Eur. J. Biochem. 14 (1970) 567-574

Influence of pH and Metal Ions on the Fluorescence of Polycyclic Hydrocarbons in Aqueous DNA Solution

Brian GREEN Chester Beatty Research Institute, London

(Received April 6, 1970)

The fluorescence of benzo[a]pyrene or perylene solubilized in DNA solutions of low ionic strength is strongly quenched either by addition of silver ions at a ratio Ag+:DNA phosphate <0.15 or by lowering the pH from 7.5 to 5. I n poly d(A-T) solutions the effects are much less marked than in DNA. Cu2+ ions are also effective quenchers in this system and Co2+ and Ni2+ have some activity but little effect is seen with Mn2+, Zn2+, Cd2+, Mg2+ or Na+ ions.

The results establish that GC pairs play the dominant role in these quenching reactions and show that the DNA bases have a specific role in the DNA/hydrocarbon interaction. They also suggest that GC-containing sites are the major sites of physicochemical fixation of hydrocarbons to DNA.

No gross difference in the general nature of the DNA binding sites was found for the car- cinogenic benzo[a]pyrene and non carcinogenic perylene.

Although polycyclic hydrocarbons were the first pure chemical compounds shown to posses carcino- genic activity, the way in which they induce heritable malignant changes in cells remains unknown. Unlike certain other classical chemical carcinogens (e.g. aromatic amines) all the metabolic derivatives which have so far been tested are less carcinogenic than the parent hydrocarbons so that it is likely that either the hydrocarbon itself or possibly a closely related species produced early in metabolism is the prox- imate carcinogen.

A reaction such as intercalation of a hydrocarbon molecule between the bases of DNA assumes import- ance as a mechanism by which a chemically inert (but relatively planar) molecule could induce an alteration in a cell’s inherited information. This could be direct (as in the case of acridines) but the reaction could also be the basis for the localization of a metabolically activated species in a biologically important site. It is now well established [l-61 that physical binding of polycyclic hydrocarbons to DNA does occur, characterized by their increased solubility in aqueous DNA solution, and an interesting distinc- tion has been observed recently [7] between the behaviour of two isomeric hydrocarbons when physically linked to DNA1. Under mild oxidising conditions up to 40°/, of the physically-bound

Unusual Abbreviations. r f , ratio of total metal ions added per DNA phosphate residue; benzopyrene refers to benzo-[a]- pyrene; poly d(A-T), 3‘4’ copolymer of deoxyadenylic and deoxythymidylic acids.

1 Does not depend on complexing.

carcinogenic benzo[a]pyrene became covalently link- ed to the DNA whereas the noncarcinogenic benzo[e]- pyrene reacted to only a very limited extent.

The nature of the physical binding of hydro- carbons to DNA is not proved beyond doubt, mainly because the low solubility of these hydrophobic molecules in aqueous media poses formidable prob- lems. To attain concentrations a t which spectral measurements can be made it is necessary to work a t high DNA concentrations (approx. 1 mM P ) and low ionic strength [1-31 but even here, when the solubility of benzo[a]pyrene is increased 100-fold compared with that in water alone, the levels are still only 1-2 pM. The evidence obtained so far, (the requirement for some stacked or double- stranded structure for the reaction [1,8-lo] the spectral shifts [ l , 2,5] and especially the orientation of the hydrocarbon molecules more or less perpendi- cular to the DNA helix axis [11,12]) is consistent with intercalation between the DNA base pairs. It seemed worthwhile to t ry to clarify further the nature of this physical DNA-hydrocarbon inter- action. One property which might be expected is that the behaviour of the hydrocarbons would readily respond to modification of the DNA bases if they are in such close contact as intercalation requires. If the binding to DNA is external or if the hydro- carbons are non-specifically enclosed in a gross micelle-like DNA structure [13] the response should be much less sensitive.

A further aim was to see if any gross difference could be distinguished between the nature of the

Eur. J. Biochem. 568 Hydrocarbon Fluorescence in Aqueous DNA

binding sites in DNA for carcinogenic and non- carcinogenic hydrocarbons since both types can bind physically to DNA[I,3,5]. A study was therefore made of the effects of pH-lowering and addition of low concentrations of metal ions on the fluorescence properties of two DNA-bound hydrocarbons, the carcinogenic benzo[u]pyrene and the non-carcino- genic perylene (Fig. 1) for which the reaction has been well characterized [12]. I n these experiments, a preliminary report of which has been presented [14], both hydrocarbons behaved similarly.

Fig. I. (A) Benzo[a]pyrene (potent carcinogen) ; (B) Perylene (non carcinogen)

MATERIALS AND METHODS

Materials DNA from either salmon sperm (Mann or BDH)

or calf thymus (Worthington, highly polymerized) was used. Salmon sperm DNA was deproteinized once or twice [I51 to reduce the protein content to less than 0.2°/0 (by amino acid analysis). The DNA was routinely made up as a O.lo /o solution in glass- distilled water and diluted for use as required. Final DNA phosphate concentrations were usually in the range 1.0-1.5 mM. The total Na+ ion concentration of such solutions is about 1 mM. Poly d(A-T), K+ salt, was purchased from Mles Laboratories, Inc. and used as supplied except where dialysed against glass- distilled water to lower the salt concentration.

The original findings were made with purified polycyclic hydrocarbons [I21 but commercial samples of benzo[u]pyrene (Eastman Kodak) were sub- sequently used, their behaviour being apparently identical. Sulphuric and nitric acids and their silver salts were Analar reagents; where possible other metal salts were also of Analar quality.

Methods pH measurements were made with a Pye pH

meter and a miniature combined electrode. In low ionic strength solutions these measurements are complicated by the considerable amount of ‘drifting’ and by the apparent change in pH during stirring. Such effects are greatly reduced in the presence of salt.

An Aminco-Bowman spectrophotofluorimeter, which could be fitted with Glan prism polarizers,

was used for fluorescence measurements and absorp- tion spectra were obtained with a Unicam SP800 spectrophotometer. Some measurements were also made with a more recent model of the Aminco- Bowman spectrophotofluorimeter fitted with the off-axis ellipsoidal mirror condensing system which provides greatly increased sensitivity.

A double-monochromator fluorometer was used to determine whether there was any shift in benzo- pyrene excitation or fluorescence peaks during quenching; none could be detected [15a]. Any such shift must be less than Znm, which is below the resolution of our Aminco-Bowman Instrument a t the slit widths used used in this study i.e. the quenching, which is measured a t fixed wavelengths above the DNA-absorbing region, represents a genuine decrease in intensity and not just a shift of the maxima away from the selected wavelengths. These were usually 3721432 nm for benzopyrene (the fluorescence peaks were not resolved) and 4281482 nm for perylene. The absorption spectrum of the bound hydrocarbon also undergoes only minor changes (< 2 nm shift) during quenching by acid or silver ions (at rf < 0.2, though these increase in magnitude a t higher rf [IS]): thus the effect represents a de- crease in relative quantum yield.

Corrections for DNA-metal ion blank solutions are very small but corrections for the contribution to the total fluorescence of unbound hydrocarbon present some difficulty. I n the case of perylene, the solubility in water ( ( 2 nM [17]) is so small that its fluorescence contribution is negligible under the conditions of measurement. I n the case of free benzopyrene the fluoresceme contribution (which is not quenched by acid or Agi- ions a t these concentra- tions) is significant, expecially where the DNA- bound hydrocarbon fluorescence is largely quenched. The relative importance of the correction obviously depends on the concentration of complexed hydro- carbon. Seven published values for the solubility of benzopyrene in water range from less than 3.5nM to 32nM. Since individual estimations may vary considerably, a single standardized correction has been adopted for this work representing the mean fluorescence level observed a t the appropriate wave- lengths after grinding benzo[a]-pyrene with five glass- distilled water samples, shaking for 48-96 h and centrifuging in quartz tubes [I81 for a t least 30 min a t 75000xg. This correction amounts in most in- stances to 5- 15O/, of the lowest fluorescence levels. There is also some contribution from scattered light for which no correction is made, so that the true ex- tent of quenching may be rather more than the recorded levels. The individual excitation and fluorescence peaks of free benzopyrene are a t 8-10 nm shorter wavelengths than those of the DNA-bound form [1,2,18,19].

Vo1.14, No..?, 1970 B. GREEN 569

RESULTS

Effect of pH During earlier spectral studies [16] it was observed

that when Ag+ ions were added, a t a ratio (rf)Ag+ ; DNA phosphate of 0.5, to a solution of DNA in glass- distilled water containing solubilized benzopyrene, the hydrocarbon fluorescence intensity was markedly decreased. Addition of this proportion of silver

4 5 6 7 4 5 6 7 PH

Fig.2. p H - i n d m d changes in the relative f lmrescew intensity of polycyclic hydrocarbons in aqueous DNA solution. (A) Ben- zo[a]pyrene in salmon sperm DNA solution (P = 1 mM). 0-0, DNA in glass-distilled water (2.7 pM benzopyrene) ; 0-0, DNA in 0.1 M Na,SO, (0.6 pM benzopyrene); A----A, DNA in 0.1 M Na,SO, heated at 100" for 10 min (1.2 pM benzopyrene). (B) Perylene (0.75 pM) in calf thymus

DNA solution in water [3]

released H+ ions [20,21] so that the pH of the solu- tion fell to approx. 4.5. When the pH was lowered in the absence of silver ions a similar degree of fluorescence quenching could be observed but, whereas this effect was largely reversed when the pH was returned to 7-8 (provided it had not been lowered enough to cause extensive denaturation of the DNA [22]) the hydrocarbon fluorescence was not restored in the Ag+-treated solutions on raising the pH to the original level. Deoxygenation did not affect the observations. The lengthened fluorescence lifetime of perylene in DNA solution is insensitive to deoxygenation [23].

Fig. 2 shows fluorescence quenching curves for benzopyrene and perylene in DNA solution in water. For unheated DNA in glass-distilled water, pH- quenching occurs mainly over the 7-5 range. Below pH 4.5-5 the cytosine protonation is presumably extensive enough to cause major collapse of the ordered structure and release of the hydrocarbon, with a corresponding increase in intensity and decrease in polarization of the hydrocarbon fluores- cence (Fig.3).

The quenching was still observed after the solu- tions were diluted 1:lO to eliminate non-specific aggregation effects but the hydrocarbon appeared to be released from the DNA a t a higher pH. This is consistent with what is known of the stability of DNA a t very low ionic strength. I n the presence of 0.1 M salt the curve is shifted to lower pH values for native DNA but the effect is small in the case of denatured DNA.

Another effect of pH-lowering is to provide more sites on the macromolecule for hydrocarbon binding.

Wavelength (nm)

Fig.3. Chnges in DNA absorptim spectrum during quenching of bound benzo[a]pyrene flmrescence by ( A ) acid and (B) Ag+ ions (pH Y.63 & 0.1). Salmon sperm DNA in water (P = 1.1 mM), shaken for 17 hours with benzopyrene and centrifuged for 30 min at 75000xg; benzopyrene = i.1 pM. The pH (A) or rj Agf (B) values are shown for each curve and the actual

quenching curves are inset. p = degree of polarization of benzopyrene fluorescence [S]

Eur. J. Biochem. 570 Hydrocarbon Fluorescence in Aqueous DNA

Native DNA in solutions of 0.1 M salt a t neutral pH displays only slight solubilizing activity towards benzo[u]pyrene [8,9]. When the pH of such a DNA solution in 0.1 M NaNO, is lowered (Fig. 4) the extent of solubilization first increases as the DNA starts to be titrated (pH 3.2) then decreases on further titra- tion as the secondary structure becomes increasingly disordered. Other experiments have confirmed this increase in solubility in DNA solutions in 0.1 to 0.2M salt a t pH 3-3.5. (At low ionic strength a smaller lowering of pH (to approx. 4.5) increases benzopyrene solubilization [3].) On raising the pH there is a rapid loss of dissolved hydrocarbon.

Wavelength (nm) 250 260 270 280 290 300 3D

I I I I 1 I I I t

1::

s 360 380400 Wavelength (nm)

Fig. 4. Solubilization of ben.w[a]pyrene in an. aqueous solution of salmon sperm DNA (P = 1.1 mM) in 0.1 M sodium nitrate at various p H values. The absorption spectra of the DNA (0.2 cm cells) and of the solubilized benzopyrene (4 cm cells) are shown; the base lines of two of the latter spectra are

raised 0.1 unit for greater clarity

Earlier studies of the effects of partial dilution- denaturation [2] and heat-denaturation [8] and the increased binding observed a t very low ionic strength lead to the suggestion that hydrocarbon binding is favoured in less well ordered regions of the DNA and the present result is in accordance with this idea since the pH levels where solubilization is maximal correspond to those a t which limited flexible single- stranded regions are produced in the DNA [24] and where the effects are partially reversible on raising the pH.

Benzopyrene shows very little binding to single- stranded DNA [1,8,9] indicating that a t least one intact base pair is required a t the binding site (cf. acridines [25]). It may be envisaged that a region in which a number of base pairs are opened would be

more flexible and hence allow more readily the deformation necessary for the insertion of a hydro- carbon between the remaining intact base pairs than a region consisting of a long rigid array of closely- stacked paired bases. Although the extent of reaction is much greater in conditions inducing flexibility in the DNA molecule and is therefore easier to study, there is nothing to indicate that the reaction is qualitatively different from that observed with native DNA under normal ionic strength conditions.

Although pH-lowering favours greater binding of hydrocarbons to DNA this can not explain the pHquenching effect. Thus in most cases no major change in the polarization of benzopyrene fluores- cence is seen after considerable quenching (Fig.3) so that there is not a shift in population from free (fluorescing) to bound (quenched) molecules : the whole process involves mainly DNA-bound hydro- carbon molecules. It is further inferred from the lack of any major change in the hydrocarbon absorp- tion spectrum (which retains the long-wavelength shift [1,2,5.19] characteristic of the DNA or purine reaction) that the extent and general nature of its binding to DNA is unaltered during pH or metal ion quenching; in particular, that there is not a shift from an internal to an external site on DNA.

Effect of Silver I o n s

The quenching effect of silver ions on DNA/ hydrocarbon solutions is illustrated in Figs.3 and 6. Again no major change occurs in fluorescence polar- ization during quenching. In Fig.3 the quenching of hydrocarbon fluorescence by addition of silver ions and by pH-lowering are compared directly on the same DNA/benzopyrene solution, together with the corresponding changes in the DNA absorption spec- trum. The pH was maintained a t 7.6 & 0.1 (Fig.3B) to avoid complication by pH effects, though no pH change was measurable in the DNA during Ag+ addition up to rf 0.1.

Release of H+ ions only becomes prominent above rf 0.15, where little quenching is observed. The report by Yamane and Davidson[20] that a t rf < 0.2 essentially all the added silver ions are bound to DNA was confirmed for the current experimental conditions by potentiometric titration (< lo/o free silver a t rf 0.15). At higher rf values the hydrocarbon fluorescence intensity appears to increase again in certain cases. This is sometimes due to the formation of a fine precipitate, presumably the result of higher free Ag+ levels [21], which causes an increased contribution from scattered light; this effect is removed after ultracentrifugation. Release of bound hydrocarbon can also occur (the fluorescence peaks are shifted to shorter wavelengths), perhaps as a consequence of changes in secondary structure [26], and this complicates the position in regions of higher

Vol. 14, N0.3, 1970 B. GREEN 57 1

r f . Cobalt (at these rf values) did not cause these effects and the fluorescence levels were not affected by ultracentrifugation. Since the hydrocarbon fluor- escence is already strongly quenched by Ag+ ions in DNA solution below these rf levels, detailed fluori- metric examination of this region will require an instrument with greater combined sensitivity and resolution.

Release of H+ ions from the DNA (above rf 0.15) implies a change in the binding of silver ions to the DNA from mainly type I to type I1 [20,21] so that these results link the hydrocarbon fluorescence quenching with the type I silver complex. The behaviour of the DNA towards increasing addition of silver ions a t pE 7 -7.5 in these low ionic strength solutions resembles that a t more acid pH (5.6) in 0.1 M salt [21] i .e. mainly type I Ag+/DNA complex is formed initially with formation of the type I1 complex occurring a t higher r f . At a comparable pH (8) in 0.1 M salt the type I1 complex is formed a t the lowest levels of bound silver [21]. A similar dependence on ionic strength applies to the protona- tion of the DNA bases during addition of acid [27].

At moderate salt concentrations it is necessary to use heat-denatured DNA to obtain useful hydro- carbon concentrations [8, 91 and in these conditions the Ag+-quenching curve shifts to higher silver concentrations in agreement with the unfavourable effect of ionic strength found by Daune et al. [28] especially for the type I complex. A further predic- tion is that silver ion quenching should be less efficient a t higher p H where type I1 complexing is favoured [21] and this is observed when quenching is compared a t pH 7 and 9.5.

The changes in the DNA absorption spectrum during silver-ion-induced quenching of benzo- pyrene fluorescence (Fig.3) confirm that type I binding predominates over the quenching range [21,28]. At pH 5.2, where the hydrocarbon fluores- cence is largely quenched, addition of Ag+ ions fails to induce further quenching (Fig.5A). It is not known whether the metal actually binds to the relevant sites where protonation has occurred but it indicates common sites of action (GC pairs) for the two quenching mechanisms.

Other Metal Ions

A series of other metal ions known to bind to DNA was examined briefly for hydrocarbon fluores- cence quenching activity to determine the specificity of the metal ion effect. The results of two such experiments appear in Fig.7. Cu2+ ions are only slightly less effective than silver ions in quenching DNA-bound benzopyrene fluorescence and a definite, though noticeably lesser, effect is seen with Co2+ and Ni2+. The other ions tested show only minor effects or none a t all a t these low concentrations.

u 0.1 0.2 0.3 0.4

' f (total Ag' and Co2')

Big.5. A. Effect of Ag+ ions on the fluorescence intensity of benzo[a]pyrene in salmon sperm DNA solution in water. 0-0, at pH7.6 & 0.3; 0-0, at pH5.4 =k 0.2, where the fluorescence is already largely quenched. B. Effect on benzopyrene fluorescence of sequential addition of Ag+ and Co2+ ions to a solution in salmon sperm DNA in glass-distilled water. P = 1.5 mM; benzopyrene = 0.9 yM; pH (initial)

= 6.8. A, Co2+ ions; e, A@ ions

4 5 6 7 8 0.1 0.2 0.3 0.4 0.5 PH 'r

Fig. 6. Comparison of quenching of benzopyrene fluorescence in aqueous DNA and polyd(A-T) solutions; (A) by acid, (B) by Ag+ ions. The results of two poly d(A-T) experiments are combined ( 0 and 0). 0-0, poly d(A-T) (expt. 1) in water, P = 0.8 mM; benzopyrene approx. 2 @I: pH (B) = 7.05 & 0.1. o-----O, poly d(A-T) (expt. 2) in water, P = 0.46 mM; benzopyrene approx. 1.4 pM (Total N&+ = 1.5 mM, K+ = 2.6-3 mM). @, solution centrifuged for 15 min a t 330OOxg before measurement: pH(B) = 7.5 f 0.15. u---a, for comparison (B) includes quenching by Co2+ ions in polyd(A-T) (expt. 2 ) : pH = 7.5 & 0.1. . .+A, DNA in water (P = 0.8 mM) ; benzopyrene = 1.8 pM: pH (B) = 7.05 f 0.15. A----A, DNA in 5 mM Na,SO, (P = 0.8 mi), total Na+ approx. 6 mM; benzo-

pyrene < 0.5 pM: pH (B) = 7.5 -J-- 0.15

Hydrocarbon Fluorescence in Aqueous DNA Eur. J. Biochem.

0.1 0.2 0.3 0.4 0.1 0.2 0.3 0.4 0.5

Fig.7. Effect of metal ions on fluorescence intensity of benzo- [alpyrene in DNA solution in glass-distilled water: p H 7.5 to 7.8. (A) Salmon sperm DNA (P = 1.5 mM); benzopyrene

= 1.2 pM; (B) Calf thymus DNA ( P = 1.1 mM);

‘f

benzopyrene = 1.6 pM

Cobalt ions, which are less effective than silver ions in quenching DNA-bound benzopyrene fluores- cence, are more effective in poly d(A-T) (Figs.6 and 7) and hence may act partly via binding to A-T pairs in DNA. However, addition of silver ions to a DNA solution in which the benzopyrene fluorescence has been quenched as far as possible with cobalt ions, causes further quenching to roughly the level achiev- ed by silver ions alone but in the reverse situation cobalt ions have little effect on a silver ion-quenched solution (Fig. 5B).

Poly d(A-T) To confirm the dominant role of GC pairs in this

quenching, similar experiments were carried out on benzopyrene solubilized in poly d(A-T) 141. The results, which are compared with those for a DNA- benzopyrene solution in Fig. 6, show that the quench- ing effect in these particular conditions is very much less in poly d(A-T). Over the major quenching range for DNA (i.e. pH 7-5 and rf < 0.12 Ag+) the effect on benzopyrene fluorescence in poly d(A-T) is less than 22O/, compared with 70--80°/, in DNA even a t higher ionic strength. Quenching appears to be starting in poly d(A-T) below pH 6 where titration of the adenine begins (see [29]). This result confirms the dominant role of GC pairs in quenching in DNA under the particular conditions used.

When the Naf and K+ concentrations of one (dialysed) poly d(A-T) test solution were measured they were found to be 1.5 and 2.6-3mM respec- tively, rather higher than for DNA. To check that

these low salt concentrations were not responsible for the marked differences between poly d(A-T) and DNA, benzopyrene was solubilized in fresh DNA in 5 mM Na,SO, (i.e. total Na+ approx. 6 mM). In this case (Fig.6) the pH-quenching curve for the hydro- carbon was slightly shifted towards lower pH but was still a t higher pH than that for poly d(A-T); the Ag+ ion-quenching was little affected.

DISCUSSION

With levels of binding as low as these (< 1 per 200 bases) one needs to be cautious in interpreting the results since the binding regions may not reflect the composition of the molecule as a whole (e.g. in the presence of minor bases or amino acids etc.). In this case the results are explicable in a straightforward way in terms of the known properties of the major bases and the evidence that the interaction is with DNA itself has been discussed previously [a, 121.

The pH levels a t which the acid quenching occurs in DNA and the small effects observed in poly d(A-T) in this range implicate the protonation of cytosine in the process. The pH quenching range corresponds to the initial protonation which (especi- ally in GC-rich regions) may give rise to conforma- tional changes but the DNA double helical structure remains intact [24,30,31] : this is confirmed by the reversibility of the quenching. On further addition of acid, where hydrocarbon is released, the effect becomes irreversible and this presumably corresponds to the production of single strands. The inter- mediate stage (limited flexible regions in DNA) corresponds to the peak levels of binding referred to earlier. Thus these various processes correspond mainly to different stages in the acid titration of DNA. The extent of the quenching suggests that most of the hydrocarbon molecules (or, a t least, most of the emitting molecules) are bound a t GC-contain- ing sites. This is hardly surprising since earlier work [4] has indicated that GC sites have a greater affinity for benzopyrene than AT sites and over To0/, of possible intercalation sites (i.e. two adjacent base pairs) contain GC in these DNA preparations. It also seems possible that cytosine residues in the binding regions are preferentially protonated since the hydro- carbon fluorescence is quenched to 20°/, of the original value before 50°/, cytosine protonation occurs (Fig. 3). This may be because less well ordered regions titrate more readily and may account for the small differences in the quenching curves for unheated DNA a t low ionic strength and heat-denatured DNA in 0.1 M salt (Fig.2).

The most active metal ions (Ag+ and Cu2+) only become effective quenchers a t these concentrations on binding to DNA : they are ineffective when benzo- pyrene is in free solution or solubilized in aqueous

Vol. 14, No. 3,1970 B. GREEN 573

solutions of a purine (caffeine) to which they do not bind [32,33].

The other metal ions were tested to determine whether the quenching of hydrocarbon fluorescence were a selective effect and clearly it is. Unfortunately, the extent and nature of the binding of all these metal ions to DNA in these particular conditions is not known but a clear inference is that the active metal ions (Ag+, Cu2+, Hg2+, Co2+ and Ni2+) all tend to bind to the bases of DNA [34,35] rather than the phos- phate groups. Those which bind principally to the phosphate groups, Na+, Mg2+ and Mna+ (under these conditions [34,35]), are inactive. Zn2+ and Cd2+ also have little activity but they do cause minor shifts (1-2 nm) in the DNA absorption spectrum, con- sistent with the report [34] that they have some base affinity so that both the nature of the metal ion and type of base binding are important.

Daune [36] has very recently discussed the bind- ing of metal ions to DNA. All those found to be active quenchers are believed to bind to N7 of the bases, whether they form a bridge to a phosphate group, an internal chelate or are sandwiched between adjacent base pairs. The binding of the two most effective quenching ions (Ag+ and Cu2+) has been investigated in great detail principally by Davidson and Daune and their colleagues. CUB+ binds preferentially to guanine, stabilizing the GC pairs [33,35,37,38] while Ag+ ions form a t least two types of complex with DNA but major quenching is associated only with the type I complex which again causes stabilization of GC pairs [26]. The silver ion is believed to be bound to the guanine (presumably N7) and may be sand- wiched between adjacent base pairs [21,28]. I n this complex a positive charge is introduced on to the base pair. The (inactive) type I1 Ag+ complex, on the other hand, is believed to involve actual replace- ment of one of the protons involved in the in&a base-pair H-bonding of GC or AT pairs, with no resultant charge alteration [21, 281.

There is a strong similarity in the changes in the DNA absorption spectrum, especially in the relative increase in absorption on the long wavelength side characteristic of cytosine protonation (Fig. 3), which accompany the quenching of benzopyrene fluores- cence during addition of acid, Ag+ and Cu2+. It has in fact been proposed [28] that binding of silver to guanine increases the mobility of a proton a t N1 along the H-bond N1 (G)---N3 (C) so that cytosine is partially positively charged as if in an acidic medium (a similar proposal has been made for the Cul+ ion [39]). GpG sequences may be favoured sites for Cu2+ chelation (at least a t higher ionic strength) [35] and GC-rich regions are also favoured sites for the double-helix-preserving conformational changes induced by pH-lowering [24,30] which may involve a modified GC H-bonding structure [30]. As a generalization, the quenching of DNA-bound hydro-

carbon fluorescence follows the introduction of a positive charge on to GC pairs.

I n free aqueous solution individual purines such as caffeine or tetramethyluric acid form stacked sandwich-type complexes with benzopyrene, with some resemblance to nucleic acid intercalation [40-421. I n neutral solution caffeine does not quench benzopyrene fluorescence while the more polar tetramethyluric acid does, but the positively charged caffeine ion (i.e. in acid solution) is an effective quencher [43].

These purine complexes have been proposed to be of the polarization-bonding type [41, 42,441 as described for plane-to-plane stacked complexes by Wallwork [45] i.e. mainly resulting from interactions between the polar groups of one component (purine) and polarizable second component (hydrocarbon). Such interactions would be strengthened by the introduction of a charge or strongly polar group on to the purine and Weil-Malherbe [43] suggested that the fluorescence quenching stemmed from this stronger interaction.

Weil-Malherbe [43] also obtained evidence that adenine and guanine show slight quenching activity for benzopyrene fluorescence in acidified goo/, aqueous ethanol and such an effect has been confirmed in aqueous solution for adenosine and deoxyguanylic acid, but not cytidine, in the present work. This may simply reflect the well-known lower affinity of pyrimidine derivatives for hydrocarbons in free aqueous solution compared with purines [40,41] and need not represent the situation in a macromolecule where they may be held in close proximity.

The exact way in which a charged GC pair quenches hydrocarbon fluorescence is unknown. The fluorescence-quenching sites for proflavine when intercalated into DNA require a GC pair next to the dye, and it has been suggested that a charge-transfer interaction is involved in the quenching process [46,47]. While interactions of this type may be unimportant in determining the extent of the normal purine or DNA reaction with hydrocarbons [42,44,1] they could be favoured if the introduction of a posi- tively charged proton or metal ion on to a GC pair, as well as strengthening the polarization forces between it and an adjacent hydrocarbon also alters the mutual orientation of the two components to give a more favourable overlap of molecular orbitals. At least with the close planer stacking of hydro- carbon and bases which is the essential feature of intercalation the possibility of such close-range electronic interactions makes quenching processes readily conceivable: it is difficult to envisage an efficient quenching mechanism for partially proto- nated DNA if the hydrocarbons are externally bound.

Finally, the observation that protonated GC pairs in DNA constitute fluorescence-quenching sites

574 B. GREEN: Hydrocarbon Fluorescence in Aqueous DNA Eur. J. Biochem.

for bound hydrocarbons allows a partial explanation of the results of fluorescence lifetime measurements of perylene in these systems [48]. It was observed that there was a lower relative quantum yield of perylene fluorescence in neutral aqueous DNA solu- tion compared with caffeine but the fluorescence lifetime was little changed (approx. 8 nsec). Because of the low extent of hydrocarbon binding only a small proportion (< l0/J of GC pairs need be pro- tonated (quite feasible a t neutral pH a t the low ionic strength used [27]) to account for the non-uniformity of emission properties of the DNA-bound perylene molecules.

I wish to thank Mr. D. R. Muirhead for skilled assis- tance with the initial observations, which were made a t the Cancer Research Laboratory, University of Western Ontario (London, Ontario, Canada). I am also indebted to Dr. J. A. McCarter and Professor E. Boyland for advice and criticism. The help of Mr. A. G. Green and Mrs. D. Pur- kiss, who carried out the metal ion and amino acid deter- minations, respectively, is gratefully acknowledged. The cooperation of Dr. K. R. Harrap in providing facilities for the use of the more sensitive version of the Aminco-Bowman spectrophotofluorimeter is also much appreciated. This research has been supported by grants to the Chester Beatty Research Institute (Institute of Cancer Research: Royal Cancer Hospital) from the Medical Research Council and the British Empire Cancer Campaign for Research.

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B. Green Chester Beatty Research Institute Institute of Cancer Research: Royal Cancer Hospital Fulham Road, London, S. W. 3, Great Britain

Address from September 1970: Biochemistry Department, University of Strathelyde, Glasgow, Cl, Great Britain