Shin-ichi Yamazaki

Kinetic analysis and mechanistic aspects of autoxidation of catechins

A peroxidase-based bioelectrochemical sensor of hydrogen peroxide (H(2)O(2)) and a Clark-type oxygen electrode were applied to continuous monitoring and kinetic analysis of the autoxidation of catechins. Four major catechins in green tea, (-)-epicatechin, (-)-epicatechin gallate, (-)-epigallocatechin, and (-)-epigallocatechin gallate, were used as model compounds. It was found that dioxygen (O(2)) is quantitatively reduced to H(2)O(2). The initial rate of autoxidation is suppressed by superoxide dismutase and H(+), but is independent of buffer capacity. Based on these results, a mechanism of autoxidation is proposed; the initial step is the one-electron oxidation of the B ring of catechins by O(2) to generate a superoxide anion (O(2)(*-)) and a semiquinone radical, as supported in part by electron spin resonance measurements. O(2)(*-) works as a stronger one-electron oxidant than O(2) against catechins and is reduced to H(2)O(2). The semiquinone radical is more susceptible to oxidation with O(2) than fully reduced catechins. The autoxidation rate increases with pH. This behavior can be interpreted in terms of the increase in the stability of O(2)(*-) and the semiquinone radical with increasing pH, rather than the acid dissociation of phenolic groups. Cupric ion enhances autoxidation; most probably it functions as a catalyst of the initial oxidation step of catechins. The product cuprous ion can trigger a Fenton reaction to generate hydroxyl radical. On the other hand, borate ion suppresses autoxidation drastically, due to the strong complex formation with catechins. The biological significance of autoxidation and its effectors are also discussed.

Highly sensitive electrochemical detection of alkaline phosphatase

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.

Role of 2-amino-3-carboxy-1,4-naphthoquinone, a strong growth stimulator for bifidobacteria, as an electron transfer mediator for NAD(P)(+) regeneration in Bifidobacterium longum.

2-Amino-3-carboxy-1,4-naphthoquinone (ACNQ) is a novel growth stimulator for bifidobacteria. The role of ACNQ as a mediator of the electron transfer from NAD(P)H to dioxygen (O(2)) and hydrogen peroxide (H(2)O(2)), proposed in our previous paper, was examined using the cell-free extract and whole cells of Bifidobacterium longum. Continuous monitoring of ACNQ, O(2) and H(2)O(2) by several amperometric techniques has revealed that ACNQ works as a good electron acceptor of NAD(P)H diaphorase and that the reduced form of ACNQ is easily autoxidized and also acts as a better electron donor of NAD(P)H peroxidase than NAD(P)H. The generation of H(2)O(2) by B. longum under aerobic conditions is effectively suppressed in the presence of ACNQ. These ACNQ-mediated reactions would play roles as NAD(P)(+)-regeneration processes. The accumulation of ACNQ in the cytosol has been also suggested. These characteristics of ACNQ seem to be responsible for the growth stimulation of bifidobacteria. Vitamin K(3), which has an extremely low growth-stimulating activity and was used as a reference compound, exhibits much lower activity as an electron transfer mediator. The difference in the activity is discussed in terms of the redox potential and partition property of the quinones.

Escherichia coli and its application in a mediated amperometric glucose sensor.

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.

Mechanistic study on the roles of a bifidogenetic growth stimulator based on physicochemical characterization

2-Amino-3-carboxy-1,4-naphthoquinone, discovered as a novel bifidogenetic growth stimulator (BGS), has been characterized by determination of redox and acid-base equilibria, partition properties, and UV-vis and electron spin resonance spectral properties. BGS is proposed to function as an electron transfer mediator from NADH to O2. BGS is reduced by NADH-reduced diaphorase (or related enzymes) and the reduced BGS is reoxidized by autoxidation and a peroxidase-catalyzed reaction. The proposed reaction would spare pyruvate as an important metabolic intermediate, and minimize the cytotoxic effects of H2O2 generated by the autoxidation. Kinetic studies were performed in model enzymatic systems using 2-methyl-1,4-naphthoquinone (VK3) as a reference compound with a very weak growth-stimulating effect. The results support our proposal and reveal the superiority of BGS to VK3 as an electron transfer mediator in the proposed reactions.

Glucose Metabolism of Lactic Acid Bacteria Changed by Quinone-mediated Extracellular Electron Transfer

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 Lactobacillus plantarum 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−.

2-Amino-3-carboxy-1,4-naphthoquinone affects the end-product profile of bifidobacteria through the mediated oxidation of NAD(P)H

Abstract. Glucose metabolism of bifidobacteria in the presence of 2-amino-3-carboxy-1,4-naphthoquinone (ACNQ), a specific growth stimulator for bifidobacteria, and ferricyanide (Fe(CN)63–) as an extracellular electron acceptor was examined using resting cells of Bifidobacterium longum and Bifidobacterium breve. NAD(P)H in the cells is oxidized by ACNQ with the aid of diaphorase activity, and reduced ACNQ donates the electron to Fe(CN)63–. Exogenous oxidation of NADH by the ACNQ/Fe(CN)63– system suppresses the endogenous lactate dehydrogenase reaction competitively, which results in the remarkable generation of pyruvate and a decrease in lactate production. In addition, a decrease in acetate generation is also observed in the presence of ACNQ and Fe(CN)63–. This phenomenon could not be explained in terms of the fructose-6-phosphate phosphoketolase pathway, but suggests rather that glucose is partially metabolized via the hexose monophosphate pathway. This was verified by NADP+-induced reduction of Fe(CN)63– in cell-free extracts in the presence of ACNQ. Effects of the ACNQ/Fe(CN)63– system on anaerobically harvested cells were also examined. Stoichiometric analysis of the metabolites from the pyruvate-formate lyase pathway suggests that exogenous oxidation of NADH is an efficient method to produce ATP in this pathway.

Mechanistic study on the role of the NAD+-NADH ratio in the glycolytic oscillation with a pyruvate sensor

Abstract 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.