acrylamide and molecular oxygen fluorescence quenching as a probe of solvent-accessibility of...

Phorochemisrry and Phorobiology Vol. 47. No. 2. pp. 181-188. 1988 Printed in Great Britain. All rights rcscrved

0031-8655188 $03.00+0.00 Copyright 0 1988 Pergamon Journals Ltd

ACRYLAMIDE AND MOLECULAR OXYGEN FLUORESCENCE QUENCHING AS A PROBE OF

FLUOROPHORES COMPLEXED WITH DNA IN RELATION TO THEIR CONFORMATIONS:

SOLVENT-ACCESSIBILITY OF AROMATIC

CORONENE-DNA AND OTHER COMPLEXES DAVID ZINGER* and NICHOLAS E. GEACINTOV~

Chemistry Department, New York University, New York, NY 10003, USA

(Received 19 May 1987; accepted 17 July 1987)

Abstract-The relationships between the accessibilities to fluorescence quenchers (0, and acrylamide) of three different aromatic molecules and their modes of binding to double-stranded DNA were investigated. Proflavin was selected for this study because it is a known intercalator, while Hoechst 33258 is known to bind externally in the minor groove of DNA; it is shown here that the relatively bulky polycyclic aromatic hydrocarbon coronene forms partial intercalation complexes, and thus represents a third class of fluorophore-DNA binding. Fluorescence quenching results suggest that Hoechst 33258 bound to DNA is fully accessible to O,, that the accessibility of partially intercalated coronene is reduced to 20% of the free-solution value, while the accessibility of intercalated proflavin to molecular oxygen is at least ten times smaller than for free polycyclic aromatic molecules in fluid solutions. In contrast, the fluorescence of all these three fluorophores bound to DNA is insensitive to acrylamide (the effective accessibilities appear to be reduced by factors greater than 50 upon binding to DNA). It is concluded that O2 is useful as a qualitative probe of the type of binding and solvent exposure of fluorophore-DNA complexes, while acrylamide appears to be of limited utility. However, acrylamide fluorescence quenching may be useful for studies of other types of fluorophore-DNA complexes since the fluorescence of the pyrene residues in covalent adducts derived from the binding of benzo(a)pyrene diol expoxide to DNA was found to be at least partially sensitive to acrylarnide.

INTRODUCTION

Molecular oxygen and acrylamide have been fre- quently employed as fluorescence quenchers to probe the solvent accessibilities of extrinsic and intrinsic fluorophores in proteins (Eftink and Ghi- ron, 1976, 1981; Lakowicz and Weber, 1973a,b; Omar and Schleich, 1981; Lakowicz, 1983). Anal- ogous experiments to probe the accessibilities of drugs, polycyclic aromatic hydrocarbons (PAH). or carcinogenic metabolites of P A H molecules bound to DNA, have been attempted (Lakowicz and Weber, 1973a; Geacintov et al., 1976; Prusik et a l . , 1979; Undeman et al . , 1980; Hogan et al., 1981; MacLeod et al., 1982; Lakowicz, 1983; Undeman et al . , 1983). The interpretation of the results, particu- larly when acrylamide was used as the fluorescence quencher, was often complicated by the lack of quenching data on model systems of fluorophore-DNA complexes of defined conformations. The aim of this work is to provide information on the oxygen and acrylamide accessibilities of three different fluorophore-DNA complexes whose conformational properties have been reason- ably well characterized. Proflavin forms intercal-

‘Present address: Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA.

tTo whom correspondence should be sent.

ation complexes, while the fluorescent stain Hoechst 33258 is believed to bind to the minor groove of DNA (“external binding”). It is shown in this work that the relatively bulky PAH compound coronene is oriented parallel to the DNA bases and is thus probably intercalatively inserted between adjacent base pairs of DNA; however, because of its relatively large size, the coronene molecules must protrude into the solvent environment, and thus constitute an example of “partial” intercalation. These three model systems (the structures of the fluorophores are shown in Fig. 1) were chosen to represent three distinctly different modes of D N A binding. Before describing the fluorescence quenching properties of these complexes, the available information concerning their conformations is reviewed.

Projlavin-DNA complexes. In the presence of a large excess of DNA (nucleotide/drug ratios > 15), more than 93% of the proflavin molecules are bound non-covalently to the DNA (Li and Crothers, 1969). The intercalation model was first proposed by Lerman (1961), but this mode of binding of proflavin to DNA is supported by a large body of experimental evidence, e.g. sedimentation studies (Cohen and Eisenberg, 1969; Lerman, 1961), X-ray fiber diffraction (Lerman, 1961), X-ray diffraction studies on proflavin-dinucleotide complexes (Aggar- wal et al., 1984; Shieh et al., 1980; Berman et al.,

181

182 DAVID ZINGER and NICHOLAS E. GEACINTOV

@ \ I - - \ / CORONENE

HOECHST 33258

Figure 1. Structures of proflavin (3,6-diaminoacridine), coronene, and Hoechst 33258 (2-[2-[4-hydroxyphenyl1-6- benzimidazoll-6-[ 1 -met hyl-4-piperazylI benzimidazole) ,

1979), unwinding of supercoiled DNA (Muller et a l . , 1973), linear dichroism studies (Hogan et al., 1979), electrofluorescence (Jennings and Ridler, 1984), and NMR studies of proflavin-polynucleotides complexes (Patel, 1979).

Hoechst 33258-DNA complexes. Hoechst 33258 is a fluorophore which binds non-covalently to DNA, and is commonly used as a stain in chromosome preparations. The conformation of the Hoechst 33258-DNA complexes have also been investigated extensively. A minor groove binding model was proposed by Mikhailov et a/. (1981) which is supported by circular dichroism (Mikhailov et al . . 1981; Comings, 1975), optical activity (Mak- arov et al., 1979), electrofluorescence (Jennings and Ridler, 1983), and by linear dichroism studies (Bon- temps et a/. , 1975). Further support for this model comes from studies of the properties of complexes with synthetic polynucleotides (Mikhailov et a/. , 1981), and the fact that the binding of Hoechst 33258 is inhibited by Distamycin A, which is known to also bind to the minor groove (Mikhailov et al . , 1981). A model, in which Hoechst 33258 is hydrogen bonded to three adjacent A-T pairs and lies in the minor groove, was recently suggested based on a crystal structure determination of a complex of the Hoechst dye with a self-complementary dodecamer (Pjura and Dickerson, 1985) and using restriction fragment footprinting techniques (Harshman and Dervan, 1985).

The Hoechst 33258-DNA complexes are characterized by strong interactions between the dye and the DNA bases, as manifested by a large (20 nm) red-shift in the excitation maximum, and a dramatic increase in the fluorescence yield of the dye upon binding to DNA (Comings, 1975).

Coronene-DNA complexes. Coronene was classi- fied by Nagata et al. (1966) as an external binder

on the basis of linear dichroism measurements. However, the linear dichroism properties of non- covalent coronene-DNA complexes were reinvesti- gated by us, and it is shown here that the planar coronene molecules appear to be partially inserted between adjacent DNA bases, rather than bound externally. The discrepancies between our data and the results of Nagata et al. (1966) are attributed to the presence of aggregates or microcrystals of coronene bound to the DNA.

MATERIALS AND METHODS

Calf thymus DNA (Worthington Biochemicals. Free- hold, NJ) was dissolved in 5 mM sodium cacodylate buffer containing 0.1 MNaCl and 3 mM ethylenediamine tetra- acetate, dialysed exhaustively against 5 mM sodium cacodylate buffer, and centrifuged at 10 000 rpm for 1 h. The thermal hyperchromicity of the DNA sample was in the range of 37-41%, as expected for DNA in the double stranded form.

Stock solutions of proflavin (Aldrich Chemical Co., Milwaukee, WI) and Hoechst 33258 (Calbiochem-Behr- ing, La Jolla, CA) in buffer solution were prepared (0.005 and 0.05 gil00 me, respectively), and the desired small aliquots were added to the DNA solutions. In determining the concentrations of these fluorophores, the following molar extinction coefficients were utilized: 36 000 A4-lcrn-l at 460 nm in the case of proflavin-DNA complexes, and 33 000 M-lcm-' at 358 nm in the case of Hoechst 33258-DNA complexes. Coronene (Aldrich Chemical Co.) stock solutions were prepared by adding a few crystals to ethanol and decanting or filtering this suspension to remove any remaining solids. An extinction coefficient of 270 000 M-km-l at 301 nm (in pentane, Sawicki eta/. , 1960) was used to estimate the concentration of coronene in the ethanol solution (about 1.5 x IO-"M). Aliquots of this stock solution were added to the aqueous DNA, so that the final ethanol content did not exceed 5-10% by volume. Because of the very low solubility of coronene in aqueous solutions, i t was necessary to main- tain these relatively low concentrations of ethanol in order to bring dissolved non-aggregated coronene molecules in contact with the DNA, and to promote monomeric coronene-DNA complex formation.

The fluorescence intensity measurements were performed using a SPEX 1902 Fluorolog fluorescence spectro- photometer (SPEX Instruments, Edison, NJ). The fluorescence yields of samples in the presence and in the absence of quencher were measured in the fluorimeter relative to a reference sample (rhodamine B in water), at 24 2 1°C.

The oxygen fluorescence quenching experiments were carried out by bubbling either 02, or NZ (deoxygenated grade), through the solutions for 15 minutes in order to saturate the aqueous samples at atmospheric pressure with either oxygen or nitrogen. Acrylamide (enzyme grade, Eastman Kodak Co.. Rochester, NY), was freshly dissolved in cacodylate buffer to a concentration of 3 M. During the experiments, small aliquots (10-200 pX) of the stock solution were added to the samples and fluorescence readings were taken following each addition. Fluorescence intensity changes due to increases in volume were taken into account when calculating the quenching ratios at the different acrylamide concentrations.

Fluorescence decay profiles were determined by the time-correlated single photon counting technique (O'Connor and Phillips, 1981). The slow fluorescence decay component of coronene-DNA was measured with a conventional apparatus as described previously (Prusik er a l . , 1979), but utilizing a 40 KHz Photochemical Research

Fluorescence of DNA complexes 183

Associates (London, Ontario) flash discharge lamp filled with hydrogen gas. The relatively fast fluorescence decays of the Hoechst 33258 dye and the Hoechst 33258-DNA complex, were measured with a time-correlated photon counting Huorimeter at the National Synchrotron Light Source at the Brookhaven National Laboratory (Laws and Sutherland. 1986).

The linear dichroism measurements were performed as described previously (Geacintov et al . , 1984). utilizing a Couette cell flow gradient technique to orient the DNA.

RESULTS AND DISCUSSION

Conformation o f coronene-D NA complexes

Absorption, fluorescence excitation and emission spectra. Upon binding to DNA, the 338 nm (in ethanol) absorption maximum is red-shifted to 347 nm (Fig. 2A); a corresponding maximum at 347 nm is also observed in the fluorescence excitation spectrum of the coronene-DNA complex (Fig. 2B). The second absorption maximum which occurs at 301 nm in ethanol, is not apparent in the absorption spectrum of the coronene-DNA complex because of the onset of the DNA absorption band (which has a considerable magnitude even at 315 nm at the DNA concentrations utilized); however, the red shift of this band in the DNA complex is clearly visible at 311 nm in the fluorescence excitation spectrum (Fig. 2B). Because of the Dhh sym- metry of coronene, there are three degenerate transition moments (Birks, 1973) oriented along the shorter axes (301-311 nm band, So + S3 transition), and the longer axes (337-348 nm band, So + S2 transition) as shown in Fig. 2A

A fluorescence emission spectrum of the coronene-DNA complexes is also shown in Fig. 2B. There is a considerable wavelength interval between the lowest-energy, detectable absorption band at 347 nm, and the first vibronic band at 432 nm in the fluorescence emission spectrum. This large apparent wavelength shift arises because the lowest energy S,, + S, transition, situated in the 360-410 nm interval. is highly forbidden (Birks, 1973). This fact also accounts for the unusually long fluorescence decay time (see below) of coronene.

The 10 nm red shift in the absorption maxima resulting from the formation of non-covalent complexes with DNA, is rather typical of PAH compounds (see for example Geacintov et al . , 1976; LeBreton, 1985). Such red shifts are usually taken as an indication of intercalative binding (LeBreton, 1985); however, such conclusions are not necessarily appropriate since Hoechst 33258, which does not bind intercalatively to DNA, also exhibits a red shift of 20 nm upon binding to DNA. Accordingly, it is necessary to utilize other experimental criteria to determine whether the PAH-DNA complex geometry is consistent with intercalation.

Linear dichroism. In the linear dichroism technique, the DNA molecules are partially oriented along the flow lines in a Couette cell (NordCn and PAP 47-2 - B

280 MO 320 340 360 WAVELENGTH ,nm

M O 350 400 450 500 550 WAVELENGTH, nm

Figure 2. (A) Absorption spectra of coronene (2.2 x 10-6M) in ethanol (-), coronene (3.8 x lo-' M ) in a 3: 1 water:ethanol mixture (--.--.-) consist- ing mostly of aggregated coronene, and a coronene-DNA complex (---); the coronene-DNA complex spectrum was obtained in 5 mM sodium cacodylate buffer (pH 7.0) containing 5% ethanol, 2.2 X M DNA (expressed in concentrations of nucleotides), and 7.5 x 10-XM coronene. The spectra are not drawn to scale-the absorbance scale should be multiplied by the appropriate factor as indicated. Also shown are representations of the transition moments of coronene. responsible for absorption at 301 nm and at 338 nm. (B) (-) fluorescence excitation and emission spectra of the coronene-DNA complex, and (---) emission spectrum of coronene aggregates in the 3:l water:ethanol mixture. The excitation spectrum is corrected for variations in intensity fluctuations of the light source utilizing a Rhodamine B quantum counter. The emission spectra are not corrected for the variations in the wavelength-dependence of the sensitivity of the detector- emission momochromator system. The Huorescence excitation spectrum was obtained with emission detected at 451 t 5 nm and a 4 nm bandpass excitation slit. Exci- tation at 311 2 5 nm and a 4 nm bandpass emission slit were used to record the fluorescence emission spectrum of the complex. In recording the fluorescence emission spectrum of the coronene aggregates, excitation was set

at 365 2 3 nm with a 5 nm bandpass emission slit.

Tjerneld, 1976; Geacintov ef al . , 1984), and the absorbance of the chromophores bound to DNA is probed with linearly polarized light. The linear dichroism signal is defined as

(1) AA = A,, - A,

where A,, and A, are the absorbances probed with the polarization vector oriented in a parallel and perpendicular orientation, respectively, with respect to the flow direction. A sign of AA can be either positive or negative, depending on the orientation of the transition moments of the chromophores


bound to the DNA. Because the axis of the DNA double helix tends to be oriented along the flow lines, the planes of the bases tend to be tilted perpendicular to the flow lines, and AA is negative within the DNA absorption band below 300 nm. Planar aromatic chromophores sandwiched between adjacent base pairs, are also characterized by negative linear dichroism signals which resemble the inverted absorption bands of these chromophores.

A typical linear dichroism spectrum of a coronene-DNA complex is shown in Fig. 3; a negative peak at about 345 nm, and a broad negative band in the 355-360 nm region are observed. A weak positive band in the 365-380 nm region is also evident. Only the well defined negative peak at 345 nm (solid line) clearly corresponds to a coronene-DNA complex absorption maximum (Fig. 2A). The longer wavelength linear dichroism bands correspond to the long-wavelength shoulder in the absorption spectrum (Fig. 2A), and are attributed to coronene aggregates (dashed line in Fig. 3) which adhere to the surface of DNA molecules, and are thus also oriented. The presence of coronene aggregates in the DNA solutions could not be avoid- ed due to the low solubility of coronene in aqueous media, even in 5-10% ethanol solutions. The presence of these aggregates is also demonstrated by fluorescence techniques (see below). Monomeric coronene bound to DNA can be easily distinguished from coronene aggregates by the structure of its fluorescence emission spectrum which displays the characteristic vibronic maxima indicated in Fig. 2B.

Externally bound molecules of coronene would be expected to display a positive linear dichroism. It may have been the positive maximum in the linear dichroism spectra of coronene-DNA complexes due to these aggregates, which led Nagata et al. (1966) to categorize coronene as an eternal binder.

a a

I I 1 I I

330 340 350 360 370 WAVELENGTH, nm

Figure 3. The flow linear dichroism spectrum of the coronene-DNA complex (solid line). The dashed-line signal is attributed to coronene aggregates bound to the

DNA. The vertical scale is in arbitrary units.

Orientation angle. As described in detail elsewhere (Fredericq and Houssier, 1973; Norden and Tjerneld 1976; Geacintov et al., 1978) the average angle of orientation 8 between a transition moment of the chromophore bound to the DNA and the helical axis can be estimated utilizing the expression for the reduced linear dichroism AAIA:

where A is the absorbance of the sample measured at the same wavelength as the linear dichroism AA, and for (with values between 0 and 1.0) expresses the degree of alignment of the DNA molecules. In practice, the orientation angle of the chromophore is estimated by comparing the values of AAIA with those of the DNA bases at 260 nm; if the sign and magnitude of the chromophore within its absorption band is the same as that of the DNA bases, then the transition moments are parallel to one another, and intercalation is indicated (Norden and Tjerneld, 1976; Geacintov et al . , 1978; Houssier, 1981).

In the case of the coronene-DNA complexes, the DNA concentrations were so high (15 optical density units at 260 nm) that accurate absolute linear dichroism measurements were not possible. Thus another approach was utilized in which the reduced dichroism of the 345-347 nm So -+ S2 coronene band was compared to that of intercalated proflavin at 460 nm. The approach is justified since the parallel orientation of proflavin with respect to the planes of the DNA bases, has been well documented by linear dichroism techniques (Ramstein et a/. , 1973; Geacintov et al., 1978; Hogan et al . , 1979). With both coronene and proflavin added to the same DNA solutions ( 2 x M ) in equivalent concentration of nucleotides), the reduced linear dichroism values were determined both at the 347 absorption maximum of complexed coronene, and at 460 nm corresponding to intercalated proflavin. The orientation angle between the in-plane transition moments of coronene and the axis of the DNA helix (assuming that this axis is oriented at 90" with respect to the planes of the bases in the Watson-Crick model) were then estimated according to the equation (Geacintov et al . , 1978):

In these experiments there was about 1 coronene molecule bound per 14,000 base pairs, and one proflavin molecule per 1000 base pairs. Using three different samples, values of 8 were found to be 85 & 3". For a perfect intercalation complex, values of 8 = 90" are expected. Thus, the three transition moments of the coronene molecule are oriented at. approximately 90" to the axis of the helix, indicating that its plane is parallel to the planes of the DNA


bases. However, the DNA intercalation sites can at most accommodate a five-membered aromatic ring system such as benzo(a)pyrene (Mantione, 1973). Coronene, which has seven aromatic rings, is too large to be accommodated at DNA intercalation sites in the classical sense (Lerman, 1961). We thus propose that coronene is only partially intercalated, with an area of the molecule equivalent to at least two aromatic rings protruding into the solvent environment.

Fluorescence properties of coronene-DNA complexes. The fluorescence decay profile of an aqueous solution of coronene-DNA complexes is shown in Fig. 4A. The decay is non-exponential with a rap- idly decaying portion contributing most of this intensity in the initial 0-20 ns range after the excitation flash, and an exponential decay component at longer time intervals. In deoxygenated solutions, the lifetime of this component is 225 ns (Fig. 4A).

In order to determine which one of these decay components can be attributed to monomolecular coronene complexed with the DNA, and which to aggregated coronene. the emission spectra corresponding to the fast and slow components were determined. This was accomplished by determining the spectral distribution of the light emitted within the first 20 ns after the flash, as well as within the

TIME, ns

2000

1500

1000

500

0

420 460 500 540 WAVELENGTH, nm

Figure 4. (A) Fluorescence decay profiles of coronene complexed with DNA in nitrogen-saturated (upper trace) and oxygen-saturated (1 atm, lower trace) solutions. The fluorescence was detected through a 490 nm interference filter, while a broad-band Corning 7-54 filter was used in the excitation beam. (B) Fluorescence emission profiles measured in the time intervals between 3 and 20 nm (0) after the onset of the 3 ns excitation pulse, and in the

150-320 ns interval (0).

150-320 ns time interval. The fast decay component is characterized by a broad, red-shifted band (Fig. 4B); this band resembles, but is not identical to the fluorescence spectrum of coronene aggregates in the 3:l water-ethanol mixture (Fig. 4B); this fast emission is probably due to aggregates of coronene bound to the DNA, and was not further investigated. The emission spectrum of the long, 225 ns component (Fig. 4B) is attributed to monomolecular coronene bound to DNA, on the basis of its similarity with the molecular emission spectrum obtained upon exciting the 31 lnm absorption band of the coronene-DNA complexes (Fig. 2B).

Comparisons of fluorescence quenching of the three different types of complexes by oxygen and by acrylamide

The fluorescence intensity ratios measured in the absence (FO) and in the presence of quencher (F) are interpreted in terms of the usual Stern-Volmer equation (Lakowicz, 1983):

where [a] is the quencher concentration, 71) the fluorescence decay time in the absence of quenchers, and K is the effective bimolecular diffusional encounter (quenching) constant.

Quenching by molecular oxygen. When molecular oxygen is used as the quencher, the value of K(OJ for various different fluorophores is about 1 x 10"' M-ls-' (Vaughan and Weber, 1970; Lakowicz and Weber,1973b; Prusik et al. ,1979; Eftink and Ghiron, 1981; Berlman, 1971).

When small fluorescent molecules are bound to DNA, K ( 0 2 ) can be reduced significantly. In the case of ethidium bromide-DNA intercalation complexes, K ( 0 2 ) is reduced by a factor of about 30 (Lakowicz and Weber, 1973a). This decrease in K ( 0 , ) is attributed to a steric blocking effect of the neighboring DNA bases which reduce the prob- ability of a bimolecular contact between intercalated ethidium bromide and 02. Correspondingly, under our experimental conditions of oxygen-saturated solutions of proflavin-DNA complexes (1.3 mM 02), the F,JF ratio is 1.00 2 0.01 (Table 1); the calculated value of K ( 0 2 ) , utilizing the value of 71)

cited in Table 1 for intercalated proflavine, is thus

In the case of Hoechst 33258-DNA complexes, the value of K ( 0 2 ) = 1.1 x 10'" M-'s-' ; it is thus concluded that the Hoechst 33258 molecules located within the minor groove of DNA are fully accessible to molecular oxygen.

In the case of the coronene-DNA complexes, the fluorescence decay time is reduced from 225 ns to 150 ns in the prescence of 1.3 mM of 0 2 (Fig. 4A). The steady-state &,IF quenching factor is 1.49 & 0.06 at the same oxygen concentration. Both

K(O2) < 1.2 x 10' M-Is-I .


Table 1. Fluorescence decay times and oxygen quenching of fluorophore-DNA complexes*

Type of Fluorophore complex

Proflavin Intercalation 6.3t 1.00 2 0.01 < 1.2 x 10'

Hoechst 33258 Minor groove 3.5 +- 0.1$ 1.05 2 0.01 (1.1 2 0.2) x 10'"

Coronenes Partial 225 1.49 2 0.06 (1.7 5 0.2) x i0' intercalation

*1.3 m M oxygen concentration, 24 2 1°C. tKubota and Steiner, 1977. $This work, synchrotron light pulse excitation. $In 5 % ethanol-buffer solution.

of these independently obtained results give values of K ( 0 , ) = (1.7 * 0.2) X 10' M-ls- l , i n accord- ance with the dynamic quenching model (Eq. 4). This value is about five times smaller than the diffusion-controlled value, but six times larger than the value characterizing intercalated ethidium bromide (Lakowicz and Weber, 1973a). The three types of DNA complexes studied are thus found to display varying accessibilities to molecular oxygen, and K decreases in the order Hoechst 33258 > coronene > proflavine and ethidium bromide. The inter- mediate value of K ( 0 2 ) in the case of coronene is consistent with a partial intercalation geometry, as suggested by the linear dichroism experiments and the bulky size of this compound.

Quenching by acrylamide. When acrylamide is utilized as the quencher, the Stern-Volmer constants K(acry1) for free proflavin and Hoechst 33258 have values of 2 x loy and 0.9 x loy M-ls-', respectively (Table 2). Because of the low solubility of coronene in aqueous solutions, values of K(acry1) for the free molecules could not be determined. In the case of a metabolite of benz(a)pyrene, the tetraol 7,8,9,10-tetrahydroxytetrahydrobenz(a)pyrene, K(acry1) = 0.75 x loy, while K ( 0 , ) = 1 x lO"'M-ls-' (Geacintov et al. , 1987). It is thus evident that acrylamide is a less efficient fluorescence quencher than 02, an effect which cannot be attributed solely to the somewhat smaller diffusion

coefficient of acrylamide in aqueous solutions (Lakowicz, 1983; Eftink and Ghiron, 1984).

The fluorescence of proflavine, Hoechst 33258 and coronene-DNA complexes is insensitive to acrylamide within experimental error (Fig. 5 and Table 2). In a previous study, MacLeod et al. (1982) reported that the fluorescence of ethidium bromide-DNA intercalation complexes was also not quenched by acrylamide even at concentrations of the latter as high as 0.8 M. These results show that even Hoechst 33258, which is located externally in the minor groove of DNA, is well protected from quenching collisions with acrylamide molecules. Even though the coronene molecules musr partially protrude into the solvent environment at the intercalation sites, their fluorescence is nearly insensitive to acrylamide. This lack of quenching of the fluor- esence of DNA-bound fluorophores, suggests that the diffusion constant of acrylamide near the DNA molecule might be significantly diminished, thus reducing its ability to quench the fluorescence of DNA-bound fluorophores.

These results further suggest that acrylamide, in contrast to 02, is not a useful quencher for distingu- ishing between different modes of binding of fluorophores to DNA. However, the fluorescence of adducts derived from the covalent binding of the carcinogen benz(a)pyrene diol epoxide (BPDE) to DNA exhibits significant sensitivity to acrylamide

Table 2. Fluorescence lifetimes and quenching constants, K(acryl), of free fluorophores and their DNA complexes*

Free Ruorophores DNA complexes

711 K(acry1) 711 K(acry1) Fluorophore (ns) (M-Is -1 ) (ns) ( M - 1s- 1 )

Proflavine 5.0t 2 x 10" 6.3t <2.9 x loh Hoechst 33258 4.8 t 0.5 0.9 f 10' 3.5 2 0.1 < 17 x loh Coronenez - - 225 < 0.8 x 10"

*Data for free fluorophores taken from Zinger (1986). tKubota and Steiner, 1977. $Because of the low solubility of coronene in aqueous solutions, quenching experiments with free molecules were not feasible.


j CORONENE

[ ACRYLAMIDE] ,M

Figure 5. Stern-Volmer acrylamide fluorescence quenching plots obtained with non-covalent proAavin-DNA, Hoechst 33258-DNA, coronene-DNA, and covalent adducts derived from the binding of 7,8-dihydroxy-9,10- epoxy-benzo(a)pyrene to DNA (the sticks in the 7,8 and 9 positions indicate -OH groups). (A) Excitation: 460 2 3 nm; emission wavelength: 500 f 3 nm. (B) Excitation: 358 * 2 nm; emission wavelength: 475 2 2 nm. (C) Exci- tation: 311 2 5 nm; emission wavelength: 451 2 5 nm. (D) Excitation: 346 ? 2 nm; emission wavelength: 398 2 5 nm; 1 x M DNA concentration with 1% of the bases modified (see Geacintov et al., 1987 for further

details).

(Fig. 5). This implies that the pyrenyl residues of the covalently bound BPDE molecules are not intercalated, and that these residues might be situated at solvent-accessible DNA binding sites, with a conformation quite different from those of the Hoechst 33258-DNA minor groove complexes. The properties of these adducts are more fully discussed elsewhere (Geacintov et al . , 1987).

Acknowledgements-This work was supported by the Department of Energy (Contract DE-AC02-78EV04959 and Grant FG-0286ER60405). Some of the fluorescence lifetime measurements were carried out at the Synchrotron Light Source, Brookhaven National laboratory, which is supported by the Department of Energy, Division of Material Sciences and Division of Chemical Sciences (Con- tract DE-AC02-76CH00016). We wish to thank Dr. Y. Mnyukh for performing the linear dichroism measurements.

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