Hydroperoxynaphthalimide Derivative-mediated Oxidation of Heme Proteins on Photoirradiation: Horseradish Peroxidase

Abstract— Horseradish peroxidase (HRP) was photoirradiated in the presence of organic peroxide (1, hydroperoxynaphthalimide derivative) at around 353 nm and 0°C. This compound bound to a heme pocket of HRP as shown by its inhibitory effect on catalysis by HRP ( K _i= 5.5 times 10⁻⁵ M) and subsequently it formed an intermediate in the same way as H202. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) suggested cleavage of the peptide chain of HRP on photoirradiation with 1. From CD spectra and SDS-PAGE, it was presumed that the destruction of both secondary structure and heme of the enzyme occurred to some extent upon photoirradiation, which resulted in a decrease in the catalytic activity. The absorption spectra also suggested that the heme group of the enzyme was destroyed, and the fluorescence spectra showed that the Trp residue in the photoirradiated HRP was oxidized to N -formylkynurenine by a hydroxyl radical generated from 1. Energy transfer from the excited naphthalimide moiety or hydrogen abstraction also seemed to make some contribution to the alteration of the heme group.     

Preparation of poly(fumaric acid) from poly(di-t-butyl fumarate) and poly(di-trimethylsilyl fumarate)

Polymerization conditions of di-t-butyl fumarate and di-trimethylsilyl fumarate were studied in detail. They cannot be polymerized by either anionic or coordination initiators, but radical and radiation polymerizations are successful. Characterization of poly(di-t-butyl fumarate), obtained thereby, with ¹H-NMR spectrum suggests that the backbone of the chain is stiff. From analysis of thermal properties of poly(di-t-butyl fumarate), it is found to be completely converted to poly(fumaric acid) by pyrolysis around 200°C. Poly(di-trimethylsilyl fumarate), on the other hand, can be quantitatively hydrolyzed with acid to the same polyacid, too. The preliminary measurement of the dissociation behaviors of poly(fumaric acid) was done by potentiometric titration, which shows that the titration curves of poly(fumaric acid) are different from those of poly(acrylic acid) and poly(maleic acid).     

Fishing of beta-amylase with a SAM of alpha-cyclodextrin-poly- (ethylene glycol) conjugate

Alpha-cyclodextrin (alpha-CD) with an amino group was conjugated to an alpha, omega-dicarboxylated poly(ethylene glycol) (PEG). The inhibition constant (Ki) of the alpha-CD-PEG conjugate for the catalysis by beta-amylase was larger than that of alpha-CD, due to a steric obstruction of the PEG moiety to the binding of alpha-CD moiety to beta-amylase. alpha-CD-PEG was further modified with cystamine (CD-PEG-Cys) or cysteamine methyl disulfide (CD-PEG-MDS), and the disulfide-carrying alpha-CD-PEG was accumulated on a gold surface as a self-assembled monolayer (SAM). The binding of beta-amylase to the alpha-CD-PEG SAM was followed by a decrease in cathodic peak current in the voltammogram of hydroquinone as a probe using a cyclic voltammetry (CV). The beta-amylase bound to the alpha-CD-PEG SAM was desorbed by the addition of free alpha-CD, and the ratio of desorbed beta-amylase from the SAM of alpha-CD-PEG-Cys to the total amount of the enzyme bound to the SAM was 40% whereas that from the alpha-CD-PEG-MDS SAM was 83-85%. The percentage of desorption was increased to 100% by the treatment of the alpha-CD-PEG-MDS SAM-carrying electrode with 2-hydroxyethyldisulfide prior to the immersion in the enzyme solution. Adsorption and desorption processes of beta-amylase to the surface of alpha-CD-PEG-MDS SAM were clearly observed using localized surface plasmon resonance absorption spectroscopy. The binding constant of the enzyme to the surface-confined alpha-CD-PEG was much larger than that to free alpha-CD, probably due to a large local concentration of the alpha-CD moiety on the gold surface.     

Influence of particle concentration on the electrorheological efficiency of polyaniline suspensions

The dependence of the efficiency of positive electrorheological (ER) phenomenon, expressed as a ratio of the low-shear electroviscosity to the field-off viscosity, on the particle concentration of polyaniline base suspensions in silicone oil has been studied. A simple analysis reveals that the dependence of the efficiency on the volume fraction of suspended particles may have a maximum at which the optimum ER performance of material is expected. Experimental results confirmed this expectation. It is demonstrated that with higher temperature the maximum is shifted to higher particle concentration.     

Digital radiation monitoring system with a personal computer

This paper describes a data processing system of monitors with a personal computer for a radioisotope laboratory. A monitoring system includes 6 area, 6 dust, and 3 gas monitors. Monitor signals through a multiplexer were fed to the personal computer which has analogue ports. Processed data were displayed on a color CRT, stored on a 5 inch mini floppy disk, and printed out as a report. We developed a data processing program with BASIC language. This system largely reduced manpower required to estimate radiation levels in the laboratory. The personal computer has sufficiently enough ability to construct a digital monitoring system for a small radioisotope laboratory.     

Continuous Determination of Trace Amounts of Humic Acid in Natural Water by Chemiluminescence Method

A chemiluminescence (CL) method for the determination of humic acid (HA) based on the oxidation of HA with hydrogen peroxide in the presence of formaldehyde in alkaline solution is described. This method is sensitive and selective for the determination of HA in natural water. HA produces strong CL in the oxidation of HA with MnO₄⁻, Br₂, ClO⁻, and Cr₂O₇²⁻, and the H₂O₂. HA-H₂O₂-HCHO system is suitable for the determination of HA because of its high sensitivity and high selectivity. The detection limit was 50 ppb and relative standard deviation for five measurements of 0.5 ppm (w/w) HA was 1.8%. Cations such as Na⁺, K⁺, Mg²⁺, Cu²⁺, and Fe3⁺ and anions such as PO₄³⁻, NO₃⁻, CO₃²⁻, SO₄²⁻, Cl⁻, and Y⁻ (EDTA-Na) did not interfere with the determination of HA. Addition of Mn(II) increased the CL intensity. The concentration of HA in natural water determined with this method is in good agreement with that determined by fluorometric analysis.     

Raman spectroscopic study on the structure of water in aqueous solution of alpha,omega-amino acids

The structure and hydrogen bonding of water in an aqueous solution of various alpha,omega-amino acids were analyzed using the contours of the OH stretching in the polarized Raman spectra. From the relative intensity of the collective band (C value) corresponding to a long-range coupling of the OH stretching in the aqueous amino acid solutions, the number of hydrogen bonds disrupted due to the presence of one amino acid molecule (N(corr) value) was evaluated. The N(corr) value for glycine was slightly positive, whereas with an increase in the number of methylene groups between ammonium and carboxylate groups, the N(corr) value gradually increased. These results suggest that the species with proximal anionic and cationic groups do not disturb the hydrogen-bonded network structure of water significantly, probably due to the counteraction of the electrostatic hydration effect attributable to the anionic and cationic groups.     

Asymmetric 1,3-dipolar cycloaddition of chiral sulphinylethenes with 1-methyl-3-oxido-pyridinium and some nitrones

1,3-Dipolar cycloaddition of (R)-(+)-p-tolyl vinyl sulphoxide 1 with 1-methyl-3-oxidopyridinium 6 proceeded in a diastereoselective manner to afford the exo and endo cycloadducts 11a,b and 12a in 36%, 7% and 29% yield, respectively. The absolute configuration of 11a was determined by its transformation to (1S)-(−)-2-tropanol (−)-15. Attempts to the cycloaddition of the sulphinylethenes 17–19 with the pyridinium 6 were nsuccessful under several conditions. The reaction of the sulphoxide 20 with pyrroline 1-oxide 21 gave an inseparable mixture of products. The cycloaddition of 20 with 3,4,5,6-tetrahydropyridine 1-oxide22 afforded a mixture of four adducts in ca. 90% yield. High level of diastereoselectivity was achieved for the endo cycloaddition affording the adduct 23 in 33% isolated yield. The absolute configuration of 23 was confirmed by a single-crystal X-ray diffraction study. The stereochemical course of the reaction was discussed based on the absolute configuration of the products.     

HYDROPEROXYNAPHTHALIMIDE DERIVATIVE-MEDIATED OXIDATION OF LYSOZYME

Abstract— Lysozyme was photoirradiated in the presence of photo-Fenton reagents (hydroperoxynaph-thalimide derivatives) at 366 nm. Enzymatic activities of photoirradiated lysozymes were lower than that of native lysozyme. Taking account of the results of amino acid analysis and of fluorescence spectra, it was probably that Trp residues in the photoirradiated lysozyme were oxidized with hydroxyl radicals. The reagents formed complexes with lysozyme as proved by the inhibitory effects of the reagents on the enzymatic activities ( K ₁= 4.7 ± 1.2 × 10⁴ M for HPO II, a hydroperoxide derivative of naphthalimide), which suggested that these reagents were bound to the active site cleft of lysozyme, and the Trp residues located in or near the active site cleft were photooxidized. Fluorescence-difference spectra of photoirradiated lysozymes showed that Trp 62 was preferentially photooxidized. Furthermore, sodium dodecyl sulfatepolyacrylamide gel electrophoresis and circular dichroism spectra showed that the photooxidation examined here induced no significant change in the molecular size but a slight change in the conformation of lysozyme, which suggests the usefulness of the reagents in the site-selective oxidation of biopolymers.