An investigation of complexes of some model tripeptides containing phenylalanine and tyrosine residues with DNA by Fourier1H NMR spectroscopy

Complexes of native and denatured DNA with model tripeptides containing phenylalanine or tyrosine residues flanked by lysine or arginine residues, respectively have been investigated by pulsed Fourier¹H NMR spectroscopy. The existence of shifts into the strong-field region of the signals of aromatic protons of the model tripeptides in the complexes both with native and with denatured DNA has been shown. Results have been obtained that indicate the possibility of the intercalation of the side chains of aromatic amino acid residues into the DNA double helix.     

Determination,by dynamic surface-tension analysis,of the molar mass of proteins denatured in guanidine thiocyanate

A drop-based dynamic surface-tension detector (DSTD) has been used to study the dynamic surface tension behavior of proteins denatured in guanidine thiocyanate (GndSCN). The dynamic surface tension at the air–liquid interface is obtained by measuring the internal pressure of drops that grow and detach at a specified rate. In the method the sample of interest is injected and subsequently flows to the DSTD-sensing capillary tip. For this work, a novel DSTD calibration procedure utilizing two distinct mobile phases is applied. Here, the mobile phases are aqueous with different constituents, for example GndSCN and phosphate buffer, either added or omitted. The dual-mobile phase calibration procedure gives the analyst the capability of making protein measurements in a GndSCN–phosphate buffer mobile phase, while measuring a calibration standard in another mobile phase, such as water, in which the surface tension of the calibration standard is readily available. Results are presented with drop volumes of either 2 L (i.e. 2-s drops) or 7 L (i.e. 7-s drops) for proteins varying in molar mass from 12,000 to 330,000 g mol^–1. We demonstrate that the DSTD can be used to determine the molar mass of proteins denatured in GndSCN. The method applies a regime where the denatured protein is detected by surface-active properties, and selectivity with regard to molar mass is contained in the dynamic component of the DSTD signal. The dynamic surface pressure signals of the denatured proteins suggest that diffusion plays a large role in the kinetics of the surface activity. The limit of detection for the denatured proteins studied ranged from 3 mg L^–1 to 14 mg L^–1. The DSTD, coupled with the novel dual-mobile phase calibration procedure, can be used to investigate the fundamental properties of proteins. Insight into the behavior at the air–liquid interface for native and denatured proteins is achieved; this is a novel tool for studying protein denaturation, complementary to other common approaches such as spectroscopy and calorimetry. Furthermore, the reported method could be widely applied to the study of effects on the interfacial properties of proteins after a variety of chemical and physical modifications that are possible with the dual-mobile phase calibration procedure.     

Spectrofluorimetric determination of nucleic acids as 8-hydroxyquinoline/ yttrium ternary complexes

The formation of nucleic acids/8-hydroxyquinoline/yttrium(III) ternary complexes and their fluorescent properties have been studied. The nucleic acids studied include native and thermally denatured calf thymus DNA, fish sperm DNA and yeast RNA. In the range of pH 7.6–8.5, controlled by NH₃-NH₄C1 buffer, ternary complexes are formed that fluoresce at different wavelengths with different nucleic acids. Based on the fluorescence reactions, sensitive spectrofluorimetric methods for nucleic acids are proposed. In optimal conditions, the calibration curves were linear in the range 0.5–4.0 gml^–1 for calf thymus DNA, 0.5–2.5 g ml^–1 for fish sperm DNA and 0.5–4.0 g ml^–1 for yeast RNA. The limits of determination (3 ) were 0.030 g ml^–1 for calf thymus DNA, 0.020 g ml^–1 for fish sperm DNA and 0.090 g ml^–1 for yeast RNA. Corresponding to the interferences of coexisting substances, six synthetic samples were constructed and the results of determination were satisfactory.     

Synthesis,DNA-binding and cleavage studies of macrocyclic copper(II) complexes

Two macrocyclic copper(II) complexes, [CuL¹](ClO₄)₂ (L¹ = 2,6,9,13-tetraparacyclophane, a Schiff base) and [CuL²]Cl₂ [L² = 3,10-bis(2-benzyl)-1,3,5,8,10,13-hexaazacyclotetradecane] have been prepared and characterized by elemental analysis, u.v.–vis., i.r. and mass spectra. Absorption, fluorescence, circular dichroic spectra and viscosity experiments have been carried out on the interaction of the two complexes with calf thymus CT DNA. The results suggest that both complexes can bind to CT DNA by intercalation via the aromatic moiety ring in the macrocycle into the base pairs of DNA. [CuL¹](ClO₄)₂ binds to CT DNA more strongly than [CuL²]Cl₂. The position of the aromatic ring in the macrocycle plays an important role in deciding the extent of binding of the complexes to DNA. Significantly, the complexes have been found to be single-strand DNA cleavers in the presence of H₂O₂ or/and 2-mercaptoethanol.     

Yeast deoxyribodipyrimidine photolyase (photoreactivating enzyme): optimum conditions for activity and competitive inhibition

Abstract— Using flash photolytic techniques and direct chemical measurements of the conversion of the substrate (conversion of thymine dimers in DNA to monomeric thymine), we have determined photolyase concentrations in partially purified preparations of soluble proteins from yeast and have determined under continuous intense light the forward rate constant k₁ for binding of the enzyme and its substrate under a variety of conditions. The ionic requirements and the sharp peak of ionic strength dependence are independent of the species of uni-univalent salts used in the assay. At infinite dilution, the k₁ for denatured DNA, and its ionic strength dependence, both appear identical to the values for native DNA. Both unirradiated denatured and unirradiated native DNA inhibit binding, denatured DNA being 10- to 20-fold more effective. These combined factors have been taken into account to devise a sensitive assay for photoreactivable lesions in unlabeled DNA by competition in a flash photoreactivation reaction. The assay is used to measure dark repair in Micrococcus luteus in complete medium. After a dose of 100 J/m² the wild type of this organism removes photoreactivable lesions (pyrimidine dimers) from its DNA with a half-time of 7 min at 35°C.     

Conformational and Database Study on the Intramolecular CH...π Aromatic Bond and Its Possible Influence on the Stereochemistry of Polypeptide Chains

The presence of intramolecular CH... aromatic bonds in organic molecules has been analyzed by means of CSD searches. In particular, molecules containing aromatic and methyl groups have been examined as a function of the length of the chain separating the interacting groups. The type of bonds in the chain and its geometry have also been taken into account. The maximum number of CH... interactions have been found for five-membered chains joining the aromatic and methyl moieties. In addition, this interaction seems favored when the chain contains two sp ² hybridized atoms. The interactions involving polypeptides have been carefully examined. Interest has focused on compounds containing both aromatic and methylic residues, i.e., a combination of phenylalanine (phe), tyrosine (tyr), and tryptophan (trp) with alanine (ala), valine (val), leucine (leu), and isoleucine (ile). The maximum number of CH... interactions have been found when five atoms constituted the chain, i.e., in the sequences phe–ala and ala–phe.     

Probing the exposure of tyrosine and tryptophan residues in partially folded proteins and folding intermediates by CIDNP pulse-labeling

A nuclear magnetic resonance (NMR) technique has been devised to probe the structures of disordered, partially folded states of proteins at the level of individual amino acid residues. Chemically induced dynamic nuclear polarization (CIDNP) is first generated in exposed aromatic side-chains of the denatured state and then transferred to the high-resolution NMR spectrum of the native state by stimulating rapid refolding of the protein. Crucial improvements in sensitivity were achieved by carrying out the polarization-producing photochemistry in a deoxygenated sample of the disordered state of the protein in a magnetic field of 4.0 T and recording the (1)H NMR spectrum of the refolded native state at 9.4 T (400 MHz). Application of this method to the low pH molten-globule state of alpha-lactalbumin reveals remarkably nativelike environments for the aromatic residues in the primary hydrophobic core of the protein. This result provides compelling evidence that the detailed fold of a protein can be established prior to the formation of the cooperative close-packed native structure.     

Electrochemical investigation of nucleic acid behaviour: Part I. Native calf thymus DNA

The polarographic behaviour of DNA extracted from the calf thymus has been studied using phase sensitive a.c. polarography. The conditions of adsorption on the dropping mercury electrode have been studied in terms of the conditions of medium (ionic strength, pH) and of the molecular weight of DNA. The ionic strength plays a deciding role in the conditions of adsorption. The adsorption also depends on the form of the macromolecule. When it is in rod form (low molecular weight) the adsorption occurs parallel to the charged surface. When it adopts a wormlike form, loops are formed which extend into the solution. The adsorption does not depend on the nature of the monovalent counterion thus the adsorption is identical in Na⁺ and NH₄⁺ media.The polarograms of native and denatured DNA present an initial rounded peak situated at the limit of the adsorption zone. The native DNA is characterised by a second peak which appears, whatever the medium and the molecular weight of the DNA may be, on the recordings of the in-phase component of the current. This peak is situated at a more positive potential than that which is normally characteristic of the denatured DNA.The characteristics of the peaks of the native DNA have been specified. The first peak corresponds to the desorption of a certain number of adsorbed elements of the macromolecule. The second peak changes its properties according to the pH. In a slightly acid medium, it corresponds to the reduction of the adsorbed bases without desorption of the reduced products. The zones of reduction are the locally destabilized, i.e. the A-T rich regions of the DNA. In alkaline medium it corresponds to the desorption of the macromolecule. A general schema of the behaviour of native DNA on the dropping mercury electrode is proposed.     

Secondary structure of DNA is recognized by slightly cross-linked cationic hydrogel

Interaction of salmon sperm DNA (300-500 bp) and ultrahigh molecular mass DNA (166 kbp) from bacteriophage T4dC with linear poly(N-diallyl-N-dimethylammonium chloride) (PDADMAC) and slightly cross-linked (#) PDADMAC (#PDADMAC) hydrogel in water has been studied by means of UV-spectroscopy, ultracentrifugation, atomic force, and fluorescence microscopy (FM). It is found that the linear polycation induced compaction of either native (double-stranded) or denatured (single-stranded) DNA by forming PDADMAC-DNA interpolyelectrolyte complexes (IPEC)s. At the same time, #PDADMAC hydrogel is able to distinguish between native and denatured DNA. Native DNA is adsorbed and captured in the hydrogel surface layer, while denatured DNA diffuses to the hydrogel interior until the whole hydrogel sample is transformed into the cross-linked IPEC. Both native and denatured DNA can be completely released from the hydrogel in appropriate conditions with no degradation by adding a low molecular salt. The data observed using conventional physicochemical methods with respect to DNA of a moderate molecular mass remarkably correlate with the pictures directly observed for ultrahigh molecular mass DNA in dynamics by using FM.     

PHOTOCHEMICALLY INDUCED BINDING OF Rh(phen)2Cl2+ TO DNA†

The photoinduced covalent binding of the title compound to native and heat denatured DNA is described. The level of binding has been measured by UV (for DNA) and atomic absorption (for Rh) analysis. Quantum efficiencies of 6.4 x 10(-4) mol Rh per mol photons and 1.6 x 10(-3) mol Rh per mol photons have been determined for binding to native and denatured calf thymus DNA, respectively. Levels of bound rhodium as high as 1 molecule per five bases have been achieved. There is no binding of the complex in the absence of light, and there is evidence that at least a portion of the binding may be due to the photolytic conversion of the complex into one or more stable intermediates. Studies with polyribonucleotides indicate a strong preference for binding to the purine bases.     

Electrochemical Sensors Based on Stationary Electrodes and Immobilized DNA or Its Fragments and the Assessment of Their Analytical Potentials

Amperometric biosensors were developed on the basis of stationary mercury-film and glassy-carbon electrodes and DNA or its fragments, oligodeoxynucleotides (ODNs), immobilized in a nitrocellulose matrix. Taking into account the high affinity of Cu(II) and Fe(III) ions to denatured DNA ((19.1 ± 0.1) ×10⁵ and (1.4 ± 0.3) × 10⁵ L/mol, respectively), a procedure was proposed for the voltammetric determination of these ions in natural materials and blood serum at a level of n × 10⁻¹¹ M. This procedure involves analyte pre-concentration on a DNA-containing biosensor. An ODN-containing biosensor (DNA probe) was used in the study of DNA hybridization for the highly specific determination of its nucleotide sequence.__________Translated from Zhurnal Analiticheskoi Khimii, Vol. 60, No. 6, 2005, pp. 639–645.Original Russian Text Copyright © 2005 by Babkina, Palecek, Jelen, Fojta.Presented at the VI All-Russia Conference (with international participation) on Electrochemical Methods of Analysis (EMA-2004, Ufa, May 23–27, 2004).     

19F NMR studies of the native and denatured states of green fluorescent protein

Biosynthetic preparation and (19)F NMR experiments on uniformly 3-fluorotyrosine-labeled green fluorescent protein (GFP) are described. The (19)F NMR signals of all 10 fluorotyrosines are resolved in the protein spectrum with signals spread over 10 ppm. Each tyrosine in GFP was mutated in turn to phenylalanine. The spectra of the Tyr --> Phe mutants, in conjunction with relaxation data and results from (19)F photo-CIDNP (chemically induced dynamic nuclear polarization) experiments, yielded a full (19)F NMR assignment. Two (19)F-Tyr residues (Y92 and Y143) were found to yield pairs of signals originating from ring-flip conformers; these two residues must therefore be immobilized in the native structure and have (19)F nuclei in two magnetically distinct positions depending on the orientation of the aromatic ring. Photo-CIDNP experiments were undertaken to probe further the structure of the native and denatured states. The observed NMR signal enhancements were found to be consistent with calculations of the HOMO (highest occupied molecular orbital) accessibilities of the tyrosine residues. The photo-CIDNP spectrum of native GFP shows four peaks corresponding to the four tyrosine residues that have solvent-exposed HOMOs. In contrast, the photo-CIDNP spectra of various denatured states of GFP show only two peaks corresponding to the (19)F-labeled tyrosine side chains and the (19)F-labeled Y66 of the chromophore. These data suggest that the pH-denatured and GdnDCl-denatured states are similar in terms of the chemical environments of the tyrosine residues. Further analysis of the sign and amplitude of the photo-CIDNP effect, however, provided strong evidence that the denatured state at pH 2.9 has significantly different properties and appears to be heterogeneous, containing subensembles with significantly different rotational correlation times.     

Characteristic and spectroscopic properties of the Schiff-base model compounds

Two series of conjugated aromatic imines (Schiff-base model compounds) with different central groups and various side-group substitutions have been synthesized and characterized by elemental analysis, differential scanning calorimetry (DSC) technique, hydrogen nuclear magnetic resonance (¹H NMR), Fourier transform infrared (FTIR) and ultra-violet and visible light (UV–vis) spectroscopy measurements. The UV–vis absorption of solutions of these compounds in dimethylacetamid (DMA), chloroform and methanol was investigated in the optical range from 240 to 450 nm, where two distinct absorption bands: at 250–280 and 315–360 nm with the different level of absorption have been observed. The influence of compound molecular structure and polarity of solvent on the absorption spectra and the possible optical transitions have been discussed. Structure of diamines in the azomethine models fundamentally affected their spectroscopic properties and conjugation of π-electrons.     

Theoretical conformational analysis of tripeptides with a glycine residue

Summary 1. The conformational states of glycine residues — R, B, L, P — in the low-energy forms of the tripeptides (I–III) are found with equal frequency.2. The conformations of the main chain in sections of protein with glycine residues correspond to the preferred forms of the tripeptide fragments (I–III).3. The close-, medium-, and long-range interactions in proteins do not contradict one another and exist at the favorable forms of the main chain.M. M. Shemyakin Institute of Bioorganic Chemistry, Academy of Sciences of the USSR. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 631–637, September–October, 1975.     

Electrochemical behavior of DNA at a silver electrode studied by cyclic and elimination voltammetry

Electrochemical characteristics of native and denatured calf thymus DNA have been studied by voltammetry on a silver electrode (AgE). Experimental results obtained from linear sweep or cyclic voltammetry (LSV or CV) have been employed in elimination voltammetry. The elimination voltammetry with linear scan (EVLS), using the linear combination of the total currents measured at different scan rates, enables one or two selected particular currents to be eliminated. The best results have been obtained by using a function eliminating the kinetic and charging currents (I(k),I(c)), and conserving the diffusion current (I(d)). This function makes it possible to increase significantly voltammetric signals of native and denatured DNAs, and to reveal processes not detectable by conventional electrochemical methods. The influence of electrochemical pretreatment of silver electrode surfaces and of starting and switching potentials on DNA voltammetric signals have been discussed. Silver electrodes coupled with elimination voltammetry represent promising tools for developing new nucleic acids biosensors.     

Synthesis and structure of amino esters of menthol

The conditions for the acylation of (–)-menthol with monochloroacetyl chloride have been studied. Amino esters of (–)-menthol have been obtained by the nucleophilic replacement of the chlorine in (–)-menthol monochloroacetate by residues of secondary amines. Their properties and their mass, IR, and¹³C NMR spectra are described.Institute of Physical Organic Chemistry, Academy of Sciences of the Belorussian SSR, Minsk. Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 728–733, November–December, 1981.     

Adsorptive Stripping Voltammetric Detection of Single‐Stranded DNA at Electrochemically Modified Glassy Carbon Electrode

Electrochemically modified glassy carbon electrode (GCE) was used to study the electrochemical oxidation and detection of denatured single‐stranded (ss) DNA by means of adsorptive stripping voltammetry. The modification of GCE, by electrochemical oxidation at +1.75 V (vs.SCE) for 10 min and cyclic sweep between +0.3 V and ?1.3 V for 20 cycles in pH 5.0 phosphate buffer, results in 100‐fold improvement in sensitivity for ssDNA detection. We speculated that the modified GCE has a high affinity to single‐stranded DNA through hydrogen bond (specific static adsorption). Single‐stranded DNA can accumulate at the GCE surface at open circuit and produce a well‐defined oxidation peak corresponding to the guanine residues at about +0.80 V in pH 5.0 phosphate buffer, while the native DNA gives no signal under the same condition. The peak currents are proportional to the ssDNA concentration in the range of 0–18.0 μg mL^?1. The detection limit of denatured ssDNA is ca. 0.2 μg mL^?1 when the accumulation time is 8 min at open circuit. The accumulation mechanism of ssDNA on the modified GCE was discussed.     

Tryptophan exposure and accessibility in the chitooligosaccharide-specific phloem exudate lectin from pumpkin (Cucurbita maxima). A fluorescence study

The exposure and accessibility of the tryptophan residues in the chitooligosaccharide-specific pumpkin (Cucurbita maxima) phloem exudate lectin (PPL) have been investigated by fluorescence spectroscopy. The emission λ_max of native PPL, seen at 338 nm was red-shifted to 348 nm upon denaturation by 6 M Gdn.HCl in the presence of 10 mM β-mercaptoethanol, indicating near complete exposure of the tryptophan residues to the aqueous medium, whereas a blue-shift to 335 nm was observed in the presence of saturating concentrations of chitotriose, suggesting that ligand binding leads to a decrease in the solvent exposure of the tryptophan residues. The extent of quenching was maximum with the neutral molecule, acrylamide whereas the ionic species, iodide and Cs⁺ led to significantly lower quenching, which could be attributed to the presence of charged amino acid residues in close proximity to some of the tryptophan residues. The Stern–Volmer plot for acrylamide was linear for native PPL and upon ligand binding, but became upward curving upon denaturation, indicating that the quenching occurs via a combination of static and dynamic mechanisms. In time-resolved fluorescence experiments, the decay curves could be best fit to biexponential patterns, for native protein, in the presence of ligand and upon denaturation. In each case both lifetimes systematically decreased with increasing acrylamide concentrations, indicating that quenching occurs predominantly via a dynamic process.     

Investigation of complexes of DNA with synthetic peptide fragments of the N-end of histone H2B

The nature of the interaction of high- and low-molecular-weight DNAs (6×10⁶ and 3×10⁵ daltons) with synthetic oligopeptides of the N-end of histone H2B having the sequences 1–21, 1–10, 1–13, 11–21, and 14–21, differing in molecular weight and amino acid composition, as a function of the amount of peptide component in the complex and the ionic strength of the solution has been studied by the methods of UV and CD spectroscopy and the spectrophotometric analysis of melting curves. It has been shown that of all the peptides studied only the 1–21 peptide possesses the capacity of condensing DNA. This capacity depends on the amount of peptide component in the complex, the molecular weight of the DNA, and the ionic strength of the solution. The interaction with peptides under all the conditions studied, without changing the conformational parameters of the DNA, stabilizes its secondary structure in relation to the action of the temperature, which depends on the number of lysine residues in the peptides.N. I. Nikitin Institute of Chemistry of the TadzhSSR Academy of Sciences, Dushanbe. Institute of Cytology of the USSR Academy of Sciences, Leningrad. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 708–713, September–October, 1987.     

4.50—Polarographic investigation of conformational changes of human serum albumin: Part I. Unfolding of human serum albumin by urea

The mechanism of the unfolding of human serum albumin by urea was studied using d.c. polarography. It was found that this reaction is a complex process which cannot be described in terms of a two-state transition model. As well as the Brdi?ka catalytic current we have also studied the reduction current of disulfide groups in native and denatured human serum albumin. The number of cystine residues accessible for electrode reduction in native and denatured protein was calculated. On the basis of these results a scheme for the unfolding of human serum albumin by urea is proposed.