Detection of a tryptophan radical as an intermediate species in the reaction of horseradish peroxidase mutant (Phe-221 --> Trp) and hydrogen peroxide

The crucial reaction intermediate in the reaction of peroxidase with hydrogen peroxide (H2O2), compound I, contains a porphyrin pi-cation radical in horseradish peroxidase (HRP), which catalyzes oxidation of small organic and inorganic compounds, whereas cytochrome c peroxidase (CcP) has a radical center on the tryptophan residue (Trp-191) and oxidizes the redox partner, cytochrome c. To investigate the roles of the amino acid residue near the heme active center in discriminating the function of the peroxidases in these two enzymes, we prepared a CcP-like HRP mutant, F221W (Phe-221 --> Trp). Although the rapid spectral scanning and stopped-flow experiments confirmed that the F221W mutant reacts with H2O2 to form the porphyrin pi-cation radical at the same rate as for the wild-type enzyme, the characteristic spectral features of the porphyrin pi-cation radical disappeared rapidly, and were converted to the compound II-type spectrum. The EPR spectrum of the resultant species produced by reduction of the porphyrin pi-cation radical, however, was quite different from that of compound II in HRP, showing typical signals from a Trp radical as found for CcP. The sequential radical formation from the porphyrin ring to the Trp residue implies that the proximal Trp is a key residue in the process of the radical transfer from the porphyrin ring, which differentiates the function of peroxidases.     

Experimental study of bubbly swirling flow in a vertical tube using ultrasonic velocity profiler (UVP) and wire mesh sensor (WMS)

Two-phase air-water bubbly swirling flow through a pipe is a complex turbulent flow and its prediction is still challenging. The present paper describes the experimental investigation of the air-water bubbly swirling flow in vertical co-current flow. Swirling flow is induced by a twisted tape in a 20 mm inner diameter pipe. The flow is investigated using Ultrasonic velocity profiler (UVP), which allows the measurement of liquid and gas velocities simultaneously. Furthermore, simultaneous measurement of void fraction is performed using Wire mesh sensor (WMS). The experimental results reveal that swirling flow has significant impact on bubbles’ distribution. In low liquid flow rate, the average bubble velocity is fairly uniform along the radial position and void fraction increases in the near wall region. However, increasing liquid flow rate at constant gas flow rate leads to increase in void fraction in the core region, this is mainly due to drift velocity which is affected by centrifugal force. Experimental findings and parametric trends based on the effects of swirling flow are summarized and discussed.     

Determination of the compositional distribution of copolymers by frontal analysis continuous capillary electrophoresis

Frontal analysis continuous capillary electrophoresis (FACCE) was carried out for a series of random copolymers of an ionic monomer, sodium 2-(acrylamido)-2-methylpropanesulfonate (AMPS), and a nonionic monomer, acrylamide (AAM). The electropherograms appeared in order of anionic content and were generally sigmoidal, in contrast to that of hyaluronic acid (HA), which was abrupt and discontinuous. This difference could be related to the compositional heterogeneity of the copolymers, completely absent in the biopolymer. Through the range of copolymer composition (10-100% AMPS) the relationship between average mobility and nominal AMPS content could be described by a calibration curve, making it possible to deduce the compositional distribution of copolymer samples.     

Dynamic flying characteristics of an air bearing slider over a disk with grooves and distribution of material properties

Bit-patterned media (BPM) consisting of land parts for read/writing and grooves, which are almost filled with non-magnetic materials to prevent magnetic interference between recording bits, are considered to be promising media for achieving ultrahigh density recording. A flying head slider flying over a BPM disk suffers from variations in both the spacing and van der Waals (vdW) attractive forces, which induce slider vibrations and spacing fluctuations. In the present study, we considered that BPM disks not only have a groove depth distribution, but that they also have a distribution of material properties (e.g., refractive index), which gives rise to a distribution in the vdW force. In the dynamic responses of sliders with small groove depths and a small variation in the refractive index, spacing fluctuations are found to be a superposition of fluctuations due to slider behaviors (1) over a disk with transverse grooves in a uniform material with a uniform refractive index (Case 1) and (2) over a flat disk with a refractive index distribution (Case 2). When the effects of Cases 1 and 2 cancel each other, the spacing fluctuations for the two cases cancel each other, reducing the total spacing fluctuation.     

Group behavior of agents with an emotional model

Recently, there has been much interest in the study of the formation of groups of agents that cause interactions between agents and invent new functions. We gave some agents an action rule based on the interactions of human feelings by using a circumplex model. It had been decided that the parameters of feelings in this model should have only two axes. In this report, eight basic action dimensions and pure feelings on four corresponding axes were given to agents as a model of feelings and actions based on the multiple factor analysis theory of R. Plutchik, and the behavioral characteristics of the group of agents were examined.     

Hydrogen bond network in the distal site of peroxidases: spectroscopic properties of Asn70 --> Asp horseradish peroxidase mutant

The distal His in peroxidases forms a hydrogen bond with the adjacent Asn, which is highly conserved among many plant and fungal peroxidases. Our previous work [Nagano, S., Tanaka, M., Ishimori, K., Watanabe, Y., & Morishima, I. (1996) Biochemistry 35, 14251-14258] has revealed that the replacement of Asn70 in horseradish peroxidase C (HRP) by Val (N70V) and Asp (N70D) discourages the oxidation activity for guaiacol, and the elementary reaction rate constants for the mutants was decreased by 10-15-fold. In order to delineate the structure-function relationship of the His-Asn couple in peroxidase activity, heme environmental structures of the HRP mutant, N70D, were investigated by CD, 1H NMR, and IR spectroscopies as well as Fe2+/Fe3+ redox potential measurements. While N70D mutant exhibited quite similar CD spectra and redox potential to those of native enzyme, the paramagnetic NMR spectrum clearly showed that the hydrogen bond between the distal His and Asp70 is not formed in the mutant. The disappearance of the splitting in the 1H NMR signal of heme peripheral 8-methyl group observed in 50% H2O/50% D2O solution of N70D-CN suggests that the hydrogen bond between the distal His and heme-bound cyanide is also disrupted by the mutation, which was supported by the low C-N vibration frequency and large dissociation constant of the heme-bound cyanide in the mutant. Together with the results from various spectroscopies and redox potentials, we can conclude that the improper positioning of the distal His induced the cleavages of the hydrogen bonds around the distal His, resulting in the substantial decrease of the catalytic activity without large structural alterations of the enzyme. The His-Asn hydrogen bond in the distal site of peroxidases, therefore, is essential for the catalytic activity by controlling the precise location of the distal His.     

Decreasing resistances of membrane-electrode assemblies containing hydrocarbon-based ionomers for improving their anodic performances

To improve methanol-oxidation performances of membrane-electrode assemblies composed of a hydrocarbon-based ionomers, the resistances involved in the reaction were decreased. Electrochemical impedance spectroscopy revealed that the proton-conductive resistance (R_i) in the anode was decreased from 0.54 to 0.40 Ω cm² by increasing a loading ratio of platinum-ruthenium to carbon support of anode catalyst from 54 to 73 wt.%. In addition, R_i was decreased to be 0.25 Ω cm² by increasing ion-exchange capacity (IEC) of the ionomer from 1.4 to 2.9 mequiv. g⁻¹. Consequently, the polarization resistance of the anode was significantly decreased, in turn, increasing current density of methanol oxidation at the potential of 0.45 V from 0.110 to 0.244 A cm⁻².     

Slug to annular flow transition during boiloff in a rod bundle under high-pressure conditions

High-pressure boiloff experiments in a wide range of bundle powers by using the Two-Phase Flow Test Facility (TPTF) were conducted. Two kinds of boiloff patterns were observed in these experiments. One is the boiloff pattern in a low bundle power, in which the dryout points of rods locate at a certain elevation in the bundle because the mixture level controls the dryout points. The other is the boiloff pattern in a high bundle power, in which the clear mixture level can not be observed and the dryout points of rods locate in a wide range of vertical directions. The vertical scatter of the dryout points is considered to be due to the break of the thin water film on the heater rods under the annular flow pattern.A simple model to predict the slug to annular flow transition in the rod bundle is proposed. In the model, the slug to annular flow transition takes place when the interferences of the water films on the neighboring rods cease. The model appeares to give good predictions of the previous flow transition experiment conducted in a rod bundle. The slug-annular transition below the dryout points was predicted with the present model in the high power boiloff experiments of TPTF. No slug-annular transition below the dryout points is predicted with the present model in the low power boiloff experiments.