Hajime Karatani

Fundamental studies for chemical speciation of iron in seawater with an improved analytical method

We improved the analytical methods for determining iron in seawater, based on our established procedure, and carried out fundamental studies for chemical speciation of iron. The blank value of the system used was ∼ 0.05 n M and the detection limit (3 SD) was 0.01 n M. To examine the dissolution of iron from suspended particles in seawater, four types of particles (aged iron colloid, biogenic particles, sediment particles and freshly deposited iron) were used. At a pH of ∼ 3, dissolution of iron from the easily leachable fraction of suspended particles was prompt, and the fraction of iron [total dissolvable iron, TD(Fe)] was thought to be the sum of labile particulate and dissolved iron. The iron fractions dissolved from suspended particles in the solutions were constant at pHs < 1.5 when solutions were heated in a microwave oven. The fraction of iron dissolved by this procedure was defined as the “leachable iron, L(Fe)”. Some known organic complexing agents were also studied as a model group to examine the possible effects of naturally occurring organic ligands on the recovery of iron with chelating resin preconcentration. Established methods were applied to analyses of seawater samples obtained from the western Southern Indian Ocean (SIO) and the East China Sea. Surface seawater samples from the SIO showed very low iron concentrations, which may be due to a lack of aeolian transport of mineral dust.

Preconcentration of chromium(III) and chromium(VI) in sea water by complexation with quinolin-8-ol and adsorption on macroporous resin

Abstract Chromium(III) is a sea water at the nanomole level was selectively collected using a column packed with macroporous polystyrene-divinylbenzene resin after complexation with quinolin-8-ol. Complex formation between ligand and inert hydrated chromium(III) ions was achieved by heating a sample solution containing a small amount of quinolin-8-ol for a short time in a microwave oven. Chromium(VI) was collected by a similar method after reducing it to chromium(III) with hydroxylamine. The effect of co-existing organic materials on the collection of chromium(III) and chromium(VI) was examined. This method was successfully applied to the determination of chromium(III) and chromium(VI) in sea water by graphite furnace AAS.

Crystal Structure of Clostridium perfringens Enterotoxin Displays Features of β-Pore-forming Toxins

Clostridium perfringens enterotoxin (CPE) is a cause of food poisoning and is considered a pore-forming toxin, which damages target cells by disrupting the selective permeability of the plasma membrane. However, the pore-forming mechanism and the structural characteristics of the pores are not well documented. Here, we present the structure of CPE determined by x-ray crystallography at 2.0 Å. The overall structure of CPE displays an elongated shape, composed of three distinct domains, I, II, and III. Domain I corresponds to the region that was formerly referred to as C-CPE, which is responsible for binding to the specific receptor claudin. Domains II and III comprise a characteristic module, which resembles those of β-pore-forming toxins such as aerolysin, C. perfringens ϵ-toxin, and Laetiporus sulfureus hemolytic pore-forming lectin. The module is mainly made up of β-strands, two of which span its entire length. Domain II and domain III have three short β-strands each, by which they are distinguished. In addition, domain II has an α-helix lying on the β-strands. The sequence of amino acids composing the α-helix and preceding β-strand demonstrates an alternating pattern of hydrophobic residues that is characteristic of transmembrane domains forming β-barrel-made pores. These structural features imply that CPE is a β-pore-forming toxin. We also hypothesize that the transmembrane domain is inserted into the membrane upon the buckling of the two long β-strands spanning the module, a mechanism analogous to that of the cholesterol-dependent cytolysins.

Selective and sensitive determination of trace manganese in sea water by flow through technique using luminol–hydrogen peroxide chemiluminescence detection

Abstract A flow through analytical method has been developed for the determination of trace amounts of manganese in sea water using luminol–hydrogen peroxide chemiluminescence (CL) detection. The method was very simple and almost all the metal ions did not interfere with the CL detection except for iron species. The iron species were thoroughly removed by passing the sample solutions through a 8-quinolinol immobilized chelating resin column. The lower and upper limits of detection were 0.029xa0nM and 4xa0μM, respectively. The relative standard deviation was 0.58% at 10xa0nM of Mn(II). The method permitted the selective and sensitive determination of sub-nM levels of Mn in sea water sample with sufficient precision.

Automated determination of vanadium(IV) and (V) in natural waters based on chelating resin separation and catalytic detection with Bindschedler’s green leuco base

An automated method for determining V(V) and V(IV) in natural waters has been developed. The method is based on the combination of a selective column extraction using two kinds of chelating resins and an air-segmented continuous flow analysis (ASCFA) using catalytic detection with Bindschedler’s green leuco base (BGL)–KBrO3 system. Based on this method, V(V) in a sample solution at pHs from 2.2 to 3.8 is selectively collected on an acetylacetone-immobilized resin and then V(IV) in the eluent is collected on an 8-quinolinole-immobilized resin separating it from iron species, which interfere with the ASCFA detection. V(V) and V(IV) are successively eluted with diluted hydrochloric acid and the resulting eluent is carried into the ASCFA. The concentrations of vanadium species are determined from the absorbance of BGL. The detection range for vanadium species was 0.05–15.0 nM and the standard deviations at 5.0 nM were 1.1% for V(IV) and 0.9% for V(V), respectively. The method was successfully applied to study the behavior of vanadium species in natural waters.

A blue fluorescent protein from a yellow-emitting luminous bacterium

Abstract— Vibrio fischeri strain Y1 emits yellow light in vivo due to the participation of a yellow fluorescent protein (YFP) in the luciferase reaction. In this study it was found that the organism also produces a protein (referred to as Y1‐BFP) emitting strong blue fluorescence. Its molecular weight, about 25 kDa, is the same as or very close to that of YFP. The fluorescence excitation and emission maxima of the purified Y1‐BFP are at 416 and 461 nm, respectively, and the fluorescence lifetime is 12.5 ns at 2°C. The molar extinction coefficient of Y1‐BFP at 416 nm was estimated to be approx. 9500. With the homologous luciferase, Y1‐BFP decreases the intensity and rate of decay in the in vitro reaction but has no effect on its emission spectrum (in contrast to YFP, which has a striking effect on the spectrum). With luciferase isolated from Vibrio harveyi, however, Y1‐BFP causes a small blue‐shift (˜10 nm) in the emission of the enzyme catalyzed reaction, whereas YFP has no effect on the emission spectrum.

Two active forms of the accessory yellow fluorescence protein of the luminous bacterium Vibrio fischeri strain Y1

Abstract In addition to the typical blue luminescence (peak wavelength, approximately 490 nm), the light emission of the luminous bacterium Vibrio fischeri strain Y1 has a yellow component (peak, approximately 540 nm). This is attributable to an accessory “yellow fluorescence protein” (YFP; Mr ≈ 25 kDa), which acts in conjunction with the enzyme luciferase and possesses a flavin chromophore. Cell extracts contain two such YFPs in approximately equal amounts. Both have activity in vitro and are otherwise similar in the several respects examined. The possibility that they represent subunits of a heterodimer was examined, but no dimeric protein could be demonstrated.

A METHOD FOR THE ELECTROCHEMICAL INITIATION OF THE in vitro BACTERIAL LUCIFERASE REACTION

Abstract— We have developed an electrochemical initiation method for the in vitro bacterial luciferase reaction using Vibrio harveyi luciferase. The cathodic conversion of oxidized riboflavin 5′‐phosphate (FMN) to its reduced form (FMNH2), on which the kinetics for the overall light‐emitting reaction depend, is followed by the bacterial luciferase reaction. The electrochemical initiation method can be used for both turnover and nonturnover luciferase reactions, depending on the FMN electroreduction methods.

Development and characterization of anodically initiated luminescent detection for alcohols and carbohydrates

Abstract We have developed and characterized a novel detection method for hydroxyl compounds with no π-electron resonance structure, such as alcohols and carbohydrates. The proposed method is based on luminescence, initiated by electro-oxidation of their hydroxyl group at a glassy carbon electrode in alkaline aqueous solution. The luminescence is elicited when a proper excitation potential is applied to the electrode and its intensity is proportional to the concentration of hydroxyl compounds. The luminescence has been applied successfully to a flow-through detection for hydroxyl compounds. In the flow-through system, a constant potential procedure is employed to trigger the luminescence. The sensitized fluorescence, triggered by the presence of a suitable fluorescer (fluorescein), is utilized to detect hydroxyl compounds in p mol amounts. With a variety of hydroxyl compounds, double logarithmic plots of luminescence intensity vs. concentration give a linear relationship in the tested range of 10μM to 1 mM. The limits of detection for alcohols (including methanol) and carbohydrates are estimated to be in the range from about 10pmol to nmol per 50 μl injection (signal-to-noise ratio of 3).

AN ELECTROCHEMICALLY TRIGGERED CHEMILUMINESCENCE FROM POLYHYDRIC ALCOHOLS

Abstract A newly found electrogenerated chemiluminescence (ecl) triggered by the electrolytic oxidation of adjacent alcoholic hydroxyl groups in an alkaline solution is reported. Cyclic voltammetric studies show that the present ecl consists of two stages depending on the applied potential. In the ecl sequence, carbonyl fragments mainly in the triplet state are considered to be formed. The gas chromatography—mass spectrometric and spectrophotometric analyses of the ecl reaction products show that an aldehyde is formed and then it is oxidized to a corresponding carboxylic acid by the anodically generated oxygen in the ecl sequence. The electronically excited aldehyde and carboxylic acid are considered to be responsible for the first-stage ecl and the second-stage ecl, respectively. The system, giving acetone instead of aldehydes, shows no clear ecl. The present ecl seems to partly resemble the sequence for the chemiluminescence from 1,2-dioxetanes.