Light activates reduction of methotrexate by NADPH in the ternary complex with Escherichia coli dihydrofolate reductase

Methotrexate (MTX), a strong inhibitor of dihydrofolate reductase (DHFR), has been widely used for chemotherapy for many types of cancer as well as for juvenile rheumatoid arthritis. It mimics folate substrates and binds tightly to the active site of DHFR, perhaps in a conformation close to the transition state of the folate catalyzed reaction. Absorption, fluorescence and ultrasensitive Raman difference spectroscopies show that light-activated MTX reacts with NADPH in the enzyme active site, producing 5,8-dihydromethotrexate (5,8-dihydro-MTX) and NADP+. The reaction, which proceeds with a hydride transfer between C4 (pro-R side) of the nicotinamide ring and N5 of the pteridine ring, is similar to that between folate and NADPH except that the hydride is transferred to C6 in this case. Hence, MTX is catalytically competent in its excited state. Most experiments were performed on the Escherichia coli enzyme, but preliminary studies show that the reaction also occurs with human DHFR.     

Incorporating dynamics in E. coli dihydrofolate reductase enhances structure-based drug discovery

Escherichia coli dihydrofolate reductase (DHFR) is a long-standing target for enzyme studies. The influence of protein motion on its catalytic cycle is significant, and the conformation of the M20 loop is of particular interest. We present receptor-based pharmacophore models-an equivalent of solvent-mapping of binding hotspots-based on ensembles of protein conformations from molecular dynamics simulations of DHFR.NADPH in both the closed and open conformation of the M20 loop. The optimal models identify DHFR inhibitors over druglike non-inhibitors; furthermore, high-affinity inhibitors of E. coli DHFR are preferentially identified over general DHFR inhibitors. As expected, models resulting from simulations with DHFR in the productive conformation with a closed M20 loop have better performance than those from the open-loop simulations. Model performance improves with increased dynamic sampling, indicating that including a greater degree of protein flexibility can enhance the quest for potent inhibitors. This was compared to the limited conformational sampling seen in crystal structures, which were suboptimal for this application.     

Determination of copper in the presence of a large excess of bismuth by differential-pulse anodic-stripping voltammetry without preliminary separation

Conditions have been found which make possible the determination of copper in the presence of a large excess of bismuth by differential-pulse and anodic-stripping voltammetry without preliminary separation. The electrochemical activity of the bismuth, which usually interferes in the determination of copper, is inhibited by using tetrabutylammonium chloride (TBAC) as surfactant. In 0.2M EDTA and 0.01M ascorbic acid at pH 4.5 as supporting electrolyte without the surfactant present, trace levels of copper (1.5 x 10(-8)M) can be determined accurately if the molar ratio of bismuth to copper is not higher than 3, but if the electrolyte also contains TBAC at 0.01M concentration, bismuth can be tolerated in concentrations up to 10(-4)M, and the height of the copper peak is unaffected.     

Determination of thallic and thallous ions by differential pulse anodic stripping voltammetry without preliminary separation

The simultaneous determination of thallic and thallous ions, without preliminary separation, has been achieved by differential pulse anodic stripping voltammetry at a hanging mercury drop electrode. The electrochemical activity of thallic ions in 0.2M EDTA at pH 4.5 +/- 0.2 is inhibited by the addition of 0.01% poly(ethyleneglycol) of M.W. 20,000 (PEG 20,000). When the electrolyte also contains ascorbic acid at 0.01M concentration, the sum of thallic and thallous species can be determined.     

Improved extraction method for avoiding the interference of ascorbic acid in the spectrophotometric determination of nitrite in meat products

In minced meat products, ascorbic acid interferes in the usual spectrophotometric determination of nitrite by the diazotisation-coupling technique with sulphanilamide and N-(1-naphthyl)ethylenediamine. In a modified extraction method, the enzyme ascorbate oxidase was shown to diminish the interference of the acid. Within 10 min the enzyme was able to reduce the level of ascorbic acid in the minced meat specimens from 1000 p.p.m. down to the tolerance limit for interference. This appeared to be superior to the extraction method involving the use of water and was comparable to a previously described method based on the use of 0.01 M sodium hydroxide. As isoascorbic acid is not approved as a food additive in Norway no attempt was made to investigate the influence of this acid.     

A molecular model of the folate binding site of Pneumocystis carinii dihydrofolate reductase

Summary The inhibition of Pneumocystis carinii dihydrofolate reductase (DHFR) continues to be the major treatment strategy for P. carinii pneumonia (PCP). The design of new anti-pneumocystis agents would be significantly enhanced by the availability of a 3D model of the methotrexate (MTX) binding site of the P. carinii DHFR. However, an X-ray crystal structure of the P. carinii DHFR is not yet available. Alignment of the amino acid sequences of P. carinii and Lactobacillus casei DHFRs indicates that the two proteins show approximately 80% homology among MTX binding-site residues. This high level of homology suggests that the L. casei DHFR MTX binding-site structure could serve as a structural template in developing a model of the P. carinii DHFR MTX binding site. Therefore, the X-ray crystal structure of L. casei DHFR was used to develop a 3D model of the methotrexate binding site of P. carinii DHFR. The molecular modeling and dynamics software QUANTA/CHARMm was used. Amino acid residue mutations and deletions were performed using QUANTA and macromolecular minimizations were achieved with CHARMm. The MTX binding-site residues of L. casei DHFR were mutated to the corresponding residues of the P. carinii DHFR sequence. The resulting structure was extensively minimized. The resulting P. carinii MTX binding-site model showed significant differences in hydrogen-bonding patterns from the L. casei MTX binding site. Also, the P. carinii site is more hydrophobic than the corresponding L. casei site. Analysis of atom-to-atom close contacts between methotrexate and protein binding-site residues indicates that the P. carinii MTX binding-site complex is primarily stabilized by hydrophobic interactions, while the L. casei complex is mostly stabilized by electrostatic interactions. The model is consistent with the observed increased sensitivity of P. carinii DHFR to lipid-soluble inhibitors and provides a rational basis for the design of new anti-pneumocystis agents.     

Application of an external contactless conductivity detector for the analysis of beverages by microchip capillary electrophoresis

Quantitative total ionic analysis of alcoholic and nonalcoholic beverages was performed by microchip capillary electrophoresis with external contactless conductivity detection. An electrolyte solution consisting of 10.5 mM histidine, 50 mM acetic acid, and 2 mM 18-crown-6 at pH 4.1 was used for the determination of NH(4) (+), K(+), Ca(2+), Na(+), and Mg(2+). Fast analysis of Cl(-), NO(3) (-), and SO(4) (2-) was achieved in 20 mM 2-(N-morpholino)ethanesulfonic acid /histidine electrolyte solution at pH 6.0 and the simultaneous separation of up to 12 inorganic and organic anions was performed in a solution containing 10 mM His and 7 mM glutamic acid at pH 5.75. Limits of detection ranged from 90 to 250 mug/L for inorganic cations and anions, and from 200 to 2000 mug/L for organic anions and phosphate. Calibration curves showed linear dependencies over one to two orders of magnitude when the stacking effect was minimized by injecting standard solutions prepared in background electrolyte solutions. Total analysis times of 35 and 90 s were achieved for the determination of 5 inorganic cations and for the simultaneous determination of 12 inorganic and organic anions, respectively, which represents a considerable reduction of analysis time compared to conventional separation methods used in food analysis.     

Ru(III)-EDTA catalyzed oxidation of ascorbic acid by hydrogen peroxide in aqueous solution

The kinetics of oxidation of ascorbic acid to dehydroascorbic acid by hydrogen peroxide catalyzed by ethylenediaminetetraacetatoruthenate(III) has been studied over the pH range 1.50 – 2.50, at 30°C and μ = 0.1 M KNO₃. The reaction has a first-order dependence on ascorbic acid and Ru(III)-EDTA concentrations, an inverse first-order dependence on hydrogen ion concentration, and is independent of hydrogen peroxide concentration in the pH range studied. A mechanism has been proposed in which ascorbate anion forms a kinetic intermediate with the catalyst in a pre-equilibrium step. Ruthenium(III) is reduced to ruthenium(II) in a rate-determining step and is reoxidized with hydrogen peroxide back to the Ru(III) complex in a fast step.     

荧光光度法二氢叶酸还原酶抑制剂筛选模型的建立和应用

本文应用二氢叶酸(DHFA)和辅酶Ⅱ(NADPH)的荧光性质,建立了二氢叶酸还原酶(DHFR)活力测定的反应体系,并对酸度等反应条件进行了优化。在该体系中,用双倒数作图法分别测定了二氢叶酸还原酶对二氢叶酸和辅酶Ⅱ的表观米氏常数。将所建立的体系应用于氨甲喋呤,甲氧苄氨嘧啶等已知抑制剂的抑制率的测定,结果满意。     

Multiple ligand-binding modes in bacterial R67 dihydrofolate reductase

Summary R67 dihydrofolate reductase (DHFR), a bacterial plasmid-encoded enzyme associated with resistance to the drug trimethoprim, shows neither sequence nor structural homology with the chromosomal DHFR. It presents a highly symmetrical toroidal structure, where four identical monomers contribute to the unique central active-site pore. Two reactants (dihydrofolate, DHF), two cofactors (NADPH) or one of each (R67•DHF•NADPH) can be found simultaneously within the active site, the last one being the reactive ternary complex. As the positioning of the ligands has proven elusive to empirical determination, we addressed the problem from a theoretical perspective. Several potential structures of the ternary complex were generated using the docking programs AutoDock and FlexX. The variability among the final poses, many of which conformed to experimental data, prompted us to perform a comparative scoring analysis and molecular dynamics simulations to assess the stability of the complexes. Analysis of ligand–ligand and ligand–protein interactions along the 4 ns trajectories of eight different structures allowed us to identify important inter-ligand contacts and key protein residues. Our results, combined with published empirical data, clearly suggest that multipe binding modes of the ligands are possible within R67 DHFR. While the pterin ring of DHF and the nicotinamide ring of NADPH assume a stacked endo-conformation at the centre of the pore, probably assisted by V66, Q67 and I68, the tails of the molecules extend towards opposite ends of the cavity, adopting multiple configurations in a solvent rich-environment where hydrogen-bond interactions with K32 and Y69 may play important roles.     

Amperometric sensor for the determination of ascorbic acid based on Cobalt hexacyanoferrate modified electrode fabricated through a new route

A new approach was attempted to prepare a chemically modified electrode using Cobalt hexacyanoferrate (CoHCF) as the redox mediator and to study its stability and electrocatalytic activity for ascorbic acid (AA) oxidation. The basic principle underlying the electrode modification is the coordination of cobalt ion with the amino nitrogen of aniline adsorbed on the surface of a graphite rod. This surface was subsequently derivatized with ferrocyanide to get CoHCF film on the electrode surface. The CoHCF modified electrode as prepared above was characterized using cyclic voltammetry. The effect of scan rate, supporting electrolyte and pH of the medium on the performance of the modified electrode was investigated. The CoHCF modified electrode exhibited good electrocatalytic activity towards the oxidation of ascorbic acid and gave a linear response from 5.52 x 10(-5) M to 3.23 x 10(-2) M with a correlation coefficient of 0.9929. The detection limit was found to be 3.33 x 10(-5) M. Hydrodynamic voltammetry and chronoamperometry studies for the oxidation of ascorbic acid were also carried out. The electrode was highly stable and exhibited good reproducibility. This modified electrode was also applied for the determination of ascorbic acid in commercial samples.     

Fabrication of multiwalled carbon nanotubes/polyaniline modified Au electrode for ascorbic acid determination

An ascorbate oxidase (AsOx) (E.C.1.10.3.3) purified from Lagenaria siceraria fruit was immobilized covalently onto a carboxylated multiwalled carbon nanotubes and polyaniline (c-MWCNT/PANI) layer electrochemically deposited on the surface of an Au electrode. The diffusion coefficient of ascorbic acid was determined as 3.05 × 10(-4) cm(2) s(-1). The behavior of different electrolytes on electro-deposition was also studied. An ascorbate biosensor was fabricated using a AsOx/c-MWCNT/PANI/Au electrode as a working electrode, Ag/AgCl (3 M/saturated KCl) as standard and Pt wire as an auxiliary electrode connected through a potentiostat. Linear range, response time and detection limit were 2-206 μM, 2 s and 0.9 μM respectively. The biosensor showed optimum response at pH 5.8 and in a broader temperature range (30-45 °C), when polarized at +0.6 V. The biosensor was employed for determination of ascorbic acid level in sera, fruit juices and vitamin C tablets. The sensor was evaluated with 91% recovery of added ascorbic acid in sera and 6.5% and 11.4% within and between batch coefficients of variation respectively for five serum samples. There was a good correlation (r = 0.98) between fruit juice ascorbic acid values by the standard 2,6-dichlorophenolindophenol (DCPIP) method and the present method. The enzyme electrode was used 200 times over a period of two months, when stored at 4 °C. The biosensor has advantages over earlier enzyme sensors in that it has no leakage of enzyme, due to the covalent coupling of enzyme with the support, lower response time, wider working range, higher storage stability and no interference by serum substances.     

Enzymatic determination of glucose in a flow system by catalytic oxidation of the nicotinamide coenzyme at a modified electrode

A chemically modified electrode for detection of dihydronicotinamide adenine dinucleotide (NADH) and dihydronicotinamide adenine dinucleotide phosphate (NADPH) is described. Graphite rods were modified by dipping them into solutions of-dimethylamino-1,2-benzophenoxzinium salt (Meldola blue). The modified electrodes were mounted in a flow-through cell in a flow-injection manifold. Samples (50 μl) of pure nicotinamide coenzymes produced strictly linear calibration graphs from 1 μM to 10 mM with a repeatability of 0.2–0.6% RSD. A packed-bed enzyme reactor (210 μl) containing immobilized glucose dehydrogenase was inserted in the manifold for glucose determinations. Oxidized coenzyme was also added to the carrier electrolyte. Straight calibration graphs were again obtained up to 1mM β-d-glucose. The detection limit was 0.25 μM β-d-glucose for 50-μl samples. The electrode was kept at ?50 to 0 m V vs. SCE which was low enough to avoid interferences from ascorbic acid, uric acid or quinones.     

Determination of inorganic anions in saliva by capillary isotachophoresis

Nitrite, nitrate, iodide and thiocyanate have been quantified in non-smoker and smoker saliva by capillary isotachophoresis (CITP). Hydrochloric acid (10 mmol l⁻¹) adjusted with histidine to pH 6.0 plus 6% poly(vinylpyrrolidone) was used as the leading electrolyte (LE) and 5 mmol l⁻¹ acetic acid as the terminating electrolyte (TE). Linearity was observed from 0.005 to 0.500 mmol l⁻¹ with a coefficient of determination (r²) of 0.999. The separation of anions was achieved in less than 19 min. The minimal sample pretreatment and relatively low running cost make isotachophoresis good alternative to existing methods.     

Mechanistic studies on the oxidation of ascorbic acid and hydroquinone by a {Mn4O6}4+ core in aqueous media

Described in this work is the kinetics of oxidation of ascorbic acid and hydroquinone by a tetranuclear Mn(IV) oxidant, [Mn(4)(μ-O)(6)(bipy)(6)](4+) (1(4+), bipy =2,2(/)-bipyridine), in aqueous solution over a wide pH range 1.5-6.0. In particular, below pH 3.0, protonation on the oxo-bridge of 1(4+) results in the formation of [Mn(4)(μ-O)(5)(μ-OH)(bipy)(6)](5+) (1H(5+)) as an additional oxidant over 1(4+). Both ascorbic acid and ascorbate whereas only hydroquinone and none of its protolytic species were found to be reactive reducing agents in these reactions. Analysis of the rate data clearly established that the oxo-bridge protonated oxidant 1H(5+) is kinetically far more superior to 1(4+) in oxidizing ascorbic acid and hydroquinone. Rates of these reactions are substantially lowered in D(2)O-enriched media in comparison to that in H(2)O media. An initial one electron one proton transfer electroprotic rate step could be mechanistically conceived. DFT studies established that among the two sets of terminal and central Mn(IV) atoms in the tetranuclear oxidant, one of the two terminal Mn(IV) is reduced to Mn(III) at the rate step that we can intuitively predict considering the probable positive charge distribution on the Mn(IV) atoms.     

Polarographic reduction of 2,4,6-trinitrobenzene-1-sulphonic acid and some 2,4,6-trinitrophenyl-amino acid derivatives

The rapid d.c. polarography of 2,4,6-trinitrobenzene-1-sulphonic acid and its derivatives with serine (I), threonine (II), glycine (III) and histidine (IV) revealed a 3-wave reduction and a marked pH dependence of the reduction potential. The polarographic waves of the derivatives (2.5 × 10^?4 M) showed appreciable changes when sulphite ions were present, with the development of a new wave at more negative potential in ?0.01 M sulphite solutions at pH 7.0. The E_1/2 values of these waves in pH 7.0 supporting electrolyte were: (I) ?1000; (II) ?1007; (III) ?1021; (IV) ?949 mV (vs. Ag/AgCl, sat. KCl). These waves were used to determine the amino acids investigated (1–4 × 10^?4 M) in the presence of excess of 2,4,6-trinitrobenzene-1-sulphonic acid, with good precision (2%).     

Separation of thorium as ascorbato complex by extraction with Aliquat 336S

Thorium was quantitatively extracted with 0.1M Aliquat 336S at pH 4.5 from 0.01M ascorbic acid. It was then stripped with 2M hydrochloric acid. Thorium arsenazo III complex was determined spectrophotometrically at 655 nm. It was separated from binary and tertiary mixtures by exploiting the difference in distribution ratios of various elements from ascorbic acid media. Some separations were accomplished by selective stripping of thorium from nitric and hydrochloric acid. The method was extended for the analysis of thorium in monazite and gas mantles.     

Electrochemical behavior of the bis(2,2'-bipyridyl)copper(II) complex immobilized on a self-assembled monolayer modified electrode for L-ascorbic acid detection

Modification of a gold electrode has been achieved by immobilizing a bis(2,2'-bipyridyl)copper(II) complex in a self-assembled monolayer (SAM) of 3-mercaptopropionic acid. The electrostatic interaction of the negatively charged SAM with a di-positive copper complex allowed the attachment. The modified electrode exhibited excellent redox behavior. The dependence of the modified electrode response was investigated in terms of pH, supporting electrolyte and ionic strength. Moreover, it showed good electrocatalytic activity for ascorbic acid oxidation, allowing convenient quantification at levels down to 8.1 x 10(-8) mol l(-1). The [Cu(bipy)2]/SAM modified electrode under optimized operational conditions (PIPES buffer 0.01 mol l(-1) at pH 6.8 and 200 mV vs. SCE) presented a linear response range between 1.0 micromol l(-1) and 100.0 micromol l(-1) for ascorbic acid. This modified electrode also presented an excellent repeatability, showing a relative standard deviation of 2.1% for a series of 12 successive measurements of a 5.0 micromol l(-1) ascorbic acid solution. Furthermore, the electroactivity was maintained over a long period (e.g., 92% after 100 determinations).     

QUENCHING OF THE TRIPLET STATE OF CHLOROPHYLL a BY ASCORBIC ACID AND ASCORBATE IN ETHANOLIC SOLUTION

Abstract— Ascorbic acid and ascorbate in chlorophyll ethanol solution were found to be fairly efficient quenchers of the chlorophyll triplet state; comparable to the efficiency of ascorbic acid as a quencher in aqueous pyridine solution.
It has been well established that ascorbic acid quenches the triplet state of chlorophyll in aqueous pyridine solution.^(1,2) The bimolecular quenching constant, kQ , is very much less than that for O₂ or quinine.^(3,4)
Information regarding the quenching of the triplet state of chlorophyll by ascorbic acid in ethanolic solution is lacking. There has been some question as to whether ascorbic acid reduces photoexcited chloro-phyll-ethanolic solution because of its high oxidation potential, or because like the ascorbate ion, it acts only as a quencher; both ascorbic acid and ascorbate in high concentrations gave low quantum yields.⁽⁵⁾ The quenching of the triplet state by ascorbic acid and ascorbate was determined by the flash-photolytic method.     

Spectrophotometric studies on the uranyl-ascorbate complex

A systematic spectrophotometric study on the complexation of uranyl ions with ascorbic acid (vitamin C) is carried out. Factors influencing the sensitivity or selectivity of uranyl ascorbate are studied, and adequate elimination or control of the procedure is suggested. Results of the absorbance of uranyl ascorbate developed in HNO₃, HCl, and H₂SO₄ acid media within the concentration range (1.0–8.0) × 10⁻N show a marked dependence on the nature and concentration of the acid used. The observed decrease in color intensity at higher acidities is explained on the basis of the decomposing effect of hydrogen ions on uranyl ascorbate and the complexing effect on uranyl ions. Study of the stoichiometry of uranium:ascorbic acid complex in acid medium by continuous variation and molar ratio methods indicated the formation of 1:1 and 1:2 complexes. The formation constant of the 1:1 complex as calculated by these two methods was found to be 4.8 × 10⁴ and 5.0 × 10⁴, respectively. Best suitable conditions for stabilizing and increasing the color intensity of uranyl ascorbate, dependent on the nature of the medium and pH of the solution, are suggested for the determination of ascorbic acid in pharmaceutical preparations using this method.