Heavy metal-free 19F NMR probes for quantitative measurements of glutathione reductase activity using silica nanoparticles as a signal quencher

For the quantitative assessment of the glutathione reductase (GR) activity with a (19)F NMR spectroscopy, we developed the heavy metal-free probes based on silica nanoparticles modified with water-soluble perfluorinated dendrimers via the disulfide linkers. Before enzymatic reaction, the molecular rotation of the perfluorinated dendrimers is highly restricted, and the magnitude of (19)F NMR signals from the perfluorinated dendrimers can be suppressed. By the reductive cleavage of the disulfide linkers with the reduced glutathione-mediated enzymatic reaction of GR, perfluorinated dendrimers can be released from the surfaces of the nanoparticles. Consequently, the (19)F NMR signals of perfluorinated dendrimers were recovered. The enzymatic activity of GR was determined from the increase of the magnitude of (19)F NMR signals. Finally, to demonstrate the feasibility of the probe in the presence of miscellaneous molecules under bio-mimetic conditions, the comparison study was executed with the cancer cell lysate. The value determined from our method showed a good agreement with that from the conventional method.     

Heat-initiated detection for reduced glutathione with 19F NMR probes based on modified gold nanoparticles

For detecting reduced glutathione (GSH) with a ¹⁹F NMR spectroscopy with time-specificity, we developed the probes based on gold nanoparticles modified with the fluorinated groups via the thermally-cleavable linkers. Before the heating treatment with the probe, the maleimide moiety as a binding site with GSH in the probe is inactivated by cycloaddition of furan. At this silent state, the magnitude of ¹⁹F NMR signals from the fluorinated groups was suppressed. By heating for the activation of the probe, the maleimide moiety was produced via retro Diels–Alder reaction, and ¹⁹F NMR signals were observed. From this moment, GSH started the reaction with the probe via Michael addition to the maleimide moiety, leading to the observation of the new peak in ¹⁹F NMR spectra. Finally, the amounts of GSH were determined from the increase of the magnitude of ¹⁹F NMR signals.     

A generic assay for phosphate-consuming or -releasing enzymes coupled on-line to liquid chromatography for lead finding in natural products

A generic continuous-flow assay for phosphate-consuming or -releasing enzymes coupled on-line to liquid chromatography (LC) has been developed. Operating the LC-biochemical assay in combination with mass spectrometry allows the fast detection and identification of inhibitors of these enzymes in complex mixtures. The assay is based on the detection of phosphate, released by the on-line continuous-flow enzymatic reaction, using a fluorescent probe. The probe consists of fluorophore-labeled phosphate-binding protein, which shows a strong fluorescence enhancement upon binding to inorganic phosphate. To detect very small changes of the phosphate concentration in a postcolumn enzymatic reaction medium, the enzymatic removal of phosphate impurities from solvents, reagents, and samples was optimized for application in continuous flow. The potential of the phosphate probe is demonstrated by monitoring the enzymatic activity, i.e., the phosphate release, from alkaline phosphatase. The selectivity of the phosphate readout, necessary to distinguish between phosphate containing substrate or product and free inorganic phosphate released after enzymatic conversion, is shown. The applicability of LC coupled to the enzymatic assay using the phosphate readout was demonstrated by detection of tetramisole in a plant extract as inhibitor of alkaline phosphatase. Parallel mass spectrometry allowed the simultaneous confirmation of the identity of the inhibitor.     

Ratiometric multimodal chemosensors based on cubic silsesquioxanes for monitoring solvent polarity

We report the multi-functionalized cubic silsesquioxane (POSS) as the ratiometric multimodal chemosensors for monitoring solvent polarity with fluorescence and (19)F NMR. The alteration of the dispersion state of the modified POSS by changing solvent polarity can be reflected into the orthogonal signal responses for the fluorescence and (19)F NMR. In addition, the ratiometric dual monitoring for the enzymatic reaction was performed using the POSS-based chemosensor.     

Molecular probe for enzymatic activity with dual output

A novel molecular probe for enzymatic activity with a dual output detection-mode has been developed. The probe effectively detected the presence of the bacterial protease penicillin-G-amidase; a single cleavage by the enzyme initiated the fragmentation of a self immolative dendritic platform to release two reporter units. The signals of the free reporters were detected by two different spectroscopic techniques, fluorescence and UV-vis. This is the first reported molecular probe with two different chromogenic reporter units activated by a specific stimulus.     

Diffusion and conformation of peptide-functionalized polyphenylene dendrimers studied by fluorescence correlation and 13C NMR spectroscopy

We report on the combined use of fluorescence correlation spectroscopy (FCS) and 1H and 13C NMR spectroscopy to detect the size and type of peptide secondary structures in a series of poly-Z-L-lysine functionalized polyphenylene dendrimers bearing the fluorescent perylenediimide core in solution. In dilute solution, the size of the molecule as detected from FCS and 1H NMR diffusion measurements matches nicely. We show that FCS is a sensitive probe of the core size as well as of the change in the peptide secondary structure. However, FCS is less sensitive to functionality. A change in the peptide secondary conformation from beta-sheets to alpha-helices detected by 13C NMR spectroscopy gives rise to a steep increase in the hydrodynamic radii for number of residues n > or = 16. Nevertheless, helices are objects of low persistence.     

A ratiometric fluorescent probe for alkaline phosphatase via regulation of excited‐state intramolecular proton transfer

A ratiometric fluorescent probe 2‐(benzimidazol‐2‐yl)phenyl phosphoric acid (1) for alkaline phosphatase (ALP) is designed and synthesized. The method employs the modulation of the excited‐state intramolecular proton transfer (ESIPT) process of 2‐(2'‐hydroxyphenyl)benzimidazole (HPBI) through the hydroxyl group protection/deprotection reaction. Upon phosphorylated with POCl₃, HPBI shows only an emission peak at 363 nm due to the blockage of ESIPT. However, once selective enzymatic hydrolysis with alkaline phosphatase (ALP) in Tris–HCl buffer occurs, the probe 1 is returned to HPBI and the ESIPT process is switched on, which results in a decrease in the emission band at 363 nm and an increase in a new fluorescence peak around 430 nm. The fluorescence intensity ratio at 430 and 360 nm (I₄₃₀/I₃₆₀) increases linearly with the activity of ALP up to 0.050 U/mL and the detection limit is 0.0013 U/mL. The proposed probe shows excellent specificity toward ALP. Copyright © 2015 John Wiley & Sons, Ltd.     

Fluorine-19 nuclear magnetic resonance study of fluorotyrosine alkaline phosphatase: the influence of zinc on protein structure and a conformational change induced by phosphate binding.

19F nuclear magnetic resonance (NMR) spectroscopy has been used to study a fully active E. coli fluorotyrosine alkaline phosphatase. The fluorotyrosine resonances provide sensitive probes of the conformational states of the protein. They were used to follow the addition of zinc or cobalt to the apoprotein, and the titration of the protein with inorganic phosphate or the inhibitor 2-hydroxy-5-nitrobenzylphosphonate. The results indicate that 2 molecules of inorganic phosphate per dimer of alkaline phosphatase are required to complete a general conformational change in the protein involving perturbations to the environment of several tyrosines. Spectra of the cobalt enzyme indicate that on specific tyrosine per subunit may be near the metal site. The 19F NMR results, combined with the 31P NMR results in the accompanying paper, lead directly to the conclusion that dissociation of noncovalently bound inorganic phosphate from the enzyme is the rate-limiting process in enzyme catalysis at high pH. The local environment of the individual fluorotyrosines is also discussed.     

A high-resolution, fluorescence-based method for localization of endogenous alkaline phosphatase activity.

We describe a high-resolution, fluorescence-based method for localizing endogenous alkaline phosphatase in tissues and cultured cells. This method utilizes ELF (Enzyme-Labeled Fluorescence)-97 phosphate, which yields an intensely fluorescent yellow-green precipitate at the site of enzymatic activity. We compared zebrafish intestine, ovary, and kidney cryosections stained for endogenous alkaline phosphatase using four histochemical techniques: ELF-97 phosphate, Gomori method, BCIP/NBT, and naphthol AS-MX phosphate coupled with Fast Blue BB (colored) and Fast Red TR (fluorescent) diazonium salts. Each method localized endogenous alkaline phosphatase to the same specific sample regions. However, we found that sections labeled using ELF-97 phosphate exhibited significantly better resolution than the other samples. The enzymatic product remained highly localized to the site of enzymatic activity, whereas signals generated using the other methods diffused. We found that the ELF-97 precipitate was more photostable than the Fast Red TR azo dye adduct. Using ELF-97 phosphate in cultured cells, we detected an intracellular activity that was only weakly labeled with the other methods, but co-localized with an antibody against alkaline phosphatase, suggesting that the ELF-97 phosphate provided greater sensitivity. Finally, we found that detecting endogenous alkaline phosphatase with ELF-97 phosphate was compatible with the use of antibodies and lectins. (J Histochem Cytochem 47:1443-1455, 1999)     

Sensitive fluorogenic substrate for alkaline phosphatase

Alkaline phosphatase serves both as a model enzyme for studies on the mechanism and kinetics of phosphomonoesterases and as a reporter in enzyme-linked immunosorbent assays (ELISAs) and other biochemical methods. The tight binding of the enzyme to its inorganic phosphate product leads to strong inhibition of catalysis and confounds measurements of alkaline phosphatase activity. We have developed an alkaline phosphatase substrate in which the fluorescence of rhodamine is triggered on P–O bond cleavage in a process mediated by a “trimethyl lock.” Although this substrate requires a nonenzymatic second step to manifest fluorescence, we demonstrated that the enzymatic first step limits the rate of fluorogenesis. The substrate enables the catalytic activity of alkaline phosphatase to be measured with high sensitivity and accuracy. Its attributes are ideal for enzymatic assays of alkaline phosphatase for both basic research and biotechnological applications.     

Conformational studies of neurotoxin II from Naja naja oxiana. Selective N-acylation, circular dichroism and nuclear-magnetic-resonance study of acylation products.

After treatment of neurotoxin II, a component part of the venom of the Middle Asian cobra Naja naja oxiana, with acetoxysuccinimide all five possible epsilon-acetylated-lysyl derivatives were obtained and the position of the label was established. Trifluoroacetylation of both the derivatives and the parent toxin yielded, respectively, the five acetyl-penta(trifluoroacetyl)-neurotoxins II and the hexa(trifluoroacetyl)-neurotoxin II, which were studied by circular dichroism (CD), 1H and 19F nuclear magnetic resonance (NMR) spectroscopy. The availability of this series of compounds made possible assignment of all six fluorine signals (from the N-terminal and the five epsilon-amino groups) in the hexa(trifluoroacetyl)-neurotoxin II NMR spectra and disclosure of the proximity of the Lys-26 and Lys-46 trifluoroacetyl groups. The pH dependence of the 19F NMR signals was determined and the pK values of the groups affecting the signal chemical shifts were calculated by a computer iterative program. In order to ascertain the relative accessibility of the lysyl side chains, the change in halfwidths of the hexatrifluoroacetylated neurotoxin II 19F signals, with addition of varying amounts of an iminoxyl spin probe, was determined. The data obtained are compared with the X-ray data on sea snake neurotoxins and the significance of the side chain interactions observed in solution is discussed.     

Fluorogenic substrates based on fluorinated umbelliferones for continuous assays of phosphatases and beta-galactosidases.

Fluorogenic substrates based on 4-methylumbelliferone (4-MU) have been widely used for the detection of phosphatase and glycosidase activities. One disadvantage of these substrates, however, is that maximum fluorescence of the reaction product requires an alkaline pH, since 4-MU has a pK(a) approximately 8. In an initial screening of five phosphatase substrates based on fluorinated derivatives of 4-MU, all with pK(a) values lower than that of 4-MU, we found that one substrate, 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), was much improved for the detection of acid phosphatase activity. When measured at the preferred acid phosphatase reaction pH (5.0), DiFMUP yielded fluorescence signals that were more than 10-fold higher than those of 4-methylumbelliferyl phosphate (MUP). DiFMUP was also superior to MUP for the detection of protein phosphatase 1 activity at pH 7 and was just as sensitive as MUP for the detection of alkaline phosphatase activity at pH 10. A beta-galactosidase substrate was also prepared based on 6, 8-difluoro-4-methylumbelliferone. This substrate, 6, 8-difluoro-4-methylumbelliferyl beta-d-galactopyranoside (DiFMUG), was found to be considerably more sensitive than the commonly used substrate 4-methylumbelliferyl beta-d-galactopyranoside (MUG), for the detection of beta-galactosidase activity at pH 7. DiFMUP and DiFMUG should have great utility for the continuous assay of phosphatase and beta-galactosidase activity, respectively, at neutral and acid pH.     

<Superscript>19</Superscript>F labelled glycosaminoglycan probes for solution NMR and non-linear (CARS) microscopy

Studying polysaccharide-protein interactions under physiological conditions by conventional techniques is challenging. Ideally, macromolecules could be followed by both in vitro spectroscopy experiments as well as in tissues using microscopy, to enable a proper comparison of results over these different scales but, often, this is not feasible. The cell surface and extracellular matrix polysaccharides, glycosaminoglycans (GAGs) lack groups that can be detected selectively in the biological milieu. The introduction of ¹⁹F labels into GAG polysaccharides is explored and the interaction of a labelled GAG with the heparin-binding protein, antithrombin, employing ¹⁹F NMR spectroscopy is followed. Furthermore, the ability of ¹⁹F labelled GAGs to be imaged using CARS microscopy is demonstrated. ¹⁹F labelled GAGs enable both ¹⁹F NMR protein-GAG binding studies in solution at the molecular level and non-linear microscopy at a microscopic scale to be conducted on the same material, essentially free of background signals.     

Membrane-associated alkaline phosphatase from Bacillus licheniformis that requires detergent for solubilization: lactoperoxidase 125I localization and molecular weight determination.

When membranes of Bacillus licheniformis MC14 were extracted exhaustively with 1 M magnesium, approximately 80% of the membrane-associated alkaline phosphatase (orthophosphoric-monoester phosphohydrolase [alkaline optimum], E.C. 3.1.3.1) was solubilized. The remaining activity could be extracted with a cationic detergent, hexadecylpyridinium chloride, without loss of enzymatic activity. The detergent-extractable alkaline phosphatase was immunoprecipitable with antibody to the salt-extractable alkaline phosphatase or the secreted alkaline phosphatase, had an approximate molecular weight of 60,000, and was localized 100% on the outer surface of the cytoplasmic membrane.     

Relation of enzyme activity to local/global stability of murine adenosine deaminase: 19F NMR studies

Adenosine deaminase (ADA, EC 3.5.4.4) is a ubiquitous (beta/alpha)8-barrel enzyme crucial for purine metabolism and normal immune competence. In this study, it was observed that loss of enzyme activity of murine ADA (mADA) precedes the global secondary and tertiary structure transition when the protein is exposed to denaturant. The structural mechanism for this phenomenon was probed using site-specific 19F NMR spectroscopy in combination with [6-19F]tryptophan labeling and inhibitor binding. There are four tryptophan residues in mADA and all are located more than 12 A from the catalytic site. The 19F NMR spectra of [6-19F]Trp-labelled mADA show that the urea-induced chemical shift change of 19F resonance of W161, one of the four tryptophan 19F nuclei, correlates with the loss of enzyme activity. The urea-induced chemical shift change of another 19F resonance of W117 correlates with the change of the apparent rate constant for the binding of transition-state analogue inhibitor deoxycoformycin to the enzyme. On the other hand, the chemical environment of the local region around W264 does not change significantly, as a consequence of perturbation by low concentrations of urea or substrate analog. The results indicate that different regions of mADA have different local stability, which controls the activity and stability of the enzyme. The results provide new insights into the relationship between the function of a protein and its conformational flexibility as well as its global stability. This study illustrates the advantage of 19F NMR spectroscopy in probing site-related conformational change information in ligand binding, enzymatic activity and protein folding.     

Copper(I)-catalysed synthesis of symmetrical perfluoroterphenyl analogues; fluorescence,antioxidant and molecular docking studies

Pentafluoroaryl analogues have been found to exhibit para specific nucleophilic aromatic substitution (S_NAr). Herein, we describe the use of S_NAr chemistry to create luminous perfluorinated symmetrical terphenyls. Both of S_NAr chemistry and copper(I)-catalysed decarboxylative cross-coupling were applied for the synthesis of the perfluorinated symmetrical terphenyls in high yields from the corresponding derivatives of aryl iodide and potassium salt of fluorobenzoate. A series of perfluorinated symmetrical terphenyls with different para alkoxy chains were synthesized. The synthesized perfluorinated terphenyl adducts were confirmed via elemental analysis, Fourier-transform infrared (FTIR), proton (¹H) carbon-13 (¹³C) and fluorine-19 (¹⁹F) nuclear magnetic resonance (NMR) spectra. The absorbance and fluorescence spectra showed solvatochromic activities. The new synthesized fluoroterphenyl hybrids were screened against antioxidant inspection over DPPH (2,2-diphenyl-1-picrylhydrazyl) performance, in assessment of vitamin C and butylated hydroxytoluene (BHT) as standard drugs exposed that fluoroterphenyl hybrid covering decyl hydrocarbons exhibited highest effectiveness through half maximal inhibitory concentration (IC₅₀) values of 21.74 μg/ml. Additionally, molecular docking procedures of the synthesized fluoroterphenyl hybrids were employed by using protein data bank (PDB ID: 5IKQ). The docking simulation displayed convenient and recognized findings with the antioxidant examination.     

Synthesis,molecular docking,and antioxidant activity of new fluorescent tetrafluoroterphenyl analogues

Nucleophilic aromatic substitution (S_NAr) chemistry has been applied to develop many functionalized pentafluorobenzene derivatives. Those compounds are highly specific at the para position of the fluorinated ring. Therefore, they are typical adducts for the preparation of antioxidant molecular systems. In this context, we report the use of S_NAr chemistry as a suitable and simple approach for the synthesis of fluorescent antioxidant perfluorinated materials bearing ether bonds in various para-substituted alkoxy chains and with high purity and excellent yields. The fluoroterphenyl core was prepared via alkylation, Cu(I)-assisted decarboxylation, and cross-coupling using the potassium salt of fluorobenzoate, followed by the reaction with different alcohols. The structures of the synthesized fluoroterphenyl adducts were investigated using FT-IR, ¹H NMR, ¹³C NMR, and ¹⁹F NMR spectroscopy. The emission spectra and absorption spectra showed solvatochromism. The newly prepared tetrafluoroterphenyl analogues were investigated by antioxidant examination using the 2,2-diphenyl-1-picrylhydrazyl assay. Results were compared with ascorbic acid and butylated hydroxytoluene as references, and revealed that the tetrafluoroterphenyl analogues containing a decyl chain had the highest activity, with an IC₅₀ value of 22.36 ± 0.19 g/ml. The produced tetrafluoroterphenyl analogues were used in molecular docking strategies with a Protein Data Bank protein ID 5IKQ. The antioxidant investigations and docking results were convergent.     

DNA hybridization assay using ATTOPHOS, a fluorescent substrate for alkaline phosphatase.

A fluorometric procedure for the detection of DNA-DNA hybrids is described. The procedure involved the detection of probe-bound alkaline phosphatase with the fluorescent substrate ATTOPHOS. This substrate is converted to ATTOFLUOR by alkaline phosphatase and fluoresces strongly at 550 nm when excited with a wavelength of 440 nm. DNA hybridization assays were performed both with dilutions of purified target plasmid DNA (pSE9 or PBR322) and whole bacterial cells. Streptavidin-alkaline phosphatase conjugates were added to react with bound probe. Fluorometric assays, as well as colorimetric assays, using 5-bromo-4-chloro-3-indolylphosphate + nitroblue tetrazolium for alkaline phosphatase activity were performed. The fluorescence of the substrate was measured at time intervals, and the slope of the regression line calculated. A slope four times greater than that of background was considered positive. One hundred femtograms or 2.2 x 10(4) molecules of homologous DNA were detected with the fluorescent assay as compared with 10,000 femtograms or 2.2 x 10(6) molecules of homologous DNA with the colorimetric assay. Similar results were obtained with whole cells. Approximately 1 x 10(3) homologous cells were detected fluorometrically and 1 x 10(5) cells were detected colorimetrically. Based on these results, we conclude that, in our hands, the DNA hybridization assay described here using ATTOPHOS as the substrate for alkaline phosphatase is a very sensitive assay for the detection of DNA-DNA hybrids.     

Dynamic reorganization of the alkaline phosphatase-containing compartment during chemotactic peptide stimulation of human neutrophils imaged by backscattered electrons

Scanning electron microscopy (EM) and cytochemical techniques were used to examine the alkaline phosphatase-containing compartment in human neutrophils after stimulation with nanomolar concentrations of N-formylmethionyl-leucyl-phenylalanine (10^–8M fMLP). Alkaline phosphatase (AlkPase) activity was demonstrated with a lead-based metal capture cytochemical method. The reaction product was visualized with the backscattered electron imaging mode of scanning EM, and analyzed by electron probe X-ray microanalysis. Alkaline phosphatase activity was detected only in fMLP-stimulated neutrophils; unstimulated neutrophils displayed no activity. Stimulation of human neutrophils with 10^–8 M fMLP induced a time-dependent intracellular redistribution of irregular round or tubular granules containing alkaline phosphatase activity, as seen by backscattering. The intracellular redistribution of alkaline phosphatase activity was accompanied by increased cytochemical activity on the cell surface. The reaction product was localized preferentially on ridges and folds of polar neutrophils. Reorganization of the AlkPase-containing compartment correlated with changes induced by fMLP in cell shape, ie, membrane ruffling and front-tail polarity, as observed with the secondary electron image mode of scanning EM. These findings demonstrate the intracellular reorganization, increase, and asymmetric distribution of alkaline phosphatase activity on the plasma membrane of human neutrophils after stimulation by chemotactic peptides.     

Structural characterization of alpha-terminal group of natural rubber. 1. Decomposition of branch-points by lipase and phosphatase treatments

Deproteinized natural rubber latex (DPNR-latex) was treated with lipase and phosphatase in order to analyze the structure of the chain-end group (alpha-terminal). The enzymatic treatment decreased the content of long-chain fatty acid ester groups in DPNR from about 6 to 2 mol per rubber molecule. The molecular weight and intrinsic viscosity were reduced to about one-third after treatment with lipase and phosphatase. The Huggins' k' constant of the enzyme-treated DPNR showed the formation of linear rubber molecules. The molecular weight distribution of DPNR changed apparently after treatment with lipase and phosphatase. (1)H NMR spectrum of rubber obtained from DPNR-latex showed small signals due to monophosphate, di-phosphate and phospholipids at the alpha-terminus. Treatment of DPNR-latex with lipase and phosphatase decreased the relative intensity of the (1)H NMR signals corresponding to phospholipids, whereas no change was observed for the signals due to mono- and diphosphates. The residual mono- and diphosphate signals as well as some phospholipid signals after lipase and phosphatase treatments indicate that mono- and diphosphate groups are directly linked at the alpha-terminus with the modified structure, expected by aggregation or linking with phospholipid molecules.