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PubMed2000

A direct method to visualise the aryl acylamidase activity on cholinesterases in polyacrylamide gels.

BACKGROUND: In vertebrates, two types of cholinesterases exist, acetylcholinesterase and butyrylcholinesterase. The function of acetylcholinesterase is to hydrolyse acetylcholine, thereby terminating the neurotransmission at cholinergic synapse, while the precise physiological function of butyrylcholinesterase has not been identified. The presence of cholinesterases in tissues that are not cholinergically innervated indicate that cholinesterases may have functions unrelated to neurotransmission. Furthermore, cholinesterases display a genuine aryl acylamidase activity apart from their predominant acylcholine hydrolase activity. The physiological significance of this aryl acylamidase activity is also not known. The study on the aryl acylamidase has been, in part hampered by the lack of a specific method to visualise this activity. We have developed a method to visualise the aryl acylamidase activity on cholinesterase in polyacrylamide gels. RESULTS: The o-nitroaniline liberated from o-nitroacetanilide by the action of aryl acylamidase activity on cholinesterases, in the presence of nitrous acid formed a diazonium compound. This compound gave an azo dye complex with N-(1-napthyl)-ethylenediamine, which appeared as purple bands in polyacrylamide gels. Treating the stained gels with trichloroacetic acid followed by Tris-HCl buffer helped in fixation of the stain in the gels. By using specific inhibitors for acetylcholinesterase and butyrylcholinesterase, respectively, differential staining for the aryl acylamidase activities on butyrylcholinesterase and acetylcholinesterase in a sample containing both these enzymes has been demonstrated. A linear relationship between the intensity of colour developed and activity of the enzyme was obtained. CONCLUSIONS: A novel method to visualise the aryl acylamidase activity on cholinesterases in polyacrylamide gels has been developed.

Amidohydrolases↗
PubMed1989

Formation of direct-acting genotoxic substances in nitrosated smoked fish and meat products: identification of simple phenolic precursors and phenyldiazonium ions as reactive products.

Epidemiological studies have associated the consumption of smoked fish and meat products with an increased risk of stomach cancer. Therefore, the reaction of such smoked foods with nitrite under acidic conditions was investigated and was shown to produce potent direct-acting genotoxic substances as detected by the SOS Chromotest. Similar genotoxic activity was observed in nitrosated samples of wood-smoke condensates. Simple phenolic compounds such as phenol, 3-methoxycatechol, catechol and vanillin were identified as the precursors of the genotoxic substances. These phenolic compounds also exhibited direct-acting genotoxicity after nitrosation. The major genotoxic substances formed after nitrosation of phenol were isolated and identified as 4- and 2-hydroxyphenyldiazonium ions. Nitrosation of various wood-smoke condensates was found to generate the same type of diazonium compounds, which in part account for the genotoxicity of nitrosated smoked foods.

Animals↗
PubMed1978

Detection of estrogen-like compounds by thin-layer chromatography.

There are different national food regulations concerning the use of hormoneactive substances in the breeding of domestic animals that serve as human food. This situation requires specific and sensitive methods for the detection of any residues in food. The hormones used in practice can be determined with improved sensitivity by coupling to the diazonium compound Fast Dark Blue R Salt. After thin-layer chromatography on high-performance thin-layer chromatographic plates with two different solvent mixtures, zeranol, estradiol, estriol and estrone are analysed as azo dyestuffs. The detection limit of these hormones is below 10 ng.

Chromatography, Thin Layer↗
PubMed1987

Covalent binding of 4-carbamoylbenzenediazonium chloride to deoxyguanine bases of DNA resulting in apparent irreversible inhibition of poly(adenosine diphosphoribose) polymerase at the nicotinamide binding site.

The poly(adenosine diphosphoribose) polymerase activity of isolated liver nuclei was inhibited by 4-carbamoylbenzenediazonium chloride, referred to as 4-diazoniobenzamide, an effect that was dependent on the time of incubation and the concentration of the diazonium compound, with inhibition following first-order kinetics. The inhibition was not reversed by reisolation of nuclei and centrifugal washing, whereas the inhibition by benzamide or 4-aminobenzamide was completely reversible under these conditions. Simultaneous incubation of 4-diazoniobenzamide with benzamide prevented enzyme inhibition. The 4-diazoniobenzoic acid analogue was not inhibitory. The mechanism of action of 4-diazoniobenzamide was traced to a specific covalent binding to dGMP of DNA to form N2-[(4-carbamoylphenyl)azo]-2'-deoxyguanosine 5'-monophosphate. Coenzymic DNA, by tight association with the polymerase protein, fixes the -C(O)NH2 moiety of the adduct at the nicotinamide-binding site of the enzyme.

Animals↗
PubMed1990

Occupational asthma and specific IgE to a diazonium salt intermediate used in the polymer industry.

This study describes sensitization to a diazonium salt intermediate, diazonium tetrafluoroborate (DTFB), produced during the manufacture of a fluorine polymer precursor. Most of the workers exposed to DTFB powder complained of respiratory and mucosal irritation. However, some of these individuals had symptoms typical of occupational asthma. Clinical and immunologic studies, including bronchial provocation testing and specific IgE measurements, were performed on two individuals with asthmatic symptoms associated with exposure to DTFB. These studies confirmed a diagnosis of occupational asthma. In an investigation of 43 other exposed workers in two separate factories handling the diazonium compound, specific IgE antibodies to DTFB-human serum albumin conjugate were found in 20% of individuals. There was a good correlation between specific IgE and exposure-related respiratory symptoms, which suggests an IgE-mediated pathogenesis. DTFB-human serum albumin conjugates were characterized by electrophoretic techniques, and it was found that conjugates prepared at pH 10 elicited optimum IgE binding in RAST. In vitro cross-linking of albumin by DTFB was demonstrated but had no effect on RAST binding. Our findings on the effect of pH and polymerization on IgE binding support proposed mechanisms for in vivo conjugation and provide information on the antigenic determinants important in IgE recognition of hapten-carrier protein conjugates.

Antibody Formation↗
PubMedDate not supplied

New reactive coenzyme analogues for affinity labeling of NAD+ and NADP+ dependent dehydrogenases.

Reactive coenzyme analogues omega-(3-diazoniumpyridinium)alkyl adenosine diphosphate were prepared by reaction of omega-(3-aminopyridinium)alkyl adenosine diphosphate with nitrous acid. In these compounds the nicotinamide ribose is substituted by hydrocarbon chains of varied lengths (n-ethyl to n-pentyl). The diazonium compounds are very unstable and decompose rapidly at room temperature. They show a better stability to 0 degree C. Lactate and alcohol dehydrogenase do not react with any of the analogues. Glyceraldehyde-3-phosphate dehydrogenase reacts rapidly with the diazoniumpentyl compound. Decreasing the length of the alkyl chain significantly decreases the inactivation velocity. 3 alpha, 20 beta-Hydroxysteroid dehydrogenase reacts at 0 degree C with the ethyl homologue and slowly with the propyl compound. The butyl- and pentyl analogues do not inactivate at 0 degree C. Tests with 14C-labeled 2-(3-diazoniumpyridinium)ethyl adenosine diphosphate show that complete loss of enzyme activity results after incorporation of 2 moles of inactivator into 1 mole of tetrameric enzyme. 4-(3-Acetylpyridinium)butyl 2'-phospho-adenosine diphosphate, a structural analogue of NADP+, was prepared by condensation of adenosine-2,3-cyclophospho-5'-phosphomorpholidate with (3-acetylpyridinium)butyl phosphate, followed by hydrolysis of the cyclic phosphoric acid with 2':3'-cyclonucleotide-3'-phosphodiesterase. Because of the redox potential (-315 mV) and the distance between the pyridinium and phosphate groups, this analogue is a hydrogen acceptor and its reduced form a hydrogen donor in tests with alcohol dehydrogenase from Thermoanaerobium brockii. The reduced form of the coenzyme analogue also is a hydrogen donor with glutathione reductase. With other NADP+-dependent dehydrogenases the compound has been shown to be a competitive inhibitor against the natural coenzyme. The acetyl group reacts with bromine to form the bromoacetyl group. This reactive bromoacetyl analogue is a specific active-site directed irreversible inhibitor of isocitrate dehydrogenase.

Affinity Labels↗
PubMed1997

Photoaffinity labeling of yeast farnesyl protein transferase and enzymatic synthesis of a Ras protein incorporating a photoactive isoprenoid.

Farnesyl protein transferase (FPTase) catalyzes the covalent attachment of a farnesyl (C15) group from farnesyl pyrophosphate (FPP) to a specific cysteine residue of Ras and several other proteins. In this report, photoactive farnesyl and geranylgeranyl pyrophosphate analogs 2-diazo-3,3,3-trifluoropropionyloxy-geranyl pyrophosphate (DATFP-GPP) and 2-diazo-3,3,3-trifluoropropionyloxy-farnesyl pyrophosphate (DATFP-FPP) were used to study the active site of Saccharomyces cerevisiae FPTase. Both analogs are substrates for the enzyme, and upon irradiation, DATFP-GPP inhibits FPTase activity in a time-dependent manner. Photoinactivation by DATFP-GPP is prevented by the presence of the natural substrate FPP. Photolysis of radiolabeled DATFP-GPP results in preferential labeling of the beta subunit of FPTase, suggesting that this subunit is involved in recognition of FPP. Of particular importance, DATFP-GPP and DATFP-FPP were used to enzymatically transfer the photoactive isoprenoid moieties to peptides and to Ras; such molecules should be useful for identifying cellular components which specifically recognize farnesylated Ras and other prenylated proteins.

Affinity Labels↗
PubMed1979

Reaction electrophoresis.

The differential mobilities of compounds in an electric field are important analytical criteria and we can use them to bring electrophoretically pure components of a mixture medium on which they are separated. To this end, the compound undergoing reaction are brought into positions on the carrier to assure optimal contact between selected fractions, within a predetermined domain of time and distance. The appearance of a product defines their reactivities, and the product's continued migration on the same carrier can provide the first key to its identity as is demonstrated and discussed. The method is called reaction electrophoresis and it will be of particular use in studies with labile components. It is illustrated here with the coupling reaction of the sodium salt of 1,4-naphthol sulfonic acid and tetrazotized o-dianisidine.

Azo Compounds↗
PubMed2002

Continuous monitoring of prostatic acid phosphatase using self-indicating substrates.

BACKGROUND: The continuous measurement of acid phosphatase (EC 3.1.3.2) activity in serum represents an analytical task not yet sufficiently accomplished. METHODS: Introducing two novel substrates-2-chloro-4-nitrophenyl phosphate (CNP-P), which was preferred, and 4-nitronaphthyl-1-phosphate (NN-P)-an alternative assay to measure enzymatic activity was developed and compared with a modification of Hillmann's method (azo coupling of released naphth-1-ol with a diazonium compound). Apart from different substrate concentrations of 2-chloro-4-nitrophenyl phosphate, 4 mmol/l, and naphthyl-1-phosphate (N-P), 8 mmol/l (with Fast Red TR, 5 mmol/l), respectively, following identical conditions were selected: Citrate, 50 mmol/l, pH 5.75; pentane-1,5-diol, 150 mmol/l; tartrate, 60 mmol/l; 37 degrees C. RESULTS: Whereas intensity and stability of the azo dye unpredictably depend on the albumin concentration of the sample, the direct test with 2-chloro-4-nitrophenyl phosphate resisted sample interferences, showed no intrinsic hydrolysis by albumin, relied on stable reagents and proved superior in sensitivity, precision and ease of handling. In measuring prostatic phosphatase, the proposed procedure closely correlated with Hillmann's method. The preliminary 0.95-reference intervals for adults were 1.2-3.9 kU/l and 5.8-14.8 U/l for total activity, respectively. CONCLUSIONS: The direct assay of the enzyme is suited as an economic, rapid and robust method for mechanized or manual use.

Acid Phosphatase↗
PubMed1994

Fluorescence of bisazo dye reaction products from the coupled tetrazonium method for proteins.

The coupled tetrazonium reaction is a well known histochemical method for proteins. This method, using Fast Blue B salt and 1-naphthol, has been applied on paraffin sections of grasshopper testis and rabbit trachea, as well as on horse blood smears. Microscopic observation under bright field illumination revealed the expected purple staining of protein-rich cell and tissue structures, which also showed a strong red fluorescence under ultraviolet, violet, violet-blue and green exciting light. Some weakly stained cell components (e.g., meiotic spindles) were easily visualized by fluorescence microscopy. Control preparations lacking either the tetrazonium or naphthol treatment, and spectroscopic studies on the bisazo dye produced in vitro (showing an emission peak at 660 nm) confirmed that the red fluorescence of stained structures arises from the protein-tetrazonium-naphthol reaction product formed in situ.

Animals↗
PubMed2001

Electronic spectral parameters of the azo indicators: methyl red, methyl orange, PAN, and fast black K-salt.

The electronic absorption spectra of some azo indicators (methyl red, methyl orange, PAN, and fast black K-salt) were taken in different solvents with different physical properties in order to study the solvent-solute interaction. The parameters used depend on the solvent dielectric constant and refractive index. The data are analyzed based on multiple regression technique.

Azo Compounds↗