Takeshi Miyakawa

Flow injection analysis to measure the production ability of superoxide with chemiluminescence detection in natural waters

In connection with the photo-chemical processes in the aquatic environment, the evaluation system for the production ability of superoxide anion was constructed. The technique was based on the flow-injection method with luminol chemiluminescence detection. The injected sample was first irradiated by a solar simulator in a vortex quartz cell (cell volume = 0.167 mL), whose vortex face was exposed to light after passing through two air-mass filters. The carrier was the aqueous solution at pH 11 adjusted by NaOH. After irradiation was finished, the carrier (with sample) flow was merged with 1.52 mM of the luminol solution and was introduced into a chemiluminescence detector. The results of the laboratory experiment show that the production of superoxide is linearly related to the concentration of humic acid up to 50 ppm, and also to that of dissolved oxygen. In addition, the chemiluminescence intensity (superoxide production) was proportionally related with the irradiation intensity of the solar simulator. By means of changing the flow rate of the carrier, the half-life of superoxide at pH 11 aqueous solution was estimated to be 15 s. ESR was measured for the sample containing 0.5% humic acid, 0.5% NaOH, and 20 μL DMPO (spin trap agent). ESR spectra were obtained after 5 min of irradiation of the solar simulator. In addition to the four sharp peaks due to OH radicals, a broad peak appeared at the middle of the OH signal. The obtained signal cannot conclusively be ascribed to superoxide, but the peak that appeared may be due to the radical produced in the humic acid molecule. The river water was collected at 18 points of the Tamagawa River located in Tokyo. Upstream, the production ability of superoxide was observed, but not downstream or in the estuarine district. Although the concentration of humic acid is much higher in the estuarine sample, some quenching mechanisms work for superoxide production.

Fluorometric Characteristics of a Wave‐Guide Cell with Low Refractive Index

Recently, Teflon AF2400 (AF 1600, and AF 1601) was commercialized (DuPont Fluoroproducts, Wilmington, DE, U.S.A), that has a refractive index (1.29) lower than water (1.33), and which means that the wave‐guiding of light is possible in water. In this study, we used Teflon AF 2400 as a waveguide capillary longpath cell for fluorometry. He‐Cd and Ar+ lasers were used as the excitation source, at 325 nm and 514.5 nm respectively. The length of the capillary wave‐guide cell was 18.70 cm. The was wound twice on a flat surface (loop diameters: 2 cm and 3 cm). The excitation was executed through the wave‐guide cell and the fluorescence from the wound capillary cell wall was collected in a perpendicular direction to the loop. With excitation at 325 nm, the fluorescence intensity at 450 nm emitted from the cell wall decreased along with the increase in the refractive index of the solvent. This can be caused by attenuation of the source light due to absorption by the solvent. In our experiment, the solvent of higher refractive index has the higher absorption at 325 nm. On the other hand, the fluorescence intensity at 590 nm, with excitation at 514.5 nm, increases with increased refractive index of the solvent. This result shows that an increase in the refractive index of the solvent is preferable for maintaining the wave‐guiding of the source light. Here, the characteristics of fluorescence spectrometry are discussed in terms of the collection of fluorescence from the wave‐guide capillary cell wall.

The simulation study of protein-protein interfaces based on the 4-helix bundle structure

Docking of two protein molecules is induced by intermolecular interactions. Our purposes in this study are: designing binding interfaces on the two proteins, which specifically interact to each other; and inducing intermolecular interactions between the two proteins by mixing them. A 4-helix bundle structure was chosen as a scaffold on which binding interfaces were created. Based on this scaffold, we designed binding interfaces involving charged and nonpolar amino acid residues. We performed molecular dynamics (MD) simulation to identify suitable amino acid residues for the interfaces. We chose YciF protein as the scaffold for the protein-protein docking simulation. We observed the structure of two YciF protein molecules (I and II), and we calculated the distance between centroids (center of gravity) of the interfaces’ surface planes of the molecules I and II. We found that the docking of the two protein molecules can be controlled by the number of hydrophobic and charged amino acid residues involved in the interfaces. Existence of six hydrophobic and five charged amino acid residues within an interface were most suitable for the protein-protein docking.

Mutual positional preference of IPMDH proteins for binding studied by coarse-grained molecular dynamics simulation

Proteins, which incorporate charged and hydrophobic amino acid residues, are useful as a material of nanotechnology. Among these proteins, IPMDH (3-isopropylmalate dehydrogenase), which has thermal stability, has potential as a material of nanofiber. In this study, we performed coarse-grained molecular dynamics simulation of IPMDH using MARTINI force fields, and we investigated the orientation for the binding of IPMDH. In simulation, we analyzed wild type of IPMDH and the mutated IPMDH proteins, where 13, 20, 27, 332, 335 and 338th amino acid residues are replaced by lysine residues which have positive charge and by glutamic acid residues which have negative charge. Since the binding of mutated IPMDH is advantageous compared with the binding of wild type for one orientation, we suggest that the Coulomb interaction for the binding of IPMDH is important.