Fluorometric determination of adenine and its derivatives by reaction with glyoxal hydrate trimer

A fluorometric determination of adenine has been devised. It was found that adenine gives strong fluorescence when reacting with glyoxal hydrate trimer in an acidic medium. The maximum wavelengths of excitation and emission spectra were 328 and 382 mμ, respectively. This reaction was successfully applied to determination of 0.2–1.0 or 2.0–10.0 mμmoles of adenine and its derivatives, in which the hydrogen atom at position 9 is substituted with ribose or substituted ribose. Other nucleic acid bases, guanine, uracil, thymine, and cytosine, did not interfere with this fluorometric determination of adenine.     

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.     

Circular dichroism of the nucleic acid monomers.

Circular dichroism spectra of the nucleic acid monomers have been measured in aqueous solution and extended into the vacuum ultraviolet region to about 166 nm. Measurements were made on ribo and deoxyribo derivatives of adenine, guanine, hypoxanthine, cytosine, thymine, and uracil derivatives both with and without the 5′-phosphate (with the exception of ribosyl thymine 5′-phosphate). Absorption spectra of the deoxyribonucleotides measured to about 175 nm are also presented. The results demonstrate that both the circular dichroism and absorption spectra observed below 200 nm are no more complicated than the spectra normally recorded above 200 nm. In most cases, the circular dichroism spectra of the various derivatives of a given base are similar, indicating that the conformations are similar. On the other hand, the differences among the circular dichroism spectra of the various derivatives of a given base are sufficient to identify a particular derivative. The average circular dichroism for the deoxyribonucleotides is compared with the circular dichroism of native E. coli DNA. The comparison reveals that the circular dichroism of DNA below 200 nm is due principally to the interaction between the bases rather than the intrinsic circular dichroism of the monomers. The monomer transitions are discussed in relationship to the absorption and circular dichroism spectra presented.     

Optical activity of simple cyclic amides in solution

The cyclic dipeptide, L -alanylglycyl anhydride, has been studied by optical rotatory dispersion; both L -alanylglycyl anhydride and the lactam, L -3-aminopyrrolidin-2-one, have been studied by circular dichroism. In hydroxylic solvents the circular dichroism spectra of 3-aminopyrrolidin-2-one can be attributed to an n–π* transition near 220 mμ and a π–π* transition near 190 mμ. In these solvents the optical activity of L -alanylglycyl anhydride can be explained as being due to contributions of n–π* transitions and a split π–π* transition. In acetonitrile, however, the circular dichroism spectrum of 3-aminopyrrolidin-2-one shows an additional apparent minimum near 200 mμ. The CD spectrum of the dipeptide is also quite distinctive in this solvent. The possible nature of the band at 200 mμ and the implications of these findings are discussed.     

A theoretical and experimental investigation of the electronic spectra and tautomerization of nucleobases

The electronic structures of all possible tautomers of uracil, thymine, cytosine, adenine and guanine have been carefully examined within the MNDO-MO frame-work. Equilibrium geometries are determined and the relative stabilities are discussed. Allowance for solvent effect on the stabilities is made by assuming a tetrahedral solvent cage with the DNA base occupying its centre. The electronic absorption spectra of the studied DNA bases, in solvents of different polarities are recorded and discussed. Assignments of the observed bands are facilitated using MNDO-CI computations. It is suggested that in solution the DNA bases are in some statistical mixtures of the most stable tautomers, and the Watson-Crick (WC) structure cannot account for the observed spectra alone.     

Steric structure of L-proline oligopeptides. II. Far-ultraviolet absorption spectra and optical rotations of L-proline oligopeptides

The results of the measurement of the far-ultraviolet absorption spectra of L -proline oligomers in water and acetonitrile are summarized as follows. The monomer has an absorption maximum at 182.5 mμ in acetonitrile. The absorption maximum of the dimer is found at 185 mμ and a shoulder appears around 200 mμ, that is, splitting of the absorption spectrum is observed in the dimer. As the degree of polymerization increases, the position of the shoulder shifts toward the wavelength of the absorption maximum of poly-L -proline II, with an accompanying increase in intensity. We may describe the absorption peak around 203 mμ of poly-L -proline II as identical with the shoulder with an increased intensity. By measurements of optical rotatory dispersion and circular dichroic spectra, it was also confirmed that the appearance of the helical conformation commences at the tetramer. When the number of residues is five or greater, the conformation of the helical structure of poly-L -proline II seems to be completed.     

Analysis of the spectra and redox properties of pure cytochromes aa3.

The findings in the current studies with pure cytochrome aa3 confirm the findings in an accompanying paper pertaining to cytochrome aa3 in mitochondria (Reddy et al., 1985). In both cases, three Nernstian titrations are seen with Em values near 200, 260, 340 mV with n values of 2, 2, and 1. Similarly, the alpha absorption features of the difference spectra in both cases were centered near 602, 605, and 607 mn. The component with Em approximately 200 mV was identified as heme a3 on the basis of experiments conducted in an atmosphere of carbon monoxide, and in both cases, the carbon monoxide-liganded species did not display an elevated Em. In the current studies, unique Soret absorbance features are added to the difference spectra for the three Nernstian transitions. Specifically, absorption peaks at 429, 446, and 448 nm go with the alpha peaks seen respectively at 602, 605, and 607 nm. Evidence was presented to support the hypothesis that the redox state of heme alpha may control the redox potential of heme a3.     

Mass spectral studies of probe pyrolysis products of intact oligoribonucleotides.

Pyrolysis of underivatized homogeneous oligoribonucleotides in the source of a mass spectrometer leads to production of simple mass spectra which resemble the spectra of the bases themselves. It is proposed that pyrolysis occurs by hydrogen transfer to the base moiety followed by elimination of the neutral base. Ionization by electron impact gives rise to the observed spectra. Mixed oligoribonucleotides pyrolyze readily to give spectra of adenine and uracil, but evidence for the presence of guanine and cytosine in mixed polymers and native RNA's is difficult to obtain presumably because of their low vapor pressure. The method may be useful for detection of modified bases in tRNA and for studies of temperature effects on RNA pyrolysis.     

Characterizing Substrate Properties of Purine-Related Compounds with Purine Metabolism Enzymes for Enzymatic Peak-Shift HPLC Method

We have extended peak-shift method for measuring purine bases to make it suitable for other purine-related compounds. We optimized the reactions of the purine metabolism enzymes 5′-nucleotidase (EC 3.1.3.5), purine nucleoside phosphorylase (PNP) (EC 2.4.2.1), xanthine oxidase (XO) (EC 1.17.3.2), urate hydroxylase (EC 1.7.3.3), adenosine deaminase (ADA) (EC 3.5.4.4), and guanine deaminase (EC 3.5.4.3) by determining their substrate specificity and reaction kinetics. These enzymes eliminate the five purine base peaks (adenine, guanine, hypoxanthine, xanthine, and uric acid) and four nucleosides (adenosine, guanosine, inosine, and xanthosine). The bases and nucleosides can be identified and accurately quantified by comparing the chromatograms before and after treatment with the enzymes. Elimination of the individual purine compound peaks was complete in a few minutes. However, when there were multiple substrates, such as for XO, and when the metabolites were purine compounds, such as for PNP and ADA, it took longer to eliminate the peaks. The optimum reaction conditions for the peak-shift assay methods were an assay mixture containing the substrate (10 μL, 0.1 mg/mL), the combined enzyme solution (10 μL each, optimum concentration), and 50 mM sodium phosphate (up to 120 μL, pH 7.4). The mixture was incubated for 60 minutes at 37°C. This method should be suitable for determining the purine content of a variety of samples, without interference from impurities.     

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.     

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.     

Syntheses and characterization of polymers containing nucleic acid bases

Two different types of polymers containing nucleic acid bases in the backbone or in the pendant groups were prepared. The polymers of the first type were polyureas obtained by the polyaddition reaction of uracil and adenine with hexamethylene diisocyanate (HMD1). The second type that is cationic polyurethanes containing nucleic acid bases in pendant groups, were obtained by the Menschutkin reaction of halogenated derivatives of uracil and adenine with a linear polyurethane containing tertiary nitrogen atoms which was based on HMD1 and N-methyldiethanolamine. Base base interactions were studied for the polymers by ultraviolet (u.r.), optical rotary dispersion (o.r.d.), and nuclear magnetic resonance (n.m.r.) spectra. A relatively high value of hypochromicity, ≈17% was observed for the mixture of the ionic polyurethane with uracil pendant and herring-sperm DNA. Complementary hydrogen bonding interactions were detected for the mixture of the ionic polyurethane with adenine pendant and with uracil pendant. The non-thrombogenic character of the polymers was examined according to the modified Lee White method. The ionic polyurethanes with adenine and uracil pendant exhibited a fairly good anti-clotting properly.     

Cross section calculations for electron scattering from DNA and RNA bases

Differential and integral cross sections for elastic electron collisions with uracil, cytosine, guanine, adenine and thymine have been calculated using the independent atom method with a static-polarization model potential for incident energies ranging from 50 to 4000 eV. Total cross sections for single electron-impact ionization of selected DNA and RNA bases have also been calculated with the binary-encounter-Bethe model from the ionization threshold up to 5000 eV. Cross sections within the investigated energy range, can be related to the molecular symmetry, the number of target electrons and molecular size; elastic and ionization processes are most efficient for guanine and adenine molecules, while the lowest cross sections were obtained for the uracil molecule. The ionization cross sections for cytosine, thymine, adenine and guanine are compared with those recently obtained with a semi-classical and binary-encounter-Bethe formalisms. No theoretical and experimental data for elastic electron scattering from DNA and RNA bases are available, but comparisons with calculations for molecules of similar size and geometry allows the validity of the theoretical approach to be verified.     

Pressure-tuning infrared and Raman microscopy study of the DNA bases: adenine,guanine, cytosine,and thymine

Diamond-anvil cell, pressure-tuning infrared (IR), and Raman microspectroscopic measurements have been undertaken to examine the effects of high pressures up to about 45?kbar on the vibrational spectra of the four DNA bases, adenine, cytosine, guanine, and thymine. Small structural changes were evident for all the four bases, viz., for adenine and cytosine at 28–31?kbar; for guanine at 16–19?kbar; and for thymine at 25–26?kbar. These changes are most likely associated with alterations in the intermolecular hydrogen-bonding interactions. The pressure dependences of the main peaks observed in the IR spectra of the two phases of guanine lie in the ?0.07–0.66 (low-pressure phase) and 0.06–0.91 (high-pressure phase) cm^?1/kbar ranges. Also, in the Raman spectra of this nucleoside base, the dν/dP values range from ?0.07–0.31 (low-pressure phase) to 0.08–0.50 (high-pressure phase) cm^?1/kbar. Similar ranges of dν/dP values were obtained for the other three nucleoside bases.     

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.     

The structure and application of oligodeoxyribonucleotides containing modified, degenerate bases.

Oligodeoxynucleotides containing modified pyrimidine bases which can stably hydrogen-bond to adenine and guanine and also purine bases which pair with thymine and cytosine residues in duplexes have been synthesised, as have oligomers with both such analogues. Structures have been investigated by melting transitions, n.m.r. spectroscopy and crystallography and then interpreted in terms of tautomeric equilibria. Applications to hybridisation probes and primers will be discussed.     

Influence of reductants upon optical characteristics of the DNA-Cu2+ complex

The addition of reducing agents, i.e., ascorbic acid or sodium borohydride, to a DNA solution containing Cu²⁺ ions causes changes in the DNA absorption spectra which are due to a new absorption band with a maximum at 280 mμ assigned to a DNA base–Cu¹⁺ complex. The stoichiometry of the complex is one Cu¹⁺ ion per four bases of DNA. The DNA–Cu¹⁺ complex has an increased melting temperature and rather different circular dichroism curve as compared with DNA itself. It is inferred that the above effects are caused by proton transfer along the hydrogen bond from guanine to cytosine under complexing of Cu¹⁺ ions with the N₇ atom of the guanine of DNA.     

Optical rotary dispersion of mucopolysaccharides. 3. Ultraviolet circular dichroism and conformational specificity in amide groups

The circular dichroism of glycosaminoglycans and ganglioside in distilled water are described, showing two bands in the region of amide and carboxyl transitions. The first, negative hand is common to all polymers and the amino sugar derivatives. The characteristics of the second band depend upon polymer structure. Those containing 4–1-linked amino sugars show a second, positive, resolved band about 190 mμ, while those with 3–1-linked amino sugars show a second, negative band less resolved from the first usually ≤185mμ, but clearly centered at 188mμ for dermatan sulfate. Ultraviolet optical absorption from 300 to ～183 mμ showed inflection regions around 190 mμ for most compounds.     

Transport of nucleic acid bases against their concentration gradients through quaternized chitosan membrane

A water-insoluble anion exchange membrane was prepared by crosslinking with ethyleneglycol diglycidylether, a membrane made of quaternized chitosan and poly(vinyl alcohol). The transports of nucleic acid bases such as uracil, cytosine, adenine, and guanine were investigated as one side of the membrane in a diaphragm cell was acidic and the other basic. Uracil was transported against its concentration gradient from the basic side to the acidic side regardless of the pH on the basic side. Cytosine, adenine, and guanine were also transported against their concentration gradients, but the direction of their transport depended upon the pH on the basic side. In particular, the transport directions for adenine and guanine were switched during identical transport experiments. Mechanisms for the transport of these nucleic acid bases against their concentration gradients through the quaternized chitosan membrane are discussed.     

Comparative study of permanganate oxidation reactions of nucleotide bases by spectroscopy

The permanganate oxidation of free nucleotide bases was successfully studied in aqueous solution of tetraethylammonium chloride using spectroscopic techniques. The reaction was highly selective toward thymine and uracil, less with cytosine, very little reaction on guanine, and no reaction on adenine.