Flow Dichroism Research Articles

Analytical model of flow dichroism in Langmuir-Blodgett films

Abstract A model of flow orientation has been developed for analyzing the dichroism in Langmuir-Blodgett (LB) films by introducing a two-dimensional fluid and a sink, which represent the monolayer and a substrate, respectively. The angular distribution function derived using the velocity gradient given by the ideal-fluid approximation satisfactorily explains the data of dichroism in the mixed LB films of a dye and a fatty acid prepared with different velocities. The results are consistent also with those of the line-shape analysis of ESR spectrum reported previously.

Kinetic flow dichroism study of conformational changes in supercoiled DNA induced by ethidium bromide and noncovalent and covalent binding of benz[a]pyrene diol epoxide.

The dynamic conformational changes due to the noncovalent intercalative binding of ethidium bromide and racemic trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), and the covalent binding of BPDE to supercoiled phi X174 DNA, have been studied by gel electrophoresis and a novel application of a kinetic flow linear dichroism technique. The magnitude of the linear dichroism (delta A) of the DNA oriented in the flow gradient is sensitive to the hydrodynamic shape of the DNA molecule which is affected by the binding of the drug or the carcinogen BPDE. While the linear dichroism of ethidium bromide supercoiled DNA is time independent, the delta A spectra of BPDE-DNA reaction mixtures vary on time scales of minutes, which correspond to the reaction rate constant of BPDE to form 7,8,9,10-tetrahydroxytetrahydrobenzo[a]pyrene hydrolysis products and covalent DNA adducts. The rapid noncovalent intercalation of BPDE causes an initial large increase in delta A (up to 250%, corresponding to the dichroism observed with relaxed circular DNA), followed by a slower decrease in the linear dichroism signal. This decrease in delta A is attributed to the removal of intercalated diol epoxide molecules and the resulting reversible increase in the number of superhelical turns. The kinetic flow dichroism spectra indicate that the noncovalent BPDE-DNA complexes are intercalative in nature, while the covalent adducts are characterized by a very different conformation in which the long axes of the pyrenyl residues are oriented at a large angle with respect to the average orientation of the planes of the DNA bases.(ABSTRACT TRUNCATED AT 250 WORDS)

Flow birefringence and dichroism of polymers. I. General theory and application to the dilute case

Birefringence of polymer solutions has two origins, the orientation of polymer segments (the intrinsic birefringence), and the anisotropy of the segment density correlation function (the form birefringence). Rigorous formulas are given for the two contributions. Also special attention is focused on the form dichroism, which seems to have potential applicability in the study of polymer dynamics. As the simplest application we examine the birefringence and dichroism of dilute solutions in weak oscillatory shear for arbitrary light wavelength.

Noncovalent intercalative complex formation and kinetic flow linear dichroism of racemic syn- and anti-benzo[a]pyrenediol epoxide-DNA solutions.

In order to gain a better understanding of the molecular basis underlying the differences in biological activities of the diastereomeric syn and anti diol epoxides of benzo[a]pyrene (trans-7,8-dihydroxy-syn-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, respectively), their interactions with DNA in aqueous solutions were studied and compared. Kinetic flow linear dichroism experiments indicate that both diastereomers (racemic mixtures) form intercalation complexes immediately after mixing; the association constant (23 degrees C, ionic strength approximately 0.005) is significantly smaller (5200 M-1) in the case of the syn than in the case of the anti diastereomer (12 200 M-1). This difference is attributed to the greater bulkiness of the 7,8,9,10 ring of the syn stereoisomer, which is in the quasi-diaxial conformation as compared to the less bulky quasi-diequatorial conformation of the anti diastereomer. In contrast, the intercalative association constants of the tetraols derived from the hydrolysis of the two diol epoxides are similar in value. Upon formation of noncovalent syn-BPDE-DNA intercalation complexes, the reaction rate constant for the formation of tetraols (approximately 98%) and covalent adducts (approximately 2%) increases from 0.009 to 0.05 s-1 at pH 9.5 in 5 mM tris(hydroxymethyl)aminomethane buffer. The conformations of the aromatic chromophores of BPDE were followed by the kinetic flow dichroism technique as a function of reaction time; while the anti diastereomer changes conformation from an intercalative to an apparently external binding site, the syn diol epoxide molecules do not appear to undergo any measurable reorientation during or after the covalent binding reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Conformations of complexes derived from the interactions of two stereoisomeric bay-region 5-methylchrysene diol epoxides with DNA.

The reaction mechanisms of two isomeric bay-region diol epoxides of 5-methylchrysene (trans-1,2-dihydroxy-anti-3,4-epoxy-1,2,3,4-tetrahydro-5-methylchrysene (DE-I) and trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydro-5-methylchrysene (DE-II) with double-stranded DNA in aqueous solutions were studied utilizing kinetic flow dichroism and fluorescence techniques. As in the case of the previously studied benzo(a)pyrene-7,8-diol-9,10-oxide isomers (BaPDE), both DE-I and DE-II rapidly form intercalation-type complexes (association constants K = 2700 and 1500 M-1 respectively in a neutral 5mM phosphate solution). The physically bound diol epoxide molecules react on time scales of minutes to form predominantly tetraols; a greater fraction (6 +/- 1%) of DE-I than of DE-II (2-3%) molecules react with the DNA to form covalent products. The DE-II isomer is characterized by a greater reactivity than DE-I, and the rates of reaction are markedly accelerated in the presence of DNA in both cases. The linear dichroism spectra of the covalent adducts reveal that the conformations of both types of adducts are similar, with the long axes of the phenanthrenyl chromophores tilted, on the average, at angles of 38-52 degrees with respect to the average orientations of the transition moments (at 260 nm) of the DNA bases. The conformations of the covalently bound DE-I and DE-II molecules resemble those observed in the case of the highly tumorigenic (+) enantiomer of anti-BaPDE.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of prior complex formation on the photoaddition of chlorpromazine to calf thymus deoxyribonucleic acid

Chlorpromazine (CPZ), 2-chloro- N-(3-dimethylaminopropyl) phenothiazine, causes cutaneous photosensitivity in man. The photoaddition of CPZ to deoxyribonucleic acid (DNA) may be an important mechanism for the phototoxicity. We have investigated the complexes formed between CPZ and calf thymus DNA prior to irradiation, related their formation to the photoaddition of CPZ to DNA and initiated studies to identify the photoadducts. In the presence of high concentrations of double-stranded DNA, the CPZ absorption maximum shifted from 305 to 340 nm with an isosbestic point at 323 nm. The CPZ fluorescence at 460 nm was quenched a maximum of 90%. The excitation and emission spectra for the unquenchable fluorescence are the same as those for free CPZ. These results together with those from flow dichroism measurements indicated that CPZ formed two complexes with double-stranded DNA. One complex involves intercalations, is non-fluorescent and absorbs at 345 nm. The second complex absorbs at 310 nm, fluoresces at 460 nm and has the phenothiazine ring parallel to the DNA axis. Non-covalent binding of CPZ to heat-denatured DNA did not shift the CPZ absorption spectrum but quenched 65% of the CPZ fluorescence. One complex between CPZ and denatured DNA will account for these results. CPZ photolysis was inhibited compared with that of free CPZ by binding to double-stranded DNA (more than 98%) or denatured DNA (65%). CPZ photoadded ten times more efficiently to denatured DNA than to double-stranded DNA. These results indicate that CPZ photolysis and photoaddition are quenched in the intercalation complex. The photoaddition to double-stranded DNA does not result from intercalated CPZ because the action spectrum maximized at 310 nm rather than at 340 nm.

Precollapse of T7 DNA by spermidine at low ionic strength: a linear dichroism and intrinsic viscosity study.

AbstractAt low salt ([Na+] = 10−3M), spermidine is capable of transforming DNA from a highly extended random coil to a compact particle. The transition takes place at a spermidine concentration of around 25 μM and the compact particle has been previously studied in considerable detail for several different DNAs. The objective of the present study is to see what effect, if any, spermidine has on T7 DNA conformation prior to collapse using flow dichroism and intrinsic viscosity. We conclude that increasing the spermidine concentration from 0 to the collapse transition point (above 20 μM) makes DNA increasingly nondraining. Furthermore, the persistence length dropped from 785 (±42) to 560 (±32) to 445 (±26) Å on increasing the ambient spermidine concentration from 0 to 1 to 10 μM. These results are in good agreement with counterion condensation theory and Odijk's theory of the electrostatic contribution to the persistence length of DNA. Nonetheless, it is concluded that counterion condensation is not entirely responsible for DNA collapse and that crosslinking promotes the transition to the compact state.

Conformations of adducts and kinetics of binding to DNA of the optically pure enantiomers of anti-benzo(a)pyrene diol epoxide

Kinetic flow dichroism studies indicate that the (+) enantiomer of 7β, 8α-dihydroxy-9α, 10α-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene physically bound at intercalative-type sites in double-stranded DNA undergoes covalent binding reactions to form adducts at external binding sites. The conformation of the non-covalent complex derived from the (−) stereoisomer is also intercalative in nature, but the conformations of the covalent adducts are heterogeneous and are characterized by both intercalative-type and external conformations. It is suggested that the distinctly higher biological activity of the (+) enantiomer relative to the activity of the (−) enantiomer may be related to the preponderance of 7,8,9-triol benzo(a)pyrene residues covalently linked to deoxyguanine and located at external binding sites in the DNA adducts.

Flow dichroism evidence for tilting of the bases when DNA is in solution.

Flow linear dichroism studies of DNAs from Clostridium perfringens, Escherichia coli, and Micrococcus luteus are extended into the vacuum UV region to 175 nm. The short-wavelength pi pi region shows a less negative reduced linear dichroism than the well-studied long-wavelength pi pi region. These results are independent of the type of DNA and the concentration of salt over the range 0.001 to 1.0 M. This proves that the bases are not perpendicular to the flow orientation. Assuming that the DNA tends to align along its helix axis, the largest angle that the bases can make with the helix axis is 73 +/- 3 degrees.

Flow dichroism of capsid DNA phages. II. Effect of DNA deletions and intercalating dyes.

AbstractThe flow linear dichroism of bacteriophage λ and its deletion mutants, λ b2 and λ b221, was determined. The hydrodynamic behavior of the three phages differed slightly, but the magnitude of the dichroism was substantially the same with 〈cos2θμp〉 = 0.364, 0.368, and 0.372, respectively. The dichroism of intercalating dyes combined with bacteriophage was used as a further probe of phage structure. The reduced dichroism from proflavin with T4 showed no change with time during the reaction, but the interpretation of the ligand dichroism is complicated by an alteration of the hydrodynamic behavior of the phage–dye complex relative to the phage alone. Ethidium with λ also produced a stable reduced dichroism, but the signal indicated an average orientation of intercalated dye that is different from the average base orientation. The reduced dichroism of ethidium changes with time as it penetrates λ b2, eventually approaching the dichroism of the nucleotide bases. The implication of these findings on the plausibility of various simple DNA packing models is discussed.

Flow dichroism of capsid DNA phages. I. Fast and slow T4B.

AbstractFlow linear dichroic studies have been conducted on phage T4B in its fast and slow forms. The behavior of the phages is well represented by an equivalent ellipsoid model using the Peterlin‐Stuart theory. The measurements permit the evaluation of the optical factor of the DNA in the phage and the rotary diffusion coefficient of the phage particle. Both these quantities change during the slow–fast conversion. The rotary diffusion results are in good agreement with those obtained by other workers with other methods. The optical factor is negative, indicating a net alignment of DNA helices parallel to the phage axis. The results exclude certain simplified models for the packaged DNA but do not lead to a unique structural conclusion. The flow dichroism experiment and its interpretation are described, and a simple method of calculating optical factors for complicated but cylindrically symmetric structures is presented.

Polynucleotide conformation from flow dichroism studies.

AbstractWe demonstrate that the isotropic absorption and linear dichroism in an unknown flow field can be used to determine base tilt in polynucleotides if three transitions are measured and the directions of the corresponding dipoles are known. The method is applied here to reach conclusions about the base tilt in poly(rA), poly(rA)+·poly(rA), and poly(rC). The respective values are: 28° tilt about the axis + 50° toward C8 from the C1′ → N9, and 25° tilt about the axis + 118° toward C5 from C1′ → N1. The results for poly(rA)+·poly(rA) are consistent with the accepted model. Spectra were measured for poly(rC)+·poly(rC), but definite conclusions must await reliable directions for transition dipoles. The dipole direction for the 218‐nm transition in rC is found to be +13° or +43° toward C5 from C1′ → N1. The CD spectra to about 168 nm are presented and discussed.

Interaction between 9-substituted benzo[alpha]phenoxazine derivatives and DNA and relationships to their antibacterial activities

Studies were made on the intermolecular interaction of ten 9-diethylaminobenzo [α]-phenoxazonium salts (BPZ) and calf-thymus deoxyribonucleic acid (DNA) and antibacterial activities for Staphylococcus aureus 209P of BPZ. (1) The interaction system of BPZ and DNA was found to show a flow dichroism in the visible region. A value of the ΔE in the equation ΔE=E11-E⊥ is found to be negative, and flow dichroism studies indicate that BPZ intercalates in the same plane as DNA base pairs. (2) The flow dichroism's ratio in the visible region produced by the interaction of BPZ with DNA paralleled in minimum inhibitory concentration (MIC) of BPZ for Staphylococcus aureus 209P.

Flow dichroism of T7 DNA as a function of salt concentration.

AbstractMeasurements have been made on the flow dichroism of T7 DNA as a function of NaCl concentration. In high salt, our results are compatible with an optical factor of −1.4 and a persistence length of 470 Å. The former is in agreement with expectations from the x‐ray diffraction structure of fibrous B–DNA, and the latter is in the midrange of recent determinations. As salt concentration is decreased, the persistence length increases. The relation of our study to other recent investigations and with current theories of the electrostatic contribution to persistence length is discussed. We note that the separation of the electrostatic expansion factor into long‐ and short‐range effects is somewhat arbitrary and might affect the interpretation of different experimental results in different ways. Finally, our hydrodynamic factors are consistent with a chain which is partially free‐draining. This goes against the traditional interpretation but is in agreement with two recent observations.

Non-covalent interaction with DNA of the mutagens 2-amino-dipyrido[1,2-a:3′,2′-d]imidazole and methyl-substituted isomers

The interactions of 2-amino-6-methyldipyrido[1,2-a:3′,2′-d]imidazole (Glu-P-1) and its analog 2-amino-dipyrido[1,2-a:3′,2′-d]imidazole (Glu-P-2) which are potent mutagens isolated from a L-glutamic acid pyrolysate, with calf thymus DNA were studied spectroscopically. Scatchard plots obtained by optical titration gave association constants, K, of 2.7 ∼ 17 × 10 3 M −1 for several derivatives. Flow dichroism studies showed that Glu-P-1 and other derivatives are oriented in parallel to the planes of base pairs of DNA. The correlation between the association constants and degrees of mutagenicity is discussed.

Studies on the interaction between steffimycins and DNA

The complexes formed between steffimycins and DNA were studied using various physicochemical techniques. The binding process has been followed spectrophotometrically or fluorimetrically. The binding parameters n and K, evaluated according to McGhee and Von Hippel, show a good affinity of these antibiotics for the macromolecule. Flow dichroism measurements showed that in the complex with DNA, the antracycline moiety of the steffimycins is intercalated between two base pairs of the macromolecule. The binding experiments with various polydeoxyribonucleotides and with various DNA samples, having different base pair compositions, suggest that an alternate sequence of A-T, such as that of poly[d(A-T)] . poly[d(A-T)], represents a good receptor site for the binding of steffimycins to DNA. The lack of in vivo activity of these antibiotics is discussed.

Interaction specificities of a platinum metallointercalation reagent to DNA

The interaction specificity of salmon sperm DNA with 2-hydroxyethanethionlato(2,2',2''-terpyridine)platinum(II),PtTS has been studied. The results of 1H and 13C nuclear magnetic resonance, flow dichroism and circular dichroism studies are found to be consistent with an intercalation mode of binding as has been proposed earlier by lippard and coworkers.

Dynamic Flow Birefringence and Flow Dichroism of Block-Copolymer Molecules in Solution with Application to the Free-Draining Limit

Dynamic Flow Birefringence and Flow Dichroism of Block-Copolymer Molecules in Solution with Application to the Free-Draining Limit

Ultraviolet flow dichroism of brain microtubule

Ultraviolet flow dichroism of brain microtubule was measured. Large positive dichroisms were observed at 255 nm, 285 nm and 292 nm. The dichroism at about 255 nm is due to bound GDP or GTP. The dichroism at 285 nm is due to tyrosine and tryptophan residues, and that at 292 nm is due to the tryptophan residues of the protein. These results show that the electronic transition moments of nucleotides and aromatic groups are both nearly parallel to the polymer axis.

Flow dichroism, flow polarized fluorescence and viscosity of the DNA · acridine complexes

The strong binding of various acridine dyes to DNA has been studied by the measurements of flow dichroism, flow polarized fluorescence and viscosity. Negative flow dichroism and percentage change in polarized fluorescence intensity show that intercalated dye molecules are oriented rather perpendicularly to the main axis of the DNA helix, like base pairs. On the other hand, viscosity measurements show that the increase of the contour length of DNA depends on the dye structure, being much smaller in the case of dyes with bulky substituents compared to that of the other dyes. This may be attributed to the formation of the outside bound complex. Further, the introduction of bulk substituents to the acridine ring leads to a little smaller values of the reduced dichroism and intensity change of polarized fluorescence. The results may be qualitatively understood if we assume that the outside bound dye lies in the groove of the DNA helix and the plane of the dye tilts from the perpendicular direction relative to the main axis of the helix.