Classification of CD and absorption spectra in the Soret band of H(2)TMPyP bound to various synthetic polynucleotides

The binding mode of porphyrins, namely meso-tetrakis(N-methyl pyridinium-4-yl)porphyrin (H(2)TMPyP), was classified in this work by absorption and circular dichroism(CD) spectroscopy. The three binding modes of intercalation, minor groove binding and external stacking exhibit their own characteristic absorption and CD spectra. Intercalation occurs for this porphyrin when bound to GC-rich polynucleotides at a low mixing ratio, as expected. This binding mode produces hypochromism and a red shift in the absorption band and a negative CD band in the Soret absorption region. When it is complexed with AT-rich polynucleotides at a low mixing ratio, hypochromism and a red shift in the absorption band and a positive CD peak is apparent, and this species can easily be assigned to the minor groove-binding mode. For both AT- and GC-rich polynucleotides at a high binding ratio, an excitonic CD was apparent. The sign of excitonic CD depends on the order of the DNA bases; the CD spectra of H(2)TMPyP complexed with non-alternating homopolymer (disregarding the nature of base pairs, i.e. AT or GC) are characterized by a positive band at short wavelengths followed by a negative band at long wavelengths. In contrast, those complexed with alternating polynucleotide were opposite to those of non-alternating homopolymers.     

Stacking interactions of ethidium bromide bound to a polyphosphate and phage DNA in situ

Ethidium bromide forms spectroscopically detectable aggregates in aqueous solution and at a high dye concentration larger than 1 × 10^?3 moles/ρ. At moderate concentration in the order of 1 × 10^?4 moles/ρ the dye interacts with inorganic polyphosphate Graham salt and with phage sd DNA in situ by formation of stacking complexes. Maximal stacking was found at a phosphate to dye ratio, P/D, of approximately 1 for Graham salt and 1.5–2 for phages. In going to a higher P/D ratio Graham salt dye complex dissociates again and free dye reappears, while phage dye binding changes from stacking (type II complex) to intercalation (type I complex). Stacking is accompanied by a decrease and intercalation by an increase of relative fluorescence intensity with respect to free dye. However, both binding types lead to hypechromism and a red shift of the dye absorption band in the visible spectral region. Thus spectral behavior of ethidium aggregates deviate clearly from that known for other dyes, i.e., acridines.     

Circular differential scattering and circular differential absorption of DNA-protein condensates and of dyes bound to DNA-protein condensates

DNA-protein condensates that give positive and negative psi-type circular dichroism (CD) spectra (psi condensates) bind intercalative and nonintercalative dyes. CD depends both on circular differential scattering and on circular differential absorption; scattering-corrected CD measurements are approximations to circular differential absorption. The circular differential scattering and scattering-corrected CD patterns observed in the DNA absorption band of psi condensates are mimicked in the induced CD band of intercalators bound to psi condensates. The induced scattering-corrected CD and circular differential scattering patterns of the groove-binding dye Hoechst 33342 bound to psi condensates are the inverse of the patterns seen with intercalative dyes, whereas the groove-binding dye manganese(III) meso-tetrakis(4-N-methylpyridyl)porphine [MnIIITMpyP-4] shows no significant induced CD patterns. The large circular differential scattering and scattering-corrected CD bands are interpreted as resulting from long-range chiral packing, rather than near-neighbor short-range interactions. Dyes intercalated into the DNA of the psi condensates have the same type of long-range chiral packing as the DNA bases. Therefore, the psi-type CD spectra seen in the UV spectra originating from the long-range packing of the DNA bases are also observed in the visible spectra when dyes are intercalated in the DNA of the psi condensates. Our interpretation comes from the observation that the induced circular differential scattering and circular differential absorption of the dye bound to the psi condensates depend only upon the sign of the circular differential absorption and the pattern of the circular differential scattering of the psi condensates without bound dye.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of methylene dimethanesulphonate (MDMS) on the conformation of DNA and its dependence on base composition.

The interaction of the alkanesulphonate, methylene dimethanesulphonate (MDMS) with DNA has been studied. Thermal denaturation studies on mixtures of MDMS and DNA showed a dose-dependent decrease of the melting temperature midpoint (Tm) of the DNA. In addition, an irreversible decrease in ultraviolet absorption (hypochromism) preceded the hyperchromic shift, the magnitude of the former being linearly related to both the relative concentration of MDMS and the G-C content of the DNA used. Neither the reduction in melting temperature nor the initial UV absorption decrease occurred after dialysis of the reaction mixture. Equimolar proportions of the hydrolysis products of MDMS did not give the same effects as observed with the unhydrolysed agent. A similar hypochromism followed by strand separation occurs when DNA is allowed to stand with MDMS at room temperature, the time of subsequent strand separation being related to the treatment level of the drug. A weak association of MDMS with DNA is considered to be involved resulting in a local compression of the helical structure in the vicinity of the G-C pairs. It is suggested that this conformational change may act as a substrate for repair enzymes in vivo.     

Binding mode of a cationic porphyrin to parallel and antiparallel thrombin binding aptamer G-quadruplex

Abstract

The spectral properties of meso-tetrakis (N-methylpyridinium-4-yl)porphyrin (TMPyP) in the presence of parallel and antiparallel G-quadruplexes formed from a thrombin-binding aptamer G-quadruplex (5′-G₃T₂G₃TGTG₃T₂G₃) were investigated in this study. Red shift and hypochromism in the Soret absorption band of TMPyP were observed after binding to both parallel and antiparallel G-quadruplexes. The extent of changes in the absorption spectra were similar for both conformers. No circular dichroism spectrum was induced in the Soret region for both parallel and antiparallel G-quadruplexes. This is suggest that there is no or very weak interaction between electric transitions of nucleobases and porphyrin molecule. The accessibility of the neutral quencher I₂ to the G-quadruplex-bound TMPyP was similar for both parallel and antiparallel G-quadruplexes. All these observations suggest that TMPyP was bound at the outside of the quadruplexes, and conceivably interacted with the phosphate group via a weak electrostatic interaction.

Communicated by Ramaswamy H. Sarma     

Binding mode of 4',6-diamidino-2-phenylindole and ethidium to poly(dG).poly(dC).poly(dC)(+) triplex and poly(dG).poly(dC) duplex

Optical spectroscopic properties of 4',6-diamidino-2-phenylindole (DAPI) and ethidium bromide complexed with poly(dG).poly(dC).poly(dC)(+) triplex and poly(dG).poly(dC) duplex were compared in this study. When complexed with both duplex and triplex, ethidium is characterized by hypochromism and a red shift in the absorption spectrum, a complicate induced circular dichroism (CD) band in the polynucleotide absorption region, and a negative reduced linear dichroism signal in both polynucleotide and drug absorption regions. The spectral properties for both duplex- and triplex-bound ethidium are identical and both can be understood by the intercalation binding mode. In contrast, the absorption and CD spectra of DAPI complexed with triplex differ from those of the DAPI-duplex complex, although both complexes can be understood by the intercalation binding mode. Considering that the third strand runs along the major groove of the template duplex, we conclude that the DAPI molecule partially intercalates near the major groove of the duplex, where the third strand can affect its spectroscopic properties.     

Interaction of ethidium bromide with DNA. Optical and electrooptical study

The binding of ethidium bromide to DNA has been studied by various optical methods. From fluorescence polarization studies, and film, electric linear dichroism, and circular dichroism spectra, we propose assignments of the absorption bands of the dye, which are discussed in connection with wave-mechanical calculations recently reported. The optical activity induced in the dye absorption bands upon binding to DNA was attributed to various origins depending on the electronic transition considered. The visible absorption band displayed a circular dichroism due to the asymmetry of the binding site and independent of the amount of binding. The transition identified at 378 nm from the circular dichroism and electric dichroism observations was thought to be due to a magnetic-dipole transition. It remained constant with increasing amounts of dye bound. The main ultraviolet band showed circular dichroism characteristics corresponding to exciton interactions between dye molecules bound to neighboring sites. The electric dichroism observed for the strongly bound dye molecules indicated that the phenanthridinium ring of ethidium bromide was probably not perfectly parallel to the DNA base planes. When the amount of dye bound to DNA exceeded the maximum amount compatible with the exclusion of adjacent binding sites, the electric dichroism decreased owing to the appearance of externally bound dye molecules with no contribution to the dichroism. Sonicated DNA was used to study the lengthening of the DNA molecule upon complexation. Although the viscosity of the complexes increased with the amount of binding, the rotational diffusion coefficient measured by the electric birefringence relaxation was not detectably affected. The absence of variation in the electric birefringence with the binding indicated that the DNA base stacking remained unaltered.     

Template synthesis of novel carboxamide dinuclear copper (II) complex: spectral characterization and reactivity towards calf-thymus DNA

Dinuclear complexes Bis [aqua 1,8-(1,2-dicarboxamido benzene) 3,6-diazaoctane copper (II)/nickel (II)] tetrachloride (1 and 2) were synthesized by a two component one-pot metal template condensation between phthalic anhydride and 1,8-diamino 3,6-diazaoctane. Elemental analysis, molar conductance measurements, electronic absorption, infra-red, electron paramagnetic resonance, nuclear magnetic resonance, atomic absorption, and electron spray mass spectral studies have been performed to probe the nature and structure of the complexes. The interaction of copper (II) complex with calf thymus (CT-DNA) has been studied by using absorption, emission and circular dichoric spectral methods, viscometry, and cyclic voltammetry. A strong hyperchromism along with a red shift in UV bands and hypochromism in the ligand field band of the complex 1 on interaction with CT-DNA imply a covalent mode of DNA binding. This is further confirmed by studying the reactivity of complex 1 using circular dichroism and viscosity measurements. The variation in relative emission intensity of DNA-bound ethidium bromide observed upon treatment with the complex 1 parallel the trend of DNA binding studies. Cyclic voltammetry studies reveal that the complex 1 prefers to bind to DNA in Cu(II) rather than Cu(I) oxidation state.     

Interaction of lysozyme with dyes. II. Binding of bromophenol blue

The binding of lysozyme with bromophenol blue (BPB) at various dye concentrations and pH was carried out at 25 degrees C by equilibrium dialysis, ultraviolet (UV) difference and circular dichroism (CD) spectral techniques. Binding isotherms at pH 5.0 show non-cooperative binding at low dye concentrations, which change over to cooperative binding at higher concentrations indicating biphasic nature. However, binding isotherms at pH 7.0 and 9.0 show cooperative binding only, at all concentrations of the dye. The number of available binding sites decreases with the increase of pH. Gibbs free energy change, calculated on the basis of Wyman's binding potential concept, decreases with the increase of pH. Binding isotherms at pH 5.0 obtained at a lower temperature of 8 degrees C, also indicate the biphasic nature similar to those observed at 25 degrees C, but with a slight decreased strength of binding. The UV difference spectra of the complex do not show any distinct peaks in the 285 to 297 nm region eliminating any possible interaction of BPB with tryptophan and tyrosine residues of the lysozyme molecule. The CD spectra of lysozyme-BPB complex show a decrease in ellipticities with reference to native lysozyme in the near UV and far UV regions. This indicates that the lysozyme-BPB complex has a lower helical content probably due to the conformational changes induced into the native enzyme. The appearance of new positive peaks at 315 nm in the near UV region and at 592 nm in the visible region of the CD spectra may be due to the induced asymmetry into the BPB molecule as a result of its binding to a cationic residue (probably a lysine residue) of lysozyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of chemical structures of acridine and triphenylmethane dyes on the induced optical activity of DNA-dye complexes

Fifteen symmetrically substituted acridine dyes, all of which are interrelated by their chemical structures, each belonging to a C_2v symmetry, and three triphenylmethane dyes with amino or dimethylamino substituents are utilized to study necessary conditions for the appearance of extrinsic Cotton effects upon their binding to native and heat-denatured deoxyribonucleic acid (DNA). Three different kinds of the DNA–dye complexes, i.e., (1) dye added to native DNA, (2) heat-denatured DNA–dye complex, and (3) dye added to preheated DNA, were examined for each dye at a fixed P/D value of about 4. Optical activity was always observed for the compelexes of type (1) in each absorption band of the dyes in the visible and near-ultraviolet region. Two exceptions are 9-acetamido- and 9-hydroxyacridine, both being nonionic in aqueous solution at a pH range of 6. Acridinium chloride was unable to exhibit any definite extrinsic Cotton effect for complexes (2) and (3). Thus, the monocationic form of a dye due to the protonation or quaternization of the ring nitrogen in acridines or exonuclear amino nitrogen in triphenylmethane dyes is concluded to be an essential factor for extrinsic Cotton effect to appear. Changes in the absorption spectra upon complex formation are also related to the structure of dyes. Hypochromism and bathochromism are associated with the induced optical activity in all cases in the presence of native and denatured DNA.     

Intercalation of bulky Delta,Delta- and Lambda,Lambda-bis-Ru(II) complex between DNA base pairs

Interaction of Δ,Δ- and Λ,Λ-bis-Ru(II) complexes with native DNA was investigated by isotropic absorption and polarized spectroscopy including circular and linear dichroism (CD and LD). Despite the steric hindrance originating from its four bulky phenanthroline ligands at both ends of the molecule, this molecule rapidly intercalates between DNA base pairs. Intercalation was judged by large hypochromism and red shift in the UV-visible absorption spectra in the absorption region of the bridging moiety as well as in the metal-to-ligand-charge transfer absorption region. Further support for the intercalation is found in the fact that the magnitude of negative reduced LD signal in the absorption region of the bridging moiety was comparable to that of the DNA absorption region, indicating that the bridge connecting the two Ru(II) complexes is nearly parallel to the DNA base planes. No difference in the binding mode between the two enantiomers was observed. In the presence of either bis-Ru(II) complex, ethidium bromide, a classical intercalator, can intercalate into the empty sites but was not able to replace the Ru(II) complexes. Near the saturation, ground state interaction between ethidium and bis-Ru(II) complex was evident by LD.     

Non-intercalative binding mode of bridged binuclear chiral Ru(II) complexes to native duplex DNA

A pair of chiral binuclear ruthenium(II) complexes were prepared and their binding affinities towards double stranded native DNA were assessed by observing isotropic absorption, polarized light spectra - circular and linear dichroism (CD and LD), fluorescence quenching and DNA thermal denaturation. Upon binding to DNA, the complexes produced LD signals consisting of positive and negative signals in the absorption region, although they exhibited red shift and hypochromism in the absorption spectrum. These contrasting observations indicated that the binding modes of the complexes are largely deviated from classical intercalative binding. Groove binding of the complexes to DNA was found to be more likely than intercalative binding. The small increase of DNA melting temperature in the presence of the complexes indicated a predominance of DNA groove binding. The absence of “molecular light switch effect” further supported non-intercalative binding. The groove binding propensity of complexes was also supported by comparison of the resulting data with the [Ru(phen)₂(dppz)]²⁺.     

R-phycoerythrins having two conformations for the same aggregate

The visible circular dichroism (CD) spectrum of an R-phycoerythrin (Porphyra tenera) is composed of several positive bands. The protein in aqueous buffer very slowly exhibits changes in the CD spectrum of its chromophores, a band at 489 nm undergoes an increase in intensity and a red shift. When the band reached a 493 nm maximum, the spectrum became very stable. The aggregation state of the protein did not change during this spectral conversion. The chromophore CD spectrum was also obtained in the presence of a low concentration of urea or sodium thiocyanate, and the identical change in the CD was noted, but the change was much faster. The visible absorption and CD in the far UV spectra were unaffected by urea. Unchanged visible absorption and protein secondary structure (61% alpha helix) contradicted by comparatively salient alterations in the visible CD spectra suggested very subtle structural changes are influencing some of the chromophores. For a second R-phycoerythrin (Gastroclonium coulteri), the CD of the chromophores had a negative band on the blue edge of the spectrum. This is the first negative CD band observed for any R-phycoerythrin. Treatment of this protein with low concentrations of urea produced a change in the visible CD with the negative band being completely converted to a positive band. Fluorescence studies showed that the treatment by urea did not affect energy migration. Deconvolution of the CD spectra were used to monitor the chromophores. The results demonstrated that the same aggregate of each R-phycoerythrin could exist in two conformations, and this is a novel finding for any red algal or cyanobacterial biliprotein. The two forms of each protein would differ in tertiary structure, but retain the same secondary structures.     

On the various types of circular dichroism induced on acridine orange bound to poly(S-carboxymethyl-L-cysteine).

Circular dichroism and absorption spectra are measured on mixed solutions of acridine orange and poly(S-carboxymethyl-L -cysteine) at different pH and P/D mixing ratios. The observed circular dichroism spectra are classified into several types, mainly based on the number and sign of circular dichroic bands in the visible region. Three of them are associated with the absorption spectra characteristic of dimeric dye or higher aggregates of dye. Type I is observed with solutions, of which the pH is acid and P/D is higher than 4, and it has an unsymmetrical pair of positive and negative dichroic bands at 470 and 430 nm. This type is induced on the dye bound to the polymer in the β-conformation. Types II and III are considered to be characteristic of randomly coiled polymers. Type II is exhibited by solutions of P/D higher than 1 at pH 5–7 and has two dichroic bands around the same wavelengths as Type I but with opposite signs and an additional positive band at 560 nm. Type III, shown by solutions of P/D 2–0.6 at pH 6–10.5, has three dichroic bands around the same wavelengths as Type II but with signs opposite to it. The other two types of circular dichroism, induced for the solutions of P/D less than 1 at slightly acid pH, are associated with the absorption spectra of monomeric dye and are observed with disordered or randomly coiled polymer. They have a pair of dichroic bands at 540 and 425 nm, and the signs of these bands are opposite to each other in these two types.     

DNA-binding studies of fluoxetine antidepressant

Fluoxetine is a selective serotonin reuptake inhibitor (SSRI) antidepressant that is widely prescribed. The DNA-binding behavior of fluoxetine antidepressant and calf thymus DNA was investigated in Tris-HCl buffer at physiological pH 7.4 with a series of techniques, including UV-Vis and circular dichroism spectroscopies, competitive study with Hoechst 33258, viscometry, and cyclic voltammetry. Fluoxetine molecules bind to DNA via groove mode as illustrated by hypochromism with no red shift in the UV absorption band of fluoxetine, decrease in Hoechst-DNA solution fluorescence, and no significant changes in viscosity of DNA. The CD spectra of DNA molecules show a little change in stacking mode of base pair but no modification changes in DNA conformation, for example, from B-DNA to A or C-DNA. The binding constant (K(b)) of DNA with fluoxetine was calculated to be 6.7 × 10(4) M(-1), which is in the range of reported and known groove binders, such as distamycin. All results showed the groove-binding mode of interaction of fluoxetine with DNA.     

Intrinsic fluorescence of B and Z forms of poly d(G-m5C).poly d(G-m5C), a synthetic double-stranded DNA: spectra and lifetimes by the maximum entropy method.

A study has been made of the fluorescence of poly d(G-m5C).poly d(G-m5C), a synthetic double-stranded DNA, in buffered neutral aqueous solution at room temperature, excited by synchrotron radiation at 280 nm and 250 nm and by a frequency-doubled pulse dye laser at 290 nm. Exciting at 280 nm, the B form shows a uni-modal UV spectrum with lambdaf(max) approximately 340 nm. The Z form has in addition a visible emission lambdaf(max) at 450 nm. The spectral positions remain unchanged on exciting at 250 nm but the relative intensities change considerably. Decay profiles have been obtained at 360 nm and 450 nm for both the B and Z forms and have been analyzed by fitting to a pseudo-continuous distribution of 100 (and occasionally 200) exponentials, ranging from 10 ps to 20 ns, by optimizing the 'entropy' of the signal (the method of maximum entropy). We find the mean lifetimes for both wavelengths of emission and for both structural forms fall into three well-separated regions in the ranges indicated tau1 approximately 0.04-0.21 ns, tau2 approximately 0.9-1.26 ns, and tau3 approximately 5.1-6.5 ns. The UV emission, from its spectral position and half-width, correlates with monomeric emission from m5C (and from C for poly d(G-C)). However the lifetime tau1 is approximately 2 orders of magnitude longer than the monomers and points to an involvement of protonated guanosine (GH+, tauf approximately 200 ps) in the overall absorption/emission sequence. In the UV the tau3 emission is predominant, with fractional time-integrated emission approximately 86% for B DNA and approximately 64% for Z. We suggest it results from exciton (stacked) absorption followed by dissociative emission. For Z DNA the visible (450 nm) emission is dominated by a tau3 species (approximately 91%) with a lifetime of 6.5 ns and we suggest it represents a hetero-excimer emission consequent upon absorption by the strongly overlapped base-stacking, which differs from that in B DNA. The weak emission corresponding to tau2 is made more apparent by scanned gated detection of the emission from laser excitation (290 nm) of single-crystal d(m5C-G)3. A central role is attributed to the tight stacking of the bases in the Z form which correlates with enhanced hypochromism at 250 nm vs. 280 nm and with the reversal of the fluorescence intensity ratios UV-visible between these wavelengths.     

Interaction of 4,5-dibromo-2,7-di-(acetatomercuri)-fluorescein with DNAs of different base composition.

The changes in absorption spectra in the visible region observed on adding different naturally occurring and synthetic DNA duplexes to solutions of 4,5-dibromo-2,7-di-(acetatomercuri)-fluorescein indicate that the mercurial reacts with polynucleotides of this type. The reaction is reversible as proved by adding excess of KCN which restores the original spectra of the free dye. The interaction is characterised also by quenching of the fluorescence of the dye and the induction of optical activity in it. The extent of these spectral effects depends strongly on the (A+T) content of the complexed DNA and decreased in the order: poly [d(A-T)], Clostridium perfringens DAN, Escherichia coli DNA, Micrococcus luteus DNA and poly(dC). From equilibrium-dialysis experiments the same order in affinity is obtained when these poly-nucleotides are at equilibrium with the same concentration of 4,5-dibromo-2,7-di-(acetatomercuri)-fluorescein. From the changes produced by different mercurials in the ORD spectra and viscosity of a DNA solution it has been concluded that 4,5-dibromo-2,7-di(acetatomercuri)-fluorescein does not cause any drastic alteration of the secondary structure of DNA.     

The interaction of calmodulin with the carbocyanine dye (Stains-all)

The dye "Stains-all" combines with calmodulin to yield a series of complex species whose absorption and circular dichroism spectra are sensitive to the presence of Ca2+ or Mg2+. At high dye:calmodulin ratios, the dominant complex formed is characterized by a strong absorption band at 600-650 nm, which is associated with a biphasic circular dichroism band. These spectral features are abolished in the presence of Ca2+.     

Induced circular dichroism and mode of binding of acridine orange adsorbed on β-form poly(S-carboxyethyl-L-cysteine) in aqueous solutions

The absorption spectra and circular dichroism (CD) have been measured for aqueous solutions of acridine orange of a constant concentration, [D] = 5 × 10^?5M, mixed with poly(S-carboxyethyl-L -cysteine) in various mixing ratios, [P]/[D], ranging from 330 to 11, at different pH. The absorption spectra of the dye–polymer solutions are hypochromic, and the main band is located at 470 nm, accompanying a shoulder at 500 nm. At alkaline pH, no CD is induced in the visible region. At neutral and acidic pH, where the polymer is in the β-conformation, CD is induced in the visible and near-uv regions. A pair of CD bands is located at the region around 450 nm, when the pH is around the neutrality, while it appears at the region around 500 nm at acidic pH. Thus, the optically active species of bound dye changes from dimer to monomer on lowering the pH. These species form dissymmetric arrays along a polypeptide chain. The fraction of bound dye forming dissymmetric sequences is not high, but most of bound dye is adsorbed randomly on the ionized carboxyl groups of polypeptide chain and gives rise to hypochromism only. A dissymmetric structure of dye–polymer complexes is presented, in which the polymer has the β-conformation and the dye cations, either dimeric or monomeric, bind to its side chains, in such a way that the longer axes of molecular planes of bound dye form a two-fold, right-handed helix along the extended polypeptide chain. A zeroth-order calculation of CD based on the coupled oscillator model leads to the result that each dissymmetric array of dye consists, on the average, of two dimeric or monomeric cations. This low number of bound cations in a dissymmetric array and the large fraction of randomly adsorbed dye suggest that the hydrophobic interaction of dye with the polymer is strong, so that dye cations are adsorbed sparsely on both sides of the extended polypeptide chain.     

Spectroscopic studies of interaction of chlorobenzylidine with DNA

Electronic absorbance and fluorescence titrations are used to probe the interaction of chlorobenzylidine with DNA. The binding of chlorobenzylidine to DNA results in hypochromism, a small shift to a longer wavelength in the absorption spectra, and emission quenching in the fluorescence spectra. These spectral characteristics suggest that chlorobenzylidine binds to DNA by an intercalative mode. This conclusion is reinforced by fluorescence polarization measurements. Scatchard plots constructed from fluorescence titration data give a binding constant of 1.3 x 10(5) M(-1) and a binding site size of 10 base pairs. This indicates that chlorobenzylidine has a high affinity with DNA. The intercalative interaction is exothermic with a Van't Hoff enthalpy of -143 kJ/mol. This result is obtained from the temperature dependence of the binding constant. The interaction of chlorobenzylidine with DNA is affected by the pH value of the solution. The binding constant has its maximum at pH 3.0. Upon binding to DNA, the fluorescence from chlorobenzylidine is quenched efficiently by the DNA bases and the fluorescence intensity tends to be constant at high concentrations of DNA when the binding is saturated. The Stern-Volmer quenching constant obtained from the linear quenching plot is 1.6 x 10(4) M(-1) at 25 degrees C. The measurements of the fluorescence lifetime and the dependence of the quenching constant on the temperature indicate that the fluorescence quenching process is static. The fluorescence lifetime of chlorobenzylidine is 1.9 +/- 0.4 ns.