Optical activity of single-stranded polydeoxyadenylic and polyriboadenylic acids; dependence of adenine chromophore cotton effects on polymer conformation

Circular dichroism (CD) curves are reported for poly dA, (pdA)₆, (pdA)₂, poly A, ApAp, ApA, AMP, dApA, pdApA, A-2′-O-methyl pA, and A-2′-O-methyl pAp. Analysis of these curves indicated the presence of single CD bands at 228–230 mμ and at 278–280 mμ in oligomers longer than dinucleotides. In the case of dinucleotides and mononucleotides (from the literature, in addition to those studied here), the 230 mμ CD of band appears but the 280 mμ CD band does not. We assign the 230 mμ band to a very weak π–π* transition at this wavelength. From theoretical considerations, we show that the 280 mμ band is not an exciton component of the strong π–π* transition at 260 mμ in adenine. We conclude that the 280 mμ CD band must be assigned to a distinct absorption, not previously reported, which we suggest arises from an n–π* transition. The fact that the n–π* CD band at 280 mμ is not seen in mononucleotides or dinucleotides is ascribed to solvation of the adenine ring by water, which shifts the band to shorter wavelengths. Therefore, only interior residues of oligomers have the 280 mμ band, and the optical activity of a polymer cannot be computed from that of a dinucleotide, by using a nearest-neighbor approximation. The existence of this end effect hag been tested, by taking it into account in computing the rotational strengths of the 278 mμ n–π* transition for several oligomers; it is pointed out that a more sensitive test of this end effect would require CD data for the oligo dA series of 3 to 5 residues. We speculate about the structural and optical differences between poly dA and poly A, and point out the need for a theoretical treatment of n–π* Cotton effects in polynucleotides.     

Induced Cotton effects of tRNA-acridine orange complex and tRNA conformation

Circular dichroism spectra of acridine orange bound to E. coli tRNA were studied at varying tRNA phosphate-to-dye (P/D) ratios for both unfractionated and purified materials in the absence of Mg⁺⁺. From the rather discrete features exhibited in the circular dichroism spectra three types of interactions were observed: (1) A high P/D ratio such as 75.2 or 49.8 indicates the interaction between the nucleotide base and dye molecule. The spectra with a large positive peak at 515 mμ are, however, quite different from that of DNA–AO complex under similar conditions. (2) With an intermediate P/D ratio (26.5 to 9.6) dye molecules bound strongly to the polynucleotide chain. (3) With low P/D ratios (≤7.5) the interaction appears to be due to the stacked dye molecules in the single-stranded part of tRNA. The spectra of the third group have an isobestic point at 477 mμ. Below a P/D ratio of 4 the spectrum shows one positive and two negative bands which may be the characteristics of circular dichroism of stacked dyes in polynucleotide chain. Although no drastic change in the conformation of tRNA itself was detectable in the presence of Mg⁺⁺ in the ultraviolet region, a dramatic change was observed in the circular dichroism of tRNA–acridine orange complex when Mg⁺⁺ concentration was increased to 10^?3M. It was inferred that certain conformational changes other than simple hydrogen bond formation occured in tRNA molecules at this high Mg⁺⁺ concentration, so that the amount of bound dye in the stacking condition was increased through the transition.     

Induced circular dichroism of DNA-dye complexes

The binding of methylene blue, proflavine, and ethidium bromide with DNA has been studied by spectrophotometric titration. Methylene blue and proflavine or methylene blue and ethidium bromide were simultaneously titrated by DNA. The results indicate that all of these dyes compete for the same bindine sites. The binding properties are discussed in terms of symmetry. The optical properties of the dye–DNA complexes have been studied as a function of DNA/dye ratio. The induced circular dichriosm due to dye–dye interaction was measured at low dye/DNA ratios for cases involving both the same dye and different dyes. A positive Cotton effect for DNA–proflavine complex may be induced at 465 mμ by eithr proflavine or ethidium bromide, whereas a netgative Cotton effect at 465 mμ may be induced by methylene blue. The limiting circular dichroism, with no dye–dye interaction, and the induced circular dichroism spectra are discussed in terms of symmetry rules.     

Tryptophol Metabolism by a Species of Arthrobacter

The stereochemistry of some dihydrofurano-isoflavones previously isolated from white lupin roots, or obtained following fungal metabolism of prenylated isoflavones, was investigated using CD spectroscopy. The osmate ester/pyridine complex of dextrorotatory lupinisoflavone A (1) exhibited a positive CD Cotton effect at 480 nm, indicating a side-structure configuration (S at C- 2″), opposite to that of natural rotenone (9), which afforded a negative Cotton effect at 474 nm (R- configuration at C-2′ on the side structure [ring E]). The stereochemistry of the laevorotatory luteone metabolite BC-1 (2) and lupinisoflavone D (4) (both ^-configuration at C-2″) was similarly determined after converting to the corresponding dehydrate (10) or trimethyl-dehydrate (1b, 10a).     

Optical rotatory dispersion and circular dichroism of carbanilyl polysaccharides

Measurements of optical rotatory dispersion (ORD) and circular dichroism (CD) have been made in the range of 600-210 mμ for the β-glycan carbanilates as for instance, 2,3,6-tricarbanilylcellulose (I), 2,3,6-tricarbanilylmannan (II), 2,3-dicarbanilylcellulose (III), and octacarbanilylcellobiose (IV) and also for the α-glycan carbanilates, such as 2,3,6-tricarbanilylamylose (V), tricarbanilylpullulan (VI), 2,3-dicarbanilylamylose (VII), and octacarbanilylmaltose (VIII). Furthermore, the 2,3,4,6-tetracarbanilyl-α-methyl-glucopyranoside (IX) and the 1,2,3,4,6-pentacarbanilylglucose (X) have been measured in dioxane at 20°C. For the β-glycans a small negative CD in the region of 238–240 mμ and nearly symmetrical ORD curve with a crossover point at 238–240 mμ are found; this indicates a simple negative Cotton effect. In the case of α-glycosides, a strong negative CD with a maximum at 240–242 mμ and a strong positive CD with a maximum at 223–225 mμ were found; the ORD curves are asymmetrical and cross the abscissa in two places, at 241–243 and 220–222 mμ. With 2,3,4,6-tetracarbanilyl-α-methylglucoside (IX) no CD and ORD in the ultraviolet region and with 1,2,3,4,6-pentacarbanilyl-glucopyranoside (X) the ORD, but not the CD, could be measured. The ORD curve is nearly symmetrical, like those of the β-glycans but is of opposite sign. It seems impossible to discuss the striking difference of the CD and ORD spectra between the α-and the β-glycans in terms of contributions of single independant chromophores influenced by their individual different steric arrangements and their spatial relation to the glycosidic bond in C₁. The exciton theory of Moffitt, which is suitable for explaining the ORD and CD spectra of helical polymers, has been applied to α- and β-glycans. A structure with helical parts is proposed for the α-glycans while a nearly planar arrangement is assumed for the β-glycans.     

Optical rotatory dispersion studies of poly-O-acetyl-gamma-hydroxy-L-proline forms I and II

Poly-O-acetyl-hydroxy-L -proline, forms I and II have been studied by optical rotatory dispersion (ORD) and ultraviolet spectrophotometry in solution and in the solid state. Cotton effects of opposite sign, but not mirror images, were observed in the 250 mμ region for the two forms (Form I, peak 278 mμ; crossover, 254 mμ; trough, 244 mμ: Form II, trough 270 mμ; crossover, 248 mμ; peak, 238 mμ). Thus, the Cotton effects for a right-handed and left-handed helix have been shown to be opposite for the proline type helices I and II. The ORD of films of form I was found to have a positive Cotton effect further into the ultraviolet region with peak at 218 mμ. Absorption spectra showed a shift of 8 mμ in the absorption peak in the 200 mμ region for the two forms (form. I, 211 mμ; form II, 203 mμ). A shoulder was demonstrated in the film absorption spectra in the 250 mμ region where the Cotton effects are found. The mixing of the n, π* and π, π* states of the amide chromophore and n, π* state of the ester chromophore was suggested as being responsible for the Cotton effects in the 250 mμ region.     

CD of ethidium bromide complexes with normal and electrophoretically anomalous DNA restriction fragments

The equilibrium binding of ethidium bromide (EB) to two small 147 base-pair (bp) DNA restriction fragments, which exhibit different mobilities in polyacrylamide gels, was investigated by CD. Two larger DNA restriction fragments and calf thymus DNA were also studied for comparison. Difference spectra were calculated by subtracting the spectrum of the pure DNA from the spectra of its DNA–EB complexes. The D/P ratios ranged from 0.03 to 1.0. The difference CD spectra of all fragments are characterized by bands with maxima near 310, 275, and 207 nm, and minima near 290, 253, 225, and 190 nm. The band near 310 nm, which has a shoulder at about 335 nm, has zero intensity at D/P ≤ 0.05, and rises to a plateau value, different for each fragment, at D/P ? 0.3 for large fragments (≥ 1400 bp), and D/P ～ 0.7 for the two small 147 bp fragments. The minimum near 290 nm is markedly blue shifted with increasing D/P, the wavelength of the extremum corresponding approximately to the wavelength of the uv absorption maximum of the DNA–EB complex. The negative amplitude of this band at D/P = 1.0 depends on the molecular weight of the DNA. The difference CD maximum near 275 nm is positive at low D/P ratios, increases and goes through a maximum at D/P = 0.06–0.1, and then becomes increasingly negative with increasing D/P. The amplitude of the negative ellipticity per added dye is constant at high D/P ratios, suggesting that the transition can be attributed to outside-bound EB molecules. The ellipticities at 310, 290, and 253 nm increase in absolute magnitude with increasing D/P at approximately the same rate, suggesting that all three bands are associated with the same optical and/or conformational transition. For the two small 147 bp fragments the fractional increases in amplitude of these bands parallel the fractional increase in length of the DNA upon binding EB, determined by electric birefringence measurements. The titration of the restriction fragments with EB was also followed by optical absorption. Two end points are observed, the first at a D/P ratio of ～ 0.1, reflecting the transition between intercalated and outside-bound dye molecules, and the second at D/P ? 1.0, the equivalence point of the titration.     

Circular dichroic spectrum of poly-L-tyrosine in aqueous solution

The UV and CD spectra of poly-L-tyrosine were investigated at pH 10.6 and pH 11.2. At pH 10.6 (μ=0.1), the CD spectrum exhibits a medium positive band at 230mμ, an extremely small negative band at 217mμ, and a large positive band at 200mμ. At pH 11.2 (μ=0.1), a new positive CD band appears at 277mμ while the bands at 230mμ and 217mμ are shifted to longer wavelengths by 15 and 10mμ respectively. These results, together with UV spectral data and a specific rotation- pH profile, suggest that at pH 10.6, poly-L-tyrosine exists in the helical conformation with only a small fraction of its side chains ionized; at pH 11.2, the polypeptide retains its helical structure but with a considerable increase in ionization.     

Conformational aspects of polypeptides. XXIX. Conformational assignments for some aromatic polypeptides by far-UV Cotton effects. New results

Recent improvements in apparatus permit the examination of circular dichroism (CD) and optical rotatory dispersion (ORD) spectra to 185 mμ. In addition, new solvents which are transparent to 185 mμ have become available for synthetic polypeptides. The spectral region 185–250 mμ is extremely important for the amide (peptide) chromophore, because of the presence at these wavelengths of the n–π* and π–π* bands,¹ and of another transition, the assignment of which remains unsettled.²     

Dielectric behavior of DNA--dye complex. II

The dielectric relaxation of native DNA and the effect of aminoacridine dyes, such as acridine orange (AO), proflavine (PF), and ethidium bromide (EB) have been investigated at different molar DNA phosphate (P) to dye (D) ratios in the frequency range 100 Hz–100 kHz. The static dielectric constant was observed to decrease with increasing binding of aminoacridines. This was interpreted as arising from the neutralization of the surface changes of the DNA molecules as a result of dye binding. At any P/D ratio the extent of charge neutralization was greatest for AO and least for the EB–DNA complex. The relaxation time (τ) for dye-bound DNA was greater compared to that for native DNA. This increase in τ was ascribed to the increase in the length of the dye-bound DNA. The maximum value of τ occurred at P/D = 20, 10, and 2 for AO-, PF-, and EB-treated DNA, respectively. The variation of τ at various levels of binding gave a qualitative idea about the conformational changes of DNA due to its binding with the dyes.     

Carbamylation of human HbF on carboxy methyl cellulose column in 8 M urea pH 6.7

Myeloperoxidase-H₂O₂-indole acetate system at pH 7.4 emitted light in visible region. Luminescent spectrum showed a weak peak at or near 480 nm and prominent peaks at or near 550, 580, and 620 nm with deep troughs near 500 and 600 nm. In some cases, no definite peak emissions near 550 and 580 nm, but a prominent broad emission between 550 and 580 nm, is observed. Such spectral patterns in the region of 510 to 620 nm were quite similar to those report for the luminescence of photo-products formed from the indole analogs (tryptophan and indole) in 50% alcohol irradiated by U.V. (365 nm) at 77°K, assuming red shift (20–25 nm) by solvent effect. Possible formation of indole acetate cation radical (a precursor of excited indole acetate) was discussed.     

Far ultraviolet optical rotatory properties of poly-L-tryptophan. I

A block copolymer [γ-Et-DL -Glu]_m [L -Trp]_n was prepared using N-carboxy anhydrides (NCA) of L -tryptohan and γ-ethyl DL -glutamate. The block copolymer, dissolved in trifluoroethanol (TFE)–dichloroacetic acid (DCA) mixtures, exhibited a sharp change in the specific rotation at 546 mμ when the solvent composition reached 70–75% DCA content. Optical rotatory dispersion (ORD) and circular dichroism (CD) measurement were carried out in TFE solution in the spectral range 180–350 mμ. Indole side-chain chromophores were found to be optically active in the polymer. On the other hand, these groups exhibit very small optical activity in the model compound C₆H₃? CH₂? O? CO? (L -Trp)₂? O? CH₃. Indole groups therefore appear to be in a dissymmetric environment only in the polymer. From these data it was concluded that poly-L -Trp is in some type of helical conformation in TFE. Strong overlapping of CD bands from side-chain chromophores and peptides chromophores in the wavelength range 185–240 mμ does not allow definite conclusions to be drawn about the type of helical conformation which exists in poly-L -Trp in TFE solution.     

The interaction of native calf thymus DNA with Fe(III)-dipyrido[3,2-a:2',3'-c]phenazine

The mono and bis dipyrido[3,2-a:2′,3′-c]phenazine (dppz) adducts of iron(III) chloride, i.e. [Fe(dppz)]Cl₃ and [Fe(dppz)₂]Cl₃, have been synthesized and characterized. The interaction of the Fe^IIIdppz hydrolyzed aquo complex with native calf thymus DNA has been monitored as a function of the metal complex-DNA molar ratio, by variable temperature UV absorption spectrophotometry, circular dichroism (CD) and fluorescence spectroscopy. The results obtained in solution at various ionic strength values give support for a tight intercalative binding of the Fe^IIIdppz cation with DNA. In particular, the appearance of induced CD bands, caused by the addition of Fe^IIIdppz, indicate the existence of a rigid metal complex-DNA-binding leading to dominating chiral organization of Fe^IIIdppz species within the DNA double helix. The trend of selected CD bands with the molar concentration of Fe^IIIdppz emphasizes that the presence of high amounts of metal complex induces also the formation of DNA-Fe^IIIdppz supramolecular aggregates in solution. The analysis of fluorescence measurements allowed us to calculate a value of the intercalative binding constant comparable to that obtained by UV spectrophotometric titration. Finally, the temperature dependence of the absorbance at 258 nm shows that the metal complex strongly increases the DNA melting temperature already at metal complex-DNA molar ratio equal to 0.25 suggesting that metal complex intercalation effectively hinders DNA denaturation. Overall, the results of the present study point out that the Fe^IIIdppz aquo complex has DNA-binding properties analogous to those previously reported for the tris-chelate Fe^II(phen)₂dppz complex (phen = 1,10-phenantroline).     

Conformation of amylose in aqueous solution: Optical rotatory dispersion and circular dichroism of amylose–iodine complexes and dependence on chain length of retrogradation of amylose

The dependence on chain length of two characteristic properties of amylose, i.e., retrogradation and complex formation with iodine, have been studied by using enzymatically synthesized, homodisperse amyloses. The association rates of amyloses in water containing 5% dimethyl sulfoxide have a sharp maximum at a degree of polymerization P?_n of 80; shorter and longer molecules are much more soluble. The iodine complexes of amylose exhibit a strong Cotton effect in the range of the long-wave absorption maximum (position depending on chain length) and two weaker Cotton effects at 480 and 350 nm. The long-wave Cotton effect is most intense at about P?_n 50 and decreases rapidly for shorter and longer chains. This behavior is unexpected and is not in accordance with the further increase of λ_max and λ_max. The experiments can best be interpreted by assuming well ordered, stiff chains in the low molecular weight range (P?_n 50–80). For longer chains, the findings are discussed in the light of current concepts of amylose conformation in aqueous solution, namely the model of the broken helical chain (alternating stiff helical segments and unordered regions) and the model of a flexible coil without a significant helical content. However, according to the results given in this paper, a wormlike helical chain seems to be the most adequate model for amylose conformation in neutral solution.     

Conformational studies of proteins with aromatic side-chain effects

We present here a brief analysis of ultraviolet isotropic absorption and related circular dichroism of the n–π* and π–π* transitions for the peptide (amide) chromophore in the 185–240 mμ region. Investigations by ultraviolet absorption and circular dichroism techniques on natural amino acids with aromatic chromophores in their side chains are also reported. By taking into account both the peptide and aromatic transitions we discuss the conformational studies of proteins with aromatic side-chain effects. Our attention is largely focused on the optical rotatory dispersion and circular dichroism spectra of these proteins in the near ultraviolet region, where characteristic aromatic side-chain bands occur. The 185–240 mμ region is also discussed when evidence exists of overlapping Cotton effects of aromatic and peptide groups.     

Induced circular dichroism of acridine orange bound to double-stranded RNA and transfer RNA

The induced circular dichroism (CD) in the visible region of acridine orange bound to the double-stranded RNA from cytoplasmic polyhedrosis virus and to yeast tRNA has been measured as a function of RNA phosphate-to-dye ratio (P/D), under the conditions of 0.01 M Na⁺ at pH 7.0. The shape of the CD spectrum of acridine orange bound to the double-stranded RNA was quite different from the spectrum of the dye bound to DNA. The CD spectral features of acridine orange bound to the double-stranded regions in tRNA closely resembled those of the double-stranded RNA-dye complex, suggesting that the dyes bind similarly to the two RNA's. It was further found that the CD spectrum of the tRNA-dye complex at sufficiently high P/D ratios, which is assignable to monomeric, intercalated dye to the base-paired parts in tRNA, is also distinct from the corresponding spectrum of the DNA-dye complex.     

Ultraviolet dichroic ratio of DNA from T2 and T5 bacteriophages

The 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.     

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.     

Effect of chemical structures of acridine and triphenylmethane dyes on the induced optical activity of DNA-dye complexes

Fifteen symmetrically substituted acridine dyes, all of which are interrelated by their chemical structures, each belonging to a C_2v symmetry, and three triphenylmethane dyes with amino or dimethylamino substituents are utilized to study necessary conditions for the appearance of extrinsic Cotton effects upon their binding to native and heat-denatured deoxyribonucleic acid (DNA). Three different kinds of the DNA–dye complexes, i.e., (1) dye added to native DNA, (2) heat-denatured DNA–dye complex, and (3) dye added to preheated DNA, were examined for each dye at a fixed P/D value of about 4. Optical activity was always observed for the compelexes of type (1) in each absorption band of the dyes in the visible and near-ultraviolet region. Two exceptions are 9-acetamido- and 9-hydroxyacridine, both being nonionic in aqueous solution at a pH range of 6. Acridinium chloride was unable to exhibit any definite extrinsic Cotton effect for complexes (2) and (3). Thus, the monocationic form of a dye due to the protonation or quaternization of the ring nitrogen in acridines or exonuclear amino nitrogen in triphenylmethane dyes is concluded to be an essential factor for extrinsic Cotton effect to appear. Changes in the absorption spectra upon complex formation are also related to the structure of dyes. Hypochromism and bathochromism are associated with the induced optical activity in all cases in the presence of native and denatured DNA.     

Optical rotatory dispersion of mononucleosides and 5′-mononucleotides

Optical rotatory dispersions between 200 and 600 mμ are presented for mononucleosides and 5′-mononucleotides in neutral, acid, and in some cases alkaline solutions. All display a single Cotton effect in the ultraviolet region (above 220–240 mμ); the purine ones are negative in sign and the pyrimidine ones positive, with the crossovers (zero rotations) in most cases close to the wavelengths of their respective absorption maxima. The visible rotatory dispersions obey the one-term Drude equation, except for TMP and UMP, which show anomalous dispersions. The rotational strengths of the dichroic bands were estimated from the Cotton effect profiles; cytosine mononucleotides and mononucleotides show the strongest rotational strengths among the compounds studied.