Fluorescent derivatization of aromatic carboxylic acids with horseradish peroxidase in the presence of excess hydrogen peroxide.

The fluorescent derivatization of aromatic carboxylic acids by the catalytic activity of horseradish peroxidase (HRP) in the presence of excess H2O2 was investigated. Four monocarboxylic acids, nine dicarboxylic acids, and two tricarboxylic acids, all of which are non- or weakly fluorescent, were effectively converted into fluorescent compounds using this new method. This technique was further developed for the fluorometric determination of trace amounts of terephthalic acid (3c) and lutidinic acid (2b), and linear calibration curves for concentrations between 2.5 and 20.0 nmol of terephthalic acid (3c) and 1.0 and 10.0 nmol of lutidinic acid (2b) were demonstrated. Compound III, an intermediate of HRP, played an essential role in this process. Additionally, lactoperoxidase and manganese peroxidase, peroxidases similar to HRP, showed successful fluorescent derivatization of nicotinic acid (1b), lutidinic acid (2b), and hemimellitic acid (4a) in the presence of excess H2O2.

Spectrophotometric determination of hydrogen peroxide, glucose, uric acid, and cholesterol using peroxidase-like activity of an Fe(III) complex of thiacalix[4]arenetetrasulfonate attached to an anion-exchanger.

An iron(III) complex of thiacalix[4]arenetetrasulfonate attached to an anion-exchanger (Fe(3+)-TCASA-500) showed high peroxidase-like catalytic activity at pH 5 - 8 for the formation of quinoid dye, following the color reaction between 3-methyl-2-benzothiazolinone hydrazone (MBTH) and N-ethyl-N-(3-sulfopropyl)aniline (ALPS) in the presence of H2O2. This catalytic activity of Fe(3+)-TCASA-500 for the MBTH-ALPS system was applied for the spectrophotometric determination of H2O2, glucose, uric acid, and cholesterol. The calibration curves were linear in the concentration range from 1.0 to 15 µg of H2O2 in a 1.0-mL sample solution, and from 5.0 to 60 µg of glucose, 2.0 to 30 µg of uric acid, and 11.6 to 116 µg of cholesterol in a 0.5-mL sample solution. The apparent molar absorptivity of H2O2 was determined as 2.31 × 10(4) L mol(-1) cm(-1), which was about 70% of that by peroxidase under the same conditions. The determination method using Fe(3+)-TCASA-500 was applied for the determination of glucose and uric acid in both control sera I and II.

Catalytic activity of thiacalix[4]arenetetrasulfonate metal complexes on modified anion-exchangers for ascorbic acid oxidation.

The catalysis of ascorbic acid (AsA) oxidation by anion-exchangers modified with metal complexes of thiacalix[4]arenetetrasulfonate (Me-TCAS[4]A-500, Me=Mn(3+), Fe(3+), Co(3+), Ce(4+), Cu(2+), Zn(2+), Ni(2+), and H2) were investigated. Me-TCAS[4]A-500 (Me=Mn(3+), Fe(3+), Ce(4+), and Cu(2+)) all exhibited the ability to catalyze the oxidative reaction of AsA to dehydroascorbic acid. However, in the presence of high concentrations of AsA, only Cu(2+)-TCAS[4]A-500 was capable of complete oxidation of the acid. Moreover, after six repeat uses, Cu(2+)-TCAS[4]A-500 maintained high and relatively constant catalytic activity. Prior treatment of glucose solutions with Cu(2+)-TCAS[4]A-500, even in the presence of high AsA concentrations, enabled the satisfactory determination of glucose without interference by AsA. Cu(2+)-TCAS[4]A-500 will therefore be applicable as an artificial substitute for ascorbate oxidase, and may be useful as a means to eliminate AsA interference during the analysis of vital compounds such as glucose and uric acid.

Photodegradation of environmental mutagens by visible irradiation in the presence of xanthene dyes as photosensitizers.

The photodegradation of environmental mutagens, such as 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAαC), and 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ), was investigated by visible irradiation in the presence of xanthene dyes as photosensitizers. Although the environmental mutagens themselves were very stable during visible irradiation under the conditions in this study, they were effectively photodegraded in the presence of the xanthene dyes (erythrosine, rose bengal, and phloxine). Moreover, photodegradation of the mutagens was further enhanced for xanthene dyes loaded onto a water-soluble diethylaminoethyl (DEAE)-dextran anion-exchanger via ionic interactions (xanthene-dyeDEX). Photodegradation was inhibited by O2 removal from the reaction solution. In ESR spin-trapping experiments using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a trapping reagent, signals characteristic of DMPO-•OH (hydroxyl radical) were observed in the presence of xanthene-dyeDEX. These results suggest that reactive oxygen species derived from O2, such as singlet molecular oxygen (•1O2) and/or •OH, were active participants in photodegradation of the mutagens in the presence of xanthene dyes or xanthene-dyeDEX.

Fluorescent derivatization of xanthurenic acid and nicotinic acid with horseradish peroxidase in the presence of excess hydrogen peroxide.

The fluorescent derivatization of tryptophan metabolites (xanthurenic acid, nicotinic acid, picolinic acid, and 3-hydroxyanthranilic acid) by the catalytic activity of horseradish peroxidase (HRP) was investigated in the presence of excess H(2)O(2). Non-fluorescent xanthurenic acid (XA) and nicotinic acid (NA) were converted into a fluorescent compound with maximum excitation and emission wavelengths at 325 and 425 nm, and 318 and 380 nm, respectively. This fluorescent derivatization was developed for the fluorometric determination of trace amounts of XA and NA. The calibration curves were linear from 1.0 to 10.0 nmol XA and from 5.0 to 20.0 nmol NA in a 1.0-mL sample solution. The UV spectra of the reaction solutions suggested that compound III as an intermediate of HRP played an essential role in this fluorescent derivatization with HRP.

Scavenging effects of metal complexes of water-soluble thiacalix[4]arenetetrasulfonate on superoxide anion radicals.

The scavenging effects of metal complexes of thiacalix[4]arenetetrasulfonate (Me-TCAS[4], Me=H2, Fe³(+), Mn³(+), Mn²(+), Cu²(+), and Zn²(+)) on superoxide anion radicals (O2⁻) generated from the xanthine-xanthine oxidase system were investigated by the nitroblue tetrazolium (NBT) method and electron spin resonance (ESR) spin-trapping method using 5,5-dimethyl-1-pyrroline-N-oxide as a trapping reagent. As a reference, calix[4]arenetetrasulfonate (H2)-CAS[4]), calix[6]arenehexasulfonate (H2-CAS[6]) and calix[8]areneoctasulfonate (H2-CAS[8]) were also examined. The results by the NBT method indicated that Fe³(+)- and Mn³(+)-TCAS[4] exhibited the highest O2⁻ scavenging activity among Me-TCAS[4] and H2-CAS[n] (n = 4, 6, 8) in this study. The IC50 values of Fe³(+)- and Mn³(+)-TCAS[4] for O2⁻ scavenging activity were estimated to be 5.3 and 7.8 µM, respectively, and were almost the same as those of tannin acid, catechin and their derivatives, which are known as very effective scavengers of O2⁻. Scavenging activities were in the order of Fe³(+)- and Mn³(+)-TCAS[4]>>Mn²(+)-, Cu²(+)-, and Zn(2+)-TCAS[4]>>H(2)-TCAS[4] and H2-CAS[n] (n=4, 6, 8). Each activity of Me-TCAS[4] (Me=Fe³(+), Mn³(+), Mn²(+), Cu²(+), and Zn²(+)) was higher than that of the corresponding metal ion, indicating that H2-TCAS[4] has the ability to raise the activity of the metal ion itself by forming a complex. Also, the ESR spin-trapping method revealed that Fe³(+)- and Mn³(+)-TCAS[4] showed high O2⁻ scavenging activities, similarly to the results by the NBT method.

Peroxidase-like catalytic activity of water-insoluble complex linked Fe(III)-thiacalix[4]arenetetrasulfonate with tetrakis(1-methylpyridinium-4-yl)porphine via ionic interaction.

A new water-insoluble Fe(3+)-TCAS[4]/TMPyP complex linked tetraanionic Fe(III)-thiacalix[4]arenetetrasulfonate (Fe(3+)-TCAS[4]) with tetracationic tetrakis(1-methylpyridinium-4-yl)porphine (TMPyP) via ionic interaction was prepared. The peroxidase-like catalytic activity of the Fe(3+)-TCAS[4]/TMPyP complex was investigated based on the dye formation reaction by oxidation of 4-aminoantipyrine and phenol with H(2)O(2) catalyzed by peroxidase. This Fe(3+)-TCAS[4]/TMPyP complex showed the highest activity in pH 5.5 acetate buffer solutions, and it was applied to the photometric determination of trace amounts of H(2)O(2). The calibration curve was linear over the range from 1.0 to 35 microg of H(2)O(2) in a 1.0 ml sample solution. Moreover, the method using glucoseoxidase and the Fe(3+)-TCAS[4]/TMPyP complex was applied to the determination of glucose, and the results were satisfactory even in control sera. The Fe(3+)-TCAS[4]/TMPyP complex can be applied to a practical sample, such as blood or urine, as an analytical reagent for the photometric determination of H(2)O(2) in place of peroxidase.

Production of an allelopathic polyacetylene in hairy root cultures of goldenrod (Solidago altissima L.).

Hairy roots of goldenrod (Solidago altissima L.) were induced by infecting axenic plants with Agrobacterium rhizogenes strain A4. Growth and allelopathic polyacetylene (cis-dehydromatricaria ester, cis-DME) production of two independent hairy root clones were examined in several culture media and light regimes. cis-DME contents in hairy roots were at the same level as those in normal roots. cis-DME production in root cultures was several-fold lower than that of native plants and greatly repressed by light.

Yokonolide B, a novel inhibitor of auxin action, blocks degradation of AUX/IAA factors.

Yokonolide B (YkB; also known as A82548A), a spiroketal-macrolide, was isolated from Streptomyces diastatochromogenes B59 in a screen for inhibitors of beta-glucoronidase expression under the control of an auxin-responsive promoter in Arabidopsis. YkB inhibits the expression of auxin-inducible genes as shown using native and synthetic auxin promoters as well as using expression profiling of 8300 Arabidopsis gene probes but does not affect expression of an abscisic acid- and a gibberellin A3-inducible gene. The mechanism of action of YkB is to block AUX/IAA protein degradation; however, YkB is not a general proteasome inhibitor. YkB blocks auxin-dependent cell division and auxin-regulated epinastic growth mediated by auxin-binding protein 1. Gain of function mutants such as shy2-2, slr1, and axr2-1 encoding AUX/IAA transcriptional repressors and loss of function mutants encoding components of the ubiquitin-proteolytic pathway such as axr1-3 and tir1-1, which display increased AUX/IAAs protein stability, are less sensitive to YkB, although axr1 and tir1 mutants were sensitive to MG132, a general proteasome inhibitor, consistent with a site of action downstream of AXR1 and TIR. YkB-treated seedlings displayed similar phenotypes as dominant AUX/IAA mutants. Taken together, these results indicate that YkB acts to block AUX/IAA protein degradation upstream of AXR and TIR, links a shared element upstream of AUX/IAA protein stability to auxin-induced cell division/elongation and to auxin-binding protein 1, and provides a new tool to dissect auxin signal transduction.