,OS 382.9715).

The location of the halogen in these products was determined by high-field two dimensional (2D) NMR analyses with reference to MV itself. Thus, for MV, the following assignments of the proton resonances in the one dimensional (1D) 'H-NMR are based on 500 MHz 2D double quantum-filtered correlated spectroscopy (DQF- COSY) and nuclear Overhauser and exchange spectroscopy (NOE- SY) spectroscopy (see below): 6 3.15 (s, 6H, 2 X NCH,), 6.58 (d, J = 2.5 Hz, lH, H-6), 6.67 (d, J = 2.5 Hz, IH, H-4), 6.83 (dd, J =

J = 9.5 Hz, lH, H-1). In the DQF-COSY spectrum (Fig. 2a), five peaks are observed on the diagonal. The doublet at 6 6.58 has a cross peak to the dd at 6 6.83 and the latter is then cross-coupled to the doublet at 6 6.67. The dd at 6 6.83 is also cross-coupled to the two doublets at 6 7.55 and 7.69. The NOESY (Fig. 2b) spectrum unequivocally confirms that the doublet at 6 6.58 and one of the dd at 6 6.83 are cross-coupled to the NCH, hydrogens through space.

For BrMV, the 500 MHz 'H-NMR spectrum (CDCI,) shows resonances at 6 3.18 (s, 6H, 2 X NCH,), 6.74 (d, J = 2.5 Hz, lH, H-6), 6.90 (dd, J = 2.5 and 9.5 Hz, IH, H-8), 7.00 (d, J = 9.5 Hz, 1H, H-2). 7.61 (d, J = 9.5 Hz, IH, H-1) and 7.77 (d, J = 9.5 Hz, IH, H-9). The DQF-COSY spectrum (not shown) contains five diagonal peaks, each corresponding to one hydrogen. A doublet at 6 6.74 has a cross peak coupled to the dd at 6 6.90 and the latter is also cross- coupled to the doublet at 6 7.77. The doublet at 6 7.00 has a cross peak to the doublet at 6 7.61. From the observed and missing values of the coupling constants relative to MV it is clear that the bromine has been introduced at either position 4 or 6. The NOESY spectrum (Fig. 2c) allows one to distinguish between these possibilities. It clearly shows two cross peaks (at 6 6.74 and 6.90) to the NCH-, resonances providing confirmation that H-6 and H-8 are still present. The bromine must therefore reside at position 4. Additional support for this conclusion is provided by the UV-visible absorption spectrum, which, in sodium phosphate buffer at pH 7, shows a A,,, at 626 nm, 6 nm red-shifted relative to MV. This small bathochromic shift is an anticipated consequence of bromine acting as an auxo- chrome. However, if bromine were at position 6, i.e. ortho to the NMe, group, we would expect a hypsochromic shift due to steric inhibition of resonance between the anilino group and the bromine. (Exactly this is observed for the dibrominated product where the ID 'H-NMR spectrum confirms that the second bromine has been introduced at position 6. In this case, the A,,, is observed at 616 nm.)

The 1D and 2D 'H-NMR spectra (not shown) of IMV are virtually identical to BrMV and the A,,, in the UV-visible absorption spectrum is 629 nm. These results are consistent with the assignment of this compound as the 4-iOdO derivative.

(CDCI,): 6 3.18 (s, 6H, 2 X NCH,), 6.74 (d, J = 2.5 Hz, IH, H-6).

H-2), 7.61 (d, J = 9.5 Hz, lH, H-I), 7.78 (d, J = 9.0 Hz, lH, H-9);

2.5, 9.5 Hz, 2H, H-2, H-8), 7.55 (d, J = 9.5 Hz, lH, H-9). 7.69 (d,

Quantum efficiency of juorescence. Fluorescence spectra and quantum efficiencies (average of two or more independent experiments) were determined in deaerated solutions (absorbance <O. 1 at the excitation wavelength of 520 nm) using Rhodamine 101 in 50 mM phosphate buffer as a standard, mf = 1.0 (6).

Singlet oxygen ,formation. The quantum yields of lo, formation for the dyes were determined using the 1,3-diphenylisobenzofuran (DPBF) bleaching method (7-9). The DPBF procedure entails irra-

Photochemistry and Photobiology, 1997, 66(2) 247

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I I ' ' I I W F I " ' I " v

7.0 7 . 1 7 .2 7 .3 7 .4 7.5 00 7.6 7 . 7 7.8 7.9

00

7.9 7 .7 7 . 5 7 .3 7 . 1 6 .9 6.7 6.5

( p p )

I

diating a solution of the sensitizing dye in the presence of the furan at a wavelength absorbed only by the sensitizer. The amount of furan consumed is followed spectrophotometrically by observing the decrease of an absorption band at 410 nm. Semiquantitative studies utilized a 500 W tungsten lamp with a A > 630 nm glass filter. The samples were kept 90 cm from the light source.

The absolute quantum yield of '0, formation was calculated by the following method. The moles of the DPBF consumed was calculated by using E,,, (410 nm) = 2.114 X lo4 M l cm-l. The amount of light (in einsteins) absorbed by the furan = 5.03 X 10l5 X A (nm) X power (W) X irradiation time (s)m (Avogadro's number). A correction was made for light scattered during irradiation by the following procedure. Power = A - C - 0S(A - B) where A

is laser power with no cell, B is laser power with cell + solvent and C is cell + reaction solution.

Typically, enough dye was added to 10 mL of absolute ethanol to give an absorbance of 0.5 at the irradiating wavelength. A 3 mL aliquot of the sensitizer solution was placed in the spectrophotometer and the absorbance was brought to 0.00 at 410 nm. An aliquot of freshly prepared DPBF in methanol (ca 30 kL) was added to give an absorbance of 1.00 at 410 nm. The sample was irradiated in a quartz cuvette and the loss of the furan was computed by measuring the decrease in the absorbance at 410 nm after irradiation.

Confirmation of the data was provided by the direct measurement of singlet oxygen emission at 1270 nm. Excitation was provided by a Nd:YAG laser emitting at 532 nm, and the sodium salt of tetra (4-sulfonatopheny1)porphyrin was used as the standard (aA = 0.72) (10).

Equilibrium dialysis. A three-chamber dialysis apparatus was used (1 1). The center chamber contained 3 mL of the sensitizer (ca 18 khf) and the two chambers on either side contained 3 mL of a pair of macromolecules (i.e. 0.5-1 .O mg/mL double-stranded [dsIDNA, bovine serum albumin [BSA], poly[A] or poly[G]). Preliminary experiments determined that equilibrium was reached when the chamber was placed on a rotomixer at 24°C for 48 h. The absorbance of each chamber was measured at the A,,, for MV in the visible region to determine the concentration of MV. For buffer, BSA, polylA], poly[C], poly[U] the A,,, = 620 nm with E = 6.5 X lo4 M - ' cm-I. For MV mixed with ca 0.8 mg/mL dsDNA and ca 0.5 rng/rnL poly[G] the A,,, = 628 and 622 nm, respectively, with E = 5.5 X lo4 and 4.7 X lo4 M-' cm-'.

Photolytic binding of dyes to DNA and polyribonucleotides. Pho- tolyses were performed using aqueous solutions of MV and DNA or polyribonucleotide (1.0 mg/mL) in 5 0 mM phosphate buffer pH 7.0. Degassed solutions were flushed with argon for a minimum of 20 min prior to photolysis. The samples were photolyzed at cu 10°C in rubber-sealed corex photolysis tubes using uranium yellow filtered light. The amount of covalent binding between MV and the DNA was determined after exhaustive dialysis of photolyzed solutions against 5 mM Tris buffer (pH 7.2) for 3 days. After dialysis, the solution was removed from the dialysis bag and the DNA assayed by measurement of absorbance at 260 nm (standard conver- sion factors of 21 and 26 absorbance units/mg were used for native and heat-denatured DNA, respectively). The assays are uncorrected for the contribution of the MV chromophore to the AZhO value. The emission spectrum of the solution was used to determine the amount of bound MV (see below). The DNA or polynucleotide was then precipitated with 0.1 vol of 2 M NaCl and 2 vol of absolute EtOH, kept overnight in a freezer and centrifuged at 2000 rpm for 20 min. The precipitate was redissolved in 5 0 mM phosphate buffer and the procedure repeated twice. Absorption and emission measurements were done after each precipitation/dissolution step to determine the extent of covalent binding, which is reported as the number of nmol of MV per mg of DNA.

Quantification of the binding of MV to DNA byjuorescence. This assay allows detection of up to 0.5 nmol/mL MV bound to DNA using a standard curve constructed as follows. Increasing aliquots of MV were added to 3 mL of 50 mM phosphate buffer, pH 7.0, and the emission spectrum for each solution was scanned in duplicate from 600 nm to 750 nm. The excitation wavelength was maintained at 520 nm and all the spectrofluorometer parameters were kept constant during each scan. Examples of MV fluorescence, both as free dye and covalently bound to DNA, are shown in Fig. 3. A standard curve (data not shown) was constructed by plotting the average fluorescence integration versus nmol of MV and gave a linear plot with a correlation coefficient of 0.97. The data are corrected for a decrease in Qf for MV from 0.017 in 50 mM phosphate buffer (pH 7.0) to a a,, of 0.008 due to dyeDNA interaction (see Results).

RESULTS Interaction of the dyes with nucleic acids in the dark

When a dilute solution of MV (22 pW) was titrated against increasing concentrations of DNA in 50 mM phosphate buff-

248 Harry Morrison eta/.

10 ....................................................................................................................

~ e 2 S ~ s 7 S r n ~

Wavelength (MI) Figure 3. Normalized emission spectra of MV in 50 mM phosphate buffer and of DNA that has been photolyzed with MV and purified.

er, pH 7.0 a bathochromic shift of 7 nm was observed at the maximum phosphate/dye (P/D) ratio of 150. There was a progressive decrease in absorbance with increasing DNA concentration, with up to 1520% hypochromicity at the highest P/D ratio ( c j Fig. 4). The titration curves are devoid of any clear isosbestic point.

In a separate study, much higher concentrations of MV and BrMV in 48 mM phosphate buffer pH 7.0/5% dimethyl sulfoxide (DMSO) [voVvol]) were mixed with 4.6 mM DNA. In this case a bathochromic shift associated with hyperchromicity was noted. The observed red shifts were 8 and 12 nm for MV and BrMV, respectively (cf. spectrum a vs b in Figs. 5 and 6).

0.24 I I

0.21

0.18

i! O . I 5 z 0.12 :: n 0.09

0.08

0.03

0.00 500 525 550 575 600 625 650 675 700

Wavelength (nm)

Figure 4. Variations in the absorption spectra of MV induced by native calf thymus DNA in SO mM phosphate buffer pH 7.0. The dye concentrations were 22 p M and the PID ratios were 0, 20, 40, 63, 80, 100, 125 and 150, from top to bottom. For purposes of clarity, the titration curves for the PID ratios of 63 and 100 have been omitted. These spectra were obtained with a commercial sample of MV, but identical observations were made with a pure sample (of MV at selected P/D ratios.

0.8

0.6

0,

C

f? 0.4

n P 4

0.2

0.0 400 500 600 700 I

Wavelength (nrn) )O

Figure 5. Optical absorption spectra of (a) 50 pM MV, (b) 50 p M MV in the presence of 4.6 m M native calf thymus DNA, (c) the MV-DNA photoadduct after 15 h of irradiation of the above MV- DNA complex followed by exhaustive dialysis and two rounds of precipitation and (d) a MV-DNA dark control treated analogously to the sample of spectrum (c). Samples in (a) and (b) were in 48 m M phosphate buffer, pH 7.0, containing 5% DMSO (vollvol); those in (c) and (d) were in SO mM Tris buffer, pH 7.2. The photoreaction was run in duplicate with similar results: only one set of data are shown here. The spectra (a) and (b) were obtained in 2 mm and those of (c) and (d) in 10 mm optical pathlength cells. (Note that the absorption maximum for MV is the same in water, phosphate buffer, pH 7.0 or Tris buffer, pH 7.2.)

Equilibrium dialysis studies involving several different macromolecules were performed in the three-chambered cell in order to test the potential selective affinity of MV to the biomolecules. The data are shown in Table 1. It is evident that MV shows preferential association with dsDNA versus BSA and a strong preference for poly[G] versus poly[A].

The quantum efficiency for MV fluorescence was determined to be 0.017 (kO.001) in 50 mM phosphate buffer, pH 7.0. This fluorescence is diminished by the presence of po- lynucleic acids, with corresponding quantum efficiencies of 0.008 ( t O . O O l ) , 0.013 (-CO.OOl) and 0.017 (20.003) in the presence of 1.2 mM dsDNA, poly[G] and poly[A], respec-

0.8

0.6

al

C

g 0.4

n a Y)

0.2

0.0 400 500 600 700 E

Wavelength (nrn) 0

Figure 6. Same as in Fig. S except that the dye was BrMV; a saturated solution of the dye was used to obtain spectrum (a).


Table 1. preferential noncovalent binding of MV* to biopolymers

Equilibrium dialysis experiments for determination of

Biopolymert Selectivity ratio$

0.35 0.97 0.21 0.41 0.63 0.95

*The MV concentration before dialysis was maintained at 18 pM. The concentration of dye was measured by A,,',, in the visible region.

TPolyribonucleotides (-0.5 mg/mL), ds DNA (0.8 mg/mL) and BSA (- 1 .0 mg/mL) in all experiments.

$Numbers represent the ratio of MV in the two dialysis chambers with biopolymers.

tively. The emission from MV in air is comparable to that seen under argon.

Photolytic covalent binding of MV to native DNA

Solutions (5 mL) of MV (50 pM) and native calf thymus DNA (2.6 mg/mL) in 50 mM phosphate buffer were irradiated in quadruplicate (A > 330 nm) at 15°C for 7.5 h under argon. The DNA adducts were isolated and purified by exhaustive dialysis and two rounds of precipitations. The re- sultant DNA pellets were clearly blue in color; a typical absorption spectrum is included in Fig. 5 (spectrum c; note spectrum c is not quantitatively comaprable to spectra a and b). A comparative irradiation, carried out under oxygen, showed much reduced MV absorption in the isolated and purified DNA (data not shown).

To quantitate the level of binding, solutions of MV (25 pM) and DNA (1 .O mg/mL) in 3.0 mL of 50 mM sodium phosphate buffer (pH 7.0) were photolyzed (X > 330 nm) under argon for 16 h at 10°C. The binding level was 3.5 nmol MV/mg DNA as estimated using the standard emission curve. The MV emission from the dark control was below the detection limit of the fluorescence assay, i.e. <0.5 nmol MV/mg DNA.

An attempt was made to determine a quantum efficiency for the binding reaction. However, when a solution of MV (50 pM) and DNA (1.62 mg/mL) was irradiated with the 620 nm (pulse energy 4.9 mJ) excitation from a Nd:YAG tunable dye laser, analysis of the isolated DNA by both absorption and emission spectral analysis gave a limiting value of < I x 1 0 - 5 .

Sephadex chromatography of isolated DNA containing covalently bound MV

Native DNA, purified by three precipitations and exhibiting a binding level of 3.5 nmol/mg, was dissolved in 1.0 mL of 50 m M Tris buffer (pH 7.2) and loaded onto a Sephadex column (1.5 X 25 cm). The sample was eluted with 50 mM phosphate buffer (pH 7.0) and collected in 3 mL fractions. Each fraction was analyzed by UV at A = 260 nm and 620 nm to determine the amount of DNA and MV present (cJ: Fig. 7).

a r 0.019

0.012 I MV

a 1 ,I 5 ~ 2 1 8 2 4 a O a G a 9 i 6 2 1 n M

Volume (ml) Volume (ml)

Figure 7. Sephadex chromatography of native DNA after photolysis with MV; 3 mL aliquots were analyzed by UV at 260 nm and 620 nm to determine the presence of DNA and MV, respectively.

Analysis of the photochemical interaction of MV with supercoiled DNA using gel electrophoresis

Solutions of supercoiled a x 1 7 4 RF I DNA with varying concentrations of MV (5-25 pA4) were irradiated (A > 520 nm) at ambient temperature under anaerobic conditions for 5 h. The samples were run on 1% neutral agarose gel, to- gether with two controls consisting of unirradiated MV (25 pM)/DNA and DNA irradiated in the absence of MV. The results are shown in Fig. 8. Neither of the controls showed any change in the DNA (lane 2 and 3). The DNA photoactivated in the presence of MV showed a decrease in supercoiled RF I (form I) and the production of a slow-migrating DNA band. The new DNA band corresponded to nicked DNA (form 11) by comparison with the marker (lane 1 ) . From Fig. 8 it is clear that the irradiated MV/DNA samples (lanes 4-8) all showed comparable levels of nicking, suggesting that the concentration of MV as low as 5 p M is effective in creating the nicks. In a time course experiment, irradiation of MV (5 pM)/DNA complex for 1-6 h under these conditions indicated that DNA is nicked in < I h (data not shown).

Photolysis of MV with the RNA polynucleotides

Solutions (3.0 mL) of MV (25 pM) and each of the RNA polynucleotides (1.1 mg/mL) were prepared in water and

form I1

Form I

Figure 8. Electrophoresis in 1% neutral agarose gel of supercoiled a x 1 7 4 phage RF 1 DNA irradiated at A > 520 nm with varying concentrations of MV for 5 h. Lane 1, relaxed a x 1 7 4 phage RF 11 DNA: lane 2, unirradiated MV (25 kLM)/DNA complex; lane 3 , DNA irradiated in the absence of MV; lanes 4-8, DNA irradiated in the presence of 5 , 10, 15, 20 and 25 p M of MV, respectively.

250 Harry Morrison eta/.

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-_._<

14 mM NaNJ

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Irradiation Time (see) Figure 9. Loss of DPBF monitored at 410 nm as a function of time. Irradiation was by a 500 W tungsten lamp with a ,630 nm glass filter. The sample was kept 90 cm from the light source. The absorption of the sensitizer was maintained at 1.0 OD.

photolyzed (A > 330 nm) under argon for 8 h at 10°C. The visible absorption spectra of the polynucleotides were obtained after purification through two precipitations. Only poly[G] showed the characteristic dye absorption between 500 and 700 nm, but its binding level was too low to be estimated by emission spectroscopy.

Photolysis of BrMV with DNA

Duplicate 4.0 mL solutions of DNA (1.52 mg/mL) and BrMV (50 I-LM> in 48 mM phosphate buffer containing 5% DMSO (voVvo1) were irradiated (A > 520 nm) under argon for 15 h at 10°C. The colored DNA was purified by exhaustive dialysis and two precipitations. Ultraviolet-visible absorption spectra showed the presence of a new A,,, at 620 nm, a result consistent with covalent binding of the dye to the DNA (see above Fig. 6, spectrum a vs c).

Singlet oxygen formation

The MV sensitization of singlet oxygen was assayed using DPBF in methanol. A progressive decrease in the DPBF was noted as a function of irradiation time. The presence of sodium azide reduced the rate of consumption of the furan, while the rate increased when the reaction was run in d3- MeOD. The data are plotted in Fig. 9.

Quantum efficiencies in absolute ethanol were determined using 532 nm excitation and found to be 0.27 (k0.02), 0.25 ( k O . l l ) and 0.49 (i0.15) for MV, BrMV and IMV, respectively. A quantum efficiency of 0.24 was independently de- iermined for MV by the measurement of singlet oxygen emission .

DISCUSSION ‘4s noted in the Introduction, MV has been found to be ef- lective in the photoinactivation of viruses (2). This is not unexpected because related phenothiazinium dyes in the MB family are generally recognized as excellent photosensitizing agents (for example, see Skripchenko et al. (2) and Cincotta ct al. (9) and leading references therein). We anticipated that

nucleic acids might be likely targets for the MV virucidal activity and the results of the current study support this pro- posal (though we have no data that exclude additional targets such as cell membranes). Thus, the absorption spectral data shown in Fig. 4 (with DNA creating a bathochromic shift in MV absorption associated with hypochromicity) clearly in- dicate that MV forms a complex with DNA. The emission data (see Results) that demonstrate that the addition of DNA diminishes MV fluorescence provide confirmatory evidence of such interaction. The source of the diminution in Qf is unclear; it could be a consequence of a reduced kf for the complexed dye andor quenching of the MV excited state by the DNA.

Interestingly, higher concentrations of MV (and BrMV), when mixed with DNA, exhibit a bathochromic shift in the long-wavelength band associated with hyperchromicity ( c j spectrum a vs b in Figs. 5 and 6). It is likely that at these concentrations the dyes are appreciably aggregated. The addition of the DNA presumably disrupts these aggregates and, in this event, interaction of the dye monomers with the mac- romolecule would be expected to have a hyperchromic effect

The absorption data in Fig. 4 do not exhibit a clear isosbestic point thus suggesting that the association is multi- modal, e.g. intercalative, groove binding involving van der Waals interactions etc. However, the observed red shift and the accompanying hypochromicity are consistent with inter- calation as a major component of MV complexation with the nucleic acid (13). This conclusion is supported by linear dichroism analysis (14) of the MV/DNA complex in which a negative band is observed in the visible region (E. Tuite, private communication). The relatively weak induced circular dichroism in MV absorption lends support to this conclusion. The complexity of the dye/DNA interaction is also seen in the equilibrium dialysis data in Table 1 that do not provide a simple, linear Scatchard plot (15,16). Interestingly, the results given in Table 1 show that MV exhibits a strong preference for noncovalent binding to DNA versus BSA, and surprisingly, a strong preference for binding to poly[G] versus poly[A].

As regards photochemistry, it is clear from the blue color of isolated DNA and from the spectral data (spectrum c in Figs. 5 and 6) that the photolysis of MV and BrMV with DNA leads to covalent binding of the dyes to the nucleic acid. The nmoVmg levels of binding observed are significant because binding levels of pmol/mg of small molecules to DNA are noteworthy. For BrMV one notes that the binding reaction is accompanied by a loss of a shoulder in the absorption band at CQ 475 nm (spectrum a in Fig. 6). In addition, MV and BrMV show a common, long-wavelength absorption band for the DNA-covalently bound dyes at 620 nm, suggestive that dehalogenation of BrMV accompanies the binding reaction. It is also interesting that this maximum matches that seen for MV in the absence of DNA rather than the bathochromically shifted maximum one observes upon mixing the dye with the DNA in the dark.

The formation of an MV-DNA covalent adduct was con- firmed by Sephadex chromatography of the DNA isolated from the cophotolysis (cJ Fig. 7). The reaction is inefficient; we estimate the quantum efficiency for covalent binding of MV to DNA to be less than using 620 nm irradiation.

(12).


Covalent binding is also observed upon photolysis of MV with poly[G] but is not observed with any of the other polyribonucleotides. It may not be coincidental that MV also exhibits a strong preference for noncovalent association with poly[G] (see above). One is tempted to speculate that electron transfer from dG, known to have the lowest ionization potential among the nucleotides (17), to the electron affinic dye plays a role in the ultimate bond-formation step(s). Con- versely, the absence of MV incorporation into poly[U] would seem to rule out a propensity for MV to undergo covalent binding through [2+2] cycloaddition chemistry. This conclusion was corroborated by the lack of release of MV from the MV-DNA photoadduct upon reirradiation with the 254 nm light. Such retrocleavage is a diagnostic test for pyrimidine cyclobutane adducts (1 8-20). The specific mechanism of covalent binding is, as yet, unknown but could involve the addition to MV of DNA radicals formed as a result of electron transfer chemistry. In fact, both DNA and poly[G] quench MV fluorescence (vide supra).

The possibility that MV might interact with DNA through photoinitiated electron-transfer chemistry naturally led to the possibility that the dye might also produce DNA nicks, and the gel studies confirm that this occurs. Specifically, MV sensitization, in a deoxygenated environment, produces a DNA with significantly altered mobility. Form I is replaced by a band corresponding to nicked DNA (form 11) and a concentration of the dye as low as 5 pM and irradiation for < 1 h were sufficient to cause the formation of the RF I1 DNA. In a separate study using the plasmid DNA p- BSSK .c-raf (eco), with a higher concentration of MV (50 pA4) in TrisEDTA buffer, pH 7.6 and a longer irradiation time (7 h), under argon at ambient temperature, we also observed a significant amount of linear DNA (form 111) result- ing from ds nicks. We did not notice a significant effect on nicking under aerobic and anaerobic conditions (data not shown).

It thus appears that covalent binding and nicking of DNA operate side by side upon photolysis of MV (and probably its bromo derivative) with the nucleic acid. We do not see a band in the gel studies that can be attributed to a dye- DNA adduct, but it is certainly possible that such adducts are simply unresolvable from one of the DNA forms seen following photolysis. In fact, it is possible that the covalent adducts play a role in the process of nicking. For example, gilvocarcin V, a photoactivated antitumor and antibiotic chemical, is very effective in forming covalent adducts with calf thymus DNA (21) but exclusively creates nicks in supercoiled pBR322 DNA (22). Likewise, DNA containing adducts formed by reaction with benzo[a]pyrene diol epoxide is readily nicked, presumably via a mechanism involving electron transfer from guanine residues to the photoexcited, bound aromatic hydrocarbon (23,24).

The photochemistry described above was conducted in the absence of oxygen. However, as expected, MV, BrMV and IMV are good sources of singlet oxygen. This is evidenced by the sensitized bleaching of DPBF, a reaction that is quenched by the addition of sodium azide and enhanced by the use of a deuterated solvent (cf: Fig. 9). In particular, IMV has a quantum efficiency for singlet oxygen formation of almost 50% (see Results), undoubtedly a consequence of the expected heavy-atom effect. Consistent with this conclusion

is the observation that oxygen does not quench MV fluorescence, i.e. the singlet oxygen must be generated by energy transfer involving the MV triplet.

SUMMARY The MV and BrMV interact with DNA in the dark to form complexes that are observable by absorption and emission spectroscopy and by equilibrium dialysis. When mixtures of the dyes and DNA are irradiated with visible light, both incorporation of the dye into the nucleic acid and nicking of the DNA are observed. These dyes generate singlet oxygen with quantum yields ranging from 0.25 for MV to 0.49 for IMV .

Acknowled~ements-Financial support by the National Heart, Lung and Blood Institute, National Institutes of Health (grant lROl HL53418), administered through the American Red Cross, is grate- fully acknowledged. We are grateful to Michael Rodgers and Ilya lvanov for measuring the quantum efficiency of singlet oxygen formation for MV, Steve Wagner for helpful comments and sugges- tions, and Ken Haher and the Purdue Chemistry Department Laser Laboratory for assistance with the laser experiments.

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