Characteristics of a protein error in determination of serum protein in the presence of inorganic salt.

There is a possibility that the color development of the dye-binding method based on a protein error of a pH indicator is affected by the coexisting inorganic salt. Thus, the author theoretically and experimentally investigated the effect of the inorganic salt on the protein error. In a theoretical analysis, the anion of an inorganic salt, like the dissociated dye and buffer anions, was assumed to react with the protein, forming a colorless anion-protein complex. The calculated results were compared with those obtained by experiments using three pH indicators and various kinds of inorganic salts. The calculated results obtained are as follows: (1) The color development decreases with increasing the concentration of the inorganic salt and the equilibrium constant of the reaction between the inorganic salt and protein; (2) The rate of the absorbance decrease is larger for a lower concentration of the inorganic salt than for a higher one; (3) The larger is the equilibrium constant, the larger is the absorbance decrease. The absorbance decrease was caused by the anion, and was increased by increasing the anion concentration. The magnitude of the effect of the anion was iodide > bromide > chloride, which was associated with their ionic radius. The difference in the effect of the anion was thought to indicate that the equilibrium constant, in other words, the bonding strength of the anion to protein is iodide > bromide > chloride.     

Mechanism of the oxidation of alcohols by oxoammonium cations

The mechanism of the oxidation of primary and secondary alcohols by the oxoammonium cation derived from 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) has been investigated computationally at the B3LYP/6-31+G* level, along with free energies of solvation, using a reaction field model. In basic solution, the reaction involves formation of a complex between the alkoxide anion and the oxoammonium cation in a pre-oxidation equilibrium wherein methoxide leads to a much larger formation constant than isopropoxide. The differences in free energy of activation for the rate-determining hydrogen transfer within the pre-oxidation complexes were small; the differences in complex formation constants lead to a larger rate of reaction for the primary alcohol, as is observed experimentally. In acidic solution, rate-determining hydrogen atom transfer from the alcohol to the oxoammonium cation had a large unfavorable free energy change and would proceed more slowly than is observed. A more likely path involves a hydride transfer that would be more rapid with a secondary alcohol than primary, as is observed. Transition states for this process were located.     

Synthesis of chromo‐ and fluorogenic poly(ortho‐diaminophenylene) chemosensors for fluoride anion

Two new poly(ortho‐diaminophenylene) derivatives containing fluorene and/or quinoxaline moieties per repeat unit in the main chain were synthesized via Suzuki coupling reaction followed by reduction process. The synthesized polymers were characterized and explored as colorimetric and fluorometric anion‐sensing materials. The polymers in dilute tetrahydrofuran (THF) solution emitted green light (about 530 nm) in their precursor benzothiadiazole forms and blue to green light (477–523 nm) in their reduced forms. The color of polymer solution was dramatically altered upon addition of fluoride anion without noticeable absorption change in UV–vis spectrum. The fluorescence was ratiometrically quenched with a linear relationship between fluorescence intensity and fluoride anion concentration implying static quenching mechanism could be applied judging from the maintenance of constant fluorescence lifetime with variable fluoride anion concentration. © 2007 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 45: 1546–1556, 2007     

Effect of the pH, the concentration and the nature of the buffer on the adsorption mechanism of an ionic compound in reversed-phase liquid chromatography. II. Analytical and overloaded band profiles on Symmetry-C18 and Xterra-C18

In a previous report, the influence of the pH, the concentration, and the nature of the buffer on the retention and overloading behavior of propranolol (pKa = 9.45) was studied on Kromasil-C18 at 2.75 < pH < 6.75, using four buffers (phosphate, acetate, phthalate, and succinate), at three concentrations, 6, 20, and 60 mM. The results showed that the propranolol cation was eluted as an ion-pair with the buffer counter-anion. A similar study was carried out with Symmetry-C18 and Xterra-C18. Two additional buffers, formate and citrate, were also used. Propranolol elution band profiles were recorded for a small (less than 1 microg) and a large (375 microg) sample size. The results are similar to those obtained with Kromasil and confirm earlier conclusions. The buffer concentration, not its pH, controls the retention time of propranolol, in agreement with the chaotropic model. The retention factor depends also on the nature of the buffer, particularly on its valence, and on the hydrophobicity of the basic anion. With the monovalent anions HCOO- (pH 3.75), H2PO4- (pH 2.75), HOOC-Ph-COO- (pH 2.75), HOOC-CH2-CH2-COO- (pH 4.16), CH3COO- (pH 4.75) and HOOC-CHCOOH-COO- (pH 3.14), at moderate loadings, and for the two larger buffer concentrations, the band profiles are well accounted for by a simple bi-Langmuir isotherm model (no adsorbate-adsorbate interactions). By contrast, these profiles are accounted for by a bi-Moreau isotherm model (i.e., with significant adsorbate-adsorbate interactions) with the bivalent anions -OOC-Ph-COO- (pH 4.75), -OOC-CH2-CH2-COO- (pH 5.61), HPO4(2-) (pH 6.75), and HOOC-CHCOO(-)-COO- (pH 4.77) and with the trivalent anion -OOC-CHCOO(-)-COO- (pH 6.39). The best values of the isotherm parameters were determined using the inverse method. The saturation capacity and the equilibrium constant on the low-energy sites increase with increasing buffer concentration, a result consistent with the formation in the mobile phase of a hydrophobic complex between the propranolol cation and the buffer anion. With bivalent and trivalent anions, adsorbate-adsorbate interactions are strong on the low-energy sites but they remain negligible on the high-energy sites. The density of the high energy sites is lower and the equilibrium constant on the low-energy sites are both higher with the bivalent and the trivalent buffer anions than with the univalent buffer anions. These results are consistent with the formation of a 2:1 and a 3:1 propranolol-buffer complex with the bivalent and the trivalent anions, respectively.     

Study on Cyclometalated Palladium-azo Complexes as Colorimetric Probes for Hazardous Gas in Water

A synthesized cyclometalated palladium-azo complex was explored as a multifunctional probe for visual detection of SO2, H2S and NH3 in water. In acidic aqueous environment, the sensing solution underwent a sharp color change from poor violet to deep blue when titrated with Na2SO3 standard solution. But the color changed from poor violet to bright yellow when titrated with Na2S standard solution. In basic environment, the sensing solution rapidly changed to magenta when titrated by NH4Cl-NH3 standard buffer solution at high concentration. However, the color of sensing solution changed to blue when titrated by NH4Cl-NH3 standard buffer solution at low concentration although the pH was kept constant during the titration. Different species of these hazardous gases at environmentally relevant concentration levels were differentiated by independent optical signal outputs, and the interference from other inorganic ions commonly existing in water was very small.     

色谱法研究杂多酸VI. 12-钨硼酸根离子溶液中降解反应的研究

本文将胶束液相色谱用于α-BW~12O~40^5-溶液中降解反应的研究。首次发现α-BW~12O~40^5-降解存在两个过程。第一步为α-BW~12O~40^5-向其异构体转化的快速平衡反应, 第二步为该异构体的缓慢降解。实验测定了杂多酸离子浓度、pH值、无机盐逍度、温度对降解反应的影响, 得到第一步反应的平衡表达式和第二步反应的速率方程式, 推断了α-BW~12O~40^5-降解反应的机理。     

Spectrophotometric determination of phosphate anion based on the formation of molybdophosphate in ethylene glycol-water mixed solution

New appropriate reaction system was found for spectrophotometric determination of phosphate anion. This spectrophotometric method is based on the color development due to the formation of yellow molybdophosphate anion in acidic ethylene glycol-water (EG-W) mixed solution containing Mo(VI) species. The solution containing e.g. 20 mM Na(2)MoO(4), 0.1 M HCl, and 40% (v/v) EG is colorless, and becomes immediately yellow by addition of phosphate anion. Thus the method is simple, rapid, and easy to carry out. Although Si(IV) species is well known to interfere with the determination of phosphate anion in many cases, the EG-W Mo(VI) solution remains colorless after addition of silicate anion at 1 mM level, indicating that no yellow molybdosilicate anion was formed in the EG-W solution. Under an optimized condition, the absorbance at e.g. 400 nm of the EG-W P(V)-Mo(VI) solution was proportional to the concentration of phosphate anion with good reproducibility, and the detection limit was 1 μM. Also the present method is less interfered by high concentrations of potassium and ammonium cations and oxidative nitrite anion as well as silicate anion.     

Buffer salt effect on pH in the interior of an anion exchange resin

The internal pH of Q Sepharose Fast Flow anion exchange resin in equilibrium with a bis-tris acetate buffer solution is investigated as a function of buffer salt concentration. Direct evidence of a resin phase pH shift is presented. At low buffer salt concentrations of 20 mM NaCl the resin phase pH is found to be as much as 1.1 pH units greater than that of the buffer phase, approaching to within 0.1 units of the buffer phase at salt concentrations greater than 250 mM. An ideal model with no adjustable parameters based on the Boltzmann distribution and the electroneutrality condition provides excellent agreement with experimental observations. The model assumes that small ions do not bind to the resin fixed charge sites and the agreement between the model predictions and observed resin internal pH suggests that strong electrolytes do not form ion pairs with the resin fixed charge sites.     

Enantiomeric separation of gemfibrozil chiral analogues by capillary electrophoresis with heptakis(2,3,6-tri-O-methyl)-beta-cyclodextrin as chiral selector

The enantiomeric separation of gemfibrozil chiral analogues was performed by capillary zone electrophoresis (CZE). Resolution of the enantiomers was achieved using heptakis(2,3,6-tri-O-methyl)-beta-cyclodextrin (TM-beta-CD) as chiral selector dissolved into a buffer solution. In order to optimize the separation conditions, type, pH and concentration of running buffer and chiral selector concentration were varied. For each pH value, the optimum chiral selector concentration that produced the resolution of the isomers was found. The migration order of labile diastereoisomers formed was valued at the optimum experimental conditions by adding a pure optical isomer to the racemic mixture. Data from 1H NMR studies confirmed host-guest interaction between TM-beta-CD and 5-(2,5-dimethylphenoxy)-2-ethylpentanoic acid sodium salt. The hypothesized stoichiometry host:guest was 1:1. An apparent equilibrium constant (Ka) was estimated monitoring the chemical shift variation as a function of TM-beta-CD concentration. Salt effect on complexation equilibrium constant was also investigated.     

Role of the buffer in retention and adsorption mechanism of ionic species in reversed-phase liquid chromatography. I. Analytical and overloaded band profiles on Kromasil-C18

The influence of the pH, the concentration, and the nature of the buffer on the retention and overloading behavior of propranolol (pKa = 9.25) on Kromasil-C18 was studied at 2.75 < pH < 6.75, using four buffers (phosphate, acetate, phthalate, and succinate), at three concentrations, 6, 20, and 60 mM. The propranolol band profiles were recorded for three sample sizes, less than 1 microg and 375 microg (sample less concentrated than the buffer), and 7500 microg (band more concentrated than the buffer). Results showed that the buffer concentration, not its pH, controls the retention time of propranolol, in agreement with the chaotropic model. The retention factor depends also on the nature of the buffer, particularly the valence of the basic anion. At moderate loading, the band profiles are well accounted for by a simple bilangmuir model (no adsorbate-adsorbate interactions) with the monovalent anions H2PO4- (pH 2.75), HOOC-Ph-COO- (pH 2.75), HOOC-CH2-CH2-COO- (pH 4.16) and CH3COO- (pH 4.75), and by a bimoreau model (significant adsorbate-adsorbate interactions) with the bivalent anions OOC-Ph-COO- (pH 4.75), OOC-CH2-CH2-COO- (pH 5.61) and HPO4(2-) (pH 6.75). The isotherm were determined using the inverse method. The results show that both the saturation capacity and the equilibrium constant on the low-energy sites increase with increasing buffer concentration, a result similar to that observed with neutral salts. For bivalent anions, the adsorbate-adsorbate interactions are much stronger on the low than on the high energy sites. The density of high energy sites is lower and the equilibrium constant on the low energy sites are higher with bivalent than with univalent anions. These results are consistent with the formation of a propranolol-buffer (2:1) complex with bivalent anions.     

Influence of pH for the determination of serum albumin by a dye-binding method in the presence of a detergent

In the dye-binding method, the absorbance increase caused by a protein error of a pH indicator is observed only in a restricted pH range. However, this pH range in the presence of a detergent has not yet been examined. Thus, the author investigated the pH (pH(UL)) where the absorbance increase becomes zero by a calculation based on the chemical equilibrium of a protein error of a pH indicator, and by experiments using four sulfonephthalein dyes. The pH(UL) value changed only with the detergent concentration, but did not change at all due to the dye, buffer solution or protein concentrations. Although the pH(UL) value was different according to the kind of dye used, it correlated well with the pK(D) values (dissociation constant) of BPB, BCG, BCP and BTB. The characteristics of pH(UL) in the reactions of the four dyes indicated good agreement with that obtained by a calculation.     

Eine allgemeine theorie der massanalytischen umschlagsschärfe

The buffer capacity of the final solution (exactly titrated to equivalence) of a titration can be calculated from. the equilibrium constant of the titrating reaction. It enables us to establish a theory of the “end point sharpness” applicable universally to the different classes of titrations (acidimetric, redox, precipitations, etc.). The results are more accurate than the approximations derived up to now from the study of the titrated solution further off the equivalence point.The use of an inproper indicator will not only produce erroneous values (high or low) in the titration but at the same time lessen considerably the sharpness of the color (etc.) change at the end point as the buffer capacity of the titrated solution is greater at any than the equivalence point.     

High pH Solubilization and Chromatography-Based Renaturation and Purification of Recombinant Human Granulocyte Colony-Stimulating Factor from Inclusion Bodies

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) is a very efficient therapeutic protein drug which has been widely used in human clinics to treat cancer patients suffering from chemotherapy-induced neutropenia. In this study, rhG-CSF was solubilized from inclusion bodies by using a high-pH solution containing low concentration of urea. It was found that solubilization of the rhG-CSF inclusion bodies greatly depended on the buffer pH employed; alkalic pH significantly favored the solubilization. In addition, when small amount of urea was added to the solution at high pH, the solubilization was further enhanced. After solubilization, the rhG-CSF was renatured with simultaneous purification by using weak anion exchange, strong anion exchange, and hydrophobic interaction chromatography, separately. The results indicated that the rhG-CSF solubilized by the high-pH solution containing low concentration of urea had much higher mass recovery than the one solubilized by 8 M urea when using anyone of the three refolding methods employed in this work. In the case of weak anion exchange chromatography, the high pH solubilized rhG-CSF could get a mass recovery of 73%. The strategy of combining solubilization of inclusion bodies at high pH with refolding of protein using liquid chromatography may become a routine method for protein production from inclusion bodies.     

Guidance for selecting the measurement conditions in the dye-binding method for determining serum protein: theoretical analysis based on the chemical equilibrium of protein error.

A methodology for selecting the measurement conditions in the dye-binding method for determining serum protein has been studied by a theoretical calculation. This calculation was based on the fact that a protein error occurs because of a reaction between the side chains of a positively charged amino acid residue in a protein molecule and a dissociated dye anion. The calculated characteristics of this method are summarized as follows: (1) Although the reaction between the dye and the protein occurs up to about pH 12, a change in the color shade, called protein error, is observed only in a pH region restricted within narrow limits. (2) Although the apparent absorbance (the absorbance of the test solution measured against a reagent blank) is lower than the true absorbance indicated by the formed dye-protein complex, the apparent absorbance correlates with the true absorbance with a correlation coefficient of 1.0. (3) At a higher dye concentration, the calibration curve is more linear at a higher pH than at a lower pH. Most of these characteristics were similarly observed experimentally in the reactions of BPB, BCG and BCP with human and bovine albumins. It is concluded that in order to ensure the linearity of the calibration curve, the measurement should be performed at a higher dye concentration and sufficiently high pH where the detection sensitivity is satisfied.     

Effect of the nature of a solvent on pseudoliving free-radical polymerization of styrene mediated by TEMPO

The kinetics and mechanism of the pseudoliving free-radical polymerization of styrene are studied for the first time under the conditions of reversible inhibition by TEMPO nitroxides. An abnormal decline in the reduced rate of polymerization, which is inconsistent with a decrease in the concentration of the monomer, and an increase in the steady-state concentration of the free nitroxide are discovered. The main quantitative characteristics of the pseudoliving process are determined, namely, the rate constants of reinitiation and reversible recombination, and the constant of pseudoliving equilibrium between dormant and growing chains. It is shown that the polarity of a medium and the concentration of the monomer determine the character of polymerization in a solution: With an increase in the polarity of the solvent, the pseudoliving equilibrium constant increases, the reduced rate of styrene polymerization decreases, and the molecularmass-distribution of the polymer formed at initial conversions narrows. The smaller the concentration of styrene in the reaction system, the more pronounced the above differences associated with the solvent nature.     

Electrochemical Studies of Pirarubicin and Its Interaction with DNA at a Co／GC Ion Implantation Modified Electrode

IntroductionIon implantation is a new material surfacemodification technique.It has been also applied tostudying the electrochemical behaviors of organicdrugs and biological materials as well as their de-terminations. This method offers good stability,reproductivity and catalytic activity[1] .Pirarubicin( THP) is an active highly effective and new antitu-moral anthracycline antibiotic,which has gainedwidespread clinical use in the chemotherapeutictreatment of a variety of human cancers.The d…     

STEADY-STATE FLUORESCENCE METHOD FOR EVALUATING EXCITED STATE PROTON REACTIONS: APPLICATION TO FLUORESCEIN

Abstract Fluorescein is a complex fluorophore in the sense that it displays four prototropic forms (cation, neutral, monoanion and dianion) in the pH range 1–9. In experiments with fluorescein-labeled proteins we have sometimes observed complex nanosecond emission kinetics, which could be due to conversion of the excited monoanion into the excited dianion through an excited state proton exchange with a proton acceptor in the labeled protein. However, the literature is ambiguous on whether this possible excited state proton reaction of fluorescein does occur in practice. In this article we describe a general steady-state fluorescence method for evaluating excited state proton reactions of simple as well as complex pH-sensitive fluorophores and apply it to evaluate excited state proton reactions of fluorescein. The method depends on finding a buffer that can serve as an excited state proton donor-acceptor but does not significantly perturb ground state proton equilibrium and especially does not form ground (or excited state complexes) with the fluorophore. Our results show that the excited monoanion-dianion proton reaction of fluorescein does occur in the presence of phosphate buffer, which serves as a proton donor-acceptor that does not significantly perturb ground state proton equilibria. The reaction becomes detectable at phosphate buffer concentrations greater than 20 mM and the reaction efficiency increases with increase in phosphate buffer concentrations. The reaction is most clearly demonstrated by adding phosphate buffer to a solution of fluorescein at constant pH 5.9 with preferential excitation of the monoanion. Under these conditions, the excited monoanion converts to the dianion during its lifetime. The conversion is detected experimentally as an increase in dianion and decrease in monoanion fluorescence intensities with increase in phosphate buffer concentration. The absorption spectrum is not significantly perturbed by the increase in phosphate buffer concentration. To quantitate the reaction, we have recorded titration graphs of fluorescence intensity versus pH for fluorescein solutions at low (5 mM) and high buffer (1 M) concentrations with preferential excitation of the monoanion and preferential detection of the dianion emission. We have also developed theoretical expressions that relate fluorescence intensity to pH in terms of the concentration of the four prototrophic forms of fluorescein, extinction coefficients, fluorescence efficiencies and ground and excited state pK_a. The theoretical expressions give very good fits to the experimental data and allow evaluation of fundamental parameters such as pK_a and fluorescence efficiencies. The analysis of the experimental data shows that the excited monoanion-dianion reaction does not significantly occur at 5 mM phosphate buffer concentration. However, at 1 M buffer concentration the reaction is sufficiently fast that it practically achieves equilibrium during the lifetimes of the excited fluorescein monoanion and dianion. The pK_a* of the excited monoanion-dianion proton reaction is around 6.3. The results and methods presented here should be useful in the development and testing of pH-sensitive labeling fluorophores and fluorescent indicators.     

Stability prediction of nafamostat mesilate in an intravenous admixture containing sodium bisulfite

The hydrolysis of nafamostat mesilate (NM) in aqueous solution was found to be accelerated by sodium bisulfite (SBS) using high-performance liquid chromatographic assay. The hydrolysis of NM catalyzed by SBS was pseudo-first-order and was considered to be a reaction between nafamostat cation (N+) and sulfite ion. The effects of SBS concentration and temperature on the hydrolysis of NM in buffer solution were examined. From the findings obtained, we estimated the compatible pH range of the intravenous admixture (mixed infusion) of NM after a constant storage time at a constant SBS concentration and temperature employing a simulated pH-profile for the mixed infusion. In order to evaluate the compatibility of the prescribed mixed infusion, the method of pH estimation for the mixed infusion was also investigated using the pH titration curve of each preparation.     

Hydrolysis of 3,4-diphenyl-1,2,5-thiadiazole-1,1-dioxide. Acid and base catalysis

We have performed a product and kinetic study of the hydrolysis of 3,4-diphenyl-1,2,5-thiadiazole-1,1-dioxide in aqueous solution. Benzil and sulfamide are the only products of hydrolysis and are formed in equimolar yields. The kinetic results indicate that a first order law is followed up to 90% conversion. The observed rate constant is independent of substrate concentration. Ionic strength and buffer concentration do not affect the rate constant, but the reaction is acid-base catalyzed. The rate-pH profile has been determined and a mechanism that fits the experimental data satisfactorily is proposed. Corresponding rate constants and equilibrium constants for the protonation of the substrate are reported.     

Protein error of pH indicators in the presence of detergents.

The characteristics of color development due to a protein error in the dye-binding method in the presence of a non-ionic detergent has been investigated by the calculations based on the chemical equilibrium of a protein error. The calculation results were compared with those obtained using three pH indicators (Bromophenol Blue, Bromocresol Green and Bromocresol Purple) and three non-ionic detergents in the pH region from 1 to 13. In the experiments, the color development increased with the lower concentrations of the detergents, but decreased at higher concentrations. The pH where the color development reached a maximum value shifted to a higher pH as the detergent was added. These experimental results were reproduced by the calculation when the molar absorptivity of the dye-protein complex was assumed to increase due to adding the detergent. Such agreement between the experimental and the calculated results indicates that the characteristics of the color development in the dye-binding method in the presence of a non-ionic detergent can be analyzed by calculations based on the chemical equilibrium of a protein error.