A simple spectrophotometric assay for arogenate dehydratase

A simple spectrophotometric assay for arogenate dehydratase, the enzyme that catalyzes the formation of L-phenylalanine from L-arogenate, is presented. The method couples the arogenate dehydratase reaction with that of an aromatic aminotransferase partially purified from Acinetobacter calcoaceticus. In the presence of 2-ketoglutarate, phenylpyruvate formation is measured at 320 nm at basic pH. The method was compared with two other methods already in use in our laboratory for arogenate dehydratase. The new method is simple, quick, fairly sensitive, and especially suitable for the screening of a large number of samples.     

A spectrophotometric assay for deacetoxycephalosporin C synthase

A continuous direct spectrophotometric assay for deacetoxycephalosporin C synthase was developed, based on the absorption at 260 nm characteristic of the dihydrothiazine moiety of cephalosporins. Km values of 0.18 mM for penicillin N and 0.16 mM for alpha-ketoglutarate were determined. A coupled assay using succinate thiokinase, pyruvate kinase and lactate dehydrogenase showed that succinate was a product of both deacetoxycephalosporin C synthase and hydroxylase reactions. The expandase reaction exhibited a 1:1.06 stoichiometry for deacetoxycephalosporin C and succinate.     

A simple and accurate spectrophotometric assay for phosphoenolpyruvate carboxylase activity

The rate of phosphoenolpyruvate carboxylase activity measured through the conventional coupled assay with malate dehydrogenase is underestimated due to the instability of oxaloacetate, which undergoes partial decarboxylation into pyruvate in the presence of metal ions. The addition of lactate dehydrogenase to the conventional assay allows the reduction of pyruvate formed from oxaloacetate to lactate with the simultaneous oxidation of NADH. Then, the enzymic determination of substrate and products shows that the combined activities of malate dehydrogenase and lactate dehydrogenase account for all the phosphoenolpyruvate consumed. The net result of the improved assay is a higher V_max with no apparent effect on K_m. The free divalent cation concentration appears to be the major factor in the control of the rate of oxaloacetate decarboxylation.     

A simple spectrophotometric assay for urinary leukocyte esterase activity

We have developed a sensitive spectrophotometric method for assaying urinary leukocyte esterase activity by employing a synthetic substrate, N-toluene sulfonyl indoxyl alanine ester. This kinetic assay can be performed with a small aliquot of urine, by following the change in absorbance of the chromophore at 385 nm. It is rapid and specific for leukocyte esterase and therefore can be used in the early diagnosis of urinary tract infection.     

A simple direct spectrophotometric assay for 24,25-dihydroxyvitamin D3

A simple reproducible assay to determine the concentration of 24,25-dihydroxyvitamin D₃ in blood plasma is described. This technique employs quantitation of the steroid by direct spectrophotometric analysis, rather than competitive protein binding assay after partition and high-pressure liquid chromatographic purification of plasma extracts. The concentration of metabolite observed in normal adult plasma is 2.4 ± 1.1 ng/ml.     

A simple spectrophotometric assay for fumarate hydratase in crude tissue extracts

A procedure is described for assaying fumarate hydratase by coupling malate formed from fumarate to NADP⁺ reduction via NADP malic enzyme. The procedure is much more sensitive than existing assay methods and cireumvents problems particularly associated with the use of these methods for determining fumarate hydratase in crude tissue extracts.     

A rapid and simple spectrophotometric assay of angiotensin-converting enzyme.

A rapid, simple, and accurate method for the chemical assay of anglotensin-converting enzyme has been developed. The method relies on previously published method for spectrophotometric assay of angiotensin-converting enzyme activity and on the use of 2,4,6-trichloro-s-triazine (TT) as a colorimetric reagent of hippuric acid (N-benzoylglycine). When 3% TT in dioxane was added to the incubation medium of the angiotensin-converting enzyme after stopping the incubation by the immersion of the test tubes in a boiling-water bath, the absorbance at 382 nm increased linearly as a function of both enzyme concentration and incubation time. Neither hippuryl-l-histidyl-l-leucine (HHL, substrate for this assay system) nor histidyl-leucine was positive in color reaction with TT. Accordingly, this method does not require any procedures for separation of hippuric acid from HHL. The enzyme activity was found to be highest at pH 8.3, at chloride ion concentration of 600 mm, and at HHL concentration of 3 mm, when the 5000g supernatant fluid of the rat lung was used.     

A simple spectrophotometric streptavidin-biotin binding assay utilizing biotin-4-fluorescein

A new assay for biotin binding capacity of Streptavidin (SA) is presented in this work. The assay is based on the large decrease in the extinction coefficient at 493 nm that accompanies binding of biotin-4-fluorescein (B4F) to SA. This decrease is attributed to formation of a charge transfer complex between the B4F-donor and one or more SA residues. We show that one may observe the stoichiometric binding via monitoring the absorbance at 493 nm using either SA or B4F as the titrant. The sensitivity of the assay is at the lower end of similar fluorimetric and photometric assays. Though the sensitivity is not substantially lower than other comparable techniques, this assay allows one added flexibility in working range and instrumentation, since the same stock solutions may be used for this new photometric assay or the fluorescence assay for which this ligand was first developed.     

A spectrophotometric assay for feruloyl esterases

We have developed a spectrophotometric assay for the quantitative determination of feruloyl esterase activity based on release of 4-nitrophenol from a novel substrate, 4-nitrophenyl ferulate in an emulsion of Triton X-100 in aqueous buffer solution. The release of 4-nitrophenol was linear with reaction time at an early stage of the reaction with various esterase preparations. The method proposed here is accurate, rapid, and easy to perform.     

A continuous spectrophotometric assay for catechol-O-methyltransferase

Catechol-O-methyl transferase (COMT) activity can be monitored continuously using a coupled enzyme assay in which the inhibitory product S-adenosylhomocysteine (SAH) is converted to S-inosylhomocysteine (SIH). A simple spectrophotometric assay for COMT is described based on the difference in the ultraviolet absorption spectra between SAH and SIH.     

A spectrophotometric method for calmodulin assay

The activity of calmodulin as an activator of cAMP phosphodiesterase was assayed. AMP was hydrolyzed by 5'-nucleotidase, and the adenosine formed was measured by both liquid scintillation counting and spectrophotometry at 265 nm. Calmodulin activities measured by the two methods were equivalent, indicating that spectrophotometric assay of calmodulin can be used in place of the isotopic method.     

A spectrophotometric assay for hypoxanthine-guanine phosphoribosyltransferase

The present paper describes a new spectrophotometric assay for HGPRTase activity which is more rapid than and as sensitive as the isotopic assays for this enzyme and which avoids the use of high-voltage electrophoresis and liquid scintillation counting.     

A spectrophotometric assay for strictosidine synthase

A spectrophotometric assay for strictosidine synthase is described. Strictosidine is extracted with ethyl acetate and, where high substrate concentrations are used, the organic extract is washed with dilute ammonia to remove coextracted secologanin; after evaporation of the solvent, the residue is heated with 5 M H2SO4 for 45 min and the A348 value is measured. Strictosidine production is calculated from the response of similarly treated standards. A minimum production of 10-25 nmol of strictosidine may be determined. The assay is demonstrated using extracts of cultured Cinchona ledgeriana cells.     

A spectrophotometric assay for dehydroascorbate reductase

A simple spectrophotometric assay for dehydroascorbate reductase based on the change in absorbance associated with the formation of ascorbic acid is described. Using a partially purified preparation from spinach leaves, the reaction was found to be linear with time and enzyme concentration. The reaction rate determined by this assay correlated well with that obtained by a high-performance liquid chromatography method. Possible advantages over currently available assays as well as potential applications are discussed.     

A rapid spectrophotometric assay for catechol-O-methyltransferase

A new assay technique for catechol-O-methyltransferase is described. 3,4-Dihydroxyacetophenone is used as the substrate for the assay and the products, 3-hydroxy-4-methoxyacetophenone and 4-hydroxy-3-methoxyacetophenone are detected spectrophotometrically at 344 nm in borate buffer, pH 10.0. This spectrophotometric procedure is simple, rapid, and inexpensive while retaining reasonably high sensitivity and precision.     

A simple spectrophotometric method of assay of forward motility of goat spermatozoa

Adult male sand rats (Psammomys obesus) were caught in the Béni-Abbès area. The highest testicular contents of androgens (ng/testis) were observed in autumn and in winter (testosterone: 7.6 +/- 1.1; androstenedione: 0.76 +/- 0.11) and the lowest in early summer (June) (testosterone: 1.5 +/- 0.3; androstenedione: 0.20 +/- 0.05). Values had increased by late July. Annual variations of the testosterone metabolic clearance rate (litres/24 h/100 g body wt) were similar to those of testicular androgens; values were high in winter (6.7 +/- 0.7) and lowest in June (3.2 +/- 0.3). The onset of testicular endocrine activity in sand rats was concomitant both with the highest temperatures and the start of reduction in photoperiod; its regression occurred when temperature and photoperiod were increasing.     

A spectrophotometric collagenase assay

A quantitative collagenase assay using Coomassie blue staining and microtiter spectrophotometry is described. Collagen is gelled and dried onto the bottom of microwells as substrate, washed, incubated with samples, washed again, and then stained. Absorbance at 590 nm increases linearly with increasing amounts of collagen in the range 5-40 micrograms. Bacterial and mammalian collagenases can be detected within 2 h, and 10 ng of bacterial collagenase may be detected in 16 h. For simple screening applications, activity may be detected by eye. The assay is safe, simple, fast, economical, and sensitive.