acid-phosphatase and 4-nitrophenol

A reversible luminescence nanoswitch through competitive hydrophobic interaction among copper nanoclusters, p-nitrophenol and α-cyclodextrin is established, and a reliable real-time luminescent assay for acid phosphatase (ACP) activity is developed on the basis of this luminescence nanoswitch. Stable and intensely luminescent copper nanoclusters (CuNCs) were synthesized via a green one-pot approach. The hydrophobic nature of CuNCs aggregate surface is identified, and further used to drive the adsorption of p-nitrophenol on the surface of CuNCs aggregate due to their hydrophobic interaction. This close contact switches off the luminescence of CuNCs aggregate through static quenching mechanism. However, the introduction of α-cyclodextrin switches on the luminescence since stronger host-guest interaction between α-cyclodextrin and p-nitrophenol causes the removal of p-nitrophenol from the surface of CuNCs. This nanoswitch in response to external stimulus p-nitrophenol or α-cyclodextrin can be run in a reversible way. Luminescence quenching by p-nitrophenol is further utilized to develop ACP assay using p-nitrophenyl phosphate ester as the substrate. Quantitative measurement of ACP level with a low detection limit of 1.3 U/L was achieved based on this specific detection strategy. This work reports a luminescence nanoswitch mediated by hydrophobic interaction, and provides a sensitive detection method for ACP level which is capable for practical detection in human serum and seminal plasma.

Topics: Acid Phosphatase; Copper; Fluorescent Dyes; Humans; Hydrophobic and Hydrophilic Interactions; Luminescence; Metal Nanoparticles; Nitrophenols; Solanum tuberosum; Spectrometry, Fluorescence; Time Factors

A multicommutated flow analysis (MCFA) system constructed of microsolenoid valves and pumps offering simultaneous determination of activity of acid phosphatase (ACP) and alkaline phosphatase (ALP) in human serum samples has been developed. The MCFA system is based on optoelectronic flow-through detector made of two light emitting diodes and operating according to paired emitter detector diode (PEDD) principle. This photometric PEDD device has been dedicated for detection of p-nitrophenol (NP) generated in the course of enzymatic hydrolysis of p-nitrophenyl phosphate and optimized for the determination of NP in human serum samples. The developed PEDD-based MCFA system allows independent optimization of conditions for reaction and detection steps of photometric ACP and ALP bioassays. Moreover, it allows elimination of photometric interferences from serum matrix components according to two-points kinetic mode of measurement. The single measurement cycle takes 12 min, consists of four measurements (two for each phosphoesterase) and enables determination of serum ACP and ALP activities at physiological and pathological levels. The real analytical utility of the developed MCFA system has been confirmed by analysis of control sera as well as real human serum samples from healthy persons and oncological patients.

Topics: Acid Phosphatase; Alkaline Phosphatase; Enzyme Assays; Equipment Design; Flow Injection Analysis; Humans; Nitrophenols; Organophosphorus Compounds; Photometry; Reproducibility of Results

The role of pharyngeal lymphoid tissue in etiopathogenesis of secretory otitis is not yet defined. The influence of tonsillar and adenoid mass, weight, obstruction of naspharyngeal orrifitium, bacterial reservoire or some immunological events are of scientific interest. Tissue nonspecific alkaline phosphatase (TNAP) and acid phosphatase (ACP) are enzymes detected in lymphoid tissue, TNAP as characteristic of B cells, ACP as a characteristic of macrophages and folucullardentritic cells. These enzymes interfere in cell metabolism by removing 5' phosphate group from nucleotides and proteins. Specific activity and kinetic properties were studied in palatinal tonsils and adenoids of children with secretory otitis (OME) and compared with children with recurrent tonsillitis without ear involvement.. Adenoid and tonsillar tissue of l7 children with OME and 30 children with recurrent tonsillitis were subjected to biochemical investigation using method of releasing of p-nitrophenol from p-nitrophenylphosphate (pNPP). Kinetic parameters as Michaelis-Menten constant were calculated by non-linear regression estimation method.. Specific activity of adenoid alkaline phosphatase was lower in children with OME in relation to children with recurrent tonsillitis (t=5.733507, p<0.01). Specific activity of adenoid acid phosphatase was also lower in children with OME (t=3.655456, p<0.01). pH optimum for both enzymes was the same in these two groups of children. Michaelis-Menten constant for both enzymes was significantly higher in adenoid of children with OME than in children with recurrent tonsillitis suggesting lower enzyme affinity for the substrate.. Differences in specific activities and kinetic properties of adenoid alkaline and acid phosphatases between children with OME and children with recurrent tonsillitis without OME were verified in this study. The results of the study are not able to explain the alteration of alkaline and acid phosphatase characteristics but could point to some possible and specific role of nasopharyngeal lymphoid tissue in pathogenesis of secretary otitis.

Topics: Acid Phosphatase; Adenoidectomy; Adenoids; Alkaline Phosphatase; B-Lymphocytes; Child; Child, Preschool; Dendritic Cells, Follicular; Female; Humans; Hydrogen-Ion Concentration; Indicators and Reagents; Macrophages; Male; Nasal Obstruction; Nitrophenols; Organophosphorus Compounds; Otitis Media with Effusion; Palatine Tonsil; Recurrence; Tonsillectomy; Tonsillitis

LC coupled to an electrochemical detector (LC-EC) operating in cathodic mode with a column-switching system realizes a high-throughput detection of p-nitrophenol (NP). The measurement-time for each NP sample was shortened to 20 s, and the successive analyses of 39 samples was completed within 13 min. In the present system, the limits of detection and quantification were 0.15 and 0.20 microM, respectively, and further, up to 25 microM, a linear calibration curve was afforded. Relative standard deviations for standard solutions of 0.20, 1.0, and 25 microM NP were 4.3, 2.0, and 1.1% (n = 5), respectively. Between-run precisions of the analysis of 5.0 and 25 microM NP over 6 days were 4.8 and 1.3%, respectively. A comparison with the commonly used Bessey-Lowry-Brock method indicates that the present LC-EC is useful for the high-throughput assay of acid and alkaline phosphatases in urine and blood samples with a p-nitrophenyl phosphate substrate.

Topics: Acid Phosphatase; Alkaline Phosphatase; Chromatography, High Pressure Liquid; Nitrophenols; Organophosphorus Compounds; Oxygen; Quality Control; Reproducibility of Results; Sensitivity and Specificity

Utilizing a commercially available helium-purging device and PEEK tubes for all tubing, especially for connection between the mobile phase and pump, high performance liquid chromatography with an electrochemical detector (ECD/HPLC) at the cathodic mode is a simple and precise method for the determination of p-nitrophenol (NP). Studies with cyclic and hydrodynamic voltammetry indicated that 25% aqueous MeOH containing 0.1% (v/v) CF(3)CO(2)H and -0.8 V vs. Ag/AgCl are the best mobile phase and detection potential for cathodic ECD/HPLC. With the present system, the limits of detection and determination were 0.2 and 0.25 microM, respectively, and up to 50 microM, a linear calibration curve was afforded. Within-day precisions for the analysis of 5 and 50 microM NP were 0.8 and 0.7% (n=6), respectively, and between-day precisions (n=6) for these samples were 3.5 and 2.2%, respectively. Compared with the commonly used Bessey-Lowry-Brock method, cathodic ECD/HPLC was useful for the assay of acid phosphatase in urine samples with p-nitrophenyl phosphate disodium salt as a substrate.

Topics: Acid Phosphatase; Chromatography, High Pressure Liquid; Electrochemistry; Electrodes; Humans; Nitrophenols

Transmission of viruses by animal sera represents a considerable risk for humans and animals particularly when the serum is used for the production of pharmaceutical products such as vaccines. Procedures applicable for inactivating large numbers of different viruses, both enveloped and non-enveloped, are therefore mandatory. For this purpose we have developed and validated UVC irradiation as the virus-inactivation procedure of choice for serum to be used in an industrial setting. Spiking experiments in foetal calf serum (FCS) were performed by independent contract laboratories and revealed constantly high clearance rates for various viruses such as bovine parvovirus, parainfluenza type III virus, bovine diarrhoea virus, foot-and-mouth disease virus and different forms of mycoplasmas. UVC-treated sera maintained their growth-promoting activities for various cell types (MRC-5, Vero, CHO). Conventional growth curves generated in the presence of 10% and 1% UVC-treated FCS differed only slightly from controls, indicating the lack of significant damage during UVC exposure. Experiments using a sensitive photometric-based acid phosphatase assay (APA), which correlates well with the more tedious cell counting procedure, confirmed these findings even in the presence of minimal serum requirements. UVC treatment of animal sera appears advantageous compared to currently recommended inactivation procedures, such as Gamma irradiation, for at least three reasons: (i) it possesses a high inactivation capacity for parvoviruses, a pathogen that cannot be destroyed easily by conventional methods; (ii) it causes no noticeable impairment in cell growth and (iii) it can be performed in a controlled manner at the production site.

Topics: Acid Phosphatase; Animals; Biological Products; Blood; Cattle; Cell Division; Chlorocebus aethiops; Culture Media; Diarrhea Viruses, Bovine Viral; DNA; Mycoplasma; Nitrophenols; Parvovirus; Photochemistry; Pyrimidines; Swine; Ultraviolet Rays; Vero Cells

In contrast to other acid phosphatases, four cytoplasmic isoforms (AP1, AP2, AP3A, and AP3B) purified from mature soybean seeds presented high activities at temperatures above 80 degrees C, when p-nitrophenylphosphate (p-NPP) was utilized as substrate. However, with tyrosine phosphate and inorganic pyrophosphate as substrates, maximum activities were observed at temperature of 60 degrees C during 10 min reaction. In the absence of substrate, enzymes lost only 20% activity after 60 min at 60 degrees C; the isoforms AP3A and AP3B retained 30% of activity at 70 degrees C after 60 min and all the isoforms were inactivated at 80 degrees C, after 5 min. Thermal inactivation studies indicated that the soybean enzymes showed different temperature dependences in relation to most plant acid phosphatases. A best protective effect was observed when the isoforms were preincubated, at 70 degrees C, with phosphate (10 mM) and p-nitrophenol (10 mM) which indicates that the enzyme inactivation was prevented only in the presence of both reaction products.

Topics: Acid Phosphatase; Cytoplasm; Diphosphates; Enzyme Stability; Glycine max; Isoenzymes; Nitrophenols; Octoxynol; Organophosphorus Compounds; Phosphates; Phosphotyrosine; Plant Proteins; Seeds; Temperature; Vanadates

Several independent experiments failed to reveal any evidence in support of the involvement of a phosphoryl-enzyme intermediate in the catalytic mechanism of pig allantoic fluid purple acid phosphatase: (i) attempts to label enzyme with phosphate derived from [32P]p-nitrophenyl phosphate were unsuccessful; (ii) values of kcat for a series of phosphate derivative varied over a wide range, with the enzyme showing a marked preference for activated ester and anhydride substrates over those with a stable leaving group; (iii) burst titrations revealed a "burst" of p-nitrophenol from p-nitrophenyl phosphate only when the enzyme was added after the substrate, suggesting that this result was an artifact of the order of addition of reagents; (iv) transphosphorylation from p-nitrophenyl phosphate to acceptor alcohols could not be detected, even under conditions where a transphosphorylation to hydrolysis ratio as low as 0.015 could have been measured; (v) enzyme-catalyzed exchange of 180 between phosphate and water was demonstrated, although at a rate much slower than that observed for other phosphatases where the involvement of a phosphoryl-enzyme intermediate in the mechanism has been clearly established. The present results are compared with those obtained in similar studies on other phosphatases, particularly the highly homologous beef spleen purple acid phosphatase, and their implications for the catalytic mechanism of the purple acid phosphatases are discussed.

Topics: Acid Phosphatase; Allantois; Aniline Compounds; Animals; Body Fluids; Catalysis; Ethanol; Glycerol; Glycoproteins; Kinetics; Magnetic Resonance Spectroscopy; Nitrophenols; Organophosphorus Compounds; Oxygen; Phosphorylation; Substrate Specificity; Swine; Trifluoroethanol

Topics: Acid Phosphatase; Animals; Arylsulfatases; beta-Galactosidase; beta-N-Acetylhexosaminidases; Circadian Rhythm; Female; Glycerophosphates; Hexosaminidases; Hydrolases; Liver; Lysosomes; Male; Mice; Nitrophenols; Parabiosis

Topics: Acid Phosphatase; Glycerol; Glycerophosphates; Hydrolysis; Kinetics; Nitrophenols; Phenol; Phenols; Phosphates; Phosphoric Monoester Hydrolases; Protein Tyrosine Phosphatases