Separation of an ε-poly-L-lysine derivative by solvent extraction under a controlled interfacial potential difference.

This paper describes the availability of a 1,2-dichloroethane (DCE)-water (W) interfacial system under a controlled interfacial potential difference for the separation of polycationic species. The system was applied to the production of polyethylene glycol-modified ε-poly-L-lysine (PEG-εPL). PEG-εPL is produced by a fermentation process, and the crude product contains a significant amount of non-modified εPL, which is hardly separated by conventional chromatographic techniques. Both εPL species exist in fully protonated forms under certain acidic conditions, and an extractant, dibenzo-18-crown-6 (DB18C6), associates with their ammonium groups to stabilize the polycations in DCE. Despite the polydispersity of the samples, the εPL and crude PEG-εPL give well-defined cyclic voltammetric waves due to the DB18C6-assisted transfer of the polycations at the polarizable DB18C6 (DCE) | (W, pH ~ 3) interface with midpoint potentials useful for a rough prediction of ion partition equilibria. Thus, the partition experiment was performed using the DB18C6, Bu4N[(CF3SO2)2N] (DCE) | crude PEG-εPL, Li[(CF3SO2)2N] (W, pH ~ 3) interfacial system, of which the potential difference was controlled to enable selective extraction of polycationic PEG-εPL by partition of the [(CF3SO2)2N]- ion. The extract could be collected from the DCE phase and was found to consist of highly purified PEG-εPL.

Determination of protamine and heparin based on their effects on a glucose oxidase enzymatic reaction.

This paper describes a sensitive method for determining protamine and heparin by utilizing a glucose oxidase enzymatic reaction. Polycationic protamine significantly promoted the enzymatic reaction rate with [Fe(CN)6]3-, so that the increase could be used to determine protamine. The promotion effect was stoichiometrically decreased by the addition of polyanionic heparin through the polyion complex formation with protamine, so that the enzymatic reaction also allowed for the determination of heparin. We thus applied the proposed method to blood plasma containing heparin and found that heparin did not stoichiometrically form a polyion complex with protamine, likely due to strong interactions between heparin and some components of the plasma. The proposed method allowed for the detection of free protamine (and/or weakly binding protamine with heparin) existing in the condition that protamine did not neutralize all of the heparin in the plasma. The method also permitted for the estimation of heparin concentrations using calibration curves. Thus, the proposed method would help reduce the risks of protamine overdose in heparin neutralization and would be a helpful tool in clinical practices that use heparin and protamine.

Determination of polyanion utilizing a promoted glucose oxidase enzymatic reaction by ε-poly-L-lysine.

In this paper, a sensitive determination method for polyanion using a glucose oxidase (GOx) enzymatic reaction with ferricyanide ion is described. We previously reported that the GOx enzymatic reaction was significantly promoted by a cationic polymer of ε-poly-L-lysine (εPL), and the enzymatic reaction could be utilized for the determination of εPL. Generally, polycation stoichiometrically forms polyion complex with polyanion. Thus, it is expected that the promotion effect of εPL on the enzymatic reaction is interfered by polyanion, and the enzymatic reaction is also applicable to the determination of polyanion. Predictably, the promotion effect of εPL was stoichiometrically interfered by polyanions, such as polyvinyl sulfate and polyacrylate, and the interference effect allowed for the determination of the polyanions. The detection limit of polyanion was estimated to be ~ 0.3 µeq L-1. As a preliminary application, the proposed method was applied to the determination of anionic polymer of heparin in a human plasma.

Tetraphenylantimony(V)-assisted transfer of hydroxide and fluoride anions across the 1,6-dichlorohexane | water interface.

The ion-transfer reaction at the 1,6-dichlorohexane (DCH) | water (W) interface in the presence of an organometallic cation, tetraphenylantimony (TPhSb+), in DCH was studied voltammetrically. When TPhSb+ salt with [(C4F9SO2)2N]- ion was added to the DCH-phase and the W-phase was buffered at pH < 6, a reversible cyclic voltammogram due to the simple transfer of TPhSb+ ion across the DCH | W interface was observed within the polarizable potential window. When the W-phase was buffered at pH > 7, the midpoint potential shifted to more positive potentials with increasing pH. The voltammogram could be attributed to the transfer of the OH- ion assisted by the formation of TPhSbOH, which is stable in DCH. Also, a 7reversible voltammogram due to the TPhSb+-assisted transfer of F- ion was observed at the TPhSb+ (DCH) | F- (W, unbuffered) interfacial system. The same results were achieved when TPhSb[(C4F9SO2)2N] in DCH was replaced by TPhSbOH or TPhSbF, indicating the applicability of the TPhSb+ and TPhSbOH (DCH) | OH- (W) interfacial system to a pH sensor for alkaline solution and that of the TPhSb+ and TPhSbF (DCH) | F- (W) interface to a F- ion sensor.

A Theoretical Approach to the Fluorophilicity of Ions via the Gibbs Energy of Ion Transfer at the Fluorous Solvent/Water Interface.

The non-Bornian solvation model has been applied to a theoretical consideration of the Gibbs free energy for the transfer of fluorinated anions, non-fluorinated cations, and non-fluorinated anions at the 2H,3H-decafluoropentane (DFP)/water (W) and 1,2-dichloroethane (DCE)/W interfaces. According to our previous experimental results, the fluorinated anions are more stable in DFP than DCE, while the non-fluorinated cations and anions are less stable in DFP. To understand this characteristic feature of DFP, energy decomposition analyses have been performed for the hypothetical transfer of ions at the DFP/DCE interface. In conclusion, the characteristics of DFP as a fluorous solvent should be explained in terms of the higher repulsive interaction of the solvent molecule with ions, particularly with non-fluorinated ions.

Fluorination Effect on the Gibbs Transfer Energy for Methylene Group from 1,2-Dichloroethane or 1,1,1,2,3,4,4,5,5,5-​Decafluoropentane to Water.

The ion-transfer reactions of alkyl and perfluoroalkyl carboxylate ions (CH3(CH2)n-2COO- and CF3(CF2)n-2COO-) at 1,2-dichloroethane (DCE) | water (W) and 1,1,1,2,3,4,4,5,5,5-decafluoropentane (DFP) | W interfaces were investigated. These ions gave reversible or quasi-reversible voltammetric waves due to their ion transfer across the interfaces, and the formal potentials and the formal Gibbs transfer energies from a non-aqueous solvent to water, ΔG°'tr,α→W (α = DCE and DFP), were determined. The ΔG°'tr,α→W for CH3(CH2)n-2COO- and CF3(CF2)n-2COO- linearly increased with n, allowing for estimating ΔG°'tr,α→W for methylene groups. The estimated value of ΔG°'tr,DCE→W for the -CH2- group was higher than that of ΔG°'tr,DCE→W for the -CH2- group, whereas the ΔG°'tr,DCE→W for the -CF2- group was lower than that of ΔG°'tr,DCE→W for the -CF2- group, indicating that the -CH2- (or -CF2-) group is more favorably (or unfavorably) solvated in the DCE phase compared to the DFP phase. From the estimated values, the fluorination effect of alkyl chains on partitioning the alkyl group between the biphase media has also been discussed.

Ion-Transfer Voltammetry at Fluorous Ether | Water Interfaces.

This paper describes that fluorous ethers, 1,1,2,2-tetrafluoroethyl-2,2,3,3-tetrafluoropropyl ether and 1H,1H,5H-octafluoropentyl-1,1,2,2-tetrafluoroethyl ether, can be used for a non-aqueous medium in electrochemistry at a liquid | liquid interface. These solvents dissolved a high concentration of tetraalkylammonium salts with highly-fluorinated anions, such as bis(nonafluorobutanesulfonyl)imide and tetrakis[3,5-bis(trifluoromethyl)phenyl]borate ions, and the solution had a high conductivity. The fluorous ether | water interfaces exhibited a substantial polarizable potential window, and various ions, including tetraphenylarsonium and tetraphenylborate ions, gave a reversible voltammetric wave due to their ion transfer across the interfaces. Using the tetraphenylarsonium-tetraphenylborate assumption, the formal potentials for the ion transfer and the formal Gibbs energies of ion transfer from the ethers to water were estimated. The Gibbs energies were close to those from a previously reported fluorous solvent, 1,1,1,2,3,4,4,5,5,5-decafluoropentane; the fluorous ethers also exhibited a higher affinity for fluorinated ions. Because of a lower volatility, the fluorous ethers would be more advantageously used in two-phase electrochemistry, particularly concerning analytical purposes.

Gibbs Transfer Energies of Ions from a Mixed Solvent of 2H,3H-Decafluoropentane and 1,2-Dichloroethane to Water.

The transfer of hydrophilic, lipophilic, and fluorophilic ions at the interface between water (W) and a mixed solvent (MIX) of 2H,3H-decafluoropentane (DFP) and 1,2-dichloroethane (DCE) was studied voltammetrically and potentiometrically, and the formal Gibbs transfer energies of the ions from MIX to W, ΔG0'tr,MIX→W, were determined. The ΔG0'tr,MIX→W values of all the ions tested were higher than those from DFP to W. Namely, the ions would exist more stably in MIX than DFP, even for fluorophilic ions. This is due to the addition of DCE, which has a higher dielectric constant. A comparison of ΔG0'tr,MIX→W with that from DCE to W showed a superior affinity of fluorophilic ions to the fluorous solvent in spite of equivolume addition of DCE. Therefore, the mixed solvent would be a practically superior extraction medium for fluorophilic ions. In practice, the MIX | methylene blue+ (W) system showed higher extractability of a fluorophilic ion C8F17SO3- than the DFP | W and DCE | W systems.

Determination of Polyhexamethylene Biguanide Utilizing a Glucose Oxidase Enzymatic Reaction.

Polyhexamethylene biguanide (PHMB) is a cationic disinfectant widely used for personal-care products and for sanitizers in swimming pools. This paper describes a promotion effect of PHMB on a glucose oxidase (GOx) enzymatic reaction with ferricyanide ion and its analytical application. The promotion effect arose from a polyion complex formation between polycationic PHMB and polyanionic GOx. The promoted GOx reaction was analyzed by Michaelis-Menten equation, and the Michalis constant and catalytic constant were estimated to be 240 µM and 31 s-1, respectively. Utilizing the promotion effect, PHMB was successfully determined in the range of 0.05 to 0.40 ppm, and the detection limit was calculated to be 0.027 ppm. The visual detection and semi-determination of PHMB with the same concentration level were also possible. As an application, the method was applied to the determination of PHMB in a contact lens detergent and its model solution.

Effect of ε-Poly-L-lysine on a Glucose Sensor Based on Glucose Oxidase and Ferricyanide Ion.

ε-Poly-L-lysine (εPL) is a homopolymer of L-lysine residues with linkages between the α-carboxyl and ε-amino groups. εPL exists as a polycationic species under acidic and neutral conditions, and can form a polyion complex with negatively charged glucose oxidase (GOx). We previously reported that εPL significantly promoted a GOx enzymatic reaction using ferricyanide ion ([Fe(CN)6]3-) as the oxidant. Here, we construct a GOx-immobilized electrode using εPL and glutaraldehyde, and show that [Fe(CN)6]3- can efficiently act as the mediator in this electrode. Ferricyanide ion failed to function adequately as the mediator when bovine serum albumin or other polyamines (e.g., polyallylamine, α-poly-L-lysine) were used instead of εPL. The GOx-immobilized electrode using εPL successfully responded to glucose even when the [Fe(CN)6]3- concentration was as low as 1 mM, and exhibited a wide dynamic range of up to several tens of mM. Thus, εPL is considered to be a useful additive for glucose sensors based on the [Fe(CN)6]4-/3--mediated GOx catalytic current.

Promotion Effect of Streptothricin on a Glucose Oxidase Enzymatic Reaction and Its Application to a Colorimetric Assay.

Previously, we reported that ε-poly-L-lysine (25 - 35 residues) significantly promoted a glucose oxidase enzymatic (GOx) reaction using ferricyanide ion as the oxidant, and that the effect was due to the formation of a polyion complex between anionic GOx and protonated (polycationic) ε-poly-L-lysine. Here, we show that streptothricins (STs), which have an L-ß-lysine oligomer (1 - 7 residues) and possess only several positive charges at most, also effectively promote the GOx enzymatic reaction. Interestingly, the promotion effect increased with the size of the lysine oligomer of STs, suggesting that the ionic valence is a key factor determining the degree of the promotion effect. The GOx enzymatic reaction is accompanied by a color change due to the reduction of yellow ferricyanide ion to a colorless reductant. A more distinctive color change can be achieved by the addition of Fe(III) ions due to the formation of Prussian blue. Thus, the promotion effect allowed for colorimetric detection of STs at the 1 mg/L level. The detection method was simple and easy to carry out, and would become a helpful tool for the detection of STs.

Colorimetric method to detect ε-poly-l-lysine using glucose oxidase.

We describe a new colorimetric assay method using glucose oxidase (GOx) to detect ε-poly-l-lysine (εPL). This method uses εPL's remarkable effect of promoting the enzymatic reaction of GOx with ferricyanide ion. This reaction reduces ferricyanide ion to ferrocyanide ion, accompanied by a color change from yellow to colorless. In this colorimetric assay, the detection limit of εPL was estimated to be approximately 0.5 mg/L when purified εPL samples were used. εPL has usually been produced by a fermentation process using Streptomyces albulus species. The components of the culture broth showed interference effects against the assay method. However, due to the high sensitivity of the assay method for εPL, εPL could be detected in the culture broth without any pretreatment. The detectable concentration of εPL in the culture broth, cPL,ac, was estimated to be approximately 20 mg/L. By combining the Berlin blue reaction with this method, the cPL,ac was reduced to 10 mg/L. In light of the proposed method's simplicity and sensitivity, it could be useful for screening εPL synthetic enzymes and microorganisms.

Mass spectrometry imaging: applications to food science.

Two-dimensional mass spectrometry (MS) analysis of biological samples by means of what is called MS imaging (MSI) is now being used to analyze analyte distribution because it facilitates determination of the existence (what is it?) and localization (where is it?) of biomolecules. Reconstruction of mass image by target signal is given after two-dimensional MS measurements on a sample section. From only one section, we can understand the existence and localization of many molecules without the need of an antibody or fluorescent reagent. In this review, we introduce the analysis of localization of functional constituents and nutrients in herbal medicine products via MSI. The ginsenosides were mainly distributed in the periderm and the tip region of the root of Panax ginseng. The capsaicin was found to be more dominantly localized in the placenta than the pericarp and seed in Capsicum fruits. We expect MSI will be a useful technique for optical quality assurance.

Promotion and suppression effects of cationic polymer ε-poly-L-lysine on the glucose oxidase reaction with ferrocene derivatives as oxidants with different charges.

The effects of the cationic polymer ε-poly-L-lysine (εPL) on the enzymatic reaction rate of glucose oxidase (GOD) with ferrocene derivatives having different ionic charges have been investigated by measuring the ferrocene derivative-mediated catalytic current of oxidation by GOD. When negatively charged ferrocenes were used, the bioelectrocatalytic current, which is related to the enzymatic reaction rate, was increased by the addition of εPL. On the other hand, the reaction rates with positively charged ferrocenes were decreased by εPL. These promotion and suppression effects of εPL were remarkable at a certain pH range, where εPL and GOD were charged positively and negatively, respectively. Within this range, the polycationic εPL would be adsorbed onto the GOD surface to enhance the electrostatic interactions of the enzyme with negatively charged substrates, and repulsion with positively charged ones. These findings should be important for practical applications of enzymes.

Analytical methods for the detection and purification of ε-poly-L-lysine for studying biopolymer synthetases, and bioelectroanalysis methods for its functional evaluation.

This article describes new analytical methods for studying biopolymer ε-poly-L-lysine (εPL). The produced amount of εPL in culture broth can be determined based on the precipitation of polycationic εPL with a colored heteropolymolybdate anion and the color change of the supernatant. The product can be separated and purified by precipitation with the tetraphenylborate anion and reprecipitation in the form of the hydrochloride salt. These methods have been applied advantageously to the screening of εPL-synthetase. Also, pyrophosphate can be determined colorimetrically based on the formation of 18-molybdopyrophosphate species. The pyrophosphate determination has been successfully applied to the assay of adenylation enzyme, which plays important roles in the biosynthetic mechanism. Under certain conditions, εPL associates with a redox enzyme, glucose oxidase. The effect of the adduction on the stability and reaction rate of the enzyme can be evaluated by measuring the bioelectrocatalytic current, which is related to the enzyme activity. Electrochemical studies showed new applications of εPL as an enzyme stabilizer and a reaction enhancer.

Silver Oxide Based Nanoparticle Assisted Laser Desorption/Ionization Mass Spectrometry for the Detection of Low Molecular Weight Compounds.

A specific property of silver oxide-based nanoparticles permits the ionization of an analyte, giving rise to various applications of a smart analytical methodology. The nanoparticles (d=6.7 nm) contained an Ag2O core. The detection of several model componds (a nucleobase and two hair growth promoters) via the use of silver oxide nanoparticles is described. Adducts were produced between the target molecules and the two silver stable isotopes (Ag(107) and Ag(109)), resulting in the formation of specific signals as well as a protonated signal. Thus, it was possible to easily determine whether the given signals were correlated with the target molecule or not.

Colorimetric determination of glucosamine and glucose based on the formation of blue molybdosilicate anion and its application to the assay of saccharifying enzyme.

A simple and convenient method of reducing monosaccharide assay is proposed. A Si(IV)-Mo(VI) solution (pH 4.9) was yellow due to the formation of the 11- and/or 12-molybdosilicate(VI) anions. By the addition of a reducing saccharide, glucosamine, the mixture turned to blue gradually, indicating that the Mo(VI) species was reduced by the saccharide to form a blue molybdosilicate anion. The molybdenum blue formation occurred more quickly when a water-miscible organic solvent, dimethyl sulfoxide, was added to the Si(IV)-Mo(VI) solution. Thus, 0.01% level glucosamine can be determined colorimetrically with microtiter plate. Oligochitosan would not interfere with the determination of the glucosamine at the same concentration. Also, a remarkable blue color development of the Si(VI)-Mo(VI) solution was observed by the addition of glucose. On the other hand, maltose, cellobiose, and water-soluble starch at the same concentration level gave no significant coloration of the reaction mixture. Thus, the present monosaccharide assay can be applied advantageously to evaluate the saccharification to produce glucosamine and glucose.

Evaluation of an electrochemical method for the analysis of enzymatic inhibition reactions.

An approximate equation for bioelectrocatalitic current was applied to an inhibition reaction analysis of bilirubin oxidase by anion (Cl(-), SCN(-), and F(-)) in order to assess the possibility of the electrochemical method for the analysis of enzymatic inhibition reactions. The approximate equation can be transformed into the Michaelis-Menten form, so that the bioelectrocatalitic current can be analyzed by the usual graphical manner, that is, Lineweaver-Burk, Hanes-Woolf, Dixon and Cornish-Bowden plots, if the rate of inhibition reaction is described as a simple Michaelis-Menten form. From the electrochemical assay, it was found that the inhibitor of Cl(-) and SCN(-) anions exhibit non-competitive inhibition while that of F(-) exhibits competitive inhibition, and their inhibition constants were 220, 45, and 22 mM, respectively. The results were essentially similar to those obtained from the conventional spectrophotometric assay.

Promotion effect by ε-poly-L-lysine on the enzymatic reaction of glucose oxidase with ferricyanide ion as an oxidant.

An enzymatic reaction rate of glucose oxidase (GOD) using ferricyanide ion ([Fe(CN)(6)](3-)) as an oxidant significantly increased by the addition of ε-poly-L-lysine (ε-PL). The bimolecular rate constant between GOD and [Fe(CN)(6)](3-) in the presence of ε-PL reached about 10000-fold relative to what it was measured without the ε-PL, and the Michaelis constant decreased. The reaction rate reached a maximum at around pH 6, where ε-PL and GOD possess highly positive and negative charges, respectively. The increment of the reaction rate by ε-PL can be attributed to the electrostatic association of the polycationic ε-PL with the negatively charged GOD to form a polyion complex soluble in the aqueous medium. The adduction of the cationic polymer may relieve the electrostatic repulsion between GOD and [Fe(CN)(6)](3-), so that the electron transfer effectively occurs between them.