A fluorimetric method for the determination of pepsin activity

An intramolecularly quenched fluorogenic peptide containing o-aminobenzoyl (Abz) and ethylenediamine 2,4-dinitrophenyl (Eddnp) groups at amino- and carboxyl-terminal amino acid residues, Abz-Lys-Pro-Ile-Glu-Phe-Phe-Arg-Leu-Eddnp, was hydrolyzed by purified human pepsin, gastricsin, and gastric juice uniquely at the Phe-Phe bond. Kinetic parameters determined for purified pepsin were K(m)=0.68+/-0.11 microM; k(cat)=6.3+/-0.16s(-1); k(cat)/K(m)=9.26s(-1) microM(-1); Gastricsin showed K(m)=2.69+/-0.18 microM; k(cat)=0.03+/-0.005s(-1); k(cat)/K(m)=0.011s(-1) microM(-1). Gastric juice (21 samples) from subjects without gastric disorders at endoscopy examination showed activities varying from 0.0008 to 9.72 micromolml(-1)min(-1). Pepstatin A inhibition of gastric juice enzymatic activity was complete at 3.4x10(-5)M (final concentration) inhibitor. In the proposed method the presence of a unique scissile bond in the synthetic substrate provides a direct ratio between enzymatic activity and amount of substrate hydrolyzed, and a unique step reaction facilitates the use of this assay for the determination of the activity of aspartic proteinases in biological fluids and during enzyme purification procedures.     

A fluorimetric guaiacol method for thyroid peroxidase activity

A sensitive fluorimetric method with guaiacol as the hydrogen donor for peroxidase activity is described. The gist of this method is measurement of the fluorescence (excitation, 300 nm; emission, 340 nm) in cyclohexane of mainly a guaiacol dimer which forms in the early phase of the color formation. Optimum conditions of the reaction were compared for horseradish peroxidase and rat thyroid peroxidase preparations. The fluorescence intensity obtained by this method using an enzyme preparation from $\text{1}\text{20}$ of a rat thyroid gland correlated well with the color development by the guaiacol method using the same preparation from whole glands of a rat.     

A fluorimetric Morgan-Elson assay method for hyaluronidase activity

Despite their physiological importance, hyaluronidases (HAases) have long been "neglected enzymes," due, presumably, in part to the lack of rapid, sensitive assays. Currently, the colorimetric Morgan-Elson assay method, which is based upon the generation of a new reducing N-acetyl-D-glucosamine terminus with each cleavage reaction, is most widely employed but is yet insensitive. We, therefore, reinvestigated the colorimetric method and established the fluorimetric Morgan-Elson assay for HAase activity, with the optimized tetraborate reagent. The fluorimetric assay, requiring neither specialized reagents nor a long time to perform, provided high sensitivity, nearly comparable to that of enzyme-linked immunosorbent assay (ELISA)-like assays, with a detection limit of 5 x 10(-3)NFU/ml of bovine testicular HAase after 1-h incubation. The increased sensitivity permitted rapid measurement of low HAase activity in biological samples such as human and rabbit serum HAases, the latter of which has not been detected either by an ELISA-like assay or by zymography. Human serum HAase was easily characterized it along with its optimum pH and kinetic parameters.     

A fluorimetric method for the determination of tryptophan in animal tissues

Tryptophan, tryptamine and peptides containing N-terminal tryptophan give two highly fluorescent products on treatment with dithiothreitol and acid ninhydrin reagent 1 or 2. The first fluorescent product (product A) gives an emission at 500nm on activation at 390–400nm and is stable for 20min. The second product (product B), which gives an emission at 530nm on activation at 470nm, is detectable within 1h after the reaction. It gives almost maximum intensity in 4h and is stable for at least 48h. Except lysine, which in equimolar amounts gives less than 1% of a product similar to product B, no other naturally occurring amino compounds give fluorescent products. A procedure is given for the determination of 0.05–34nmol of tryptophan in tissue extracts. By using this procedure rat brain was found to contain 17.56±0.76 (s.e.m.) nmol/g wet wt.     

A fluorimetric method for measuring the activity of soil enzymes

Summary A fluorimetric method is described for the measurement of the activity of a range of soil enzymes. The method is based on the measurement of 4-methylumbelliferone (MUB), a fluorescent product liberated on hydrolysis of the enzyme substrate. The main advantage of the method over colorimetric techniques is that separation of MUB from the soil is unnecessary and the method is therefore suitable for routine, automated analyses. The method was used to measure the activity of β-cellobiase, β-galactosaminidase, β-glucosidase and β-xylosidase over a wide range of substrate concentration and in a range of soils. Kinetic parameters are reported for these enzymes. The method was also shown to be suitable for the assay of arylsulphatase and acid and alkaline phosphatase in soil. The technique should be applicable to a wide range of soil hydrolases, using the same assay methods.     

Quality-control method for the determination of biological activity of engineered calcineurin subunit B

The aim of this study was to establish a quality-control method for calcineurin subunit B(CNB) biological activity determinations. CNB enhances the p-nitrophenylphosphate(p NPP) dephosphorylating activity of calcineurin subunit A Δ316 mutant(CNAΔ316). A series of CNB concentrations were fitted to a four-parameter equation to calculate the corresponding p NPP maximum dephosphorylation rates. Values were calculated based on biological activity references using a parallel line method. The method was then validated for accuracy, precision, linearity, linear range, sensitivity, specificity, and robustness. The recovery results were greater than 98%. Intra-plate precision was 6.7%, with inter-plate precision of 10.8%. The coefficient of determination was greater than 0.98. The linear range was 0.05–50 μg m L?1, with sensitivity of 50 μg m L?1. Tested cytokines did not induce CNAΔ316 dephosphorylation of p NPP. The chosen CNAΔ316 concentration range did not affect activity determinations.     

A novel inhibitory kinetic fluorimetric method for the determination of trace methomyl in environmental samples

A novel inhibitory kinetic fluorimetric method for the determination of trace methomyl was proposed. It was shown that the Fenton reagent oxidized rhodamine B in acid medium which enabled the fluorescence quenching of the latter. The presence of trace methomyl clearly inhibited the reaction. Upon addition of EDTA, a good linear relationship between the inhibitory effect and the concentration of methomyl was observed, together with improved stabilization and sensitivity. Factors affecting the determination of trace methomyl were investigated systematically. Under the optimum conditions, the linear range for the determination of methomyl was 0.04–2.2 µg/mL; the detection limit and the quantification limit for methomyl were 0.011 and 0.037 µg/mL, respectively. The proposed method was applied to the determination of methomyl in four environmental soil samples, six environmental water samples and one synthetic sample; the results were compared with those determined by the HPLC method. The recoveries and the relative errors were 83.5–101.2 and 0.47–2.02%, respectively. The possible reaction mechanism has also been discussed. Copyright © 2009 John Wiley & Sons, Ltd.     

A kinetic method for determination of arylsulfatase activity

A new assay for arylsulfatase activity is described, which consists of direct kinetic measurements of pseudo-first-order rate constants by means of a spectrophotometric procedure. The assay is applicable for reactions occurring at different pH conditions and it can be used for a wide range of activities.     

Novel post-column derivatization method for the fluorimetric determination of norepinephrine and epinephrine

A novel method is described in which catecholamines are converted into fluorescent products by heating in alkaline borate buffer. The method was applied to the determination of norepinephrine and epinephrine after separation by high-performance liquid chromatography using a pellicular, strong cation exchanger. The new system is simpler than the system based on the trihydroxyindole reaction. It is suitable for the measurement of catecholamines in the range 0.25–20 ng. The assay of catecholamines in human urine is also described.     

A continuous fluorimetric assay for ATPase activity.

A new continuous coupled fluorimetric assay is described for ATPases in general. Thus phosphate released from ATP hydrolysis is coupled to the nucleoside phosphorylase reaction using 7-methylguanosine as a fluorescent substrate for the nucleoside phosphorylase reaction. The hydrolysis of 7-methylguanosine leads to 7-methylguanine, which has lower quantum yield and hence can be used to monitor ATP hydrolysis continuously. The method has the potential to be extended to GTPase and nucleotidyltransferase assays.     

A microplate fluorimetric assay for measuring dehalogenase activity

A fluorimetric assay was developed to measure halide release from halogenated compounds being degraded by microbes. The method relies on the property of halides to quench the fluorescence of 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ) by collision quenching. The assay shows a wide response to halide concentration (1-500 mM) and tolerates a wide pH range. Furthermore, it is simple to use, has the potential for automation and uses an inexpensive non-toxic reagent.     

A sensitive fluorimetric microassay for the determination of glutathione peroxidase activity. Application to human blood platelets

The method proposed for measuring glutathione peroxydase (GSH-Px) activity is based on the determination of oxidized glutathione (GSSG) using o-phtalaldehyde (OPT) as a fluorescent reagent. This method makes it possible to study the kinetics of both substrates (peroxide and reduced glutathione, GSH), and allosteric kinetics were found for GSH, with human platelets as the source of GSH-Px. Different methods for platelet disruption were compared. The reference values obtained for GSH-Px activity in human blood platelets by this fluorimetric procedure and the conventional enzymatic method were very similar and significantly higher than those previously reported; the reasons for this difference are discussed.     

A rapid method for determination of ribonucleotide reductase activity

Ribonucleoside diphosphate reductase activity is determined in centrifuged homogenates by following the conversion of cytosine ribonucleotide to cytosine deoxyribonucleotide. The enzymatic reaction is measured by monitoring the radioactivity of the reaction products separated by thin layer chromatography on PEI-cellulose plates. The method is rapid and permits the simultaneous processing of multiple samples.