Interaction of nucleic acids with electrically charged surfaces. VI. A comparative study on the electrochemical behaviour of native and denatured DNAs at graphite electrodes.

Adsorption and electrochemical oxidation of deoxyribonucleic acid (DNA) at a pyrolytic graphite electrode (PGE) and a paraffin wax-impregnated spectroscopic graphite electrode (WISGE) were studied using differential pulse voltammetry. DNA is adsorbed at the surface of the graphite electrodes in a broad range of potentials including the potentials of electrochemical oxidation of DNA. Both native and denatured DNAs yield two single, well-defined and separated peaks, G and A, on the differential pulse voltammograms at the PGE and WISGE. The more negative peak, G, corresponds to electrochemical oxidation of adenine residues. Peaks G and A of native DNA occur at the same potentials as peaks G and A of denatured DNA. However, electrochemical oxidation of adenine and guanine residues at graphite electrodes is markedly suppressed in native DNA. The heights of the peaks G and A represent a sensitive indicator of the helix-coil transition of DNA. An analysis of the product of interaction of a sample of native DNA with a large pyrolytic graphite electrode in the presence of formaldehyde at approximately neutral pH did not prove changes in the secondary structure of native DNA due to its interaction with the graphite electrode. It is suggested that the decreased differential pulse-voltammetric activity of native DNA is connected with its decreased flexibility.     

Interaction of nucleic acids with electrically charged surfaces: II. Conformational changes in double-helical polynucleotides

The influence of adsorption of double-stranded (ds) DNA, ds RNA and homopolymeric pairs at a mercury electrode on conformation of these polynucleotides was studied. Changes in the polarographic reducibility of polynucleotides, which were followed by means of normal pulse polarography and linear sweep peak voltammetry at the dropping mercury electrode were exploited to indicate conformational changes. It was found that, as a consequence of adsorption of ds polynucleotides on the negatively charged electrode conformational changes similar to denaturation take place in a narrow potential region around ?1.2 V (the region U). After sufficiently long time of the contact with the electrode (under our conditions about 10 s) these changes reach limiting values, which can approach total denaturation. Upon adsorption of ds polynucleotides on the electrode charged to more positive potentials than the region U either (1) no conformational changes occur or (2) only a small part of the polynucleotide (probably labile regions of the ds molecule) is very quickly denatured - the remainder of the molecule preserves its ds structure. Conformational changes of adsorbed ds polynucleotides are influenced by factors which change the stability of ds polynucleotides in solution. It is supposed that denaturation of ds polynucleotides in the region U might result from the strains connected with the repulsion of certain segments of the molecule anchored on the electrode from the negatively charged surface.     

Interaction of nucleic acids. VI. Interaction of purine with nucleic acids

The interaction of purine with DNA, tRNA, poly A, poly C, and poly A. poly U complex was investigated. In the presence of purine, the nucleic acids in coil form (such as denatured DNA, poly A and poly C in neutral solutions, or tRNA) have lower optical rotations. In addition, hydrodynamic studies indicate that in purine solutions the denatured DNA has a higher viscosity and a decreased sedimentation coefficient. These findings indicate that through interaction with purine, the bases along the poly-nucleotide chain are unstacked and are separated farther from each other, resulting in increased assymmetry (and possibly volume) of the whole polymer. Thus, the de-naturation effect of purine reported previously can be explained by this preferential interaction of purine with the bases of nucleic acids in coil form through a hydrophobic-costacking mechanism. Results from studies on optical rotation and helix-coil transition show that the interaction of purine is greater with poly A than with poly C. The influence of temperature, Mg⁺⁺ concentration, ionic strength, and purine concentration on the effect of purine on nucleic acid conformation has also been investigated. In all these situations the unraveling of nucleic acid conformation occurs at much lower temperatures (20–40°C lower) in the presence of purine (0.2–0.6M).     

Adsorption of peptide nucleic acid and DNA decamers at electrically charged surfaces.

Adsorption behavior of peptide nucleic acid (PNA) and DNA decamers (GTAGATCACT and the complementary sequence) on a mercury surface was studied by means of AC impedance measurements at a hanging mercury drop electrode. The nucleic acid was first attached to the electrode by adsorption from a 5-microliter drop of PNA (or DNA) solution, and the electrode with the adsorbed nucleic acid layer was then washed and immersed in the blank background electrolyte where the differential capacity C of the electrode double layer was measured as a function of the applied potential E. It was found that the adsorption behavior of the PNA with an electrically neutral backbone differs greatly from that of the DNA (with a negatively charged backbone), whereas the DNA-PNA hybrid shows intermediate behavior. At higher surface coverage PNA molecules associate at the surface, and the minimum value of C is shifted to negative potentials because of intermolecular interactions of PNA at the surface. Prolonged exposure of PNA to highly negative potentials does not result in PNA desorption, whereas almost all of the DNA is removed from the surface at these potentials. Adsorption of PNA decreases with increasing NaCl concentration in the range from 0 to 50 mM NaCl, in contrast to DNA, the adsorption of which increases under the same conditions.     

Specific recognition of single-stranded nucleic acids. Interaction of tryptophan-containing peptides with native, denatured, and ultraviolet-irradiated DNA

The binding of the tripeptide Lys-Trp-Lys to native, denatured, and ultraviolet-irradiated DNAs has been investigated by fluorescence spectroscopy. Two types of complexes are formed which both involve electrostatic interactions. Only one of them involves a stacking of the tryptophyl ring with nucleic acid bases. Quantitative analysis of fluorescence data shows that this stacking interaction is strongly favored in denatured as compared to native DNA. In ultraviolet-irradiated DNA, the peptide Lys-Trp-Lys binds selectively to unpaired regions around thymine dimers. Due to the stacking interaction of the aromatic amino acid with nucleic acid bases, this simple tripeptide is therefore able to discriminate between single-stranded and double-stranded regions in a nucleic acid.     

Interaction of nucleic acids with electrically charged surfaces. VII. The effect of ionic strength of neutral medium on the conformation of dna adsorbed on the mercury electrode

Triangular-wave direct current (d.c.) voltammetry at a hanging mercury drop electrode and phase-selective alternating current (a.c.) polarography at a dropping mercury electrode were used for the investigation of adsorption of double-helical (ds) DNA at mercury electrode surfaces from neutral solutions of 0.05-0.4 M HCOONH4. It was found for the potential region T (from -0.1 V up to ca. -1.0 V) that the height of voltammetric peaks of ds DNA is markedly influenced by the initial potential only at relatively low ionic strength (mu) (from 0.05 up to ca. 0.3). Also a decrease of differential capacity (measured by means of a.c. polarography) in the region T depended markedly on the electrode potential only at relatively low ionic strength. The following conclusions were made concerning the interaction of ds DNA with a mercury electrode charged to potentials of the region T in neutral medium of relatively low ionic strength mu < 0.3). (i) When ds DNA is adsorbed, a significantly higher number of DNA segments is anchored in the positively charged electrode surface than in the surface bearing a negative charge, (ii) In the region T, especially adsorbed labile regions of ds DNA are opened in the electrode surface, which are present in ds DNA already in the bulk of the solution, (iii) In the narrow region of potentials in the Vicinity of the zero charge potential a higher number of ds DNA segments can be opened, probably as a consequence of the strain which could act on the ds DNA molecule in the course of the segmental adsorption/desorption process.     

Electrochemical behaviour of proteins at graphite electrodes. II. Electrooxidation of amino acids

Electrochemical oxidation of L,alpha-amino acids at a paraffin-wax impregnated spectroscopic graphite electrode (WISGE) was studied by means of linear sweep, cyclic, phase-sensitive alternating current and differential pulse voltammetric techniques. It was found that out of the amino acids usually occurring in proteins only tyrosine, tryptophan, histidine, cystine, cysteine and methionine were oxidized at the WISGE. At relatively low concentrations of amino acids (up to ca. 2 x 10(-4) M) the electrode process in which the amino acids are oxidized at the WISGE has the characteristics of an irreversible reaction controlled by diffusion. Coulometric measurements showed that oxidation of tyrosine and tryptophan at the WISGE, i.e. of amino acids which are responsible for the oxidizability of proteins at graphite electrodes, is a two-electron process. At higher concentrations of tyrosine-and tryptophan (above ca. 2 x 10(-4) M) adsorption of the oxidation product of these amino adds was demonstrated.     

Interaction of aminoacridines with nucleic acids. A pulse-radiolysis study.

Pulse radiolysis has been used to study the interaction of aminoacridines with nucleic acids. The data confirm that there are two modes of binding. These are: a weak interaction which has a maximum binding ratio of one site per dye; and a strong binding process effected by both electrostatic and Van der Waals interactions. The limit of this latter, strong binding mode is approximately six sites per dye. The radiation-induced transient absorption spectrum of benzoflavine is characterized by a pronounced bleaching at 440 nm, which is quenched by the addition of nucleic acids. Mechanisms have been proposed for the reactions of both eaq-and .OH with benzoflavine which account both for the observed bleaching of benzoflavine solutions and for the protective effect of nucleic acids. It is proposed that eaq-reacts with benzoflavine to form a stable benzoflavine semiquinone radical and that .OH reacts with subsequent formation of a very stable benzoflavine hydroxycyclohexadienyl radical.     

In-situ FTIR study on adsorption and oxidation of native and thermally denatured calf thymus DNA at glassy carbon electrodes

In-situ Fourier transform infra-red (FTIR) spectra of native and thermally denatured calf thymus DNA (CT DNA) adsorbed and/or oxidized at a glassy carbon (GC) electrode surface are reported. The adsorption of native DNA occurs throughout the potential range (- 0.2 approximately 1.3 V) studied, and the adsorbing state of DNA at electrode surface is changed from through the C=O band of bases and pyrimidine rings to through the C=O of cytosine and imidazole rings while the potential shifts negatively from 1.3 V to -0.2 V. An in-situ FTIR spectrum of native CT DNA adsorbed at GC electrode surface is similar to that of the dissolved DNA, indicating that the structure of CT DNA is not distorted while it is adsorbed at the GC electrode surface. In the potential range of -0.2 approximately1.30 V, the temperature-denatured CT DNA is adsorbed at the electrode surface first, then undergoes electrochemical oxidation reaction and following that, diffuses away from the electrode surface.     

Theoretical studies on protein-nucleic acid interactions. I. Interaction of positively charged amino acids with nucleic acid fragments

Coulombic interactions between the side chains of charged amino acids (Arg⁺, Lys⁺, and His⁺) and negatively charged phosphate groups of nucleic acid fragments have been studied theoretically. Diribose monophosphate and dideoxyribose monophosphate are chosen as model systems for single-stranded RNA and DNA, respectively. The interaction energies have been calculated by second-order perturbation theory using simplified formulas for individual terms. The interaction energy in this formalism is a sum of electrostatic, polarization, dispersion, and repulsive energies. Our results show that about 90% of the total interaction energy is contributed by the electrostatic term alone. Contribution from the repulsive term exceeds that from the dispersion term. Calculated interaction energies suggest that Lys⁺ and His⁺ form more stable complexes with RNA than with single-stranded DNA. On the other hand, Arg⁺ has a higher affinity for DNA than for RNA. The affinity of nucleic acids for the three amino acids is in the order Lys⁺ > His⁺ > Arg⁺. Further, the basic amino acid residues form more stable complexes with A-DNA than with B-DNA. The role of the Coulombic interactions in the specific recognition of nucleic acids by proteins is discussed.     

Interaction of native and apo-carbonic anhydrase with hydrophobic adsorbents: A comparative structure-function study

Our previous studies indicated that native carbonic anhydrase does not interact with hydrophobic adsorbents and that it acquires this ability upon denaturation. In the present study, an apo form of the enzyme was prepared by removal of zinc and a comparative study was performed on some characteristic features of the apo and native forms by far- and near-UV circular dichroism (CD), intrinsic fluorescent spectroscopy, 1-anilino naphthalene-8-sulfonate (ANS) binding, fluorescence quenching by acrylamide, and Tm measurement. Results indicate that protein flexibility is enhanced and the hydrophobic sites become more exposed upon conversion to the apo form. Accordingly, the apo structure showed a greater affinity for interaction with hydrophobic adsorbents as compared with the native structure. As observed for the native enzyme, heat denaturation of the apo form promoted interaction with alkyl residues present on the adsorbents and, by cooling followed by addition of zinc, catalytically-active immobilized preparations were obtained.     

烟草愈伤组织继代培养与分化期间核酸代谢的比较研究

Changes in the contents of DNA and RNA, RNA species, the synthesis rates of DNA and RNA, and the activity of DNase and RNase were investigated in the callus of tobacco (Nicotiana tabacum L. cv. Willow Leaf) during subculture and differentiation. The contents of DNA and RNA were higher in differentiating callus than that in subcultured callus. After day 12, the contents of DNA and RNA in differentiating callus rose continuously while the contents of DNA and RNA in subcultured callus remained constant. Changes in RNA species and its relationship to total RNA level were also analyzed. At the stage of shoot primordium formation in differentiating callus, the activity of RNase increased markedly and the synthesis rate of RNA increased continuously; while the RNase activity and the synthesis rate of RNA in subcultured callus were much lower during the same period. During the period of shoot growth, the synthesis rate of DNA in differentiating callus was elevated compared to that in subcultured callus. The results above suggested that the metabolism of nucleic acids in differentiating callus was more active than that in subcultured callus.     

Isoelectric focusing of proteins in the native and denatured states. Anomalous behaviour of plasma albumin

1. An analytical technique of isoelectric focusing in thin layers of polyacrylamide gel has been used to determine the isoelectric point, pI, of several proteins in the presence and in the absence of concentrated urea. 2. The presence of urea did not greatly affect pI except for bovine plasma albumin, where an increase of approx. 1pH unit was found. 3. Evidence is presented that this change in the pI of bovine plasma albumin is due to the normalization of certain ionizable groups on unfolding of the protein in urea. 4. Evidence is also presented that prolonged exposure of bovine plasma albumin to urea results in intramolecular disulphide interchange and that, on removal of urea, the new patterns of disulphide bonding stabilize abnormal conformations with pI values intermediate between those of the native and denatured states. 5. The studies demonstrate heterogeneity in bovine plasma albumin based on primary-sequence differences. 6. Isoelectric focusing of proteins in urea appears to be useful in the study of various aspects of protein structure.     

Interaction of metal ions with nucleic acids. Interaction of copper(II) with guanosine and its derivatives.

Interaction of copper(II) with guanosine, 2'-deoxyguanosine, 1-methylguanosine, 7-methylguanosine and GMP was studied withe use of spectroscopic and magneto-chemical methods. The main site of copper(II) binding in guanosine is nitrogen N-7; participation of N-1 is not excluded. The involvement of carbonyl oxygen in copper binding or copper chelation to N-7 and 0-6 is rather unlikely. A crystalline complex of copper(II) with GMP [Cu(C10H12O8N5P) .(H2O)3] was obtained, and it was demonstrated that copper(II) is bound with N-7 and the phosphate group.     

Interaction of metal ions with nucleic acids. Interaction of copper(II) with adenosine and its derivatives.

The interaction of copper(II) with adenosine, 2'-deoxyadenosine, 1-methyladenosine, 7-deazaadenosine and AMP was studied by spectroscopic and magnetochemical methods. In non-aqueous medium, copper(II) interacts with adenosine and AMP at N-7 and N-1, and with 1-methyladenosine at N-7 and N-3. The copper ion is not bound to the NH2 group. In aqueous solution, copper(II) interacts both with N-7 and N-1 of adenosine, and in AMP additionally with the phosphate group. The interaction of copper(II) with the heterocyclic part, but not withthe phosphate group, is dependent on the extent of protonation of the molecular. A crystalline AMP-copper(II) complex [Cu(C10H12N5O7P).(H2O)2] was obtained; the phosphate group and probably N-7 are involved in the complex formation.