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.

First direct evidence for direct cell-membrane penetrations of polycationic homopoly(amino acid)s produced by bacteria.

Bacteria produce polycationic homopoly(amino acid)s, which are characterized by isopeptide backbones. Although the biological significance of polycationic homopoly(amino acid)s remains unclear, increasing attention has recently been focused on their potential use to achieve cellular internalization. Here, for the first time, we provide direct evidence that two representative bacterial polycationic isopeptides, ε-poly-L-α-lysine (ε-PαL) and ε-oligo-L-ß-lysine (ε-OßL), were internalized into mammalian cells by direct cell-membrane penetration and then diffused throughout the cytosol. In this study, we used clickable ε-PαL and ε-OßL derivatives carrying a C-terminal azide group, which were enzymatically produced and then conjugated with a fluorescent dye to analyze subcellular localization. Interestingly, fluorescent proteins conjugated with the clickable ε-PαL or ε-OßL were also internalized into cells and diffused throughout the cytosol. Notably, a Cre recombinase conjugate with ε-PαL entered cells and mediated the Cre/loxP recombination, and ε-PαL was found to deliver a full-length IgG antibody to the cytosol and nucleus.

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 Synthetic Model for the Possible FeIV2(µ-O)2 Core of Methane Monooxygenase Intermediate Q Derived from a Structurally Characterized FeIIIFeIV(µ-O)2 Complex.

A bis(µ-oxo)diiron(IV,IV) complex as a model for intermediate Q in the methane monooxygenase reaction cycle has been prepared. The precursor complex with a [FeIIIFeIV(µ-O)2] core was fully characterized by X-ray crystallography and other spectroscopic analyses and was converted to the [FeIV2(µ-O)2] complex via electrochemical oxidation at 1000 mV (vs Ag/Ag+) in acetone at 193 K. The UV-vis spectral features, Mössbauer parameters (ΔEQ = 2.079 mm/s and δ = -0.027 mm/s), and EXAFS analysis (Fe-O/N = 1.73/1.96 Å and Fe···Fe = 2.76 Å) support the structure of the low-spin (S = 1, for each Fe) [FeIV2(µ-O)2] core. The rate constants of the hydrogen abstraction reaction from 9,10-dihydroanthracene at 243 K suggest the high reactivity of these synthetic bis(µ-oxo)diiron complexes supported by simple N4 tripodal ligand.

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.

Structure and electrochemical properties of (µ-O)2Mn2(iii,iii) and (µ-O)2Mn2(iii,iv) complexes supported by pyridine-, quinoline-, isoquinoline- and quinoxaline-based tetranitrogen ligands.

Seven new bis(µ-oxo)dimanganese complexes with Mn2(iii,iii) or Mn2(iii,iv) oxidation states were prepared using quinoline- and isoquinoline-based tetraamine ligands. The structures of the ligands include ethylenediamine, trans-1,2-cyclohexanediamine and tripodal amine, bearing two or three nitrogen-containing heteroaromatics. Regardless of the skeleton and number of aliphatic nitrogen atoms in the ligands, quinoline complexes stabilize the Mn2(iii,iii) oxidation state, whereas, isoquinoline ligands afford Mn2(iii,iv) complexes. A systematic comparison of the differences in structural parameters and redox potentials of a total of 14 complexes with a (µ-O)2Mn2 diamond core, which includes corresponding pyridine and quinoxaline derivatives as supporting ligands, highlights the distinct deviation of quinoline and tripodal amine motifs in this ligand series.

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.

A Solubility-based Separation of Group B Soyasaponins from the Whole Soybean Flour.

This note describes a simple and rapid method to separate specific soyasaponins, including the major and multi-functional bioactive species soyasaponin Bb, from the whole soybean flour. The method is based on the difference in solubility of the soyasaponins and other components in methanol, aqueous borax solution, and 1-octanol. First, the whole soybean flour was mixed with methanol to extract hydrophobic components, and the methanol solution was concentrated. Second, the methanol solution was mixed with a larger volume of aqueous borax solution, and the supernatant was acidified to obtain a precipitate. The precipitate was found to be containing mainly four group B soyasaponins. Two of those are non-conjugated molecules, soyasaponins Bb and Bc, and the other two are 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one conjugated ones, soyasaponins ßg and ßa. All of them have two cis-diol groups in their structure. Therefore, although they are fundamentally insoluble in water, they became soluble in the aqueous medium in the form of divalent anionic species by the borate ester formation, and that they were re-precipitated by the hydrolysis. Furthermore, the non-conjugated group B soyasaponins could be separated by washing with 1-octanol to remove highly hydrophobic soyasaponins ßg and ßa. The solubility-based technique would be useful as pretreatment in the purification of these group B soyasaponins.

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.

Imaging mass spectrometry analysis of ubiquinol localization in the mouse brain following short-term administration.

We analyzed the localization of ubiquinol, the reduced form of coenzyme Q10 (Re-CoQ10), in mouse brain sections using matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance imaging mass spectrometry (IMS) to evaluate the effect of dietary Re-CoQ10 in mouse brain. Mice were orally administered Re-CoQ10 for 14 days and brain Re-CoQ10 content was subsequently quantified using liquid chromatography-mass spectrometry. IMS was employed to visualize Re-CoQ10 at a resolution of 150 µm in the mouse brain. Increased Re-CoQ10 was observed in the corpus callosum, hippocampus, midbrain, cerebellum, brain stem, substantia nigra and striatum. These regions are related to movement, memory and vital life functions. Thus, we demonstrated the effect of Re-CoQ10 administration on the specific localization of Re-CoQ10 in the brain.

Nanoparticle-Assisted Laser Desorption/Ionization Mass Spectrometry (Nano-PALDI MS) with Py-Tag for the Analysis of Small Molecules.

We compared two ionization methods, matrix assisted laser desorption/ionization (MALDI) and nanoparticle assisted laser desorption/ionization (Nano-PALDI) mass spectrometry (MS), for the analysis of amino acids derivatized with Py-Tag™ that consists pyrylium-based compound. Py-Tag is a useful stable derivatization reagent due to wide mass differences (using 13C as the sole stable labelling isotope). For Py-Tag labelled lysine, sensitive signals that showed less noise with a ten times higher sensitivity, showed a wider mass difference by Nano-PALDI MS compared to MALDI MS.

Multi-imaging of Cytokinin and Abscisic Acid on the Roots of Rice (Oryza sativa) Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry.

Plant hormones act as important signaling molecules that regulate responses to abiotic stress as well as plant growth and development. Because their concentrations of hormones control the physiological responses in the target tissue, it is important to know the distributions and concentrations in the tissues. However, it is difficult to determine the hormone concentration on the plant tissue as a result of the limitations of conventional methods. Here, we report the first multi-imaging of two plant hormones, one of cytokinin [i.e., trans-zeatin (tZ)] and abscisic acid (ABA) using a new technology, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) imaging. Protonated signals of tZ (m/z 220.1) and ABA (m/z 265.3) were chosen on longitudinal sections of rice roots for MS imaging. tZ was broadly distributed about 40 mm behind the root apex but was barely detectable at the apex, whereas ABA was mainly detected at the root apex. Multi-imaging using MALDI-TOF-MS enabled the visualization of the localization and quantification of plant hormones. Thus, this tool is applicable to a wide range of plant species growing under various environmental conditions.

Solubility-based Separation and Purification of Long-Chain Chitin Oligosaccharides with an Organic-Water Mixed Solvent.

A simple and rapid method for separation and purification of chitin oligosaccharides, (GlcNAc)n, with n ≥ 5 is presented. A commercially available chitin oligosaccharides sample, consisting of (GlcNAc)n with n = 1 - 7, was used as the starting material. Ten milligrams of the material was mixed with 100 µL of the 1 mol/L HCl. All the (GlcNAc)n species were dissolved in the aqueous medium. The aqueous solution was mixed with 900 µL of EtOH; the mixture was centrifuged, and the supernatant was removed to obtain a precipitate. The precipitate was found to consist mainly of (GlcNAc)n with n ≥ 5, indicating the significant difference in solubility between the short-chain (GlcNAc)n species with n ≤ 3 and the longer ones. By the repetition of the operations, a high purity long-chain (GlcNAc)n sample with n ≥ 5 could be prepared successfully. Since the long-chain (GlcNAc)n species are known to have excellent elicitor activity, this sample would be useful in the study of plant pathology, as well as chitin and chitosan chemistry.

Colorimetric Microtiter Plate Assay of Polycationic Aminoglycoside Antibiotics in Culture Broth Using Amaranth.

We present a colorimetric method for the detection of aminoglycoside antibiotics such as neomycin (NEO) using a reddish anionic dye, amaranth (AR3-). Under acidic conditions, at which NEO exists in fully protonated form (NEOH6+), the AR3- anion associates with the NEOH6+ cation to form a precipitate, NEOH(AR)2. The precipitate was soluble in a buffer solution of pH 8.5, yielding a reddish solution with an absorption maximum at around 520 nm. Tobramycin and gentamycin, which exist as pentavalent cations under acidic conditions, gave almost the same results. On the other hand, kanamycin, amikacin and streptomycin, which would exist as tri- and tetravalent cations, were not precipitated. Thus, the AR3- anion could be considered to be an analytical reagent for specific aminoglycosides with polycationic functionality. However, since the precipitation reaction was considerably affected by other anions, a separation method using the tetraphenylborate anion was employed as a pretreatment. The separation method involves precipitating the polycationic aminoglycosides with the tetraphenylborate anion, washing the precipitate with acetonitrile, and re-precipitating the aminoglycosides as hydrochloride salts. Thus, the present method was applied to a microtiter plate assay of the products in an NEO-producing culture broth.