Supramolecular order following binding of the dichroic birefringent sulfonic dye Ponceau SS to collagen fibers

The optical anisotropies (linear dichroism or LD and birefringence) of crystalline aggregates of the sulfonic azo-dye Ponceau SS and of dye complexed with chicken tendon collagen fibers were investigated in order to assess their polarizing properties and similarity to liquid crystals. In some experiments, the staining was preceded by treatment with picric acid. Crystalline fibrous aggregates of the dye had a negative LD, and their electronic transitions were oriented perpendicular to the filamentary structures. The binding of Ponceau SS molecules to the collagen fibers altered the LD signal, with variations in the fiber orientation affecting the resulting dichroic ratios. The long axis of the rod-like dye molecule was assumed to be bound in register, parallel to the collagen fiber. Picric acid did not affect the oriented binding of the azo dye to collagen fibers. There were differences in the optical anisotropy of Ponceau SS-stained tendons from 21-day-old and 41-day-old chickens, indicating that Ponceau SS was able to distinguish between different ordered states of macromolecular aggregation in chicken tendon collagen fibers. In the presence of dichroic rod-like azo-dye molecules such as Ponceau SS, collagen also formed structures with a much higher degree of orientation. The presence of LD in the Ponceau SS-collagen complex even in unpolarized light indicated that this complex can act as a polarizer.     

Polarization microscopy and microspectrophotometry of Sirius Red, Picrosirius and Chlorantine Fast Red aggregates and of their complexes with collagen

Summary A detailed quantitative analysis of the anisotropic properties of Sirius Red F3B, Picrosirius, and Chlorantine Fast Red crystals, and of their complexes with a macromolecularly oriented protein either in a pure form or as part of a tissue structure was carried out. Collagen I was used as the protein model. Linear dichroism and dispersion of birefringence were investigated in dye aggregates, in stained filaments of collagen I and in collagen bundles in sections of tendon. A positive linear dichroism, the characteristics of which varied as a function of the dye type used, was demonstrated for the dye aggregates and stained substrates. However, even thin regions of the stained tendon collagen bundles showed very high absorbances, differing from the pattern reported previously, for collagen stained with another sulphonated azo dye, Xylidine Ponceau. Consequently, not all these dyes enable protein concentration and orientation to be determined in collagen-containing structures. From the linear dichroism patterns it is assumed that the long axis of the molecules of these azo dye is mostly parallel to that of filaments of pure collagen I and statistically parallel to the long axis of collagen bundles of tendon sections. The dye aggregates and, stained pure collagen I and tendon collagen bundles exhibited birefringent images with interference colours that varied as a function of thickness and packing state of the preparations, which is in agreement with reports in the literature. The optical retardations of the collagen bundles increased by a factor of 5–6 times after staining with Picrosirius. From data on form dichroism it is concluded that when studying the macromolecular orientation of collagen preparations stained with azo dyes, the choice of the mounting medium deserves consideration.     

Comparisons between hog intestinal peroxidase and bovine lactoperoxidase-compound I formation and inhibition by benzhydroxamic acid.

The bihelical polydeoxyribonucleotides DNA and poly (deoxyadenylate-deoxythymidylate form at least two distinct complexes with the dye Hoechst 33258. The nonfluorescent complex formed at low polymer/dye ratios is replaced at high polymer/dye ratios by an intensely fluorescent complex. The transition is accompanied by pronounced changes in circular dichroism and absorption spectra and may be interpreted in terms of a noncooperative replacement of dye molecules bound in proximity by isolated molecules of bound dye. In the case of the bihelical polyribonucleotides the transition exhibits positive cooperativity and major differences from the deoxyribose polymers exist in the circular dichroism spectra, suggesting a different geometry for the complex species.     

Base tilt of B-form poly[d(G)]-poly[d(C)] and the B- and Z-conformations of poly[d(GC)]-poly[d(GC)] in solution

We have measured the CD, isotropic absorption, and linear dichroism (LD) in the vacuum-uv spectral region for the B-conformations of poly[d(G)]-poly[d(C)] and poly[d(GC)]-poly[d(GC)], and for the Z-conformation of poly[d(GC)]-poly[d(GC)] formed in 70% trifluoroethanol. The reduced dichroism (LD divided by isotropic absorption) for all conformations varied with wavelength, indicating that the bases are not perpendicular to the helix axis. Since the directions of the transition dipoles are known, the inclinations and axes of inclination of each base can be determined from the wavelength dependence of the reduced dichroism spectra. The results indicate that the base normals of the (G + C) polymers in the B- and Z-conformations are tilted at angles greater than 19° with respect to the helix axis. The guanine and cytosine bases have different inclinations, and the tilt axes are not parallel. Therefore, the bases for all the (G + C) polymer conformations studied are buckled and propeller twisted.     

Orientation of pigments and pigment-protein complexes in the green photosynthetic bacterium Prosthecochloris aestuarii

The orientation of pigments and pigment-protein complexes of the green photosynthetic bacterium Prosthecochloris aestuarii was studied by measurement of linear dichroism spectra at 295 and 100 K. Orientation of intact cells and membrane vesicles (Complex I) was obtained by drying on a glass plate. The photochemically active pigment-protein complexes (photosystem-protein complex and reaction center pigment-protein complex) and the antenna bacteriochlorophyll a protein were oriented by pressing a polyacrylamide gel. The data indicate that the near-infrared transitions (Q_y) of bacteriochlorophyll c and most bacteriochlorophyll a molecules have a relatively parallel orientation to the membrane, whereas the Q_y transitions of the bacteriochlorophyll a in the antenna protein are oriented predominantly perpendicularly to the membrane. Carotenoids and the Q_x transitions (590–620 nm) of bacteriochlorophyll a, not belonging to the bacteriochlorophyll a protein, have a relatively perpendicular orientation to the membrane. The absorption and linear dichroism spectra indicate the existence of different pools of bacteriochlorophyll c in the chlorosomes and of carotenoid and bacteriopheophytin c in the cell membrane. The results suggest that the photosystem-protein and reaction center pigment-protein complexes are oriented with their short axes approximately perpendicular to the plane of the membrane. The symmetry axis of the bacteriochlorophyll a protein has an approximately perpendicular orientation.     

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.     

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.     

Effect of pulsed and reversing electric fields on the orientation of linear and supercoiled DNA molecules in agarose gels

When linear or supercoiled DNA molecules are imbedded in agarose gels and subjected to electric fields, they become oriented in the gel matrix and give rise to an electric birefringence signal. The sign of the birefringence is negative, indicating that the DNA molecules are oriented parallel to the electric field lines. If the DNA molecules are larger than about 1.5 kilobase pairs, a delay is observed before the birefringence signal appears. This time lag, which is roughly independent of DNA molecular weight, decreases with increasing electric field strength. The field-free decay of the birefringence is much slower for the DNA molecules imbedded in agarose gels than observed in free solution, indicating that orientation in the gel is accompanied by stretching. Both linear and supercoiled molecules become stretched, although the apparent change in conformation is much less pronounced for supercoiled molecules. When the electric field is rapidly reversed in polarity, very little change in the birefringence signal is observed for linear or supercoiled DNAs if the equilibrium orientation (i.e., birefringence) had been reached before field reversal. Apparently, completely stretched, oriented DNA molecules are able to reverse their direction of migration with little or no loss of orientation. If the steady-state birefringence had not been reached before the field reversal, complicated orientation patterns are observed after field reversal. Very large, partially stretched DNA molecules exhibit a rapid decrease in orientation at field reversal. The rate of decrease of the birefringence signal in the reversing field is faster than the field-free decay of the birefringence and is approximately equal to the rate of orientation in the field (after the lag period).(ABSTRACT TRUNCATED AT 250 WORDS)     

Concentration and temperature effects in interactions of the dye pyronine G with polynucleotides

The interaction of poly(A) and poly(A).poly(U) with pyronine G dye depending on the concentration of components and temperature was studied spectrophotometrically in the visible and UV ranges at pH (6.86). It was found that the interaction of pyronine G with poly(A) and poly(A).poly(U) results in the formation of two types of complexes. The relation of the equilibrium concentrations of these complexes depends on the initial concentrations of the components in solution. The formation of complex I results in shifting the spectrum towards the short wave range with regard to the monomer band and reflects the aggregation of the dye cations. Complex II is characterized by the shift towards the long wave range. Complex II is formed in considerable amounts for poly(A).pyronine G system at large P/D and for poly(A).poly(U).pyronine G system at P/D = 5-6 and is probably due to the interaction between the dye and polynucleotides of the intercalation type or reflects the interaction between the dye and two negatively charged phosphate groups. Analysis of temperature measurements of spectra confirms the formation of various types of complexes in the system studied.     

Geometry of DNA–dye intercalation complexes from the study of linear dichroic spectra of stretched films

Films of DNA–dye complexes were combined with films of pure DNA deposited on poly(vinyl alcohol) support and stretched. Reproducible dichroic spectra were obtained after equilibration of the stretched films at 93% relative humidity. Dye diffusion into the supporting poly(vinl alcohol) matrix was eliminated. The long axis of intercalated acriflavine is perpendicular to the DNA helix; proflavine deviates slightly and 9-aminoacridine significantly from such an intercalation geometry. The dichroism of two mutually perpendicularly polarized transitions of 9-aminoacridine enabled us to determine both the angles of tilt and twist of the plane of the dye relative to the DNA helix in the complex.