Unwinding of DNA induced by fluorescein mercuric acetate

Fluorescein mercuric acetate causes the unwinding of DNA and binds to the separated bases. This unwinding process can be followed by measuring absorption changes of this reagent. For untreated calf thymus DNA, the initial rate was very slow, and the shape of the kinetic curve was sigmoidal. When double-strand breaks of DNA were produced by DNase II treatment or sonication, the initial rate increased and the sigmoidal character disappeared. The initial rate was shown to be proportional to the concentration of helix ends. From this relation the rate of unwinding was estimated to be 2.0 base pairs/sec at 1.0 × 10^?5M fluorescein mercuric acetate and 25°C. DNase I treatment, which produces single-strand breaks and a smaller number of double-strand breaks, also increased the initial rate. However, this increase was due only to the double-strand breaks, that is, single-strand breaks had no significant effect on the initial rate. Also, uv irradiation increased the initial rate linearly with uv dose, at least up to 2 × 10⁵ erg/mm², suggesting that this increase is due to photoproducts other than usual pyrimidine dimers. We discuss the usefulness of this kinetic method in structural studies of DNA.     

Understanding nucleosomal histone and DNA interactions: a biophysical study

Histones are associated with DNA to form nucleosome essential for chromatin structure and major nuclear processes like gene regulation and expression. Histones consist of H1, H2A, H2B and H3, H4 type proteins. In the present study, combined histones from calf thymus were complexed with ct DNA and their binding affinities were measured fluorimetrically. All the five histones were resolved on SDS page and their binding with DNA was visualized. The values of biding affinities varied with pH and salt concentration. Highest affinity (4.0?×?10⁵ M^?1) was recorded at pH 6.5 in 50 mM phosphate buffer and 1.5?×?10⁴ M^?1 in 2 M NaCl at pH 7.0. The CD spectra support the highest binding affinity with maximum conformational changes at pH 7.0. The time-resolved fluorescence data recorded two life times for histone tyrosine residues at 300 nm emission in phosphate buffer pH 6.5. These life times did not show much change upon binding with DNA in buffer as well as in 2 M NaCl. The isothermal calorimetric studies yielded thermodynamic parameters ΔG, ΔH and ΔS as ?1.6?×?10⁵ cal/mol, ?1.13?×?10³ cal/mol and ?3.80 cal/mol/deg, respectively, evidencing a spontaneous exothermic reaction. The dominant binding forces in building the nucleosome are electrostatic interactions.     

Catalysis by polymer complexes. I. Enhanced esterolytic reactivity of hydroxamate–polymer complexes

N-methylmyristohydroxamic acid (1) bound to polymer micelles of laurylated poly(2- and 4-vinylpyridines) (lauryl group contet: 2VP-L, 30 mol%; 4VP-L, 33 mol%) quantitatively reacted with p-nitrophenyl acetate (NpAc) within a few seconds at 30°C, pH 8.95. Second order rate constants k_a were 34,000 M^?1 sec^?1 for 1–2VP-L and 11,400 M^?1 sec^?1 for 1–4VP-L at μ = 0.5, and they were pronouncedly improved by a decrease in ionic strength (k_a = 27,500–80,200 M^?1 sec^?1 at μ = 0.08). In contrast, poly(N-ethyl-4-vinylpyridinium bromide) hardly affected the nucleophilicity of the hydroxamate ion. Therefore, the enhancement was considered to be associated with some micellar characteristics. Typical saturation phenomena of the reaction rate were observed for p-nitrophenyl hexanoate (NpOCOPe) and 3-nitro-4-acetoxybenzoic acid (NpAcCOOH). It was suggested that binding of NpOCOPe is caused by the hydrophobic interaction, while that of NpAcCOOH is probably induced by the electrostatic interaction. It is demonstrated that the cationic polymer micelle enormously activates the bound hydroxamate anion, and these complexes would be of much interest as a biomimetic system for enzyme catalysis.     

Trypsin inhibitors from ridged gourd (Luffa acutangula Linn.) seeds: Purification,properties, and amino acid sequences

Two trypsin inhibitors, LA-1 and LA-2, have been isolated from ridged gourd (Luffa acutangula Linn.) seeds and purified to homogeneity by gel filtration followed by ion-exchange chromatography. The isoelectric point is atpH 4.55 for LA-1 and atpH 5.85 for LA-2. The Stokes radius of each inhibitor is 11.4 å. The fluorescence emission spectrum of each inhibitor is similar to that of the free tyrosine. The biomolecular rate constant of acrylamide quenching is 1.0×10⁹ M^?1 sec^?1 for LA-1 and 0.8 × 10⁹ M^?1 sec^?1 for LA-2 and that of K₂HPO₄ quenching is 1.6×10¹¹ M^?1 sec^?1 for LA-1 and 1.2×10¹¹M^?1 sec^?1 for LA-2. Analysis of the circular dichroic spectra yields 40%α-helix and 60%Β-turn for La-1 and 45%α-helix and 55%Β-turn for LA-2. Inhibitors LA-1 and LA-2 consist of 28 and 29 amino acid residues, respectively. They lack threonine, alanine, valine, and tryptophan. Both inhibitors strongly inhibit trypsin by forming enzymeinhibitor complexes at a molar ratio of unity. A chemical modification study suggests the involvement of arginine of LA-1 and lysine of LA-2 in their reactive sites. The inhibitors are very similar in their amino acid sequences, and show sequence homology with other squash family inhibitors.     

Quenching Effect of Tocopherols on the Methyl Linoleate Photooxidation and Their Oxidation Products

The quenching effect of α-, γ- and δ-tocopherols on the methylene blue sensitized photo- oxidation of methyl linoleate was investigated, and the ¹O₂. quenching ability of tocopherols was determined. The ¹O₂ quenching rate constants for α-, γ- and δ-tocopherols in ethanol were estimated to be 2.6 × 10⁸ m^?1 sec^?1, 1.8 × 10⁸ m^?1 sec^?1 and 1.0 × 10⁸ m^?1 sec^?1, respectively. And the rate constants for the chemical reaction between each tocopherol and ¹O₂ were 6.6 × 10⁶ m^?1 sec^?1, 2.6 × 10⁶ m^?1 sec^?1 and 0.7 × 10⁶ m^?1 sec^?1 for α-, γ- and δ-tocopherols, respectively. The results show that α-tocopherol is the most effective compound toward ¹O₂ among the three tocopherols. The photooxidation of each tocopherol produced two peroxides which, after chemical reduction, were identified to be tocopherol hydroquinone by gas chromatography-mass spectrometry analysis. The photooxidation mechanism of these tocopherols was assumed to be different from that of autoxidation.     

The reaction of cytochalasin a with sulfhydryl groups

The reaction of cytochalasin A with sulfhydryl groups was examined. Cysteine and glutathione reacted readily with cytochalasin A at pH 7.0, 20°C, following second-order kinetics with rate constants of 7,600 M^?1 sec^?1 and 870 × 10³ M^?1 sec^?1. No reaction of cytochalasin B could be demonstrated under the same conditions. The reaction of cytochalasin A with the amino group of glycine ethyl ester had a second-order rate constant of 0.02 M^?1 sec^?1. Cytochalasin A did not react with sufhydryl groups of native ovalbumin or lactic dehydrogenase but reacted with an least 2 and 12 groups respectively when the proteins were denatured in 0.1% SDS. The reactivity of cytochalasin A with sulfhydryl groups is attributable to the α,β-unsaturated ketone groups it contains.     

Transient electric birefringence of the bacteriophages T3 and T7

Electric birefringence measurements of suspensions of T3 and T7 bacteriophages in 10^?2 M phosphate buffer, pH 6.9, show that there is a difference in their rotational diffusion coefficient. The values corrected to 25°C and water viscosity are D_25,w = 4630 ± 130 sec^?1 and D_25,w = 5290 ± 260 sec^?1 for T3 and T7, respectively. The value obtained from shell model calculations (according to Filson and Bloomfield) is D_25,w = 4500 ± 600 sec^?1. The apparent permanent dipole moments are 4.5 × 10^?26 C·m and 1.7 × 10^?26 C·m for T3 and T7, respectively. For both phage particles the intrinsic optical anisotropy is +7.2 × 10^?3. It is shown that this anisotropy is mainly due to the DNA molecule inside the head of the phage. Its positive value means that there exists an excess orientation of the DNA helix perpendicular to the symmetry axis of the particle. For T7 an unexpectedly large increase of Δn_s and K_sp occurs at a glycerol concentration of about 30% (v/v). This increase is interpreted as being caused by a change of the shape of the particle and/or a change in the secondary structure of the DNA inside the head of the bacteriophage.     

Equilibrium studies on neutral red-DNA binding.

Spectrophotometric binding studies were undertaken on the interaction of neutral red with native and heat-denatured, sonicated, calf thymus DNA in a 0.2M ionic strength buffer containing Tris–sodium acetate–potassium chloride at 25°C. The pK_A of neutral red was found to be 6.81. At pH 5 the binding of protonated neutral red was complicated even at low concentration ratios of dye to DNA. In the pH range 7.5–8.5 the tight binding process could be studied and it was found that both protonated and free base species of neutral red significantly bind with DNA having association constants (in terms of polynucleotide phosphate) of 5.99 × 10³ M^?1 and 0.136 × 10³ M^?1, respectively, for native DNA and 7.48 × 10³ M^?1 and 0.938 × 10³ M^?1, respectively, for denatured DNA. The pK_A value of the neutral red–DNA complexes were 8.46 for native DNA and 7.72 for denatured DNA. These results are discussed in terms of possible binding mechanisms.     

Cooperative nonenzymic base recognition. Kinetics of the binding of a base monomer to a complementary polynucleotide template

The kinetics of double-helix formation by poly U and the complementary monomer N-6,9-dimethyladenine (m⁶m⁹A) has been measured using a new fast temperature-jump apparatus. The cooperative binding kinetics are complicated by the extensive self-association of the monomers, but a satisfactory analysis using average relaxation times was possible in terms of three different models. Application of a model which considers only monomer binding yields the upper limit for the binding rate constant of an m⁶m⁹A monomer next to an already bound monomer on a poly U strand: (2 ± 0.4) × 10⁸ M^?1sec^?1. A lower limit is found by using a model which allows for binding of all m⁶m⁹A stacks to poly U with equal rate constants: (3 ± 0.3) × 10⁷ M^?1sec^?1. A third model with “weighted” rate constants consistent with the data: (7.5 ± 1.0) × 10⁷ M^?1sec^?1. The rate of cooperative binding of m⁶m⁹A to the trimer UpUpU has also been measured. The rate constants obtained with the trimer agree with those obtained with the polymer for each of the three models within experimental error.     

Human enteropeptidase light chain: Bioengineering of recombinants and kinetic investigations of structure and function

The serine protease enteropeptidase exhibits a high level of substrate specificity for the cleavage sequence DDDDK～ X, making this enzyme a useful tool for the separation of recombinant protein fusion domains. In an effort to improve the utility of enteropeptidase for processing fusion proteins and to better understand its structure and function, two substitution variants of human enteropeptidase, designated R96Q and Y174R, were created and produced as active (>92%) enzymes secreted by Pichia pastoris with yields in excess of 1.7 mg/Liter. The Y174R variant showed improved specificities for substrates containing the sequences DDDDK (k_cat/K_M = 6.83 × 10⁶ M^?1 sec^?1) and DDDDR (k_cat/K_M = 1.89 × 10⁷ M^?1 sec^?1) relative to all other enteropeptidase variants reported to date. BPTI inhibition of Y174R was significantly decreased. Kinetic data demonstrate the important contribution of the positively charged residue 96 to extended substrate specificity in human enteropeptidase. Modeling shows the importance of the charge–charge interactions in the extended substrate binding pocket.     

Hydrolysis of p-nitrotrifluoroacetanilide catalyzed by water and imidazole

The hydrolyses of p-nitrotrifluoroacetanilide catalyzed by water and imidazole were examined at 70°C. The pH-rate constant profile of the hydrolysis in H₂O was examined in the pH range 0.0–11.4. The hydrolysis was independent of pH in the region from pH 1.0 to 4.5, presumably a water-catalyzed reaction. The rate constant and the D₂O solvent isotope effect for this reaction were 1.0 × 10^?4 sec^?1 and 3.7, respectively. Both natural imidazole and imidazolium cation catalyzed hydrolysis. The rate constant of the hydrolysis catalyzed by neutral imidazole was determined to be 5.4 × 10^?3M^?1 sec^?1 and the D₂O solvent isotope effect was 1.8.     

Insight into the binding of a non-toxic,self-assembling aromatic tripeptide with ct-DNA: Spectroscopic and viscositic studies

The report describes the synthesis, self-association and DNA binding studies of an aromatic tripeptide H-Phe-Phe-Phe-OH (FFF). The peptide backbone adopts β—sheet conformation both in solid and solution. In aqueous solution, FFF self-assembles to form nanostructured aggregates. Interactions of this peptide with calf-thymus DNA (ct-DNA) have been studied using various biophysical techniques including ultraviolet (UV) absorption spectroscopy, fluorescence spectroscopy and circular dichroism (CD) spectroscopy. The value of mean binding constant calculated from UV and fluorescence spectroscopic data is (2.914 ± 0.74) x 10³ M^?1 which is consistent with an external binding mode. Fluorescence intercalator displacement (FID) assay, iodide quenching study, viscosity measurement and thermal denaturation study of DNA further confirm the groove binding mode of peptide, FFF with ct-DNA. MTT cell survival assay reveals very low cytotoxicity of the peptide toward human lung carcinoma cell line A549.     

The kinetics of the anation reaction of aquopentaamminecobalt(III) by acetate in aqueous acidic solution

The anation reaction of aquopentaamminecobalt(III) by acetate has been studied in the temperature range 60–80°C and acidity range 1.0 ≦ pH ≦ 5.5 for total acetate concentrations ≦ 0.5 M and at ionic strength 1.0 M. The anation by acetic acid follows second-order kinetics (k₀), whereas the kinetic results for the anation by acetate (Q₁, k₁) provide evidence for the formation of an ion-pair with the complex ion. Typical experimental results at 70°C are k₀ = 5.33 X 10⁻⁵ M⁻¹ sec⁻¹, Q₁ = 5.87 M⁻¹ and k₁ = 1.46 X 10⁻⁴sec⁻¹. The activation parameters for the different reaction paths are reported and the results discussed with reference to various other anation reactions of Co(III) complexes.     

Peroxidase-like activities of iron(III)—Porphyrins: kinetics of the reduction of a peroxidatically active derivative of deuteroferriheme by anilines

The kinetics of the reduction by aniline and a series of substituted anilines of a peroxidatically active intermediate, formed by oxidation of deuteroferriheme with hydrogen peroxide, have been studied by stopped-flow spectrophotometry. The reaction with aniline was first order with respect to [intermediate] and showed first-order saturation kinetics with respect to [aniline]. The second-order rate constant was 2.0 ± 0.2 × 10⁵ M^?1 sec^?1 at 25°C (independent of pH in the range 6.60–9.68) compared with the value of 2.4 × 10⁵ M^?1 sec^?1 for the reaction of aniline with horseradish peroxidase Compound I. The effect of aniline substituents upon reactivity towards the heme intermediate closely paralled those reported for reaction with the enzymic intermediate. Anilines bearing electron-donating substituents reacted more rapidly and those bearing electron-withdrawing substituents more slowly than the unsubstituted amine. The rate constants for the heme intermediate reactions (k_dfh)found to be related to those for the enzymic reactions (k_hrp) by the equation:log k_DFH= 0.65log k_HRP+ 1.96 with a correlation coefficient of 0. 98.     

Oxidation of ascorbic acid with superoxide anion generated by the xanthine-xanthine oxidase system.

Ascorbic acid was found to be oxidized by O₂^$\text{?}$ which was generated by the xanthine-xanthine oxidase system. From a kinetic analysis of the inhibition of this reaction by superoxide dismutase, the second-order rate constant for the reaction between ascorbic acid and O₂^? at pH 7.4 was estimated to be 2.7 × 10⁵ M^?1 sec^?1. A function of ascorbic acid as a defense against O₂^? is presented.     

Reaction of imidazole and hydroquinone with oxymyoglobin

Oxymyoglobin reacts with imidazole, substituted imidazoles, and hydroquinone to give metmyoglobin. The kinetics of these reactions have been studied. The rates are first order in both reactants, and second-order rate constants are reported. At pH 8.2, k₁ for imidazole is 2.5 ± 0.3 × 10^?3 M^?1 sec^?1 and for hydroquinone is 4 ± 0.4 × 10^?1 M^?1 sec^?1. The rates are independent of pH for imidazole but increase rapidly with pH for hydroquinone. The mechanism for all these reactions is thought to involve the two-electron reduction of molecular oxygen to peroxide with concurrent oxidation of both the protein and the reactant. An analogous mechanism has been suggested previously [1] for the reaction of oxyhemoglobin with hydroquinone. It has previously been shown [6] that imidazole can mediate the transfer of electrons to heme proteins by forming a transient reduced radical. The present results indicate that it can also form a transient oxidized radical under mild conditions. This dual capability may be important in biological electron-transfer processes.     

Purification and partial characterization of the extradiol dioxygenase, 2′-carboxy-2,3-dihydroxybiphenyl 1,2-dioxygenase,in the fluorene degradation pathway from Rhodococcus sp. strain DFA3

Type II extradiol dioxygenase, 2′-carboxy-2,3-dihydroxybiphenyl 1,2-dioxygenase (FlnD1D2) involved in the fluorene degradation pathway of Rhodococcus sp. DFA3 was purified to homogeneity from a heterologously expressing Escherichia coli. Gel filtration chromatography and SDS-PAGE suggested that FlnD1D2 is an α₄β₄ heterooctamer and that the molecular masses of these subunits are 30 and 9.9 kDa, respectively. The optimum pH and temperature for enzyme activity were 8.0 and 30 °C, respectively. Assessment of metal ion effects suggested that exogenously supplied Fe²⁺ increases enzyme activity 3.2-fold. FlnD1D2 catalyzed meta-cleavage of 2′-carboxy-2,3-dihydroxybiphenyl homologous compounds, but not single-ring catecholic compounds. The K_m and k_cat/K_m values of FlnD1D2 for 2,3-dihidroxybiphenyl were 97.2 μM and 1.5 × 10^?2 μM^?1sec^?1, and for 2,2′,3-trihydroxybiphenyl, they were 168.0 μM and 0.5 × 10^?2 μM^?1sec^?1, respectively. A phylogenetic tree of the large and small subunits of type II extradiol dioxygenases suggested that FlnD1D2 constitutes a novel subgroup among heterooligomeric type II extradiol dioxygenases.     

A proton NMR spin-lattice relaxation study of the imino proton exchange kinetics in calf-thymus DNA

The results of semiselective ¹H-nmr inversion recovery experiments on sonicated calf thymus DNA fragments are reported. The measurements were conducted in aqueous solutions containing 85% D₂O, in order to reduce the dipolar contribution to the observed relaxation rates. In solutions containing 0.2M NaCl, 0.4 mM EDTA, and 10 mM cacodylate at pH = 7.0, the exchange rates of the imino protons in A-T base pairs confirm values published earlier in the literature, extrapolating to 0.25 s^?1 at 25°C. Corresponding values for the G-C base pairs are published for the first time, and are about sixfold slower. The addition of up to 0.1M Tris buffer (pH = 7.3 at 25°C), caused a striking increase in the measured exchange rates for both the A-T and G-C imino protons, resembling the effect recently observed for poly(rA)-poly(rU) and poly(rI)-poly(rC), and suggesting that the exchange rates measured for nucleic acid duplexes in low buffer concentrations at neutral pH do not reflect base-pair opening rates as assumed in the past. Lower limits to the base-pair opening rates could be estimated from extrapolation of the experimental data to infinite buffer concentration, and are 1 × 10³ s^?1 for the A-T, and 50 s^?1 for the G-C, base paris at 62°C.     

Physical binding of pyrene and phenanthrene to native and denatured DNA: Measurements by spectral and coupled-column liquid chromatography methods

The physical (noncovalent) binding of pyrene and phenanthrene to calf-thymus DNA in aqueous NaCl solutions was measured by a spectral method (analysis of absorption spectra by Benesi-Hildebrand plots) and a coupled-column liquid chromatography method (equilibration of DNA solutions with solid hydrocarbon in a generator column and analysis of dissolved hydrocarbon by liquid chromatography). The measurements yielded values of an affinity constant K′ = nK, where n is the apparent number of binding sites per nucleotide and K is the apparent binding constant. The affinity of native DNA for pyrene decreases monotonically with increasing NaCl concentration, whereas the affinity of heat-denatured DNA exhibits a maximum at 0.10M NaCl.K′ for the binding of phenanthrene to native DNA is an order of magnitude lower than K′ for pyrene. The molar enthalpy for the binding of pyrene to native DNA in 10 mM NaCl is (?34.0 ± 1.0) kJ mol^?1. The spectral method data indicate that 50 is an upper limit for the average number of base pairs between intercalation sites for pyrene along the DNA helix and that only a fraction of these sites are bound at the highest binding ratios.     

Inactivation of taste receptor cell function by two cationic protein modification reagents

Two cationic protein modification reagents, 1-cyclohexyl-3-(2-morpholinylethyl) carbodiimide (CMCD) and dimethyl (2-hydroxy-5-nitrobenzyl) sulfonium bromide (HNB-dmS), inhibit taste receptor cell stimulation by NaCl, sucrose, and HCl. Modified inactive derivatives of the reagents under the same conditions are ineffective. Inactivation by HNB-dmS is essentially irreversible. The effects of inactivation by CMCD are reversible after about 10–15 minutes of a water rinse, however, when applied in the presence of glycine methyl ester, the inhibited response is stabilized and only recovers after about 1.5–3 hours. Glycine methyl ester alone has no inhibitory properties. The kinetics of inactivation by both HNB-dmS and CMCD are consistent with a second-order reaction with rate constants of 0.041 ± 0.001 M^?1 sec^?1 and 0.121 ± 0.012 M^?1 sec^?1, respectively. The rate of inactivation by both compounds is independent of NaCl concentration as well as degree of receptor stimulation. This, together with the observation that the response to stimulation by all effectors examined is altered, suggests the inactivation occurs at an event which is common to the transduction of the response from all three stimuli. The ether:water partition coefficients, as well as previous results from inactivation by N–substituted maleimides, indicate that hydrophilic reagents do not cross the cell membrane in significant concentrations within the time period of application. This suggests the site of modification by the cationic protein modification reagents is at the surface of the cell membrane. Significant residual NaCl, sucrose, and HCl activity remains after total inactivation. To account for this, a two-state membrane receptor system is postulated.