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.     

An ultrasensitive, continuous assay for xylanase using the fluorogenic substrate 6,8-difluoro-4-methylumbelliferyl beta-D-xylobioside

We describe a fluorescence-based assay for the analysis of xylanase activity using a novel fluorogenic substrate, 6,8-difluoro-4-methylumbelliferyl beta-D-xylobioside (DiFMUX(2)). Generation of fluorescent 6,8-difluoro-4-methylumbelliferone (DiFMU) from the substrate corresponded directly to xylanase activity. High-performance liquid chromatography analysis of the digestion products showed that xylanase hydrolyzed DiFMUX(2) directly to DiFMU and xylobiose. The assay provides the speed, sensitivity, and convenience required for measuring xylanase activity or for screening xylanase inhibitors in a high-throughput format and is suitable for the kinetic assay of xylanases from a variety of sources.     

Determination of Neuraminidase Kinetic Constants Using Whole Influenza Virus Preparations and Correction for Spectroscopic Interference by a Fluorogenic Substrate

The influenza neuraminidase (NA) enzyme cleaves terminal sialic acid residues from cellular receptors, a process required for the release of newly synthesized virions. A balance of NA activity with sialic acid binding affinity of hemagglutinin (HA) is important for optimal virus replication. NA sequence evolution through genetic shift and drift contributes to the continuous modulation of influenza virus fitness and pathogenicity. A simple and reliable method for the determination of kinetic parameters of NA activity could add significant value to global influenza surveillance and provide parameters for the projection of fitness and pathogenicity of emerging virus variants. The use of fluorogenic substrate 2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUNANA) and cell- or egg-grown whole influenza virus preparations have been attractive components of NA enzyme activity investigations. We describe important criteria to be addressed when determining K_m and V_max kinetic parameters using this method: (1) determination of the dynamic range of MUNANA and 4-methylumbelliferone product (4-MU) fluorescence for the instrument used; (2) adjustment of reaction conditions to approximate initial rate conditions, i.e. ≤15% of substrate converted during the reaction, with signal-to-noise ratio ≥10; (3) correction for optical interference and inner filter effect caused by increasing concentrations of MUNANA substrate. The results indicate a significant interference of MUNANA with 4-MU fluorescence determination. The criteria proposed enable an improved rapid estimation of NA kinetic parameters and facilitate comparison of data between laboratories.     

In-gel protein phosphatase assay using fluorogenic substrates

We developed a method for the detection of phosphatase activity using fluorogenic substrates after polyacrylamide gel electrophoresis. When phosphatases such as Ca²⁺/calmodulin-dependent protein kinase phosphatase (CaMKP), protein phosphatase 2C (PP2C), protein phosphatase 5 (PP5), and alkaline phosphatase were resolved by polyacrylamide gel electrophoresis in the absence of SDS and the gel was incubated with a fluorogenic substrate such as 4-methylumbelliferyl phosphate (MUP), all of these phosphatase activities could be detected in situ. Although 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) as well as MUP could be used as a fluorogenic substrate for an in-gel assay, MUP exhibited lower background fluorescence. Using this procedure, several fluorescent bands that correspond to endogenous phosphatases were observed after electrophoresis of various crude samples. The in-gel phosphatase assay could also be used to detect protein phosphatases resolved by SDS-polyacrylamide gel electrophoresis. In this case, however, the denaturation/renaturation process of resolved proteins was necessary for the detection of phosphatase activity. This procedure could be used for detection of renaturable protein phosphatases such as CaMKP and some other phosphatases expressed in cell extracts. The present fluorescent in-gel phosphatase assay is very useful, since no radioactive compounds or no special apparatus are required.     

Glycosylation and sulfation of 4-methylumbelliferone by <Emphasis Type="Italic">Gliocladium deliquescens</Emphasis> NRRL 1086

The aim of the study was to expand the substrate scope of Gliocladium deliquescens NRRL 1086 and generate coumarin glycosides using 4-methylumbelliferone (4-MU) as a substrate. The obtained results indicated that 4-MU can be metabolized to its glucoside (M1) and sulfate conjugate (M2), and the structures of the metabolites were elucidated by spectroscopic or enzymatic methods. Time course experiments detected that nearly 90% of 4-MU could be metabolized within 24 h, and the maximum yield of M1 could reach as high as 32%. Further tests revealed that the glucose concentration in the medium had little effect on the yield of M1 but time of 4-MU-adding could markedly affect the glycosylation procedure, and the favorable time to accumulate M1 was in the 12 or 24 h-old stage II culture, while in the 36 h-old or even older stage II culture, the substrate was almost metabolized to M2. The attempts to alter the ratio between M1 and M2 were performed by addition of quercetin and S-tetrahydroberberrubine or reduction of the sulfate concentration in the culture medium. Herein we describe, to the best of our knowledge, the first example of simultaneously microbial glycosylation and sulfation of coumarins.     

Surface phosphophilicity of aluminum-containing adjuvants probed by their efficiency for catalyzing the P--O bond cleavage with chromogenic and fluorogenic substrates

Aluminum-containing adjuvants are widely used in a variety of vaccine products, such as recombinant proteins, virus-like particles, conjugated polysaccharides, and recently DNA vaccines. Aluminum-containing adjuvants are also known to have a high affinity to inorganic phosphate and its mono- or diesters. Since phosphate groups are present in many antigens as well as the natural physiological environment, a better understanding of the interactions between phosphate and phospho-containing species could help in the design of improved vaccines. This report describes a convenient and novel continuous procedure to measure the avidity denoted by the new term "phosphophilicity" of phosphate and phosphate esters to the surface of aluminum-containing adjuvants. The assay measures the rate of hydrolysis of a fluorogenic substrate-6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP)-with a microplate reader. This method was based on the fundamental bioorganic phenomenon that when a tight binding event occurs, the effective concentration of nucleophile(s) will be significantly increased in the proximity of the P atom for a nucleophilic reaction (i.e., the cleavage of the P&bond;O bond) to take place. A very good leaving group (pK(a) of DiFMU approximately 4.7) in the phosphate monoester substrate makes the assay highly sensitive. Top reading of the nascent fluorescence makes the assay very convenient with no need to separate the particulate adjuvants from the reaction mixtures. The results from this assay are consistent with catalysis of the chromogenic phosphate mono- or diesters.     

The quenching effect of flavonoids on 4-methylumbelliferone, a potential pitfall in fluorimetric neuraminidase inhibition assays

Many assays aimed to test the inhibitory effects of synthetic molecules, and naturally occurring products on the neuraminidase activity exploit the hydrolysis of 2'-O-(4-methylumbelliferyl)-N-acetylneuraminic acid (4-MUNANA). The amount of the released product, 4-methylumbelliferone (4-MU), is then measured fluorimetrically. The authors attempted an analysis of the inhibitory properties of 35 naturally occurring flavonoids on neuraminidase N3, where only 29 of them were sufficiently soluble in the assay medium. During the analysis, the authors noticed a strong quenching effect due to the test compounds on the fluorescence of 4-MU. The quenching constants for the flavonoids were determined according to the Stern-Volmer approach. The extent of fluorescence reduction due to quenching and the magnitude of the fluorescence reduction measured in the inhibition assays were comparable: for 11 of 29 compounds, the two values were found to be coincident within the experimental uncertainty. These data were statistically analyzed for correlation by calculating the pertinent Pearson correlation coefficient. Inhibition and quenching were found to be positively correlated (r = 0.71, p(uncorr) = 1.5 × 10(-5)), and the correlation was maintained for the whole set of tested compounds. Altogether, the collected data imply that all of the tested flavonoids could produce false-positive results in the neuraminidase inhibition assay using 4-MUNANA as a substrate.     

Studies on ram acrosin. Fluorimetric titratiion of operational molarity with 4-methylumbelliferyl p-guanidinobenzoate.

1. Titration in sodium barbiturate buffer of acrosin, a serine proteinase from sperm acrosomes, with the ester substrate 4-methylumbelliferyl p-guanidinobenzoate gave rise to an incomplete 'burst' of 4-methylumbelliferone. Studies of the effects on the reaction of activators of acrosin (Ca2+, water-miscible solvents) showed that titrations carried out in barbiturate buffer containing 1M-CaCl2 and diluted with 0.2 vol. of dimethylsulphoxide produced a rapid quantitative burst within 4 min at 20 degrees C. 2. The net post-burst production of 4-methylumbelliferone was neglibible because (a) the acyl-enzyme was very stable, and (b) the slow post-burst formation of 4-methylumbelliferone (turnover of acyl-enzyme) was virtually equal to the slow photolytic destruction of 4-methylumbelliferone that was liberated during the burst. 3. The standard procedure permits titrations of 20-100pmol of acrosin, i.e. amounts normally taken for conventional rate assays, and with these amounts the impurities present in crude enzme fractions did not interfere. The burst was judged to be quantitative on the basis of comparisons with titrations of acrosin with p-nitrophenyl p'-quanidinobenzoate. 4. The burst reaction of trypsin with the 4-methylumbelliferyl ester was inhibited by high Ca2+ concentrations and by dimethyl sulphoxide. 5. The association and dissociation of complexes of both acrosin and trypsin with protein-type inhibitors (Kunitz pancreatic trypsin inhibitor and a spermatozoal acrosin inhibitor) are rather slow. It is thus possible, in certain cases, to use the ester to titrate both total enzyme in an inhibitor-enzyme mixture and net enzyme, i.e. the stoicheiometric excess of enzyme over inhibitor.     

Purification and properties of arylsulfatase B from high- and low-activity mouse strains

Arylsulfatase B (arylsulfate sulfohydrolase; EC 3.1.6.1) activities in C57BL/6J, SWR/J, and A/J mouse liver approximate a 5:3:1 ratio. Each enzyme was purified to apparent homogeneity, and the properties of the three purified enzymes were compared. The purified enzyme behaved as a monomer with an apparent molecular weight of 50,000. The purified enzyme catalyzed the hydrolysis of p-nitrocatechol sulfate (pNCS), 4-methylumbelliferyl sulfate (4MUS), and chondroitin-4-sulfate (C4S) heptasaccharide. Purified SWR/J arylsulfatase B possessed a higher relative electrophoretic mobility at pH 4.0 than the A/J and C57BL/6J isozymes, and the SWR/J enzyme was more thermostable than either the C57BL/6J or the A/J enzyme. No differences were observed among the three enzymes with respect to their Michaelis constants for 4MUS and pNCS, isoelectric points, responses to inhibitors, pH optima, or electrophoretic mobilities at pH 8.3. The relative in vivo rates of synthesis of C57BL/6J, A/J, and SWR/J arylsulfatase B were comparable.     

A spectrofluorimetric assay of calmodulin-dependent protein phosphatase using 4-methylumbelliferyl phosphate

A continuous spectrofluorimetric assay of calmodulin-dependent phosphatase is described. The assay monitors the formation of a fluorescent 4-methylumbelliferone from the dephosphorylation of 4-methylumbelliferyl phosphate and detects as little as 1 pmol of 4-methylumbelliferone. The phosphatase shows a Km of 1.3 mM for the substrate and a Vmax of 100 nmol/mg/min.     

Characterisation of an ionisable group involved in binding and catalysis by sialidase from influenza virus.

The effect of pH on the kinetics of sialidase purified from influenza virus (A/Tokyo/3/67, H2N2) was investigated. A pK of 9.0 for inhibition of the enzyme by three competitive inhibitors, due to an ionisable group in the active site, was observed. A similar pK was observed for V/Km for the fluorogenic substrate 2-(4-methylumbelliferyl)-N-acetyl-alpha-D-neuraminic acid. However, the shape of the V/Km profile indicates that this substrate is sticky. Solvent perturbation experiments indicated that the observed ionisable active site group is likely to be a cationic amino acid. The results provide evidence against the hypothesis that Glu 276 acts as a proton donor in the enzyme reaction and supports the proposal of a role for one of the active site cationic amino acids in binding and catalysis.     

A continuous fluorimetric assay for glycosidase activity: Human N-acetyl-β-d-hexosaminidase

The fluorescence intensity of 4-methylumbelliferone (λ_excitation = 320 nm, λ_emission = 450 nm) is approximately 120-fold greater than those of 4-methylumbelliferyl glycosides over a pH range from 3 to 7. Therefore, continuous fluorimetric monitoring of the enzymatic release of 4-methylumbelliferone from its corresponding glycoside can be performed. The technique we developed is suitable for kinetic studies of various glycosidases operating within the indicated pH range. This procedure was found to be accurate, sensitive, and rapid, as shown using N-acetyl-β-d-hexosaminidases A and B isolated from human placenta.     

Fluorimetric Assay of the Activity of Extracellular Lipases of Pseudomonas fluorescens and Serratia marcescens

A fluorimetric assay was carried out on the activity of extracellular lipase concentrations obtained from Serratia marcescens and Pseudomonas fluorescens using as substrates fatty acyl esters of 4-methylumbelliferone (4-methylumbelliferone elaidate, 4-methylumbelliferone nonanoate, 4-methylumbelliferone butyrate, 4-methylumbelliferone palmitate and 4-methylumbelliferone oleate) at pH 4.0, 6.0, 8.0 and 10.0. The Ser. marcescens and Ps. fluorescens were cultured in Pope and Skerman's basal medium (Skerman 1957) supplemented with 0.5% (w/v) of a commercial medium. The extracellular lipases were isolated and purified by ammonium sulphate precipitation. The assay was carried out by relating the fluorescent intensity emitted by two lipase concentrations on five substrates against four standard curves. These standard curves were prepared by estimating the intensity of fluorescence given by varying dilutions of 4-methylumbelliferone at the four pH levels. The results indicated that the oleic ester of 4-methylumbelliferone was a suitable substrate at pH 8.0 and pH 10.0. These pH values were also optimum for fluorescent intensity on substrates of 4-methylumbelliferone elaidate, 4-methylumbelliferone butyrate and 4-methylumbelliferone palmitate. However, on substrate 4-methylumbelliferone nonanoate, the optimum pH was 4.0.     

Loss of phosphatase activity in Ptp69D alleles supporting axon guidance defects

PTP69D is a receptor protein tyrosine phosphatase that was identified as a key regulator of neuromuscular axon guidance in Drosophila, and has subsequently been shown to play a similar role in the central nervous system and retina. Three Ptp69D alleles with mutations involving catalytically important residues exhibit a high degree of phenotypic variation with viability of mutant adult flies ranging from 0 to 96%, and ISNb motor nerve defects ranging from 11 to 57% [Desai and Purdy, 2003]. To determine whether mutations in Ptp69D affecting axon guidance and viability demonstrate losses of phosphatase activity and whether differences in catalytic potential underlie phenotypic variability, we expressed full-length wild-type and mutant PTP69D protein in Schneider 2 cells, and assessed phosphatase activity using the fluorogenic substrate 6,8-difluoro-4-methylumbelliferone phosphate (DiFMUP). Detailed biochemical characterization of wild-type PTP69D, including an examination of sensitivity to various inhibitors, in vitro catalytic efficiency, and the pH-k(cat) profile of the enzyme, suggests a common tyrosine phosphatase reaction mechanism despite lack of sequence conservation in the WPD loop. Analysis of mutant proteins revealed that every mutant had less than 1% activity relative to the wild-type enzyme, and these rates did not differ significantly from one another. These results indicate that mutations in Ptp69D resulting in axon guidance defects and lethality significantly compromise catalytic activity, yet the range of biological activity exhibited by Ptp69D mutants cannot be explained by differences in catalytic activity, as gauged by their ability to hydrolyze the substrate DiFMUP.     

Quantification of Staphylococcus aureus and Escherichia coli in the liquid medium by fluorimetry and its use in phagocytosis assay

A fluorimetric technique was compared with the plate counting method for quantification of viable cells of Staphylococcus aureus and Escherichia coli in the liquid medium. The fluorimetric assay measures the release of fluorogenic 4-methylumbelliferone (4-MU) from 4-methylumbelliferyl phosphate by the bacterial phosphatases. The increase in fluorescence was dependent on the size of bacterial inocula. Setting the fluorescence threshold at the middle of the logarithmic growth phase resulted in good linear relationship between bacterial counts and fluorescence ( r = 0·99 for both Staph. aureus and E. coli ). There was also an excellent correlation between the fluorimetric assay and the plate counting method in quantifying viable bacteria in saline ( r = 0·99). Both methods were further compared for evaluation of extracellular bacteria following phagocytosis. The fluorimetric technique, in general, gave a higher percentage of phagocytosis than the plate counting method with statistical significance for E. coli.     

Preparation and application of a fluorogenic substrate for endo-beta-xylosidase

The present paper describes a fluorometric assay for galactosaminoglycan-degrading endo-beta-xylosidase, utilizing glycosaminoglycan chains bearing a 4-methylumbelliferyl group at the reducing terminus as a substrate. This fluorogenic substrate is synthesized by human skin fibroblasts cultured in the presence of a fluorogenic xyloside, 4-methylumbelliferyl-beta-D-xyloside. The assay is based on measurement of the fluorescence of 4-methylumbelliferone, enzymatically liberated from the synthetic substrate by endo-beta-xylosidase. We examined the applicability of the assay for analysis of endo-beta-xylosidase activity.     

Synthesis of a fluorogenic mucopolysaccharide by chondrocytes in cell culture with 4-methylumbelliferyl β-d-xyloside

Culture of chondrocytes in the presence of 4-methylumbelliferyl β-d-xyloside resulted in a synthesis of protein-free, fluorogenic chondroitin sulfate which was heterogeneous on DEAE-cellulose chromatography. Degradation of the major chromatographic fraction with chondroitinase-ABC yielded, in addition to a large quantity of Δ⁴-glucuronic acid-containing disaccharides, two flurogenic oligosaccharides of different size. Quantitative analysis showed that Δ⁴-glucuronic acid, galactose, xylose, and 4-methylumbelliferone were present in the small oligosaccharide fragment in a molar ratio of 1:2:1:1. Since these analytical data are analogous to those reported for glycopeptides derived from proteochondroitin sulfates, it may be suggested that 4-methylumbelliferyl β-d-xyloside replaces the need for xylosyl protein core in the normal synthesis of proteochondroitin sulfate with a resultant production of the unusual polysaccharide bearing the added xyloside at the reducing end.     

Detection of arylsulfatase A activity after electrophoresis in polyacrylamide gels: problems and solutions.

When arylsulfatase extracted from normal human skin fibroblasts was electrophoresed in polyacrylamide gels with a Tris-glycine buffer at pH 8.4–8.6, two problems occurred. First, no arylsulfatase A activity was detected. Second, an artifactual fluorescent spot was generated when the gels were stained for arylsulfatase activity with 4-methylumbelliferyl sulfate as substrate. The artifact simulated arylsulfatase A activity in mobility but also appeared when 4-methylumbelliferyl substrates for other enzymes were used. It can be eliminated by prerunning or prolonged storage of the gets before use. The arylsulfatase A activity, however, could be recovered only when a low pH buffer system (pH 58–68) was used for electrophoresis. The highest percentage recovery (70%) of activity was obtained in acrylamide gels polymerized with ammonium persulfate, prerun for 0.5 h before use and electrophoresed with an anode buffer of acetic acid-triethanolamine at pH 5.8.     

A microfluidics-based mobility shift assay to discover new tyrosine phosphatase inhibitors

Protein tyrosine phosphatases (PTPs) play key roles in regulating tyrosine phosphorylation levels in cells. Since the discovery of PTP1B as a major drug target for diabetes and obesity, PTPs have emerged as a new and promising class of signaling targets for drug development in a variety of therapeutic areas. The routine use of generic substrate 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) in our hands led to the discovery of very similar and often not very selective molecules. Therefore, to increase the chances to discover novel chemical scaffolds, a side-by-side comparison between the DiFMUP assay and a chip-based mobility shift assay with a specific phosphopeptide was performed, on 1 PTP, using a focused set of compounds. Assay robustness and sensitivity were comparable for both the DiFMUP and mobility shift assays. The off-chip mobility shift assay required a longer development time because of identification, synthesis, and characterization of a specific peptide, and its cost per point was higher. However, although most potent scaffolds found with the DiFMUP assay were confirmed in the mobility shift format, the off-chip mobility shift assay led to the identification of previously unidentified chemical scaffolds with improved druglike properties.