Characterization of transferrin metal-binding sites by diffusion-enhanced energy transfer

The distance from the protein surface to ferric or manganic ions in the two specific metal-binding sites of human serum transferrin has been estimated by measuring energy transfer from freely diffusing terbium chelaters in aqueous solution to transferrin-bound metal ions. In addition, both monoferric forms of the protein were studied, as well as the diferric complex formed by using oxalate instead of (bi)carbonate as the auxiliary anion in binding of iron(III) to transferrin. Second-order rate constants for energy transfer between electrically neutral terbium(III)--N-(2-hydroxy-ethyl)ethylenediaminetriacetate and the FeA, FeB, and Fe2 forms of transferrin were 0.9 X 10(5) M-1 S-1, 1.4 X 10(5) M-1 S-1, and 2.6 X 10(5) M-1 S-1, respectively (based on iron concentraton). For the Fe2 species, substitution of oxalate for (bi)carbonate has the effect of decreasing the accessibility of both electrically neutral and negatively charged terbium chelates to the protein-bound iron chromophores. Theoretical considerations of the effect of acceptor location in the protein on energy transfer suggest that the iron chromophores are not on the surface of the protein but are less than 1.7 nm below the surface. The use of diterbium transferrin as energy donor to a small cobalt chelate in solution or to diferric transferrin corroborates these results.     

DNA Bifunctional intercalators. 2. Fluorescence properties and DNA binding interaction of an ethidium homodimer and an acridine ethidium heterodimer

An ethidium homodimer and acridine ethidium heterodimer have been synthesized (Gaugain, B., Barbet, J., Oberlin, R., Roques, B. P., & Le Pecq, J. B. (1978) Biochemistry 17 (preceding paper in this issue)). The binding of these molecules to DNA has been studied. We show that these dimers intercalate only one of their chromophores in DNA. At high salt concentration (Na+ greater than 1 M) only a single type of DNA-binding site exists. Binding affinity constants can then be measured directly using the Mc Ghee & Von Hippel treatment (Mc Ghee, J. D., & Von Hippel, P. H. (1974) J. Mol. Biol. 86, 469). In these conditions the dimers cover four base pairs when bound to DNA. Binding affinities have been deduced from competition experiments in 0.2 M Na+ and are in agreement with the extrapolated values determined from direct DNA-binding measurements at high ionic strength. As expected, the intrinsic binding constant of these dimers is considerably larger than the affinity of the monomer (ethidium dimer K = 2 X 10(8) M-1; ethidium bromide K = 1.5 X 10(5) M-1 in 0.2 M Na+). The fluorescence properties of these molecules have also been studied. The efficiency of the energy transfer from the acridine to the phenanthridinium chromophore, in the acridine ethidium heterodimer when bound to DNA, depends on the square of the AT base pair content. The large increase of fluorescence on binding to DNA combined with a high affinity constant for nucleic acid fluorescent probes. In particular, such molecules can be used in competition experiments to determine the DNA binding constant of ligands of high binding affinity such as bifunctional intercalators.     

Electron donor properties of the antitumour drug amsacrine as studied by fluorescence quenching of DNA-bound ethidium

The effect of the antitumour acridine derivative amsacrine [4'-(9-acridinylamino)methanesulphon-m-anisidide] on the fluorescence lifetime of DNA-bound ethidium has been investigated using a synchronously pumped cavity dumped dye laser producing picosecond pulses for sample excitation and a time-correlated single photon counting detection system. As the proportion of DNA-bound amsacrine on the synthetic DNA polymer poly[deoxyadenylic-thymidylic acid] is increased, the fluorescence decay curve of ethidium can be accurately resolved into two exponential components. The short lifetime component, whose proportion increases with increasing proportions of DNA-bound amsacrine, has a lifetime of between 3 and 4 ns, significantly longer than that of ethidium in aqueous solution (1.63 ns). The magnitude of the long lifetime component decreases from 25.4 to 14 ns with increasing proportions of bound amsacrine. It is concluded that a new fluorescence state of ethidium (lifetime 3-4 ns) is present, probably resulting from reversible electron transfer between ethidium and amsacrine. The ability of various 9-anilinoacridine derivatives to quench the fluorescence of DNA-bound ethidium appears to be related to the electron donor properties of the substituents on the anilino ring, as well as to experimental antitumour activity. The electron donor properties of DNA-bound amsacrine may therefore be relevant to its antitumour action.     

Interaction of Two Peptide-Acridine Conjugates Containing the SPKK Peptide Motif with DNA and Chromatin

Abstract

The interaction between DNA and two peptide-acridine conjugates containing one (1) or two (2) moieties of the Ser-Pro-Lys-Lys (SPKK) minor groove-binding peptide motif has been studied by a combination of hydrodynamic, biochemical and spectroscopic methods including diffusion-enhanced luminescence energy transfer (DELET) measurements with a Tb(III) lanthanide chelate as donor. Viscometric titrations do not reveal any significant difference between the two hybrid molecules which both unwind (by about 15°) and extend the DNA similarly. DELET measurements show that the acridinyl chromophore of compounds 1 and 2 is much more accessible than that of a simple monointercalating drug such as acridine orange or ethidium. The accessibility factor increases proportionally with the peptide length, reflecting the extent of perturbation imposed upon the intercalating chromophore by the binding to DNA of the peptide moiety of the hybrids. Experiments with the osmium tetroxide- bispyridine reagent indicate that the two hybrid compounds both affect the local conformation of DNA rendering certain thymine residues conspicuously accessible to the probe. The drug-induced sites of hyperreactivity towards OsO₄ in DNA are very similar with the exception of a short run of three T residues which is attacked more strongly in the presence of tetrapeptide-acridine conjugate 1 than with the octapeptide-acridine conjugate 2. These results are fully in agreement with previous footprinting studies and support the view that a minimum of two SPKK motifs is required to mimic the AT-specific minor groove binding antibiotic netropsin. On the basis of the DNA-binding properties of these two peptide-acridine hybrids, we present DNA-binding models in which the acridinyl moiety of compound 1 protrudes slightly outside the double helix but remains more or less parallel to the plane of the base-pairs. In contrast, with compound 2, where the octapeptide SPKKSPKK is bound to the minor groove, we postulate that the chromophore lies only partially overlapped with the base pairs in the intercalation site and, in addition, the heterocyclic chromophore is significantly tilted with respect to the double helix axis. Electric linear dichroism and DELET measurements with chromatin reveal that the presence of histone proteins affects the intercalative binding of compound 2 while it has practically no effect on the binding of compound 1.     

A reexamination of the problem of resonance energy transfer between DNA intercalated chromophores using bisintercalating compounds.

The rate of energy transfer between DNA intercalated ethidium cations calculated by Paoletti and Le Pecq1 using the Forster theory differs from the measured one by a factor of twenty two, if the proper geometrical factors are taken into account. By changing some of the parameters used in the calculation, the discrepancy can be reduced but not eliminated. This led us to the study of other systems where experimental and calculated results can be more directly compared. The apparent rate of energy transfer between ethidium and one of its non fluorescent analogues and between various pairs of intercalated chromophores has been studied. The fluorescence anisotropy decay of acridine dimers in glycerol or bisintercalated in DNA has been measured. These studies show that the Forster theory of energy transfer does not apply to the case of identical chromophores when they are relatively close to each other.     

Heterodimeric DNA-binding dyes designed for energy transfer: synthesis and spectroscopic properties.

Heterodimeric dyes are described which bind tightly to double-stranded (dsDNA) with large fluorescence enhancements. These dyes are designed to exploit energy transfer between donor and acceptor chromophores to tune the separation between excitation and emission wavelengths. The dyes described here absorb strongly at the 488 nm argon ion line, but emit at different wavelengths, and can be applied to multiplex detection of various targets. The chromophores in these dyes, a thiazole orange-thiazole blue heterodimer (TOTAB), two different thiazole orange-ethidium heterodimers (TOED1 and TOED2), and a fluorescein-ethidium heterodimer (FED), are in each case linked through polymethyleneamine linkers. The emission maxima of the DNA-bound dyes lie at 662 (TOTAB), 614 (TOED 2), and 610 nm (FED). The dyes showed a > 100 fold enhancement of the acceptor chromophore fluorescence on binding to dsDNA and no sequence selectivity. In comparison with direct 488 nm excitation of the constituent monomeric dyes, in the heterodimers the fluorescence of the acceptor chromophores was greatly enhanced and the emission of the donor chromophores quenched by over 90%. The acceptor emission per DNA-bound dye molecule was constant from 100 DNA bp:dye to 20 bp:dye and decreased sharply at higher dye:DNA ratios.     

Synthesis, structure and DNA interaction of cobalt(III) bis-complexes of 1,3-bis(2-pyridylimino)isoindoline and 1,4,7-triazacyclononane

The complex [CoL(2)](ClO(4)).MeOH (1), where HL is the tridentate 3N ligand 1,3-bis(2-pyridylimino)isoindoline, has been isolated and its X-ray crystal structure successfully determined. It possesses a distorted octahedral structure in which both the ligands are coordinated meridionally to cobalt(III) via one deprotonated isoindoline (L(-)) and two pyridine nitrogen atoms. Interestingly, the average dihedral angle between pyridine and isoindoline rings is 25.9 degrees , indicating that the ligand is twisted upon coordination to cobalt(III). The interaction of the complex with calf-thymus DNA has been studied using various spectral methods and viscosity and electrochemical measurements. For comparison, the DNA interaction of [Co(tacn)(2)]Cl(3) (2), where tacn is facially coordinating 1,4,7-triazacyclononane, has been also studied. The ligand-based electronic spectral band of 1 and the N(sigma)-->Co(III) charge transfer band of 2 exhibit moderate hypochromism with small or no blue shift on interaction with DNA. The intrinsic binding constants calculated reveal that the monopositive complex ion [CoL(2)](+) exhibits a DNA-binding affinity lower than the tripositive complex ion [Co(tacn)(2)](3+). The steric clashes with DNA exterior caused by the second L(-) ligand bound to cobalt(III), apart from the lower overall positive charge on the [CoL(2)](+) complex, dictates its DNA-binding mode to be surface binding rather than partial intercalative interaction expected of the extended aromatic chromophore of deprotonated isoindoline anion. An enhancement in relative viscosity of CT DNA on binding to 1 is consistent with its DNA surface binding. On the other hand, a slight decrease in viscosity of CT DNA was observed on binding to 2 revealing that the smaller cation leads to bending (kinking) and hence shortening of DNA chain length. The electrochemical studies indicate that the DNA-bound complexes are stabilised in the higher Co(III) rather than the lower Co(II) oxidation state, suggesting the importance of electrostatic forces of DNA interaction.     

Spectroscopic and molecular modeling studies of caffeine complexes with DNA intercalators.

Recent studies have demonstrated that caffeine can act as an antimutagen and inhibit the cytoxic and/or cytostatic effects of some DNA intercalating agents. It has been suggested that this inhibitory effect may be due to complexation of the DNA intercalator with caffeine. In this study we employ optical absorption, fluorescence, and molecular modeling techniques to probe specific interactions between caffeine and various DNA intercalators. Optical absorption and steady-state fluorescence data demonstrate complexation between caffeine and the planar DNA intercalator acridine orange. The association constant of this complex is determined to be 258.4 +/- 5.1 M-1. In contrast, solutions containing caffeine and the nonplanar DNA intercalator ethidium bromide show optical shifts and steady-state fluorescence spectra indicative of a weaker complex with an association constant of 84.5 +/- 3.5 M-1. Time-resolved fluorescence data indicate that complex formation between caffeine and acridine orange or ethidium bromide results in singlet-state lifetime increases consistent with the observed increase in the steady-state fluorescence yield. In addition, dynamic polarization data indicate that these complexes form with a 1:1 stoichiometry. Molecular modeling studies are also included to examine structural factors that may influence complexation.     

The transverse location of the retinal chromophore in the purple membrane by diffusion-enhanced energy transfer

We have used fluorescence energy transfer in the rapid-diffusion limit (RDL) to estimate the trans-membrane depth of retinal in the purple membrane (PM). Chelates of Tb(III) are excellent energy donors for the retinal chromophore of PM, having a maximum Ro value for F?rster energy transfer of approximately 62 A (assuming a donor quantum yield of 1). Energy transfer rates were measured from the time-resolved emission kinetics of the donor. The distance of closest approach between chelates and the chromophore was estimated by simulating RDL energy-transfer rate constants according to geometric models of either PM sheets or membrane vesicles. The apparent rate constant for RDL energy transfer between Tb(III)HED3A and retinal in PM sheets is 1.5(+/- 0.1) x 10(6) M-1 s-1, corresponding to a depth of approximately 10 +/- 2 A for the retinal chromophore. Cell envelope vesicles (CEVs) from Halobacterium halobium were studied by using RDL energy transfer to assess the proximity of retinal to either the extracellular or intracellular face of the PM. The estimated depth of retinal from the extravesicular face of the PM is 10 +/- 3 A, based on the RDL energy-transfer rate constant. Energy-transfer levels to retinal in the PM were estimated by an indirect method with energy donors trapped in the inner-aqueous space of CEVs. The rate constants derived for this arrangement are too low to be consistent with the shortest depth of retinal deduced for PM sheets. Thus, the intravesticular face of CEVs, corresponding to the cytoplasmic face of cells, is the more distant surface from the chromophore of bacteriorhodopsin.     

Thiol-reactive luminescent chelates of terbium and europium

Thiol-reactive lanthanide complexes have been synthesized that are luminescent when bound to terbium and/or europium. The complexes consist of a diethylenetriaminepentaacetate (DTPA) chelate covalently joined through one amide bond to a chromophore, carbostyril 124, and via a second amide bond to a maleimide, bromoacetamide, or pyridyldithio moiety. Site-specific attachment and characterization of the complexes attached to DNA-activating protein NtrC, to various sites on myosin, or to DNA are presented. The compounds coordinate a surprisingly large number of ligation sites of terbium when a hydrazide spacer is used between the chelate and thiol-reactive moiety, although this extra ligation can cause quenching when europium is used. Synthesis is a simple two- or three-step reaction, and purification is straightforward. The compounds should be useful as nonisotopic replacements, as long-lifetime probes in imaging, and as donors in luminescence resonance energy transfer. They are examples of a wide class of chelates that can be made conjugatable via readily available hetero- or homo-bifunctional linkers.     

PMR studies of the self-association of dna intercalating ellipticine derivatives in aqueous solution

In aqueous solution DNA intercalating ellipticine derivatives aggregate in n-mers. The self-association constants K are higher than those of 2-nicthoxy-6-chloro-9-[3-diniethylaminopropyl-amino]-acridine and ethidium bromide. They are of the same order as that of actinomycin D but inferior to that of acridine orange. The increase of the 9-hydroxy-ellipticine constant by addition of sodium chloride shows the importance of anion participation in the mechanism of stacking in accordance with the high energy of self-association. In the stacked n-mets the ellipticine rings aie inverted. The geometry shows the importance of the orientation of the quadiupole axis in the intermotecular association of the intercalating drugs.     

Diffusion-enhanced energy transfer investigation of histone H5 in chromatin with a fluorescently-labelled antibody fragment Fab'.

The location of chicken erythrocyte H5 histone relative to the axis the 30 nm chromatin fibre axis has been investigated by diffusion-enhanced energy transfer. In this investigation, a neutral lanthanide chelate as donor and a fluorescent probe specific to H5 as acceptor have been used. The acceptor probe consists of H5 antibody Fab' fragment, which has been labeled with 5-iodoacetamidofluorescein (5-IAF). Using H5 fragments we have shown by ELISA that the antibodies recognized the N- and C-terminal ends of this histone. A neutral chelate of terbium (TbHED3A) was chosen as a suitable donor for energy transfer with IAF-labelled Fab' (Fab'-IAF) bound to H5 in various chromatin structures. The ionic strength dependence of the energy transfer from TbHED3A to chromatin-bound Fab'-IAF was used to estimate the accessibility and the location of the Fab' in chromatin. The rate constants for energy transfer, obtained from the lifetimes of the TbHED3A excited state in presence and absence of acceptor, indicated a decrease in transfer efficiency upon increase of salt concentration from 5 to 80 mM NaCl. This can be correlated with the chromatin folding occurring in this ionic strength range and is consistent with the location of at least some of the N and C-termini of H5 within the condensed chromatin structure.     

Transverse location of the retinal chromophore of rhodopsin in rod outer segment disc membranes

Diffusion-enhanced fluorescence energy transfer was used to study the structure of photoreceptor membranes from bovine retinal rod outer segments. The fluorescent energy donor was Tb³⁺ chelated to dipicolinate and the acceptor was the 11-cis retinal chromophore of rhodopsin in vesicles made from disc membranes. The rapid-diffusion limit for energy transfer was attained in these experiments because of the long excited state lifetime of the terbium donor (~2 ms). Under these conditions, energy transfer is very sensitive to a, the distance of closest approach between the donor and acceptor (Thomas et al., 1978). Vesicles containing terbium dipicolinate in their inner aqueous space were prepared by sonicating disc membranes in the presence of this chelate and chromatographing this mixture on a gel filtration column. The sidedness of rhodopsin in these vesicles was the same as in native disc membranes. The transfer efficiency from terbium to retinal in this sample was 43%. For an R₀ value of 46.7 Å and an average vesicle diameter of 650 Å, this corresponds to an a value of 22 Å from the inner aqueous space of the vesicle. The distance of closest approach from the external aqueous space, determined by adding terbium dipicolinate to a suspension of already formed vesicles, was found to be 28 Å. These values of a show that the retinal chromophore is far from both aqueous surfaces of the disc membrane. Hence, the transverse location of the retinal chromophore is near the center of the hydrophobic core of the disc membrane. These findings suggest that conformational changes induced by photoisomerization are transmitted through a distance of at least 20 Å within rhodopsin to trigger subsequent events in visual excitation.     

Studies of electron migration in DNA in aqueous solution using intercalating dyes.

The reactions of the hydrated electron (e-aq) and of the hydroxyl radical (OH) with double-stranded DNA in aqueous solution at room temperature have been studied through the use of the intercalating dyes, proflavine and ethidium. These dyes react with e-aq with rate constants of (2.5 +/- 0.2) - 10(10) M-1 - s-1 and (3.0 +/- 0.3) - 10(10) M-1 - s-1, respectively; the rate constant for the reaction of OH with proflavine is (1.0 +/- 0.2) - 10(10) M-1 - s-1. When these molecules are bound within the DNA structure both the yields and the rate constants of reaction with e-aq are reduced in a manner entirely consistent with a simple competition between the DNA bases and restricted dye molecules reacting with a bimolecular rate constant of about 2 - 10(9) M-1 - s-1. No evidence of free electron migration in the DNA was obtained, and an upper limit of five base pairs for the range of such migration was derived. Reactions of the hydroxyl radical with DNA-bound proflavine also lead to a rate constant of about 2 - 10(9) M-1 - s-1. These rate constants are in good agreement with rate predictions (per base unit) for a diffusion-controlled reaction with the DNA structure.     

Self-catalyzed cyclization of the intervening sequence RNA of Tetrahymena: inhibition by intercalating dyes.

The intervening sequence (IVS) excised from the pre-rRNA of Tetrahymena undergoes a self-catalyzed cleavage-ligation reaction to form a covalently closed circular RNA. This cyclization reaction is kinetically inhibited by ethidium bromide (50% inhibition at 22 +/- 14 microM, greater than 99% inhibition at 53 +/- 16 microM for a 20 minute reaction). The dye does not alter the sites of the cyclization reaction, but it does increase the relative amount of reaction at a minor site 19 nucleotides from the 5' end of the IVS. The reversibility of the inhibition and the relative inhibitory strength of acridine orange, ethidium and proflavine suggest that inhibition is due to intercalation of the dye in functionally important secondary or tertiary structures of the IVS. The concentration of dye required to inhibit cyclization is much higher than expected from the known binding constants of such dyes to tRNA. At high Mg2+ to Na+ ratios, conditions which should stabilize RNA structure, a subpopulation of the IVS RNA molecules is resistant to ethidium inhibition, even at 200 microM ethidium. These data are interpreted as reflecting two conformational isomers of the IVS that differ in their reactivity and in their sensitivity to dye binding.     

10N-nonyl acridine orange interacts with cardiolipin and allows the quantification of this phospholipid in isolated mitochondria.

The acridine orange derivative, 10N-nonyl acridine orange, is an appropriate marker of the inner mitochondrial membrane in whole cells. We use membrane model systems to demonstrate that 10N-nonyl acridine orange binds to negatively charged phospholipids (cardiolipin, phosphatidylinositol and phosphatidylserine). The stoichiometry has been found to be 2 mol 10N-nonyl acridine orange/mol cardiolipin and 1 mol dye/mol phosphatidylserine or phosphatidylinositol, while, with zwitterionic phospholipids, significant binding could not be detected. The affinity constants were 2 x 10(6) M-1 for cardiolipin-10N-nonyl-acridine-orange association and only 7 x 10(4) M-1 for that of phosphatidylserine and phosphatidylinositol association. The high affinity of the dye for cardiolipin may be explained by two essential interactions; firstly an electrostatic interaction between the quaternary ammonium of nonyl acridine orange and the ionized phosphate residues of cardiolipin and secondly, hydrophobic interactions between adjacent chromophores. A linear relationship was demonstrated between the cardiolipin content of model membranes and the incorporated dye. Consequently, a convenient and rapid method for cardiolipin quantification in membranes was established and applied to the cardiolipin-containing organelle, the mitochondrion.     

Fluorescence decay and quantum yield characteristics of acridine orange and proflavine bound to DNA

Fluorescence properties (quantum yield, decay curve, lifetime and polarization) of acridine orange and proflavine bound to DNA were examined as a function of nucleotide to dye (P/D) ratio. First, mean fluoiescence lifetimes were determined by the phase-shift measurements. The lifetime and quantum yield of acridine orange increased in a parallel fashion with increasing P/D ratio. There was no parallel relation between the lifetime and quantum yield for proflavine; the lifetime showed a minimum around P/D = 10. Next, fluorescence decay curves were measured by the monophoton counting technique and analyzed with the aid of the method of moments and the Laplace transform method. The results showed that the fluorescence decay of bound acridine orange was exponential above P/D = 10. On the other hand, the decay of bound proflavine was exponential above P/D = 100, but markedly deviated from exponentiality with decreasing P/D ratio. The results of fluorescence polarization suggested that this phenomenon is the result of Förster energy transfer between proflavine molecules bound to the fluorescent site (AT pair) and bound to the quenching site (GC pair). Critical transfer distances were 26-4 and 37.0 Å, respectively, for bound proflavine and acridine orange.     

Spectrophotometric and NMR analysis of the interaction of fluorinated mono- and bifunctional intercalators with DNA, poly(dA-dT), and poly(dG-dC)

We have synthesized and investigated the DNA binding properties of three fluorinated acridine derivatives—a monomer (I), a short dimer (II) and a long dimer (III). Only III has a sufficiently long chain bridging the two acridine nuclei to permit binding by bisintercalation. Analysis of the equilibrium and kinetic binding properties of these compounds to poly(dA-dT) demonstrates that they behave very similarly to their unfluorinated parent compounds. Helix extension, as determined by viscosity measurements, shows that both compounds I and II bind by monointercalation while III binds by bisintercalation. These results are confirmed by ¹⁹F-nmr analysis, which indicates, in particular, that the two chromophores of III share the same molecular environment as that of I in the presence of either calf thymus DNA or poly(dA-dT). Negative nuclear Overhauser effects in the presence of DNA indicate tight binding such that the motion of the ligands is governed by the polynucleotide dynamics. Optical titrations establish that in 4M NaCl, both I and III bind to calf thymus DNA, but no binding was observed with poly(dG-dC). This result is in contrast to those for dimers of ethidium, which show substantial binding to polynucleotides under high salt conditions. Nuclear magnetic resonance experiments, however, carried out at considerably higher concentrations, show that compound I does indeed bind to poly(dG-dC) under these high salt conditions, albeit weakly, and leads to a conversion of the polynucleotide from a left-handed to a right-handed conformation.     

Quenching of DNA-ethidium fluorescence by amsacrine and other antitumor agents: a possible electron-transfer effect

The antitumor agent amsacrine, 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), when bound to double-stranded DNA, particularly poly(deoxyadenylicthymidylic acid), reduced the fluorescence of bound ethidium without physically displacing it from DNA. Fluorescence lifetime measurements showed that the reduction of fluorescence was not due to reduction of the lifetime of the excited state of ethidium. Rather, a proportion of the DNA-bound ethidium changed to a state where the fluorescence was highly quenched. Several other 9-anilinoacridine derivatives, and also 9-hydroxyellipticine, caused quenching of ethidium-DNA fluorescence, whereas 9-aminoacridine, proflavin, and ellipticine had no effect. Resonance energy transfer (F?rster transfer) is not responsible for the effect since there is no spectral overlap between the absorption spectrum of any of the agents and the fluorescence emission spectrum of ethidium. It is suggested that quenching may occur as a result of reversible formation of electron-transfer complexes between the intercalating drug and the excited state of ethidium.     

Heavy-atom effects on energy transfer from polynucleotides to terbium (III)

Energy transfer in nucleic acids or polynucleotides at room temperature can be studied by using the fluorescence of complexed terbium (III) as a tool. Complexing the heavy atom thallium (I) enhances energy transfer from poly(G) to terbium (III). Thallium has no effect on transfer from GMP to terbium and a small negative effect on the transfer from single-stranded DNA to terbium. Use of the Medinger-Wilkinson model to analyze the poly(G) results provides an estimate of the room-temperature intersystem crossing constant.