Electric Dichroism Measurements Research Articles

Surface Charge Movements of Purple Membrane During Light-Dark Adaptation

The difference in the surface charge distribution between light-adapted and dark-adapted purple membranes was investigated with electric dichroism measurements from approximately pH 5 to pH 11. Purple membrane sheets in solution are oriented in a weak electric field by their permanent dipole moment, which is due to the charge distribution of the membrane surfaces and/or within the membrane. The degree of orientation of purple membrane sheets was obtained from the measurement of "electrical anisotropy" of retinal chromophore in the membranes. At about pH 7, there was no difference in the "electric anisotropy" between light- and dark-adapted purple membranes. At about pH 9, the electric anisotropy of dark-adapted purple membrane was larger than that of light-adapted purple membrane. But at around pH 6 the difference was opposite. Linear dichroism experiments did not show any change of retinal tilt angle with respect to the membrane normal between the two forms from approximately pH 5 to pH 10. This result indicates that the changes in the "electric anisotropy" are not due to the change of retinal tilt angle, but due to the change in the permanent dipole moment of the membrane. To estimate the change in surface charges from the permanent dipole moment, we investigated the difference of the permanent dipole moment between the native purple membrane and papain-treated purple membrane in which negative charges in the cytoplasmic-terminal part are removed. This estimation suggests that this light-dark difference at around pH 9 can be accounted for by a change of approximately 0.5 electric charge per bacteriorhodopsin (bR) molecule at either of the two surfaces of the membrane. We also found from pH electrode measurements that at about pH 8 or 9 light adaptation was accompanied by an uptake of approximately 0.1 protons per bR. A possible movement of protons during light-dark adaptation is discussed. The direction of the permanent dipole moment does not change with papain treatment. The permanent dipole moment in papain-treated purple membrane is estimated to be 27 +/-2 debye/bR.

Structural information from electric dichroism measurements of DNA and alternating GC nucleic acids in solution: the question of base tilt.

AbstractValues of the apparent intrinsic dichroism of the 260‐nm transition of duplex poly(dG‐dC) and poly(dG‐m5dC) in both B‐ and Z‐conformations are determined from electric dichroism (ED) measurements by extrapolation of the data at high field strengths. These values are compared to each other and to values of the intrinsic dichroism calculated for the Z‐ and B‐forms generated respectively from the crystal structure coordinates [(1981) A. H. J. Wang, G. J. Quigley, F. J. Kolpak, G. Van der Marel, T. H. Van Boom & A. Rich, Science 211, 171–176.] of the d(CpGpCpGpCpGp) hexamer and from the coordinates [(1982) A. V. Fratini, M. L. Kopka, H. R. Drew & R. E. Dickerson, J. Biol. Chem. 257, 14686–14707.]. of the terminal CpGpCpGp base pairs of the crystal of straight B‐form dodecamer d(CpGpCpGpApApTpTpCpGpCpGp). Direct association of the dichroism with the orientation of the optical transition moments with respect to the helix axis of poly(dG‐dC) implies that the average tilt of the bases is much different than the values calculated from the structural coordinates. The experimental values for both the B‐ and Z‐forms are, however, almost identical to those observed with fragments of “random” sequence B‐form DNA of the same molecular length. It is argued that this descrepancy and other anomalies reported in the literature are due to the theoretically predicted unusual high field behavior associated with diffuse ion atmosphere polarization. Unlike either permanent dipoles or induced moments based on bond polarizabilities, the orienting dipole energy from the polarization of the counterions surrounding DNA approaches a finite limiting value, dependent on DNA length, at very high field strengths. This can result in extrapolated dichroisms that do not reflect structure at perfect orientation, but rather structure at some limiting orientation. Consequently, we believe, the conclusion drawn from ED measurements on short fragments of DNA that the bases are highly tilted from the perpendicular to the helix axis by propeller twisting or otherwise is suspect.

Laser-Based Instrument for Electric Dichroism Spectroscopy of Small Molecules

While the chemical information provided by electric dichroism spectroscopy can be useful for studying the conformation and electronic structures of molecules in solution, the dichroism signals produced at fields approaching the dielectric strength of organic solvents are extremely small and difficult to detect. A double-beam, laser-based instrument for electric dichroism measurements is described which allows detection of dichroism signals corresponding to changes in absorbance of less than 10−6 with only a 1.2-kV high-voltage source.

Racemic adsorption of a bis(chelated)cobalt(III) complex by colloidally dispersed sodium montmorillonite

An optically active bis(chelated)cobalt(III) complex, Co(PAN) 2 +, (PAN = 1-(2-pyridylazo)-2-naphthol), was adsorbed by colloidally dispersed sodium montmorillonite. When Co(PAN) 2 + was added as a pure enantiomer such as (+) or (-)-Co(PAN) 2 +, a complex ion occupied one cation exchange site per chelate. Contrary to this, when the same chelate was adsorbed from racemic mixture, a complex ion occupied 0.5 cation-exchange site per chelate. The binding states of the enantiomeric and racemic complex ions were studied with the electronic spectra, electric dichroism, and stopped-flow measurements. It was concluded that the racemic ions were stacked stereoregularly in an alternating sequence of the opposite enantiomers on a silicate sheet of clay, while the enantiomeric ions were arranged less densely due to the steric interference between the ligands of neighboring chelates.

Electric dichroism studies of the bonding of Co(III) chelate with synthetic polyelectrolytes

The interactions of bis-2-(2-pyridylazo)-1-naphthol Co(III), [Co(III) (αPAN) 2 +], with five kinds of synthetic polyelectrolytes have been studied by spectrophotometric and transient electric dichroism measurements. The polyelectrolytes were: poly(styrene sulphonic acid) (PSS); poly(acrylic acid) (PAA); poly( l-glutamic acid) (PLG); poly(N e ,N e - dicarboxylmethyl- l-lysine (PDCML); and poly( l-lysine) (PLL). The equilibrium constant of the reaction: ▪ with P = polyelectrolyte residue was determined spectrophotometrically: K 1 is > 10 7 M −1 (PSS); (1.4 ± 0.2) × 10 4 M −1 (PAA); (4.0 ± 0.4) × 10 3 M −1 (PLG); (1.4 ± 0.2) × 10 6 M −1 (PDCML); and < 10 2 M −1 (PLL) at pH 6–8. From transient electric dichroism, the angle (ψ) between the αPAN plane and the polymer axis was determined to be 65° (PSS); 52° (PAA); 55° (PLG); and 52° (PDCML). The large K 1 and ψ values for PSS are ascribed to the hydrophobic interaction between the aromatic αPAN ring and the styrene sulphonate residues of PSS. Using stopped-flow electric dichroism measurements, rapid transfer of a bound Co(III) chelate from the PDCML to PSS chains was shown to occur.

Specific interactions between dye cations in premicellar aggregates as revealed by electric dichroism measurements

The electronic absorption and electric dichroism spectra are compared between an SDS solution containing one kind of cationic dye ( D + SDS ) and an SDS solution containing two kinds of cationic dyes (D 1 +/D 2 +/SDS). Here D 1 + and D 2 + are chosen from acridine orange cation (AOH +), N-nonylacridine orange cation (AO +(CH 2) 9H) and thionine cation (Th +). At the investigated SDS concentration (≈5 × 10 −5 M), the aggregate of a 1:1 dye-SDS ion pair is formed, which remains stable in a solution for several hours. In any of the three cases studied, the absorbance and dichroism amplitude for D 1 +/D 2 +/SDS are not expressed by the sum of the ones for D 1 + SDS and D 2 + SDS . The results indicate that the coexistence of two kinds of dyes in the same aggregate induces the rearrangement and orientational change of dye-SDS pairs.

Unfolding of 175-base-pair nucleosomes.

Calf thymus nucleosomes containing 175 base pairs of DNA unfold in two steps as the salt concentration is lowered, as detected by electric dichroism measurements. The transition midpoints are at 2.9 and 1.1 mM ionic strength at 7 degrees C with at most a small dependence on temperature. We identify the product of the 2.9 mM transition as an expanded disklike structure similar to the product of the 1.0--1.3 mM unfolding transition of 146-base-pair nucleosomes. The product of the 1.1 mM transition in 175-base-pair nucleosomes is elongated into a more asymmetric particle.

Length changes in solution accompanying the B-Z transition of poly (dG-m5dC) induced by Co(NH3)63+.

Transient electric dichroism measurements have been used to observe the rotational relaxation times of 145 base pair fragments of poly (dGm5dC) and random sequence DNA to solution. From these the lengths of the fragments are calculated and the interbase pair separation or rise per base pair (RPB) calculated. The observations show that even in low salt, the addition of very low concentrations of trivalent Co(NH3)63+ results in a transition of the dGm5dC polymer from B-form to Z-form with a change in the RPB from 3.4 +/- .06A to 3.7 +/- .06A, the latter form defined by the criterion of an inverted circular dichroism spectra similar to that observed at high salt in the absence of Co(NH3)63+. The 145 base pair DNA and poly (dGm5dC) are found to be essentially fully extended rods in low salt (0.2 - 2 mM Na+) solutions.

The structural organization of dinucleosomes and oligonucleosomes. Electric dichroism and birefringence study.

The spatial organization of nucleosomes and linker DNA in dinucleosomes and oligonucleosomes of various chain lengths has been investigated through electric dichroism, birefringence and relaxation times measurements at low ionic strengths (0.5 to 2.2 mM). From the negative dichroism observed for all the samples, it is concluded that the nucleosome subunits in the oligonucleosome chain must lie with their disc planes closely parallel to the fibre axis. The large increase of the negative dichroism of dinucleosomes upon Hl removal is interpreted by the unwinding of the DNA tails and the internucleosomal segment. All the samples displayed, under bipolar pulses, a predominantly induced orientation mechanism.

Evidence for the binding states of copper(II)-serum albumin complexes as revealed by transient electric dichroism measurements.

AbstractTransient electric dichroism has been measured for a Cu(II)–bovine serum albumin (BSA)–2‐(2‐pyridylazo)‐1‐naphthol (αPAN) complex at pH 5.5–12. From the magnitude of the reduced linear dichroism and the disorientation rate of the oriented chromophore, at least three kinds of binding states of Cu(αPAN)+ complex exist. They are present predominantly at pH 5.5–10, 7.5–10, and 10–12, with the αPAN plane approximately parallel, vertical, and parallel with respect to the oriented axis of a BSA molecule.

Electric dichroism and sedimentation velocity studies of DNA-Hg(II) and DNA-Ag(I) complexes

The cause of the induced CD bands of DNA-Hg 2+ and DNA-Ag + complexes has been interpreted as a condensed ψ state (Walter, A. and Luck, G. (1977) Nucl. Acids Res. 4, 539–550). Electric dichroism and sedimentation velocity experiments indicate that there is no tertiary structure formation of DNA-heavy-metal-ion complexes to support a ψ state. The increase of the sedimentation coefficient of DNA-heavy-metal-ion complexes can be accounted for by an increase in molecular weight and a decrease in the partial specific volume upon the binding of Hg 2+ and Ag +. Electric dichroism measurements show that the new optical bands produced by the binding of Hg 2+ to DNA and the mode I binding of Ag + to DNA result in transition moments polarized in the plane of the bases. Binding mode II of the DNA-Ag + complex at 265 nm gives an optical perturbation where the transition moment has an out-of-plane contribution. The CD studies reported in the following paper give further insight into the binding sites and binding modes of these two metal ions.

Unfolding of nucleosomes by ethidium binding.

We report spectroscopic, hydrodynamic, and biochemical studies on the complex of ethidium bromide with 140 base pair nucleosomal core particles. Fluorescence titration indicates a greater intrinsic affinity of ethidium for nucleosomes than for DNA, and fluorescence depolarization measurements imply increased immobilization of ethidium bound to nucleosomes, but with more extensive dye-dye energy transfer compared to DNA-bound dye. Ethidium intercalated into DNA in nucleosomes has a limiting reduced linear dichroism of -0.45 at 320 nm and -0.25 at 530 nm. Both the energy transfer and dichroism results are consistent with clustering of the nucleosome-bound dye molecules. Electric dichroism measurements and ultracentrifugation studies reveal that structural distortion of the nucleosome accompanies ethidium binding, occurring in the range of r (ethidium residues per base pair) values from 0.02 to 0.06. The distortion transition is characterized by an increase in the negative limiting reduced dichroism from 0.29 to 0.45 at 265 nm, an increase in the field-induced viscosity-limited rotational orientation time from 0.8 to 3 mus, and a decrease in sedimentation coefficient from 10.5 to 8.2 S. The complex was modeled hydrodynamically as a cylinder of 335-A length and 67-A diameter, containing 1.4 superhelical turns of DNA. Dimethylsuberimidate cross-linked nucleosomes, or native nucleosomes in the presence of Mg2+, bind ethidium weakly and are not distorted. The periodicity of cutting sites produced by DNase II digestion of nucleosomes remains constant as ethidium is added, but the bandwidth increases. A thermodynamic model is proposed to interpret the binding isotherm, based on enhancement of drug binding affinity due to release of superhelical stress in the nucleosome-ethidium complex.

Transient electric dichroism of rod-like DNA molecules.

We report transient electric dichroism studies on monodisperse rod-like DNA molecules. By using restriction fragments and DNAs of known length, it is shown that the orientation time is accurately predicted by the theoretically calculated rotational diffusion coefficient. The field dependence of the steady-state dichroism values is not consistent with the induced electric dipole orientation mechanism, and the time dependence is not consistent with the presence of a permanent dipole moment. In order to explain the dependence of the dichroism on the electric field, the ionic strength of the medium, and the length of the macromolecule, we propose a new model in which anisotropic ion flow produces an asymmetric ion atmosphere around the polyelectrolyte, resulting in an orienting torque. From the limiting dichroism at high field, we estimate that the DNA bases are inclined at an angle of 73 degrees or less relative to the helix axis, in good agreement with the revised model of B-form DNA suggested by Levitt, in which the base pairs have a propeller-like twist. Our results establish transient electric dichroism measurements as a technique well suited for study of alterations in the length and base pair inclination of rod-like DNA molecules.

Binding of ethidium bromide to ribosomal RNA: Absorption, fluorescence, circular and electric dichroism study

The interaction between ethidium bromide and ribosomal RNA has been studied by means of absorption, fluorescence, circular and electric dichroism measurements in the near ultraviolet and visible regions at low ionic strengths (1 · 10 −3 and 6 · 10 −3). The results have been interpreted on the basis of a model of interaction involving the intercalation of the phenanthridinium ring of the dye in the double-stranded regions of the RNA molecule, resulting in an increase of the dye-dye interactions as compared to DNA, and a stiffening of the intercalation regions.

The reaction of anthramycin with DNA: III. Properties of the complex

Anthramycin has a high degree of specificity for reaction with helical DNA. This specificity must reflect some special configurational relationship between anthramycin and helical DNA. Several physical properties reflecting configuration of anthramycin—DNA complexes were studied. Intrinsic viscosity and radius of gyration were found to be increased in the complex, but only at high anthramycin DNA ratio, and only at sufficiently high DNA molecular weight. There was no significant change in sedimentation constant. These findings argue that at high extents of binding, the DNA helix becomes stiffened but not lengthened. Electric dichroism measurements indicated that the chromophore portion of the anthramycin molecule is oriented at an angle of approx. 36° relative to the helix axis. Anthramycin therefore is not intercalated between base-pairs. Circular dichroism measurements supported the suggestion that anthramycin in the complex resembles the deprotonated form. The patterns for complexes with native and denatured DNA were basically similar. From this and previous data, it is suggested that the major binding of an anthramycin molecule with helical DNA involves only one strand.

Electro-optical properties of nucleic acids and nucleoproteins: V. Study of sonicated DNA and nucleohistone solutions

The ultrasonic degradation of DNA and nucleohistone samples has been studied at different frequencies and for different sonication times, by means of electric birefringence and dichroism measurements. No significant alteration in the secondary structure of these macromolecules has been detected. The histones appear to have a temporary protective effect on the degradation of the nucleohistone. The influence of the sonication time on the electro-optical behaviour of the two molecules is studied with respect to the different orientation theories. The orientation of molecular segments is considered to account for the electro-optical behaviour of the native samples. These segments, oriented at high field strengths in the high molecular weight samples, could correspond to the whole sonicated molecules. At low field strengths, the highly sonicated samples nearly obey the Kerr law contrary to the native and slightly sonicated ones. The electro-optical signals observed under reversing pulses demonstrate that the orientation mechanism of DNA and nucleohistone is mainly an induced polarization mechanism. The small transient at the field reversal is attributed to a small contribution of permanent dipole moment parallel to the symmetry axis rather than to a slow polarizability along the same direction.

Properties of Nuclease-resistant Fragments of Calf Thymus Chromatin

Abstract Nuclease-resistant chromatin fragments have been isolated on the basis of their insolubility in 0.15 m NaCl after limited digestion of calf thymus chromatin with micrococcal nuclease. Such fragments exhibit an unusual circular dichroism spectrum in the range from 260 to 300 nm, which is unlike that of B form DNA or undigested chromatin and most closely resembles that of C form DNA. Electric dichroism and sedimentation velocity measurements suggest that the DNA in these fragments is folded into a compact, perhaps superhelical, configuration. The average A258 nm: A230 nm ratio for six preparations was 1.27 ± 0.02, indicating a well defined composition of approximately 1.85 mg of protein per mg of DNA.

Molecular orientation in quantasomes: I. Electric dichroism and electric birefringence of quantasomes from spinach chloroplasts

Quantasomes (lamella subunits) isolated from spinach chloroplasts have been shown to exhibit both intrinsic electric birefringence (Kerr effect) and intrinsic electric dichroism. Measurements of Kerr constants and of rotational relaxation times suggest that the birefringence arises primarily from the proteins in the preparations. The smallest particles studied give rise to rotational relaxation times in the range 20 to 200 μsec. The shortest times may be associated with a particle having a permanent dipole moment. The spherical chromatophore subunits from Rhodospirillum rubrum do not exhibit detectable electric birefringence, as measured in the apparatus of this study. The dichroic ratio spectrum from 330 to 730 mμ of spinach quantasomes has been determined by electric dichroism measurements. The measured dichroic ratios of 1·03 to 1·10 throughout most of the spectrum suggest that the chlorophyll and carotenoid molecules in the quantasome are largely unoriented. A pronounced peak occurs in the dichroic ratio spectrum at 695 mμ, about 17 mμ to the long wavelength side of the strong chlorophyll a absorption maximum in this region. The dichroic ratio increment is at least five times greater at 695 than at 678 mμ. The dichroism anomaly is interpreted as resulting from a small fraction (∼5%) of the chlorophyll a which is strongly oriented at a particular site (the quantatrope) in the quantasome. It is suggested that an oriented cytochrome and one or more bound acceptor molecules are present at this site as well. A mechanism is postulated whereby electronic excitation energy resulting from the absorption of light by the pigments of the quantasome is converted at the quantatrope to chemical potential associated with electron transport processes.