Insight into multicopper oxidase laccase from Myrothecium verrucaria ITCC-8447: a case study using in silico and experimental analysis.

The oxidation activity of multicopper-oxidases overlaps with different substrates of laccases and bilirubin oxidases, thus in the present study an integrated approach of bioinformatics using homology modeling, docking, and experimental validation was used to confirm the type of multicopper-oxidase in Myrothecium verrucaria ITCC-8447. The result of peptide sequence of M. verrucaria ITCC-8447 enabled to predict the 3 D-structure of multicopper-oxidase. It was overlapped with the structure of laccase and root mean square deviation (RMSD) was 1.53 Å for 533 and, 171 residues. The low binding energy with azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (-5.64) as compared to bilirubin (-4.39) suggested that M. verrucaria ITCC-8447 have laccase-like activity. The experimental analysis confirmed high activity with laccase specific substrates, phenol (18.3 U/L), ampyrone (172.4 U/L) and, ampyrone phenol coupling (50 U/L) as compared to bilirubin oxidase substrate bilirubin (16.6 U/L). In addition, lowest binding energy with ABTS (-5.64), syringaldazine SYZ (-4.83), guaiacol GCL (-4.42), and 2,6-dimethoxyphenol DMP (-4.41) confirmed the presence of laccase. Further, complete remediation of two hazardous model pollutants i.e., phenol and resorcinol (1.5 mM) after 12 h of incubation and low binding energy of -4.32 and, -4.85 respectively confirmed its removal by laccase. The results confirmed the presence of laccase in M. verrucaria ITCC-8447 and its effective bioremediation potential.

Directed aryl sulfotransferase evolution toward improved sulfation stoichiometry on the example of catechols.

Sulfation is an important way for detoxifying xenobiotics and endobiotics including catechols. Enzymatic sulfation occurs usually with high chemo- and/or regioselectivity under mild reaction conditions. In this study, a two-step p-NPS-4-AAP screening system for laboratory evolution of aryl sulfotransferase B (ASTB) was developed in 96-well microtiter plates to improve the sulfate transfer efficiency toward catechols. Increased transfer efficiency and improved sulfation stoichiometry are achieved through the two-step screening procedure in a one-pot reaction. In the first step, the p-NPS assay is used (detection of the colorimetric by-product, p-nitrophenol) to determine the apparent ASTB activity. The sulfated product, 3-chlorocatechol-1-monosulfate, is quantified by the 4-aminoantipyrine (4-AAP) assay in the second step. Comparison of product formation to p-NPS consumption ensures successful directed evolution campaigns of ASTB. Optimization yielded a coefficient of variation below 15% for the two-step screening system (p-NPS-4-AAP). In total, 1760 clones from an ASTB-SeSaM library were screened toward the improved sulfation activity of 3-chlorocatechol. The turnover number (kcat = 41 ± 2 s-1) and catalytic efficiency (kcat/KM = 0.41 µM-1 s-1) of the final variant ASTB-M5 were improved 2.4- and 2.3-fold compared with ASTB-WT. HPLC analysis confirmed the improved sulfate stoichiometry of ASTB-M5 with a conversion of 58% (ASTB-WT 29%; two-fold improvement). Mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR) confirmed the chemo- and regioselectivity, which yielded exclusively 3-chlorocatechol-1-monosulfate. For all five additionally investigated catechols, the variant ASTB-M5 achieved an improved kcat value of up to 4.5-fold and sulfate transfer efficiency was also increased (up to 2.3-fold).

Pharmacokinetic investigations of the marker active metabolites 4-methylamino-antipyrine and 4-amino-antipyrine after intramuscular injection of metamizole in healthy piglets.

Metamizole (MT) is a pyrazolone nonsteroidal anti-inflammatory drug labelled for humans and animals. The aim of this study was to assess the pharmacokinetics of its active metabolites 4-methylamino-antipyrine (MAA) and 4-amino-antipyrine (AA) in male piglets after a single intramuscular injection of MT. Eight healthy male piglets were administered MT (100 mg/kg) intramuscularly. Blood was sampled at scheduled time intervals, and drug plasma concentrations evaluated by a validated HPLC method. MAA and AA plasma concentrations were quantitatively detectable from 0.25 to 48 h and 0.50 to 72 h, respectively, in 6 of 8 and 7 of 8 animals. The average maximum concentrations of MAA and AA were of 47.59 and 4.94 mg/mL, respectively. The average half-lives were 8.57 and 13.3 h for MAA and AA, respectively. This study showed that the amount of MAA and AA produced in piglets is different to that in the animal species previously investigated. Further studies are necessary to understand whether these differences in MAA and AA plasma concentrations between animal species necessitate diverse therapeutic drug dosing.

Pyrazolones metabolites are relevant for identifying selective anaphylaxis to metamizole.

Non-steroidal anti-inflammatory drugs (NSAIDs) are the most common cause of hypersensitivity reactions, with pyrazolones the most frequent drugs inducing selective reactions. Immediate selective hypersensitivity to pyrazolones is thought to be mediated by specific-IgE. Sensitivity of in vitro diagnostic tests is low and this may be due to the incomplete characterization of the structures involved. Here we investigated whether main metabolites of metamizole (dipyrone) in human could be involved in the immune response using the basophil activation test (BAT). We studied subjects with confirmed selective immediate hypersensitivity to metamizole and performed BAT with metamizole and its metabolites: 4-methylamino-antipyrine (MAA), 4-aminoantipyrine (AA), 4-acetylamino-antipyrine (AAA) and 4-formylamino-antipyrine (FAA). BAT results showed an increase of positive results from 37.5% to 62.5% using metamizole plus metabolites as compared with the BAT carried out only with the parent drug, demonstrating that metamizole metabolites have a role in the reaction and can induce specific basophil activation in patients with immediate hypersensitivity to this drug. Our findings indicate that pyrazolone metabolites are useful for improving the in vitro diagnosis of allergic reactions to metamizole.

A first continuous 4-aminoantipyrine (4-AAP)-based screening system for directed esterase evolution.

Esterases hydrolyze ester bonds with an often high stereoselectivity as well as regioselectivity and are therefore industrially employed in the synthesis of pharmaceuticals, in food processing, and in laundry detergents. Continuous screening systems based on p-nitrophenyl- (e.g., p-nitrophenyl acetate) or umbelliferyl-esters are commonly used in directed esterase evolution campaigns. Ongoing challenges in directed esterase evolution are screening formats which offer a broad substrate spectrum, especially for complex aromatic substrates. In this report, a novel continuous high throughput screening system for indirect monitoring of esterolytic activity was developed and validated by detection of phenols employing phenyl benzoate as substrate and p-nitrobenzyl esterase (pNBEBL from Bacillus licheniformis) as catalyst. The released phenol directly reacts with 4-aminoantipyrine yielding the red compound 1,5-dimethyl-4-(4-oxo-cyclohexa-2,5-dienylidenamino)-2-phenyl-1,2-dihydro-pyrazol-3-one. In this continuous B. licheniformis esterase activity detection system (cBLE-4AAP), the product formation is followed through an increase in absorbance at 509 nm. The cBLE-4AAP screening system was optimized in 96-well microtiter plate format in respect to standard deviation (5 %), linear detection range (15 to 250 µM), lower detection limit (15 µM), and pH (7.4 to 10.4). The cBLE-4AAP screening system was validated by screening a random epPCR pNBEBL mutagenesis library (2000 clones) for improved esterase activity at elevated temperatures. Finally, the variant T3 (Ser378Pro) was identified which nearly retains its specific activity at room temperature (WT 1036 U/mg and T3 929 U/mg) and shows compared to WT a 4.7-fold improved residual activity after thermal treatment (30 min incubation at 69.4 °C; WT 170 U/mg to T3 804 U/mg).

Dipyrone metabolite 4-MAA induces hypothermia and inhibits PGE2 -dependent and -independent fever while 4-AA only blocks PGE2 -dependent fever.

BACKGROUND AND PURPOSE: The antipyretic and hypothermic prodrug dipyrone prevents PGE2 -dependent and -independent fever induced by LPS from Escherichia coli and Tityus serrulatus venom (Tsv) respectively. We aimed to identify the dipyrone metabolites responsible for the antipyretic and hypothermic effects. EXPERIMENTAL APPROACH: Male Wistar rats were treated i.p. with indomethacin (2 mg·kg(-1) ), dipyrone, 4-methylaminoantipyrine (4-MAA), 4-aminoantipyrine (4-AA) (60-360 mg·kg(-1) ), 4-formylaminoantipyrine, 4-acethylaminoantipyrine (120-360 mg·kg(-1) ) or vehicle 30 min before i.p. injection of LPS (50 µg·kg(-1) ), Tsv (150 µg·kg(-1) ) or saline. Rectal temperatures were measured by tele-thermometry and dipyrone metabolite concentrations determined in the plasma, CSF and hypothalamus by LC-MS/MS. PGE2 concentrations were determined in the CSF and hypothalamus by elisa. KEY RESULTS: In contrast to LPS, Tsv-induced fever was not followed by increased PGE2 in the CSF or hypothalamus. The antipyretic time-course of 4-MAA and 4-AA on LPS-induced fever overlapped with the period of the highest concentrations of 4-MAA and 4-AA in the hypothalamus, CSF and plasma. These metabolites reduced LPS-induced fever and the PGE2 increase in the plasma, CSF and hypothalamus. Only 4-MAA inhibited Tsv-induced fever. The higher doses of dipyrone and 4-MAA also induced hypothermia. CONCLUSIONS AND IMPLICATIONS: The presence of 4-MAA and 4-AA in the CSF and hypothalamus was associated with PGE2 synthesis inhibition and a decrease in LPS-induced fever. 4-MAA was also shown to be an antipyretic metabolite for PGE2 -independent fever induced by Tsv suggesting that it is responsible for the additional antipyretic mechanism of dipyrone. Moreover, 4-MAA is the hypothermic metabolite of dipyrone.

Exploring DNA binding and nucleolytic activity of few 4-aminoantipyrine based amino acid Schiff base complexes: a comparative approach.

A series of novel Co(II), Cu(II), Ni(II) and Zn(II) complexes were synthesized from Schiff base(s), obtained by the condensation of 4-aminoantipyrine with furfural and amino acid (glycine(L1)/alanine(L2)/valine(L3)) and respective metal(II) chloride. Their structural features and other properties were explored from the analytical and spectral methods. The binding behaviors of the complexes to calf thymus DNA were investigated by absorption spectra, viscosity measurements and cyclic voltammetry. The intrinsic binding constants for the above synthesized complexes are found to be in the order of 10(2) to 10(5) indicating that most of the synthesized complexes are good intercalators. The binding constant values (Kb) clearly indicate that valine Schiff-base complexes have more intercalating ability than alanine and glycine Schiff-base complexes. The results indicate that the complexes bind to DNA through intercalation and act as efficient cleaving agents. The in vitro antibacterial and antifungal assay indicates that these complexes are good antimicrobial agents against various pathogens. The IC50 values of [Ni(L1)2] and [Zn(L1)2] complexes imply that these complexes have preferable ability to scavenge hydroxyl radical.

Binding and conformational changes of human serum albumin upon interaction with 4-aminoantipyrine studied by spectroscopic methods and cyclic voltammetry.

The interactions of 4-aminoantipyrine (AAP) with human serum albumin (HSA) have been studied by UV-visible spectroscopy, fluorescence spectroscopy and cyclic voltammetry. The binding of 4-aminoantipyrine quenches the HSA fluorescence, revealing a 1:1 interaction with a binding constant of about 10(5) M(-1). The experimental results showed that AAP effectively quenched the intrinsic fluorescence of HSA via dynamic type of quenching. In addition, according to the synchronous fluorescence spectra of HSA in presence of 4-aminoantipyrine, the tryptophan residue of the proteins are most perturbed by the binding process. The number of binding sites, the binding constant, site probe study, some common metal ions effect and the thermodynamic parameters were calculated.

Fully automated analytical procedure for propofol determination by sequential injection technique with spectrophotometric and fluorimetric detections.

In this work, an application of an enzymatic reaction for the determination of the highly hydrophobic drug propofol in emulsion dosage form is presented. Emulsions represent a complex and therefore challenging matrix for analysis. Ethanol was used for breakage of a lipid emulsion, which enabled optical detection. A fully automated method based on Sequential Injection Analysis was developed, allowing propofol determination without the requirement of tedious sample pre-treatment. The method was based on spectrophotometric detection after the enzymatic oxidation catalysed by horseradish peroxidase and subsequent coupling with 4-aminoantipyrine leading to a coloured product with an absorbance maximum at 485 nm. This procedure was compared with a simple fluorimetric method, which was based on the direct selective fluorescence emission of propofol in ethanol at 347 nm. Both methods provide comparable validation parameters with linear working ranges of 0.005-0.100 mg mL(-1) and 0.004-0.243 mg mL(-1) for the spectrophotometric and fluorimetric methods, respectively. The detection and quantitation limits achieved with the spectrophotometric method were 0.0016 and 0.0053 mg mL(-1), respectively. The fluorimetric method provided the detection limit of 0.0013 mg mL(-1) and limit of quantitation of 0.0043 mg mL(-1). The RSD did not exceed 5% and 2% (n=10), correspondingly. A sample throughput of approx. 14 h(-1) for the spectrophotometric and 68 h(-1) for the fluorimetric detection was achieved. Both methods proved to be suitable for the determination of propofol in pharmaceutical formulation with average recovery values of 98.1 and 98.5%.

DNA binding mode of novel tetradentate amino acid based 2-hydroxybenzylidene-4-aminoantipyrine complexes.

Few transition metal complexes of tetradentate N(2)O(2) donor Schiff base ligands containing 2-hydroxybenzylidene-4-aminoantipyrine and amino acids (alanine/valine) abbreviated to KHL(1)/KHL(2) have been synthesized. All the metal complexes have been fully characterized with the help of elemental analyses, molecular weights, molar conductance values, magnetic moments and spectroscopic data. The Schiff bases KHL(1)/KHL(2) are found to act as tetradentate ligands using N(2)O(2) donor set of atoms leading to a square-planar geometry for the complexes around the metal ions. The binding behaviors of the complexes to calf thymus DNA have been investigated by absorption spectra, viscosity measurements and cyclic voltammetry. The DNA binding constants reveal that all these complexes interact with DNA through minor groove binding mode. The studies on mechanism of photocleavage reveal that singlet oxygen ((1)O(2)) and superoxide anion radical (O(2)(-)) may play an important role in the photocleavage. The Schiff bases and their metal complexes have been screened for their in vitro antibacterial activities against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus epidermidis, Klebsiella pneumoniae and antifungal activities against Aspergillus niger, Fusarium solani, Culvularia lunata, Rhizoctonia bataicola and Candida albicans by MIC method.

DNA template-assisted modulation of horseradish peroxidase activity.

The precise alignment of DNA molecules by Watson-Crick base-pairing combined with its polymeric characteristics have allowed DNA to be used as a template or scaffold for assembling materials. In this work, we investigate the role of calf-thymus DNA as a template for enhancing the horseradish peroxidase (HRP)-mediated oxidation of phenol and phenolic derivatives. The HRP-catalyzed oxidation of phenol into polyphenolic products and in presence of 4-aminoantipyrine into quinoneimine dye complexes is studied. Visible spectroscopy reveals an increased yield of both products of the enzymatic reaction in presence of calf-thymus DNA and is attributed to the prearrangement of the corresponding substrates on the DNA. The concentrations of calf-thymus DNA and the substrates are found to affect the nature of prearrangement and subsequent formation of polymeric or co-oxidation products. Also, phenolic derivatives with different aromatic substitutions display divergent propensities towards product formation in presence of the DNA template. Our results demonstrate the ability of calf-thymus DNA to modulate the activity of HRP and exercise control on the nature of products formed. This work highlights the potential of using DNA as a template for influencing enzymatic reactions involving aromatic substrates.

Scavenging activity of aminoantipyrines against hydroxyl radical.

The pyrazolone derivatives antipyrine and 4-(N,N-dimethyl)-aminoantipyrine (aminopyrine) have long been used as analgesic, antipyretic and anti-inflammatory drugs. However, in spite of its recognized therapeutic benefits, the use of pyrazolones has been associated with agranulocytosis. Though the oxidation of aminopyrine by neutrophil-generated hypochlorous acid (HOCl), leading to the formation of a cation radical, has been considered responsible for the potential bone marrow toxicity, the reaction mechanisms of pyrazolones against other reactive oxygen species (ROS) remains elusive. Thus, the reactions of 4-aminoantipyrine and methylated derivatives with hydroxyl radicals (HO*) were studied as a model of their reactivity against ROS. The results show that 4-(N,N-dimethyl)-aminoantipyrine (aminopyrine) undergoes demethylation when reacting with HO. radical, leading to 4-(N-methyl)-aminoantipyrine, which is further demethylated to 4-aminoantipyrine. In addition, it was also observed that another favorable reaction of 4-aminoantipyrines in these conditions is the hydroxylation on the aromatic ring, a reaction that is common to aminopyrine, 4-(N-methyl)-aminoantipyrine, and 4-aminoantipyrine. Whether these reaction mechanisms give rise to harmful reactive intermediates requires further chemico-biological evaluation.

Spectroscopic investigation on the toxicological interactions of 4-aminoantipyrine with bovine hemoglobin.

The effects of 4-aminoantipyrine (AAP) on bovine hemoglobin (BHb) were investigated by fluorescence spectroscopy, synchronous fluorescence spectroscopy, ultraviolet-visible absorption spectroscopy and circular dichroism spectroscopy (CD) under simulated physiological conditions. The experimental results showed that AAP effectively quenched the intrinsic fluorescence of BHb via static quenching. The number of binding sites, the binding constant K(a), and the thermodynamic parameters (DeltaH(o), DeltaS(o) and DeltaG(o)) were measured at two different temperatures. Van der Waals' interactions and hydrogen bonds were the predominant intermolecular forces in stabilizing the BHb-AAP complex. The experiment results confirmed micro-environmental and conformational changes of BHb in the presence of AAP. The alpha-helix content decreased, indicating that AAP destroys some of the hydrogen bonding networks in the polypeptide chain.

[Effect of herbicide tralkoxydim and 2-acylcyclohexane-1,3-diones on peroxidase activity].

Effect of 2-acylcyclohexane-1,3-dione derivatives (tralkoxydim and its diketone precursors) on peroxidase-catalyzed oxidation of 3,3',5,5'-tetramethylbenzidine (TMB), o-phenylenediamine (PDA), and the phenol-4-aminoantipyrine (4-AAP) couple has been studied. This effect varies from horseradish peroxidase (HRP) inactivation to activation in the reactions of peroxidation ofTMB, PDA, and, to a lesser extent, the phenol-4-AAP couple. The diketone-mediated HRP activation depends strongly on pH, presence of dimethylformamide, the structures of tralkoxydim and other diketones, and the substrate nature. The type of activation in the course of peroxidation with the presence of tralkoxydim can be noncompetitive (PDA and TMB) or mixed (TMB) depending on conditions. The maximal level of the HRP activation mediated by diketones depends on their structure. It can reach 4000% of the initial HRP-catalyzed peroxidation rate for TMB and ca. 1000% for PDA. A test system is proposed for quantitative tralkoxydim assay at millimolar concentration. It includes HRP and TMB as the substrate with spectrometrical monitoring of the TMB peroxidation product at 655 nm.

Inhibition of cyclooxygenases by dipyrone.

BACKGROUND AND PURPOSE: Dipyrone is a potent analgesic drug that has been demonstrated to inhibit cyclooxygenase (COX). In contrast to classical COX-inhibitors, such as aspirin-like drugs, dipyrone has no anti-inflammatory effect and a low gastrointestinal toxicity, indicating a different mode of action. Here, we aimed to investigate the effects of dipyrone on COX. EXPERIMENTAL APPROACH: The four major metabolites of dipyrone, including the two pharmacologically active metabolites, 4-methyl-amino-antipyrine (MAA) and amino-antipyrine (AA), were used to characterise their binding to COX and haem as well as their effects on the biochemical properties of COX. Mass spectrometry, UV and visible photometry were used to study binding and prostaglandin production. Levels of anti-oxidant enzymes were assessed by Western blotting. KEY RESULTS: The pharmacologically active metabolites of dipyrone, MAA and AA, did not inhibit COX activity in vitro like classical COX inhibitors, but instead redirected the prostaglandin synthesis, ruling out inhibition of COX through binding to its active site. We found that MAA and AA formed stable complexes with haem and reacted with hydrogen peroxide in presence of haem, ferrous ions (Fe(2+)) or COX. Moreover, MAA reduced Fe(3+) to Fe(2+) and accordingly increased lipid peroxidation and the expression of anti-oxidant enzymes in cultured cells and in vivo. CONCLUSIONS AND IMPLICATIONS: Our data suggest that the pharmacologically active metabolites of dipyrone inhibit COX activity by sequestering radicals which initiate the catalytic activity of this enzyme or through the reduction of the oxidative states of the COX protein.

Extracellular peroxidase production by Coprinus species from urea-treated soil.

Thirteen strains of inky-cap mushroom Coprinus species were evaluated for the production of extracellular peroxidase. The liquid fermentation was carried out in shake flasks containing 1% glucose, 0.5% peptone, 0.3% yeast extract, and 0.3% malt extract broth at 25 degrees C. Peroxidase activity was detected in the liquid culture of several Coprinus species, including C. echinosporus NBRC 30630; C. macrocephalus NBRC 30117; Coprinus spp. UAMH 10065, UAMH 10066, UAMH 10067, and 074, after 10 days of growth. Peroxidase production by Coprinus sp. UAMH 10067, a Coprinus species isolated from urea-treated soil, was comparable to that of C. cinereus and reached 15 U.mL(-1) after 10 days. In addition, the peroxidase from Coprinus sp. UAMH 10067 was apparently more thermally stable than the enzyme produced by C. cinereus.

Catalytic spectrofluorimetric determination of superoxide anion radical and superoxide dismutase activity using N,N-dimethylaniline as the substrate for horseradish peroxidase (HRP).

The coupled reaction of N,N-dimethylaniline (DMA) with 4-aminoantipyrine (4-AAP) using superoxide anion radical (O2-) as oxidizing agent under the catalysis of horseradish peroxidase (HRP) was studied. Based on the reaction, O2- produced by irradiating Vitamin B2, (VB2) was spectrophotometricly determined at 554 nm. The linear range of this method was 1.8 x 10(-6)-1.2 x 10(-4) mol l(-1) with a detection limit of 5.3 x 10(-7) mol l(-1). The effect of interferences on the determination of O2- was investigated. The proposed method was successfully applied to the determination of superoxide dismutase (SOD) activity in human blood and mouse blood.

[Co-oxidation of phenols and 4-aminoantipyrene catalyzed by microperoxidase and their complexes with proteins]. / Sopriazhennoe okislenie fenolov i 4-aminoantipirina, kataliziruemoe mikroperoksidazami i ikh kompleksami s belkami.

The kinetic parameters of 4-aminoantipyrine (AAP) co-oxidation with phenol or p-iodophenol in a wide range of hydrogen peroxide concentrations were compared for microperoxidases MP-8, MP-9, MP-11, horseradish peroxidase (HRP) and peroxidase of Arthromyces ramosus (PAR). The peroxidatic activity increased in the following order: HRP > PAR > MP-9 > MP-8 > MP-11. Microperoxidase complexes with human serum albumin (1:1) retained their peroxidatic activity at high H2O2 concentrations, protecting haem from destruction by active radicals. Monoclonal antibodies against porphyrin decreased the peroxidatic activities of three microperoxidases during co-oxidation of AAP and phenol and protected haem. Inert proteins (BSA, ovalbumin) had little effect on the HRP activity, whereas enzyme-specific antibodies strongly activated HRP at high concentrations of hydrogen peroxide, thereby forming an immune complex protecting HRP from its dissociation down to haem and the apoenzyme.