Showing papers on "Circular dichroism published in 1973"

Different conformations of double‐stranded nucleic acid in solution as revealed by circular dichroism

Abstract: Conformation of two-stranded DNA in H2O–methanol, H2O–ethanol, H2O–isopropanol, and H2O–dioxane solutions at different concentrations of alkaline ions has been studied with the aid of circular dichroism. The following conclusions are drawn: The conformation of DNA in H2O and H2O–methanol belongs to a family of B forms (B, C, T forms are the representatives of the family). The magnitude of the winding angle between adjacent base pairs (θ) is determined by the concentration and type of the cations. In H2O the cation action is nonspecific and leads to an increase in θ value. In 80% methanol the ions act specifically, Cs+ being to stabilize a form with a greater θ value, and Li+ being with a lesser one. The total θ change is likely within the limits of 33° ⩽ θ ⩽ 45°. At high content of ethanol, isopropanol, or dioxane (∼80%), but not with methanol, and in low ionic strength the conformation of DNA belongs to a family of A forms (A form is one of the members of the family) and is specified by the concentration and type of cation involved. The two-stranded regions of RNA in H2O are also of A type and winds with the rise of cation concentration. The range of θ variation is not narrower than 30° ⩽ θ 33°. The conformational transitions within the families (induced by ions) are of non-cooperative pattern, wheras the transitions between the families (induced by nonpolar component) are of cooperative pattern. The effect of cations, when specific, is discussed on the basis of steric correspondence between the width of DNA narrow groove and the size of a hydrated cation.

Circular dichroism and optical rotatory dispersion of proteins and polypeptides.

Abstract: Publisher Summary This chapter discusses the methodology of circular dichroism (CD) and optical rotatory dispersion (ORD) data gathering and analysis in the rapidly changing field of protein structure, making use of synthetic polypeptide studies when necessary. ORD is the measurement, as a function of wavelength, of a molecule's ability to rotate the plane of linearly polarized light. CD is similar data evaluating the molecule's unequal absorption of right- and left-handed circularly polarized light. CD and ORD can yield useful estimates of protein secondary structure. Although all the amino acids except glycine contain at least one asymmetric carbon atom, most amino acids display only small ORD and CD bands. It is the conformation of the protein—that is, the asymmetric and periodic arrangement of peptide units in space, which gives rise to their characteristic ORD and CD spectra. In recent years, X-ray diffraction analysis has lead to the complete mapping of the peptide backbone and side-chain positions of lysozyme, several other enzymes, and quite a few other proteins in the solid state.

Alteration of the Relative Stability of dA·dT and dG·dC Base Pairs in DNA

Abstract: Several small alkylammonium ions can eliminate, or even reverse, the usual dependence of the DNA transition temperature on base composition. For example, in 3 M tetramethylammonium chloride, or 2.4 M tetraethylammonium chloride, DNAs of different base compositions all melt at a common temperature, and with a greatly decreased breadth of transition reflecting only the sequence-independent components of melting cooperativity. At still higher concentrations of such additives, dG·dC-rich DNAs melt at lower temperatures than dA·dT-rich molecules. Circular dichroism spectra show that these additives alter the structure of the DNA double helix very little at room temperature. This differential (base-specific) effect on helix stability is investigated with several small additives related to the tetraalkylammonium ions. Additives larger than tetraethylammonium ion have little differential effect on helix stability. Preferential binding of ions to dA·dT base pairs, requiring fit into a “groove” of DNA, is consistent with these data and with equilibrium binding studies. These differential effects can be distinguished from general destabilizing effects, which are independent of specific features of macromolecular conformation or chemistry. Possible experimental uses of this ability to alter the base-composition-dependent components of the stability of the DNA helix are discussed, as well as the insight this phenomenon provides into the molecular basis for the differential stability of dA·dT and dG·dC base pairs.

Characterization of Cd, Zn‐Thionein (Metallothionein) Isolated from Rat and Chicken Liver

Abstract: The preparation of pure metallothionein from Cd-pretreated rats and chicken was performed using short-time treatment with chloroform/ethanol followed by ion-exchange and gel chromatography. The protein contained 7 g-atoms metal ions per 12000 g protein. The Zn to Cd ratio was 1:2.4 ± 0.1. The Cd, Zn-thionein remained homogeneous during Sephadex G-25, G-50 and G-75 gel filtration, during polyacrylamide disc-gel electrophoresis and in the analytical ultracentrifuge. The high content of cysteine residues (approx. one-third of the total residues) was consistent with the cysteine content of metallothionein isolated from human or equine tissues. The Cd, Zn-thionein was of considerable temperature stability. The physicochemical properties were examined by ultraviolet spectroscopy, circular dichroism (CD) measurements and X-ray photoelectron spectroscopy. The millimolar absorption coefficient of the native protein was at 250 nm ɛ250= 80.6 mM−1· cm−1. Virtually no aromatic amino acids were present. The ɛ250 value of the apoprotein prepared by metal displacement using HCl was ɛ250= 13.2 mM−1· cm−1. When the ultraviolet spectrum of the apoprotein was recorded at pH 6.6 a distinct peak was detectable at 255 nm (ɛ250= 18.7 m M−1· cm−1). The existence of inter- or intramolecular disulphide formation was confirmed by CD measurements employing the polyethyleneglycol esters of di-tert-butyloxy-carbonyl-l-cystine and tert-butyloxycarbonyl-l-cysteine as model compounds for high-molecular-weight and water-soluble cysteine compounds, respectively. From CD data it was concluded that Cd, Zn-thionein exists mainly as a random-coil peptide. Some indication of the binding of Cd and Zn in the native protein with ⊖S–R moieties was obtained from ultraviolet data. Final proof of the exclusive coordination of these metal ions with cysteine sulphur was successful using both circular dichroism measurements and especially X-ray photo-electron spectroscopy. The last method proved most convenient for determining the binding energies of the core electrons of Cd (Cd 3d3/2= 411.0 eV and Cd 3d5/2= 404.4 eV), Zn (2 p3/2= 1021.0 eV) and S (2p = 161.7 eV). The corresponding binding energies for S in the cystine complexes with Zn2+ and Cd2+ were 163.0 eV and 162.8 eV, respectively.

Interaction of metallic cations with DNA VI. Specific binding of Mg++ and Mn++

Abstract: The binding of Mg2+ and Mn2+ by DNA by a divalent cation specific electrode and by ultracentrifugation. Both techniques give similar results for the stoichiometry of the reaction. An oscillating densiemete allowed us to detect small changes of volume accompanying the binding. The reaction was also followed by circular dichroism measurements. Interpretation of the results is only possible if one assumes an electrostate site-binding of Mg2+ to phosphate group, and a chelation Mn2+ between the phosphate group and the N7 of the guanine. Physical modifications accompanying these two types of binding are discused and compared to the role of these cations in some biological systems involving DNA.

The interaction of bilirubin with human serum albumin.

Abstract: The interaction of bilirubin with de-fatted human serum albumin has been studied by circular dichroism and spectrofluorimetry. From the dependence of the induced circular dichroism of the bilirubin on the ratio of protein to bilirubin, it is shown that in the presence of salt (0.5 M sodium chloride) there are two binding sites with markedly different optical characteristics. From an analysis of the profiles of ellipticity as a function of protein: bilirubin ratio, association constants of 7 and 0.3 μM−1 at pH 8.5 were derived. At low salt concentrations qualitatively similar binding profiles are obtained with a marked maximum of ellipticity at a protein: bilirubin ratio of about 1. These cannot however be adequately accounted for with only two binding sites. The addition of a third site allows the data to be fitted over the entire concentration range at all wavelengths tested, with association constants falling in the range 10–0.01 μM−1. When bilirubin is bound to albumin, the fluorescence of the protein is quenched and that of the bilirubin is enhanced. The Fluorescence characteristics of the binding sites are shown to be different: the weaker sites, which have lower fluorescence, but higher circular dichroism than the stronger, are relatively much more labile to salt. The pH-dependence of the optical properties reveals the presence of a state defined by a plateau at between pH 8 and 9, another by a plateau centred at about pH 6.5, and another, the appearance of which is associated with the N–F transition of the protein, and which exists only within an extremely narrow pH range, at about pH 4. The pH at which the minimum occurs undergoes a large shift with salt concentration. This state is characterised by an extremely large circular dichroism (effective molar ellipticity -5 × 105 deg. cm2· dmol−1), opposite in sign to the system of Cotton effects observed at higher pH. This complex shows no fluorescence enhancement. The system shows large hysteresis, satisfactory results being obtained only when the complex is first formed by equilibration at pH 8–9 at low ionic strength. Under other conditions the self-association of the bilirubin evidently prevents the attainment of equilibrium, and vitiates any attempts at analysis of the binding equilibria. The competition of oleate ions with the bilirubin binding sites has been studied. Circular dichroism, under physiological conditions, of human serum containing bilirubin shows that the binding of the bilirubin by albumin alone at the corresponding ionic strength and pH defines the state of the ligand in the plasma.

Extended conformations of polypeptides and proteins in urea and guanidine hydrochloride

Abstract: By analyzing the effect of urea and guanidine hydrochloride on the circular dichroism of many polypeptides and proteins, it is concluded that under conditions of high concentration of the perturbant and at low temperatures the resultant state approached is that of a local extended helix structure instead of a completely random coil. Intensification by urea and guanidine hydrochloride of the circular dichroism bands of poly-L-proline II leads to the proof that the mechanism of interaction of urea and guanidine hydrochloride with proteins is through hydrogen bonding to the backbone carbonyl group.

A Near‐Infrared Circular Dichroism and Magnetic Circular Dichroism Instrument

Abstract: We report a high sensitivity, near‐infrared circular dichroism (CD) instrument CD is measured by the standard modulation method; phase modulation is provided by an Infrasil quartz photoelastic modulator The spectral range 08–30 μ is spanned Incorporation of a superconducting magnet allows magnetic circular dichroism (MCD) measurements to be made The natural CD of nickel and samarium tartrate complexes and the MCD of a single crystal of Co/MgO at 12 K and 406 kG are reported Performance of the instrument is comparable to visible‐ultraviolet CD instruments

Circular dichroism and rotatory dispersion near absorption bands of cholesteric liquid crystals

Abstract: A macroscopic theory is developed to explain the intense and highly structured circular dichroism (CD) and optical rotary dispersion (ORD) measurements of cholesteric liquid crystals in spectral regions of absorption, e.g., in the infrared. Oseen’s helical model of the dielectric tensor of the liquid crystal is used. The effects of each absorption band are represented by frequency-dependent, complex terms in the dielectric tensor. Assuming reasonable values for the absorptive parameters of the band, we are able to explain the magnitude and shape of the CD and ORD spectra, as well as their trends with the pitch of the helix. The calculated sign of the Cotton effect is shown to depend upon the direction of polarization of the absorption band with respect to the background birefringence. The observed preponderance of bands with one sign is qualitatively explained on the basis of the molecular structure of the liquid crystal.

Experimental Differential Light-Scattering Correction to the Circular Dichroism of Bacteriophage T2

Abstract: Experimental techniques are presented that can be used to assay and correct for differential light scattering effects in circular dichroism spectra of biological macrostructures. The assay is based upon use of variable detector geometries that collect light over large solid angles. Disrupted T2 virus suspensions and purified T2 phage DNA exhibit geometry-independent spectra; the spectrum of intact T2 virus is highly sensitive to detection geometry. On the basis of spectra obtained after light-scattering correction, the structure of T2 DNA in the phage particle is assigned to the C form. We conclude that: (i) The measured circular dichroism of a light-scattering specimen may be highly sensitive to light-detection geometry of the instrument. This effect is indicative of differential scattering intensity for left and right circularly polarized light. (ii) Some optically active particles, although they scatter light intensely, exhibit circular dichroism that is independent of detection geometry and, therefore, apparently uninfluenced by differential light scattering. We infer that whether differential light scattering arises may depend upon the presence or absence of ordered asymmetry in the organization of the scattering particle. (iii) The circular dichroism of any light-scattering specimen should be measured again in apparatus designed for differential light-scattering correction as a prerequisite to meaningful structural conclusions. (iv) Differential scattering effects in circular dichroism may be potentially useful as a probe for large-order organization of the scattering particle.

Induced optical activity in poly-L-lysine-methyl orange system.

Abstract: The absorption and cicular dichroism spectra of the complex of poly-L-lysine (PLL) in the random coil form with methyl orange (MO) have been measured in aqueous solution. A new absorption band is observed at the shorter wavelength compared with that of the free dye. Although MO does not show a formation of dimer or aggregation with an increase in concentration, circular dichroism bands are observed at the wavelength corresponding to the wavelength of the new absorption band. These induced circular dichroism bands may arise from the dimeric MO molecules bound to PLL in the random coil form. The main contribution to the interaction between MO molecules is shown to be the electro static interaction. The observed circular dichroism spectra and the configuration of dimeric MO molecules bound to PLL can be explained by the dipole couping mechanism.

Interactions of metallic ions with DNA. V. DNA Renaturation Mechanism in the Presence of Cu2

Abstract: New experimental data were obtained by means of circular dichroism, melting, renaturation, and kinetic experiments, upon Cu2+ binding to DNA, poly dAT, and poly dGdC. They enable us to propose a model of binding giving a satisfactory explanation to all of the data found in the literature. Two types of binding sites are proposed: (a) a “sandwich” of Cu2+ between two adjacent G-C pairs giving a charge-transfer complex, and (b) a chelate between a phosphate group and a nitrogen atom of the bases (N7 of guanine and N3 of cytosine at room temperature, N3 of adenine after thermal opening of A-T pair). Type (a) stabilizes the helix and keeps the two strands linked. Type (b) destabilizes the helix and explains why the kinetic rate of renaturation is the same as that of copper release.

Vibrational circular dichroism observed in crystalline α‐NiSO4 · 6H2O and α‐ZnSeO4 · 6H2O between 1900 and 5000 cm−1

Abstract: Axial circular dichroism (CD) of crystalline α‐NiSO4 · 6H2O and α‐ZnSeO4 · 6H2O is measured between 1900 and 5000 cm−1 at room temperature. Three CD bands with rotational strengths of 7 × 10−44, 3 × 10−44, and 1 × 10−42 erg · cm3 per molecule of water are observed at 5000, 4050, and 2300 cm−1, respectively, in α‐NiSO4 · 6H2O. These CD bands are due to combination bands of H2O vibrations in the crystals. Substitution of H2O with D2O on α‐NiSO4 · 6H2O shifts the 5000 cm−1 band to a frequency lower by the expected factor of 2. The 5000 and 4050 cm−1 CD bands are also observed in α‐ZnSeO4 · 6H2O with the same intensities as in α‐NiSO4 · 6H2O. Their positions are, however, shifted by 50 cm−1 toward higher energy. Due to strong absorption, the CD of fundamental vibrational modes of H2O in the crystals could not be measured. The CD is interpreted as a consequence of the linear k dependence of the effective charge of E mode optical phonons near k = 0.