Flow Dichroism Research Articles

Fluorescence and flow dichroism of F-actin-epsilon-ADP; the orientation of the admine plane relative to the long axis of F-actin.

The excitation polarization spectrum of epsilon-ADP bound to F-actin shows that two absorption dipoles at 260 nm and 340 nm are oriented in different directions relative to the emission dipole. On the other hand, the linear dichroism of F-actin-epsilon-ADP gives that the dichroic ratio of the bound epsilon-ADP is approximately constant (about-0.5) in the wavelength region form 250 to 350nm. Furthermore, the fluorescence polarization of epsilon-ADP bound to F-actin which is oriented in the field of flow shows that the emission dipole is nearly perpendicular to the long axis of F-actin. From these observations we conclude that the adenine plane of the bound nucleotide is almost perpendicular to the long axis of F-actin.

Stereospecific binding of diastereomeric peptides to salmon sperm DNA.

Studies of the interaction specificities of L-lysyl-L-phenylalaninamide (1) and the diastereomeric dipeptide amide, L-lysyl-D-phenylalaninamide (2), with salmon sperm DNA reveal distinct differences in the binding site of the aromatic ring of the phenylalanine residue. The results of 1H nuclear magnetic resonance (NMR), spin-lattice relaxation rates, viscometric, and flow dichroism studies indicate the aromatic ring of 1 is "partially" inserted between base pairs of DNA whereas the aromatic ring of 2 points outward toward the solution. The terminal L-lysyl residue presumably interacts stereospecifically with DNA helix thus dictating the positioning of the aromatic ring of the C-terminal phenylalanine residue. In the accompanying paper (E. J. Gabbay et al. (1976), Biochemistry, following paper in this issue), the interaction of several oligopeptide amides (containing the N-terminal L-Lys-L-Phe residue) with DNA is examined. The results are found to be consistent with stereospecific binding of the terminal L-lysyl residue, and in addition, the evidence suggests that oligopeptides may bind to DNA via a modified single-stranded beta-sheet structure which is wrapped around the nucleic acid helix in a manner similar to that described by M. H. F. Wilkins (1956), Cold Spring Harbor Symp. Quant. Biol. 21, 75).

Optical anisotropy of absorbing particles: Light scattering depolarization, electric- and flow birefringence and dichroism of suspensions of carbon blacks

The use of a rather: elongated spheroid, the compacity of which decreases with its weight, equivalent to the aggregates allows to explain the effects of depolarized light scattering and of birefringence. In particular, a very unusual negative macroform birefringence has been found. Moreover the theoretical expressions concerning the absorbing particles which simultaneously exhibit an electric dichroism and birefringence have been written. They were tested by means of suspensions of carbon blacks.

Complex formation between naphthothiopheneethanolamines and deoxyribonucleic acids.

Reduced viscosities, viscometric titrations, fluorescence, and flow dichroism measurements indicate that the aromatic ring system of naphthothiopheneethanolamines (NA) is intercalated on binding to DNA. No marked change in binding was found on substituting a dibutylamino group for a piperidyl ring on the side chain. Ionic strength studies, however, did indicate that the positive charge on the side chain is quite important for binding. Both the binding equilibrium constant and the number of binding sites, as determined from a Scatchard plot of spectrophotometric results, decreased on increasing the sodium chloride concentration. The increase in Tm for several DNA samples, caused by adding NA to DNA at a fixed ratio, showed a marked dependence on the percentage of AT base pairs. The conclusion drawn from these data is that the DNA-NA complex involves high specificity of the NA for AT base pairs, intercalation of the aromatic ring system, and binding of the NA side chain in the DNA minor groove with strong interaction between the positively charged side chain and the DNA phosphate groups.

Electric dichroism of chromatin

The linear dichroism of sheared calf thymus chromatin, oriented in solution by a pulsed electric field, has been measured. The limiting value of this dichroism is considerably less negative than that of calf thymus DNA, but does not approach the positive values predicted if chromatin were uniformly supercoiled in the manner suggested by X-ray studies. The decay of the dichroism of chromatin, after termination of the electric pulse, is similar to that of DNA, indicating that chromatin retains a high degree of chain flexibility. These data, as well as previous flow dichroism studies (Ohba, 1966; Smart & Bonner, 1971), suggest that chromatin is not predominantly supercoiled in solutions of low ionic strength. Evidence for a structurally heterogeneous model for chromatin is discussed.

Colloidal Studies on Surface Active Agents. XXIV.

In order to obtain information on the mode of solubilization of water-insoluble dyes into the micelles of nonionic surfactants, polyoxyethylene alkyiphenylethers, the solubilization ratios of dyes to the micelle and their flow dichroism have been investigated. Dyes used are Orange OT and 1, 4-diaminoanthraquinone. Experimental results showed that in every case one molecule of dye is solubilized per micelle at saturation. Above 100g/l of the surfactant concentration flow dichroism was clearly observed and constant differential dichroism was obtained. These facts suggest that above this concentration the shape of the surfactant micelles deviates markedly from sphere and the solubilized dyes orientate in the non-spherical micelles.

Comparison of the UV flow dichroism spectra of TMV and several of its mutants.

AbstractThe dichroic ratio spectra of TMV and four of its mutants (YTAMV, GTAMV, HR, and CV4) were determined from 320 to 240 nm. Measurements were also made on particles of ATMV (TMV protein reconstituted with polyadenylic acid) and rods of repolymerized TMV, YTAMV and GTAMV protein, respectively. These cylindrical molecules were oriented in a flow gradient with their degree of orientation being determined from anisotropic light scattering measurements. Taking length distributions into account, the dichroic ratios were then extrapolated to values characteristic of perfect particle alignment.Due to the large number of overlapping absorbing bands in the whole viruses, it is very difficult to separate out specific contributions to the total spectra. Since, however, the spectra for the whole viruses were similar, this would suggest an overall likeness in structure for all the virus particles studied. Because the repolymerized proteins did not have the four RNA chromophoric groups present, their spectra would be more sensitive to protein contributions to the total spectrum. Repolymerized rods of YTAMVP yielded results similar to those for TMVP, while the spectrum for repolymerized rods of GTAMVP was significantly different from that obtained for TMVP.Dichroic ratio spectra of the nucleic acids, as they exist within the whole particles, were also calculated by subtracting protein absorptivities from their respective viral absorptivities. The spectra indicates similar results for the various nucleic acids in all the particles studied.

Light scattering and flow dichroism studies on DNA after the photoreaction with psoralen.

Light scattering measurements performed on DNA after irradiation in the presence of psoralen clearly show that inter strand cross linkings are present in the macromolecule. In fact after heat denaturation and successive cooling irradiated macromolecule shows a molecular weight practically unchanged while a DNA sample after the same treatment shows a molecular weight half of the intact native DNA. Also the general conformation of irradiated DNA undergoes practically to no modifications after the same heat treatment while native DNA shows itself to have been strongly modified. Moreover, on the basis of flow dichroism determinations, DNA cross-linked by psoralen after heat denaturation showed to be able to restore its ordered double helix structure, during the successive cooling.

Studies on the role of histones in the structure of chromatin

The effects of selective removal of histones from chromatin on physical properties of the nucleoprotein complex have been examined using two dissociating agents: (1) sodium deoxycholate, which removes histone II at lowest concentrations of dissociating agent, followed by histones III and IV at slightly higher concentrations, and lastly, at still higher concentrations, by histone I; and (2) sodium chloride, which removes histone I at lowest concentrations of dissociating agent, followed by histones II, III and IV at higher concentrations. Properties studied include thermal denaturation, sedimentation velocity, flow dichroism, and the irreversible disruption of a 130 s cross-linked component of sheared chromatin. None of the structural and chemical parameters measured depends on the interaction of any one specific kind of histone with DNA. Rather, all of the properties measured are conferred upon DNA by all species of histones.

Polymer Dynamics. V. The Shear Dependent Properties of Linear Polymers Including Intrinsic Viscosity, Flow Dichroism and Birefringence, Relaxation, and Normal Stresses

Under the physical requirement that the contour length of a polymer molecule remains constant irrespective of shear rate in the Rouse and Zimm theories and the Harris–Hearst theory, the shear rate dependence of intrinsic viscosity, flow birefringence, flow dichroism, normal stresses, the relaxation of flow birefringence and flow dichroism, and some quantities at zero shear rate have been calculated for molecules of different stiffness ranging from the rod to the flexible coil in θ solvents. Changes of hydrodynamic interaction with shear rate are not included. Comparing the present theory with the experimental results there is satisfactory agreement for stiff molecules such as the cellulose derivatives and DNA at low shear.

Modification of the flow dichroism spectrum of rat liver nuclear DNA by in vivo alkylation with hepatocarcinogenic dialkylnitrosamines.

Modification of the flow dichroism spectrum of rat liver nuclear DNA by in vivo alkylation with hepatocarcinogenic dialkylnitrosamines.

Flow Visualization by Using Flow Dichroism

Flow Visualization by Using Flow Dichroism

Quantitative flow dichroism. II. Form dichroism at ultraviolet wavelengths.

AbstractForm dichroism has been predicted to make an important contribution to the dichroic spectra of rod‐shaped macromolecules. Ultraviolet flow dichroism experiments on tobacco mosaic virus and bacteriophage fd in solvents of different refractive indices were unable to detect the phenomenon, indicating that for these viruses form dichroism is much less important than had been postulated.

Quantitative flow dichroism. I. Correction for disorientation in a solution of rods.

AbstractFlow dichroism suffers from two ambiguities which have prevented it from being a useful analytical technique. One is imperfect particle orientation, and the other is the unknown importance of form dichroism. This paper presents a method for calculating from experimental data the dichroism which would be observed from a solution in which all rods were aligned parallel to one another. The procedure utilizes anisotropic light scattering as an independent measure of particle orientation and requires only that the solute particles may be approximated by thin rods.

Study on the binding nature of acridine orange to DNA by means of flow dichroism.

Abstract The binding nature of acridine orange to DNA was studied by means of equilibrium dialysis and flow dichroism with spectrophotometric analysis. The adsorption isotherm of the acridine orange-DNA system was obtained by means of equilibrium dialysis. From the experiment, the binding constant (K) and the fractional amount of the binding site to the total number of nucleotides (f) were estimated for native- and heat-denatured DNA. Under a shear gradient, the acridine orange-DNA complex showed a negative dichroism in visible region as well as in the ultraviolet region. This implies that acridine orange is oriented on DNA rather perpendicularly to its main axis, like base pairs. The magnitude of the dichroism of the complex at 500 mμ, near the visible absorption peak of acridine orange, was larger than each value of the complex and that of the DNA itself (260 mμ). The transition moment corresponding to the absorption at 500 mμ may be oriented in a more orderly manner and/or more nearly perpendicularly to the main axis of DNA than that corresponding to 260 mμ.

Berberine: complex with DNA.

A complex of calf-thymus DNA with berberine sediments in the analytical ultracentrifuge. The DNA produced systematic changes in the absorption spectrum of berberine which suggest that single alkaloid molecules bind to DNA. Flow dichroism of purines and pyrimidines and of berberine in the complex with DNA had the same signs and magnitudes. Berberine shifted the thermal strand separation profile of DNA to higher temperatures. Therefore, the alkaloid forms a complex with DNA, probably by intercalation.

Hydrodynamic relaxation times of DNA from decay of flow dichroism measurements

AbstractThe relaxation of the flow dichorism signal of native DNA molecules after rapid (∼0.004 sec) stoppage of the flow has been studied in the narrow‐channel flow dichroism apparatus previously described. The flow dichroism signal decays with a spectrum of relaxation times. The slowest relaxation time accounts for about 0.40 of the total signal for shear gradients in the range of 100–300 sec−1 and for about 0.25 of the total signal for shear gradients in the range of 5,000– 20,000 sec−1. This longest relaxation time in dilute aqueous salt solution at 25°C for T4‐DNA molecules is measured as 0.45 sec; in the molecular weight range from 125 × 106 to 12 × 106, the relaxation time varies as. M(1.6±0.1). The relaxation time is proportional to η/T for large (sixfold) changes in solvent viscosity and for temperature changes between 25 and 0°C, showing that the molecular relaxation is hydrodynamically limited. An accurate analysis of the data at short times to determine the shorter relaxation times was not possible. A theoretical prediction based on the Zimm‐Rouse theory of polymer dynamics for the longest relaxation time is in good agreement with the observed value, but the theory predicts that 87%, of the total signal should decay with this relaxation time, whereas experimentally only a smaller fraction of the total signal does so. This discrepancy occurs because the theory is applicable to low hydrodynamic shear stress flows and the experiments were made at higher shear stresses.

Flow dichroism of DNA: a new apparatus and further studies.

AbstractA new apparatus for the study of flow dichroism of macromolecules is described. The flow is down a long, narrow channel and an unpolarized light beam propagates along the flow direction. For a molecule such as DNA, in which the transition moments of the chromophores are perpendicular to the axis of orientation, an increase of absorbance is observed during flow. The apparatus is best suited for macromolecules which are readily orientable or at high shear gradients so that the extinction angle is close to 0°. The apparatus has the following advantages: dilute macromolecule solutions can be used; high shear gradients are easily obtained; only small volumes of solution are needed. The flow can be stopped rapidly so that relaxation times for disorientation can be studied. The flow dichorism of native, two‐stranded DNA has been measured for the molecular weight range of 0.6 × 106 to 125 × 106, and for the shear gradient range (in aqueous solution at 25°C) from 200 sec−1 to 21000 sec−1. At a fixed gradient the dichroism increases with molecular weight, but the curve is concave downwards. At a given molecular weight the dichroism increases with increasing shear gradient, but the curve is concave downwards. When the solvent viscosity and temperature are varied, the dichroism is a function of η〈G〉/T showing that the orientation is due to hydro‐dynamic shear stress and that the flexibility of DNA in a flow field is not due to local denaturation. The Zimm‐Rouse theory with no parameters taken from flow optical data predicts the correct order of magnitude of the dichroism but the experimentally observed shear gradient and molecular weight dependence do not fit the theory. This is an expected result, since the theory is believed to be applicable only at small distortions and extensions of the macromolecule.

Interaction of 2-Amino-1, 4-naphthoquinone Imine with Nucleic Acid

On 2-amino-1, 4-naphthoquinone imine-HCl (ANQI), which had previously been reported to interact with nucleic acid and to inhibit the synthesis of nucleic acid and protein, especially of DNA, in Ehrlich mouse ascites tumor cells in vitro, the details of the interaction with nucleic acid were investigated by the methods of melting experiment for DNA, difference spectrum, equilibrium dialysis, and flow dichroism. The results are as follows : 1. The sites in DNA for attachment of ANQI were the purine base moieties, i.e., both of adenine and guanine. ANQI interacted with a highly polymerized native DNA at the highest degree and hardly interacted with such monomers as bases, nucleosides, and nucleotides. 2. ANQI was found to be able to bind to calf thymus DNA up to the ratio of 2 moles per 4 nucleotides by the method of equilibrium dialysis. This means that ANQI can attach with all the purine bases in DNA. 3. Such bivalent metal ions as Mg2+, Ca2+, Mn2+, Cu2+, Zn2+, and Ba2+ interfered with the interaction between ANQI and DNA. On the other hand, Hg2+ increased the extent of interaction. 4. It was found from the measurement of flow dichroism on DNA-bound ANQI that the orientation of ANQI was nearly parallel with the planes of DNA bases.

Ultraviolet dichroic ratio of DNA from T2 and T5 bacteriophages

AbstractThe dichroic ratios of T5st‐O and T2H bacteriophage DNA molecules were measured throughout the ultraviolet region of the spectrum. Two methods of DNA orientation were studied: (1) orientation in solution in a Shimadzu flow dichroism instrument attached to a Beckman DU spectrophotometer, and (2) alcohol precipitation of the DNA from solution and orientation in a thin film on the quartz face of a humidity chamber. Spectra in the latter case were recorded using a Gary Model 14 spectrophotomcter fitted with Glan prisms. The lower wavelength limit was 215 mμ in both systems. The DNA preparations were carefully characterized as to spectral purity, sedimentation coefficient, hyperchromicity, protein content, and DNA content. In addition, the structure of the DNA oriented in films was inferred from x‐ray diffraction patterns of fibers of the precipitated DXA. The A and B configurations of DNA in films could not be distinguished by the dichroic ratio measuiements. The dichroic ratio obtained for the film‐oriented DNA at high relative humidity shows the same wavelength dependence as for the flow‐oriented DNA. The same wavelength dependence for DNA in the fibrous state and in solution, when considered together with the x‐ray diffract ion results, indicates that DNA in solution maintains an orientation of bases which is similar to that in fibers. I1Or both solutions and films of DNA, the dichroic ratio is constant from 290mμ to 240 mμ and increases at wavelengths below 240 mμ. The increased parallel absorption below 240 mμ is consistent with the existence of an n→π* transition. The inherent molecular dichroic ratio is found to be the same for T5st‐O DNA and T2H DNA in solution, and is a maximum of 0.09 ± 0.02 at 260 mμ.