Structure and optical activity of the DNA-aminoacridine complex

The electronic absorption and circular dichroism spectra of the DNA-acridine orange complex have been measured over a range of ionic strength, pH, and DNA phosphate to dye (P/D) ratios. Three circular dichroism bands associated with the long wavelength absorption band of acridine orange are induced on complex formation with DNA. Two of the dichroism bands, due mainly to dimeric dye molecules, are favored by low ionic strength, low pH (3.2), and a low P/D ratio (～3), while the third, deriving primarily from monomeric dye, is optimum at high ionic strength, neutral pH, and a larger P/D ratio (9). The data suggest that monomeric acridine orange binds to DNA in the form of a left-handed helical array with four dye molecules per turn, while the bound dimer has a skewed sandwich conformation which is itself dissymmetric. The stereochemical relations between the bound monomer dye and the DNA are consistent with a modified intercalation model for the DNA-acridine complex.     

Induced optical activity of acridine orange bound to poly-S-carboxymethyl-L-cysteine

The absorption and rotatory properties of acridine orange-poly-S-carboxymethyl-L -cysteine system in water and in 0.2 M NaCl have been measured at different pH and polymer-to-dye mixing ratios. The absorption spectra indicate that the dyes are bound to the polymer in dimeric or highly aggregated forms. At neutral pH where the polymer is randomly coiled, no optical activity is induced on the absorption bands of bound acridine orange. At acid pH where the polymer has the β-conformation, a pair of positive and negative circular dichroic bands occur at each of the absorption bands, centered around 458 and 261 mμ. The signs of those bands are opposite to those found for α-helical poly-L -glutamic acid. A model for the binding of dye to the β-form polymer is presented, in which dimeric dyes are attached to ionized carboxyl groups and slack one another to form linear arrays on both sides of an extended polypeptide chain. The observed circular dichroism spectra can be explained by the Tinoco's exciton mechanism, based on this model. Low molecular weight poly-S-carboxymethyl-L -cysteine induces quite a different circular dichroism on bound acridine orange.     

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.     

Electronic spectrum,optical activity,and structure of the acridine orange complex with poly-α,L-glutamic acid

The electronic absorption and circular dichroism spectra of the complex formed by acridine orange with poly-α,L -glutamic acid in the α-helix conformation have been measured in aqueous solution over a range of glutamate residue-to-dye ratios. Three Cotton effects (circular dichroism bands) associated with the long wavelength absorption band of acridine orange at 4950 A. are induced by complex formation between the dye and the polypeptide, and further circular dichroism bands are observed in the ultraviolet region associated with the 2700 A., but not with the 2950 A. absorption band of the dye. The induced optical activity is found to be relatively insensitive to the glutamate residue-to-dye ratio and to be more dependent upon the ionic strength of the solution. By Measuring the circular dichroism spectrum of the complex in aqueous solution under streaming conditions with the light propagated along the direction of flow the observed circular dichroism bands are assigned to electronic transitions polarized parallel or perpendicular to the axis of the polypeptide α-helix. From the spectroscopic data it is inferred that the dye aggregate in the L -PGA–AO complex has the form of a left-handed superhelix bound to the core of the right-handed α-helix of poly-α,L -glutamic acid. It is shown that the longer and the shorter of the in-plane axes of the dye molecule are probably orientated respectively at a small angle, and radially, with respect to the axis of the α-helix in the complex.     

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.     

A study of the interaction between D-amino acid oxidase and quasi-substrates.

To study the interaction between D-amino acid oxidase [EC 1.4.3.3] and quasi-substrates such as benzoate and o-, m-, and p-aminobenzoate, visible circular dichroism spectra (CD spectra) were measured and the binding rate and affinity of o-aminobenzoate to the enzyme were observed by following the absorption changes at various wavelengths. We found a new CD band around 560 nm, corresponding to the charge-transfer complexes which result from the formation of aminobenzoate complexes with the enzyme. The ellipticity of this band was positive for the p-aminobenzoate complex, but negative for the o- and m-aminobenzoate complexes. Crossover points in CD spectra were observed at 470 nm for the m-aminobenzoate complex and at 475 nm for the o-aminobenzoate complex. They probably resulted from overlapping of the positive CD band of FAD bound with the enzyme and the negative CD band of the charge-transfer complex. We propose that the amino group in aminobenzoate, not the pi-electrons of the benzene ring, is the electron donor in the charge-transfer complex and that the position of the amino group is very important for the charge-transfer interaction. The binding rate and affinity of o-aminobenzoate to the enzyme were determined using the absorption changes at 370 nm (380 nm), caused by the modification of electronic states of FAD bound with the enzyme, and at 550 nm (565 nm), caused by the formation of the charge-transfer complex of o-aminobenzoate with the enzyme. No differences between these parameters with wavelength were observed. This independence of wavelength simplifies discussion of the experimental data obtained from absorption changes.     

Complexes produced by associated forms of porphyrin bound with hydrophobic-hydrophilic copolymer and transition metal ions

Properties of protonated dimeric forms of meso-tetraphenylporphine (TPP) and meso-tetra(p-aminophenyl)porphine (TAPP) bound with copolymer and also complexes produced by associated TAPP bound with copolymer, Mn2+, and Fe3+ are investigated by absorption, luminescence, and Raman spectroscopy. According to absorption spectra of protonated dimers of TPP, three dimeric forms of the porphyrin are observed in the ground state. However, selective excitation of these forms according to the fluorescence spectra reveals only two dimeric forms in the excited state. In contrast, similar selective excitation of TAPP bound with copolymer in aqueous-dioxane solution results in weak changes in the fluorescence spectra, nevertheless, there is strong interaction between porphyrin and macromolecular carboxyl groups in the ground state. In the case of the formation of the complexes between associated TAPP bound with copolymer, Mn2+ and Fe3+, a new band in the near IR region with a maximum at 840 nm is built up in the fluorescence spectrum. However, this near IR emission is completely quenched when new strong vibrational bands at approximately 1800 and 1900 cm-1 are revealed in the resonance Raman spectra of the complexes. The observed effects are explained in terms of direct participation of water molecules involved in the water-porphyrin dimeric complex in the processes of transformation of excitation energy. The involvement of water in this dimeric complex can lead to redistribution of flows of the energy degradation when transition metal ions play a role of the agent which enhances the trapping properties of the porphyrin-metal-ions complexes.     

Spectroscopic properties of complexes of acridine orange with glycosaminoglycans. I. Soluble complexes.

The interaction of acridine orange with dermatan and chondrotin sulfates results in the formation of complexes containing bound dye molecules ordered into dissymmetric arrays. Complexes containing an excess of available disaccharide residues compared to dye are completely soluble, and exhibit biphasic circular dichroism bands. Analysis of the dependence of the extrinsic circular dichrosim on dye aggregation indicates the presence of extended dye stacks bound to the glycosaminoglycan. Complexes formed in solutions containing an excess of dye are only partially soluble, and exhibit circular dichroism spectra having band shifts and intensity changes relative to the soluble complexes. The latter complexes show a sharp drop in induced circular dichroism with temperature, due to a cooperative change in the structure of the complex. The structural order of the dye–glycosaminoglycan complex may differ from the intrinsic structure of the glycosaminoglycan itself in dilute solution.     

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)     

Spectroscopy on the B850 band of individual light-harvesting 2 complexes of Rhodopseudomonas acidophila. I. Experiments and Monte Carlo simulations

The electronic structure of the circular aggregate of 18 bacteriochlorophyll a (BChl a) molecules responsible for the B850 absorption band of the light-harvesting 2 (LH2) complex of the photosynthetic purple bacterium Rhodopseudomonas acidophila has been studied by measuring fluorescence-excitation spectra of individual complexes at 1.2 K. The spectra reveal several well-resolved bands that are obscured in the single, broad B850 band observed in conventional absorption measurements on bulk samples. They are interpreted consistently in terms of the exciton model for the circular aggregate of BChl a molecules. From the energy separation between the different exciton transitions a reliable value of the intermolecular interaction is obtained. The spectra of the individual complexes allow for a distinction between the intra- and the intercomplex disorder. In addition to the random disorder, a regular modulation of the interaction has to be assumed to account for all the features of the observed spectra. This modulation has a C(2) symmetry, which strongly suggests a structural deformation of the ring into an ellipse.     

Characterization of the anion transport channel protein in human erythrocytes. Induced circular dichroism of inhibitors bound to the anion transport channel

The induced circular dichroism (CD) of erythrocyte ghosts with anion-transport inhibitors has been studied. A ghost-EITC (eosin 5-isothiocyanate) system shows an induced CD spectrum at the wavelength region corresponding to the absorption bands of EITC. Also a ghost-EMI (eosin 5-maleimide) system shows induced CD, but has bands of opposite sign to the EITC system. From the change of the CD intensity, the number of EITC molecules bound to one erythrocyte was estimated to be about 1.4 X 10(6), being close to the number of band 3 copies per ghost. The CD spectra of EITC and EMI systems show that a configurational structure of the moiety anchoring the EMI molecule is the reverse to that of EITC. The preferred conformation of bound EITC may be twisted in a right-handed sense. From the signs of the induced CD bands in ghost-stilbene disulfonate systems, the chirality of twisted stilbene derivatives seems to be a left-handed sense, as is the case for the EMI derivative. The CD spectra of EITC in the presence of DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonate) shows that the binding site of EITC may not be identical with that of DIDS. The results observed in this study reflect the ternary arrangement of the functional amino groups in anion recognition sites.     

Interaction between acridine orange and polyriboadenylic acid

The absorption spectra and circular dichroism of aqueous solutions of acridine orange mixed with polY(riboadenylic acid) [poly(rA)] have been measured for different mixing ratios at acid and neutral pH. The binding ratio of dye to poly(rA) has been determined by equilibrium dialysis. At acid pH where poly(rA) is in a double-stranded helix, monomeric dye molecules are intercalated between base pairs, first sparsely and then at neighbouring sites with mutual coupling, as the nucleotide-to-dye mixing ratio decreases. In the presence of excess dye, dimeric dye molecules of antiparallel type are bound to phosphate groups electrostatically and stack together to form linear sequences along a poly(rA) chain. At neutral pH where poly(rA) is single-stranded, isolated intercalation of monomeric dye molecules can occur in the helical parts. At intermediate mixing ratios, half-intercalated dimeric dye molecules are bound to adjacent sites and electronically coupled, inducing characteristic circular dichroism. In the presence of higher amounts of dye, external stacking of dimeric dye molecules of antiparallel type occurs along a poly(rA) chain. The binding of dye cations is suppressed to some degree at acid pH compared to that at neutral pH, owing to the repulsion exerted by protonated adenine bases.     

Noncovalent interactions of peptides with porphyrins in aqueous solution: conformational study using vibrational CD spectroscopy.

Noncovalent interactions of poly(L-lysine) (PL), oligopeptides L-lysyl-L-alanyl-L-alanine and (L-lysyl-L-alanyl-L-alanine)(2) with meso-tetrakis(4-sulfonatophenyl)porphine (TPPS), and poly(L-glutamic acid) (PLGA) with meso-tetrakis(1-methyl-4-pyridyl)porphine tetra-p-tosylate (TMPyP) in aqueous solutions have been studied using combination of spectroscopic methods: Vibrational circular dichroism (VCD) spectroscopy in the mid-infrared region provides a direct information on conformational changes of the polypeptides and oligopeptides caused by interactions with porphyrins; ultraviolet-visible absorption, fluorescence, and electronic circular dichroism (ECD) reveal the aggregation characterization of the porphyrin part of the complexes. Interactions of TPPS with tripeptide, hexapeptide, and PL containing about ten amino acid residues in the molecular chain are accompanied with the changes of VCD patterns in the amide I' region. In these cases, the conformation of the oligopeptide part of complexes is obviously influenced by interactions with TPPS and partial changes of random coil structure are observed in VCD. When PL was composed of the hundreds of lysine residues, just a weak intensity decrease was detected and the shape of VCD spectrum typical for the random coil structure was preserved. As follows from the uv-vis absorption and fluorescence spectra, porphyrin molecules are attached to peptides by electrostatic interaction as a monomer or dimer and interaction between porphyrin and peptide depends on the polypeptide chain length. For the PLGA-TMPyP system with PLGA containing from tens to hundreds of glutamic acid residues in the chain, the VCD spectra were unchanged when TMPyP was presented in the aqueous solution of PLGA and random coil conformation of PLGA-TMPyP aggregates was preserved.     

Isolation and characterization of the 7S protein from glanded cottonseed (Gossypium herbacium)

The major protein from glanded cottonseed has been isolated in a homogeneous form. Its S²⁰ w value at 1 protein concentration is 6S in 1 M NaCl solution. It contains 1 carbohydrate and is free from phosphorus, gossypol (bound or free) and nucleic acid impurities. It consists of atleast seven non-identical subunits. The protein has an ultraviolet absorption maximum at 278 nm and fluorescence excitation and emission maxima at 280 nm and 325 nm respectively. Optical rotatory dispersion and circular dichroism measurements indicate that the protein consists predominantly of Β-structure and random coil. The observed near-ultraviolet circular dichroic bands can be attributed to tyrosine, phenylalanine and tryptophan residues of the protein.     

FTIR spectra of solid poly-l-lysine in the stretching NH mode range

Three bands at 3270 cm(-1), 3200 cm(-1) and 3030 cm(-1) are found in the IR stretching proton (nu(1)) mode spectral range in spectra of solid poly-l-lysine (PLL). Strong quantitative changes of these bands are observed in samples dried from water solutions with different pH. The narrow band at 3270 cm(-1), which is strong in the spectrum of PLL precipitated from pH=12 alkaline medium, is assigned to the nu(1) peptide proton mode of NH-CO (amide A) of the beta-sheet structure type. The band at 3200 cm(-1), which is intensified in PLL precipitated from pH=1 acidic medium, relates to the nu(1) peptide mode in the random coil structure. The band at 3030 cm(-1), whose peak intensity increases two-fold in going from alkaline to acidic medium, is assigned to the nu(1) modes of protonated NH(3)(+) side chain groups. The frequencies of all bands were used for estimating H-bond energy relying on an empirical correlation between this property and the red shift of the nu(1) band. The enthalpy of the secondary structure transition from beta-sheet to the random coil, which is observed in PLL at the change of pH from 11 to 1 amounts to 4.7 kJ mol(-1).     

Circular dichroism of collagen, gelatin, and poly(proline) II in the vacuum ultraviolet.

Circular dichroism spectra for acid-soluble calfskin collagen, gelatin, and poly(proline) II in solution have been extended into the vacuum ultraviolet region. The extended spectrum of gelatin reveals that the circular dichroism of this unordered polymer is more closely related to the spectrum of charged polypeptides than might be evident from near ultraviolet work. A short-wavelength band is found at about 172 nm, which corresponds in position, magnitude, and sign to a band recorded earlier for poly(L -glutamic acid) at pH 8.0. This band is observed in a helical structure for the first time in the vacuum ultraviolet circular dichroism and absorption spectra of poly(proline) II. Both circular dichroism and absorption spectra point to the assignement of this band as the nσ*. Neither the nσ* nor the expected positive lobe of the ππ* helix band is observed in the extended circular dichroism spectrum of collagen. We postulate that these two bands cancel here in analogy to the case of α-helical poly(L -glutamic acid).     

Circular dichroism of metallothioneins. A structural approach.

A comprehensive study on circular dichroism of metallothioneins containing Zn, Cd and Cu was carried out. The contributions of the metals, the sulphur and the polypeptide chain to the observed Cotton effects was shown. From the pH dependency of the extrinsic Cotton effects which are due to the metal-thiolate chromophore the stability of the metal clusters was found to decrease in the order Cu greater than Cd greater than Zn. The pH values corresponding to the dissociation of half of the bound metal ions are 0.44 for Cu-thionein, 3.05 for Cd-thionein and 4.6 for Zn-thionein. The extrinsic Cotton effects of Cd, Zn-thioneins of varying Cd to Zn ratio could be simulated using the difference circular dichroic spectra of Cd-thionein (bands at 227, 242.5 and 262 nm), Zn-thionein (bands at 225 and 244 nm) and the circular dichroic spectrum of cysteine-thionein (band at 200 nm, shoulder at 225 nm). Since during the dissociation of the metals the circular dichroic spectra exhibited changes only in amplitude and not in shape we can conclude that the dissociation of the metal ions involves the complete sequential degradation of metal clusters. In the near-ultraviolet region the metal-free proteins show only Cotton effects attributable to a disulphide chromophore. Thus Cotton bands are observed for cystine-thionein at 282.5 and 260 nm. From the intrinsic circular dichroism of Cd- and Zn-thionein (negative Cotton effect at 200 nm, shoulder at 225 nm) it follows that the protein conformation consists of less than 5% helical or pleated sheet structure and therefore has to be classified as unordered structure or "fixed" random coil     

Ceruloplasmin-anion interaction. A circular dichroism spectroscopic study.

The effect of anion binding to ceruloplasmin has been studied using absorption and cirbular dichroism spectral data. At anion to ceruloplasmin molar ratios approaching infinite, OCN-, N3- and SCN- bind to ceruloplasmin giving rise to similar alterations in circular dichroism and absorption spectra. The positive bands at 610 and 520 nm in circular dichroism spectra disappear, a negative one apperars at 600 nm and the peak at 450 nm is only slightly modified. There is a new negative band at 410 nm well-defined in OCN- ceruloplasmin spectra. The decrease in absorption at 610 nm is ascribed to the disruption of one type I Cu-S(cysteine) bond owing presumably to the changes induced by anions in the protein secondary structure. The new band at 410 nm is assigned to a charge transfer transition from the ligand replacing cysteine at its binding site. Both absorption and circular dichroism spectra show isobestic points indicating that anion binding to the enzyme, disruption of one of the two type I Cu-S bonds and coordination of this Cu to another protein residue take place simultaneously.     

Spectral control of metal admixtures in tetracycline]

Analysis of circular dichroism spectra made it possible to offer a method for estimation of tetracycline solutions contamination with metal ions. By its sensitivity the method is much superior to the spectrophotometric one used at present for determination of the antibiotic purity. In the latter method formation of complexes with metals is traced by batochromic displacement of the absorption spectra. The new method is rapid, relatively selective and requires comparatively small quantities of the substance for the analysis, which provides its use under both laboratory and manufacture conditions. The method is based on identification of the circular dichroism spectra of tetracycline complexes with metals in the long wavelength region. The presence of the circular dichroism concervative bands with strictly defined extremums in the spectra of tetracycline low acid solutions contaminated by multiply charged metal ions allowed vs. the circular dichroism spectra of pure tetracycline sample to conclude that the solution contained admixtures and to suggest their nature. It was shown that the charge, ion radius and tetracycline:metal relation were the factors defining the mark and location of the dichroism band extremums. At lambda(extr)-410-415 nm the tetracycline complexes with light metal ions such as Mg2+, Al3+ and Ca2+ were detected by the circular dichroism negative band in the spectra, while the complexes with heavy metal ions such as Sc3+, Sr3+, Cu3+, Cd3+, Ba2+, Y3+ and the cerium subgroup lanthanides were detected by the circular dichroism positive band. The tetracycline complexes with the lanthanides of the second half of the yttrium subgroup (Ho(3+)-Lu3+) were characterized by the presence of the circular dichroism minimum at lambda(min)-425 nm. When the tetracycline concentration was above 1.5 x 10(-3) M, multiligand complexes with circular dichroism negative extremum at lambda(min)-400 nm formed.     

Circular dichroism studies of complexes of sequential histidine polypeptides with methyl orange

The induced circular dichroism (c.d.) spectra of poly(l-histidine) and sequential histidine polypeptide-dye complexes were measured. Two dichroic bands associated with the blue shifted absorption shoulder of methyl orange at around 370 nm were observed by complex formation between the polypeptides and the dye. Induced c.d. arose from the dye bound to the polypeptide in random coil structure, the optimal pH being 4.1. Added sodium chloride decreases the intensity of the induced c.d. Intramolecular interaction was assumed from the relationship between the concentrations of the polypeptide-dye complex and the intensities of the induced c.d. The intensity of the induced c.d. decreases with increasing distance between the intramolecular histidine residues. The induced c.d. spectrum of the poly(l-histidine)-dye complex shows the irreversible thermal change when heating.