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In Japan, successful cases of a bridge to lung transplantation (BTT) by extracorporeal membrane oxygenation (ECMO) are rare. We present the case of a man in his thirties, diagnosed with interstitial pneumonia 6 years prior and registered for lung transplant 1 year prior due to disease progression despite treatment. Due to the patient's worsening respiratory failure, he was transferred to our hospital for BTT by ECMO. Since long-term management was expected and pulmonary hypertension was present, veno-arterial (V-A) ECMO was conducted using the right atrial blood outflow via the right internal jugular vein and right axillary artery inflow via a vascular graft. After tracheostomy, he was managed as "Awake ECMO". In addition, interprofessional collaboration such as physiotherapist rehabilitation, nurses, and liaison teams prevented muscle weakness and supported the mental aspect. We were able to minimize complications such as severe infections and bleeding. A compatible brain-dead donor was found on day 108 after introducing ECMO, and the patient was transferred to a transplant facility on day 109. The peripheral upper V-A ECMO is one of the configurations suitable for long-term BTT management.

Background Iliopsoas hematoma occasionally occurs in patients receiving anticoagulation therapy. It may be a life-threatening complication and can cause disseminated intravascular coagulation, which could develop into abdominal compartment syndrome. The incidence of and factors associated with iliopsoas hematoma during venovenous extracorporeal membrane oxygenation (ECMO) have not been well studied. Objectives To describe the incidence of iliopsoas hematoma and associated factors among patients undergoing venovenous ECMO. Methods A retrospective cohort study was conducted at Nippon Medical School Hospital from April 2015 to October 2018. All patients (>18 years old) with iliopsoas hematoma received a diagnosis based on computed tomography. Results During the study period, 54 patients were supported with venovenous ECMO. Iliopsoas hematoma occurred in 8 of those patients (15%), none of whom had disseminated intravascular coagulopathy or abdominal compartment syndrome develop. Univariate analysis indicated that management of ECMO while the patient was awake and mobilization beyond sitting on the edge of the bed were significantly different (P < .05) in patients with and patients without iliopsoas hematoma. Mortality, however, did not differ significantly between the 2 groups. Conclusions Our findings emphasize that recognizing factors associated with iliopsoas hematoma and detecting them early are crucial during venovenous ECMO in order to treat patients with iliopsoas hematoma appropriately.

A mediated amperometric enzyme electrode, which is constructed by immobilizing an enzyme behind a semipermeable membrane on the surface of a carbon paste electrode containing a mediator compound, has allowed a variety of bioelectrochemical studies in both applied and fundamental aspects such as biosensors, biofuel cells, and enzyme kinetics. This paper describes such bioelectrochemical studies in which we have been involved in these 40 years.

It has been shown that polyammonium cations comprising quaternary ammonium and hydrophilic groups such as amide and hydroxyl groups stabilize a redox enzyme bilirubin oxidase (BOD). The BOD catalyzes the reaction: 4[Fe(CN)6]4- + 4H+ + O2 --4[Fe(CN)6]3- + 2H2O, and has been a promising enzyme for use as a cathode catalyst in biofuel cells. In this study, the stabilizing effect of poly[oxyethylene(dimethylimino)propyl(dimethylimino)ethylene] (PA1) on BOD has been investigated. The sample solution containing BOD and the PA1 salt was kept at a given temperature, and the loss of the enzymatic activity was detected after given stored times. The activity decreased exponentially with stored time so that the first-order rate-constant of inactivation was determined. The inactivation rate-constant lowered with increasing the concentration of the PA1 salt, suggesting that BOD was stabilized by the association with the PA1 cation. The PA1 cation may act like a protective colloid or decrease the local disorder of BOD by its wrapping. A membrane-covered electrode containing BOD, PA1, and [Fe(CN)6](4-/3-) in the internal solution phase was examined in air-saturated aqueous solution. The electrode gave a well-defined current-potential curve with a steady state limiting current due to the PA1-[Fe(CN)6](4-/3-) polyion complex-mediated bioelectrocatalytic current for the reduction of O2. The decreasing of the steady state limiting current became slower in the presence of the PA1 salt, indicating again the stabilizing effect of PA1 cation on BOD.

Quinohemoprotein amine dehydrogenase (QH-AmDH) from Paracoccus denitrificans is an alphabetagamma heterotrimeric enzyme catalyzing oxidative deamination of amines with large substitutes, such as butylamine and benzylamine. The smallest gamma subunit has cross-linking cysteine tryptophylquinone (CTQ) as a catalytic center. A hemoprotein similar to QH-AmDH in molecular mass, subunit structure, and UV-vis spectral property, but having no enzymatic activity, was isolated. The enzymatically silent form (sQH-AmDH) was activated slowly by the substrates of QH-AmDH, and quickly by reductants, dithionite and dithiothreitol. Electrolysis of sQH-AmDH yielded the active form at potentials more negative than -0.17 V (vs. SHE). The activated protein reacted with a carbonyl reagent, 4-nitrophenylhydrazine, giving a typical spectrum of 4-nitrophenylhydrazone, while sQH-AmDH did not give such a spectrum. The gamma subunit of sQH-AmDH showed a sharp peak at 390 nm in UV-vis spectrum clearly distinguishable from that of QH-AmDH. Electrospray ionization mass spectrometric analysis showed that the molecular mass of the gamma subunit of sQH-AmDH was larger than that of QH-AmDH by 15.6. The data suggest that the CTQ-like moiety of sQH-AmDH is an oxime. This hypothesis was confirmed by subsequent hydroxylamine treatment of QH-AmDH. QH-AmDH was treated with hydroxylamine yielding an oxime derivative. The UV-vis spectral properties of the gamma subunit of hydroxylamine-treated QH-AmDH were identical to those of sQH-AmDH. The hydroxylamine-treated QH-AmDH was also reactivated by butylamine, as in the case of sQH-AmDH.

Biofuel cells are a next-generation energy device because they use renewable fuels with high energy density and safety. We have developed passive-type biofuel cell units, which generate a power over 100 mW (80 cm3, 39.7 g). Our biofuel cell, in which two-electron oxidation of glucose and four-electron reduction of O2 occurs at pH 7 in mediated bioelectrochemical processes under quiescent conditions, accomplished the maximum power density of 1.45 ± 0.24 mW cm−2 at 0.3 V. This performance was achieved by introducing three technologies: (1) Enzymes and mediator are densely entrapped on carbon-fiber electrodes with the enzymatic activity retained, (2) the concentration of buffer in electrolyte solution was optimized for the immobilized enzymes, and (3) the cathode structure was designed to supply O2 efficiently. The cell units with a multi-stacked structure successfully operate a radio-controlled car (16.5 g), which demonstrates the potential of biofuel cells in practical applications.

Cyclic voltammograms of the aqueous solution containing polyammonium cations and the [Fe(CN)6]4-. anion at a plastic-formed carbon or platinum electrode were recorded. Polyammonium salts comprising quaternary ammonium in the main chain or sub chain and those comprising primary ammonium were tested. Under certain conditions, well-developed anodic and cathodic peak currents with the midpoint potentials different from that when the polyammonium cation is absent, that is, the midpoint potential of [Fe(CN)6]3- + e- = [Fe(CN)6]4- were exhibited, indicating that the [Fe(CN)6]4-/3- anionic species associate with the polyammonium cations tested to form the polycation-[Fe(CN)6]4-/3- complex species. The polycation complex species can be easily localized around the electrode surface with a dialysis membrane. An application of the polycation complex species as an electron-transfer mediator in the catalytic electrode with a redox enzyme was examined.

The kinetics of glucoamylase-catalyzed hydrolysis of starch granules from six different botanical sources (rice, wheat, maize, cassava, sweet potato, and potato) was studied by the use of an electrochemical glucose sensor. A higher rate of hydrolysis was obtained as a smaller size of starch granules was used. The adsorbed amount of glucoamylase on the granule surface per unit area did not vary very much with the type of starch granules examined, while the catalytic constants of the adsorbed enzyme (k(0)) were determined to be 23.3+/-4.4, 14.8+/-6.0, 6.2+/-1.8, 7.1+/-4.1, 4.6+/-3.0, and 1.6+/-0.6 s(-1) for rice, wheat, maize, cassava, sweet potato, and potato respectively, showing that k(0) was largely influenced by the type of starch granules. A comparison of the k(0)-values in relation to the crystalline structure of the starch granules suggested that k(0) increases as the crystalline structure becomes dense.

Direct electron transfer (DET)-type electrocatalytic properties were examined for a site-directed mutant (M467Q) of Myrothecium verrucaria bilirubin oxidase (BOD), in which Met467 as the axial ligand of the type I copper site is replaced by Gln. Although the enzymatic activity of the mutant is very low toward bilirubin [A. Shimizu, et al., J. Biochem. 125 (1999) 662–668], it works as a good catalyst in DET-type bioelectrocatalytic reduction of dioxygen into water. The wave appears around the formal potential of the type I Cu site, which is about 0.23 V more negative than that of the wild-type recombinant (rBOD), and the catalytic limiting current density is larger than that of rBOD. Detailed analysis of the voltammograms and the enzyme kinetics suggests that the electrons are transferred from electrodes to the type I Cu site and that the mutation does not injure the kinetics of the three processes: the heterogeneous electron transfer, the intramolecular electron transfer from the type I Cu site to the trinuclear type II–III Cu cluster, and the catalytic process to reduce dioxygen at the type II–III Cu cluster. The overall catalytic constant is increased by the mutation. This result can be interpreted in terms of the increased Gibbs energy difference in the electron transfer reaction from the type I Cu site to the type II–III Cu cluster.

An amperometric biosensor for the detection of cellobiose has been introduced to study the kinetics of enzymatic hydrolysis of crystalline cellulose by cellobiohydrolase. By use of a sensor in which pyrroloquinoline quinone-dependent glucose dehydrogenase was immobilized on the surface of electrode, direct and continuous observation of the hydrolysis can be achieved even in a thick cellulose suspension. The steady-state rate of the hydrolysis increased with increasing concentrations of the enzyme to approach a saturation value and was proportional to the amount of the substrate. The experimental results can be explained well by the rate equations derived from a three-step mechanism consisting of the adsorption of the free enzyme onto the surface of the substrate, the reaction of the adsorbed enzyme with the substrate, and the liberation of the product. The catalytic constant of the adsorbed enzyme was determined to be 0.044+/-0.011s(-1).

A novel FAD-dependent glucose dehydrogenase (FAD-GDH) was found and its enzymatic property for glucose sensing was characterized. FAD-GDH oxidized glucose in the presence of some artificial electron acceptors, except for O2, and exhibited thermostability, high substrate specificity and a large Michaelis constant for glucose. FAD-GDH was applied to an amperometric glucose sensor with Fe(CN)6(3-) as a soluble mediator. The use of a relatively high concentration of Fe(CN)6(3-) resulted in a good linearity between the current response and the glucose concentration, taking into account a large Michaelis constant for Fe(CN)6(3-). The glucose sensor was completely insensitive to O2 and responded linearly to glucose up to 30 mM. Compared to glucose, the response to other saccharides was negligible. The sensor can be stored at room temperature in a desiccator for at least one month without any change in the response or activity.

One-compartment bulk electrolysis and simultaneous spectroscopic measurements are realized in a conventional spectroscopic cuvette without separator by using a mesh-type working electrode with extremely large surface area and a wire-type counter electrode with very small surface area. Spectrophotometric monitoring revealed complete electrolysis in a first-order kinetics. This technique was applied to mediated titration of cytochrome c and bilirubin oxidase for determining their redox potentials. Kinetics for the solution redox reaction between protein and mediator is described. The subtraction of spectral background due to mediator adsorption is very easy because of high reproducibility. The experiments can be done under completely anaerobic conditions. Low-absorbance protein samples (of low concentrations or small absorption coefficients) and hydrophobic proteins (such as membrane-bound proteins) are acceptable for measurements.

Bilirubin oxidase (BOD) was immobilized as a multiple layer in a cationic polymer (poly- l -lysine) matrix on an electrode surface. The BOD-modified electrode catalyzed four-electron reduction of dioxygen (O 2 ) to water, without any mediator, to produce a diffusion-controlled voltammogram for the O 2 reduction in a quiescent solution. The voltammogram was successfully analyzed by the theory of irreversible voltammograms. Under convective conditions where O 2 depletion was negligible near the electrode surface, a steady-state voltammogram was obtained with a limiting current density of about 1 mA cm −2 . The steady-state voltammogram was explained by an equation derived on the basis of a reaction layer model, in which BOD was considered to be diffusible in the immobilized layer.

The hmd gene of histamine dehydrogenase from Nocardioides simplex was overexpressed in Escherichia coli, and the resulting enzyme was purified to homogeneity. The purified recombinant enzyme is almost identical with the native enzyme in view of molecular weight and specific activity, and is stoichiometrically assembled with the three cofactors 6-S-cysteinyl FMN, 4Fe-4S cluster, and ADP.

Histamine dehydrogenase from Nocardioides simplex is a homodimeric enzyme and catalyzes oxidative deamination of histamine. The gene encoding this enzyme has been sequenced and cloned by polymerase chain reactions and overexpressed in Escherichia coli. The sequence of the complete open reading frame, 2073 bp coding for a protein of 690 amino acids, was determined on both strands. The amino acid sequence of histamine dehydrogenase is closely related to those of trimethylamine dehydrogenase and dimethylamine dehydrogenase containing an unusual covalently bound flavin mononucleotide, 6-S-cysteinyl-flavin mononucleotide, and one 4Fe-4S cluster as redox active cofactors in each subunit of the homodimer. The presence of the identical redox cofactors in histamine dehydrogenase has been confirmed by sequence alignment analysis, mass spectral analysis, UV-vis and EPR spectroscopy, and chemical analysis of iron and acid-labile sulfur. These results suggest that the structure of histamine dehydrogenase in the vicinity of the two redox centers is almost identical to that of trimethylamine dehydrogenase as a whole. The structure modeling study, however, demonstrated that a putative substrate-binding cavity in histamine dehydrogenase is quite distinct from that of trimethylamine dehydrogenase.

Intact hydrogenase in Desulfovibrio vulgaris (Hildenborough) cells exhibits catalytic activity for the reaction H2+Mox2H++Mred (here Mox/Mred is a redox couple serving as an electron acceptor/donor). Dependence on pH of the catalytic activity was studied in view of the expected pH dependence of the substrate concentration [H+] and the standard free energy change of the overall reaction ΔG°. It has been revealed that the conventional Michaelis-Menten type analysis for the H2 evolution reaction leads an erroneous conclusion with respect to the dependence of the reaction rate on [H+] and that the pH dependence of the hydrogenae-catalyzed H2 evolution and consumption reactions reflect the pH-dependent change of ΔG°.

Aromatic amine dehydrogenase was purified and characterized from Alcaligenes xylosoxidans IFO13495 grown on beta-phenylethylamine. The molecular mass of the enzyme was 95.5 kDa. The enzyme consisted of heterotetrameric subunits (alpha2beta2) with two different molecular masses of 42.3 kDa and 15.2 kDa. The N-terminal amino acid sequences of the alpha-subunit (42.3-kDa subunit) and the beta-subunit (15.2-kDa subunit) were DLPIEELXGGTRLPP and APAAGNKXPQMDDTA respectively. The enzyme had a quinone cofactor in the beta-subunit and showed a typical absorption spectrum of tryptophan tryptophylquinone-containing quinoprotein showing maxima at 435 nm in the oxidized form and 330 nm in the reduced form. The pH optima of the enzyme activity for histamine, tyramine, and beta-phenylethylamine were the same at 8.0. The enzyme retained full activity after incubation at 70 degrees C for 40 min. It readily oxidized various aromatic amines as well as some aliphatic amines. The Michaelis constants for phenazine methosulfate, beta-phenylethylamine, tyramine, and histamine were 48.1, 1.8, 6.9, and 171 microM respectively. The enzyme activity was strongly inhibited by carbonyl reagents. The enzyme could be stored without appreciable loss of enzyme activity at 4 degrees C for one month at least in phosphate buffer (pH 7.0).

Combinations of bilirubin oxidase and metal complexes: [W(CN)8]3−/4−, [Os(CN)6]3−/4− and [Mo(CN)8]3−/4− (the formal potentials, E0′(M), being 0.320, 0.448, and 0.584 V vs. Ag|AgCl, respectively, at pH 7.0), allowed bioelectrocatalytic reduction of O2 to water at their formal potentials near neutral pH. The O2 reduction current appeared even at the standard potential of the O2/H2O redox couple, E0′(O2/H2O), when [Mo(CN)8]3−/4− was used at pH 7.4, though the magnitude was small. The magnitude of the bioelectrocatalytic current systematically decreased with the decrease in the potential difference between E0′(O2/H2O) and E0′(M). A limiting current as large as 17 mA/cm2 of a projected electrode surface area was obtained at 0.25 V (−0.37 V vs. E0′(O2/H2O)) for the O2 reduction at pH 7.0 with a carbon felt electrode modified with electrostatically entrapped bilirubin oxidase and [W(CN)8]3−/4− at the electrode rotation rate of 4000 rpm. Keywords: Bioelectrocatalysis, Bilirubin oxidase, Dioxygen reduction, [W(CN)8]3−/4−, [Os(CN)6]3−/4−, [Mo(CN)8]3−/4−

Escherichia coli cells, which contain apo-glucose dehydrogenase, were used in constructing a mediated amperometric glucose sensor. The E. coli modified glucose sensor, which was prepared by immobilizing E. coli cells behind a dialysis membrane on a carbon paste electrode containing 2,3-dimethoxy-5-methyl-1,4-benzoquinone (Q(0)), produced a current for the electrocatalytic oxidation of glucose with Q(0) as an electron transfer mediator only after the addition of a trace amount of pyrroloquinoline quinone (PQQ), the cofactor of the enzyme. This allows a novel method of glucose measurements free from the interference of the redox active substances, if contained, in a sample solution. The glucose sensor was insensitive to dioxygen; the currents measured under anaerobic and aerobic conditions, and even under dioxygen saturated conditions were almost the same in magnitude at a given concentration of glucose over the range of 0.2-10 mM. Response time of the glucose sensor was 2 min to attain 90% level of the steady-state current. The E. coli modified glucose sensor was reusable when treated with ethylenediaminetetraacetic acid (EDTA). When E. coli cells were lyophilized, they could be stored at room temperature in a dry box for more than six months without loss of the catalytic activity.

An analytical equation for the steady-state catalytic current of mediated bioelectrocatalysis has been derived as a function of the mediator concentration and the electrode potential for the case where the concentration of a substrate is sufficiently higher than the Michaelis constant for the substrate and the redox reaction of a mediator to obey the Nernst equation at an electrode surface. The equation can be reduced to the equations so far proposed for limited cases. The equation representing the limiting steady-state catalytic current as a function of the mediator concentration is useful to evaluate the catalytic constant of the enzyme and the Michaelis constant for the mediator, separately.

Quinohemoprotein amine dehydrogenase (QH-AmDH) from Paracoccus denitrificans has a novel cofactor cysteine tryptophylquinone (CTQ) in the smallest gamma subunit and two hemes c in the largest alpha subunit [Datta, S., Mori, Y., Takagi, K., Kawaguchi, K., Chen, Z., Okajima, T., Kuroda, S., Ikeda, T., Kano, K., Tanizawa, K., and Mathews, F. S. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 14268-14273]. The spectral change of QH-AmDH was assigned to the redox reaction of the hemes c alone. The redox potentials of the two hemes c with His and Met as the second axial ligands, respectively, were determined to be 0.149 and 0.235 V versus SHE at pH 7.0 by a mediator-assisted continuous-flow column electrolytic spectroelectrochemistry (MCES). The monomeric gamma subunit of QH-AmDH was isolated from urea-treated QH-AmDH. The fully oxidized and reduced forms of the gamma subunit exhibited a unique absorption band centered at 380 nm and a shoulder band around 315 nm, respectively, at neutral pH. The two-electron redox potential of CTQ in the isolated gamma subunit was evaluated to be 65 mV at pH 7.0 by MCES. The redox reaction was linked to the two-proton transfer at pH8.6 and to a single-proton transfer at pH8.6. The pK(a) value (K(a) being the acid dissociation constant) of 8.6 was assigned to one of the phenolic OH groups of the quinol form. Upon deprotonation, the red shift of the shoulder band was observed. The gamma subunit adsorbed on a glassy carbon electrode, and gave a direct but quasi-reversible electrochemical signal. Intra- and interprotein electron transfers of QH-AmDH are discussed from thermodynamic and structural points of view.

Mediated biosensors consisting of an oxidase and peroxidase (POx) have attracted increasing attention because of their wide applicability. However, since most of oxidases utilize artificial electron acceptors in place of dioxygen, the competition between O2 and the electron acceptor in the mediated sensors is anticipated. This has been evidenced with a glucose oxidase (GOx)- and POx-coentrapped and ferrocene-embedded carbon paste electrode, which exhibits peak-shaped current-time curves at increased concentrations of glucose and also gives a peak-shaped calibration curve. Digital simulation has been applied to clarify the cause of such unusual responses, by taking into account the ping-pong enzyme kinetics on two- and three-substrate models for POx and GOx, respectively. The simulation has well reproduced such unusual responses and has clearly revealed that the depletion of O2 in the enzyme layer is the most important factor responsible for such unusual responses. To overcome such a drawback of oxidase/POx bienzyme sensors, it is expected to be essential to decrease the rate of the oxidase reaction. In contrast, increase in the POx activity is useful to improve the sensitivity. According to the simulation-based expectation, the GOx and POx concentrations in the bienzyme sensor are adjusted to exhibit normal behavior with high sensitivity.

微生物全細胞のグルコース酸化活性を電気化学的に測定する方法を用いて, 微生物に及ぼす薬剤の影響について調べた. Escherichia coli IFO3301 (E. coli) やLactobacillus plantarum IFO14711 (L. plantarum) の細胞にエタノール, 過酸化水素, 次亜塩素酸のような薬剤を作用させると, 薬剤濃度の増加とともにグルコース酸化活性及びコロニー形成能が低下した. 両菌のグルコース酸化活性は, コロニー形成能に比べて薬剤感受性が高かった. Pseudomonas putida IFO14164 (P. putida) のグルコース酸化活性は, E. coli やL. plantarum と異なり, 薬剤感受性が低かった. P. putida 細胞に26%エタノール又は0.5%過酸化水素を30℃, 10分間作用させると, コロニー形成能は消失するが, グルコース酸化活性は95%エタノール又は3%過酸化水素処理でも約20%残存した. 対数増殖期のP. putida やE. coli 細胞のコロニー形成能及びグルコース酸化活性は, 定常期に比べて次亜塩素酸に対する感受性が高かった. 低濃度の次亜塩素酸でP. putida 細胞を処理すると, コロニー形成能の薬剤耐性は増大したが, グルコース酸化活性の耐性は低下した. E. coli では, 次亜塩素酸による前処理はコロニー形成能とグルコース酸化活性の両方の薬剤耐性を増大させた.

It can be expected that extracellular electron transfer to regenerate NAD+ changes the glucose metabolism of the homofermentative lactic acid bacteria. In this work, the glucose metabolism of Lactobacillusplantarum and Lactococcus lactis was examined in resting cells with 2-amino-3-carboxy-1,4-naphthoquinone (ACNQ) as the electron transfer mediator and ferricyanide (Fe(CN)6(3-)) as the extracellular electron acceptor. NADH in the cells was oxidized by ACNQ with the aid of diaphorase, and the reduced ACNQ was reoxidized with Fe(CN)6(3-). The extracellular electron transfer system promoted the generation of pyruvate, acetate, and acetoin from glucose, and restricted lactate production. Diaphorase activity increased when cultivation was aerobic, and this increased the concentrations of pyruvate, acetate, and acetoin relative to the concentration of lactate to increase in the presence of ACNQ and Fe(CN)6(3-)

A bioelectrochemical pyruvate sensor is applied to the monitoring of the glycolytic oscillation. This method allows acetaldehyde (or NADH) perturbation experiments. A moderate extent of acetaldehyde addition causes the phase reset of the oscillation of pyruvate as well as NADH. The phenomenon is reasonably explained by our model, whereby the NADH consumption catalyzed by alcohol dehydrogenase (ADH) activates glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transiently and then ADP is effectively phosphorylated by the succeeding 3-phosphoglycerate kinase (3-PKG) reaction, which decelerates the phosphofructokinase (PFK) reaction and then the glycolytic flux. The fast transmission of information from the increase in the NAD + –NADH ratio by acetaldehyde addition to the decrease in the ADP–ATP ratio was verified experimentally by a model enzyme system composed of ADH, GAPDH, and 3-PKG. The acetaldehyde-driven ADP removal in the ADH–GAPDH–3-PKG system is proposed to play an important role in the glycolytic oscillation. The significance of the NAD + –NADH ratio on the period of the glycolytic oscillation is also discussed.

Fluorescence superquenching is investigated for polyelectrolytes consisting of cyanine dye pendant polylysines ranging in number of polymer repeat units (N(PRU)) from 1 to 900, both in solution and after adsorption onto silica nanoparticles. As N(PRU) increases, the absorption and fluorescence evolve from monomer spectra to red-shifted features indicative of molecular J aggregates. In solution, the superquenching sensitivity toward an anionic electron acceptor increases by more than a millionfold over the N(PRU) range from 1 to 900. The dramatic increase is attributed to enhanced equilibrium constants for binding the quenchers, and the amplified quenching of a delocalized exciton of approximately 100 polymer repeat units. The self-assembly of monomer onto silica and clay nanoparticles leads to formation of J aggregates, and surface-activated superquenching enhanced 10,000x over the monomer in solution, indicating the formation of "self-assembled polymers" on the nanoparticle surface. Utilization of these self-assembled polymers as high-sensitivity biosensors is demonstrated.

A bacterial cell mixture ot Cellulomonas cartae KYM-7 and Agrobacterium tumefaciens KYM-8 was analyzed by capillary zone electrophoresis (CZE) and capillary gel electrophoresis (CGE). Both pherograms showed two discrete peaks. The cells in the peaks were collected, Gram stained, and examined with a microscope. The cells of the two strains were well separated by OGE, and each OGE peak consisted almost entirely of cells of one strain (greater than 98% purity), whereas each CZE peak contained cells of both strains (about 90% purity). In the concentration range of 1.0 x 10(10) to 1.0 x 10(12) cells/mL, the area of CGE peaks was proportional to the amount of cells. The growth of the two strains in mixed culture was measured by OGE. The OGE quantification data were in good agreement with those obtained using fluorescence in situ hybridization. The CGE analyses were accomplished in 1 h, using a relatively uncomplicated procedure. Thus, OGE exhibited great advantages in accuracy, rapidity, and simplicity.

A novel photosynthetic bioelectrochemical cell that utilizes biocatalysts in both anode and cathode compartments was constructed for the first time. in the anodic half-cell, some parts of the electrons produced by the oxidation of water in the photosystem of cyanobacteria are transferred to the carbon felt anode through quinonoid electron transfer mediators. The electron is passed to dioxygen to regenerate water in the cathodic half-cell reaction with an aid of bilirubin oxidase reaction via a mediator. The maximum electric power was about 0.3–0.4 W m-2 for the projective electrode surface area at an apparent efficiency of the light energy conversion of 2–2.5%. The factors governing the cell output are discussed on the basis of the potential-current curves of each half-cell.

Spectroelectrochemical studies were performed on the interaction between Ca2+ and pyrroloquinoline quinone (PQQ) in soluble glucose dehydrogenase (sGDH) and in the free state by applying a mediated continuous-flow column electrolytic spectroelectrochemical technique. The enzyme forms used were holo-sGDH (the holo-form of sGDH from Acinetobacter calcoaceticus) and an incompletely reconstituted form of this, holo-X, in which the PQQ-activating Ca2+ is lacking. The spectroelectrochemical and ESR data clearly demonstrated the generation of the semiquinone radical of PQQ in holo-sGDH and in the free state in the presence of Ca2+. In contrast, in the absence of Ca2+ no semiquinone was observed, either for PQQ in the free state (at pH7.0) or in the enzyme (holo-X). Incorporation of Ca2+ into the active site of holo-X, yielding holo-sGDH, caused not only stabilization of the semiquinone form of PQQ but also a negative shift (of 26.5mV) of the two-electron redox potential, indicating that the effect of Ca2+ is stronger on the oxidized than on the reduced PQQ. Combining these data with the observations on the kinetic and chemical mechanisms, it was concluded that the strong stimulating effect of Ca2+ on the activity of sGDH can be attributed to facilitation of certain kinetic steps, and not to improvement of the thermodynamics of substrate oxidation. The consequences of this conclusion are discussed for the oxidative as well as for the reductive part of the reaction of sGDH.

Bioelectrocatalytic sensor of histamine has been constructed using quinohemoprotein amine dehydrogenase (QH-AmDH) as an enzyme and its native electron acceptor cytochrome c-550 (Cyt c-550) as a mediator. Highly reversible electron transfer of Cyt c-550 is achieved at bis(4-pyridyl)disulfide-modified Au electrodes. The electron transfer from substrate-reduced QH-AmDH to oxidized Cyt c-550 is in the diffusion-controlled region, in spite of relatively small redox potential difference. QH-AmDH and Cyt c-550 can be co-entrapped on the electrode surface with a suitable dialysis membrane. As a result, Cyt c-550 is found to be an excellent mediator compared with other artificial ones. The sensor allows the histamine detection down to 500 nM (S/N=3) and the linearity is retained up to 1.5 mM with a relative standard deviation of 4.8 % at 10 µM histamine (n=6). The performance of the histamine biosensor is discussed in view of clinical chemistry.

A mediated bioelectrocatalysis using a bacterial cell-modified electrode was applied to the evaluation of heat resistance of Escherichia coli and Lactobacillus plantarum. The first derivative of the time course of the bioelectrocatalysis current for the bacterial D-glucose oxidation had a maximum dI/dtmax at about 10 to 60 s after the addition of D-glucose to the solution. The dI/dtmax value decreased in different manners depending on the conditions of heat treatment of the bacterial cells prior to their immobilization on electrode. The decreasing tendency in the dI/dtmax value was similar to that in the colony formation unit measured by a conventional colony counting method. The dI/dtmax method, which takes only 2 h, is much more time saving compared with the conventional method taking 24 h incubation for E. coli and 72 h incubation for L. plantarum. In some cases, however, there was a difference in the magnitude of relative decrease between the two methods, which could be attributed to the appearance of injured cells that were active in the dI/dtmax measurement but not in the colony formation.

Electrochemical reduction of dioxygen to water proceeds very effectively at 0.4 V versus Ag ∣ AgCl in pH 7.0 solution at an ambient temperature through the 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonate) (ABTS2−)-mediated and bilirubin oxidase (BOD) [EC 1.3.3.5]-catalyzed reaction of dioxygen. Electrochemistry of the ABTS2− oxidation and the indirect catalytic reduction of dioxygen with ABTS2− and BOD have been studied in detail to elucidate fully the bioelectrocatalytic behavior. The bioelectrocatalytic system using a carbon felt electrode has been examined and discussed in view of the cathode reaction in a biofuel cell.

An approximate equation is derived for the steady-state limiting current of mediated bioelectrocatalysis as a function of the bulk mediator concentration in the case where enzyme and mediator are in the soluble state and the substrate concentration is sufficiently higher than the Michaelis constant of the substrate. The error of the approximate equation has been evaluated by digital simulation and an empirical equation to correct the error has been proposed to describe the steady-state current more precisely. These equations have been applied successfully to typical mediated bioelectrocatalytic systems to evaluate the catalytic constant and the Michaelis constant of the mediator.

The physiological electron acceptor of quinohemoprotein amine dehydrogenase (QH-AmDH) from Paracoccus denitrificans IFO 12442 was identified by biochemical and electrochemical methods. Of three types of heme c-containing proteins purified together with QH-AmDH from the periplasm of n-butylamine-grown cells, only constitutive cytochrome c-550 was reduced by the addition of QH-AmDH and n-butylamine. Reconstitution of the respiratory chain revealed that cytochrome c-550 mediates the electron transfer from QH-AmDH to the terminal oxidase. This is a new pathway of the amine oxidation respiratory chain of P. denitrificans.

A biochemical fuel cell was constructed using H2 as fuel to produce H2O in the reaction with O2 at neutral pH and ambient temperature. The cell uses carbon felt as an electrode material for both the anode and the cathode and an anion exchange membrane as a separator. The anodic oxidation of H2 was accelerated by methyl viologen-mediated electrocatalysis with bacterial cells Desulfoibrio ulgaris (Hildenborough) as catalysts, and the cathodic reduction of O2 was accelerated by 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate)-mediated electrocatalysis with bilirubin oxidase as a catalyst. The bioelectrocatalytic systems allowed the cell to operate at 1.0 V with current 0.9 mA at an electrode of size 1.5 × 1.5 × 0.1 cm3. The cell voltage attained 1.17 V at open circuit, which is close to the standard electromotive force 1.23 V. The cell voltage–current behavior is interpretable by linear sweep voltammetry using the same electrode system. On this basis, the electrochemistry behind the performance of the biochemical fuel cell is discussed.

The electron transfer from the photosynthetic system in cyanobacteria, Synechococcus sp. PPC7942 to exogenous electron acceptors was examined using several electrochemical techniques. 1,4-Benzoquinone (BQ) and 2,6-dimethyl-1,4-benzoquinone (DMBQ) were found to function as good exogenous electron acceptors for the photosystem. Kinetic analysis with rotating disk amperometry revealed that the photoreduction of these quinones proceeds in Michaelis–Menten type kinetics for the concentration of the quinones and the light intensity. The electron transfer rate of the BQ reduction was as high as 68% compared with that of the photosynthetic oxygen evolution. Synechococcus sp. cell-entrapped and DMBQ-embedded carbon paste electrodes provided steady-state current ascribed to the photoelectrochemical oxidation of water. Although several inhibitors against the photosynthetic system suppressed the photoelectrochemical response, phenylmercury acetate, which inhibits ferredoxin and ferredoxin–NADP oxidoreductase, was found to enhance the photocurrent. Some electrochemical aspects of this system are discussed.

Desulfovibrio vulgaris (Hildenborough) cells catalyzed the oxidation of hydrogen with several quinone compounds as exogenous electron acceptors, in which hydrogenase existing in the periplasmic space of the bacterial cells functioned as the enzyme to catalyze the reaction. The rates of the hydrogen oxidation and quinone reduction were analyzed by a Michaelis−Menten type equation to yield the values of the catalytic constant of a D. vulgaris cell, kB,cat, and the bimolecular reaction rate constants for hydrogen, kB,cat/KB,H, and for quinone, kB,cat/KB,Q. They were in the ranges of kB,cat = (1.1−5.3) × 107 s-1, kB,cat/KB,H = (1.8−2.2) × 1012 M-1 s-1 and kB,cat/KB,Q = (0.97−10) × 1010 M-1 s-1 for the reactions with four kinds of quinone compounds. The mass transfer process involved in the bacterial cell-catalyzed reaction was considered by a model taking account of the substrate diffusion to and through the cross-membrane channels (composed of proteins called porins) distributed in the bacterial outer membra...

Alkaline phosphatase (ALP), a labeling enzyme frequently used for enzyme immunoassay, was determined at the attomole level using a mushroom tyrosinase (TN)-embedded carbon paste electrode. The detection scheme consists of two successive amplifications. The first reaction is the ALP-catalyzed hydrolysis of phenyl phosphate to accumulate phenol as a product. The second is the bioelectrocatalytic detection of phenol with the TN-embedded electrode at 0 V versus Ag/AgCl, which allowed sensitive detection of phenol down to 35 nM. The two-enzymatic reactions of ALP and TN were made to proceed successively in one electrochemical cell by adjusting pH to their optimum ones. This method provided a linear relation between the ALP concentration and the steady-state current and allowed the ALP detection as low as 7 amol/10 μl sample. The ALP detection system was applied to a streptavidin-biotin binding assay. The lowest detection limit of biotin was 4 fmol/10 μl sample with an ALP reaction time of 50 min.

Recent progress in mediated bioelectrocatalysis is reviewed. In a section concerning fundamentals, special attention is paid to describing the property of a steady-state current in this system from thermodynamic and kinetic viewpoints. Practical applications of the system includes biosensors for enzyme substrates, amplified detection of mediators, bioreactors, biofuel cells, enzyme kinetic measurements, and protein redox potential measurements.

Apo-glucose dehydrogenase existing in Escherichia coli is converted to the holoenzyme with exogenous pyrroloquinoline quinone (PQQ) and Mg(2+). Catalytic behaviour of the E. coli cells with the holoenzyme is characterized by a Michaelis-Menten-type equation with a catalytic constant of the cell and apparent Michaelis constants for D-glucose and an artificial electron acceptor added to the E. coli suspension. The catalytic constant is expressed as the product of the number of molecules of the enzyme contained in an E. coli cell (z) and the catalytic constant of the enzyme (k(cat)), which were determined to be 2.2x10(3) and 6.8+/-0.8x10(3) s(-1) (phenazine methosulphate as an electron acceptor) respectively. Kinetics of the in vivo holoenzyme formation can be followed by an enzyme-electrochemical method developed by us. The rate constants for the reactions of apoenzyme with PQQ (k(f,PQQ)) and with Mg(2+) (k(f,Mg)) were determined to be 3.8+/-0.4x10(4) M(-1).s(-1) and 4. 1+/-0.9 M(-1).s(-1) respectively. Equilibrium constants for the binding of apoenzyme to PQQ and Mg(2+) were determined as the dissociation constants K(d,PQQ(Mg)) and K(d,Mg) to be 1.0+/-0.1 nM and 0.14+/-0.01 mM respectively. The dissociation constants for Ca(2+) were also determined. The holoenzyme, once formed in E. coli, returns gradually to the apoenzyme in the absence of PQQ and/or Mg(2+) in solution. EDTA was effective to remove Mg(2+) from the enzyme in the cells to deactivate the enzyme completely, while PQQ remained in the E. coli cells.