Fluorometric assay for 1-deoxyfructose

A sensitive fluorometric assay has been developed for the measurement of 1-deoxyfructose in biological fluids. Samples containing 1-deoxyfructose are incubated with an equal volume of 0.01 m 3,5-diaminobenzoic acid in 5.0 m phosphoric acid in a boiling-water bath for 15 min. The fluorescent product has an emission maximum at 502 nm and an excitation maximum at 396 nm. Fluorescence is proportional to 1-deoxyfructose concentrations over a range from 0.002 to 1.0 mm. The method can be used to detect as little as 0.03 μmol of 1-deoxyfructose in deproteinized blood and in urine and no interference is observed with glucose, fructose, pyruvate, or ketone bodies. The method will also detect 1-deoxytagatose, 2-deoxyaldohexoses, and -pentoses, 2,5-anhydromannose, and a number of 2-, 3-, 4-, and 5-mono- or bis(hydroxymethyl)furans. The fluorescence properties of the products formed from all of the above compounds are similar suggesting structural similarities of the adducts formed and possible mechanistic similarities of the reactions involved.     

Fluorometric assay for intestinal peptidases

A fluorometric assay for intestinal peptidases has been developed. Amino acids liberated by hydrolysis are estimated by use of l-amino-acid oxidase, peroxidase, and the fluorogenic reagent p-hydroxyphenylacetic acid, which yields a highly fluorescent compound on oxidation. During development of fluorescence, continued hydrolysis of peptides by peptidases which contaminate available preparations of l-amino-acid oxidase is prevented by the use of two inhibitors, 1,10-phenanthroline and p-hydroxymercuribenzoate. The assay is at least 10 times more sensitive than comparable spectrophotometric methods which employ the potentially carcinogenic chromogen o-dianisidine for detection of amino acids.     

Fluorometric assay for alcohol sulfotransferase

A sensitive fluorometric assay was developed for alcohol sulfotransferase (AST). This was the first continuous fluorometric assay reported for AST. It used 3'-phosphoadenosine 5'-phosphosulfate regenerated from 3-phosphoadenosine 5'-phosphate by a recombinant phenol sulfotransferase (PST) using 4-methylumbelliferyl sulfate as the sulfuryl group donor. The recombinant PST did not use the alcohol substrate under the designed condition, and the sensitivity for AST activity was found to be comparable to that of radioactive assay as reported in the literature. The change of fluorescence intensity of 4-methylumbelliferone corresponded directly to the amount of active AST and was sensitive enough to measure nanogram or picomole amounts of the enzyme activity. This fluorometric assay was used to determine the activities of AST as purified form and in crude extracts of pig liver, rat liver, and Escherichia coli. Some properties of human dehydroepiandrosterone sulfotransferase were determined by this method and were found to be comparable to published data. Under similar assay conditions, the contaminated activities of arylsulfatase in crude extracts were also determined. This method not only is useful for the routine and detailed kinetic study of this important class of enzymes but also has the potential for the development of a high-throughput procedure using microplate reader.     

Polyvinylferrocenium modified Pt electrode for the design of amperometric choline and acetylcholine enzyme electrodes

A simple method of enzyme immobilization was investigated, which is useful for development of enzyme electrodes based on polyvinylferrocenium perchlorate coated Pt electrode surface. Enzymes were incorporated into the polymer matrix via ion exchange process by immersing polyvinylferrocenium perchlorate coated Pt electrode in enzyme solution for several times. Choline and acetylcholine enzyme electrodes were developed by co-immobilizing choline oxidase and acetylcholinesterase in polyvinylferrocenium perchlorate matrix coated on a Pt electrode surface. The amperometric responses of the enzyme electrodes were measured at +0.70 V versus SCE, which was due to the electrooxidation of enzymatically produced H2O2. The effects of the thickness of the polymeric film, pH, temperature, substrate and enzyme concentrations on the response of the enzyme electrode were investigated. The optimum pH was found to be pH 7.4 at 25 degrees C. The steady-state current of these enzyme electrodes were reproducible within +/-5.0% of the relative error. Response time was found to be 30-50s and upper limit of the linear working portions was found to be 1.2mM choline and acetylcholine concentrations in which produced detectable currents were 1.0 x 10(-6)M substrate concentrations. The apparent Michaelis-Menten constant and the activation energy of this immobilized enzyme system were found to be 1.74 mM acetylcholine and 14.9 kJ mol(-1), respectively. The effects of interferents and stability of the enzyme electrodes were also investigated.     

Fluorometric assay for ADP-ribosylarginine cleavage enzymes

A continuous fluorometric assay for enzyme activities which remove ADP-ribose linked to proteins at arginine was developed. The substrate analog, N alpha-dansyl-N omega-(1,N6-etheno-ADP-ribosyl)arginine methyl ester, was used to assay the catalytic activities of dinitrogenase reductase activating glycohydrolase from Rhodospirillum rubrum and nucleotide pyrophosphatase from Crotalus adamanteus. The assay is based on the increase in fluorescent emission by ethenoadenine accompanying the enzyme-catalyzed hydrolysis of the substrate. The assay has been used to detect activities of 10 fmol substrate cleaved per minute. The substrate anomerizes to give a 40:60 equilibrium of alpha:beta ribosylguanidinium anomers, allowing the determination of enzyme stereospecificity. The substrate was used to determine the kinetic parameters and products of the N-glycohydrolase and the pyrophosphatase.     

Fluorometric assay for pancreatic cholesterylester hydrolase

A fluorescent cholesterylester analogue, cholesteryl 6-pyrenylhexanoate (ChPH), was used as a substrate for pancreatic cholesterylester hydrolase (CEH, EC 3.1.1.13). The substrate consisted of ChPH in egg phosphatidylcholine stabilized microemulsion with the aqueous phase containing deoxycholate below its critical micellar concentration. Due to the high local concentration of the pyrene moiety in the ChPH phase the fluorescence emission due to monomeric pyrene (IM) is greatly exceeded by the excimer fluorescence intensity (IE). Upon reacting with CEH 6-pyrenylhexanoic acid and free cholesterol are formed. The fluorescent product, 6-pyrenylhexanoic acid, is transferred into the aqueous phase containing deoxycholate, thus resulting in an enhanced fluorescence due to monomeric pyrene. CEH activity can thus be assessed directly by monitoring IM vs. time without product separation. Useful assay conditions were found to be 10 microM ChPH, 0.1 microM egg phosphatidylcholine, 2 mM sodium deoxycholate at 25 degrees C and pH 6.5-7.0.     

Differential assay for choline acetyltransferase

A rapid and sensitive radiochemical assay for choline acetyltransferase (EC 2.3.1.6) is reported. The assay allows for the fact that during incubation of [¹⁴C]acetyl-CoA and choline with a cell homogenate, at least one product is formed besides [¹⁴C]acetylcholine, which passes an anion exchange column. In contrast to [¹⁴C]acetylcholine, this major contaminant ([¹⁴C]acetylcarnitine) is not hydrolyzed apparently by Electrophorus acetylcholinesterase. Therefore, two types of assays are performed, the one in the presence of an acetylcholinesterase inhibitor, the other in the presence of acetylcholinesterase from Electrophorus. After passing the reaction mixtures over anion exchange columns, the radioactivities of the effluents are determined. Their difference is proportional to the choline acetyltransferase activity.     

Enzymic radioassay for acetylcholine and choline in brain

This assay for acetylcholine (ACh) or choline in extracts of rat brain involves the isolation of the choline ester by high-voltage paper electrophoresis, alkaline hydrolysis of ACh to choline, and the quantitative enzymic conversion of choline to a radioactive derivative, P³²-phosphorylcholine. The method is specific, is applicable to large numbers of tissue samples, and has a blank value of about 3 nanograms of choline.     

Fluorometric assay for determining epoxide hydrolase activity

We have developed a rapid screening procedure that enables the screening of hundreds of enzyme samples or variants for epoxide hydrolase activity towards any substrate. The procedure detects the products of the enzymatic reaction via periodate cleavage and remaining fluorescence of carboxyfluorescein.     

Spectrophotometric assay for choline acetyltransferase

A rapid and simple spectrophotometric assay for choline acetyltransferase is described. The method employs 4,4′-dithiodipyridine to measure the coenzyme A produced by the enzymic reaction. The conditions of the assay are described. The results are compared with those obtained by the radiochemical assay of the enzyme.     

Fluorometric assay for tissue transglutaminase-mediated transamidation activity

Herein, we report the development of a direct discontinuous fluorometric transamidation assay for determining tissue transglutaminase (TG2) activity. In the assay reaction, TG2 catalyzes the formation of a biotin-fluorophore conjugate, using a fluorescent, high affinity γ-glutamyl donor substrate and a biotinylated amine as a γ-glutamyl acceptor substrate. After the reaction, the conjugate is fixed on streptavidin-coated beads and excess substrate is washed away, allowing the transamidation activity to be quantified by fluorescence measurement. This method was used to detect the activity of as little as 0.6 mU of purified TG2, and can be used for detection of activity from crude cellular lysates. Furthermore, this assay can be used for screening potential inhibitors and synthetic substrates, the latter of which was demonstrated herein.     

Fluorometric assay of peroxisomal oxidases

The present paper deals with the adaptation of the fluorometric measurement of H2O2 originally described by Guilbault et al. (1967, Anal. Chem. 39, 271) for the assay of the peroxisomal oxidation of D-amino acids, L-alpha-hydroxyacids, uric acid, and acyl-CoA esters. The present work essentially covers three facets: (i) the general kinetics of the assay of peroxisomal oxidases and the influence of each component of the assay medium on these kinetics; (ii) the measurement of peroxisomal oxidase activities in subcellular fractions and tissues from human and untreated and clofibrate-treated rodents; and (iii) the comparison between the oxidase activities measured by the fluorometric and spectrophotometric methods.