Influence of cyanine dye structure on self-aggregation and interaction with nucleic acids: a kinetic approach to TO and BO binding

Kinetics and equilibria of cyanine dyes thiazole orange (TO) and benzothiazole orange (BO) self-aggregation and binding to CT-DNA are investigated in aqueous solution at 25 degrees C and pH 7. Absorbance spectra and T-jump experiments reveal that BO forms J-aggregates while TO forms more stable H-aggregates. Fluorescence and absorbance titrations show that TO binds to DNA more tightly than BO. TO stacks externally to DNA for low polymer-to-dye concentration ratios (C(P)/C(D)) while dye intercalation occurs for high values of C(P)/C(D). T-jump and stopped-flow experiments performed at high C(P)/C(D) agree with reaction scheme D+S <=> D,S <=> DS(I) <=> DS(II) where the precursor complex D,S evolves to a partially intercalated complex DS(I) which converts to the more stable intercalate DS(II). Non-electrostatic forces play a major role in D,S stabilization. Last step is similar for both dyes suggesting accommodation of the common benzothiazole residue between base pairs. Experiments using poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC) confirm base pair preference for TO.     

Conformational studies on the pyridoxal Schiff bases of polypeptides

The pyridoxal Schiff bases of the polypeptides poly(L -lysine), poly(L -ornithine), and poly(L -α,γ-diaminobutyric acid) were prepared and investigated in water/methanol by CD spectra and equilibrium dialysis experiments. Only the poly(L -α,γ-diaminobutyric acid) derivative is characterized by a relevant optical activity similar to that found in pyridoxal enzymes. The stereospecific interactions between the pyridoxylideneimine group and the polypeptide chain prevent the hydrolysis reaction of the aldimine bond.     

Kinetic and thermodynamic investigation of the (dien)Pd(II)-polycytidylic acid system

The reaction between (dien)PdCl+ and polycytidylic acid was studied using spectroscopic and stopped-flow methods. In neutral solution, the palladium complex binds at the N3 site of the cytosine base and causes a noncooperative disruption of the ordered helical structure of poly(C). Interaction at the phosphate group of the polynucleotide was also demonstrated by using the dye acridine orange as an indicator. The results of this study show that the mechanism previously proposed for cytidine and CMP can be applied to poly(C), taking into account particular features of the polymer (polyelectrolytic nature, structure, etc.). In particular, electrostatic effects seem to play a major role in the interaction with metal ion complexes like (dien)Pd(II).     

Induced optical activity in poly-L-lysine-methyl orange system

The absorption and cicular dichroism spectra of the complex of poly-L -lysine (PLL) in the random coil form with methyl orange (MO) have been measured in aqueous solution. A new absorption band is observed at the shorter wavelength compared with that of the free dye. Although MO does not show a formation of dimer or aggregation with an increase in concentration, circular dichroism bands are observed at the wavelength corresponding to the wavelength of the new absorption band. These induced circular dichroism bands may arise from the dimeric MO molecules bound to PLL in the random coil form. The main contribution to the interaction between MO molecules is shown to be the electro static interaction. The observed circular dichroism spectra and the configuration of dimeric MO molecules bound to PLL can be explained by the dipole couping mechanism.     

Induced CD and interaction of some porphyrin derivatives with α-helical polypeptides in aqueous solutions

Absorption spectra and induced CD have been measured on aqueous solutions of water-soluble porphyrins with α-helical poly(L -glutamic acid) or α-helical poly (L -lysine) at different mixing ratios. For the former, porphyrin is porphine-meso-tetra (4-N-methylpyridinium) (TMpyP), and for the latter, it is porphine-meso-tetra (4-benzenesulfonate) (TPPS) or porphine-meso-tetra(4-benzoate) (TPPC). All the solutions of porphyrin-polypeptide systems show hypochromism in the Soret band and induced CD in the Soret region. The CD is characterized by a positive band at a shorter wavelength and a stronger negative band at a longer wavelength. The hypochromicity and the magnitude of molar ellipticities are much larger for the TPPS– and TPPC–poly (L -lysine) systems than for the TMpyP–poly (L -glutamic acid) system. Porphyrin ions bind to the α-helix electrostatically, and the two components of the Soret transition of porphyrin are subject to dissymmetric perturbation. TMpyP ions bind to the α-helix at isolated sites, while TPPS ions and TPPC ions are in pairs on the α-helix, that is, two ions bind consecutively and dissymmetrically. In the TMpyP–poly (L -glutamic acid) system a single CD band is associated with each of the two components of the Soret transition, and these are of opposite sign. In the TPPS– and TPPC–poly (L -lysine) systems, a pair of positive and negative CD bands is associated with each of the two components, thus giving apparently a single pair of CD bands with a shoulder, owing to partial overlapping.     

Premelting and the hydrogen-exchange open state in synthetic RNA duplexes

Here we attempt to relate equilibrium temperature-dependent spectral changes in two synthetic RNA homopolymer duplexes—poly(rA) · poly(rU) and poly(rI) · poly(rC)—to the conformational opening detected in stopped-flow hydrogen–deuterium exchange experiments on these molecules. We are concerned with changes in several spectral properties that occur well below onset of the thermally induced helix-coil “melting” transition in these systems. These are known as “premelting” transitions, and can be detected in uv CD spectra as well as in vibrational bands of the bases in the ir. Both CD and ir spectra exhibit isoelliptic or isosbestic points consistent with a well-defined two-state premelting process. Application of a least-squares analysis to two-state models for premelting using data from different bands in the CD and ir shows that the enthalpies are substantially greater than that of the hydrogen-exchange opening. Thus the hydrogen-exchange open state represents only one premelting reaction among several that lead to equilibrium changes in helix geometry or base vibrational modes. The latter include processes that occur on a rapid time scale, including potential base-pair openings not productive for the exchange reaction. It appears that the former, and not the hydrogen-exchange opening, dominates the premelting alterations monitored by ir and CD spectroscopy.     

Solid-state conformation of copolymers of β-benzyl-L-aspartate with L-alanine,L-leucine,L-valine, γ-benzyl-L-glutamate,or ϵ-carbobenzoxy-L-lysine

The solid-state conformation of copolymers of β-benzyl-L -aspartate [L -Asp(OBzl)] with L -leucine (L -Leu), L -alanine (L -Ala), L -valine (L -Val), γ-benzyl-L -glutamate [L -Glu(OBzl)], or ?-carbobenzoxy-L -lysine (Cbz-L -Lys) has been studied by ir spectroscopy and circular dichroism (CD). The ir spectra in the region of the amide I and II bands and in the region of 700–250 cm^?1 have been determined. The results from the ir studies are in good agreement with data obtained by CD experiments. Incorporation of the amino acid residues mentioned above into poly[L -Asp(OBzl)] induces a change from the left-handed into the right-handed α-helix. This conformational change for the poly[L -Asp(OBzl)] copolymers was observed in the following composition ranges: L -Leu, 0–15 mol %; L -Ala, 0–32 mol %; L -Val, 0–8 mol %; L -Glu(OBzl), 3–10 mol %; and Cbz-L -Lys, 0–9 mol %.     

New aspects of the interaction of the antibiotic coralyne with RNA: coralyne induces triple helix formation in poly(rA)?poly(rU)

The interaction of coralyne with poly(A)•poly(U), poly(A)•2poly(U), poly(A) and poly(A)•poly(A) is analysed using spectrophotometric, spectrofluorometric, circular dichroism (CD), viscometric, stopped-flow and temperature-jump techniques. It is shown for the first time that coralyne induces disproportionation of poly(A)•poly(U) to triplex poly(A)•2poly(U) and single-stranded poly(A) under suitable values of the [dye]/[polymer] ratio (C_D/C_P). Kinetic, CD and spectrofluorometric experiments reveal that this process requires that coralyne (D) binds to duplex. The resulting complex (AUD) reacts with free duplex giving triplex (UAUD) and free poly(A); moreover, ligand exchange between duplex and triplex occurs. A reaction mechanism is proposed and the reaction parameters are evaluated. For C_D/C_P> 0.8 poly(A)•poly(U) does not disproportionate at 25°C and dye intercalation into AU to give AUD is the only observed process. Melting experiments as well show that coralyne induces the duplex disproportionation. Effects of temperature, ionic strength and ethanol content are investigated. One concludes that triplex formation requires coralyne be only partially intercalated into AUD. Under suitable concentration conditions, this feature favours the interaction of free AU with AUD to give the AUDAU intermediate which evolves into triplex UAUD and single-stranded poly(A). Duplex poly(A)•poly(A) undergoes aggregation as well, but only at much higher polymer concentrations compared to poly(A)•poly(U).     

Circular dichroism studies of complexes of sequential histidine polypeptides with methyl orange

The induced circular dichroism (c.d.) spectra of poly(l-histidine) and sequential histidine polypeptide-dye complexes were measured. Two dichroic bands associated with the blue shifted absorption shoulder of methyl orange at around 370 nm were observed by complex formation between the polypeptides and the dye. Induced c.d. arose from the dye bound to the polypeptide in random coil structure, the optimal pH being 4.1. Added sodium chloride decreases the intensity of the induced c.d. Intramolecular interaction was assumed from the relationship between the concentrations of the polypeptide-dye complex and the intensities of the induced c.d. The intensity of the induced c.d. decreases with increasing distance between the intramolecular histidine residues. The induced c.d. spectrum of the poly(l-histidine)-dye complex shows the irreversible thermal change when heating.     

Induced circular dichroism and mode of binding of acridine orange adsorbed on β-form poly(S-carboxyethyl-L-cysteine) in aqueous solutions

The absorption spectra and circular dichroism (CD) have been measured for aqueous solutions of acridine orange of a constant concentration, [D] = 5 × 10^?5M, mixed with poly(S-carboxyethyl-L -cysteine) in various mixing ratios, [P]/[D], ranging from 330 to 11, at different pH. The absorption spectra of the dye–polymer solutions are hypochromic, and the main band is located at 470 nm, accompanying a shoulder at 500 nm. At alkaline pH, no CD is induced in the visible region. At neutral and acidic pH, where the polymer is in the β-conformation, CD is induced in the visible and near-uv regions. A pair of CD bands is located at the region around 450 nm, when the pH is around the neutrality, while it appears at the region around 500 nm at acidic pH. Thus, the optically active species of bound dye changes from dimer to monomer on lowering the pH. These species form dissymmetric arrays along a polypeptide chain. The fraction of bound dye forming dissymmetric sequences is not high, but most of bound dye is adsorbed randomly on the ionized carboxyl groups of polypeptide chain and gives rise to hypochromism only. A dissymmetric structure of dye–polymer complexes is presented, in which the polymer has the β-conformation and the dye cations, either dimeric or monomeric, bind to its side chains, in such a way that the longer axes of molecular planes of bound dye form a two-fold, right-handed helix along the extended polypeptide chain. A zeroth-order calculation of CD based on the coupled oscillator model leads to the result that each dissymmetric array of dye consists, on the average, of two dimeric or monomeric cations. This low number of bound cations in a dissymmetric array and the large fraction of randomly adsorbed dye suggest that the hydrophobic interaction of dye with the polymer is strong, so that dye cations are adsorbed sparsely on both sides of the extended polypeptide chain.     

Conformational studies on Cu(II) polycomplexes of pyridoxal Schiff bases of polypeptides

Structures of Cu(II) complexes of pyridoxal Schiff bases with poly(L -lysine), poly(L -ornithine), and poly(L -α,γ-diaminobutyric acid) were investigated by absorption spectra, CD, and conformational analysis. Although the polypeptides retain their typical right-handed α-helical conformation, opposite Cotton effects were found for the poly(L -lysine) and poly(L -ornithine) polycomplexes in the whole range of wavelengths from 600 to 250 nm. As in the analogous derivatives of salicyladehyde, this effect seems to be due to a stereospecific binding of the square planar Cu(II)-bis-pyridoxylideneimine group to the α-helical matrix. Circular dichroism spectrum of poly(L -α,γ-diaminobutyric acid) polycomplex is similar to that found for poly(L -lysine) derivative, but indicates large tetrahedral distortion of the square-planar coordination of copper ion.     

Stopped-flow kinetic, 1H, and 31P NMR analysis of the intercalation of ethidium with poly d(G-C) X d(G-C)

In a low salt buffer (0.011 M Na+) stopped-flow kinetic results for the SDS driven dissociation of an ethidium-Poly d(G-C) X d(G-C) complex are 8.7, 23, and 58.5 s-1 at 20, 30, and 40 degrees C, respectively. These results predict that in NMR experiments at high field strengths, ethidium should be in slow exchange among polymer binding sites. This has been found to be the case for both 31P (109 MHz) and 1H (imino proton spectra in H2O at 270 MHz) experiments. At higher salt, and/or higher temperature, and/or lower field, the bound and free peaks are no longer resolved in the NMR spectra. Good agreement is obtained between the stopped-flow kinetic results and the coalescence temperature observed in NMR experiments. Imino protons in base pairs on both sides of the intercalated ethidium are shifted approximately one ppm upfield while only the phosphate groups at the intercalation site experience large chemical shifts.     

Hydrodynamic behavior and molecular conformation of poly(L-lysine HBr) in carbonate buffer solution

In carbonate buffer at pH 10.5, a transparent solution of poly(L -lysine HBr) was obtained up to fairly high concentration of 3 g/dl at room temperature. The hydrodynamic behavior of the solution has been studied by sedimentation analyses and viscosity measurements. A dimer form for high concentrations and a monomer form for low concentrations were inferred. The dimer and monomer forms were assigned to a β-structure and α-helix, respectively, based on the CD and optical rotary dispersion spectra. Using CD spectroscopy, a reversible transition between α-helix and β-structure was observed as a function of either poly(L -lysine HBr) concentration or temperature. An aggregated form which was assigned to the antiparallel pleated sheet appeared at 50°C on the basis of its ir spectrum.     

Kinetic studies on mammalian cytochrome c modified with 2-hydroxy-5-hydroxy-5-nitrobenzyl bromide.

The reduction of 2-hydroxy-5-nitrobenzyl tryptophyl cytochrome c by the chromous ion was studied by stopped-flow techniques. At pH6.5 the reduction of 2-hydroxy-5-nitrobenzyl tryptophyl cytochrome c is complex, showing the presence of three distinct phases. Two chromium concentration-dependent phases are observed (1.1 X 10(5) M-1-S-1, phase 1; 1.25 X 10(4)M-1-S-1, phase 2) and one slow first-order process (0.25S-1, phase 3). A comparison of the static and kinetic difference spectra, along with the data from the reduction of the reoxidized reduced protein, suggests that the slow chromium concentration-independent phase is due to a slow conformational event after fast reduction of the NO2 group. The rates of the chromium concentration-dependent phases show a marked variation with pH above 7.5. The activation energies for the three processes were also measured at 33.2, 38.6 and 69.7 kJ-mol-1 for phases 1, 2 and 3 respectively. The reaction of reduced 2-hydroxy-5-nitrobenzyl tryptophyl cytochrome c with CO was foollowed by means of both stopped-flow and flash photolysis. The combination with CO at pH 6.8 as measured in stopped-flow experiments showed two phases, one CO-dependent phase (phase 2, 2.4 X 10(2)M-1-S-1) and one CO-independent phase (phase 1, 0.015S-1). Investigation of the pH-dependence of the phases showed both the rates and amounts of each phase to be pH-invariant. CO recombination, after photolytic removal, was found to be biphasic; a CO-dependent phase (phase 2, 2.4 X 10(2)M-1-S-1) and a CO-independent phase (phase 1, 1.0s-1) were observed. A tentative model which can accommodate these observations is proposed.     

Conformational studies of sequential polypeptides containing lysine and tyrosine

The conformation of three sequential copolypeptides, poly(L -tyrosyl-L -lysine), poly(L -tyrosyl-L -lysyl-L -lysine), and poly[L -tyrosyl-(L -lysyl)₂-L -lysine] have been studied by a variety of techniques, including CD, ir spectroscopy, analytical ultracentrifugation, and x-ray diffraction. Depending upon the pH and sovent composition, poly(L -tyrosyl-L lysyl-L -lysine) and poly [L -tyrosyl-(L lysyl)₂-L -lysine] can adopt either the α-helical or random-coil conformation, while poly(L -tyrosyl-L -lysine) forms either inter- or intramolecular β-structures.     

Induced optical activity in the complex of poly(L-arginine) with azo dyes

The absorption and CD spectra of the complexes of poly(L -arginine) (PLA) and azo dyes have been measured in aqueous solution. On complexation, Blue-shifted additional absorption bands were observed. In the wide pH 4–11 range, induced CD was observed at the visible wavelengths corresponding to the blue-shifted absorption bands. The induced CD arose from the dimeric dye molecules bound to PLA in the α-helical structure. When a modified analysis of induced CE is made by the excition chirality method, the origin of the induced CD can be assigned to the dipole coupling. The PLA–dye complexes showed the counterlockwise (negative, S) chirality of the transition dipole moments of dyes.     

Spin-label studies of the pH-induced random coil to α-helix conformational transition in poly(L-lysine)

Poly(L -lysine) of various molecular weights between 2700 and 475,000 was spin-labeled. From the electron spin resonance spectra, the degree of freedom of the nitroxide was determined by calculation of the rotational correlation time as the poly(L -lysine) underwent the pH-induced random coil to α-helix conformational transition. In general, the rotational correlation time of the nitroxide increased as the pH was increased, indicating a more restricted environment for the spin label when poly(L -lysine) is deprotonated. For the high-molecular-weight poly(L -lysine) this corresponds to the formation of the α-helix and indicates that the side chain–side chain interaction and decreased segmental motion of the backbone (slightly) restricts the motion of the spin label. For the 2700-molecular-weight poly(L -lysine), previously shown not to assume a helical conformation at high pH, the increase in the rotational correlation time of the spin label indicates that the side chain–side chain interaction takes place after deprotonation but without helix formation. This may indicate that helix formation per se is not needed to produce the observed effect even with the high-molecular-weight polymers. The rotational correlation time of the spin label at a particular pH did not depend on the molecular weight of the poly(L -lysine) over the 200-fold range of molecular weights. This indicates that the rotational correlation time reflects the rotational mobility of the spin label in a localized environment and not the rotational diffusion of the entire macromolecule.     

刺芹侧耳产漆酶条件优化及对偶氮染料甲基橙的脱色

漆酶是一种含铜的单电子多酚氧化酶,能够催化氧化各种酚类及多种染料,在处理染料废水方面具有巨大的潜力。刺芹侧耳Pleurotus eryngii具有较强的产漆酶能力,但漆酶产量在较大程度上受环境条件限制。本文研究了氮源含量、pH、温度、金属离子等环境条件对刺芹侧耳产漆酶能力的影响,优化了其产漆酶条件,并用其粗酶液对典型偶氮类染料甲基橙进行脱色,结果表明,在氮源0.5%（W/W）、pH 5.5、温度28℃、添加5.0mmol/L Mg ²⁺的培养条件下,刺芹侧耳产漆酶能力最强,培养6d时,漆酶酶活可达78.0U/L。用优化培养的刺芹侧耳粗酶液对偶氮染料甲基橙进行脱色,28h后脱色率可达90%,脱色反应为准一级动力学反应,甲基橙并未完全矿化,而是生成小分子中间产物。     

The importance of coulombic interactions for the induction of beta structure in lysine oligomers by sodium dodecyl sulfate.

Circular dichroism spectra have been obtained for tri(L -lysine), tetra(L -lysine), and penta(L -lysine) in aqueous sodium dodecyl sulfate at 25°C. None of the oligomers are affected significantly by sodium dodecyl sulfate at detergent concentrations exceeding 0.01 M. Literature results show that the high-molecular-weight polymer forms a β strucure under these conditions. At detergent concentrations near 3.5 × 10^?4 M the penta(L -lysine), but not the smaller oligomers, undergoes a conformational change. Its circular dichroism under these conditions is essentially identical to that observed with poly(L -lysine) when it forms a β structure in sodium dodecyl sulfate. Solutions of the penta(L -lysine), which exhibit this modified circular dichroism, are also turbid, leading to the conclusion that the oligomer has formed an intermolecular β structure. When these experiments are conducted in the presence of 0.1 M sodium hydroxide, the sodium dodecyl sulfate produces neither turbidity nor a modified circular dichroism spectrum. These observations provide compelling evidence that Coulombic interaction between the anionic detergent head and the cationic lysyl amino groups is essential for the conformational change induced in penta(L -lysine) by sodium dodecyl sulfate.     

The reaction of Pseudomonas aeruginosa cytochrome c-551 oxidase with oxygen.

The reaction of ascorbate-reduced Pseudomonas cytochrome oxidase with oxygen was studied by using stopped-flow techniques at pH 7.0 and 25 degrees C. The observed time courses were complex, the reaction consisting of three phases. Of these, only the fastest process, with a second-order rate constant of 3.3 X 10(4) M-1.S-1, was dependent on oxygen concentration. The two slower processes were first-order reactions with rates of 1.0 +/- 0.4s-1 and 0.1 +/- 0.03s-1. A kinetic titration experiment revealed that the enzyme had a relatively low affinity constant for oxygen, approx. 10(4)M-1. Kinetic difference spectra were determined for all three reaction phases, showing each to have different characteristics. The fast-phase difference spectrum showed that changes occurred at both the haem c and haem d1 components of the enzyme during this process. These changes were consistent with the haem c becoming oxidized, but with the haem d1 assuming a form that did not correspond to the normal oxidized state, a situation that was not restored even after the second kinetic phase, which reflected further changes in the haem d1 component. The results are discussed in terms of a kinetic scheme.