Potassium permanganate–acridine yellow chemiluminescence system for the determination of fluvoxamine,isoniazid and ceftriaxone

Based on the oxidation of acridine yellow by permanganate in basic medium, a new chemiluminescence system was developed for the sensitive determination of some important drugs. The remarkable inhibiting effect of fluvoxamine, ceftriaxone and isoniazid on this reaction was applied to their detection. A possible mechanism was proposed for this system based on chemiluminescence emission wavelengths and experimental observations. Under optimum conditions, calibration graphs were obtained for 1 × 10^?9 to 1 × 10^?6 mol/L of fluvoxamine; 2 × 10^?8 to 8 × 10^?6 mol/L of ceftriaxone and 5 × 10^?8 to 4 × 10^?5 mol/L of isoniazid. This proposed method was satisfactorily used in the determination of these drugs in pharmaceutical samples and human urine and serum. Copyright © 2014 John Wiley & Sons, Ltd.     

Analysis of the binding of phenylalanine to phenylalanyl-tRNA synthetase

Using the complete rate equation for the PP_i-ATP exchange reaction at equilibrium, the dissociation constants of phenylalanine (10^?5m), phenylalanine butyl ester (8 × 10^?5m), benzyl alcohol (6 × 10^?4m), phenylalaninol (2 × 10^?4m), hydrocinnamic acid (3 × 10^?3m) and glycine (>1 m) with the phenylalanyl-tRNA synthetase (Escherichia coli K12) were determined. Taking the model of Koshland (1962) for the estimation of the configurational free energy change due to proximity and orientation, and decomposing the process of binding into several thermodynamic steps, the contribution to binding of the benzyl group, glycine unit, protonated amino group, carboxylate group and joint interactions were estimated. The results are: (1) the standard free energy contributions for binding phenylalanine are benzyl group (?8.2 kcal/mol), glycine unit (?2.5 kcal/mol), protonated amino group (?0.8 kcal/mol) and carboxylate group (1 kcal/mol). (2) The standard free energy change due to the change in the interaction between the protonated amino group and carboxylate group when they are transferred from the aqueous environment to the enzyme environment is ?2.7 kcal/mol. (3) A dissociation constant for glycine of 7.5 m is calculated without the hypothesis that a conformational change occurs in the enzyme when the benzyl unit of phenylalanine binds, permitting an interaction of the enzyme with the protonated amino and/or carboxylate groups.The detection of E·AA² and E·ATP shows that a sequential addition of substrates is not necessary for binding. A comparison of the dissociation constants of E·AA (10^?5m), E·ATP (1.5 × 10^?3m), E·PP (5.5 × 10^?4m), E·I (8 × 10^?5m) and the mixed complexes E·I·ATP (6 × 10^?8m²), E·I·PP (5 × 10^?8m²) and E·AA·PP (7 × 10^?9m²), with phenylalanine butyl ester as the inhibitor, indicates no strong interaction between the binding of ATP or PP_i with the binding of phenylalanine.     

A critical reexamination of the continous spectrophotometric assay for adenosine deaminase

The continuous spectrophotometric assay for adenosine deaminase has been reinvestigated, using both adenosine and 9-β-d-arabinofuranosyladenine as substrates. This assay is based on the reported decrease in absorbance at or near 265 nm between the adenine nucleoside substrate and the hypoxanthine nucleoside product. In the substrate concentration range 1,5 – 8.0 × 10^?4m, the progress of the reaction is associated with an anomalous sigmoidal dependence of absorbance on time, and the overall change in absorbance decreases with increasing substrate concentration. Near 8 × 10^?4m substrate, the deamination proceeds with no change in absorbance, while at higher concentrations, small increases in absorbance are observed. These effects, if ignored, generate initial “rate” data exhibiting an apparent substrate inhibition whieh, however, is completely an artifact induced by the spectral anomalies. Over the entire concentration range 5 × 10^?6–1 × 10^?3m, alternative assay methods (e.g., discontinuous detection of the product, ammonia) yeld normal Michaelis-Menten kineties. The anomalous behavior manifested in the continuous spectrophotometric assay is due to large negative deviations from Beer's law. These deviations are observed for all four of the nucleosides tested, viz., adenosine, 9-β-d-arabinofuranosyladenine, inosine, and 9-β-d-arabinofuranosylhypoxanthine. The departure from Beer's law is detectable anywhere in the concentration range 5 × 10^?6–1 × 10^?3m, but is most marked at concentrations above 1 × 10^?4m. Thus, the continuous spectrophotometric assay for adenosine deaminase should be utilized withextreme caution, and should not be employed at concentrations exceeding 1 × 10^?4m, irrespective of the K_m value for the substrate. Specific recommendations are given for future assays.     

Polarographic studies of the oxidation of vitamin K5 in aqueous solution.

Electrochemical behavior of 4-amino-2-methyl-1-naphthol hydrochloride (VK₅) has been studied by de and ac polarography in order to determine the applicability of these methods to the determination of VK₅ in aqueous solution. VK₅ exhibits two well-defined anodic waves on dc polarograms, one of which is due to the oxidation of VK₅ to the corresponding quinoimine and the other, to the formation of mercurous chloride on a dropping mercury electrode. Complete preremoval of dissolved oxygen makes the former wave reproducible, and its limiting current is found to be proportional to the VK₅ concentration ranging from 1.0 × 10^?5 to 1.0 × 10^?3m. On ac polarograms, electrooxidation of VK₅ also gives rise to a distinct peak whose height is proportional to the VK₅ concentration in the range of 5.0 × 10^?7 to 1.0 × 10^?5m. These facts can be applied to the microdetermination of VK₅ present in concentrations as low as the 0.1-ppm range.     

Sensitive determination of enoxacin in pharmaceutical formulations by its quench effect on the fluorescence of glutathione‐capped CdTe quantum dots

A sensitive and simple method for the determination of enoxacin (ENX) was developed based on the fluorescence quenching effect of ENX for glutathione (GSH)‐capped CdTe quantum dots (QDs). Under optimum conditions, a good linear relationship was obtained from 4.333 × 10^?9 mol?L^?1 to 1.4 × 10^?5 mol?L^?1 with a correlation coefficient (R) of 0.9987, and the detection limit (3σ/K) was 1.313 × 10^?9 mol?L^?1. The corresponding mechanism has been proposed on the basis of electron transfer supported by ultraviolet–visible (UV) light absorption, fluorescence spectroscopy, and the measurement of fluorescence lifetime. The method has been applied to the determination of ENX in pharmaceutical formulations (enoxacin gluconate injections and commercial tablets) with satisfactory results. The proposed method manifested several advantages such as high sensitivity, short analysis time, low cost and ease of operation. Copyright © 2015 John Wiley & Sons, Ltd.     

Salt-induced structural changes of nucleosome core particles.

The effects of sodium chloride concentration on the structure of chicken erythrocyte nucleosome core particles have been studied by the use of fluorescently labelled histones. Histone H3 was modified with two sulfhydryl-specific dyes and reconstituted into core nucleosomes. Between 10^?4 m and 0.6 M-NaCl four different states were observed by the fluorescent techniques of collisional quenching, polarization and energy transfer. Below 5 × 10^?4 m-NaCl the nucleosome is flexible, with the single cysteine residues of the two H3 species about 48 Å apart and somewhat exposed. Between 5 × 10^?3 m and 10^?1 m-NaCl the nucleosome is rigid and non-spherical. The cysteine residues are close together and buried. Between 10^?1 m and 4 × 10^?1 m-NaCl, the cysteines become slightly more exposed but remain close together. At 6 × 10^?1 m-NaCl the nucleosome is very flexible. The cysteines are more than 70 Å apart and are quite exposed. The dramatic structural changes that are observed in core nucleosomes are consistent with the variety of functions in which they must participate in the cell.     

Analysis of phenolic compounds in health care products by low‐pressure liquid‐chromatography with monolithic column and chemiluminescent detection

This paper presents a new application for monolithic columns with low‐pressure chromatographic separation using an flow injection analysis configuration with chemiluminescent detection for the determination of a mixture of phenolic compounds: phloroglucinol, 2,4‐dihydroxybenzoic acid, salicylic acid, methyl paraben and n‐propyl gallate. The procedure consists of the separation of these compounds on a reverse‐phase ultra‐short monolithic column with pH 3.0 acetate buffer and 5% acetonitrile as carrier phase. The detection is based on a chemiluminescence measurement coming from Ce(IV)–Rhodamine 6G chemistry with the incorporation of two different chemiluminescent chemical conditions in the chromatographic setup in order to enhance the sensitivity for the different phenolic compounds. All separation and detection variables were optimized to propose a determination method. The analysis is performed in 280?s, with the sampling frequency being some 13 h^?1. The calibration function is a double reciprocal function obtaining good results within two orders of magnitude. The limits of detection were 8.8 × 10 ^?8 m (phloroglucinol), 2.7 × 10 ^?8 m (2,4‐dihydroxybenzoic acid); 2.3 × 10 ^?8 m (salicylic acid); 5.2 × 10 ^?8 m (methyl paraben) and 4.1 × 10 ^?6 m (n‐propyl gallate), and the relative standard deviations at a medium level of the linear range were 4.4% (phloroglucinol), 2.8% (2,4‐dihydroxybenzoic acid), 5.2% (salicylic acid), 3.6% (methyl paraben) and 6.8% (n‐propyl gallate). The method was applied and validated satisfactorily for the determination of these compounds in healthcare products, comparing the results against an HPLC reference method. Copyright © 2009 John Wiley & Sons, Ltd.     

Some physicochemical factors relevant to cellular interactions

Colloidal stability theory is discussed to accomodate the conditions imposed by biological systems. It is shown that to obtain potential curves with secondary minima, Hamaker's constant must be in the range of 1–5 × 10^?14 ergs. The effect of increasing the dielectric constant is shown in theory to lower the surface potential and electrophoretic mobility but to increase the total energy of interaction. Calculations made from the theory predict the forces between model cells to be ca. 4.0 × 10^?7 dynes. By cone-plate shearing of cell aggregates, the most successful of several techniques tried and discussed, at shear rates approaching 1 × 10^?4 second^?1 (1.5 × 10^?4 dynes) semi-complete disaggregation was achieved although cell disruption was apparent; analysis of blood viscosity data indicates 5–10 × 10^?7 dynes are required to separate red cells suspended in plasma. Colloidal stability theory, while not applicable to cell systems associated by special areas of attachment, seems to describe the physicochemical interaction of freely moving or reversibly adherent cells.     

High‐sensitivity spectrofluorimetric determination of tiopronin based on inhibition of hemoglobin

A highly sensitive and simple spectrofluorimetric method for the determination of tiopronin based on its inhibitory effect on the hemoglobin‐catalyzed reaction of H₂O₂ and l ‐tyrosine was developed. The concentration of tiopronin is linear with decreased fluorescence (ΔF) of the system under the optimal experimental conditions. The calibration graph is linear in the range 1.23 × 10^?8 to 3.06 × 10^?5 mol L^?1 with a detection limit of 6.13 × 10^?9 mol L^?1. The relative standard deviation was 4.38% for 11 determinations of 6.13 × 10^?6 mol L^?1. This method can be used for the determination of tiopronin in pharmaceuticals with satisfactory results. Copyright © 2010 John Wiley & Sons, Ltd.     

MEMBRANE THIAMINE TRTPHOSPHATASE FROM RAT BRAIN: INHIBITION BY ATP AND ADP

—The hydrolysis of ThTP by rat brain membrane-bound ThTPase is inhibited by nucleoside diphosphates and triphosphates. ATP and ADP are most effective, reducing hydrolysis by 50% at concentrations of 2 × 10^?5m and 7·5 × 10^?5m respectively. Nucleoside monophosphates and free nuclcosides as well as P_i have no effect on enzyme activity. ThMP and ThDP also fail to inhibit hydrolysis in concentrations up to 5 × 10^?3m . Non-hydrolysable methylene phosphate analogs of ATP and ADP were used in further kinetic studies with the ThTPase. The mechanism of inhibition by these analogs is shown to be of mixed non-competitive nature for both compounds. An observed K_i, of 4 × 10^?5m for the ATP analog adenosine-PPCP and 9 × 10^?5m for the ADP analog adenosine-PCP is calculated at pH 6·5. Formation of the true enzyme substrate, the [Mg²⁺. ThTP] complex, is not significantly affected by concentrations of analogs producing maximal (>95%) inhibition of enzyme activity. Likewise the relationships between pH and observed K_m and pH and V_max are not shifted by the presence of similar concentrations of inhibitor.     

Catalytic properties of three lactate dehydrogenases from potato tubers (Solanum tuberosum)

Two l-lactate dehydrogenase isoenzymes and one dl-lactate dehydrogenase could be separated from potato tubers by polyacrylamide-gel electrophoresis. The enzymes are specific for lactate, while β-hydroxybutyric acid, glycolic acid, and glyoxylic acid are not oxidized. Their pH optima are pH 6.9 for the oxidation and 8.0 for the reduction reaction.The K_m values for l-lactate for the two isoenzymes are 2.00 × 10^?2 and 1.82 × 10^?2, m. In the reverse reaction the affinities for pyruvate are 3.24 × 10^?4 and 3.34 × 10^?4, m. Both enzymes have similar affinities for NAD and NADH (3.00 × 10^?4; 4.00 × 10^?4, and 8.35 × 10^?4; 5.25 × 10^?4, m).The dl-lactate oxidoreductase may transfer electrons either to NAD or N-methyl-phenazinemethosulfate. The K_m values of this enzyme for l-lactate are 4.5 × 10^?2, m and for d-lactate 3.34 × 10^?2, m. Its affinity for pyruvate is 4.75 × 10^?4, m. The enzyme is inhibited by excess NAD (K_m = 1.54 × 10^?4, M) and has an affinity toward NADH (K_m = 5.00 × 10^?3, M) which is about one tenth of that of the two isoenzymes of l-lactate dehydrogenase.     

Terbium‐sensitized fluorescence method for the determination of deferasirox in biological fluids and tablet formulation

A novel, rapid and sensitive spectroflurimetric method was developed and validated for the determination of deferasirox in urine, serum and tablet samples based on sensitization of terbium fluorescence. The excitation and emission wavelengths were 328 and 545 nm, respectively. The optimum conditions for the determination of deferasirox were investigated considering the effects of various parameters. The method was quantitatively evaluated in terms of linearity, recovery, reproducibility and limit of detection. Under the optimal conditions, the fluorescence intensities were linear with the concentration of deferasirox in the range of 5 × 10^?9 to 5×10^?6 mol L^?1, with a detection limit of 1.5 × 10^?9 mol L^?1 and a relative standard deviation of 1.1–2.3%. Linearity, reproducibility, recovery and limit of detection made the method suitable for determination of deferasirox in urine, serum and tablets samples. Copyright © 2010 John Wiley & Sons, Ltd.     

A serotonin sensitive adenylate cyclase in mature rat brain synaptic membranes

Results from this study have indicated serotonin-sensitive adenylate cyclase activity in adult rat brain. The enzyme is localized in the synaptosomal plasma membrane and apparently has multiple activation sites for serotonin with specific activity maxima occuring at serotonin concentrations of 5 × 10^?10, 5 × 10^?9, 1 × 10^?8, and 5 × 10^?8 moles/liter. The production of cyclic AMP at these sites appears to be unaffected by 1 × 10^?5M fluphenazine, while 1 × 10^?5M tryptamine, methysergide, and ergonovine decreased the stimulatory effect of 1 × 10^?8M 5-HT by 30 percent, 80 percent, and 57 percent respectively.     

Evaluation of the effect of Tb(IV)-NR complex on herring sperm DNA genetic information by mean of spectroscopic

Abstract

The interaction between Tb(IV)-NR complex and herring sperm DNA in buffer solution of Tris-HCl was investigated with the use of acridine orange(AO) as a spectral probe. The binding modes and other information were provided by the UV–spectrophotometry and fluorescence spectroscopy. The thermodynamic functions expressed that the binding constants of Tb(IV)-NR complex with DNA was K^θ_298.15K = 4.03?×?10⁵?L·mol^?1, K^θ_310.15K =1.30?×?10⁷?L·mol^?1, and the Δ_rGθ m _298.15?K＝?3.20?×?10⁴ J·mol^?1. The scatchard equation suggested that the interaction mode between Tb(IV)-NR complex and herring sperm DNA is electrostatic and weak intercalation bindings. FTIR spectroscopy results also indicate that there is a specific interaction between the Tb(IV)-NR complex and the A and G bases of DNA.     

Spectrophotometric determination of bromopyruvate by reaction with 2-nitro-5-thiobenzoic acid

A spectrophotometric method for determination of bromopyruvate, based on the reaction with 2-nitro-5-thiobenzoic acid, is described. The reaction is complete in 30 min at room temperature in 0.1 m Tris-MES, 1 mm EDTA, pH 8.0. The method is sensitive to at least 1 × 10^?5m bromopyruvate. Reagents are stable, easy to prepare, and specific for β-halopyruvate. Bromopyruvate solutions must be prepared fresh daily. Solutions of bromopyruvate at pH 8.0 and 23°C have a half-life of 3 hr.     

Oligosaccharides of human milk. Isolation and characterization of three new disialyfucosyl hexasaccharides.

Renal mitochondrial 25-hydroxyvitamin D₃-1-hydroxylase (1-hydroxylase) is sensitive to inhibition by 2 × 10^?5m calcium and 5 × 10^?3m phosphate when hydroxylation is supported by either malate or NADPH. This sensitivity to ion inhibition is observed in mitochondria from both vitamin D-deficient and repleted chicks and remains when mitochondria are frozen and thawed or are incubated in a hypotonic medium. The ionophore A23187 inhibits the 1-hydroxylase but partially reverses the inhibition exerted by 2, 5, or 7.5 × 10^?5m calcium. Addition of a kidney soluble cell fraction (37,000g supernatant) to isolated mitochondria did not enhance the 1-hydroxylase activity under conditions of varied substrate concentration, osmolarity of the incubation medium, or mitochondrial washes. It is concluded that a soluble cellular component is not involved in the regulation of the 1-hydroxylase but that intramitochondrial calcium and phosphate may well play a role in its regulation.     

Quantum‐dots‐based fluoroimmunoassay for the rapid and sensitive detection of avian influenza virus subtype H5N1

The continuous spread of highly pathogenic avian influenza virus (AIV) subtype H5N1 is threatening the poultry industry and human health worldwide. Rapid and sensitive diagnostic methods are required for the H5N1 surveillance. In this study, the fluorescent (FL) probe of CdTe quantum dots (QDs) was designed using covalently linked rabbit anti‐AIV H5N1 antibody. Based on these QD–antibody conjugates, a novel sandwich FL‐linked immunosorbent assay (sFLISA) was developed for H5N1 viral antigen detection. The sFLISA allowed for H5N1 viral antigen determination in a linear range of 8.0 × 10^?3 to 5.1 × 10^?1 μg mL^?1 with the limit of detection (LOD) of 1.5 × 10^?4 μg mL^?1. In comparison with virus isolation for 103 clinic samples, the sensitivity and specificity of sFLISA were found to be 93.6 and 91.1% respectively. The sFLISA supplied a novel approach to rapid and sensitive detection of AIV subtype H5N1 and showed great potential for biological applications in immunoassays. Copyright © 2009 John Wiley & Sons, Ltd.     

The interaction of substrate-related ketals with bacterial and viral neuraminidases

Arthrobacter sialophilus neuraminidase catalyzes the hydration of 5-acetamido-2,6-anhydro-3, 5-dideoxy-d-glycero-d-galacto-non-2-enonic acid (2,3-dehydro-AcNeu) with K_m and k_cat values of 8.9 × 10^?4m and 6.40 × 10^?4 s^?1, respectively. The methyl ester of 2,3-dehydro-AcNeu as well as 2,3-dehydro-4-epi-AcNeu are also hydrated by the enzyme. The product resulting from the enzymatic hydration of 2,3-dehydro-AcNeu is N-acetylneuraminic acid. A series of derivatives of 2,3-dehydro-AcNeu (K_I 1.60 × 10^?6m) including 2,3-dehydro-4-epi-AcNeu (2.10 × 10^?4m) and 2,3-dehydro-4-keto-AcNeu (K_I = 6.10 × 10^?5 m) were each competitive inhibitors of the enzyme. The methyl esters of these ketal derivatives were also competitive enzyme inhibitors. Dissociation constants for these ketals were determined independently by fluorescence enzyme titrations which gave values similar to those found kinetically. These six relatives of 2,3-dehydro-AcNeu were also competitive inhibitors for the influenza viral neuraminidases. For the viral neuraminidases, the dissociation constant for 2,3-dehydro-AcNeu and its methyl ester were 2.40 × 10^?6 and 1.17 × 10^?3m, respectively. The interpretation placed upon the K_I values determined for these ketals against the Arthrobacter versus influenza neuraminidases is that the bacterial enzyme has a more flexible glycone binding site.     

Rabbit brain purine nucleoside phosphorylase. Physical and chemical properties. Inhibition studies with aminopterin, folic acid and structurally related compounds.

Rabbit brain purine nucleoside phosphorylase used in this study was purified 6000-fold to apparent homogeneity and a specific activity or 50 μmol min^?1 mg ^?1 protein. A molecular weight of 70.000 daltons was determined for the native enzyme by gel filtration on Sephadex. Electrophoresis on polyacrylamide gel, in presence of sodium dodecyl sulfate, gave a subunit molecular weight of 34,500 daltons, suggesting that the enzyme is dimeric with, probably, identical subunits. The relationship of the structure of certain biologically active substances to their inhibitory action on the enzyme was examined. Folic acid and the compound d,l-6-methyl 5,6,7,8-tetrahydropterine, with similar substituents on their primary ring structure, were competitive inhibitors of the enzyme. The inhibition constants calculated were 3.37 × 10^?5M for folic acid and 3.80 × 10^?5m for d,l-6-methyl 5,6,7,8-tetrahydropterine. Aminopterin and the purine analog 8-aza-2,6-diaminopurine, with similar substituents on their primary ring structure, were noncompetitive inhibitors of the enzyme. Their respective inhibition constants were 1.50 × 10^?4 and 1.95 × 10^?4m. Erythro-9-(2-hydroxy-3-nonyl) adenine, an adenosine deaminase inhibitor, was also examined for inhibitory potency with mammalian purine nucleoside phosphorylase, and was observed to be a competitive inhibitor of this enzyme, with an inhibition constant of 1.90 × 10^?4m. The Michaelis constant for the substrate guanosine was near 6.0 × 10^?5m. Physical probe of the nature of the functional groups which participate in enzymic catalysis implicated both histidine and cysteine as the essential catalytic species. Photooxidation studies suggested a pH-dependent sensitivity of an essential catalytic group, and its probable location at the active site.     

Determination of carbamazepine in human urine and serum samples by high‐performance liquid chromatography with post‐column Ru(bipy)‐Ce(SO4)2 chemiluminescence detection

A novel, sensitive and rapid CL method coupled with high‐performance liquid chromatography separation for the determination of carbamazepine is described. The method was based on the fact that carbamazepine could significantly enhance the chemiluminescence of the reaction of cerium sulfate and tris(2,2‐bipyridyl) ruthenium(II) in the presence of acid. The chromatographic separation was performed on a Kromasil® (Sigma‐Aldrich) TM RP‐C18 column (id: 150 mm × 4.6 mm, particle size: 5 µm, pore size: 100 Å) with a mobile phase consisting of methanol–water‐glacial acetic acid (70:29:1, v/v/v) at a flowrate of 1.0 mL/min, the total analysis time was within 650 s. Under optimal conditions, CL intensity was linear for carbamazepine in the range 2.0 × 10^?8 ~ 4.0 × 10^?5 g/mL, with a detection limit of 6.0 × 10^?9 g/mL (S/N = 3) and the relative standard detection was 2.5% for 2.0 × 10^?6 g/mL (n = 11). This method was successfully applied to the analysis of carbamazepine in human urine and serum samples. The possible mechanism of the CL reaction is also discussed briefly. Copyright © 2012 John Wiley & Sons, Ltd.