19F-NMR study on the interaction of fluorobenzoate with porcine kidney D-amino acid oxidase

The interactions of competitive inhibitors, o-, m-, and p-fluorobenzoates, with porcine kidney D-amino acid oxidase (DAO) were studied by 19F-NMR spectroscopy. The 19F-signals of DAO-bound fluorobenzoates were observed as considerably broadened peaks. The chemical shifts, which are referenced to 20 mM NaF in 50 mM sodium phosphate, pH 7.0, were 6.0, 8.2, and 11.9 ppm for free o-, m-, and p-fluorobenzoates, respectively, while those of o-, m-, and p-fluorobenzoates bound to DAO were 12.5, 5.4, and 13.1 ppm, respectively. The 19F-signals of bound o- and p-fluorobenzoates were downfield-shifted relative to those of the free species, whereas the 19F-resonance of m-fluorobenzoate was up-field shifted from that of the free ligand. The magnitude of the chemical shift difference between the free and bound forms decreases in the order of o-, m-, and p-fluorobenzoates. The remarkably large downfield shift of the o-fluorobenzoate when bound to DAO was attributed to the close proximity of the ortho-fluorine atom to the flavin nucleus in comparison with meta- or para-fluorine. The pH-dependences of the 19F-resonances of o-, m-, and p-fluorobenzoates were observed and the pKa values of 3.33, 3.80, and 4.05 were obtained for the carboxyl groups of o-, m-, and p-fluorobenzoates, respectively. It was observed that the 19F-resonances of o- and p-fluorobenzoates are highly sensitive to the ionic state of the carboxyl group, while that of m-fluorobenzoate was moderately sensitive.     

EDTA bis-(ethyl phenylalaninate): a novel transition metal-ion chelating hydroxyl radical scavenger with a potential anti-inflammatory role

Conjugation of ethylenediaminetetraacetic acid (EDTA) to ethyl phenylalaninate generates a novel radical scavenging metal-ion chelator EDTA bis-(ethyl phenylalaninate) (EBEP). The oxidation products o-, m- and p-tyrosine were isolated from hydrolysed, aqueous and aerated solutions containing EBEP, Fe(II) and H(2)O(2). Data obtained demonstrate the potential of EBEP to act as a radical scavenging, iron-ion chelating antioxidant under physiologically relevant conditions.     

Analysis of Acidic Metabolites of Biogenic Amines in Bovine Retina and Vitreous and Aqueous Humour by Gas Chromatography-Negative Ion Chemical Ionisation Mass Spectrometry

Acidic metabolites of a number of biogenic amines have been identified and quantified by reaction with either acetic or propionic anhydride in the aqueous phase followed by extraction into ethyl acetate, esterification of carboxyl groups with ditrifluoromethylbenzyl bromide (DTFMBzBr), and then conversion of the remaining free hydroxyl groups to acetates. Subsequent analysis of these derivatives revealed that most (greater than 60%) of the ion current was carried by the ion resulting from the loss of DTFMBz from the molecular ion. This made the method highly specific and practical--limits of detection were established at approximately 200 pg with a potential limit of detection below the picogram level. This method establishes unequivocally that the metabolites of tyramine, dopamine, and adrenaline/noradrenaline (4-hydroxyphenylacetic acid, 3,4-dihydroxyphenylacetic acid, and dihydroxymandelic acid, respectively) are present in bovine retina and in vitreous and aqueous humour. In addition, high concentrations of the dopamine metabolite homovanillic acid were found in retina and vitreous, but not in aqueous humour. p-Hydroxymandelic acid, the acidic metabolite of p-octopamine/p-synephrine, was identified in vitreous and in aqueous humour.     

Synthesis and cytogenetic studies of structure--biological activity relationship of esters of Hecogenin and aza-homo-Hecogenin with N,N-bis(2-chloroethyl)aminocinnamic acid isomers

The esters of Hecogenin and aza-homo-Hecogenin with N,N-bis(2-chloroethyl)aminocinnamic acid isomers have been prepared and their cytogenetic studies of structure-biological activity relationship were evaluated. The cytogenetic effects (sister chromatid exchanges (SCEs) induction and proliferation rate indices (PRIs) depression) by o-, m- and p-[N,N-bis(2-chloroethyl)amino] cinnamic acid were also investigated. Among the above compounds tested, those of the m-[N,N-bis(2-chloroethyl)amino] cinnamic acid and of the o-[N,N-bis(2-chloroethyl)amino] cinnamic acid ester of aza-homo-Hecogenin were more active in comparison to the others.     

Simultaneous determination of 3-nitro tyrosine, o-, m-, and p-tyrosine in urine samples by liquid chromatography-ultraviolet absorbance detection with pre-column cloud point extraction

Stable 3-nitro tyrosine (3-NO(2)-Tyr), o-, m-, and p-tyrosine isomers induced by oxidation of tyrosine residues in protein were considered important biomarkers for the existence of toxic oxidizing agents peroxynitrite (ONOO(-)) and OH*, which could lead to such diseases as acute lung injury, neurodegenerative disorders, atherosclerosis, cancers and many other diseases. Therefore, development of an accurate, simple and sensitive method to simultaneously detect o-, m-, and p-tyrosine and 3-NO(2)-Tyr is necessary. Fluorescence detection is highly sensitive to o-, m-, and p-tyrosine, but it cannot be used to detect 3-NO(2)-Tyr, due to the strong fluorescence-quenching characteristic of the NO(2) group. In this study, we developed a highly sensitive reversed HPLC-UV method, combined with pre-column cloud point extraction (CPE), to simultaneously determine o-, m-, and p-tyrosine and 3-NO(2)-Tyr. The procedure included derivatization of a sample with 6-aminoquinolyl-N-hydroxy-succinimidyl carbomate (AccQ) at 0.20 mol/l borate buffer (pH 8.80) for 30 min at 70 degrees C, and pre-concentration with surfactant cloud point extraction. The surfactant-rich phase was then diluted with deionized water and injected directly into the to HPLC column for analysis. A C(18) column (3.9 mm i.d. x 300 mm) was used for gradient elution separation at 25 degrees C and the detection wavelength was at 254 nm. Nineteen general amino acids showed no interference. The detection limits of p-, o-, m-Tyr and 3-NO(2)-Tyr were between 5 and 15 nmol/l. The linear range was from 0.05 to approximately 100 micromol/l.     

Anaerobic biodegradation of phenolic compounds in digested sludge.

We examined the anaerobic degradation of phenol and the ortho, meta, and para isomers of chlorophenol, methoxyphenol, methylphenol (cresol), and nitrophenol in anaerobic sewage sludge diluted to 10% in a mineral salts medium. Of the 12 monosubstituted phenols studied, only p-chlorophenol and o-cresol were not significantly degraded during an 8-week incubation period. The phenol compounds degraded and the time required for complete substrate disappearance (in weeks) were: phenol (2), o-chlorophenol (3), m-chlorophenol (7), o-methoxyphenol (2), m- and p-methoxyphenol (1), m-cresol (7), p-cresol (3), and o-, m-, and p-nitrophenol (1). Complete mineralization of phenol, o-chlorophenol, m-cresol, p-cresol, o-nitrophenol, p-nitrophenol, and o-, m-, and p-methoxyphenol was observed. In general, the presence of Cl and NO2 groups on phenols inhibited methane production. Elimination or transformation of these substituents was accompanied by increased methane production, o-Chlorophenol was metabolized to phenol, which indicated that dechlorination was the initial degradation step. The methoxyphenols were transformed to the corresponding dihydroxybenzene compounds, which were subsequently mineralized.     

13C-NMR studies of porcine kidney D-amino acid oxidase reconstituted with 13C-enriched flavin adenine dinucleotide. Effects of competitive inhibitors

D-Amino acid oxidase (DAO) from porcine kidney was reconstituted with FAD's which were enriched with 13C at the 2-, 4-, 4a-, and 10a-positions of the isoalloxazine moiety, and 13C-NMR spectra of the reconstituted DAO were measured in the absence and presence of various competitive inhibitors. When compared to the corresponding chemical shifts of FMN at infinite dilution (Moonen, C.T.W. et al. (1984) Biochemistry 23, 4859-4867), the resonances of C(2), C(4), C(4a), and C(10a) of FAD bound to apoDAO were all shifted to higher field. However, when the reconstituted DAO was complexed with o-, m-, p-aminobenzoate, or benzoate, each of the four 13C-signals underwent a different change in chemical shift. In these changes we observed no characteristics which distinguish DAO-aminobenzoate complexes from DAO-benzoate complex, even though o-, m-, and p-aminobenzoates are known to form charge-transfer complexes with DAO. The signals due to 2- and 4-13C were more sensitive to the formation of the inhibitor-DAO complexes than those of the other carbon atoms. These findings suggest the modulation of the hydrogen bonds at the oxygen atoms of C(2) = O and C(4) = O with the protein moiety as the result of the inhibitor-binding.     

Thermodynamic insight into the origin of the inclusion of monosulfonated isomers of triphenylphosphine into the beta-cyclodextrin cavity

Formation of inclusion complexes between the beta-cyclodextrin and the potassium salt of o-, m-, and p-substituted monosulfonated triphenylphosphine derivatives was investigated in aqueous solution by NMR spectroscopy. Titration and continuous variation plots obtained from 31P and 1H NMR data indicate the formation of a 1:1 inclusion complex for the three phosphine isomers. T-ROESY NMR experiments show that in all cases a non-sulfonated aromatic ring was included into the hydrophobic cavity of beta-cyclodextrin from the secondary hydroxyl groups side. The standard enthalpy and entropy of the inclusion process were estimated from the temperature dependence of the association constant. All inclusion complexes were enthalpy stabilized, but highly entropy destabilized.     

Determination of betaxolol in human aqueous humour by high-performance liquid chromatography with fluorescence detection

A reversed-phase high-performance liquid chromatographic method is described for the determination of betaxolol in human aqueous humour. Betaxolol and the internal standard metoprolol were extracted with cyclohexane and separated on a reversed-phase column (Luna C(18), 250 x 4.6 mm, 5 microm) with a mobile phase containing acetonitrile-phosphate buffer (40:60, v/v) at a flow-rate of 0.8 ml/min. The column effluent was monitored with a fluorescence detector at 227 nm (excitation) and 301 nm (emission). The retention times for metoprolol and betaxolol were 3.55 and 5.63 min, respectively. The recovery from aqueous humour was found to be 71.6% for betaxolol at 1.25 microg/ml. The within-day and day-to-day accuracy values were in the range of 96.17-105.2% for betaxolol at 0.1, 4 and 12 microg/ml (n=6), within-day and day-to-day precision values were less than 10% for betaxolol at the concentrations given above. The detection limit corresponding to the signal-to-noise ratio of 3:1 was 15 ng/ml. The presented method was suitable for measuring betaxolol levels in human aqueous humour samples obtained from patients after topical administration.     

Degradation of high concentrations of cresols by Pseudomonas sp. CP4

Pseudomonas sp. CP₄, a potent phenol-degrading laboratory isolate could mineralize all three isomers of cresol. This strain readily utilized up to 1.4, 1.1 and 2.2 g/l of o- m- and p-cresol, respectively as the sole sources of carbon and energy. These are the highest concentrations of cresols reported to be degraded by a bacterial strain. The rates of degradation of the three isomers were in the order: o- > p- > m-cresol. All the isomers of cresol were catabolized through a meta-cleavage pathway. Fairly high catechol 2,3-dioxygenase (C230) activity against catechol was observed in the cell-free extracts of the culture grown on these compounds and were in the order: m- > o- > p-cresol.     

Photo-induced DNA scission by Cu(ii)-meso-tetrakis(n-N-methylpyridiniumyl)porphyrins (n = 2, 3, 4) and their binding modes to supercoiled DNA

The photo-induced cleavage of pGEM-7zf-NIS super-coiled DNA by Cu(ii)-meso-tetrakis(n-N-methylpyridiniumyl)porphyrins (n = 2, 3, 4 referred to as o-, m- and p-CuTMPyP, respectively) and their binding mode were investigated in this study. m-CuTMPyP was most efficient in cleavage than o- and p-CuTMPyP isomers. Cleavage was suppressed by N(2) bubbling, suggesting that the cleavage occurred by an oxidative cleavage mechanism. Sodium azide, an (1)O(2) quencher, and DMSO, a hydroxyl radical scavenger, inhibited cleavage, indicating that hydroxyl radicals and singlet oxygen were likely reactive species responsible for the cleavage. Reduced linear dichroism spectroscopy showed angles of o-CuTMPyP's electric transition moments, in which the periphery pyridinium ring was prevented from free rotation, of 59° and 61° with respect to the local DNA helix axis. The spectra of m- and p-CuTMPyP complexed with pGEM-7zf-NIS DNA were characterized by large signals in the Soret band, coincident with those of known intercalated porphyrins.     

High-performance liquid chromatographic assay of bromocriptine in plasma and eye tissue of the rabbit

A reliable reversed-phase high-performance liquid chromatographic method has been developed for the determination of bromocriptine (BCT) in plasma and eye tissues. The BCT and propranolol, added as an internal standard (I.S.), were extracted by a liquid–liquid technique followed by an aqueous back-extraction, allowing injection of an aqueous solvent into a 4-μm Nova-Pak C₁₈ column (150×3.9 mm I.D.). The mobile phase was a mixture of 30 parts of acetonitrile and 70 parts of 0.2% triethylamine (pH 3) at a flow-rate of 1 ml/min. Fluorescence detection was at an excitation wavelength of 330 nm and an emission wavelength of 405 nm. The retention times of I.S. and BCT were 4.1 and 11.6 min, respectively. The calibration curve was linear over the concentration range 0.2–10 μg/l for plasma (r>0.999) and vitreous humour (r>0.997) and 1–50 μg/l for aqueous humour (r>0.985). The limit of quantification was 0.2 μg/l for plasma and vitreous humour using a 1-ml sample and was 1 μg/l for aqueous humour using a 0.2-ml sample. The quality control samples were reproducible with acceptable accuracy and precision. The within-day recovery (n=3) was 100–102% for plasma, 91–106% for aqueous humour and 96–111% for vitreous humour. The between-day recovery (n=9) was 90–114% for plasma, 83–115% for aqueous humour and 90–105% for vitreous humour. The within-day precision (n=3) and the between-day precision (n=9) were 1.7–7.0% and 8.1–13.6%, respectively. No interferences from endogenous substances were observed. Taken together, the above simple, sensitive and reproducible high-performance liquid chromatography assay method was suitable for the determination of BCT in plasma and eye tissues following ocular application of BCT for the therapy of myopia.     

Ability of different biomaterials to enantioselectively catalyze oxidation and reduction reactions

We studied the ability of different biomaterials to enantioselectively catalyze oxidation or reduction reactions with the help of substrate rac-1-m or p-ArCH(OH)Me and the 1-o-ArC(O)Me derivatives. Apoenzyme (NAD(P)(+)-dependent secondary alcohol dehydrogenase(NAD(P)-E)) and cofactor (NAD(P)(+)) were activated by preincubating immobilized aqueous plant leaf (e.g., young wheat leaves), cereal tissue (wheat bran), vegetable (e.g., carrot), and seaweed (e.g., wakame seaweed) solutions, and the NAD(P)-E oxidized only (R)-isomers highly enantioselectively. Thus, greater than 99% ee(s) of (S)-isomers (1m-5m and 1p-5p) can be obtained from corresponding rac-1-m or p-ArCH(OH)Me. Further, immobilized chlorella cells and immobilized baker's yeast can reduce highly stereoselectively; greater than 99% ee(s) of (S)-isomers (1o-5o) can be obtained from corresponding 1-o-ArC(O)Me. Specific use of each isomer ((S)-6 and (R)-6) with greater than 99% ee(s) of racemic-1-2-NpCH(OH)Me becomes possible through selective use of NAD(P)-E eluted from artemisia vulgaris indica leaves and young wheat leaves. We suggest that the pH of the reaction media can determine not only the direction of NAD(P)-E, toward enantioselectively catalyzed oxidation (pH > 7.0) or reduction reaction (pH < 7.0), but also the regioselective reactivity of NAD(P)-E to the substrate o- (pH < 7.0), m-, and p-substituted groups (pH > 7.0). Thus, in comparison to current biocatalysts, several biomaterials can serve as asymmetric reagent bases, providing easily obtained, low-cost natural catalysts with stereoselectivity, regioselectivity, and substrate specificity that work under mild conditions for asymmetric synthesis of organic compounds.     

Proteomic analysis of human cataract aqueous humour: Comparison of one-dimensional gel LCMS with two-dimensional LCMS of unlabelled and iTRAQ®-labelled specimens

In this study, we report a comparative and quantitative analysis by mass spectrometry of the protein content of aqueous humour from cataract (control) patients. In addition to protein profiling, the approach is layered with quantitative proteomics using the iTRAQ? methodology. Aqueous humour from ten clinically-matched patients was collected and depleted of albumin and immunoglobulin G. Pairs of patient material were pooled and divided into three aliquots for subsequent analysis by alternative proteomic approaches. Excluding keratin, trypsin, residual albumin and immunoglobulins, a total of 198 protein groups were identified across the entire study. Relative protein quantitation with iTRAQ? revealed that 88% of the proteins had a maximal ±2-fold differential regulation between 3 of the 4 labelled samples, indicating minimal variation. The identified proteins were categorised by gene ontology and one third of the proteins were annotated as extracellular. The major molecular functions of the proteins in aqueous humour are binding (protein, metal ion, heparin, and DNA) and inhibition of proteolytic activity. Complementary to molecular function, the predominant biological processes for the proteins in aqueous humour are assigned to inflammatory and immune responses, and transport.     

New reaction system for hydrocarbon oxidation by chloroperoxidase

A novel reaction system was developed to maximize the catalytic efficiency of chloroperoxidase (CPO, from Caldariomyces fumago) toward the oxidation of hydrocarbons. The reaction system consisted of an organic/aqueous emulsion comprising pure substrate and aqueous buffer supplemented with the surfactant dioctyl sulfosuccinate. The emulsion system attenuated not only the destabilizing effects of the substrate and product on the enzyme by emulsifying the compounds, but also oxidant toxicity (oxidative stress) by increasing substrate availability. As a result, CPO exhibited total turnover numbers (TTNs, defined as the amount of product produced over the catalytic lifetime of the enzyme) of ca. 20,000 mol product/mol enzyme for the oxidation of styrene, toluene, and o-, m-, p-xylenes. The TTNs are over 10-fold higher than those previously reported for the oxidation of benzylic hydrocarbons by CPO. This study represents a significant step toward the development of CPO as a practical catalyst for large-scale organic syntheses.     

Synthesis and evaluation of 19F labeled sulfonyl fluorides as probes of protease structure: alpha-chymotrypsin

Active site Ser-195-fluorine-labeled derivatives of alpha-chymotrypsin were prepared from a series of N-(trifluoromethylphenyl)-fluorosulfonylphenyl carboxamides whose synthesis is described. The six new 19F spin labels varied in the position of the -CF3 substituent (o-, m-, and p-) and the fluorosulfonyl substituent (m- or p-). The chemical shifts of these covalently bound analogs of "tosyl-chymotrypsin" were each uniquely sensitive to their environment in the catalytic center as evidenced by differences in resonance line position for each label. Upon titrating these derivatives with the reversible competitive inhibitor, indole, a downfield shift was observed (with all but one label), which could be fit in each case to an apparent dissociation constant for indole binding. Indole binding to the p-sulfonyl derivatives was essentially unaltered from that for the native enzyme, while the m-sulfonyl derivatives required some additional free energy of binding to saturate the enzyme. The results are consistent with a partial embedding of the phenylsulfonyl moiety in the aromatic specificity pocket.     

A study of the interaction between D-amino acid oxidase and quasi-substrates.

To study the interaction between D-amino acid oxidase [EC 1.4.3.3] and quasi-substrates such as benzoate and o-, m-, and p-aminobenzoate, visible circular dichroism spectra (CD spectra) were measured and the binding rate and affinity of o-aminobenzoate to the enzyme were observed by following the absorption changes at various wavelengths. We found a new CD band around 560 nm, corresponding to the charge-transfer complexes which result from the formation of aminobenzoate complexes with the enzyme. The ellipticity of this band was positive for the p-aminobenzoate complex, but negative for the o- and m-aminobenzoate complexes. Crossover points in CD spectra were observed at 470 nm for the m-aminobenzoate complex and at 475 nm for the o-aminobenzoate complex. They probably resulted from overlapping of the positive CD band of FAD bound with the enzyme and the negative CD band of the charge-transfer complex. We propose that the amino group in aminobenzoate, not the pi-electrons of the benzene ring, is the electron donor in the charge-transfer complex and that the position of the amino group is very important for the charge-transfer interaction. The binding rate and affinity of o-aminobenzoate to the enzyme were determined using the absorption changes at 370 nm (380 nm), caused by the modification of electronic states of FAD bound with the enzyme, and at 550 nm (565 nm), caused by the formation of the charge-transfer complex of o-aminobenzoate with the enzyme. No differences between these parameters with wavelength were observed. This independence of wavelength simplifies discussion of the experimental data obtained from absorption changes.     

Chromatographic identification of a biochemical alteration in the aqueous humour of megalophthalmic Black Moor goldfish

Purpose: To observe if any biochemical abnormalities exist between the eye of megalophthalmic and non-megalophthalmic goldfish by high-performance liquid chromatography (HPLC). Method: Aqueous humour and sera from megalophthalmic and non-megalophthalmic goldfish were subjected to HPLC and monitored by photodiode array detection (Waters, MA, USA). Results: An unusual accumulation of a compound with a UV absorption maximum at 290 nm was observed in the aqueous humour of megalophthalmic eye. This compound was also present in the sera of both normal goldfish and one of its megalophthalmic mutant. However, it was significantly elevated in the aqueous humour of the megalophthalmic eye only. This compound concentration was very high in the eye of small fish and its concentration increased only slightly with the expansion of the eye in larger fish. Conclusions: The presence of this compound in the serum and aqueous humour indicates a specific systemic metabolic variation in Black Moor goldfish not seen other animal species we had studied (humans, bovine, chick, rabbits and rats). The marked elevation of this compound in the megalophthalmic eye indicates a possible association of this compound with the metabolic variation accounting for the expansion of the eye in megalophthalmic goldfish.     

Collagen type IX and HNK-1 epitope in tears of patients with pseudoexfoliation syndrome

Pseudoexfoliation syndrome (PEX) is an age-related condition, which may cause open-angle glaucoma and has increasing interest since it seems to affect additional human tissues, i.e., cardiovascular tissue, skin, and still lacks elucidated pathogenesis. Collagen type IX and HNK-1 epitope have been considered characteristic constituents of the aqueous humour of PEX patients, since their amounts were increased in PEX aqueous humour compared to normal eyes. Since it has been proposed that the initial manifestations of PEX syndrome occur in conjunctiva, the present study was undertaken to investigate the presence of the same antigens in tears of PEX patients and their possible use as the biochemical markers for early diagnosis. Tears of PEX patients and healthy individuals were subjected to western blotting analysis for various basement membrane components identified in aqueous humour. It was found that collagen type IX and HNK-1 epitope were present in tears, the amount of the former being increased 2.7 times compared to normal (P<0.05), surprisingly high as compared with total protein or lysozyme activity in tears, which were found to be increased in PEX patients about 25% with no statistical differences (P approximately 0.4). The results suggest the possible use of tears' collagen type IX for the diagnosis of PEX syndrome.     

Influence of position of substituent groups on removal of chlorophenols and cresols by horseradish peroxidase and determination of optimum conditions

Enzymatic treatment of o-, m-, and p-chlorophenols and o-, m-, and p-cresols from artificial wastewater was undertaken through the enzymatic conversion into the corresponding phenoxy radicals with horseradish peroxidase (HRP) and nonenzymatic radical coupling reaction. The concentration of chlorophenols and cresols decreased sharply over the reaction time and water-insoluble oligomer precipitates were generated. The optimum conditions were determined to be the H2O2 concentration of 2.5 mM and poly(ethylene glycol) with molecular mass of 1.0 x 10(4) (10K-PEG) of 0.10 mg/cm3 at 30 degrees C for treatment of p-chlorophenol at 2.5 mM. The optimum pH values depended on the relative position of a chlorine atom for chlorophenols and on a methyl group for cresols. Concentrations of HRP and 10K-PEG were increased to 1.0 U/cm3 and 1.0 mg/cm3 respectively to treat m-chlorophenol highly. For o-chlorophenol, a decrease in the pH value to 3.0 after the enzymatic treatment led to the enhancement of the aggregation of oligomer precipitates. The % residual value for o-cresol effectively decreased by absorbing water-soluble intermediates on the chitosan films. These results indicate that chlorophenols and cresols were removed to a great degree by this technique, although the detailed procedure depended on the position of substituent groups of chlorophenols and cresols.